研究者詳細 - 吉村　昭彦

論文 - 吉村　昭彦

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Significant association between joint ultrasonographic parameters and synovial inflammatory factors in rheumatoid arthritis

Kondo Y., Suzuki K., Inoue Y., Sakata K., Takahashi C., Takeshita M., Kassai Y., Miyazaki T., Morita R., Niki Y., Kaneko Y., Yasuoka H., Yamaoka K., Yoshimura A., Takeuchi T.

Arthritis Research and Therapy （Arthritis Research and Therapy） 21 （ 1 ） 2019年10月

ISSN 14786354

© 2019 The Author(s). Background: Ultrasonography (US) can directly demonstrate joint inflammation, including grayscale (GS) signs of synovial hypertrophy and power Doppler (PD) techniques to demonstrate increased blood flow and vascularization. Recently, echogenicity, especially hypoechoic synovium, has also been associated with local inflammatory activity. However, only a few studies have demonstrated correlation between histopathologic and immunopathologic evaluation and US findings. The aim of this study was to clarify whether joint US findings including synovial hypertrophy, vascularity, and echogenicity can accurately characterize synovial pathophysiology in patients with active rheumatoid arthritis (RA). Methods: A total of 44 patients with RA were included, both treated (n = 25) and untreated (n = 19) and scheduled for US examination of the knee joint with synovial fluid (SF) aspiration and two treated patients also underwent synovial biopsy. US images were quantitatively analyzed using grayscale assessment of synovial hypertrophy and PD for vascularity and echogenicity. Levels of nine SF cytokines and growth factors were also measured. Results: Both US synovial hypertrophy and PD vascularity significantly correlated with SF inflammatory cytokine levels such as IL-6, IL-8, IL-1β and IL-10 in untreated patients. Angiogenic factors, including vascular endothelial growth factor (VEGF), only correlated with PD vascularity. In the treated patients, the associations between synovial hypertrophy and any cytokines were diminished, although synovial vascularity and echogenicity correlated with IL-6 and VEGF (p < 0.05). Histopathologic analysis revealed that hypoechogenicity of the synovium correlated with marked infiltration of lymphocytes and hypervascularity. Conclusions: We demonstrated the pathophysiological origins of US findings in the joint. The degree of US vascularity of the synovium correlated with local inflammatory cytokine levels and angiogenetic factors in patients with active RA. Synovial echogenicity, and not hypertrophy, correlated with inflammation, especially in treated patients with RA.

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miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity

Łyszkiewicz M., Winter S., Witzlau K., Föhse L., Brownlie R., Puchałka J., Verheyden N., Kunze-Schumacher H., Imelmann E., Blume J., Raha S., Sekiya T., Yoshimura A., Frueh J., Ullrich E., Huehn J., Weiss S., Gutierrez M., Prinz I., Zamoyska R., Ziętara N., Krueger A.

PLoS biology （PLoS biology） 17 （ 3 ） 2019年03月

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.

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Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis

Nakamoto N., Sasaki N., Aoki R., Miyamoto K., Suda W., Teratani T., Suzuki T., Koda Y., Chu P., Taniki N., Yamaguchi A., Kanamori M., Kamada N., Hattori M., Ashida H., Sakamoto M., Atarashi K., Narushima S., Yoshimura A., Honda K., Sato T., Kanai T.

Nature Microbiology （Nature Microbiology） 4 （ 3 ） 492 - 503 2019年03月

© 2019, The Author(s), under exclusive licence to Springer Nature Limited. Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and its frequent complication with ulcerative colitis highlights the pathogenic role of epithelial barrier dysfunction. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC, yet its underlying mechanism remains unknown. Here, we identify Klebsiella pneumonia in the microbiota of patients with PSC and demonstrate that K. pneumoniae disrupts the epithelial barrier to initiate bacterial translocation and liver inflammatory responses. Gnotobiotic mice inoculated with PSC-derived microbiota exhibited T helper 17 (T H 17) cell responses in the liver and increased susceptibility to hepatobiliary injuries. Bacterial culture of mesenteric lymph nodes in these mice isolated K. pneumoniae, Proteus mirabilis and Enterococcus gallinarum, which were prevalently detected in patients with PSC. A bacterial-organoid co-culture system visualized the epithelial-damaging effect of PSC-derived K. pneumoniae that was associated with bacterial translocation and susceptibility to T H 17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the T H 17 immune response induced by PSC-derived microbiota. These results highlight the role of pathobionts in intestinal barrier dysfunction and liver inflammation, providing insights into therapeutic strategies for PSC.

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Pulmonary phagocyte-derived NPY controls the pathology of severe influenza virus infection

Fujiwara S., Hoshizaki M., Ichida Y., Lex D., Kuroda E., Ishii K., Magi S., Okada M., Takao H., Gandou M., Imai H., Hara R., Herzog H., Yoshimura A., Okamura H., Penninger J., Slutsky A., Uhlig S., Kuba K., Imai Y.

Nature Microbiology （Nature Microbiology） 4 （ 2 ） 258 - 268 2019年02月

© 2018, The Author(s), under exclusive licence to Springer Nature Limited. Crosstalk between the autonomic nervous system and the immune system by means of the sympathetic and parasympathetic pathways is a critical process in host defence. Activation of the sympathetic nervous system results in the release of catecholamines as well as neuropeptide Y (NPY). Here, we investigated whether phagocytes are capable of the de novo production of NPY, as has been described for catecholamines. We show that the synthesis of NPY and its Y1 receptor (Y1R) is increased in phagocytes in lungs following severe influenza virus infection. The genetic deletion of Npy or Y1r specifically in phagocytes greatly improves the pathology of severe influenza virus infection, which is characterized by excessive virus replication and pulmonary inflammation. Mechanistically, it is the induction of suppressor of cytokine signalling 3 (SOCS3) via NPY–Y1R activation that is responsible for impaired antiviral response and promoting pro-inflammatory cytokine production, thereby enhancing the pathology of influenza virus infection. Thus, direct regulation of the NPY–Y1R–SOCS3 pathway on phagocytes may act as a fine-tuner of an innate immune response to virus infection, which could be a therapeutic target for lethal influenza virus infection.

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IL-6, IL-17 and Stat3 are required for auto-inflammatory syndrome development in mouse

Oike T., Kanagawa H., Sato Y., Kobayashi T., Nakatsukasa H., Miyamoto K., Nakamura S., Kaneko Y., Kobayashi S., Harato K., Yoshimura A., Iwakura Y., Takeuchi T., Matsumoto M., Nakamura M., Niki Y., Miyamoto T.

Scientific Reports （Scientific Reports） 8 （ 1 ） 2018年12月

© 2018, The Author(s). Auto-inflammatory syndrome, a condition clinically distinct from rheumatoid arthritis, is characterized by systemic inflammation in tissues such as major joints, skin, and internal organs. Autonomous innate-immune activation is thought to promote this inflammation, but underlying pathological mechanisms have not been clarified nor are treatment strategies established. Here, we newly established a mouse model in which IL-1 signaling is conditionally activated in adult mice (hIL-1 cTg) and observed phenotypes similar to those seen in auto-inflammatory syndrome patients. In serum of hIL-1 cTg mice, IL-6 and IL-17 levels significantly increased, and signal transducer and activator of transcription 3 (Stat3) was activated in joints. When we crossed hIL-1 cTg with either IL-6- or IL-17-deficient mice or with Stat3 conditional knockout mice, phenotypes seen in hIL-1 cTg mice were significantly ameliorated. Thus, IL-6, IL-17 and Stat3 all represent potential therapeutic targets for this syndrome.

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Multi-omics monitoring of drug response in rheumatoid arthritis in pursuit of molecular remission

Tasaki S., Suzuki K., Kassai Y., Takeshita M., Murota A., Kondo Y., Ando T., Nakayama Y., Okuzono Y., Takiguchi M., Kurisu R., Miyazaki T., Yoshimoto K., Yasuoka H., Yamaoka K., Morita R., Yoshimura A., Toyoshiba H., Takeuchi T.

Nature Communications （Nature Communications） 9 （ 1 ） 2018年12月

© 2018 The Author(s). Sustained clinical remission (CR) without drug treatment has not been achieved in patients with rheumatoid arthritis (RA). This implies a substantial difference between CR and the healthy state, but it has yet to be quantified. We report a longitudinal monitoring of the drug response at multi-omics levels in the peripheral blood of patients with RA. Our data reveal that drug treatments alter the molecular profile closer to that of HCs at the transcriptome, serum proteome, and immunophenotype level. Patient follow-up suggests that the molecular profile after drug treatments is associated with long-term stable CR. In addition, we identify molecular signatures that are resistant to drug treatments. These signatures are associated with RA independently of known disease severity indexes and are largely explained by the imbalance of neutrophils, monocytes, and lymphocytes. This high-dimensional phenotyping provides a quantitative measure of molecular remission and illustrates a multi-omics approach to understanding drug response.

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Development and Functional Modulation of Regulatory T Cells by Transcription Factors and Epigenetics

Ito M., Iizuka-Koga M., Ando M., Yoshimura A.

Cornea （Cornea） 37 S42 - S49 2018年11月

Regulatory T cells (Tregs) are essential for the maintenance of immune homeostasis. Studies of Treg are not only necessary for understanding the mechanism of immune homeostasis but also extremely useful for the development of treatments of various immune diseases. Forkhead box P3 (Foxp3) was identified as the master gene responsible for the immune-suppressing activity of Tregs. The promoter region and several intronic enhancers, designated conserved noncoding sequence (CNS) 0, 1, 2, and 3, at the Foxp3 gene locus have important roles in Foxp3 expression and Treg development. We demonstrated that transcription factors Nr4a and Smad2/3 are required for development of thymic Tregs and induced Tregs, respectively. In addition to transcription factors, Treg-specific DNA demethylation has been shown to be important for Treg stability. In particular, DNA demethylation of CNS2 was implicated in Treg stability, and members of the ten-eleven translocation family of demethylation factors were recently demonstrated to have important roles in 5'-C-phosphate-G-3' demethylation at CNS2. This article summarizes recent findings regarding the roles of transcription factors and epigenetic modifications in the differentiation, maintenance, and function of Tregs. This review will facilitate clinical application of Tregs to diseases in the field of ophthalmology, including uveitis and age-related macular degeneration.

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Pivotal role of innate myeloid cells in cerebral post-ischemic sterile inflammation

Tsuyama J., Nakamura A., Ooboshi H., Yoshimura A., Shichita T.

Seminars in Immunopathology （Seminars in Immunopathology） 40 （ 6 ） 523 - 538 2018年11月

ISSN 18632297

© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. Inflammatory responses play a multifaceted role in regulating both disability and recovery after ischemic brain injury. In the acute phase of ischemic stroke, resident microglia elicit rapid inflammatory responses by the ischemic milieu. After disruption of the blood-brain barrier, peripheral-derived neutrophils and mononuclear phagocytes infiltrate into the ischemic brain. These infiltrating myeloid cells are activated by the endogenous alarming molecules released from dying brain cells. Inflammation after ischemic stroke thus typically consists of sterile inflammation triggered by innate immunity, which exacerbates the pathologies of ischemic stroke and worsens neurological prognosis. Infiltrating immune cells sustain the post-ischemic inflammation for several days; after this period, however, these cells take on a repairing function, phagocytosing inflammatory mediators and cellular debris. This time-specific polarization of immune cells in the ischemic brain is a potential novel therapeutic target. In this review, we summarize the current understanding of the phase-dependent role of innate myeloid cells in ischemic stroke and discuss the cellular and molecular mechanisms of their inflammatory or repairing polarization from a therapeutic perspective.

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NQO1 inhibits the TLR-dependent production of selective cytokines by promoting IκB-ζ degradation

Kimura A., Kitajima M., Nishida K., Serada S., Fujimoto M., Naka T., Fujii-Kuriyama Y., Sakamato S., Ito T., Handa H., Tanaka T., Yoshimura A., Suzuki H.

Journal of Experimental Medicine （Journal of Experimental Medicine） 215 （ 8 ） 2197 - 2209 2018年09月

ISSN 00221007

© 2018 Kimura et al. NAD(P)H:quinone oxidoreductase 1 (NQO1) protects cells against oxidative stress and toxic quinones. In this study, we found a novel role of NQO1 in suppressing Toll-like receptor (TLR)–mediated innate immune responses. NQO1-deficient macrophages selectively produced excessive amounts of IL-6, IL-12, and GM-CSF on LPS stimulation, and the deletion of NQO1 in macrophages exacerbated LPS-induced septic shock. NQO1 interacted with the nuclear IκB protein IκB-ζ, which is essential for the TLR-mediated induction of a subset of secondary response genes, including IL-6, and promoted IκB-ζ degradation in a ubiquitin-dependent manner. We demonstrated that PDLIM2, known as the ubiquitin E3 ligase, participates in NQO1-dependent IκB-ζ degradation. NQO1 augmented the association between PDLIM2 and IκB-ζ, resulting in increased IκB-ζ degradation. Collectively, this study describes a mechanism of the NQO1–PDLIM2 complex as a novel and important regulator in the innate immune signaling and suggests the therapeutic potential of NQO1 in TLR-mediated inflammation and disorders.

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Nr4a Receptors Regulate Development and Death of Labile Treg Precursors to Prevent Generation of Pathogenic Self-Reactive Cells

Sekiya T., Hibino S., Saeki K., Kanamori M., Takaki S., Yoshimura A.

Cell Reports （Cell Reports） 24 （ 6 ） 1627 - 1638.e6 2018年08月

© 2018 The Author(s) Regulatory T (Treg) cells develop from a self-reactive, CD4-single positive (CD4SP) precursor cell pool. Thus, Treg-fated developing thymocytes are expected to possess the potential to generate pathogenic self-reactive cells. However, no such pathogenic conversion has been observed, indicating mechanisms of defense to prevent such a deleterious event. Here, we show that, after the initial developmental phase, the Nr4a family of nuclear receptors promotes the development of Treg cells by cooperating with other Treg cell developmental machineries, as well as by forming a reinforcing loop with Foxp3. Nr4a-deficient Treg-fated thymocytes survive and can elicit autoimmunity, highlighting their roles in elimination of developing Treg precursors that fail to complete their development. Our findings reveal that the defective development of Treg-fated thymocytes is a potential route for the generation of pathogenic self-reactive cells, which is normally suppressed by Nr4a factors at both developmental and cell death levels. Sekiya et al. find that developing precursors of Treg cells are a labile population compared to mature Treg cells and can convert to pathogenic self-reactive cells. They show that Nr4a family nuclear receptors prevent this pathogenic conversion of Treg precursors at the level of both development and cell death.

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Generation and application of human induced-stem cell memory T cells for adoptive immunotherapy

Kondo T., Imura Y., Chikuma S., Hibino S., Omata-Mise S., Ando M., Akanuma T., Iizuka M., Sakai R., Morita R., Yoshimura A.

Cancer Science （Cancer Science） 109 （ 7 ） 2130 - 2140 2018年07月

ISSN 13479032

© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Adoptive T-cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen-specific stem cell memory T (TSCM) cells is expected to overcome this shortcoming as TSCM cells are close to naïve T cells, but are also highly proliferative, long-lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into TSCM-like cells (iTSCM) by coculturing with OP9 cells expressing Notch ligand, Delta-like 1 (OP9-hDLL1). Here we show the methodological parameters of human CD8+ iTSCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti-CD3/CD28 antibodies or by antigen-presenting cells, human iTSCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by coculture with OP9-hDLL1 cells, interleukin (IL)-7 and IL-15 (but not IL-2 or IL-21) could efficiently generate iTSCM cells. Epstein–Barr virus-specific iTSCM cells showed much stronger antitumor potentials than conventionally activated T cells in humanized Epstein–Barr virus transformed-tumor model mice. Thus, adoptive T-cell therapy with iTSCM offers a promising therapeutic strategy for cancer immunotherapy.

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Negative regulation of cytokine signaling in immunity

Yoshimura A., Ito M., Chikuma S., Akanuma T., Nakatsukasa H.

Cold Spring Harbor Perspectives in Biology （Cold Spring Harbor Perspectives in Biology） 10 （ 7 ） 2018年07月

© 2018 Cold Spring Harbor Laboratory Press; all rights reserved. Cytokines are key modulators of immunity. Most cytokines use the Janus kinase and signal transducers and activators of transcription (JAK-STAT) pathway to promote gene transcriptional regulation, but their signals must be attenuated by multiple mechanisms. These include the suppressors of cytokine signaling (SOCS) family of proteins, which represent a main negative regulation mechanism for the JAK-STAT pathway. Cytokine-inducible Src homology 2 (SH2)-containing protein (CIS), SOCS1, and SOCS3 proteins regulate cytokine signals that control the polarization of CD4+ T cells and the maturation of CD8+ T cells. SOCS proteins also regulate innate immune cells and are involved in tumorigenesis. This review summarizes recent progress on CIS, SOCS1, and SOCS3 in T cells and tumor immunity.

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Inhibition of Nr4a receptors enhances antitumor immunity by breaking treg-mediated immune tolerance

Hibino S., Chikuma S., Kondo T., Ito M., Nakatsukasa H., Omata-Mise S., Yoshimura A.

Cancer Research （Cancer Research） 78 （ 11 ） 3027 - 3040 2018年06月

ISSN 00085472

© 2018 American Association for Cancer Research. Enhanced infiltration of regulatory T cells (Treg) into tumor tissue is detrimental to patients with cancer and is closely associated with poor prognosis as they create an immunosuppressive state that suppresses antitumor immune responses. Therefore, breaking Treg-mediated immune tolerance is important when considering cancer immunotherapy. Here, we show that the Nr4a nuclear receptors, key transcription factors maintaining Treg genetic programs, contribute to Treg-mediated suppression of antitumor immunity in the tumor microenvironment. Mice lacking Nr4a1 and Nr4a2 genes specifically in Tregs showed resistance to tumor growth in transplantation models without exhibiting any severe systemic autoimmunity. The chemotherapeutic agent camptothecin and a common cyclooxygenase-2 inhibitor were found to inhibit transcriptional activity and induction of Nr4a factors, and they synergistically exerted antitumor effects. Genetic inactivation or pharmacologic inhibition of Nr4a factors unleashed effector activities of CD8þ cytotoxic T cells and evoked potent antitumor immune responses. These findings demonstrate that inactivation of Nr4a in Tregs breaks immune tolerance toward cancer, and pharmacologic modulation of Nr4a activity may be a novel cancer treatment strategy targeting the immunosuppressive tumor microenvironment. Significance: This study reveals the role of Nr4a transcription factors in Treg-mediated tolerance to antitumor immunity, with possible therapeutic implications for developing effective anticancer therapies

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Regulation of Pathogenic T Helper 17 Cell Differentiation by Steroid Receptor Coactivator-3

Tanaka K., Martinez G., Yan X., Long W., Ichiyama K., Chi X., Kim B., Reynolds J., Chung Y., Tanaka S., Liao L., Nakanishi Y., Yoshimura A., Zheng P., Wang X., Tian Q., Xu J., O'Malley B., Dong C.

Cell Reports （Cell Reports） 23 （ 8 ） 2318 - 2329 2018年05月

© 2018 The Author(s) T helper 17 (Th17) cell development is programmed by the orphan nuclear receptor RORγt, but the underlying mechanism is not well understood. Nuclear receptor-mediated transcriptional activation depends on coactivators. Here, we show that steroid receptor coactivator-3 (SRC-3) critically regulates Th17 cell differentiation. Reduced incidence of experimental autoimmune encephalitis (EAE) associated with decreased Th17 cell generation in vivo was observed in mice with SRC-3 deletion specifically in T cells. In vitro, SRC-3 deficiency did not affect TGF-β/IL-6-induced Th17 cell generation but severely impaired pathogenic Th17 differentiation induced by IL-1/IL-6/IL-23. Microarray analysis revealed that SRC-3 not only regulates IL-17A but also IL-1R1 expression. SRC-3 bound to Il17a and Il1r1 loci in a RORγt-dependent manner and was required for recruitment of the p300 acetyltransferase. Thus, SRC-3 is critical for RORγt-dependent gene expression in Th17 cell-driven autoimmune diseases. The unique transcriptional programs in IL-1-induced inflammatory Th17 cells have remained unclear. Tanaka et al. demonstrate that SRC-3 in CD4+ T helper cells functions as a specific coactivator of RORγt, a master transcription factor of Th17 cells, by regulating IL-1-IL1R1 signaling.

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Spred1 Safeguards Hematopoietic Homeostasis against Diet-Induced Systemic Stress

Tadokoro Y., Hoshii T., Yamazaki S., Eto K., Ema H., Kobayashi M., Ueno M., Ohta K., Arai Y., Hara E., Harada K., Oshima M., Oshima H., Arai F., Yoshimura A., Nakauchi H., Hirao A.

Cell Stem Cell （Cell Stem Cell） 22 （ 5 ） 713 - 725.e8 2018年05月

ISSN 19345909

© 2018 Elsevier Inc. Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesis due to compensatory upregulation of Spred2. In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal that diet-induced stress disrupts fine-tuning of Spred1-mediated signals to govern HSC homeostasis. Tadokoro et al. show that Spred1 negatively regulates HSC self-renewal in a manner supported by ROCK activity and that Spred1 safeguards HSC homeostasis under high-fat diet (HFD) conditions by regulating HSC self-renewal. The gut microbiota dysbiosis induced by HFD disrupts the fine-tuning of Spred1-mediated signals that govern HSC homeostasis.

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STAT3 expression by myeloid cells is detrimental for the T- cell-mediated control of infection with Mycobacterium tuberculosis

Gao Y., Basile J., Classon C., Gavier-Widen D., Yoshimura A., Carow B., Rottenberg M.

PLoS Pathogens （PLoS Pathogens） 14 （ 1 ） 2018年01月

ISSN 15537366

© 2018 Gao et al. STAT3 is a master regulator of the immune responses. Here we show that M. tuberculosis-infected stat3fl/fllysm cre mice, defective for STAT3 in myeloid cells, contained lower bacterial load in lungs and spleens, reduced granuloma extension but higher levels of pulmonary neutrophils. STAT3-deficient macrophages showed no improved control of intracellular mycobacterial growth. Instead, protection associated to elevated ability of stat3fl/fllysm cre antigen-presenting cells (APCs) to release IL-6 and IL-23 and to stimulate IL-17 secretion by mycobacteria-specific T cells. The increased IL-17 secretion accounted for the improved control of infection since neutralization of IL-17 receptor A in stat3fl/fllysm cre mice hampered bacterial control. APCs lacking SOCS3, which inhibits STAT3 activation via several cytokine receptors, were poor inducers of priming and of the IL-17 production by mycobacteria-specific T cells. In agreement, socs3fl/flcd11c cre mice deficient of SOCS3 in DCs showed increased susceptibility to M. tuberculosis infection. While STAT3 in APCs hampered IL-17 responses, STAT3 in mycobacteria-specific T cells was critical for IL-17 secretion, while SOCS3 in T cells impeded IL-17 secretion. Altogether, STAT3 signalling in myeloid cells is deleterious in the control of infection with M. tuberculosis.

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Reprogramming of Th1 cells into regulatory T cells through rewiring of the metabolic status

Kanamori M., Nakatsukasa H., Ito M., Chikuma S., Yoshimura A.

International Immunology （International Immunology） 30 （ 8 ） 357 - 373 2018年

ISSN 09538178

© The Japanese Society for Immunology. 2018. T helper type 1 (Th1) cells form one of the most stable CD4 T-cell subsets, and direct conversion of fully differentiated Th1 to regulatory T (Treg) cells has been poorly investigated. Here, we established a culture method for inducing Foxp3 from Th1 cells of mice and humans. This is achieved simply by resting Th1 cells without T-cell receptor ligation before stimulation in the presence of transforming growth factor-beta (TGF-ß). We named the resulting Th1-derived Foxp3+ cells Th1reg cells. Mouse Th1reg cells showed an inducible Treg-like phenotype and suppressive ability both in vitro and in vivo. Th1reg cells could also be induced from in vivo-developed mouse Th1 cells. Unexpectedly, the resting process enabled Foxp3 expression not through epigenetic changes at the locus, but through metabolic change resulting from reduced mammalian target of rapamycin complex 1 (mTORC1) activity. mTORC1 suppressed TGF-ß-induced phosphorylation of Smad2/3 in Th1 cells, which was restored in rested cells. Our study warrants future research aiming at development of immunotherapy with Th1reg cells.

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The mechanism underlying the resolution of sterile inflammation through scavenger receptor

Shchita T., Yoshimura A.

Seikagaku （Seikagaku） 90 （ 1 ） 99 - 102 2018年

ISSN 00371017

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Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells

吉村昭彦

EPIGENETICS & CHROMATIN 10 59 - 70 2017年05月

研究論文（学術雑誌）, ISSN 1756-8935

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Notch-mediated conversion of activated T cells into stem cell memory-like T cells for adoptive immunotherapy

吉村昭彦

NATURE COMMUNICATIONS 8 400 - 400 2017年05月

研究論文（学術雑誌）, ISSN 2041-1723

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Suppressors of cytokine signaling: Potential immune checkpoint molecules for cancer immunotherapy

吉村昭彦

CANCER SCIENCE 108 （ 4 ） 574 - 580 2017年04月

研究論文（学術雑誌）, ISSN 1349-7006

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Suppressors of cytokine signaling: Potential immune checkpoint molecules for cancer immunotherapy

吉村昭彦

CANCER SCIENCE 108 （ 4 ） 574 - 580 2017年04月

研究論文（学術雑誌）, ISSN 1349-7006

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Mafb prevents excess inflammation after ischemic stroke by accelerating clearance of danger signals through MSR1

吉村昭彦

JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 37 400 - 400 2017年04月

研究論文（学術雑誌）, ISSN 0271-678X

High-mobility group box I is an important mediator of microglial activation induced by cortical spreading depression

吉村昭彦

JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 37 （ 3 ） 890 - 901 2017年03月

研究論文（学術雑誌）, ISSN 0271-678X

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Role of scavenger receptors as damage-associated molecular pattern receptors in Toll-like receptor activation

吉村昭彦

INTERNATIONAL IMMUNOLOGY 29 （ 2 ） 59 - 70 2017年02月

研究論文（学術雑誌）, ISSN 0953-8178

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Control of IFN-γ production and regulatory function by the inducible nuclear protein IκB-ζ in T cells

MaruYama Takashi, Kobayashi Shuhei, Ogasawara Kouetsu, Yoshimura Akihiko, Chen WanJun, Muta Tatsushi

Journal of Leukocyte Biology 98 （ 3 ） 385 - 393 2015年09月

ISSN 0741-5400

The transcriptional regulator IκB-ζ is important for the control of apoptosis in keratinocytes. Thus, IκB-ζ-deficient mice develop autoimmune diseases, such as Sjögren’s syndrome. However, T cells also play a pivotal role in Sjögren’s syndrome. To study the role of IκB-ζ in T cells, we generated T cell-specific, IκB-ζ-deficient mice. We observed increased numbers of peripheral effector/memory CD4+ cells and IFN-γ-producing CD4+ cells in 3-week-old mice. We found that IκB-ζ can be up-regulated by TGF-β1 in naïve CD4+ T cells and that it negatively regulates IFN-γ expression. In addition, we generated Treg-specific, IκB-ζ deficient mice and found that IκB-ζ is dispensable for the plasticity and stability of T<inf>regs</inf>. However, T<inf>regs</inf> from T cell-specific, IκB-ζ-deficient mice have reduced immunoregulatory function. Thus, our data reveal a previously unappreciated role for IκB-ζ in IFN-γ production in T cells and the immunoregulatory function of T<inf>regs</inf>.

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Number of circulating follicular helper 2 T cells correlates with IgG4 and interleukin-4 levels and plasmablast numbers in IgG4-related disease

Akiyama Mitsuhiro, Suzuki Katsuya, Yamaoka Kunihiro, Yasuoka Hidekata, Takeshita Masaru, Kaneko Yuko, Kondo Harumi, Kassai Yoshiaki, Miyazaki Takahiro, Morita Rimpei, Yoshimura Akihiko, Takeuchi Tsutomu

Arthritis and Rheumatology 67 （ 9 ） 2476 - 2481 2015年09月

ISSN 2326-5191

Objective To elucidate the pathologic role of follicular helper T (Tfh) cells and their subsets in active, untreated IgG4-related disease. Methods Fifteen patients with active, untreated, biopsy-proven IgG4-related disease, 24 patients with primary Sjögren's syndrome (SS), 12 patients with allergic rhinitis, and 23 healthy controls were evaluated. Tfh cells were defined as CD3+CD4+CXCR5+CD45RA- cells. Circulating Tfh cell subsets among CXCR5+CD45RA-CD4+ T cells were defined as Tfh17 cells (CXCR3-CCR6+), Tfh1 cells (CXCR3+CCR6-), or Tfh2 cells (CXCR3-CCR6-). CD19+CD20-CD27+CD38+ cells were defined as plasmablasts. Serum cytokine levels (interleukin-4 [IL-4], IL-10, IL-21, and IL-33) were measured by cytometric bead array or enzyme-linked immunosorbent assay. Results Patients with IgG4-related disease had significantly increased levels of Tfh2 cells compared to healthy controls or patients with primary SS or allergic rhinitis. Increased Tfh2 levels were strongly associated with increased serum IgG4 levels and the IgG4:IgG ratio in IgG4-related disease. A positive correlation was observed between Tfh2 counts, plasmablast counts, and serum IL-4 levels. Interestingly, levels of plasmablasts and serum IL-4 and IgG4 decreased after treatment with glucocorticoids, whereas no obvious change was observed in Tfh2 cell counts. Conclusion The Tfh2 cell count was specifically increased in IgG4-related disease and was correlated with elevated serum levels of IgG4 and IL-4 and plasmablast counts. Tfh2 cells were the only component that was not affected by glucocorticoid treatment, suggesting that Tfh2 cells are the cell type implicated in IgG4-related disease.

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Control of IFN-γ production and regulatory function by the inducible nuclear protein IκB-ζ in T cells

MaruYama Takashi, Kobayashi Shuhei, Ogasawara Kouetsu, Yoshimura Akihiko, Chen WanJun, Muta Tatsushi

Journal of Leukocyte Biology 98 （ 3 ） 385 - 393 2015年09月

ISSN 0741-5400

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Number of circulating follicular helper 2 T cells correlates with IgG4 and interleukin-4 levels and plasmablast numbers in IgG4-related disease

Arthritis and Rheumatology 67 （ 9 ） 2476 - 2481 2015年09月

ISSN 2326-5191

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Spred1, a suppressor of the ras-ERK pathway, negatively regulates expansion and function of group 2 innate lymphoid cells

Suzuki Mayu, Morita Rimpei, Hirata Yasuko, Shichita Takashi, Yoshimura Akihiko

Journal of Immunology 195 （ 3 ） 1273 - 1281 2015年08月

ISSN 0022-1767

Cytokines from group 2 innate lymphoid cells (ILC2s) have been implicated in acute allergic responses, such as papain-induced lung inflammation. However, the means of homeostatic regulation of ILC2s have not been established. In this study, we demonstrated that Spred1, a negative regulator of the Ras-ERK pathway, plays an important role in the proliferation and apoptosis of ILC2s and in cytokine secretion from ILC2s. Intranasal administration of papain stimulated IL-5 and IL-13 production in the lung, which was enhanced when Spred1 was deleted. In vitro, Spred1-/- ILC2s proliferated faster than wild type ILC2s did and produced higher levels of cytokines in response to IL-33. On the contrary, a MEK inhibitor suppressed ILC2 proliferation and cytokine production. Spred1 deficiency resulted in stabilization of GATA3, which has been shown to play essential roles in the maintenance and cytokine production of ILC2. These data suggest that Spred1 negatively regulates ILC2 development and functions through the suppression of the Ras-ERK pathway.

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Spred1, a suppressor of the ras-ERK pathway, negatively regulates expansion and function of group 2 innate lymphoid cells

Suzuki Mayu, Morita Rimpei, Hirata Yasuko, Shichita Takashi, Yoshimura Akihiko

Journal of Immunology 195 （ 3 ） 1273 - 1281 2015年08月

ISSN 0022-1767

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Smad2 and Smad3 Inversely Regulate TGF-β Autoinduction in Clostridium butyricum-Activated Dendritic Cells

Kashiwagi Ikkou, Morita Rimpei, Schichita Takashi, Komai Kyoko, Saeki Keita, Matsumoto Makoto, Takeda Kiyoshi, Nomura Masatoshi, Hayashi Atsushi, Kanai Takanori, Yoshimura Akihiko

Immunity 43 （ 1 ） 65 - 80 2015年07月

ISSN 1074-7613

Colonization with a mixture of Clostridium species has been shown to induce accumulation of induced regulatory T (iTreg) cells in the colon. Transforming growth factor-β (TGF-β) is an essential factor for iTreg cell induction; however, the relationship between Clostridium species and TGF-β remains to be clarified. Here we demonstrated that a gram-positive probiotic bacterial strain, Clostridium butyricum (C. butyricum), promoted iTreg cell generation in the intestine through induction of TGF-β1 from lamina propria dendritic cells (LPDCs). C. butyricum-mediated TGF-β1 induction was mainly Toll-like receptor 2 (TLR2) dependent, and the ERK-AP-1 kinase pathway played an important role. In addition, the autocrine TGF-β-Smad3 transcription factor signal was necessary for robust TGF-β expression in DCs, whereas Smad2 negatively regulated TGF-β expression. Smad2-deficient DCs expressed higher concentrations of TGF-β and were tolerogenic for colitis models. This study reveals a novel mechanism of TGF-β induction by Clostridia through a cooperation between TLR2-AP-1 and TGF-β-Smad signaling pathways.

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Smad2 and Smad3 Inversely Regulate TGF-β Autoinduction in Clostridium butyricum-Activated Dendritic Cells

Kashiwagi Ikkou, Morita Rimpei, Schichita Takashi, Komai Kyoko, Saeki Keita, Matsumoto Makoto, Takeda Kiyoshi, Nomura Masatoshi, Hayashi Atsushi, Kanai Takanori, Yoshimura Akihiko

Immunity 43 （ 1 ） 65 - 80 2015年07月

ISSN 1074-7613

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STAP-2 protein expression in B16F10 melanoma cells positively regulates protein levels of tyrosinase, which determines organs to infiltrate in the body

Sekine Yuichi, Togi Sumihito, Muromoto Ryuta, Kon Shigeyuki, Kitai Yuichi, Yoshimura Akihiko, Oritani Kenji, Matsuda Tadashi

Journal of Biological Chemistry 290 （ 28 ） 17462 - 17473 2015年07月

ISSN 0021-9258

Melanoma is the most serious type of skin cancer, with a highly metastatic phenotype. In this report, we show that signal transducing adaptor protein 2 (STAP-2) is involved in cell migration, proliferation, and melanogenesis as well as chemokine receptor expression and tumorigenesis in B16F10 melanoma cells. This was evident in mice injected with STAP-2 shRNA (shSTAP-2)-expressing B16F10 cells, which infiltrated organs in a completely different pattern from the original cells, showing massive colonization in the liver, kidney, and neck but not in the lung. The most important finding was that STAP-2 expression determined tyrosinase protein content. STAP-2 colocalized with tyrosinase in lysosomes and protected tyrosinase from protein degradation. It is noteworthy that B16F10 cells with knocked down tyrosinase showed similar cell characteristics as shSTAP-2 cells. These results indicated that tyrosinase contributed to some cellular events beyond melanogenesis. Taken together, one possibility is that STAP-2 positively regulates the protein levels of tyrosinase, which determines tumor invasion via controlling chemokine receptor expression.

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STAP-2 protein expression in B16F10 melanoma cells positively regulates protein levels of tyrosinase, which determines organs to infiltrate in the body

Sekine Yuichi, Togi Sumihito, Muromoto Ryuta, Kon Shigeyuki, Kitai Yuichi, Yoshimura Akihiko, Oritani Kenji, Matsuda Tadashi

Journal of Biological Chemistry 290 （ 28 ） 17462 - 17473 2015年07月

ISSN 0021-9258

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Polarization diversity of human CD4+ stem cell memory T cells

Takeshita Masaru, Suzuki Katsuya, Kassai Yoshiaki, Takiguchi Maiko, Nakayama Yusuke, Otomo Yuki, Morita Rimpei, Miyazaki Takahiro, Yoshimura Akihiko, Takeuchi Tsutomu

Clinical Immunology 159 （ 1 ） 107 - 117 2015年07月

ISSN 1521-6616

T cells are considered to develop through three stages, from naïve T (Tn) into central memory T (Tcm) and finally into effector memory T (Tem). Among the subsets of Tn, stem cell memory T (Tscm) were recently found to be the least developed memory subset. While this subset was revealed to possess self-reproducibility and multipotentiality, little is known about the relationship between development and polarity. We conducted transcriptome analysis of human CD4+ T subsets and found that Tscm was a clearly distinct subset, located between Tn and Tcm. Surface antigen analysis and differentiation assay showed that the flexibility of polarity and the cytokine production progressively changed as the differentiation of CD4+ T cells advanced. Interestingly, we found that most cells of the CD45RO-CCR7+CCR6+ subset, hitherto considered the naïve precursor of Th17, were in fact Tscm. These findings may advance our understanding of the highly heterogeneous human helper T cells.

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Polarization diversity of human CD4+ stem cell memory T cells

Takeshita Masaru, Suzuki Katsuya, Kassai Yoshiaki, Takiguchi Maiko, Nakayama Yusuke, Otomo Yuki, Morita Rimpei, Miyazaki Takahiro, Yoshimura Akihiko, Takeuchi Tsutomu

Clinical Immunology 159 （ 1 ） 107 - 117 2015年07月

ISSN 1521-6616

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Bruton's tyrosine kinase is essential for NLRP3 inflammasome activation and contributes to ischaemic brain injury

Ito Minako, Shichita Takashi, Okada Masahiro, Komine Ritsuko, Noguchi Yoshiko, Yoshimura Akihiko, Morita Rimpei

Nature Communications 6 2015年06月

Inflammasome activation has been implicated in various inflammatory diseases including post-ischaemic inflammation after stroke. Inflammasomes mediate activation of caspase-1, which subsequently induces secretion of pro-inflammatory cytokines such as IL-1β and IL-18, as well as a form of cell death called pyroptosis. In this study, we report that Bruton's tyrosine kinase (BTK) is an essential component of the NLRP3 inflammasome, in which BTK physically interacts with ASC and NLRP3. Inhibition of BTK by pharmacological or genetic means severely impairs activation of the NLRP3 inflammasome. The FDA-approved BTK inhibitor ibrutinib (PCI-32765) efficiently suppresses infarct volume growth and neurological damage in a brain ischaemia/reperfusion model in mice. Ibrutinib inhibits maturation of IL-1β by suppressing caspase-1 activation in infiltrating macrophages and neutrophils in the infarcted area of ischaemic brain. Our study indicates that BTK is essential for NLRP3 inflammasome activation and could be a potent therapeutic target in ischaemic stroke.

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Bruton's tyrosine kinase is essential for NLRP3 inflammasome activation and contributes to ischaemic brain injury

Ito Minako, Shichita Takashi, Okada Masahiro, Komine Ritsuko, Noguchi Yoshiko, Yoshimura Akihiko, Morita Rimpei

Nature Communications 6 2015年06月

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A gp130-Src-YAP module links inflammation to epithelial regeneration

Taniguchi Koji, Wu Li Wha, Grivennikov Sergei I., De Jong Petrus R., Lian Ian, Yu Fa Xing, Wang Kepeng, Ho Samuel B., Boland Brigid S., Chang John T., Sandborn William J., Hardiman Gary, Raz Eyal, Maehara Yoshihiko, Yoshimura Akihiko, Zucman-Rossi Jessica, Guan Kun Liang, Karin Michael

Nature 519 （ 7541 ） 57 - 62 2015年03月

ISSN 0028-0836

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

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A gp130-Src-YAP module links inflammation to epithelial regeneration

Nature 519 （ 7541 ） 57 - 62 2015年03月

ISSN 0028-0836

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Anti-IL-1^-/-3 p40 antibody attenuates experimental chronic graft-versus-host disease via suppression of IFN-γ/IL-17-producing cells

Okamoto Sachiyo, Fujiwara Hideaki, Nishimori Hisakazu, Matsuoka Ken Ichi, Fujii Nobuharu, Kondo Eisei, Tanaka Takehiro, Yoshimura Akihiko, Tanimoto Mitsune, Maeda Yoshinobu

Journal of Immunology 194 （ 3 ） 1357 - 1363 2015年02月

ISSN 0022-1767

Chronic graft-versus-host disease (GVHD) is a major cause of late death and morbidity after allogeneic hematopoietic cell transplantation. Recently, in addition to Th2 cells, Th1 and Th17 cells have been shown to contribute to chronic GVHD progression. IL-12 induces Th1 cells and IL-23 plays a role in stabilizing and/or amplifying Th17 cells, as well as in inducing IFN-γ/IL-17 double-producing cells. Because mAb targeting the p40 subunit common to both IL-12 and IL-23 can inhibit both IL-12R and IL-23R-mediated signaling, we investigated the effects of anti-p40 mAb on a well-defined chronic GVHD mice model. Treatment of anti-p40 mAb in allogeneic recipients significantly reduced the severity of clinical and pathological chronic GVHD. Intracellular staining revealed that IFN-γ single-positive (IL-17^-) and IFN-γ/IL-17 double-positive cells were suppressed in anti-p40 mAb-treated allogeneic recipients compared with control recipients. The cytokine levels of IFN-γ and IL-17 were also decreased in serum from anti-p40 mAb-treated allogeneic recipients. T-bet expression of donor IL-17⁺ CD4⁺ T cells was reduced significantly in anti-p40 mAb-treated recipients, and this reduction in T-bet expression was associated with IL-22 production by donor T cells. These results suggested that anti-p40 mAb attenuated chronic GVHD via suppression of IFN-γ/IL-17-producing cells, and that targeting the IL-12/IL-23 pathway may represent a promising therapeutic strategy for preventing and treating chronic GVHD.

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Anti-IL-1^-/-3 p40 antibody attenuates experimental chronic graft-versus-host disease via suppression of IFN-γ/IL-17-producing cells

Okamoto Sachiyo, Fujiwara Hideaki, Nishimori Hisakazu, Matsuoka Ken Ichi, Fujii Nobuharu, Kondo Eisei, Tanaka Takehiro, Yoshimura Akihiko, Tanimoto Mitsune, Maeda Yoshinobu

Journal of Immunology 194 （ 3 ） 1357 - 1363 2015年02月

ISSN 0022-1767

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ETS transcription factor ETV2 directly converts human fibroblasts into functional endothelial cells

Morita Rimpei, Suzuki Mayu, Kasahara Hidenori, Shimizu Nana, Shichita Takashi, Sekiya Takashi, Kimura Akihiro, Sasaki Ken Ichiro, Yasukawa Hideo, Yoshimura Akihiko

Proceedings of the National Academy of Sciences of the United States of America 112 （ 1 ） 160 - 165 2015年01月

ISSN 0027-8424

Transplantation of endothelial cells (ECs) is a promising therapeutic approach for ischemic disorders. In addition, the generation of ECs has become increasingly important for providing vascular plexus to regenerated organs, such as the liver. Although many attempts have been made to generate ECs from pluripotent stem cells and nonvascular cells, the minimum number of transcription factors that specialize in directly inducing vascular ECs remains undefined. Here, by screening 18 transcription factors that are important for both endothelial and hematopoietic development, we demonstrate that ets variant 2 (ETV2) alone directly converts primary human adult skin fibroblasts into functional vascular endothelial cells (ETVECs). In coordination with endogenous FOXC2 in fibroblasts, transduced ETV2 elicits expression of multiple key endothelial development factors, including FLI1, ERG, and TAL1, and induces expression of endothelial functional molecules, including EGFL7 and von Willebrand factor. Consequently, ETVECs exhibits EC characteristics in vitro and forms mature functional vasculature in Matrigel plugs transplanted in NOD SCID mice. Furthermore, ETVECs significantly improve blood flow recovery in a hind limb ischemic model using BALB/c-nu mice. Our study indicates that the creation of ETVECs provides further understanding of human EC development induced by ETV2.

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ETS transcription factor ETV2 directly converts human fibroblasts into functional endothelial cells

Morita Rimpei, Suzuki Mayu, Kasahara Hidenori, Shimizu Nana, Shichita Takashi, Sekiya Takashi, Kimura Akihiro, Sasaki Ken Ichiro, Yasukawa Hideo, Yoshimura Akihiko

Proceedings of the National Academy of Sciences of the United States of America 112 （ 1 ） 160 - 165 2015年01月

ISSN 0027-8424

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Distinct effects of endogenous interleukin-23 on eosinophilic airway inflammation in response to different antigens

Ogawa Rika, Suzuki Yusuke, Kagawa Shizuko, Masaki Katsunori, Fukunaga Koichi, Yoshimura Akihiko, Fujishima Seitaro, Terashima Takeshi, Betsuyaku Tomoko, Asano Koichiro

Allergology International 2015年01月

ISSN 1323-8930

Background: The role of interleukin (IL)-23 in asthma pathophysiology is still controversial. We examined its role in allergic airway inflammation in response to two distinct antigens using IL-23-deficient mice. Methods: Allergic airway inflammation was evaluated in wild-type and IL-23p19 -/- mice. Mice were sensitized to ovalbumin (OVA) or house dust mite (HDM) by intraperitoneal injection of antigen and their airways were then exposed to the same antigen. Levels of antigen-specific immunoglobulins in serum as well as cytokines in bronchoalveolar or peritoneal lavage fluid and lung tissue were determined by enzyme-linked immunosorbent assay and/or quantitative polymerase chain reaction. Results: Deficiency of IL-23p19 decreased eosinophils and Th2 cytokines in bronchoalveolar lavage fluid (BALF) of OVA-treated mice, while it increased BALF eosinophils of HDM-treated mice. Peritoneal injection of OVA with alum, but not of HDM, induced local synthesis of IL-6, IL-10, and IL-23. Systemic production of antigen-specific IgG<inf>1</inf> was partially dependent on IL-23. In contrast, airway exposure to HDM, but not to OVA, induced IL-23p19 mRNA expression in the lungs. In IL-23p19-deficient mice, HDM-exposed lungs did not exhibit the induction of IL-17A, which negatively regulates eosinophilic inflammation. Conclusions: Different antigens induced IL-23 at different part of the body in our similar asthma models. Endogenous IL-23 production at the site of antigen sensitization facilitates type-2 immune responses, whereas IL-23 production and subsequent IL-17A synthesis in the airways suppresses allergic inflammation.

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Distinct effects of endogenous interleukin-23 on eosinophilic airway inflammation in response to different antigens

Ogawa Rika, Suzuki Yusuke, Kagawa Shizuko, Masaki Katsunori, Fukunaga Koichi, Yoshimura Akihiko, Fujishima Seitaro, Terashima Takeshi, Betsuyaku Tomoko, Asano Koichiro

Allergology International 2015年01月

ISSN 1323-8930

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Suppressor of cytokine signaling 3 is an inducible host factor that regulates virus egress during Ebola virus infection

Okumura Atsushi, Rasmussen Angela L., Halfmann Peter, Feldmann Friederike, Yoshimura Akihiko, Feldmann Heinz, Kawaoka Yoshihiro, Harty Ronald N., Katze Michael G.

Journal of Virology 89 （ 20 ） 10399 - 10406 2015年

ISSN 0022-538X

Ebola virus (EBOV) initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal EBOV infections. The VP40 matrix protein is a key viral structural protein that is critical for virion egress. Physical and functional interactions between VP40 and host proteins such as Tsg101 and Nedd4 facilitate efficient release of VP40-driven viruslike particles (VLPs) and infectious virus. Here, we show that host suppressor of cytokine signaling 3 (SOCS3) can also bind to EBOV VP40, leading to enhanced ubiquitinylation and egress of VP40. Indeed, titers of infectious EBOV derived from SOCS3 knockout mouse embryonic fibroblasts (MEFs) were significantly reduced compared to those from wild-type (WT) MEFs at 24 and 48 h postinfection. Importantly, this reduced virus yield could be rescued back to WT levels by exogenously expressing SOCS3. Lastly, we show that SOCS3 expression is induced by EBOV glycoprotein (GP) expression and that VLPs containing EBOV VP40 and GP induced production of proinflammatory cytokines, which induced SOCS3 for negative-feedback regulation. These data indicate that host innate immune protein SOCS3 may play an important role in budding and pathogenesis of EBOV.

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Resolution of elevated circulating regulatory T cells by corticosteroids in patients with IgG4-related dacryoadenitis and sialoadenitis

Akiyama Mitsuhiro, Suzuki Katsuya, Kassai Yoshiaki, Miyazaki Takahiro, Morita Rimpei, Yoshimura Akihiko, Takeuchi Tsutomu

International Journal of Rheumatic Diseases 2015年

ISSN 1756-1841

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Resolution of elevated circulating regulatory T cells by corticosteroids in patients with IgG4-related dacryoadenitis and sialoadenitis

Akiyama Mitsuhiro, Suzuki Katsuya, Kassai Yoshiaki, Miyazaki Takahiro, Morita Rimpei, Yoshimura Akihiko, Takeuchi Tsutomu

International Journal of Rheumatic Diseases 2015年

ISSN 1756-1841

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The lysosome rupture-activated TAK1-JNK pathway regulates NLRP3 inflammasome activation

Okada Masahiro, Matsuzawa Atsushi, Yoshimura Akihiko, Ichijo Hidenori

Journal of Biological Chemistry 289 （ 47 ） 32926 - 32936 2014年11月

ISSN 0021-9258

Results: Inhibition of CaMKII, TAK1, or JNK specifically suppressed lysosome rupture-induced NLRP3 inflammasome activation.Background: The mechanisms underlying lysosome rupture-mediated inflammasome activation are not understood.Conclusion: Activation of the Ca²⁺-CaMKII-TAK1-JNK pathway in lysosome rupture is necessary for complete activation of the NLRP3 inflammasome.Significance: Our results suggest novel roles for the Ca²⁺-CaMKII-TAK1-JNK pathway in the regulation of the inflammasome and propose potential therapeutic targets for inflammatory diseases.

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The lysosome rupture-activated TAK1-JNK pathway regulates NLRP3 inflammasome activation

Okada Masahiro, Matsuzawa Atsushi, Yoshimura Akihiko, Ichijo Hidenori

Journal of Biological Chemistry 289 （ 47 ） 32926 - 32936 2014年11月

ISSN 0021-9258

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Post-ischemic inflammation regulates neural damage and protection

Shichita Takashi, Ito Minako, Yoshimura Akihiko

Frontiers in Cellular Neuroscience 8 （ OCT ） 2014年10月

ISSN 1662-5102

Post-ischemic inflammation is important in ischemic stroke pathology. However, details of the inflammation process, its resolution after stroke and its effect on pathology and neural damage have not been clarified. Brain swelling, which is often fatal in ischemic stroke patients, occurs at an early stage of stroke due to endothelial cell injury and severe inflammation by infiltrated mononuclear cells including macrophages, neutrophils, and lymphocytes. At early stage of inflammation, macrophages are activated by molecules released from necrotic cells [danger-associated molecular patterns (DAMPs)], and inflammatory cytokines and mediators that increase ischemic brain damage by disruption of the blood–brain barrier are released. After post-ischemic inflammation, macrophages function as scavengers of necrotic cell and brain tissue debris. Such macrophages are also involved in tissue repair and neural cell regeneration by producing tropic factors. The mechanisms of inflammation resolution and conversion of inflammation to neuroprotection are largely unknown. In this review, we summarize information accumulated recently about DAMP-induced inflammation and the neuroprotective effects of inflammatory cells, and discuss next generation strategies to treat ischemic stroke.

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Post-ischemic inflammation regulates neural damage and protection

Shichita Takashi, Ito Minako, Yoshimura Akihiko

Frontiers in Cellular Neuroscience 8 （ OCT ） 2014年10月

ISSN 1662-5102

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Loss of Sprouty4 in T cells ameliorates experimental autoimmune encephalomyelitis in mice by negatively regulating IL-1β receptor expression

Fukaya Tomohiro, Someya Kazue, Hibino Sana, Okada Masahiro, Yamane Humitsugu, Taniguchi Koji, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 447 （ 3 ） 471 - 478 2014年05月

ISSN 0006-291X

Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-β for early differentiation. To gain pathogenicity, however, Th17 cells require IL-1β and IL-23. The underlying mechanism by which these confer pathogenicity is not well understood. Here we show that Sprouty4, an inhibitor of the PLCγ-ERK pathway, critically regulates inflammatory Th17 (iTh17) cell differentiation. Sprouty4-deficient mice, as well as mice adoptively transferred with Sprouty4-deficient T cells, were resistant to experimental autoimmune encephalitis (EAE) and showed decreased Th17 cell generation in vivo. In vitro, Sprouty4 deficiency did not severely affect TGF-β/IL-6-induced Th17 cell generation but strongly impaired Th17 differentiation induced by IL-1/IL-6/IL-23. Analysis of Th17-related gene expression revealed that Sprouty4-deficient Th17 cells expressed lower levels of IL-1R1 and IL-23R, while RORγt levels were similar. Consistently, overexpression of Sprouty4 or pharmacological inhibition of ERK upregulated IL-1R1 expression in primary T cells. Thus, Sprouty4 and ERK play a critical role in developing iTh17 cells in Th17 cell-driven autoimmune diseases. © 2014 Elsevier Inc.

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Loss of Sprouty4 in T cells ameliorates experimental autoimmune encephalomyelitis in mice by negatively regulating IL-1β receptor expression

Fukaya Tomohiro, Someya Kazue, Hibino Sana, Okada Masahiro, Yamane Humitsugu, Taniguchi Koji, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 447 （ 3 ） 471 - 478 2014年05月

ISSN 0006-291X

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Signal-transducing adaptor protein-2 controls the IgE-Mediated, mast cell-mediated anaphylactic responses

Sekine Yuichi, Nishida Keigo, Yamasaki Satoru, Muromoto Ryuta, Kon Shigeyuki, Kashiwakura Jun Ichi, Saitoh Kodai, Togi Sumihito, Yoshimura Akihiko, Oritani Kenji, Matsuda Tadashi

Journal of Immunology 192 （ 8 ） 3488 - 3495 2014年04月

ISSN 0022-1767

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that regulates immune and inflammatory responses through interactions with a variety of signaling and transcriptional molecules. In the current study, we clarified the physiological role of STAP-2 in mast cell function, a key mediator of IgE-associated allergic responses. STAP-2 is constitutively expressed in mast cells. STAP-2 deficiency in mast cells greatly enhances FcRI-mediated signals, resulting in the increased tyrosine phosphorylation of the phospholipase C-g isoform, calcium mobilization, and degranulation. Of importance, STAP-2-deficient mice challenged with DNP-BSA after passive sensitization with anti-DNP IgE show more severe rectal temperature decrease than do wild-type mice. STAP-2-deficient mice also show increased vascular permeability and more severe cutaneous anaphylaxis after DNP-BSA injection. These regulatory functions performed by STAP-2 indicate that there is an interaction between STAP-2 and FcRI. In addition, our previous data indicate that STAP-2 binds to the phospholipase C-g isoform and IkB kinase-b. Therefore, our data described in this article strongly suggest that manipulation of STAP-2 expression in mast cells may control the pathogenesis of allergic diseases and have the potential for treating patients with allergy. Copyright © 2014 by The American Association of Immunologists, Inc.

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Signal-transducing adaptor protein-2 controls the IgE-Mediated, mast cell-mediated anaphylactic responses

Sekine Yuichi, Nishida Keigo, Yamasaki Satoru, Muromoto Ryuta, Kon Shigeyuki, Kashiwakura Jun Ichi, Saitoh Kodai, Togi Sumihito, Yoshimura Akihiko, Oritani Kenji, Matsuda Tadashi

Journal of Immunology 192 （ 8 ） 3488 - 3495 2014年04月

ISSN 0022-1767

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A proteomic approach for the elucidation of the specificity of ectodomain shedding

Shirakabe Kyoko, Shibagaki Yoshio, Yoshimura Akihiko, Koyasu Shigeo, Hattori Seisuke

Journal of Proteomics 98 233 - 243 2014年02月

ISSN 1874-3919

Ectodomain shedding (shedding) is a posttranslational modification mechanism, which liberates extracellular domains of membrane proteins through juxtamembrane processing. Because shedding alters cell characteristics in a rapid and irreversible manner, it must be strictly regulated. However, the regulatory mechanisms of shedding in response to environmental changes remain obscure. To evaluate the regulatory mechanisms of endogenous shedding, we previously developed a proteomic screening system to identify shedding targets. This system revealed a comprehensive picture of membrane proteins shed under defined conditions. In this study, we have improved the screening system to compare the shedding patterns in a mouse macrophage cell line treated with two different shedding inducers, lipopolysaccharide (LPS) and 12. O-tetradecanoylphorbol 13-acetate (TPA). We show here that LPS simultaneously activates the shedding of multiple membrane proteins. We further show that TPA specifically activates the shedding of αM/β2 integrin (Mac-1), which was not shed upon LPS-stimulation of macrophages. These results clearly demonstrate that the regulation of endogenous membrane protein shedding is both stimulus- and substrate-specific. Biological significance: The shedding targets reported to date play pivotal roles in a variety of biological phenomena, including the immunological response, cell growth, cell adhesion and cell movement. In addition, several disease-related membrane proteins are shedding targets. Thus, understanding the regulation of shedding is important for the elucidation of pathogenesis and the development of therapeutic strategies. We submit that a comprehensive characterization of endogenous shedding is indispensable for understanding the regulatory mechanisms of shedding, and thus have developed a proteomic screening system to identify shedding targets. In this study, using our screening system, we demonstrate that different extracellular stimuli activate different types of shedding, even in a single cell. Our results prove that this proteomic approach is quite effective for the elucidation of the regulatory mechanisms of shedding. © 2014 Elsevier B.V.

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A proteomic approach for the elucidation of the specificity of ectodomain shedding

Shirakabe Kyoko, Shibagaki Yoshio, Yoshimura Akihiko, Koyasu Shigeo, Hattori Seisuke

Journal of Proteomics 98 233 - 243 2014年02月

ISSN 1874-3919

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Prevention of allogeneic cardiac graft rejection by transfer of ex vivo expanded antigen-specific regulatory T-cells

Takasato Fumika, Morita Rimpei, Schichita Takashi, Sekiya Takashi, Morikawa Yasuhide, Kuroda Tatsuo, Niimi Masanori, Yoshimura Akihiko

PLoS ONE 9 （ 2 ） 2014年02月

The rate of graft survival has dramatically increased using calcineurin inhibitors, however chronic graft rejection and risk of infection are difficult to manage. Induction of allograft-specific regulatory T-cells (Tregs) is considered an ideal way to achieve long-term tolerance for allografts. However, efficient in vitro methods for developing allograft-specific Tregs which is applicable to MHC full-mismatched cardiac transplant models have not been established. We compared antigen-nonspecific polyclonal-induced Tregs (iTregs) as well as antigen-specific iTregs and thymus-derived Tregs (nTregs) that were expanded via direct and indirect pathways. We found that iTregs induced via the indirect pathway had the greatest ability to prolong graft survival and suppress angiitis. Antigen-specific iTregs generated ex vivo via both direct and indirect pathways using dendritic cells from F1 mice also induced long-term engraftment without using MHC peptides. In antigen-specific Treg transferred models, activation of dendritic cells and allograft-specific CTL generation were suppressed. The present study demonstrated the potential of ex vivo antigen-specific Treg expansion for clinical cell-based therapeutic approaches to induce lifelong immunological tolerance for allogeneic cardiac transplants. © 2014 Takasato et al.

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Prevention of allogeneic cardiac graft rejection by transfer of ex vivo expanded antigen-specific regulatory T-cells

Takasato Fumika, Morita Rimpei, Schichita Takashi, Sekiya Takashi, Morikawa Yasuhide, Kuroda Tatsuo, Niimi Masanori, Yoshimura Akihiko

PLoS ONE 9 （ 2 ） 2014年02月

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Macrophages and dendritic cells emerge in the liver during intestinal inflammation and predispose the liver to inflammation

Mikami Yohei, Mizuno Shinta, Nakamoto Nobuhiro, Hayashi Atsushi, Sujino Tomohisa, Sato Toshiro, Kamada Nobuhiko, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Ebinuma Hirotoshi, Hibi Toshifumi, Yoshimura Akihiko, Kanai Takanori

PLoS ONE 9 （ 1 ） 2014年01月

The liver is a physiological site of immune tolerance, the breakdown of which induces immunity. Liver antigen-presenting cells may be involved in both immune tolerance and activation. Although inflammatory diseases of the liver are frequently associated with inflammatory bowel diseases, the underlying immunological mechanisms remain to be elucidated. Here we report two murine models of inflammatory bowel disease: RAG-2^-/- mice adoptively transferred with CD4⁺CD45RB^high T cells; and IL-10 ^-/- mice, accompanied by the infiltration of mononuclear cells in the liver. Notably, CD11b^-CD11c^lowPDCA-1⁺ plasmacytoid dendritic cells (DCs) abundantly residing in the liver of normal wild-type mice disappeared in colitic CD4⁺CD45RB^high T cell-transferred RAG-2^-/- mice and IL-10^-/- mice in parallel with the emergence of macrophages (Mφs) and conventional DCs (cDCs). Furthermore, liver Mφ/cDCs emerging during intestinal inflammation not only promote the proliferation of naïve CD4⁺ T cells, but also instruct them to differentiate into IFN-γ-producing Th1 cells in vitro. The emergence of pathological Mφ/cDCs in the liver also occurred in a model of acute dextran sulfate sodium (DSS)-induced colitis under specific pathogen-free conditions, but was canceled in germ-free conditions. Last, the Mφ/cDCs that emerged in acute DSS colitis significantly exacerbated Fas-mediated hepatitis. Collectively, intestinal inflammation skews the composition of antigen-presenting cells in the liver through signaling from commensal bacteria and predisposes the liver to inflammation. © 2014 Mikami et al.

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Enhancement of leptin receptor signaling by SOCS3 deficiency induces development of gastric tumors in mice

Inagaki-Ohara K., Mayuzumi H., Kato S., Minokoshi Y., Otsubo T., Kawamura Y. I., Dohi T., Matsuzaki G., Yoshimura A.

Oncogene 33 （ 1 ） 74 - 84 2014年01月

ISSN 0950-9232

Leptin acts on its receptor (ObR) in the hypothalamus to inhibit food intake and energy expenditure. Leptin and ObR are also expressed in the gastrointestinal tract; however, the physiological significance of leptin signaling in the gut remains uncertain. Suppressor of cytokine signaling 3 (SOCS3) is a key negative feedback regulator of ObR-mediated signaling in the hypothalamus. We now show that gastrointestinal epithelial cell-specific SOCS3 conditional knockout (T3b-SOCS3 cKO) mice developed gastric tumors by enhancing leptin production and the ObRb/signal transducer and activator of transcription 3 (STAT3) signaling pathway. All T3b-SOCS3 cKO mice developed tumors in the stomach but not in the bowels by 2 months of age, even though the SOCS3 deletion occurred in both the epithelium of stomach and bowels. The tumors developed in the absence of the inflammatory response and all cKO mice died within 6 months. These tumors displayed pathology and molecular alterations, such as an increase in MUC2 (Mucin 2, oligomeric mucus/gel-forming) and TFF3 (trefoil factor 3), resembling human intestinal-type gastric tumors. Administration of antileptin antibody to T3b-SOCS3 cKO mice reduced hyperplasia of gastric mucosa, which is the step of the initiation of gastric tumor. These data suggest that SOCS3 is an antigastric tumor gene that suppresses leptin overexpression and ObRb/STAT3 hyperactivation, supporting the hypothesis that the leptin/ObRb/STAT3 axis accelerates tumorigenesis and that it may represent a new therapeutic target for the treatment of gastric cancer. © 2014 Macmillan Publishers Limited.

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Macrophages and dendritic cells emerge in the liver during intestinal inflammation and predispose the liver to inflammation

PLoS ONE 9 （ 1 ） 2014年01月

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Enhancement of leptin receptor signaling by SOCS3 deficiency induces development of gastric tumors in mice

Inagaki-Ohara K., Mayuzumi H., Kato S., Minokoshi Y., Otsubo T., Kawamura Y. I., Dohi T., Matsuzaki G., Yoshimura A.

Oncogene 33 （ 1 ） 74 - 84 2014年01月

ISSN 0950-9232

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A bidirectional crosstalk between iNKT cells and adipocytes mediated by leptin modulates susceptibility for T cell mediated hepatitis

Venken Koen, Seeuws Sylvie, Zabeau Lennart, Jacques Peggy, Decruy Tine, Coudenys Julie, Verheugen Eveline, Windels Fien, Catteeuw Dominiek, Drennan Michael, Van Calenbergh Serge, Lambrecht Bart N., Yoshimura Akihiko, Tavernier Jan, Elewaut Dirk

Journal of Hepatology 60 （ 1 ） 175 - 182 2014年01月

ISSN 0168-8278

Background & Aims Immunometabolism is an emerging field of clinical investigation due to the obesity epidemic worldwide. A reciprocal involvement of immune mediators in the body energy metabolism has been recognized for years, but is only partially understood. We hypothesized that the adipokine leptin could provide an important modulator of iNKT cells. Methods The expression of leptin receptor (LR) on resting and activated iNKT cells was measured by flow cytometry. FACS-sorted hepatic iNKT cells were stimulated with anti-CD3/CD28Ab coated beads in the absence or presence of a neutralizing anti-leptin Ab. Furthermore, we evaluated the outcome of LR blocking nanobody treatment in ConA induced hepatitis and towards metabolic parameters in WT and iNKT cell deficient mice. Results The LR is expressed on iNKT cells and leptin suppresses iNKT cell proliferation and cytokine production in vitro. LR deficient iNKT cells are hyper-responsive further enforcing the role of leptin as an important inhibitor of iNKT cell function. Consistently, in vivo blockade of LR signaling exacerbated ConA hepatitis in wild-type but not in iNKT cell deficient mice, through both Janus kinase (JAK)2 and mitogen-activated protein kinase (MAPK) dependent mechanisms. Moreover, LR inhibition altered fat pad features and was accompanied by insulin resistance, only in wild-type mice. Curiously, this interaction was strictly dependent on MAPK mediated LR signaling in iNKT cells and uncoupled from the more central effects of leptin. Conclusions Our data support a new concept of immune regulation by which leptin protects towards T cell mediated hepatitis via modulation of iNKT cells. © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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Protective role of JAK/STAT signaling against renal fibrosis in mice with unilateral ureteral obstruction

Koike Kiyomi, Ueda Seiji, Yamagishi Sho ichi, Yasukawa Hideo, Kaida Yusuke, Yokoro Miyuki, Fukami Kei, Yoshimura Akihiko, Okuda Seiya

Clinical Immunology 150 （ 1 ） 78 - 87 2014年01月

ISSN 1521-6616

Inflammation is involved in renal fibrosis, a final common pathway for kidney diseases. To clarify how JAK/STAT/SOCS system was involved in renal fibrosis, UUO was induced in BALB/c or SOCS3^+/- mice in the presence or absence of JAK inhibitor-incorporated nanoparticle (pyridine6-PGLA). UUO increased pSTAT3 and subsequently elevated SOCS3 levels in the obstructed kidneys. pSTAT3 levels were further increased in SOCS3^+/- mice. UUO-induced renal fibrosis was markedly suppressed in SOCS3^+/- mice, while it was aggravated by pre-treatment with pyridine6-PGLA. Although there were no differences in renal mRNA levels of TGF-β and collagens between wild and SOCS3^+/- mice, MMP-2 activity was enhanced in SOCS3^+/- UUO mice. Activated MMP-2 was completely suppressed by pyridine6-PGLA-pre-treatment. TNF-α one of JAK/STAT activators, increased pSTAT3 levels and subsequently induced MMP-2 activation in proximal tubular cells. These results suggest that JAK/STAT3 signaling may play a role in repair process of renal fibrosis in UUO partly via MMP-2 activation. © 2013 Elsevier Inc.

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A bidirectional crosstalk between iNKT cells and adipocytes mediated by leptin modulates susceptibility for T cell mediated hepatitis

Journal of Hepatology 60 （ 1 ） 175 - 182 2014年01月

ISSN 0168-8278

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Protective role of JAK/STAT signaling against renal fibrosis in mice with unilateral ureteral obstruction

Koike Kiyomi, Ueda Seiji, Yamagishi Sho ichi, Yasukawa Hideo, Kaida Yusuke, Yokoro Miyuki, Fukami Kei, Yoshimura Akihiko, Okuda Seiya

Clinical Immunology 150 （ 1 ） 78 - 87 2014年01月

ISSN 1521-6616

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Aryl hydrocarbon receptor plays protective roles in cona-induced hepatic injury by both suppressing IFN-γ expression and inducing IL-22

Abe Hiromi, Kimura Akihiro, Tsuruta Sanae, Fukaya Tomohiro, Sakaguchi Ryota, Morita Rimpei, Sekiya Takashi, Shichita Takashi, Chayama Kazuaki, Fujii-Kuriyama Yoshiaki, Yoshimura Akihiko

International Immunology 26 （ 3 ） 129 - 137 2014年

ISSN 0953-8178

The aryl hydrocarbon receptor (AhR), a ligand-activated nuclear transcription factor, is known to mediate the toxic and carcinogenic effects of various environmental pollutants, while AhR has been shown to protect animals from various types of tissue injury. ConA-induced hepatitis is known as a mouse model of acute liver injury. Here, we found a protective role of AhR in ConA-induced hepatitis. AhR is induced in the liver during ConA-induced hepatitis, and Ahr^-/- mice were highly sensitive to this model. Bone marrow chimera experiments indicate that Ahr^-/- hematopoietic cells are responsible for hypersensitivity to ConA-induced hepatitis. We found that IFN-γ from invariant NKT cells was up-regulated and IL-22 from innate lymphoid cells (ILCs) was abolished in Ahr^-/- mice. In addition, IL-22 production was still observed in Rag2^-/- mice but it was severely reduced in Ahr^-/-Rag2^-/- mice. ConA-induced IL-22 production was also dependent on retinoic acid-related orphan receptor γt. These results show that AhR has crucial protective roles in ConA-induced liver injury via promoting IL-22 production from ILCs and suppressing IFN-γ expression from NKT cells. © The Japanese Society for Immunology. 2013. All rights reserved.

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Aryl hydrocarbon receptor protects against bacterial infection by promoting macrophage survival and reactive oxygen species production

Kimura Akihiro, Abe Hiromi, Tsuruta Sanae, Chiba Sayuri, Fujii-Kuriyama Yoshiaki, Sekiya Takashi, Morita Rimpei, Yoshimura Akihiko

International Immunology 26 （ 4 ） 209 - 220 2014年

ISSN 0953-8178

Aryl hydrocarbon receptor (AhR) is crucial for various immune responses. The relationship between AhR and infection with the intracellular bacteria Listeria monocytogenes (LM) is poorly understood. Here, we show that in response to LM infection, AhR is required for bacterial clearance by promoting macrophage survival and reactive oxygen species (ROS) production. AhR-deficient mice were more susceptible to listeriosis, and AhR deficiency enhances bacterial growth in vivo and in vitro. On the other hand, pro-inflammatory cytokines were increased in AhR-deficient macrophages infected with LM despite enhanced susceptibility to LM infection in AhR-deficient mice. Subsequent studies demonstrate that AhR protects against macrophage cell death induced by LM infection through the induction of the antiapoptotic factor, the apoptosis inhibitor of macrophages, which promotes macrophage survival in the setting of LM infection. Furthermore, AhR promotes ROS production for bacterial clearance. Our results demonstrate that AhR is essential to the resistance against LM infection as it promotes macrophage survival and ROS production. This suggests that the activation of AhR by its ligands may be an effective strategy against listeriosis. © The Japanese Society for Immunology. 2013. All rights reserved.

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Spred-2 deficiency exacerbates lipopolysaccharide-induced acute lung inflammation in mice

Xu Yang, Ito Toshihiro, Fushimi Soichiro, Takahashi Sakuma, Itakura Junya, Kimura Ryojiro, Sato Miwa, Mino Megumi, Yoshimura Akihiko, Matsukawa Akihiro

PLoS ONE 9 （ 9 ） e108914 2014年

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe and life-threatening acute lung injury (ALI) that is caused by noxious stimuli and pathogens. ALI is characterized by marked acute inflammation with elevated alveolar cytokine levels. Mitogen-activated protein kinase (MAPK) pathways are involved in cytokine production, but the mechanisms that regulate these pathways remain poorly characterized. Here, we focused on the role of Sprouty-related EVH1-domain-containing protein (Spred)-2, a negative regulator of the Ras-Raf-extracellular signal-regulated kinase (ERK)-MAPK pathway, in lipopolysaccharide (LPS)-induced acute lung inflammation.METHODS: Wild-type (WT) mice and Spred-2(-/-) mice were exposed to intratracheal LPS (50 µg in 50 µL PBS) to induce pulmonary inflammation. After LPS-injection, the lungs were harvested to assess leukocyte infiltration, cytokine and chemokine production, ERK-MAPK activation and immunopathology. For ex vivo experiments, alveolar macrophages were harvested from untreated WT and Spred-2(-/-) mice and stimulated with LPS. In in vitro experiments, specific knock down of Spred-2 by siRNA or overexpression of Spred-2 by transfection with a plasmid encoding the Spred-2 sense sequence was introduced into murine RAW264.7 macrophage cells or MLE-12 lung epithelial cells.RESULTS: LPS-induced acute lung inflammation was significantly exacerbated in Spred-2(-/-) mice compared with WT mice, as indicated by the numbers of infiltrating leukocytes, levels of alveolar TNF-α, CXCL2 and CCL2 in a later phase, and lung pathology. U0126, a selective MEK/ERK inhibitor, reduced the augmented LPS-induced inflammation in Spred-2(-/-) mice. Specific knock down of Spred-2 augmented LPS-induced cytokine and chemokine responses in RAW264.7 cells and MLE-12 cells, whereas Spred-2 overexpression decreased this response in RAW264.7 cells.CONCLUSIONS: The ERK-MAPK pathway is involved in LPS-induced acute lung inflammation. Spred-2 controls the development of LPS-induced lung inflammation by negatively regulating the ERK-MAPK pathway. Thus, Spred-2 may represent a therapeutic target for the treatment of ALI.

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Signal-transducing adaptor protein-2 regulates macrophage migration into inflammatory sites during dextran sodium sulfate induced colitis

Fujita Natsuko, Oritani Kenji, Ichii Michiko, Yokota Takafumi, Saitoh Norimitsu, Okuzaki Daisuke, Sekine Yuichi, Kon Shigeyuki, Muromoto Ryuta, Saitoh Kodai, Yoshimura Akihiko, Matsuda Tadashi, Kanakura Yuzuru

European Journal of Immunology 44 （ 6 ） 1791 - 1801 2014年

ISSN 0014-2980

Signal-transducing adaptor protein-2 (STAP-2) was cloned as a c-fms/M-CSF receptor interacting protein. STAP-2 is an adaptor protein carrying pleckstrin homology and Src homology 2 like domains, as well as a YXXQ motif. STAP-2 has been indicated to have an ability to bind and modulate a variety of signaling and transcriptional molecules. Especially, our previous in vitro studies showed that STAP-2 is crucial for immune and/or inflammatory responses. Here, we have investigated the role of STAP-2 in intestinal inflammation in vivo. The disruption of STAP-2 attenuates dextran sodium sulfate induced colitis via inhibition of macrophage recruitment. To study whether hematopoietic or epithelial cell derived STAP-2 is required for this phenomenon, we generated BM chimeric mice. STAP-2-deficient macrophages impair the ability of CXCL12-induced migration. Intriguingly, STAP-2 also regulates production of proinflammatory chemokines and cytokines such as CXCL1 and TNF-α from intestinal epithelial cells. Therefore, STAP-2 has a potential to regulate plural molecular events during pathological inflammatory responses. Furthermore, our findings not only indicate that STAP-2 is important in regulating intestinal inflammation, but also provide new insights toward the development of novel therapeutic approaches. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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SOCS1 and regulation of regulatory T cells plasticity

Takahashi Reiko, Yoshimura Akihiko

Journal of immunology research 2014 943149 2014年

Several reports have suggested that natural regulatory T cells (Tregs) lose Forkhead box P3 (Foxp3) expression and suppression activity under certain inflammatory conditions. Treg plasticity has been studied because it may be associated with the pathogenesis of autoimmunity. Some studies showed that a minor uncommitted Foxp3(+) T cell population, which lacks hypomethylation at Treg-specific demethylation regions (TSDRs), may convert to effector/helper T cells. Suppressor of cytokine signaling 1 (SOCS1), a negative regulator of cytokine signaling, has been reported to play an important role in Treg cell integrity and function by protecting the cells from excessive inflammatory cytokines. In this review, we discuss Treg plasticity and maintenance of suppression functions in both physiological and pathological settings. In addition, we discuss molecular mechanisms of maintaining Treg plasticity by SOCS1 and other molecules. Such information will be useful for therapy of autoimmune diseases and reinforcement of antitumor immunity.

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Role of inflammatory cytokines in brain ischemia

Yoshimura Akihiko

Therapeutic Research 35 （ 9 ） 801 - 803 2014年

ISSN 0289-8020

Aryl hydrocarbon receptor plays protective roles in cona-induced hepatic injury by both suppressing IFN-γ expression and inducing IL-22

Abe Hiromi, Kimura Akihiro, Tsuruta Sanae, Fukaya Tomohiro, Sakaguchi Ryota, Morita Rimpei, Sekiya Takashi, Shichita Takashi, Chayama Kazuaki, Fujii-Kuriyama Yoshiaki, Yoshimura Akihiko

International Immunology 26 （ 3 ） 129 - 137 2014年

ISSN 0953-8178

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Aryl hydrocarbon receptor protects against bacterial infection by promoting macrophage survival and reactive oxygen species production

Kimura Akihiro, Abe Hiromi, Tsuruta Sanae, Chiba Sayuri, Fujii-Kuriyama Yoshiaki, Sekiya Takashi, Morita Rimpei, Yoshimura Akihiko

International Immunology 26 （ 4 ） 209 - 220 2014年

ISSN 0953-8178

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Spred-2 deficiency exacerbates lipopolysaccharide-induced acute lung inflammation in mice

Xu Yang, Ito Toshihiro, Fushimi Soichiro, Takahashi Sakuma, Itakura Junya, Kimura Ryojiro, Sato Miwa, Mino Megumi, Yoshimura Akihiko, Matsukawa Akihiro

PLoS ONE 9 （ 9 ） e108914 2014年

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Signal-transducing adaptor protein-2 regulates macrophage migration into inflammatory sites during dextran sodium sulfate induced colitis

European Journal of Immunology 44 （ 6 ） 1791 - 1801 2014年

ISSN 0014-2980

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SOCS1 and regulation of regulatory T cells plasticity

Takahashi Reiko, Yoshimura Akihiko

Journal of immunology research 2014 943149 2014年

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Role of inflammatory cytokines in brain ischemia

Yoshimura Akihiko

Therapeutic Research 35 （ 9 ） 801 - 803 2014年

ISSN 0289-8020

Regulation of human autoimmune regulator (AIRE) gene translation by miR-220b

Matsuo Tomohito, Noguchi Yukiko, Shindo Mieko, Morita Yoshifumi, Oda Yoshie, Yoshida Eiko, Hamada Hiroko, Harada Mine, Shiokawa Yuichi, Nishida Takahiro, Tominaga Ryuji, Kikushige Yoshikane, Akashi Koichi, Kudoh Jun, Shimizu Nobuyoshi, Tanaka Yuka, Umemura Tsukuru, Taniguchi Taketoshi, Yoshimura Akihiko, Kobayashi Takashi, Mitsuyama Masao, Kurisaki Hironori, Katsuta Hitoshi, Nagafuchi Seiho

Gene 530 （ 1 ） 19 - 25 2013年11月

ISSN 0378-1119

Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4⁺ T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein-Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3'UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3'UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3'UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3'UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation. © 2013 Elsevier B.V.

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Regulation of human autoimmune regulator (AIRE) gene translation by miR-220b

Gene 530 （ 1 ） 19 - 25 2013年11月

ISSN 0378-1119

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IL-23 protection against Plasmodium berghei infection in mice is partially dependent on IL-17 from macrophages

Ishida Hidekazu, Imai Takashi, Suzue Kazutomo, Hirai Makoto, Taniguchi Tomoyo, Yoshimura Akihiko, Iwakura Yoichiro, Okada Hiroko, Suzuki Tomohisa, Shimokawa Chikako, Hisaeda Hajime

European Journal of Immunology 43 （ 10 ） 2696 - 2706 2013年10月

ISSN 0014-2980

Although IL-12 is believed to contribute to protective immune responses, the role played by IL-23 (a member of the IL-12 family) in malaria is elusive. Here, we show that IL-23 is produced during infection with Plasmodium berghei NK65. Mice deficient in IL-23 (p19KO) had higher parasitemia and died earlier than wild-type (WT) controls. Interestingly, p19KO mice had lower numbers of IL-17-producing splenic cells than their WT counterparts. Furthermore, mice deficient in IL-17 (17KO) suffered higher parasitemia than the WT controls, indicating that IL-23-mediated protection is dependent on induction of IL-17 during infection. We found that macrophages were responsible for IL-17 production in response to IL-23. We observed a striking reduction in splenic macrophages in the p19KO and 17KO mice, both of which became highly susceptible to infection. Thus, IL-17 appears to be crucial for maintenance of splenic macrophages. Adoptive transfer of macrophages into macrophage-depleted mice confirmed that macrophage-derived IL-17 is required for macrophage accumulation and parasite eradication in the recipient mice. We also found that IL-17 induces CCL2/7, which recruit macrophages. Our findings reveal a novel protective mechanism whereby IL-23, IL-17, and macrophages reduce the severity of infection with blood-stage malaria parasites. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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IL-23 protection against Plasmodium berghei infection in mice is partially dependent on IL-17 from macrophages

Ishida Hidekazu, Imai Takashi, Suzue Kazutomo, Hirai Makoto, Taniguchi Tomoyo, Yoshimura Akihiko, Iwakura Yoichiro, Okada Hiroko, Suzuki Tomohisa, Shimokawa Chikako, Hisaeda Hajime

European Journal of Immunology 43 （ 10 ） 2696 - 2706 2013年10月

ISSN 0014-2980

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Th17 cells carrying TCR recognizing epidermal autoantigen induce psoriasis-like skin inflammation

Nishimoto Shuhei, Kotani Hitoshi, Tsuruta Sanae, Shimizu Nana, Ito Minako, Shichita Takashi, Morita Rimpei, Takahashi Hayato, Amagai Masayuki, Yoshimura Akihiko

Journal of Immunology 191 （ 6 ） 3065 - 3072 2013年09月

ISSN 0022-1767

Psoriasis is considered a Th17-type autoimmune skin inflammatory disease; however, involvement of an autoantigen-specific TCR has not been established. In this study, we show that psoriasis-like skin inflammation can be induced by autoreactive Th17 cells.We previously developed the desmoglein 3-specific TCR-transgenic (Dsg3H1) mouse, in which CD4+ T cells recognize physiological epidermal autoantigen. T cells from Dsg3H1 mice were polarized into Th17 cells in vitro and then adoptively transferred into Rag2^-/- mice. Dsg3H1-Th17 cells induced severe psoriasis-like skin inflammation within 2 wk after transfer in the tissues in which desmoglein 3 is expressed. Such pathology was not observed when wild-type Th17 cells or Th1-skewed Dsg3H1 T cells were transferred, and it was strongly suppressed by anti-IL-12/23 and anti-IL-17 Abs. Although IFN-γ⁺/IL-17⁺ T cells accumulated in the skin lesions of mice that received Dsg3H1-Th17 cells, IFN-γ-deficient Dsg3H1-Th17 cells were fully pathogenic. These results demonstrate that cutaneous psoriasis-like immunopathology can be developed by epidermis-specific recognition of Th17 cells, which is strictly dependent on IL-17 but not IFN-γ. Copyright © 2013 by The American Association of Immunologists, Inc.

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Th17 cells carrying TCR recognizing epidermal autoantigen induce psoriasis-like skin inflammation

Nishimoto Shuhei, Kotani Hitoshi, Tsuruta Sanae, Shimizu Nana, Ito Minako, Shichita Takashi, Morita Rimpei, Takahashi Hayato, Amagai Masayuki, Yoshimura Akihiko

Journal of Immunology 191 （ 6 ） 3065 - 3072 2013年09月

ISSN 0022-1767

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TRAF6 Is Essential for Maintenance of Regulatory T Cells That Suppress Th2 Type Autoimmunity

Muto Go, Kotani Hitoshi, Kondo Taisuke, Morita Rimpei, Tsuruta Sanae, Kobayashi Takashi, Luche Hervé, Fehling Hans Joerg, Walsh Matthew, Choi Yongwon, Yoshimura Akihiko

PLoS ONE 8 （ 9 ） 2013年09月

Regulatory T cells (Tregs) maintain immune homeostasis by limiting inflammatory responses. TRAF6 plays a key role in the regulation of innate and adaptive immunity by mediating signals from various receptors including the T-cell receptor (TCR). T cell-specific deletion of TRAF6 has been shown to induce multiorgan inflammatory disease, but the role of TRAF6 in Tregs remains to be investigated. Here, we generated Treg-specific TRAF6-deficient mice using Foxp3-Cre and TRAF6-flox mice. Treg-specific TRAF6-deficient (cKO) mice developed allergic skin diseases, arthritis, lymphadenopathy and hyper IgE phenotypes. Although TRAF6-deficient Tregs possess similar in vitro suppression activity compared to wild-type Tregs, TRAF6-deficient Tregs did not suppress colitis in lymphopenic mice very efficiently due to reduced number of Foxp3-positive cells. In addition, the fraction of TRAF6-deficient Tregs was reduced compared with wild-type Tregs in female cKO mice without inflammation. Moreover, adoptive transfer of Foxp3 ⁺ Tregs into Rag2^-/- mice revealed that TRAF6-deficient Tregs converted into Foxp3^- cells more rapidly than WT Tregs under lymphopenic conditions. Fate-mapping analysis also revealed that conversion of Tregs from Foxp3⁺ to Foxp3^- (exFoxp3 cells) was accelerated in TRAF6-deficient Tregs. These data indicate that TRAF6 in Tregs plays important roles in the maintenance of Foxp3 in Tregs and in the suppression of pathogenic Th2 type conversion of Tregs. © 2013 Muto et al.

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TRAF6 Is Essential for Maintenance of Regulatory T Cells That Suppress Th2 Type Autoimmunity

Muto Go, Kotani Hitoshi, Kondo Taisuke, Morita Rimpei, Tsuruta Sanae, Kobayashi Takashi, Luche Hervé, Fehling Hans Joerg, Walsh Matthew, Choi Yongwon, Yoshimura Akihiko

PLoS ONE 8 （ 9 ） 2013年09月

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Smad2/3 and IRF4 play a cooperative role in IL-9-producing T cell induction

Tamiya Taiga, Ichiyama Kenji, Kotani Hitoshi, Fukaya Tomohiro, Sekiya Takashi, Shichita Takashi, Honma Kiri, Yui Katsuyuki, Matsuyama Toshifumi, Nakao Takako, Fukuyama Satoru, Inoue Hiromasa, Nomura Masatoshi, Yoshimura Akihiko

Journal of Immunology 191 （ 5 ） 2360 - 2371 2013年09月

ISSN 0022-1767

IL-9 is a pleiotropic cytokine that can regulate autoimmune and allergic responses. Th9 cells can develop from naive T cells or Th2 cells through stimulation by TGF-β in vitro. In this study, we demonstrated that Smad2 and Smad3 are necessary for IL-9 production from T cells in an OVA-induced asthma model using T cell-specific Smad2- and Smad3-deficient mice. Smad2 and Smad3 were also redundantly essential for TGF-β signaling to induce histone modifications for Il9 transcription. Although Smad2/3 was recruited to the Il9 promoter by TGF-β stimulation, they are not sufficient to activate the Il9 promoter. By the screening the transcription factors, we found that IFN regulatory factor 4 (IRF4) was essential for the Smad2/3-mediated Il9 promoter activation. In addition, Smad2/3 physically interacted with IRF4, and Smad2/3 did not bind to the Il9 promoter and could not induce Th9 in IRF4-deficient T cells. Similarly, IRF4 could not stimulate Il9 transcription in the absence of Smad2/3, and TGF-β enhanced IRF4 recruitment to the Il9 promoter in a Smad2/3-dependent manner. We propose that Smad2/3 and IRF4 cooperatively transactivate the Il9 promoter and play an important role in regulating allergic immune responses by inducing Th9 cells. Copyright © 2013 by The American Association of Immunologists, Inc.

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Smad2/3 and IRF4 play a cooperative role in IL-9-producing T cell induction

Journal of Immunology 191 （ 5 ） 2360 - 2371 2013年09月

ISSN 0022-1767

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[Post-ischemic innate immunity and its application for novel therapeutic strategy targeting brain inflammation].

Ito Minako, Kondo Taisuke, Shichita Takashi, Yoshimura Akihiko

Nihon rinsho. Japanese journal of clinical medicine 71 （ 7 ） 1291 - 1301 2013年07月

ISSN 0047-1852

Stroke or brain ischemia is one of the major causes of death and disability worldwide. Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. In a mouse stroke model, we have reported that IL-23 produced from infiltrating macrophages induces IL-17 producing T cells. IL-17 is mainly produced from gammadeltaT cells and promotes delayed (day 3-4) ischemic brain damage. We also demonstrated that peroxiredoxin (Prx) family proteins released extracellularly from necrotic brain cells induce expression of inflammatory cytokines including IL-23 in macrophages through activation of Toll-like receptor 2(TLR2) and TLR4, thereby promoting neural cell death. We thus propose that regulation of the IL-23-IL-17 axis including gammadeltaT cells, macrophages, and extracellular Prxs could be a potent neuroprotective tool.

CIS

Yoshimura Akihiko

Nature Immunology 14 （ 7 ） 692 - 694 2013年07月

ISSN 1529-2908

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Critical and Independent Role for SOCS3 in Either Myeloid or T Cells in Resistance to Mycobacterium tuberculosis

Carow Berit, Reuschl Ann Kathrin, Gavier-Widén Dolores, Jenkins Brendan J., Ernst Matthias, Yoshimura Akihiko, Chambers Benedict J., Rottenberg Martin E.

PLoS Pathogens 9 （ 7 ） 2013年07月

ISSN 1553-7366

Suppressor of cytokine signalling 3 (SOCS3) negatively regulates STAT3 activation in response to several cytokines such as those in the gp130-containing IL-6 receptor family. Thus, SOCS3 may play a major role in immune responses to pathogens. In the present study, the role of SOCS3 in M. tuberculosis infection was examined. All Socs3^fl/fl LysM cre, Socs3^fl/fl lck cre (with SOCS3-deficient myeloid and lymphoid cells, respectively) and gp130^F/F mice, with a mutation in gp130 that impedes binding to SOCS3, showed increased susceptibility to infection with M. tuberculosis. SOCS3 binding to gp130 in myeloid cells conveyed resistance to M. tuberculosis infection via the regulation of IL-6/STAT3 signalling. SOCS3 was redundant for mycobacterial control by macrophages in vitro. Instead, SOCS3 expression in infected macrophages and DCs prevented the IL-6-mediated inhibition of TNF and IL-12 secretion and contributed to a timely CD4+ cell-dependent IFN-γ expression in vivo. In T cells, SOCS3 expression was essential for a gp130-independent control of infection with M. tuberculosis, but was neither required for the control of infection with attenuated M. bovis BCG nor for M. tuberculosis in BCG-vaccinated mice. Socs3^fl/fl lck cre mice showed an increased frequency of γδ+ T cells in different organs and an enhanced secretion of IL-17 by γδ+ T cells in response to infection. Socs3^fl/fl lck cre γδ+ T cells impaired the control of infection with M. tuberculosis. Thus, SOCS3 expression in either lymphoid or myeloid cells is essential for resistance against M. tuberculosis via discrete mechanisms. © 2013 Carow et al.

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[Post-ischemic innate immunity and its application for novel therapeutic strategy targeting brain inflammation].

Ito Minako, Kondo Taisuke, Shichita Takashi, Yoshimura Akihiko

Nihon rinsho. Japanese journal of clinical medicine 71 （ 7 ） 1291 - 1301 2013年07月

ISSN 0047-1852

CIS

Yoshimura Akihiko

Nature Immunology 14 （ 7 ） 692 - 694 2013年07月

ISSN 1529-2908

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Critical and Independent Role for SOCS3 in Either Myeloid or T Cells in Resistance to Mycobacterium tuberculosis

Carow Berit, Reuschl Ann Kathrin, Gavier-Widén Dolores, Jenkins Brendan J., Ernst Matthias, Yoshimura Akihiko, Chambers Benedict J., Rottenberg Martin E.

PLoS Pathogens 9 （ 7 ） 2013年07月

ISSN 1553-7366

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A single strain of Clostridium butyricum induces intestinal IL-10-producing macrophages to suppress acute experimental colitis in mice

Hayashi Atsushi, Sato Toshiro, Kamada Nobuhiko, Mikami Yohei, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Hibi Toshifumi, Roers Axel, Yagita Hideo, Ohteki Toshiaki, Yoshimura Akihiko, Kanai Takanori

Cell Host and Microbe 13 （ 6 ） 711 - 722 2013年06月

ISSN 1931-3128

Imbalance in gut bacterial composition provokes host proinflammatory responses causing diseases such as colitis. Colonization with a mixture of Clostridium species from clusters IV and XIVa was shown to suppress colitis through the induction of IL-10-producing regulatory T (Treg) cells. We demonstrate that a distinct Clostridium strain from cluster I, Clostridium butyricum (CB), prevents acute experimental colitis in mice through induction of IL-10, an anti-inflammatory cytokine. However, while CB treatment had no effect on IL-10 production by T cells, IL-10-producing F4/80⁺CD11b ⁺CD11c^int macrophages accumulated in the inflamed mucosa after CB treatment. CB directly triggered IL-10 production by intestinal macrophages in inflamed mucosa via the TLR2/MyD88 pathway. The colitis-preventing effect of CB was negated in macrophage-specific IL-10-deficient mice, suggesting that induction of IL-10 by intestinal macrophages is crucial for the probiotic action of CB. Collectively, CB promotes IL-10 production by intestinal macrophages in inflamed mucosa, thereby preventing experimental colitis in mice. © 2013 Elsevier Inc.

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A single strain of Clostridium butyricum induces intestinal IL-10-producing macrophages to suppress acute experimental colitis in mice

Hayashi Atsushi, Sato Toshiro, Kamada Nobuhiko, Mikami Yohei, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Hibi Toshifumi, Roers Axel, Yagita Hideo, Ohteki Toshiaki, Yoshimura Akihiko, Kanai Takanori

Cell Host and Microbe 13 （ 6 ） 711 - 722 2013年06月

ISSN 1931-3128

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CDK inhibitors suppress Th17 and promote iTreg differentiation, and ameliorate experimental autoimmune encephalomyelitis in mice

Yoshida Hideyuki, Kotani Hitoshi, Kondo Taisuke, Tani Ito, Wei Xuetao, Tsuruta Sanae, Kimura Akihiro, Asakawa Mayako, Ito Minako, Nagai Shigenori, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 435 （ 3 ） 378 - 384 2013年06月

ISSN 0006-291X

Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-β for early differentiation. Several Smad-independent pathways including the JNK and the RhoA-ROCK pathways have been implicated in the induction of RORγt, the master regulator of Th17, however, molecular mechanisms underlying Smad-independent pathway remain largely unknown. To identify novel pathways involved in Th17 differentiation, we screened 285 chemical inhibitors for known signaling pathways. Among them, we found that Kenpaullone, a GSK3-β and CDK inhibitor, efficiently suppressed TGF-β-mediated RORγt induction and enhanced Foxp3 induction in primary T cells. Another CDK inhibitor, Roscovitine, but not other GSK3-β inhibitors, suppressed Th17 differentiation and enhanced iTreg development. Kenpaullone and Roscovitine suppressed experimental autoimmune encephalomyelitis (EAE), a typical Th17-mediated autoimmune disease model. These two compounds enhanced STAT5 phosphorylation and restored IL-2 production in the presence of TGF-β. These data suggest that CDK inhibitors modulate TGF-β-signaling pathways, which restore TGF-β-mediated suppression of IL-2 production, thereby modifying the Th17/iTreg balance. © 2013 Elsevier Inc.

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CDK inhibitors suppress Th17 and promote iTreg differentiation, and ameliorate experimental autoimmune encephalomyelitis in mice

Yoshida Hideyuki, Kotani Hitoshi, Kondo Taisuke, Tani Ito, Wei Xuetao, Tsuruta Sanae, Kimura Akihiro, Asakawa Mayako, Ito Minako, Nagai Shigenori, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 435 （ 3 ） 378 - 384 2013年06月

ISSN 0006-291X

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CLP1 links tRNA metabolism to progressive motor-neuron loss

Hanada Toshikatsu, Weitzer Stefan, Mair Barbara, Bernreuther Christian, Wainger Brian J., Ichida Justin, Hanada Reiko, Orthofer Michael, Cronin Shane J., Komnenovic Vukoslav, Minis Adi, Sato Fuminori, Mimata Hiromitsu, Yoshimura Akihiko, Tamir Ido, Rainer Johannes, Kofler Reinhard, Yaron Avraham, Eggan Kevin C., Woolf Clifford J., Glatzel Markus, Herbst Ruth, Martinez Javier, Penninger Josef M.

Nature 495 （ 7442 ） 474 - 480 2013年03月

ISSN 0028-0836

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1^K/K) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1^K/K mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53. © 2013 Macmillan Publishers Limited. All rights reserved.

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CLP1 links tRNA metabolism to progressive motor-neuron loss

Nature 495 （ 7442 ） 474 - 480 2013年03月

ISSN 0028-0836

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IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality

Zhang Hong, Neuhöfer Patrick, Song Liang, Rabe Björn, Lesina Marina, Kurkowski Magdalena U., Treiber Matthias, Wartmann Thomas, Regnér Sara, Thorlacius Henrik, Saur Dieter, Weirich Gregor, Yoshimura Akihiko, Halangk Walter, Mizgerd Joseph P., Schmid Roland M., Rose-John Stefan, Algül Hana

Journal of Clinical Investigation 123 （ 3 ） 1019 - 1031 2013年03月

ISSN 0021-9738

Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis-associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI.

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IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality

Journal of Clinical Investigation 123 （ 3 ） 1019 - 1031 2013年03月

ISSN 0021-9738

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Overexpression of SOCS3 in T lymphocytes leads to impaired interleukin-17 production and severe aortic aneurysm formation in mice-brief report

Romain Mélissa, Taleb Soraya, Dalloz Marion, Ponnuswamy Padmapriya, Esposito Bruno, Pérez Nicolas, Wang Yu, Yoshimura Akihiko, Tedgui Alain, Mallat Ziad

Arteriosclerosis, Thrombosis, and Vascular Biology 33 （ 3 ） 581 - 584 2013年03月

ISSN 1079-5642

Objective-Mutations of signal transducer and activator of transcription 3 (STAT3) are responsible for autosomal dominant hyperimmunoglobulin E syndrome. Recently, we reported frequent vascular abnormalities, including aneurysms in these patients, and demonstrated that STAT3 inhibition promoted aneurysm in mice. The purpose of this study was to investigate the role of cell-specific STAT3 signaling in the susceptibility to aneurysm. Methods and Results-C57BL/6 wild-type mice were irradiated and repopulated with bone marrow cells isolated from either wild-type mice or from mice with defective STAT3 signaling as a result of overexpression of suppressor of cytokine signaling 3 (SOCS3-Tg mice). Mice were then subjected to a validated model of abdominal aortic aneurysm induced by angiotensin II infusion for 28 days, along with repetitive injections of a neutralizing antitransforming growth factor-β antibody. We found that overexpression of SOCS3 in bone marrow-derived cells significantly increased aneurysm severity (P=0.04). In contrast, overexpression of SOCS3 in the vessel wall had no effect on the disease process. Surprisingly, deletion of STAT3 signaling in macrophages did not affect aneurysm development. Interestingly, however, defective STAT3 signaling in SOCS3-Tg T cells markedly increased aneurysm severity (P=0.01) and mortality from aneurysm rupture (P=0.008). Overexpression of SOCS3 in T cells significantly decreased interleukin-17 production (P<0.0001) and was associated with a reduction of its plasma levels (P=0.02). Conclusion-These findings clearly identify a central role for T cell-specific STAT3 signaling in the promotion of vascular aneurysm and support previous work on interleukin-17 protective role in this process. © 2013 American Heart Association, Inc.

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[Peroxiredoxin triggers post-ischemic inflammation].

Yoshimura Akihiko, Ito Minako, Kondo Taisuke, Shichita Takashi

Seikagaku. The Journal of Japanese Biochemical Society 85 （ 3 ） 179 - 186 2013年03月

ISSN 0037-1017

Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis

Sekiya Takashi, Kashiwagi Ikkou, Yoshida Rei, Fukaya Tomohiro, Morita Rimpei, Kimura Akihiro, Ichinose Hiroshi, Metzger Daniel, Chambon Pierre, Yoshimura Akihiko

Nature Immunology 14 （ 3 ） 230 - 237 2013年03月

ISSN 1529-2908

Regulatory T cells (T reg cells) develop from progenitor thymocytes after the engagement of T cell antigen receptors (TCRs) with high-affinity ligands, but the underlying molecular mechanisms are still unclear. Here we show that the Nr4a nuclear receptors, which are encoded by immediate-early genes upregulated by TCR stimulation in thymocytes, have essential roles in T reg cell development. Mice that lacked all Nr4a factors could not produce T reg cells and died early owing to systemic autoimmunity. Nr4a receptors directly activated the promoter of the gene encoding the transcription factor Foxp3, and forced activation of Nr4a receptors bypassed low-strength TCR signaling to drive the T reg cell developmental program. Our results suggest that Nr4a receptors have key roles in determining CD4 + T cell fates in the thymus and thus contribute to immune homeostasis. © 2013 Nature America, Inc. All rights reserved.

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Overexpression of SOCS3 in T lymphocytes leads to impaired interleukin-17 production and severe aortic aneurysm formation in mice-brief report

Romain Mélissa, Taleb Soraya, Dalloz Marion, Ponnuswamy Padmapriya, Esposito Bruno, Pérez Nicolas, Wang Yu, Yoshimura Akihiko, Tedgui Alain, Mallat Ziad

Arteriosclerosis, Thrombosis, and Vascular Biology 33 （ 3 ） 581 - 584 2013年03月

ISSN 1079-5642

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[Peroxiredoxin triggers post-ischemic inflammation].

Yoshimura Akihiko, Ito Minako, Kondo Taisuke, Shichita Takashi

Seikagaku. The Journal of Japanese Biochemical Society 85 （ 3 ） 179 - 186 2013年03月

ISSN 0037-1017

Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis

Sekiya Takashi, Kashiwagi Ikkou, Yoshida Rei, Fukaya Tomohiro, Morita Rimpei, Kimura Akihiro, Ichinose Hiroshi, Metzger Daniel, Chambon Pierre, Yoshimura Akihiko

Nature Immunology 14 （ 3 ） 230 - 237 2013年03月

ISSN 1529-2908

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IL-23-independent induction of IL-17 from γδT cells and innate lymphoid cells promotes experimental intraocular neovascularization

Hasegawa Eiichi, Sonoda Koh Hei, Shichita Takashi, Morita Rimpei, Sekiya Takashi, Kimura Akihiro, Oshima Yuji, Takeda Atsunobu, Yoshimura Takeru, Yoshida Shigeo, Ishibashi Tatsuro, Yoshimura Akihiko

Journal of Immunology 190 （ 4 ） 1778 - 1787 2013年02月

ISSN 0022-1767

Choroidal neovascularization (CNV) is a characteristic of age-related macular degeneration. Genome-wide association studies have provided evidence that the immune system is involved in the pathogenesis of age-related macular degeneration; however, the role of inflammatory cytokines in CNV has not been established. In this study, we demonstrated that IL-17 had a strong potential for promoting neovascularization in a vascular endothelial growth factor-independent manner in laser-induced experimental CNV in mice. Infiltrated γδT cells and Thy-1⁺ innate lymphoid cells, but not Th17 cells, were the main sources of IL-17 in injured eyes. IL-23 was dispensable for IL-17 induction in the eye. Instead, we found that IL-1β and high-mobility group box 1 strongly promoted IL-17 expression by γδT cells. Suppression of IL-1β and high-mobility group box 1, as well as depletion of γδT cells, reduced IL-17 levels and ameliorated experimental CNV. Our findings suggest the existence of a novel inflammatory cytokine network that promotes neovascularization in the eye. Copyright © 2013 by The American Association of Immunologists, Inc.

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IL-23-independent induction of IL-17 from γδT cells and innate lymphoid cells promotes experimental intraocular neovascularization

Hasegawa Eiichi, Sonoda Koh Hei, Shichita Takashi, Morita Rimpei, Sekiya Takashi, Kimura Akihiro, Oshima Yuji, Takeda Atsunobu, Yoshimura Takeru, Yoshida Shigeo, Ishibashi Tatsuro, Yoshimura Akihiko

Journal of Immunology 190 （ 4 ） 1778 - 1787 2013年02月

ISSN 0022-1767

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Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis

Sekiya Takashi, Kashiwagi Ikkou, Yoshida Rei, Fukaya Tomohiro, Morita Rimpei, Kimura Akihiro, Ichinose Hiroshi, Metzger Daniel, Chambon Pierre, Yoshimura Akihiko

Nature Immunology 2013年01月

ISSN 1529-2908

Regulatory T cells (T_reg cells) develop from progenitor thymocytes after the engagement of T cell antigen receptors (TCRs) with high-affinity ligands, but the underlying molecular mechanisms are still unclear. Here we show that the Nr4a nuclear receptors, which are encoded by immediate-early genes upregulated by TCR stimulation in thymocytes, have essential roles in T_reg cell development. Mice that lacked all Nr4a factors could not produce T_reg cells and died early owing to systemic autoimmunity. Nr4a receptors directly activated the promoter of the gene encoding the transcription factor Foxp3, and forced activation of Nr4a receptors bypassed low-strength TCR signaling to drive the T_reg cell developmental program. Our results suggest that Nr4a receptors have key roles in determining CD4⁺ T cell fates in the thymus and thus contribute to immune homeostasis.

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Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis

Sekiya Takashi, Kashiwagi Ikkou, Yoshida Rei, Fukaya Tomohiro, Morita Rimpei, Kimura Akihiro, Ichinose Hiroshi, Metzger Daniel, Chambon Pierre, Yoshimura Akihiko

Nature Immunology 2013年01月

ISSN 1529-2908

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Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging

Nishimura Hirohito, Ritchie Ken, Kasai Rinshi S., Goto Miki, Morone Nobuhiro, Sugimura Hiroyuki, Tanaka Koichiro, Sase Ichiro, Yoshimura Akihiko, Nakano Yoshitaro, Fujiwara Takahiro K., Kusumi Akihiro

Journal of Cell Biology 202 （ 6 ） 967 - 983 2013年

ISSN 0021-9525

Fluorescence microscopy is used extensively in cellbiological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for =10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1-2 μm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed. ©2013 Nishimura et al.

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Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging

Journal of Cell Biology 202 （ 6 ） 967 - 983 2013年

ISSN 0021-9525

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TAK1 (MAP3K7) Signaling Regulates Hematopoietic Stem Cells through TNF-Dependent and -Independent Mechanisms

Takaesu Giichi, Inagaki Maiko, Takubo Keiyo, Mishina Yuji, Hess Paul R., Dean Gregg A., Yoshimura Akihiko, Matsumoto Kunihiro, Suda Toshio, Ninomiya-Tsuji Jun

PLoS ONE 7 （ 11 ） 2012年11月

A cytokine/stress signaling kinase Tak1 (Map3k7) deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs) harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2) using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1⁺, c-Kit⁺ (LSK) cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6-18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC. © 2012 Takaesu et al.

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TAK1 (MAP3K7) Signaling Regulates Hematopoietic Stem Cells through TNF-Dependent and -Independent Mechanisms

Takaesu Giichi, Inagaki Maiko, Takubo Keiyo, Mishina Yuji, Hess Paul R., Dean Gregg A., Yoshimura Akihiko, Matsumoto Kunihiro, Suda Toshio, Ninomiya-Tsuji Jun

PLoS ONE 7 （ 11 ） 2012年11月

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Novel therapeutic strategies targeting innate immune responses and early inflammation after stroke

Shichita Takashi, Ago Tetsuro, Kamouchi Masahiro, Kitazono Takanari, Yoshimura Akihiko, Ooboshi Hiroaki

Journal of Neurochemistry 123 （ SUPPL. 2 ） 29 - 38 2012年11月

ISSN 0022-3042

Post-ischemic inflammation is an essential step in the progression of ischemic stroke. This review focuses on the function of infiltrating immune cells, macrophages, and T cells, in ischemic brain injury. The brain is a sterile organ; however, the activation of Toll-like receptor (TLR) 2 and TLR4 is pivotal in the beginning of post-ischemic inflammation. Some endogenous TLR ligands are released from injured brain cells, including high mobility group box 1 and peroxiredoxin family proteins, and activate the infiltrating macrophages and induce the expression of inflammatory cytokines. Following this step, T cells also infiltrate into the ischemic brain and mediate post-ischemic inflammation in the delayed phase. Various cytokines from helper T cells and _γδT cells function as neurotoxic (IL-23/IL-17, IFN- _γ) or neuroprotective (IL-10, IL-4) mediators. Novel neuroprotective strategies should therefore be developed through more detailed understanding of this process and the regulation of post-ischemic inflammation. © 2012 International Society for Neurochemistry.

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Novel therapeutic strategies targeting innate immune responses and early inflammation after stroke

Shichita Takashi, Ago Tetsuro, Kamouchi Masahiro, Kitazono Takanari, Yoshimura Akihiko, Ooboshi Hiroaki

Journal of Neurochemistry 123 （ SUPPL. 2 ） 29 - 38 2012年11月

ISSN 0022-3042

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STAP-2 interacts with and modulates BCR-ABL-mediated tumorigenesis

Sekine Y., Ikeda O., Mizushima A., Ueno Y., Muromoto R., Yoshimura A., Kanakura Y., Oritani K., Matsuda T.

Oncogene 31 （ 40 ） 4384 - 4396 2012年10月

ISSN 0950-9232

In chronic myeloid leukemia (CML), the BCR-ABL fusion oncoprotein activates multiple pathways involved in cell survival, growth promotion and disease progression. In this report, we show that the signal-transducing adaptor protein-2 (STAP-2) is involved in BCR-ABL activity. We demonstrate that STAP-2 bound to BCR-ABL, and BCR and ABL proteins, depending on the STAP-2 Src homology 2-like domain. BCR-ABL phosphorylates STAP-2 Tyr250 and the phosphorylated STAP-2 in turn upregulated BCR-ABL phosphorylation, leading to enhanced activation of downstream signaling molecules including ERK (extracellular- signal-regulated kinase), STAT5 (signal transducer and activator of transcription 5), BCL-xL (B-cell lymphoma-extra large) and BCL-2(B-cell lymphoma 2). In addition, STAP-2 interacts with BCR-ABL to alter chemokine receptor expression leading to downregulation of CXCR4 and upregulation of CCR7. The interaction between STAP-2 and BCR-ABL plays a crucial role in conferring a growth advantage and resistance to imatinib, a BCR-ABL inhibitor, as well as tumor progression. Notably, mice injected with BCR-ABL/STAP-2-expressing Ba/F3 cells developed lymph node enlargement and hepatosplenomegaly. Moreover, suppression of STAP-2 in K562 CML cells resulted in no tumor formation in mice. Our results demonstrate a critical contribution of STAP-2 in BCR-ABL activity, and suggest that STAP-2 might be an important candidate for drug development for patients with CML. Furthermore, the expression of STAP-2 provides useful information for estimating the characteristics of individual CML clones. © 2012 Macmillan Publishers Limited.

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STAP-2 interacts with and modulates BCR-ABL-mediated tumorigenesis

Sekine Y., Ikeda O., Mizushima A., Ueno Y., Muromoto R., Yoshimura A., Kanakura Y., Oritani K., Matsuda T.

Oncogene 31 （ 40 ） 4384 - 4396 2012年10月

ISSN 0950-9232

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Deleterious effect of the IL-23/IL-17A axis and γδT cells on left ventricular remodeling after myocardial infarction.

Yan Xiaoxiang, Shichita Takashi, Katsumata Yoshinori, Matsuhashi Tomohiro, Ito Hideyuki, Ito Kentaro, Anzai Atsushi, Endo Jin, Tamura Yuichi, Kimura Kensuke, Fujita Jun, Shinmura Ken, Shen Weifeng, Yoshimura Akihiko, Fukuda Keiichi, Sano Motoaki

Journal of the American Heart Association 1 （ 5 ） 2012年10月

Left ventricular (LV) remodeling leads to chronic heart failure and is a main determinant of morbidity and mortality after myocardial infarction (MI). At the present time, therapeutic options to prevent LV remodeling are limited. We created a large MI by permanent ligation of the coronary artery and identified a potential link between the interleukin (IL)-23/IL-17A axis and γδT cells that affects late-stage LV remodeling after MI. Despite the finsinf that infarct size 24 hours after surgery was similar to that in wild-type mice, a deficiency in IL-23, IL-17A, or γδT cells improved survival after 7 days, limiting infarct expansion and fibrosis in noninfarcted myocardium and alleviating LV dilatation and systolic dysfunction on day 28 post-MI. M(1) macrophages and neutrophils were the major cellular source of IL-23, whereas >90% of IL-17A-producing T cells in infarcted heart were CD4(-) TCRγδ(+) (γδT) cells. Toll-like receptor signaling and IL-1β worked in concert with IL-23 to drive expansion and IL-17A production in cardiac γδT cells, whereas the sphingosine-1-phosphate receptor and CCL20/CCR6 signaling pathways mediated γδT cell recruitment into infarcted heart. IL-17A was not involved in the acute inflammatory response, but it functioned specifically in the late remodeling stages by promoting sustained infiltration of neutrophils and macrophages, stimulating macrophages to produce proinflammatory cytokines, aggravating cardiomyocyte death, and enhancing fibroblast proliferation and profibrotic gene expression. The IL-23/IL-17A immune axis and γδT cells are potentially promising therapeutic targets after MI to prevent progression to end-stage dilated cardiomyopathy.

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Deleterious effect of the IL-23/IL-17A axis and γδT cells on left ventricular remodeling after myocardial infarction.

Journal of the American Heart Association 1 （ 5 ） 2012年10月

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Spred-2 deficiency exacerbates acetaminophen-induced hepatotoxicity in mice

Wakabayashi Hiroshi, Ito Toshihiro, Fushimi Soichiro, Nakashima Yuki, Itakura Jyunya, Qiuying Liu, Win Min Min, Cuiming Sun, Chen Cao, Sato Miwa, Mino Megumi, Ogino Tetsuya, Makino Hirofumi, Yoshimura Akihiko, Matsukawa Akihiro

Clinical Immunology 144 （ 3 ） 272 - 282 2012年09月

ISSN 1521-6616

MAPKs are involved in acetaminophen (APAP)-hepatotoxicity, but the regulatory mechanism remains unknown. Here, we explored the role of Spred-2 that negatively regulates Ras/ERK pathway in APAP-hepatotoxicity. Spred-2 knockout (KO) mice demonstrated exacerbated liver injury, an event that was associated with increased numbers of CD4 ⁺ T, CD8 ⁺ T and NK cells in the liver compared to the control. Levels of CXCL9/CXCL10 that attract and activate these cells were increased in Spred-2 KO-liver. Kupffer cells isolated from Spred-2 KO mice after APAP challenge expressed higher levels of CXCL9/CXCL10 than those from the control. Upon stimulation with APAP or IFNγ, naïve Kupffer cells from Spred-2 KO mice expressed higher levels of CXCL9/CXCL10. NK cell-depletion attenuated APAP-hepatotoxicity with lowered hepatic IFNγ and decreased numbers of not only NK cells but also CD4 ⁺ T and CD8 ⁺ T cells in the liver. These results suggest that Spred-2 negatively regulates APAP-hepatotoxicity under the control of Kupffer cells and NK cells. © 2012 Elsevier Inc.

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Spred-2 deficiency exacerbates acetaminophen-induced hepatotoxicity in mice

Clinical Immunology 144 （ 3 ） 272 - 282 2012年09月

ISSN 1521-6616

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Loss of Pdk1-Foxo1 signaling in myeloid cells predisposes to adipose tissue inflammation and insulin resistance

Kawano Yoshinaga, Nakae Jun, Watanabe Nobuyuki, Fujisaka Shiho, Iskandar Kristy, Sekioka Risa, Hayashi Yoshitake, Tobe Kazuyuki, Kasuga Masato, Noda Tetsuo, Yoshimura Akihiko, Onodera Masafumi, Itoh Hiroshi

Diabetes 61 （ 8 ） 1935 - 1948 2012年08月

ISSN 0012-1797

Chronic inflammation in adipose tissue contributes to obesity-related insulin resistance. The 3-phosphoinositide-dependent protein kinase 1 (Pdk1)/forkhead transcription factor (Foxo1) pathway is important in regulating glucose and energy homeostasis, but little is known about this pathway in adipose tissue macrophages (ATMs). To investigate this, we generated transgenic mice that carried macrophage/granulocyte-specific mutations, including a Pdk1 knockout (LysMPdk1 ^-/-), a Pdk1 knockout with transactivation- defective Foxo1 (Δ256LysMPdk1 ^-/-), a constitutively active nuclear (CN) Foxo1 (CNFoxo1 ^LysM), or a transactivation-defective Foxo1 (Δ256Foxo1 ^LysM). We analyzed glucose metabolism and gene expression in ATM populations isolated with fluorescence-activated cell sorting. The LysMPdk1 ^-/- mice exhibited elevated M1 macrophages in adipose tissue and insulin resistance. Overexpression of transactivation-defective Foxo1 rescued these phenotypes. CNFoxo1 ^LysM promoted transcription of the C-C motif chemokine receptor 2 (Ccr2) in ATMs and increased M1 macrophages in adipose tissue. On a high-fat diet, CNFoxo1 ^LysMmice exhibited insulin resistance. Pdk1 deletion or Foxo1 activation in bone marrow-derived macrophages abolished insulin and interleukin-4 induction of genes involved in alternative macrophage activation. Thus, Pdk1 regulated macrophage infiltration by inhibiting Foxo1-induced Ccr2 expression. This shows that the macrophage Pdk1/Foxo1 pathway is important in regulating insulin sensitivity in vivo. © 2012 by the American Diabetes Association.

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Loss of Pdk1-Foxo1 signaling in myeloid cells predisposes to adipose tissue inflammation and insulin resistance

Diabetes 61 （ 8 ） 1935 - 1948 2012年08月

ISSN 0012-1797

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Dysregulation of suppressor of cytokine signaling 3 in keratinocytes causes skin inflammation mediated by interleukin-20 receptor-related cytokines

Uto-Konomi Ayako, Miyauchi Kosuke, Ozaki Naoko, Motomura Yasutaka, Suzuki Yoshie, Yoshimura Akihiko, Suzuki Shinobu, Cua Daniel, Kubo Masato

PLoS ONE 7 （ 7 ） 2012年07月

Homeostatic regulation of epidermal keratinocytes is controlled by the local cytokine milieu. However, a role for suppressor of cytokine signaling (SOCS), a negative feedback regulator of cytokine networks, in skin homeostasis remains unclear. Keratinocyte specific deletion of Socs3 (Socs3 cKO) caused severe skin inflammation with hyper-production of IgE, epidermal hyperplasia, and S100A8/9 expression, although Socs1 deletion caused no inflammation. The inflamed skin showed constitutive STAT3 activation and up-regulation of IL-6 and IL-20 receptor (IL-20R) related cytokines, IL-19, IL-20 and IL-24. Disease development was rescued by deletion of the Il6 gene, but not by the deletion of Il23, Il4r, or Rag1 genes. The expression of IL-6 in Socs3 cKO keratinocytes increased expression of IL-20R-related cytokines that further facilitated STAT3 hyperactivation, epidermal hyperplasia and neutrophilia. These results demonstrate that skin homeostasis is strictly regulated by the IL-6-STAT3-SOCS3 axis. Moreover, the SOCS3-mediated negative feedback loop in keratinocytes has a critical mechanistic role in the prevention of skin inflammation caused by hyperactivation of STAT3. © 2012 Uto-Konomi et al.

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Dysregulation of suppressor of cytokine signaling 3 in keratinocytes causes skin inflammation mediated by interleukin-20 receptor-related cytokines

Uto-Konomi Ayako, Miyauchi Kosuke, Ozaki Naoko, Motomura Yasutaka, Suzuki Yoshie, Yoshimura Akihiko, Suzuki Shinobu, Cua Daniel, Kubo Masato

PLoS ONE 7 （ 7 ） 2012年07月

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Forced expression of stabilized c-Fos in dendritic cells reduces cytokine production and immune responses in vivo

Yoshida Ryoko, Suzuki Mayu, Sakaguchi Ryota, Hasegawa Eiichi, Kimura Akihiro, Shichita Takashi, Sekiya Takashi, Shiraishi Hiroshi, Shimoda Kouji, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 423 （ 2 ） 247 - 252 2012年06月

ISSN 0006-291X

Intracellular cyclic adenosine monophosphate (cAMP) suppresses innate immunity by inhibiting proinflammatory cytokine production by monocytic cells. We have shown that the transcription factor c-Fos is responsible for cAMP-mediated suppression of inflammatory cytokine production, and that c-Fos protein is stabilized by IKKβ-mediated phosphorylation. We found that S308 is one of the major phosphorylation sites, and that the S308D mutation prolongs c-Fos halflife. To investigate the role of stabilized c-Fos protein in dendritic cells (DCs) in vivo, we generated CD11c-promoter-deriven c-FosS308D transgenic mice. As expected, bone marrow-derived DCs (BMDCs) from these Tg mice produced smaller amounts of inflammatory cytokines, including TNF-α, IL-12, and IL-23, but higher levels of IL-10, in response to LPS, than those from wild-type (Wt) mice. When T cells were co-cultured with BMDCs from Tg mice, production of Th1 and Th17 cytokines was reduced, although T cell proliferation was not affected. Tg mice demonstrated more resistance to experimental autoimmune encephalomyelitis (EAE) than did Wt mice. These data suggest that c-Fos in DCs plays a suppressive role in certain innate and adaptive immune responses. © 2012 Elsevier Inc.

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Forced expression of stabilized c-Fos in dendritic cells reduces cytokine production and immune responses in vivo

Yoshida Ryoko, Suzuki Mayu, Sakaguchi Ryota, Hasegawa Eiichi, Kimura Akihiro, Shichita Takashi, Sekiya Takashi, Shiraishi Hiroshi, Shimoda Kouji, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 423 （ 2 ） 247 - 252 2012年06月

ISSN 0006-291X

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Signal-transducing adaptor protein-2 modulates fas-mediated T cell apoptosis by interacting with caspase-8

Sekine Yuichi, Yamamoto Chikako, Kakisaka Michinori, Muromoto Ryuta, Kon Shigeyuki, Ashitomi Dai, Fujita Natsuko, Yoshimura Akihiko, Oritani Kenji, Matsuda Tadashi

Journal of Immunology 188 （ 12 ） 6194 - 6204 2012年06月

ISSN 0022-1767

We found that an adaptor protein, signal-transducing adaptor protein (STAP)-2, is a new member of the Fas-death-inducing signaling complex and participates in activation-induced cell death in T cells. STAP-2 enhanced Fas-mediated apoptosis and caspase-8 aggregation and activation in Jurkat T cells. Importantly, STAP-2 directly interacted with caspase-8 and Fas, resulting in enhanced interactions between caspase-8 and FADD in the Fas-death-inducing signaling complex. Moreover, STAP-2 protein has a consensus caspase-8 cleavage sequence, VEAD, in its C-terminal domain, and processing of STAP-2 by caspase-8 was crucial for Fas-induced apoptosis. Physiologic roles of STAP-2 were confirmed by observations that STAP-2-deficient mice displayed impaired activation-induced cell death and superantigen-induced T cell depletion. Therefore, STAP-2 is a novel participant in the regulation of T cell apoptosis after stimulation. Copyright © 2012 by The American Association of Immunologists, Inc.

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Signal-transducing adaptor protein-2 modulates fas-mediated T cell apoptosis by interacting with caspase-8

Sekine Yuichi, Yamamoto Chikako, Kakisaka Michinori, Muromoto Ryuta, Kon Shigeyuki, Ashitomi Dai, Fujita Natsuko, Yoshimura Akihiko, Oritani Kenji, Matsuda Tadashi

Journal of Immunology 188 （ 12 ） 6194 - 6204 2012年06月

ISSN 0022-1767

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Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain

Shichita Takashi, Hasegawa Eiichi, Kimura Akihiro, Morita Rimpei, Sakaguchi Ryota, Takada Ichiro, Sekiya Takashi, Ooboshi Hiroaki, Kitazono Takanari, Yanagawa Toru, Ishii Tetsuro, Takahashi Hideo, Mori Shuji, Nishibori Masahiro, Kuroda Kazumichi, Akira Shizuo, Miyake Kensuke, Yoshimura Akihiko

Nature Medicine 18 （ 6 ） 911 - 917 2012年06月

ISSN 1078-8956

Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. However, the mechanism that activates infiltrating macrophages in the ischemic brain remains to be clarified. Here we demonstrate that peroxiredoxin (Prx) family proteins released extracellularly from necrotic brain cells induce expression of inflammatory cytokines including interleukin-23 in macrophages through activation of Toll-like receptor 2 (TLR2) and TLR4, thereby promoting neural cell death, even though intracellular Prxs have been shown to be neuroprotective. The extracellular release of Prxs in the ischemic core occurred 12 h after stroke onset, and neutralization of extracellular Prxs with antibodies suppressed inflammatory cytokine expression and infarct volume growth. In contrast, high mobility group box 1 (HMGB1), a well-known damage-associated molecular pattern molecule, was released before Prx and had a limited role in post-ischemic macrophage activation. We thus propose that extracellular Prxs are previously unknown danger signals in the ischemic brain and that its blocking agents are potent neuroprotective tools. © 2012 Nature America, Inc. All rights reserved.

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Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain

Nature Medicine 18 （ 6 ） 911 - 917 2012年06月

ISSN 1078-8956

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Preferential induction of Th17 cells in vitro and in vivo by Fucogalactan from Ganoderma lucidum (Reishi)

Yoshida Hideyuki, Suzuki Mayu, Sakaguchi Ryota, Tani Ito, Kotani Hitoshi, Shudo Norimasa, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 422 （ 1 ） 174 - 180 2012年05月

ISSN 0006-291X

The mushroom known as Reishi (Ganoderma lucidum) has been used as an herbal medicine for tumor treatment and immune system activation. Because its effects on the differentiation of effector T helper cells have not yet been fully understood, we investigated the effects of Reishi and those of its principal ingredient, β-glucan, on the activation of dendritic cells and the differentiation of Th17 cells. Reishi extracts as well as purified β-glucan (Curdran) activated DCs and caused them to produce large amounts of IL-23. β-glucan also enhanced and sustained the transcription of IL-23p19. The MEK-ERK signaling pathway positively regulates IL-23p19 transcription in β-glucan-stimulated DCs. In a mixed leukocyte reaction, Reishi-stimulated DCs preferentially induced Th17 cells. Furthermore, orally-administrated Reishi increased the percentages of Th17 cells and the transcription levels of antimicrobial peptides. Our results show that Reishi and β-glucan activate DCs to produce large amounts of IL-23, which induces Th17 differentiation both in vitro and in vivo. © 2012 Elsevier Inc.

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Preferential induction of Th17 cells in vitro and in vivo by Fucogalactan from Ganoderma lucidum (Reishi)

Yoshida Hideyuki, Suzuki Mayu, Sakaguchi Ryota, Tani Ito, Kotani Hitoshi, Shudo Norimasa, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 422 （ 1 ） 174 - 180 2012年05月

ISSN 0006-291X

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CCL2-induced migration and SOCS3-mediated activation of macrophages are involved in cerulein-induced pancreatitis in mice

Saeki Keita, Kanai Takanori, Nakano Masaru, Nakamura Yuji, Miyata Naoteru, Sujino Tomohisa, Yamagishi Yoshiyuki, Ebinuma Hirotoshi, Takaishi Hiromasa, Ono Yuuichi, Takeda Kazuyoshi, Hozawa Shigenari, Yoshimura Akihiko, Hibi Toshifumi

Gastroenterology 142 （ 4 ） 2012年04月

ISSN 0016-5085

Background & Aims: Acute pancreatitis is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with recruitment of leukocytes. We investigated the pathologic roles of innate immune cells, especially macrophages, in cerulein and L-arginineinduced acute pancreatitis in mice. Methods: Acute pancreatitis was induced by sequential peritoneal administration of cerulein to mice. We determined serum concentrations of amylase and lipase, pancreatic pathology, and features of infiltrating mononuclear cells. We performed parabiosis surgery to assess the hemodynamics of pancreatic macrophages. Results: Almost all types of immune cells, except for CD11b ^highCD11c ^- cells, were detected in the pancreas of healthy mice. However, activated CD11b ^highCD11c ^- cells, including Gr-1 ^low macrophages and Gr-1 ^high cells (granulocytes and myeloid-derived suppressor cells), were detected in damaged pancreas after cerulein administration. CCL2 ^-/- mice given cerulein injections developed significantly less severe pancreatitis, with less infiltration of CD11b ^highCD11c ^-Gr-1 ^low macrophages, but comparable infiltration of myeloid-derived suppressor cells, compared with cerulein-injected wild-type mice. Parabiosis and bone marrow analyses of these mice revealed that the CD11b ^highCD11c ^-Gr-1 ^low macrophages had moved out of the bone marrow. Furthermore, mice with macrophage-specific deletion of suppressor of cytokine signaling 3 given injections of cerulein developed less severe pancreatitis and Gr-1 ^low macrophage produced less tumor necrosis factor-α than wild-type mice given cerulein, although the absolute number of CD11b ^highCD11c ^-Gr-1 ^low macrophages was comparable between strains. Induction of acute pancreatitis by L-arginine required induction of macrophage migration by CCL2, via the receptor CCR2. Conclusions: Cerulein induction of pancreatitis in mice involves migration of CD11b ^highCD11c ^-Gr-1 ^low macrophage from the bone marrow (mediated by CCL2 via CCR2) and suppressor of cytokine signaling 3dependent activation of macrophage. These findings might lead to new therapeutic strategies for acute pancreatitis. © 2012 AGA Institute.

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CCL2-induced migration and SOCS3-mediated activation of macrophages are involved in cerulein-induced pancreatitis in mice

Gastroenterology 142 （ 4 ） 2012年04月

ISSN 0016-5085

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JAK's SOCS

Yoshimura Akihiko, Yasukawa Hideo

Immunity 36 （ 2 ） 157 - 159 2012年02月

ISSN 1074-7613

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JAK's SOCS

Yoshimura Akihiko, Yasukawa Hideo

Immunity 36 （ 2 ） 157 - 159 2012年02月

ISSN 1074-7613

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Low dose CP-690,550 (tofacitinib), a pan-JAK inhibitor, accelerates the onset of experimental autoimmune encephalomyelitis by potentiating Th17 differentiation

Yoshida Hideyuki, Kimura Akihiro, Fukaya Tomohiro, Sekiya Takashi, Morita Rimpei, Shichita Takashi, Inoue Hiromasa, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 418 （ 2 ） 234 - 240 2012年02月

ISSN 0006-291X

Th17 cells, which have been implicated in autoimmune diseases, require STAT3 signaling activated by IL-6 or IL-23 for their development. Other Th1 and Th2 cytokines such as IL-2, IFN-γ and IL-4 strongly suppress Th17 development. Recently, CP-690,550 (tofacitinib), originally developed as a JAK3 inhibitor, has been shown to be effective in phase III clinical trials of rheumatoid arthritis and collagen-induced arthritis (CIA) models, but the precise mechanism of the effect, especially with respect to Th17 cells, is poorly understood. To our surprise, a low dose CP-690,550 was found to accelerate the onset of experimental autoimmune encephalomyelitis (EAE) at a concentration that suppressed CIA. At an early stage after immunization, more IL-17 production was observed in 15. mg/kg body weight CP-690,550-treated mice than in untreated mice. In vitro, CP-690,550 inhibited both Th1 and Th2 development, while promoting Th17 differentiation at 10-50. nM concentrations. Enhancement of Th17 by CP-690,550 is probably due to suppression of IL-2 signaling, because anti-IL-2 antibodies cancel the Th17-promoting effect of CP-690,550. CP-690,550 selectively inhibited IFN--induced STAT1, IL-4-induced STAT6 and IL-2-induced STAT5 at 3-30. nM, while suppression of IL-6-induced STAT3 phosphorylation required a concentration greater than 100. nM. In HEK293T cells, CP-690,550 less effectively suppressed JAK1-mediated STAT3 phosphorylation compared with JAK3. These results suggest that CP-690,550 has a different effects among JAKs and STATs, thereby affecting helper T cell differentiation, and murine autoimmune disease models. © 2012 Elsevier Inc.

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Low dose CP-690,550 (tofacitinib), a pan-JAK inhibitor, accelerates the onset of experimental autoimmune encephalomyelitis by potentiating Th17 differentiation

Yoshida Hideyuki, Kimura Akihiro, Fukaya Tomohiro, Sekiya Takashi, Morita Rimpei, Shichita Takashi, Inoue Hiromasa, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 418 （ 2 ） 234 - 240 2012年02月

ISSN 0006-291X

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TGFβ-activated kinase 1 (TAK1)-binding proteins (TAB) 2 and 3 negatively regulate autophagy

Takaesu Giichi, Kobayashi Takashi, Yoshimura Akihiko

Journal of Biochemistry 151 （ 2 ） 157 - 166 2012年02月

ISSN 0021-924X

Transforming growth factor β-activated protein kinase 1 (TAK1)-binding protein 2 (TAB2) and its close homolog TAB3 are initially characterized as adapter proteins essential for TAK1 activation in response to interleukin-1β and tumour necrosis factor-α. However, the physiological roles of TAB2 and TAB3 are still not fully understood. Here we report that TAB2 and TAB3 bind to Beclin1 and colocalize in the cytoplasm. TAB2 also interacts with ATG13 and is phosphorylated by ULK1. Overexpression of TAB2 or TAB3 induces punctate localization of ATG5 under the normal culture condition. Knockdown of TAB2 and TAB3 results in the decrease in endogenous protein level of p62/SQSTM1 under the normal culture condition, while overexpression of TAB2 results in the accumulation of p62/SQSTM1 independently of TAK1. The decrease of p62/SQSTM1 induced by the knockdown of TAB2 and TAB3 is largely dependent on ATG5. These results suggest that TAB2 and TAB3 negatively regulate autophagy independently of TAK1 activity. © The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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TGFβ-activated kinase 1 (TAK1)-binding proteins (TAB) 2 and 3 negatively regulate autophagy

Takaesu Giichi, Kobayashi Takashi, Yoshimura Akihiko

Journal of Biochemistry 151 （ 2 ） 157 - 166 2012年02月

ISSN 0021-924X

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SOCS, inflammation, and autoimmunity

Yoshimura Akihiko, Suzuki Mayu, Sakaguchi Ryota, Hanada Toshikatsu, Yasukawa Hideo

Frontiers in Immunology 3 （ MAR ） 2012年

Cytokines play essential roles in innate and adaptive immunity. However, excess cytokines or dysregulation of cytokine signaling will cause a variety of diseases, including allergies, autoimmune diseases, inflammation, and cancer. Most cytokines utilize the so-called Janus kinase-signal transducers and activators of transcription pathway. This pathway is negatively regulated by various mechanisms including suppressors of cytokine signaling (SOCS) proteins. SOCS proteins bind to JAK or cytokine receptors, thereby suppressing further signaling events. Especially, suppressor of cytokine signaling-1 (SOCS1) and SOCS3 are strong inhibitors of JAKs, because these two contain kinase inhibitory region at the Nterminus. Studies using conditional knockout mice have shown that SOCS proteins are key physiological as well as pathological regulators of immune homeostasis. Recent studies have also demonstrated that SOCS1 and SOCS3 are important regulators of helper T cell differentiation and functions. This review focuses on the roles of SOCS1 and SOCS3 in T cell mediated inflammatory diseases. © 2012 Yoshimura, Suzuki, Sakaguchi, Hanada and Yasukawa.

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Smad2 and smad3 are redundantly essential for the suppression of inos synthesis in macrophages by regulating IRF3 and STAT1 pathways

Sugiyama Yuki, Kakoi Kyosuke, Kimura Akihiro, Takada Ichiro, Kashiwagi Ikko, Wakabayashi Yu, Morita Rimpei, Nomura Masatoshi, Yoshimura Akihiko

International Immunology 24 （ 4 ） 253 - 265 2012年

ISSN 0953-8178

Although transforming growth factor (TGF)-β1 is a well-known immunosuppressive cytokine, little is known about the role of its downstream transcription factors, Smad2 and Smad3, in the suppression of macrophage activation. Previous studies have demonstrated that Smad3 is critical for the suppression of LPS-mediated inducible nitric oxide (NO) synthase (iNOS) induction, although the role of Smad2 remains to be investigated. In this study, we found that iNOS induction was enhanced in Smad2-deficient bone marrow-derived macrophages (BMDMs) and peritoneal macrophages in vitro and tumor-associated macrophages in vivo, compared with wild-type (WT) macrophages. However, TGF-β1 still suppressed iNOS induction in Smad2-deficient macrophages. In Smad2/3 double knockout (KO) (Smad2/3 DKO) BMDMs, LPS-mediated NO/iNOS induction was more strongly elevated than in Smad2 or Smad3 single KO BMDMs, and its suppression by exogenous TGF-β1 was severely impaired. These data suggest that Smad2 and Smad3 redundantly regulate iNOS induction. Similarly, the production of IL-6 and TNFα, but not IL-10 was augmented in Smad2/3 DKO BMDMs, suggesting that Smad2 and Smad3 also redundantly suppressed some cytokines production. In Smad2/3 DKO macrophages, TLR3- as well as TLR4-mediated IRF3 activation and IFN-β production were strongly augmented, which resulted in hyper STAT1 phosphorylation. Furthermore, IFN-β- and IFN-γ-induced iNOS induction in the absence of TLR signaling and STAT1 transcriptional activity were augmented in Smad2/3 DKO BMDMs. These results suggest that Smad2 and Smad3 negatively regulate iNOS induction in macrophages by suppressing multiple steps in the IRF3-IFN-β-STAT1 pathway. © The Japanese Society for Immunology. 2012. All rights reserved.

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Foreward

Yoshimura Akihiko

Seikagaku 84 （ 4 ） 245 2012年

ISSN 0037-1017

Post-ischemic inflammation in the brain

Shichita Takashi, Sakaguchi Ryota, Suzuki Mayu, Yoshimura Akihiko

Frontiers in Immunology 3 （ MAY ） 2012年

Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. In this review, we focus on the post-ischemic inflammation triggered by infiltrating immune cells, macrophages, and T lymphocytes. Brain ischemia is a sterile organ, but injury-induced inflammation is mostly dependent on Toll-like receptor (TLR) 2 andTLR4. Some endogenousTLR ligands, high mobility group box 1 (HMGB1) and peroxiredoxin family proteins, in particular, are implicated in the activation and inflammatory cytokine expression in infiltrating macrophages. Following macrophage activation, T lymphocytes infiltrate the ischemic brain and regulate the delayed phase inflammation. IL17-producing γδT lymphocytes induced by IL-23 from macrophages promote ischemic brain injury, whereas regulatory T lymphocytes suppress the function of inflammatory mediators. A deeper understanding of the inflammatory mechanisms of infiltrating immune cells may lead to the development of novel neuroprotective therapies. © 2012 Shichita, Sakaguchi, Suzuki and Yoshimura.

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SOCS, inflammation, and autoimmunity

Yoshimura Akihiko, Suzuki Mayu, Sakaguchi Ryota, Hanada Toshikatsu, Yasukawa Hideo

Frontiers in Immunology 3 （ MAR ） 2012年

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Smad2 and smad3 are redundantly essential for the suppression of inos synthesis in macrophages by regulating IRF3 and STAT1 pathways

Sugiyama Yuki, Kakoi Kyosuke, Kimura Akihiro, Takada Ichiro, Kashiwagi Ikko, Wakabayashi Yu, Morita Rimpei, Nomura Masatoshi, Yoshimura Akihiko

International Immunology 24 （ 4 ） 253 - 265 2012年

ISSN 0953-8178

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Foreward

Yoshimura Akihiko

Seikagaku 84 （ 4 ） 245 2012年

ISSN 0037-1017

Post-ischemic inflammation in the brain

Shichita Takashi, Sakaguchi Ryota, Suzuki Mayu, Yoshimura Akihiko

Frontiers in Immunology 3 （ MAY ） 2012年

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Pyridone 6, a pan-JAK inhibitor, ameliorates allergic skin inflammation of NC/Nga mice via suppression of Th2 and enhancement of Th17

Nakagawa Ryusuke, Yoshida Hideyuki, Asakawa Mayako, Tamiya Taiga, Inoue Naoko, Morita Rimpei, Inoue Hiromasa, Nakao Atsuhito, Yoshimura Akihiko

Journal of Immunology 187 （ 9 ） 4611 - 4620 2011年11月

ISSN 0022-1767

Atopic dermatitis (AD) is a common pruritic inflammatory disease triggered by a defective skin barrier and immunodysregulation. AD has been considered a typical example of a Th2 response associated with allergic disease. In the early phases of the disease, symptoms include IgE hyperproduction, eosinophil accumulation, and mast cell activation; in the chronic phase, a Th1- dominant immune response is also observed at the sites of AD skin lesions. The role of IL-17-producing Th (Th17) cells in AD has not been established. In the current study, we found that pyridone 6 (P6), a pan-JAK inhibitor, delayed the onset and reduced the magnitude of skin disease in an AD-like skin-disease model of NC/Nga mice. P6 reduced IFN-γ and IL-13, whereas it enhanced IL- 17 and IL-22 expression. In vitro, P6 also inhibited both Th1 and Th2 development, whereas it promoted Th17 differentiation from naive T cells when present within a certain range of concentrations. This was probably because P6 strongly inhibited STAT1, STAT5, and STAT6 phosphorylation, whereas STAT3 phosphorylation was less efficiently suppressed by P6 at the same concentration. Furthermore, IL-22 protects keratinocytes from apoptosis induced by IFN-γ, and administration of IL-17 and IL-22 partially ameliorated skin diseases in NC/Nga mice. These results suggested that the JAK inhibitor P6 is therapeutic for AD by modulating the balance of Th2 and Th17. Copyright © 2011 by The American Association of Immunologists, Inc.

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Pyridone 6, a pan-JAK inhibitor, ameliorates allergic skin inflammation of NC/Nga mice via suppression of Th2 and enhancement of Th17

Nakagawa Ryusuke, Yoshida Hideyuki, Asakawa Mayako, Tamiya Taiga, Inoue Naoko, Morita Rimpei, Inoue Hiromasa, Nakao Atsuhito, Yoshimura Akihiko

Journal of Immunology 187 （ 9 ） 4611 - 4620 2011年11月

ISSN 0022-1767

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IL-1β and TNFα-initiated IL-6-STAT3 pathway is critical in mediating inflammatory cytokines and RANKL expression in inflammatory arthritis

Mori Tomoaki, Miyamoto Takeshi, Yoshida Hideyuki, Asakawa Mayoko, Kawasumi Miyuri, Kobayashi Takashi, Morioka Hideo, Chiba Kazuhiro, Toyama Yoshiaki, Yoshimura Akihiko

International Immunology 23 （ 11 ） 701 - 712 2011年11月

ISSN 0953-8178

Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes irreversible joint damage and significant disability. However, the fundamental mechanisms underlying how inflammation and joint destruction in RA develop and are sustained chronically remain largely unknown. Here, we show that signal transducer and activator of transcription 3 (STAT3) is the key mediator of both chronic inflammation and joint destruction in RA. We found that inflammatory cytokines highly expressed in RA patients, such as IL-1β, tumor necrosis factor alpha and IL-6, activated STAT3 either directly or indirectly and in turn induced expression of IL-6 family cytokines, further activating STAT3 in murine osteoblastic and fibroblastic cells. STAT3 activation also induced expression of receptor activator of nuclear factor kappa B ligand (RANKL), a cytokine essential for osteoclastogenesis, and STAT3 deficiency or pharmacological inhibition promoted significant reduction in expression of both IL-6 family cytokines and RANKL in vitro. STAT3 inhibition was also effective in treating an RA model, collagen-induced arthritis, in vivo through significant reduction in expression of IL-6 family cytokines and RANKL, inhibiting both inflammation and joint destruction. Leukemia inhibitory factor expression and STAT3 activation by IL-1β were mainly promoted by IL-6 but still induced in IL-6-deficient cells. Thus, our data provide new insight into RA pathogenesis and provide evidence that inflammatory cytokines trigger a cytokine amplification loop via IL-6-STAT3 that promotes sustained inflammation and joint destruction. © The Japanese Society for Immunology. 2011. All rights reserved.

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IL-1β and TNFα-initiated IL-6-STAT3 pathway is critical in mediating inflammatory cytokines and RANKL expression in inflammatory arthritis

Mori Tomoaki, Miyamoto Takeshi, Yoshida Hideyuki, Asakawa Mayoko, Kawasumi Miyuri, Kobayashi Takashi, Morioka Hideo, Chiba Kazuhiro, Toyama Yoshiaki, Yoshimura Akihiko

International Immunology 23 （ 11 ） 701 - 712 2011年11月

ISSN 0953-8178

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Histone 3 lysine 9 (H3K9) methyltransferase recruitment to the interleukin-2 (IL-2) promoter is a mechanism of suppression of IL-2 transcription by the transforming growth factor-β-smad pathway

Wakabayashi Yu, Tamiya Taiga, Takada Ichiro, Fukaya Tomohiro, Sugiyama Yuki, Inoue Naoko, Kimura Akihiro, Morita Rimpei, Kashiwagi Ikko, Takimoto Tomohito, Nomura Masatoshi, Yoshimura Akihiko

Journal of Biological Chemistry 286 （ 41 ） 35456 - 35465 2011年10月

ISSN 0021-9258

Suppression of IL-2βproduction from T cells is an important process for the immune regulation by TGF-β. However, the mechanism by which this suppression occurs remains to be established. Here, we demonstrate that Smad2 and Smad3, two major TGF-β-downstream transcription factors, are redundantly essential for TGF-β-mediated suppression of IL-2 production in CD4 ⁺ T cells using Smad2- and Smad3-deficient T cells. Both Smad2 and Smad3 were recruited into the proximal region of the IL-2 promoter in response to TGF-β. We then investigated the histone methylation status of the IL-2 promoter. Although both histone H3 lysine 9 (H3K9) and H3K27 trimethylation have been implicated in gene silencing, only H3K9 trimethylation was increased in the proximal region of the IL-2 promoter in a Smad2/3-dependent manner, whereas H3K27 trimethylation was not. The H3K9 methyltransferases Setdb1 and Suv39h1 bound to Smad3 and suppressed IL-2 promoter activity in collaboration with Smad3. Overexpression of Suv39h1 in 68-41 T cells strongly inhibited IL-2 production in response to T cell receptor stimulation irrespective of the presence or absence of TGF-β, whereas Setdb1 overexpression only slightly suppressed IL-2 production. Silencing of Suv39h1 by shRNA reverted the suppressive effect of TGF-β on IL-2 production. Furthermore, TGF-β induced Suv39h1 recruitment to the proximal region of the IL-2 promoter in wild type primary T cells; however, this was not observed in Smad2 ^-/-Smad3 ^+/- T cells. Thus, we propose that Smads recruit H3K9 methyltransferases Suv39h1 to the IL-2 promoter, thereby inducing suppressive histone methylation and inhibiting T cell receptor-mediated IL-2 transcription. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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Histone 3 lysine 9 (H3K9) methyltransferase recruitment to the interleukin-2 (IL-2) promoter is a mechanism of suppression of IL-2 transcription by the transforming growth factor-β-smad pathway

Wakabayashi Yu, Tamiya Taiga, Takada Ichiro, Fukaya Tomohiro, Sugiyama Yuki, Inoue Naoko, Kimura Akihiro, Morita Rimpei, Kashiwagi Ikko, Takimoto Tomohito, Nomura Masatoshi, Yoshimura Akihiko

Journal of Biological Chemistry 286 （ 41 ） 35456 - 35465 2011年10月

ISSN 0021-9258

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Suppression of SOCS3 in macrophages prevents cancer metastasis by modifying macrophage phase and MCP2/CCL8 induction

Hiwatashi Kiyokazu, Tamiya Taiga, Hasegawa Eiichi, Fukaya Tomohiro, Hashimoto Masayuki, Kakoi Kyosuke, Kashiwagi Ikko, Kimura Akihiro, Inoue Naoko, Morita Rimpei, Yasukawa Hideo, Yoshimura Akihiko

Cancer Letters 308 （ 2 ） 172 - 180 2011年09月

ISSN 0304-3835

Inflammation has been demonstrated to play important roles in tumorigenesis, tumor progression, and metastasis. STAT3 has been shown to be frequently activated in a variety of human cancer cells and STAT3 signaling promotes the growth and survival of tumor cells. However, the role of STAT3 of myeloid cells associated with tumors is currently unknown. Suppressor of cytokine signaling-3 (SOCS3) has been shown to be a negative regulator of STAT3. In this study, we used macrophage specific SOCS3 conditional knockout (cKO) mice to investigate the effect of the hyperactivation of STAT3 in macrophages on tumor development and metastasis. In a subcutaneous transplantation model of B16F10 melanoma cells, although tumor sizes were not significantly different, SOCS3-cKO mice survived longer than wild-type (WT) mice did. SOCS3-cKO mice exhibited fewer lung and liver metastatic tumor nodules than WT mice when B16F10 was challenged intravenously. SOCS3^-/- macrophages stimulated with tumor lysates in vitro exhibited prolonged STAT3 phosphorylation and produced less amount of TNFα and IL-6, and higher amount of MCP2/CCL8 than WT macrophages. MCP/CCL8 was induced via STAT3 and exhibited anti-tumor metastatic effect in WT mice. These data suggest that hyperactivation of STAT3 in myeloid cells simultaneously exerted an anti-inflammatory as well as anti-tumor effects. Thus, the targeted inhibition of SOCS3 activity in macrophages may be therapeutic for the suppression of tumor metastasis. © 2011 Elsevier Ireland Ltd.

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Suppression of SOCS3 in macrophages prevents cancer metastasis by modifying macrophage phase and MCP2/CCL8 induction

Hiwatashi Kiyokazu, Tamiya Taiga, Hasegawa Eiichi, Fukaya Tomohiro, Hashimoto Masayuki, Kakoi Kyosuke, Kashiwagi Ikko, Kimura Akihiro, Inoue Naoko, Morita Rimpei, Yasukawa Hideo, Yoshimura Akihiko

Cancer Letters 308 （ 2 ） 172 - 180 2011年09月

ISSN 0304-3835

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SOCS1 is essential for regulatory T cell functions by preventing loss of Foxp3 expression as well as IFN-γ and IL-17A production

Takahashi Reiko, Nishimoto Shuhei, Muto Go, Sekiya Takashi, Tamiya Taiga, Kimura Akihiro, Morita Rimpei, Asakawa Mayako, Chinen Takatoshi, Yoshimura Akihiko

Journal of Experimental Medicine 208 （ 10 ） 2055 - 2067 2011年09月

ISSN 0022-1007

Regulatory T cells (T_reg cells) maintain immune homeostasis by limiting inflammatory responses. SOCS1 (suppressor of cytokine signaling 1), a negative regulator of cytokine signaling, is necessary for the suppressor functions of T_reg cells in vivo, yet detailed mechanisms remain to be clarified. We found that Socs1^-/- Treg cells produced high levels of IFN-γ and rapidly lost Foxp3 when transferred into Rag2^-/- mice or cultured in vitro, even though the CNS2 (conserved noncoding DNA sequence 2) in the Foxp3 enhancer region was fully demethylated. Socs1^-/- T_reg cells showed hyperactivation of STAT1 and STAT3. Because Foxp3 expression was stable and STAT1 activation was at normal levels in Ifnγ^-/-Socs1^-/- T_reg cells, the restriction of IFN-γ-STAT1 signaling by SOCS1 is suggested to be necessary for stable Foxp3 expression. However, Ifnγ^-/-Socs1^-/- Treg cells had hyperactivated STAT3 and higher IL-17A (IL-17) production compared with Ifnγ^-/-Socs1^+/+ Treg cells and could not suppress colitis induced by naive T cells in Rag2^-/- mice. In vitro experiments suggested that cytokines produced by Socs1^-/- T_reg cells and Ifnγ^-/-Socs1^-/- T_reg cells modulated antigen-presenting cells for preferential Th1 and Th17 induction, respectively. We propose that SOCS1 plays important roles in T_reg cell integrity and function by maintaining Foxp3 expression and by suppressing IFN-γ and IL-17 production driven by STAT1 and STAT3, respectively. © 2011 Takahashi et al.

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SOCS1 is essential for regulatory T cell functions by preventing loss of Foxp3 expression as well as IFN-γ and IL-17A production

Takahashi Reiko, Nishimoto Shuhei, Muto Go, Sekiya Takashi, Tamiya Taiga, Kimura Akihiro, Morita Rimpei, Asakawa Mayako, Chinen Takatoshi, Yoshimura Akihiko

Journal of Experimental Medicine 208 （ 10 ） 2055 - 2067 2011年09月

ISSN 0022-1007

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Desmoglein 3-specific CD4⁺ T cells induce pemphigus vulgaris and interface dermatitis in mice

Takahashi Hayato, Kouno Michiyoshi, Nagao Keisuke, Wada Naoko, Hata Tsuyoshi, Nishimoto Shuhei, Iwakura Yoichiro, Yoshimura Akihiko, Yamada Taketo, Kuwana Masataka, Fujii Hideki, Koyasu Shigeo, Amagai Masayuki

Journal of Clinical Investigation 121 （ 9 ） 3677 - 3688 2011年09月

ISSN 0021-9738

Pemphigus vulgaris (PV) is a severe autoimmune disease involving blistering of the skin and mucous membranes. It is caused by autoantibodies against desmoglein 3 (Dsg3), an adhesion molecule critical for maintaining epithelial integrity in the skin, oral mucosa, and esophagus. Knowing the antigen targeted by the autoantibodies renders PV a valuable model of autoimmunity. Recently, a role for Dsg3-specific CD4⁺ T helper cells in autoantibody production was demonstrated in a mouse model of PV, but whether these cells exert cytotoxicity in the tissues is unclear. Here, we analyzed 3 Dsg3-specific TCRs using transgenic mice and retrovirus induction. Dsg3-specific transgenic (Dsg3H1) T cells underwent deletion in the presence of Dsg3 in vivo. Dsg3H1 T cells that developed in the absence of Dsg3 elicited a severe pemphigus-like phenotype when cotransferred into immunodeficient mice with B cells from Dsg3^-/- mice. Strikingly, in addition to humoral responses, T cell infiltration of Dsg3-expressing tissues led to interface dermatitis, a distinct form of T cell-mediated autoimmunity that causes keratinocyte apoptosis and is seen in various inflammatory/autoimmune skin diseases, including paraneoplastic pemphigus. The use of retrovirally generated Dsg3-specific T cells revealed that interface dermatitis occurred in an IFN-γ- and TCR avidity-dependent manner. This model of autoimmunity demonstrates that T cells specific for a physiological skin-associated autoantigen are capable of inducing interface dermatitis and should provide a valuable tool for further exploring the immunopathophysiology of T cell-mediated skin diseases.

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Desmoglein 3-specific CD4⁺ T cells induce pemphigus vulgaris and interface dermatitis in mice

Journal of Clinical Investigation 121 （ 9 ） 3677 - 3688 2011年09月

ISSN 0021-9738

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Regulatory T cells suppress development of colitis, blocking differentiation of T-helper 17 into alternative T-helper 1 cells

Sujino Tomohisa, Kanai Takanori, Ono Yuichi, Mikami Yohei, Hayashi Atsushi, Doi Tomomitsu, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Takaishi Hiromasa, Ogata Haruhiko, Yoshimura Akihiko, Littman Dan R., Hibi Toshifumi

Gastroenterology 141 （ 3 ） 1014 - 1023 2011年09月

ISSN 0016-5085

Background & Aims: Although T-helper (Th) 17 and Th1 cells are involved in pathogenesis of intestinal inflammation, their developmental pathways and sufficiency to promote disease are not known; nor are the roles of CD4 ⁺CD25 ⁺ regulatory T (T _R) cells in their development. Methods: We performed adoptive transfer experiments to investigate the induction and suppression of colitis using nave CD4 ⁺CD45RB ^high T cells and/or CD4 ⁺CD25 ⁺ T _R cells that were obtained from retinoid-related orphan receptor gamma t (RORγt) gfp/ ⁺ or Ly5.1/Ly5.2 congenic mice. Results: We observed 3 types of colitogenic CD4 ⁺ Th1 cells (interleukin [IL]-17A ^-interferon [IFN]-γ ⁺): RORγt classical Th1 cells that differentiated directly from nave T cells; RORγt ⁺ Th1-like cells; and RORγt alternative Th1 cells that were terminally differentiated from RORγt ⁺ cells via Th17 (IL-17A ⁺IFN-γ), Th17/Th1 (IL-17A ⁺IFN- γ ⁺), or Th1-like (IL-17AIFN-γ ⁺) cells. In this pathway, CD4 ⁺CD25 ⁺ T _R cells suppress the development of not only classical Th1 cells, but also alternative Th1 cells at the transition of Th17/Th1 into alternative Th1 cells, resulting in accumulation of Th17 and Th17/Th1 cells in mice in which the development of colitis was suppressed. Furthermore, T _R cells regulated the established balance of Th17 and Th1 cells under colitic conditions to yield a high ratio of Th17 and Th17/Th1 cells to Th1 cells in noncolitic conditions. Conclusions: Th17 and Th17/Th1 cells become colitogenic alternative Th1 cells via Th17, Th17/Th1, and Th1-like cells, independently of classical Th1 cells. T _R cells suppress this pathway, resulting in accumulation of Th17 and Th17/Th1 cells. © 2011 AGA Institute.

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1,25-Dihydroxyvitamin D ₃ ameliorates Th17 autoimmunity via transcriptional modulation of interleukin-17A

Joshi Sneha, Pantalena Luiz Carlos, Liu Xikui K., Gaffen Sarah L., Liu Hong, Rohowsky-Kochan Christine, Ichiyama Kenji, Yoshimura Akihiko, Steinman Lawrence, Christakos Sylvia, Youssef Sawsan

Molecular and Cellular Biology 31 （ 17 ） 3653 - 3669 2011年09月

ISSN 0270-7306

A new class of inflammatory CD4 ⁺ T cells that produce interleukin-17 (IL-17) (termed Th17) has been identified, which plays a critical role in numerous inflammatory conditions and autoimmune diseases. The active form of vitamin D, 1,25-dihydroxyvitamin D ₃ [1,25(OH) ₂D ₃], has a direct repressive effect on the expression of IL-17A in both human and mouse T cells. In vivo treatment of mice with ongoing experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis) diminishes paralysis and progression of the disease and reduces IL-17A-secreting CD4 ⁺ T cells in the periphery and central nervous system (CNS). The mechanism of 1,25(OH) ₂D ₃ repression of IL-17A expression was found to be transcriptional repression, mediated by the vitamin D receptor (VDR). Transcription assays, gel shifting, and chromatin immunoprecipitation (ChIP) assays indicate that the negative effect of 1,25(OH) ₂D ₃ on IL-17A involves blocking of nuclear factor for activated T cells (NFAT), recruitment of histone deacetylase (HDAC), sequestration of Runt-related transcription factor 1 (Runx1) by 1,25(OH) ₂D ₃/VDR, and a direct effect of 1,25(OH) ₂D ₃ on induction of Foxp3. Our results describe novel mechanisms and new concepts with regard to vitamin D and the immune system and suggest therapeutic targets for the control of autoimmune diseases. © 2011, American Society for Microbiology.

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Regulatory T cells suppress development of colitis, blocking differentiation of T-helper 17 into alternative T-helper 1 cells

Gastroenterology 141 （ 3 ） 1014 - 1023 2011年09月

ISSN 0016-5085

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1,25-Dihydroxyvitamin D ₃ ameliorates Th17 autoimmunity via transcriptional modulation of interleukin-17A

Joshi Sneha, Pantalena Luiz Carlos, Liu Xikui K., Gaffen Sarah L., Liu Hong, Rohowsky-Kochan Christine, Ichiyama Kenji, Yoshimura Akihiko, Steinman Lawrence, Christakos Sylvia, Youssef Sawsan

Molecular and Cellular Biology 31 （ 17 ） 3653 - 3669 2011年09月

ISSN 0270-7306

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Silencing suppressor of cytokine signaling-1 (SOCS1) in macrophages improves Mycobacterium tuberculosis control in an interferon-γ (IFN-γ)-dependent manner

Carow Berit, Ye Xiang Qun, Gavier-Wide Dolores, Bhuju Sabin, Oehlmann Wulf, Singh Mahavir, Sköld Markus, Ignatowicz Lech, Yoshimura Akihiko, Wigzell Hans, Rottenberg Martin E.

Journal of Biological Chemistry 286 （ 30 ） 26873 - 26887 2011年07月

ISSN 0021-9258

Protection against infection with Mycobacterium tuberculosis demands IFN-γ. SOCS1 has been shown to inhibit responses to IFN-γ and might thereby play a central role in the outcome of infection. We found that M. tuberculosis is a highly efficient stimulator of SOCS1 expression in murine and human macrophages and in tissues from infected mice. Surprisingly, SOCS1 reduced responses to IL-12, resulting in an impaired IFN-γ secretion by macrophages that in turn accounted for a deteriorated intracellular mycobacterial control. Despite SOCS1 expression, mycobacteria-infected macrophages responded to exogenously added IFN-γ. SOCS1 attenuated the expression of the majority of genes modulated by M. tuberculosis infection of macrophages. Using a conditional knockdown strategy in mice, we found that SOCS1 expression by macrophages hampered M. tuberculosis clearance early after infection in vivo in an IFN-γ-dependent manner. On the other hand, at later time points, SOCS1 expression by non-macrophage cells protected the host from infection-induced detrimental inflammation. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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Silencing suppressor of cytokine signaling-1 (SOCS1) in macrophages improves Mycobacterium tuberculosis control in an interferon-γ (IFN-γ)-dependent manner

Carow Berit, Ye Xiang Qun, Gavier-Wide Dolores, Bhuju Sabin, Oehlmann Wulf, Singh Mahavir, Sköld Markus, Ignatowicz Lech, Yoshimura Akihiko, Wigzell Hans, Rottenberg Martin E.

Journal of Biological Chemistry 286 （ 30 ） 26873 - 26887 2011年07月

ISSN 0021-9258

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miR126 positively regulates mast cell proliferation and cytokine production through suppressing Spred1

Ishizaki Takuma, Tamiya Taiga, Taniguchi Koji, Morita Rimpei, Kato Reiko, Okamoto Fuyuki, Saeki Kazuko, Nomura Masatoshi, Nojima Yoshihisa, Yoshimura Akihiko

Genes to Cells 16 （ 7 ） 803 - 814 2011年07月

ISSN 1356-9597

The protein known as Spred1 (Sprouty-related Ena/VASP homology-1 domain-containing protein) has been identified as a negative regulator of growth factor-induced ERK/mitogen-activated protein kinase activation. Spred1 has also been implicated as the target of microRNA-126 (miR126), a miRNA located within the Egfl7 gene, and is involved in the regulation of vessel development through its role in regulating VEGF signaling. In this study, we examined the role of miR126 and Spred1 in the hematopoietic system, as miR126 has been shown to be overexpressed in leukemic cells. miR126 levels were down-regulated during mast cell differentiation from bone marrow cells, whereas Spred1 expression was inversely up-regulated. Overexpression of miR126 suppressed Spred1 expression and enhanced ERK activity in primary bone marrow cells and MC9 mast cells, which were associated with elevated FcεRI-mediated cytokine production. To confirm the effect of Spred1 reduction in vivo, we generated hematopoietic cell-specific Spred1-conditional knockout mice. These mice showed increased numbers of mast cells, and Spred1-deficient bone marrow-derived mast cells were highly activated by cross-linking of Fcε-R stimulation as well as by IL-3 and SCF stimulation. These results suggest that Spred1 negatively regulates mast cell activation, which is modulated by miR126. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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miR126 positively regulates mast cell proliferation and cytokine production through suppressing Spred1

Ishizaki Takuma, Tamiya Taiga, Taniguchi Koji, Morita Rimpei, Kato Reiko, Okamoto Fuyuki, Saeki Kazuko, Nomura Masatoshi, Nojima Yoshihisa, Yoshimura Akihiko

Genes to Cells 16 （ 7 ） 803 - 814 2011年07月

ISSN 1356-9597

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Transcription Factor Smad-Independent T Helper 17 Cell Induction by Transforming-Growth Factor-β Is Mediated by Suppression of Eomesodermin

Ichiyama Kenji, Sekiya Takashi, Inoue Naoko, Tamiya Taiga, Kashiwagi Ikko, Kimura Akihiro, Morita Rimpei, Muto Go, Shichita Takashi, Takahashi Reiko, Yoshimura Akihiko

Immunity 34 （ 5 ） 741 - 754 2011年05月

ISSN 1074-7613

Transforming growth factor-β (TGF-β) has been shown to be required for Th17 cell differentiation via Smad-independent mechanisms. The molecular mechanism underlying this pathway remains to be clarified, however. We searched for genes regulated by TGF-β through the Smad-independent pathway by using Smad2 and Smad3 double-deficient T cells and identified the transcription factor Eomesodermin (Eomes), whose expression was suppressed by TGF-β via the c-Jun N-terminal kinase (JNK)-c-Jun signaling pathway. Inhibition of JNK strongly suppressed disease in an in vivo EAE model as well as in vitro Th17 cell induction. Overexpression of Eomes substantially suppressed Th17 cell differentiation, whereas ablation of Eomes expression could substitute for TGF-β in Th17 cell induction in primary T cells. Eomes suppressed Rorc and Il17a promoters by directly binding to the proximal region of these promoters. In conclusion, the suppression of Eomes by TGF-β via the JNK pathway is an important mechanism for Smad-independent Th17 cell differentiation. © 2011 Elsevier Inc.

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Transcription Factor Smad-Independent T Helper 17 Cell Induction by Transforming-Growth Factor-β Is Mediated by Suppression of Eomesodermin

Ichiyama Kenji, Sekiya Takashi, Inoue Naoko, Tamiya Taiga, Kashiwagi Ikko, Kimura Akihiro, Morita Rimpei, Muto Go, Shichita Takashi, Takahashi Reiko, Yoshimura Akihiko

Immunity 34 （ 5 ） 741 - 754 2011年05月

ISSN 1074-7613

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Neuronal suppressor of cytokine signaling-3 deficiency enhances hypothalamic leptin-dependent phosphatidylinositol 3-kinase signaling

Metlakunta Anantha S., Sahu Maitrayee, Yasukawa Hideo, Dhillon Sandeep S., Belsham Denise D., Yoshimura Akihiko, Sahu Abhiram

American Journal of Physiology - Regulatory Integrative and Comparative Physiology 300 （ 5 ） 2011年05月

ISSN 0363-6119

Suppressor of cytokine signaling-3 (SOCS3) is thought to be involved in the development of central leptin resistance and obesity by inhibiting STAT3 pathway. Because phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in transducing leptin action in the hypothalamus, we examined whether SOCS3 exerted an inhibition on this pathway. We first determined whether leptin sensitivity in the hypothalamic PI3K pathway was increased in brain-specific Socs3-deficient (NesKO) mice. In NesKO mice, hypothalamic insulin receptor substrate-1 (IRS1)-associated PI3K activity was significantly increased at 30 min and remained elevated up to 2 h after leptin intraperitoneal injection, but in wild-type (WT) littermates, the significant increase was only at 30 min. Hypothalamic p-STAT3 levels were increased up to 5 h in NesKO as opposed to 2 h in WT mice. In food-restricted WT mice with reduced body weight, leptin increased hypothalamic PI3K activity only at 30 min, and p-STAT3 levels at 30 -120 min postinjection. These results suggest increased leptin sensitivity in both PI3K and STAT3 pathways in the hypothalamus of NesKO mice, which was not due to a lean phenotype. In the next experiment with a clonal hypothalamic neuronal cell line expressing proopiomelanocortin, we observed that whereas leptin significantly increased IRS1-associated PI3K activity and p-JAK2 levels in cells transfected with control vector, it failed to do so in SOCS3- overexpressed cells. Altogether, these results imply a SOCS3 inhibition of the PI3K pathway of leptin signaling in the hypothalamus, which may be one of the mechanisms behind the development of central leptin resistance and obesity. © 2011 the American Physiological Society.

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Suppressors of cytokine signaling (SOCS) proteins and JAK/STAT pathways

Tamiya Taiga, Kashiwagi Ikko, Takahashi Reiko, Yasukawa Hideo, Yoshimura Akihiko

Arteriosclerosis, Thrombosis, and Vascular Biology 31 （ 5 ） 980 - 985 2011年05月

ISSN 1079-5642

Various cytokines are involved in the regulation of the immune system and inflammation. Dysregulation of cytokine signaling can cause a variety of diseases, including allergy, autoimmune diseases, inflammation, and cancer. Most cytokines use the so-called janus kinase/signal transducer and activator of transcription pathway, and this pathway is negatively regulated by suppressors of cytokine signaling (SOCS) proteins. SOCS proteins bind to janus kinase and to certain cytokine receptors and signaling molecules, thereby suppressing further signaling events. Studies have shown that SOCS proteins are key physiological regulators of inflammation. Recent studies have also demonstrated that SOCS1 and SOCS3 are important regulators of adaptive immunity. Copyright © 2011 American Heart Association. All rights reserved.

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Neuronal suppressor of cytokine signaling-3 deficiency enhances hypothalamic leptin-dependent phosphatidylinositol 3-kinase signaling

Metlakunta Anantha S., Sahu Maitrayee, Yasukawa Hideo, Dhillon Sandeep S., Belsham Denise D., Yoshimura Akihiko, Sahu Abhiram

American Journal of Physiology - Regulatory Integrative and Comparative Physiology 300 （ 5 ） 2011年05月

ISSN 0363-6119

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Suppressors of cytokine signaling (SOCS) proteins and JAK/STAT pathways

Tamiya Taiga, Kashiwagi Ikko, Takahashi Reiko, Yasukawa Hideo, Yoshimura Akihiko

Arteriosclerosis, Thrombosis, and Vascular Biology 31 （ 5 ） 980 - 985 2011年05月

ISSN 1079-5642

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Stat3/Socs3 Activation by IL-6 Transsignaling Promotes Progression of Pancreatic Intraepithelial Neoplasia and Development of Pancreatic Cancer

Lesina Marina, Kurkowski Magdalena U., Ludes Katharina, Rose-John Stefan, Treiber Matthias, Klöppel Günter, Yoshimura Akihiko, Reindl Wolfgang, Sipos Bence, Akira Shizuo, Schmid Roland M., Algül Hana

Cancer Cell 19 （ 4 ） 456 - 469 2011年04月

ISSN 1535-6108

Physiological levels of Kras^G12D are sufficient to induce pancreatic intraepithelial neoplasias (PanINs); the mechanisms that drive PanIN progression are unknown. Here, we establish that, in addition to oncogenic Kras^G12D, IL-6 transsignaling-dependent activation of Stat3/Socs3 is required to promote PanIN progression and pancreatic ductal adenocarcinoma (PDAC). Myeloid compartment induces Stat3 activation by secreting IL-6; consequently, IL-6 transsignaling activates Stat3 in the pancreas. Using genetic tools, we show that inactivation of IL-6 transsignaling or Stat3 inhibits PanIN progression and reduces the development of PDAC. Aberrant activation of Stat3 through homozygous deletion of Socs3 in the pancreas accelerates PanIN progression and PDAC development. Our data describe the involvement of IL-6 transsignaling/Stat3/Socs3 in PanIN progression and PDAC development. © 2011 Elsevier Inc.

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Stat3/Socs3 Activation by IL-6 Transsignaling Promotes Progression of Pancreatic Intraepithelial Neoplasia and Development of Pancreatic Cancer

Cancer Cell 19 （ 4 ） 456 - 469 2011年04月

ISSN 1535-6108

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T cell treatment with small interfering RNA for suppressor of cytokine signaling 3 modulates allergic airway responses in a murine model of asthma

Moriwaki Atsushi, Inoue Hiromasa, Nakano Takako, Matsunaga Yuko, Matsuno Yukiko, Matsumoto Takafumi, Fukuyama Satoru, Kan-o Keiko, Matsumoto Koichiro, Tsuda-Eguchi Miyuki, Nagakubo Daisuke, Yoshie Osamu, Yoshimura Akihiko, Kubo Masato, Nakanishi Yoichi

American Journal of Respiratory Cell and Molecular Biology 44 （ 4 ） 448 - 455 2011年04月

ISSN 1044-1549

CD4+ T cells, particularly T helper (Th) 2 cells, play a pivotal role in the pathophysiology of allergic asthma. Suppressor of cytokine signaling (SOCS) proteins control the balance of CD4⁺ T cell differentiation. Mice that lack SOCS3 in T cells by crossing SOCS3-floxed mice with Lck-Cre-transgenic mice have reduced allergen-induced eosinophilia in the airways. Here, we studied the effects of SOCS3 silencing with small interfering (si) RNA in primary CD4⁺ T cells on Th2 cell differentiation and on asthmatic responses in mice. Th2 cells were generated from ovalbumin (OVA)-specific T cell receptor-transgenic mice in vitroandtransferred into recipient mice. Transfection of SOCS3-specific siRNA attenuated Th2 response in vitro. Adoptive transfer of SOCS3-siRNAT cells exhibited markedly suppressed airway hyperresponsiveness and eosinophilia after OVA challenge, with a concomitant decrease in OVA-specific CD4⁺ T cell accumulation in the airways. To investigate the mechanism of this impaired CD4⁺ T cell accumulation, we inactivated SOCS3 of T cells by crossing SOCS3-floxed (SOCS3 ^flox/flox) mice with CD4-Cre transgenic mice. CD4-Cre x SOCS3 ^flox/flox mice exhibited fewer IL-4-producing cells and more reduced eosinophil infiltration in bronchoalveolar lavage fluids than control mice in a model of OVA-induced asthma. Expression of CCR3 and CCR4 in CD4⁺ T cells was decreased in CD4-Cre x SOCS3^flox/flox mice. CCR4 expression was also decreased in CD4⁺ T cells after transfer of SOCS3 siRNA-treated T cells. These findings suggest that the therapeutic modulation of SOCS3 expression in CD4⁺ T cells might be effective in preventing the development of allergic asthma. Copyright © 2011 American Thoracic Society.

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T cell treatment with small interfering RNA for suppressor of cytokine signaling 3 modulates allergic airway responses in a murine model of asthma

American Journal of Respiratory Cell and Molecular Biology 44 （ 4 ） 448 - 455 2011年04月

ISSN 1044-1549

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Involvement of STAP-2 in Brk-mediated phosphorylation and activation of STAT5 in breast cancer cells

Ikeda Osamu, Mizushima Akihiro, Sekine Yuichi, Yamamoto Chikako, Muromoto Ryuta, Nanbo Asuka, Oritani Kenji, Yoshimura Akihiko, Matsuda Tadashi

Cancer Science 102 （ 4 ） 756 - 761 2011年04月

ISSN 1347-9032

Signal-transducing adaptor protein (STAP)-2 is a recently identified adaptor protein that contains Pleckstrin homology and Src homology 2-like domains, and is also known to be a substrate of breast tumor kinase (Brk). In a previous study, we found that STAP-2 upregulated Brk-mediated activation of signal transducer and activator of transcription (STAT) 3 in breast cancer cells. Here, we examined the involvement of STAP-2 in Brk-mediated STAT5 activation in breast cancer cells. Ectopic expression of STAP-2 induced Brk-mediated transcriptional activity of STAT5. Furthermore, STAP-2-knockdown in T47D breast cancer cells induced a marked decrease in proliferation that was as strong as that after Brk- or STAT5b-knockdown. Regarding the mechanism, the Pleckstrin homology domain of STAP-2 is likely to participate in the process by which Brk phosphorylates and activates STAT5. Taken together, our findings provide insights toward the development of novel therapeutic strategies as well as novel prognostic values in breast carcinomas. © 2011 Japanese Cancer Association.

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Involvement of STAP-2 in Brk-mediated phosphorylation and activation of STAT5 in breast cancer cells

Ikeda Osamu, Mizushima Akihiro, Sekine Yuichi, Yamamoto Chikako, Muromoto Ryuta, Nanbo Asuka, Oritani Kenji, Yoshimura Akihiko, Matsuda Tadashi

Cancer Science 102 （ 4 ） 756 - 761 2011年04月

ISSN 1347-9032

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Raftlin is involved in the nucleocapture complex to induce poly(I

Watanabe Ayako, Tatematsu Megumi, Saeki Kazuko, Shibata Sachiko, Shime Hiroaki, Yoshimura Akihiko, Obuse Chikashi, Seya Tsukasa, Matsumoto Misako

Journal of Biological Chemistry 286 （ 12 ） 10702 - 10711 2011年03月

ISSN 0021-9258

The double-stranded RNA analog, poly(I:C), extracellularly activates both the endosomal Toll-like receptor (TLR) 3 and the cytoplasmic RNA helicase, melanoma differentiation-associated gene 5, leading to the production of type I interferons (IFNs) and inflammatory cytokines. The mechanism by which extracellular poly(I:C) is delivered to TLR3-positive organelles and the cytoplasm remains to be elucidated. Here, we show that the cytoplasmic lipid raft protein, Raftlin, is essential for poly(I:C) cellular uptake in human myeloid dendritic cells and epithelial cells. When Raftlin was silenced, poly(I:C) failed to enter cells and induction of IFN-β production was inhibited. In addition, cellular uptake of B-type oligodeoxynucleotide that shares its uptake receptor with poly(I:C) was suppressed in Raftlin knockdown cells. Upon poly(I:C) stimulation, Raftlin was translocated from the cytoplasm to the plasma membrane where it colocalized with poly(I:C), and thereafter moved to TLR3-positive endosomes. Thus, Raftlin cooperates with the uptake receptor to mediate cell entry of poly(I:C), which is critical for activation of TLR3. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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Raftlin is involved in the nucleocapture complex to induce poly(I

Watanabe Ayako, Tatematsu Megumi, Saeki Kazuko, Shibata Sachiko, Shime Hiroaki, Yoshimura Akihiko, Obuse Chikashi, Seya Tsukasa, Matsumoto Misako

Journal of Biological Chemistry 286 （ 12 ） 10702 - 10711 2011年03月

ISSN 0021-9258

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Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling

Matsumura Kaori, Taketomi Takaharu, Yoshizaki Keigo, Arai Shinsaku, Sanui Terukazu, Yoshiga Daigo, Yoshimura Akihiko, Nakamura Seiji

Biochemical and Biophysical Research Communications 404 （ 4 ） 1076 - 1082 2011年01月

ISSN 0006-291X

Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation. © 2010 Elsevier Inc.

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Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling

Matsumura Kaori, Taketomi Takaharu, Yoshizaki Keigo, Arai Shinsaku, Sanui Terukazu, Yoshiga Daigo, Yoshimura Akihiko, Nakamura Seiji

Biochemical and Biophysical Research Communications 404 （ 4 ） 1076 - 1082 2011年01月

ISSN 0006-291X

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Loss of Sprouty2 partially rescues renal hypoplasia and stomach hypoganglionosis but not intestinal aganglionosis in Ret Y1062F mutant mice

Miyamoto Rieko, Jijiwa Mayumi, Asai Masato, Kawai Kumi, Ishida-Takagishi Maki, Mii Shinji, Asai Naoya, Enomoto Atsushi, Murakumo Yoshiki, Yoshimura Akihiko, Takahashi Masahide

Developmental Biology 349 （ 2 ） 160 - 168 2011年01月

ISSN 0012-1606

The glial cell line-derived neurotrophic factor (GDNF)/RET tyrosine kinase signaling pathway plays crucial roles in the development of the enteric nervous system (ENS) and the kidney. Tyrosine 1062 (Y1062) in RET is an autophosphorylation residue that is responsible for the activation of the PI3K/AKT and RAS/MAPK signaling pathways. Mice lacking signaling via Ret Y1062 show renal hypoplasia and hypoganglionosis of the ENS although the phenotype is milder than the Gdnf- or Ret-deficient mice. Sprouty2 (Spry2) was found to be an antagonist for fibroblast growth factor receptor (FGFR) and acts as an inhibitory regulator of ERK activation. Spry2-deficient mice exhibit hearing loss and enteric nerve hyperplasia. In the present study, we generated Spry2-deficient and Ret Y1062F knock-in (tyrosine 1062 is replaced with phenylalanine) double mutant mice to see if abnormalities of the ENS and kidney, caused by loss of signaling via Ret Y1062, are rescued by a deficiency of Spry2. Double mutant mice showed significant recovery of ureteric bud branching and ENS development in the stomach. These results indicate that Spry2 regulates downstream signaling mediated by GDNF/RET signaling complex in vivo. © 2010 Elsevier Inc.

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Loss of Sprouty2 partially rescues renal hypoplasia and stomach hypoganglionosis but not intestinal aganglionosis in Ret Y1062F mutant mice

Miyamoto Rieko, Jijiwa Mayumi, Asai Masato, Kawai Kumi, Ishida-Takagishi Maki, Mii Shinji, Asai Naoya, Enomoto Atsushi, Murakumo Yoshiki, Yoshimura Akihiko, Takahashi Masahide

Developmental Biology 349 （ 2 ） 160 - 168 2011年01月

ISSN 0012-1606

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Effects of a Janus kinase inhibitor, pyridone 6, on airway responses in a murine model of asthma

Matsunaga Yuko, Inoue Hiromasa, Fukuyama Satoru, Yoshida Hideyuki, Moriwaki Atsushi, Matsumoto Takafumi, Matsumoto Koichiro, Asai Yukari, Kubo Masato, Yoshimura Akihiko, Nakanishi Yoichi

Biochemical and Biophysical Research Communications 404 （ 1 ） 261 - 267 2011年01月

ISSN 0006-291X

Th2 cytokines and their downstream Janus kinase (JAK)-signal transducer and activation of transcription (STAT) pathways play a critical role in allergic asthma. We studied the effects of a pan-JAK inhibitor, pyridone 6 (P6), on asthmatic responses in a mouse model and investigated the mechanism for its biological effects. Mice were sensitized and challenged by ovalbumin (OVA). P6 treatment during the challenge phase suppressed eosinophilia in bronchoalveolar lavage (BAL) fluids but did not affect airway hyperresponsiveness (AHR). To improve the efficacy of the JAK inhibitor, P6 was encapsulated in polylactic- coglycolic acid nanoparticles (P6-PLGA). P6-PLGA treatment just before OVA challenge suppressed both airway eosinophilia and AHR. Although the IL-13 levels in BAL fluids and the OVA-specific IgE levels in serum after the challenge phase treatment with P6-PLGA were similar to those after a sham treatment, the eotaxin levels in BAL fluids and lung mCLCA3/Gob-5 expression were decreased in P6-PLGA-treated mice. Interestingly, the local IL-13 levels and serum OVA-specific IgE were decreased, while IL-17-producing T cells were increased by P6-PLGA treatment during the sensitization plus challenge phases. In vitro, P6 strongly suppressed the differentiation of Th2 from naive CD4 T cells, but it partly enhanced Th17 differentiation. P6 potently suppressed IL-13-mediated STAT6 activation and mCLCA3/Gob-5 expression in mouse tracheal epithelial cells. These findings suggest that the JAK inhibitor P6 suppresses asthmatic responses by inhibiting Th2 inflammation and that application of PLGA nanoparticles improves the therapeutic potency of P6. © 2010 Elsevier Inc.

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Effects of a Janus kinase inhibitor, pyridone 6, on airway responses in a murine model of asthma

Matsunaga Yuko, Inoue Hiromasa, Fukuyama Satoru, Yoshida Hideyuki, Moriwaki Atsushi, Matsumoto Takafumi, Matsumoto Koichiro, Asai Yukari, Kubo Masato, Yoshimura Akihiko, Nakanishi Yoichi

Biochemical and Biophysical Research Communications 404 （ 1 ） 261 - 267 2011年01月

ISSN 0006-291X

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Smad2 and Smad3 are redundantly essential for the TGF-β-mediated regulation of regulatory T plasticity and Th1 development (Journal of Immunology (2010) 185, (842-855))

Takimoto T., Wakabayashi Y., Sekiya T., Inoue N., Morita R., Ichiyama K., Takahashi R., Asakawa M., Muto G., Mori T., Hasegawa E., Shizuya S., Hara T., Nomura M., Yoshimura A.

Journal of Immunology 186 （ 1 ） 632 2011年01月

ISSN 0022-1767

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Smad2 and Smad3 are redundantly essential for the TGF-β-mediated regulation of regulatory T plasticity and Th1 development (Journal of Immunology (2010) 185, (842-855))

Takimoto T., Wakabayashi Y., Sekiya T., Inoue N., Morita R., Ichiyama K., Takahashi R., Asakawa M., Muto G., Mori T., Hasegawa E., Shizuya S., Hara T., Nomura M., Yoshimura A.

Journal of Immunology 186 （ 1 ） 632 2011年01月

ISSN 0022-1767

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Legius syndrome in fourteen families

Denayer Ellen, Chmara Magdalena, Brems Hilde, Kievit Anneke Maat, Van Bever Yolande, Van Den Ouweland Ans M W, Van Minkelen Rick, De Goede-Bolder Arja, Oostenbrink Rianne, Lakeman Phillis, Beert Eline, Ishizaki Takuma, Mori Tomoaki, Keymolen Kathelijn, Van Den Ende Jenneke, Mangold Elisabeth, Peltonen Sirkku, Brice Glen, Rankin Julia, Van Spaendonck-Zwarts Karin Y., Yoshimura Akihiko, Legius Eric

Human Mutation 32 （ 1 ） 2011年01月

ISSN 1059-7794

Legius syndrome presents as an autosomal dominant condition characterized by café-au-lait macules with or without freckling and sometimes a Noonan-like appearance and/or learning difficulties. It is caused by germline loss-of-function SPRED1 mutations and is a member of the RAS-MAPK pathway syndromes. Most mutations result in a truncated protein and only a few inactivating missense mutations have been reported. Since only a limited number of patients has been reported up until now, the full clinical and mutational spectrum is still unknown. We report mutation data and clinical details in fourteen new families with Legius syndrome. Six novel germline mutations are described. The Trp31Cys mutation is a new pathogenic SPRED1 missense mutation. Clinical details in the 14 families confirmed the absence of neurofibromas, and Lisch nodules, and the absence of a high prevalence of central nervous system tumors. We report white matter T2 hyperintensities on brain MRI scans in 2 patients and a potential association between postaxial polydactyly and Legius syndrome. © 2010 Wiley-Liss, Inc.

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[Suppression of leptin and insulin signaling by SOCS3].

Yoshimura Akihiko

Nihon rinsho. Japanese journal of clinical medicine 69 Suppl 1 782 - 789 2011年01月

ISSN 0047-1852

Legius syndrome in fourteen families

Human Mutation 32 （ 1 ） 2011年01月

ISSN 1059-7794

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[Suppression of leptin and insulin signaling by SOCS3].

Yoshimura Akihiko

Nihon rinsho. Japanese journal of clinical medicine 69 Suppl 1 782 - 789 2011年01月

ISSN 0047-1852

Prostaglandin E2 and SOCS1 have a role in intestinal immune tolerance

Chinen Takatoshi, Komai Kyoko, Muto Go, Morita Rimpei, Inoue Naoko, Yoshida Hideyuki, Sekiya Takashi, Yoshida Ryoko, Nakamura Kazuhiko, Takayanagi Ryoichi, Yoshimura Akihiko

Nature Communications 2 （ 1 ） 2011年

Interleukin 10 (IL-10) and regulatory T cells (Tregs) maintain tolerance to intestinal microorganisms. However, Il10^-/- Rag2^-/- mice, which lack IL-10 and Tregs, remain healthy, suggesting the existence of other mechanisms of tolerance. Here, we identify suppressor of cytokine signalling 1 (SOCS1) as an essential mediator of immune tolerance in the intestine. Socs1^-/- Rag2^-/- mice develop severe colitis, which can be prevented by the reduction of microbiota and the transfer of IL-10-sufficient Tregs. Additionally, we find an essential role for prostaglandin E2 (PGE2) in the maintenance of tolerance within the intestine in the absence of Tregs. Socs1^-/- dendritic cells are resistant to PGE2-mediated immunosuppression because of dysregulated cytokine signalling. Thus, we propose that SOCS1 and PGE2, potentially interacting together, act as an alternative intestinal tolerance mechanism distinct from IL-10 and Tregs. © 2011 Macmillan Publishers Limited. All rights reserved.

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The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4⁺ T cells

Sekiya Takashi, Kashiwagi Ikkou, Inoue Naoko, Morita Rimpei, Hori Shohei, Waldmann Herman, Rudensky Alexander Y., Ichinose Hiroshi, Metzger Daniel, Chambon Pierre, Yoshimura Akihiko

Nature Communications 2 （ 1 ） 2011年

Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms. Although the Forkhead transcription factor Foxp3 defines the Treg cell lineage and functions, the molecular mechanisms of Foxp3 induction and maintenance remain elusive. Here we show that Foxp3 is one of the direct targets of Nr4a2. Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications. Ectopic expression of Nr4a2 imparts Treg-like suppressive activity to naïve CD4⁺ T cells by inducing Foxp3 and by repressing cytokine production, including interferon-γ and interleukin-2. Deletion of Nr4a2 in T cells attenuates induction of Tregs and causes aberrant induction of Th1, leading to the exacerbation of colitis. Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo. Thus, Nr4a2 has the ability to maintain T-cell homoeostasis by regulating induction, maintenance and suppressor functions of Tregs, and by repression of aberrant Th1 induction. © 2011 Macmillan Publishers Limited. All rights reserved.

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TGF-β function in immune suppression.

Yoshimura Akihiko, Muto G.

Current topics in microbiology and immunology 350 127 - 147 2011年

ISSN 0070-217X

Transforming growth factor-β (TGF-β) has been shown to play an essential role in establishing immunological tolerance, yet recent studies have revealed the pro-inflammatory roles of TGF-β in inflammatory responses. TGF-β induces Foxp3-positive regulatory T cells (iTregs), while in the presence of IL-6, it induces pathogenic IL-17 producing Th17 cells. TGF-β inhibits the proliferation of T cells as well as cytokine production via Foxp3-dependent and independent mechanisms. On the one hand, little is known about molecular mechanisms involved in immune suppression via TGF-β; however, recent studies suggest that Smad2 as well as Smad3 play essential roles in Foxp3 induction and cytokine suppression, whereas Th17 differentiation is promoted via the Smad-independent pathway. Mutual suppression of signaling between TGF-β and inflammatory cytokines has been shown to be necessary for the balance of immunity and tolerance.

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Aberrant IL-4 production by SOCS3-over-expressing T cells during infection with Leishmania major exacerbates disease manifestations

Nakaya Mako, Hamano Shinjiro, Kawasumi Miyuri, Yoshida Hiroki, Yoshimura Akihiko, Kobayashi Takashi

International Immunology 23 （ 3 ） 195 - 202 2011年

ISSN 0953-8178

Suppressor of cytokine signaling (SOCS) 3 is a major negative feedback regulator of signal transducer and activator of transcription 3-activating cytokines. Studies using T-cell-specific SOCS3-deficient mice indicate that the absence of SOCS3 in T cells results in exacerbation of disease progression after infection by Leishmania major due to skewing of the T_h3 cell phenotype accompanied by hyper-production of IL-10 and transforming growth factor β (TGF-β). Here we show that transgenic mice over-expressing the SOCS3 gene in T cells (Lck-SOCS3 Tg mice) are also susceptible to infection by L. major. Forced expression of SOCS3 in T cells did not affect the production of the anti-inflammatory cytokines IL-10 and TGF-β or that of the protective T_h1 type cytokine IFN-γ, which is required for parasite clearance. CD⁴1 T cells isolated from infected-Lck-SOCS3 Tg mice produced much higher levels of IL-4 when they were restimulated with L. major antigen in vitro. Exacerbation of disease progression in Lck-SOCS3 Tg mice was completely reversed by administration of a neutralizing antibody against IL-4. These data suggest that tight regulation of SOCS3 expression in T_h cells is crucial for disease control during infection by L. major. © The Japanese Society for Immunology. 2011. All rights reserved.

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SOCS1 regulates type I/type II NKT cell balance by regulating IFNγ signaling

Hashimoto Masayuki, Hiwatashi Kiyokazu, Ichiyama Kenji, Morita Rimpei, Sekiya Takashi, Kimura Akihiro, Sugiyama Yuki, Sibata Toshikatsu, Kuroda Kazumichi, Takahashi Reiko, Yoshimura Akihiko

International Immunology 23 （ 3 ） 165 - 176 2011年

ISSN 0953-8178

Suppressor of cytokine signaling-1 (SOCS1) has been shown to be an essential negative regulator of cytokine responses, including those of IFNγ, IL-2, IL-4 and IL-7. SOCS1 deficiency resulted in hyperactivation not only of T cells in general but also of NKT cells specifically. Consistent with previous reports, T- and NKT-cell-specific deletion of Socs1 in mice resulted in enhanced sensitivity to ConA-induced hepatitis. Compared with wild-type (WT) NKT cells, SOCS1-deficient NKT cells produced larger quantities of IFNγg in response to ConA and proliferated faster in response to IL-2 and IL-15. To our surprise, however, SOCS1-deficient NKT cells did not respond to the synthetic glycolipid ligand alpha-galactosylceramide (α-GalCer), though they did respond to sulfatide. α-GalCer-CD1d-tetramer-positive type I NKT [invariant NKT (iNKT)] cells were marginally detected in the periphery of SOCS1-conditional knockout (cKO) mice, suggesting that most of the SOCS1-deficient NKT cells at the periphery were type II NKT cells. Consistently, invariant Vα14 expression was much lower in SOCS1-deficient NKT cells than in WT NKT cells, indicating that iNKT cell homeostasis was abnormal in SOCS1-cKO mice. This reduction in iNKT cells was not observed in mice of an IFNγ-deficient background. These results suggest that SOCS1 is an important regulator of the balance between type I and type II NKT cells at the periphery. © The Japanese Society for Immunology. 2011. All rights reserved.

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Prostaglandin E2 and SOCS1 have a role in intestinal immune tolerance

Chinen Takatoshi, Komai Kyoko, Muto Go, Morita Rimpei, Inoue Naoko, Yoshida Hideyuki, Sekiya Takashi, Yoshida Ryoko, Nakamura Kazuhiko, Takayanagi Ryoichi, Yoshimura Akihiko

Nature Communications 2 （ 1 ） 2011年

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The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4⁺ T cells

Sekiya Takashi, Kashiwagi Ikkou, Inoue Naoko, Morita Rimpei, Hori Shohei, Waldmann Herman, Rudensky Alexander Y., Ichinose Hiroshi, Metzger Daniel, Chambon Pierre, Yoshimura Akihiko

Nature Communications 2 （ 1 ） 2011年

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TGF-β function in immune suppression.

Yoshimura Akihiko, Muto G.

Current topics in microbiology and immunology 350 127 - 147 2011年

ISSN 0070-217X

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Aberrant IL-4 production by SOCS3-over-expressing T cells during infection with Leishmania major exacerbates disease manifestations

Nakaya Mako, Hamano Shinjiro, Kawasumi Miyuri, Yoshida Hiroki, Yoshimura Akihiko, Kobayashi Takashi

International Immunology 23 （ 3 ） 195 - 202 2011年

ISSN 0953-8178

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SOCS1 regulates type I/type II NKT cell balance by regulating IFNγ signaling

Hashimoto Masayuki, Hiwatashi Kiyokazu, Ichiyama Kenji, Morita Rimpei, Sekiya Takashi, Kimura Akihiro, Sugiyama Yuki, Sibata Toshikatsu, Kuroda Kazumichi, Takahashi Reiko, Yoshimura Akihiko

International Immunology 23 （ 3 ） 165 - 176 2011年

ISSN 0953-8178

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Interactions of STAP-2 with Brk and STAT3 participate in cell growth of human breast cancer cells

Ikeda Osamu, Sekine Yuichi, Mizushima Akihiro, Nakasuji Misa, Miyasaka Yuto, Yamamoto Chikako, Muromoto Ryuta, Nanbo Asuka, Oritani Kenji, Yoshimura Akihiko, Matsuda Tadashi

Journal of Biological Chemistry 285 （ 49 ） 38093 - 38103 2010年12月

ISSN 0021-9258

STAP-2 (signal transducing adaptor protein-2) is a recently identified adaptor protein that contains pleckstrin homology (PH) and Src homology 2-like domains, as well as a STAT3-binding motif in its C-terminal region. STAP-2 is also a substrate of breast tumor kinase (Brk). In breast cancers, Brk expression is deregulated and promotes STAT3-dependent cell proliferation. In the present study, manipulated STAP-2 expression demonstrated essential roles of STAP-2 in Brk-mediated STAT3 activation. STAP-2 interacts with both Brk and STAT3. In addition, small interfering RNA-mediated reduction of endogenous STAP-2 expression strongly decreased Brk-mediated STAT3 activation in T47D breast cancer cells. The PH domain of STAP-2 is involved in multiple steps: the binding between Brk and STAP-2, the activation and tyrosine phosphorylation of STAT3, and the activation of Brk. Notably, a STAP-2 PH-Brk fusion protein exhibited robust kinase activity and increased activation and tyrosine phosphorylation of STAT3. Finally, STAP-2 knockdown in T47D cells induced a significant decrease of proliferation, as strong as that of Brk or STAT3 knockdown. Taken together, our findings are likely to inform the development of a novel therapeutic strategy, as well as the determination of novel prognostic values, in breast carcinomas. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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Interactions of STAP-2 with Brk and STAT3 participate in cell growth of human breast cancer cells

Ikeda Osamu, Sekine Yuichi, Mizushima Akihiro, Nakasuji Misa, Miyasaka Yuto, Yamamoto Chikako, Muromoto Ryuta, Nanbo Asuka, Oritani Kenji, Yoshimura Akihiko, Matsuda Tadashi

Journal of Biological Chemistry 285 （ 49 ） 38093 - 38103 2010年12月

ISSN 0021-9258

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Development of experimental cerebral malaria is independent of IL-23 and IL-17

Ishida Hidekazu, Matsuzaki-Moriya Chikako, Imai Takashi, Yanagisawa Kunio, Nojima Yoshihisa, Suzue Kazutomo, Hirai Makoto, Iwakura Yoichiro, Yoshimura Akihiko, Hamano Shinjiro, Shimokawa Chikako, Hisaeda Hajime

Biochemical and Biophysical Research Communications 402 （ 4 ） 790 - 795 2010年11月

ISSN 0006-291X

Cerebral malaria (CM) is the most severe complication of Plasmodium infection. Although inappropriate immune responses to Plasmodium falciparum are reported as the major causes of CM, the precise mechanisms for development remain unclear. IL-23 and IL-17 have critical roles in the onset of autoimmunity and inflammatory diseases triggered by microbial infections. Thus, we investigated the influence of IL-23 and IL-17 on experimental CM (ECM) using Plasmodium berghei ANKA infection of C57BL/6 mice. Both IL-23 deficient mice and wild-type (WT) mice developed ECM. IL-17 deficient mice also developed ECM, while IL-17 producing cells other than CD4 ⁺ T cells (Th17) were increased in WT mice that developed ECM. In conclusion, this study showed that IL-23 and IL-17 are not involved in ECM development. © 2010 Elsevier Inc.

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Suppressor of cytokine signaling 3 inhibits LPS-induced IL-6 expression in osteoblasts by suppressing CCAAT/enhancer-binding protein

Yan Chunguang, Cao Jay, Wu Min, Zhang Wei, Jiang Tao, Yoshimura Aihiko, Gao Hongwei

Journal of Biological Chemistry 285 （ 48 ） 37227 - 37239 2010年11月

ISSN 0021-9258

Suppressor of cytokine signaling 3 (SOCS3) is an important intracellular protein that inhibits cytokine signaling in numerous cell types and has been implicated in several inflammatory diseases. However, the expression and function of SOCS3 in osteoblasts are not known. In this study, we demonstrated that SOCS3 expression was transiently induced by LPS in osteoblasts, and apparently contributed to the inhibition of IL-6 induction by LPS treatment. We found that tyrosine 204 of the SOCS box, the SH2 domain, and the N-terminal kinase inhibitory region (KIR) of SOCS3 were all involved in its IL-6 inhibition. Furthermore, we demonstrated that CCAAT/enhancerbinding protein (C/EBP) β was activated by LPS (increased DNA binding activity), and played a key role in LPS-induced IL-6 expression in osteoblasts. We further provided the evidence that SOCS3 functioned as a negative regulator for LPS response in osteoblasts by suppressing C/EBPβ DNA binding activity. In addition, tyrosine 204 of the SOCS box, the SH2 domain, and the N-terminal kinase inhibitory region (KIR) of SOCS3 were all required for its C/EBPβ inhibition. These findings suggest that SOCS3 by interfering with C/EBPβ activation may have an important regulatory role during bone-associated inflammatory responses.

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Development of experimental cerebral malaria is independent of IL-23 and IL-17

Biochemical and Biophysical Research Communications 402 （ 4 ） 790 - 795 2010年11月

ISSN 0006-291X

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Suppressor of cytokine signaling 3 inhibits LPS-induced IL-6 expression in osteoblasts by suppressing CCAAT/enhancer-binding protein

Yan Chunguang, Cao Jay, Wu Min, Zhang Wei, Jiang Tao, Yoshimura Aihiko, Gao Hongwei

Journal of Biological Chemistry 285 （ 48 ） 37227 - 37239 2010年11月

ISSN 0021-9258

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Therapeutic effect of IL-12/23 and their signaling pathway blockade on brain ischemia model

Konoeda Fumie, Shichita Takashi, Yoshida Hideyuki, Sugiyama Yuki, Muto Go, Hasegawa Eiichi, Morita Rinpei, Suzuki Norihiro, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 402 （ 3 ） 500 - 506 2010年11月

ISSN 0006-291X

Recently, T cell cytokines such as IL-17 and IFN-γ have been shown to play important roles in the progression of brain injury induced by ischemia. We have shown that IL-23 from infiltrated macrophages activates γδT cells, thereby inducing IL-17 from these cells. However, deletion of the IL-23 gene in mice showed a more dramatic protective effect against brain ischemia reperfusion (I/R) model than γδT cell depletion did, suggesting that IL-23 plays some other pivotal role in brain injury in addition to its role in IL-17 induction. To develop therapeutic methods based on these findings, we examined the effect of the JAK kinase inhibitor CP-690550 and an anti-IL12/23 monoclonal antibody on an I/R model. CP-690550 efficiently inhibited IL-17 production from memory T cells in vitro and partly suppressed infarct volume increase after I/R. Anti-p40 antibody, which blocks both IL-12 and IL-23, efficiently suppressed I/R injury and improved recovery of neurological deficits. The number of IL-17-producing cells was decreased by anti-p40 antibody treatment. Thus the JAK inhibitor and anti-p40 antibody, both of which have already been under trial for the treatment of several human inflammatory diseases, appear to be promising therapeutic agents for the amelioration of stroke. © 2010 Elsevier Inc.

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Therapeutic effect of IL-12/23 and their signaling pathway blockade on brain ischemia model

Konoeda Fumie, Shichita Takashi, Yoshida Hideyuki, Sugiyama Yuki, Muto Go, Hasegawa Eiichi, Morita Rinpei, Suzuki Norihiro, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 402 （ 3 ） 500 - 506 2010年11月

ISSN 0006-291X

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Membrane molecules mobile even after chemical fixation

Tanaka Kenji A K, Suzuki Kenichi G N, Shirai Yuki M., Shibutani Shusaku T., Miyahara Manami S H, Tsuboi Hisae, Yahara Miyako, Yoshimura Akihiko, Mayor Satyajit, Fujiwara Takahiro K., Kusumi Akihiro

Nature Methods 7 （ 11 ） 865 - 866 2010年11月

ISSN 1548-7091

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Membrane molecules mobile even after chemical fixation

Tanaka Kenji A K, Suzuki Kenichi G N, Shirai Yuki M., Shibutani Shusaku T., Miyahara Manami S H, Tsuboi Hisae, Yahara Miyako, Yoshimura Akihiko, Mayor Satyajit, Fujiwara Takahiro K., Kusumi Akihiro

Nature Methods 7 （ 11 ） 865 - 866 2010年11月

ISSN 1548-7091

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Function of miR-146a in Controlling Treg Cell-Mediated Regulation of Th1 Responses

Lu Li Fan, Boldin Mark P., Chaudhry Ashutosh, Lin Ling Li, Taganov Konstantin D., Hanada Toshikatsu, Yoshimura Akihiko, Baltimore David, Rudensky Alexander Y.

Cell 142 （ 6 ） 914 - 929 2010年09月

ISSN 0092-8674

Foxp3⁺ regulatory T (Treg) cells maintain immune homeostasis by limiting different types of inflammatory responses. Here, we report that miR-146a, one of the miRNAs prevalently expressed in Treg cells, is critical for their suppressor function. The deficiency of miR-146a in Treg cells resulted in a breakdown of immunological tolerance manifested in fatal IFNγ-dependent immune-mediated lesions in a variety of organs. This was likely due to augmented expression and activation of signal transducer and activator transcription 1 (Stat1), a direct target of miR-146a. Likewise, heightened Stat1 activation in Treg cells subjected to a selective ablation of SOCS1, a key negative regulator of Stat1 phosphorylation downstream of the IFNγ receptor, was associated with analogous Th1-mediated pathology. Our results suggest that specific aspects of Treg suppressor function are controlled by a single miRNA and that an optimal range of Stat1 activation is important for Treg-mediated control of Th1 responses and associated autoimmunity. © 2010 Elsevier Inc.

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Function of miR-146a in Controlling Treg Cell-Mediated Regulation of Th1 Responses

Lu Li Fan, Boldin Mark P., Chaudhry Ashutosh, Lin Ling Li, Taganov Konstantin D., Hanada Toshikatsu, Yoshimura Akihiko, Baltimore David, Rudensky Alexander Y.

Cell 142 （ 6 ） 914 - 929 2010年09月

ISSN 0092-8674

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Smad2 and Smad3 are redundantly essential for the TGF-β-mediated regulation of regulatory T plasticity and Th1 development

Takimoto Tomohito, Wakabayashi Yu, Sekiya Takashi, Inoue Naoko, Morita Rimpei, Ichiyama Kenji, Takahashi Reiko, Asakawa Mayako, Muto Go, Mori Tomoaki, Hasegawa Eiichi, Shizuya Saika, Hara Toshiro, Nomura Masatoshi, Yoshimura Akihiko

Journal of Immunology 185 （ 2 ） 842 - 855 2010年07月

ISSN 0022-1767

Although it has been well established that TGF-β plays a pivotal role in immune regulation, the roles of its downstream transcription factors, Smad2 and Smad3, have not been fully clarified. Specifically, the function of Smad2 in the immune system has not been investigated because of the embryonic lethality of Smad2-deficient mice. In this study, we generated T cell-specific Smad2 conditional knockout (KO) mice and unexpectedly found that Smad2 and Smad3 were redundantly essential for TGF-β-mediated induction of Foxp3-expressing regulatory T cells and suppression of IFN-γ production in CD4⁺ T cells. Consistent with these observations, Smad2/Smad3-double KO mice, but not single KO mice, developed fatal inflammatory diseases with higher IFN-γ production and reduced Foxp3 expression in CD4⁺ T cells at the periphery. Although it has been suggested that Foxp3 induction might underlie TGF-β-mediated immunosuppression, TGF-β still can suppress Th1 cell development in Foxp3-deficient T cells, suggesting that the Smad2/3 pathway inhibits Th1 cell development with Foxp3-independent mechanisms. We also found that Th17 cell development was reduced in Smad-deficient CD4⁺ T cells because of higher production of Th17-inhibotory cytokines from these T cells. However, TGF-β-mediated induction of RORγt, a master regulator of Th17 cell, was independent of both Smad2 and Smad3, suggesting that TGF-β regulates Th17 development through Smad2/3-dependent and -independent mechanisms. Copyright © 2010 by The American Association of Immunologists, Inc.

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Smad2 and Smad3 are redundantly essential for the TGF-β-mediated regulation of regulatory T plasticity and Th1 development

Journal of Immunology 185 （ 2 ） 842 - 855 2010年07月

ISSN 0022-1767

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Cellular and molecular basis for the regulation of inflammation by TGF-β

Yoshimura Akihiko, Wakabayashi Yu, Mori Tomoaki

Journal of Biochemistry 147 （ 6 ） 781 - 792 2010年06月

ISSN 0021-924X

Transforming growth factor-β (TGF-β) has been shown to play an essential role in the suppression of inflammation, yet recent studies have revealed the positive roles of TGF-β in inflammatory responses. For example, TGF-β induces Foxp3-positive regulatory T cells (iTregs) in the presence of interleukin-2 (IL-2), while in the presence of IL-6, it induces pathogenic IL-17 producing Th17 cells. TGF-β inhibits the proliferation of immune cells as well as cytokine production via Foxp3-dependent and -independent mechanisms. Little is known about molecular mechanisms involved in immune suppression via TGF-β; however, Smad2/3 have been shown to play essential roles in Foxp3 induction as well as in IL-2 and IFN-γ suppression, whereas Th17 differentiation is promoted via the Smad-independent pathway. Interaction between TGF-β and other cytokine signaling is important in establishing the balance of immunity and tolerance. © The Authors 2010. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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Cellular and molecular basis for the regulation of inflammation by TGF-β

Yoshimura Akihiko, Wakabayashi Yu, Mori Tomoaki

Journal of Biochemistry 147 （ 6 ） 781 - 792 2010年06月

ISSN 0021-924X

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Interaction between Ebola virus glycoprotein and host Toll-like receptor 4 leads to induction of proinflammatory cytokines and SOCS1

Atsushi Okumura, Pitha Paula M., Yoshimura Akihiko, Harty Ronald N.

Journal of Virology 84 （ 1 ） 27 - 33 2010年01月

ISSN 0022-538X

Ebola virus initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal Ebola virus infections. Here we report that host Toll-like receptor 4 (TLR4) is a sensor for Ebola virus glycoprotein (GP) on virus-like particles (VLPs) and that resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines and suppressor of cytokine signaling 1 (SOCS1) in a human monocytic cell line and in HEK293-TLR4/MD2 cells stably expressing the TLR4/MD2 complex. Ebola virus GP was found to interact with TLR4 by immunoprecipitation/Western blot analyses, and Ebola virus GP on VLPs was able to stimulate expression of NF-κB in a TLR4-dependent manner. Interestingly, we found that budding of Ebola virus VLPs was more pronounced in TLR4-stimulated cells than in unstimulated control cells. In sum, these findings identify the host innate immune protein TLR4 as a sensor for Ebola virus GP which may play an important role in the immunopathogenesis of Ebola virus infection. Copyright © 2010, American Society for Microbiology.

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Interaction between Ebola virus glycoprotein and host Toll-like receptor 4 leads to induction of proinflammatory cytokines and SOCS1

Atsushi Okumura, Pitha Paula M., Yoshimura Akihiko, Harty Ronald N.

Journal of Virology 84 （ 1 ） 27 - 33 2010年01月

ISSN 0022-538X

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Role of T lymphocytes in ischemic brain injury

Shichita Takashi, Konoeda Fumie, Yoshimura Akihiko

Clinical Neurology 50 （ 11 ） 878 - 880 2010年

ISSN 0009-918X

Recently, inflammation has been implicated in the progression of cerebral ischemic injury. Especially, T lymphocytes have been shown to infiltrate into the ischemic brain 24 hours after the onset, however, the function and specific subpopulation of these infiltrated T lymphocytes have not been fully understood. By using cytokine-deficient mice with transient focal ischemia model, we have shown that IL-23 and IL-17 but not IL-6 or IFN-γ play pivotal roles in the ischemic brain injury. We found that IL-23, which was mainly produced from infiltrated macrophages, induced IL-17-producing T lymphocytes in the ischemic brain. IL-23 and IL-17 expression increased 1 day or 3 day after ischemic injury, respectively, and promoted inflammatory responses by increasing the expression of neurotoxic factors. To our surprise, IL-17 was mainly produced by γδT lymphocytes, but not helper T cells. Based on these findings, we have tried to develop new therapeutic strategy for the progression of brain infarction. We found that administration of FTY720 (Fingolimod) and antibody against γδT lymphocytes attenuated ischemic brain damage. Furthermore, antibody against p40 which neutralizes both IL-23 and IL-12 simultaneously was also effective. Therefore, anti-cytokine therapy will be applicable for neuroprotection in the delayed phase of brain ischemia.

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TGF-β function in immune suppression

Yoshimura Akihiko, Muto Go

Current topics in microbiology and immunology 350 127 - 147 2010年

ISSN 0070-217X

Transforming growth factor-β (TGF-β) has been shown to play an essential role in establishing immunological tolerance, yet recent studies have revealed the pro-inflammatory roles of TGF-β in inflammatory responses. TGF-β induces Foxp3-positive regulatory T cells (iTregs), while in the presence of IL-6, it induces pathogenic IL-17 producing Th17 cells. TGF-β inhibits the proliferation of T cells as well as cytokine production via Foxp3-dependent and independent mechanisms. On the one hand, little is known about molecular mechanisms involved in immune suppression via TGF-β however, recent studies suggest that Smad2 as well as Smad3 play essential roles in Foxp3 induction and cytokine suppression, whereas Th17 differentiation is promoted via the Smad-independent pathway. Mutual suppression of signaling between TGF-β and inflammatory cytokines has been shown to be necessary for the balance of immunity and tolerance. © Springer-Verlag Berlin Heidelberg 2011.

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Role of T lymphocytes in ischemic brain injury

Shichita Takashi, Konoeda Fumie, Yoshimura Akihiko

Clinical Neurology 50 （ 11 ） 878 - 880 2010年

ISSN 0009-918X

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TGF-β function in immune suppression

Yoshimura Akihiko, Muto Go

Current topics in microbiology and immunology 350 127 - 147 2010年

ISSN 0070-217X

Transforming growth factor-β (TGF-β) has been shown to play an essential role in establishing immunological tolerance, yet recent studies have revealed the pro-inflammatory roles of TGF-β in inflammatory responses. TGF-β induces Foxp3-positive regulatory T cells (iTregs), while in the presence of IL-6, it induces pathogenic IL-17 producing Th17 cells. TGF-β inhibits the proliferation of T cells as well as cytokine production via Foxp3-dependent and independent mechanisms. On the one hand, little is known about molecular mechanisms involved in immune suppression via TGF-β however, recent studies suggest that Smad2 as well as Smad3 play essential roles in Foxp3 induction and cytokine suppression, whereas Th17 differentiation is promoted via the Smad-independent pathway. Mutual suppression of signaling between TGF-β and inflammatory cytokines has been shown to be necessary for the balance of immunity and tolerance. © Springer-Verlag Berlin Heidelberg 2011.

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Erratum

Yoshida Ryoko, Takaesu Giichi, Yoshida Hideyuki, Okamoto Fuyuki, Yoshioka Tomoko, Choi Yongwon, Akira Shizuo, Kawai Taro, Yoshimura Akihiko, Kobayashi Takashi

Journal of Biological Chemistry 284 （ 52 ） 36720 2009年12月

ISSN 0021-9258

Signal-transducing adaptor protein-2 regulates stromal cell-derived factor-1α-induced chemotaxis in T cells

Sekine Yuichi, Ikeda Osamu, Tsuji Satoshi, Yamamoto Chikako, Muromoto Ryuta, Nanbo Asuka, Oritani Kenji, Yoshimura Akihiko, Matsuda Tadashi

Journal of Immunology 183 （ 12 ） 7966 - 7974 2009年12月

ISSN 0022-1767

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains, as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 regulates integrin-mediated T cell adhesion. In the present study, we find that STAP-2 expression affects Jurkat T cell migration after stromal cell-derived factor-1α (SDF-1α)-treatment. Furthermore, STAP-2-deficient T cells exhibit reduced cell migration after SDF-1α-treatment. Importantly, overexpression of STAP-2 in Jurkat T cells induces activation of small guanine triphosphatases, such as Rac1 and Cdc42. Regarding the mechanism for this effect, we found that STAP-2 associates with Vav1, the guanine-nucleotide exchanging factor for Rac1, and enhances downstream Vav1/Rac1 signaling. These results reveal a novel STAP-2-mediated mechanism for the regulation of SDF-1α-induced chemotaxis of T cells via activation of Vav1/Rac1 signaling. Copyright © 2009 by The American Association of Immunologists, Inc.

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SOCS3 in T and NKT cells negatively regulates cytokine production and ameliorates ConA-induced hepatitis

Nakaya Mako, Hashimoto Masayuki, Nakagawa Ryusuke, Wakabayashi Yu, Ishizaki Takuma, Takada Ichiro, Komai Kyoko, Yoshida Hiroki, Yoshimura Akihiko

Journal of Immunology 183 （ 11 ） 7047 - 7053 2009年12月

ISSN 0022-1767

Suppressor of cytokine signaling 3 (SOCS3), a negative-feedback molecule for cytokine signaling, has been implicated in protection against liver injury. Previous studies have shown that overexpression of SOCS3 in the liver by adenovirus or membrane permeable recombinant protein protected the liver from various injuries. However it remained uncertain in which type of cells SOCS3 suppresses liver injury. In this study, we demonstrated that forced expression of SOCS3 in T and NKT cells suppressed ConA-induced hepatitis using T and NKT cell-specific SOCS3 transgenic (Lck-SOCS3 Tg) mice. IFN-γ and IL-4 production was reduced in Lck-SOCS3 Tg mice as well as splenocytes treated with ConA. IFN-γ and IL-4 levels were also reduced in Lck-SOCS3 Tg mice administrated with α-galactosylceramide, suggesting that SOCS3 in NKT cells has suppressive function. Sustained expression of SOCS3 in an NKT cell line also resulted in reduced expression of various cytokines and transcription factors. In contrast, T and NKT cell-specific SOCS3 conditional knockout (Lck-SOCS3 cKO) mice were hypersensitive to ConA-mediated hepatitis. Isolated SOCS3-deficient NKT cells produced higher levels of IFN-γ and IL-4. These data indicate that SOCS3 plays a negative regulatory role in NKT cell activation and that forced expression of SOCS3 in NKT cells is effective in preventing hepatitis. Copyright © 2009 by The American Association of Immunologists, Inc.

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Loss of SOCS3 expression in T cells reveals a regulatory role for interleukin-17 in atherosclerosis

Taleb Soraya, Romain Mélissa, Ramkhelawon Bhama, Uyttenhove Catherine, Pasterkamp Gerard, Herbin Olivier, Esposito Bruno, Perez Nicolas, Yasukawa Hideo, Van Snick Jacques, Yoshimura Akihiko, Tedgui Alain, Mallat Ziad

Journal of Experimental Medicine 206 （ 10 ） 2067 - 2077 2009年09月

ISSN 0022-1007

Atherosclerosis is an inflammatory vascular disease responsible for the first cause of mortality worldwide. Recent studies have clearly highlighted the critical role of the immunoinflammatory balance in the modulation of disease development and progression. However, the immunoregulatory pathways that control atherosclerosis remain largely unknown. We show that loss of suppressor of cytokine signaling (SOCS) 3 in T cells increases both interleukin (IL)-17 and IL-10 production, induces an antiinflammatory macrophage phenotype, and leads to unexpected IL-17-dependent reduction in lesion development and vascular inflammation. In vivo administration of IL-17 reduces endothelial vascular cell adhesion molecule-1 expression and vascular T cell infiltration, and significantly limits atherosclerotic lesion development. In contrast, overexpression of SOCS3 in T cells reduces IL-17 and accelerates atherosclerosis. We also show that in human lesions, increased levels of signal transducer and activator of transcription (STAT) 3 phosphorylation and IL-17 are associated with a stable plaque phenotype. These results identify novel SOCS3-controlled IL-17 regulatory pathways in atherosclerosis and may have important implications for the understanding of the increased susceptibility to vascular inflammation in patients with dominant-negative STAT3 mutations and defective Th17 cell differentiation. © 2009 Taleb et al.

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Sprouty4 deficiency potentiates Ras-independent angiogenic signals and tumor growth

Taniguchi Koji, Ishizaki Takuma, Ayada Toranoshin, Sugiyama Yuki, Wakabayashi Yu, Sekiya Takashi, Nakagawa Ryusuke, Yoshimura Akihiko

Cancer Science 100 （ 9 ） 1648 - 1654 2009年09月

ISSN 1347-9032

Sprouty proteins have been shown to negatively regulate a variety of receptor tyrosine kinase (RTK) signaling pathways and are considered to be tumor suppressor proteins. The pathophysiological functions of Sproutys in vivo remain to be investigated. In this study, we examined the physiological function of Sprouty4 as an angiogenic regulator, using Sprouty4 knockout (KO) mice and cells.We found that transplanted tumor cells grow much faster in Sprouty4 KO mice than in wild type (WT) mice, which we associate with enhanced neovascularization in the tumors transplanted into Sprouty4 KO mice. Moreover, vascular endothelial growth factor (VEGF)-A-induced angiogenesis and vascular permeability in vivo were enhanced in Sprouty4 KO mice compared with WT mice. Ex vivo angiogenesis, which we induced by VEGF-A, basic fibroblast growth factor (bFGF), and sphingosine-1-phosphate (S1P), was also enhanced in the aortas of Sprouty4 KO mice. We demonstrated that Sprouty4 suppresses Ras-independent VEGF-A and S1P signaling,while it does not affect Ras-dependent VEGF-C signaling. These data indicate that Sprouty4 selectively suppresses Ras-independent angiogenic factor signals and is an important negative regulator of pathophysiological angiogenesis. © 2009 Japanese Cancer Association.

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Pivotal role of cerebral interleukin-17-producing T cells in the delayed phase of ischemic brain injury

Shichita Takashi, Sugiyama Yuki, Ooboshi Hiroaki, Sugimori Hiroshi, Nakagawa Ryusuke, Takada Ichiro, Iwaki Toru, Okada Yasunori, Iida Mitsuo, Cua Daniel J., Iwakura Yoichiro, Yoshimura Akihiko

Nature Medicine 15 （ 8 ） 946 - 950 2009年08月

ISSN 1078-8956

Lymphocyte recruitment and activation have been implicated in the progression of cerebral ischemia-reperfusion (I/R) injury, but the roles of specific lymphocyte subpopulations and cytokines during stroke remain to be clarified. Here we demonstrate that the infiltration of T cells into the brain, as well as the cytokines interleukin-23 (IL-23) and IL-17, have pivotal roles in the evolution of brain infarction and accompanying neurological deficits. Blockade of T cell infiltration into the brain by the immunosuppressant FTY720 reduced I/R-induced brain damage. The expression of IL-23, which was derived mostly from infiltrated macrophages, increased on day 1 after I/R, whereas IL-17 levels were elevated after day 3, and this induction of IL-17 was dependent on IL-23. These data, together with analysis of mice genetically disrupted for IL-17 and IL-23, suggest that IL-23 functions in the immediate stage of I/R brain injury, whereas IL-17 has an important role in the delayed phase of I/R injury during which apoptotic neuronal death occurs in the penumbra. Intracellular cytokine staining revealed that T lymphocytes, but not CD4 ⁺ helper T cells, were a major source of IL-17. Moreover, depletion of T lymphocytes ameliorated the I/R injury. We propose that T lymphocytes, including T lymphocytes, could be a therapeutic target for mitigating the inflammatory events that amplify the initial damage in cerebral ischemia.

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Cyclic Adenosine Monophosphate Suppresses the Transcription of Proinflammatory Cytokines via the Phosphorylated c-Fos Protein (DOI

Koga Keiko, Takaesu Giichi, Yoshida Ryoko, Nakaya Mako, Kobayashi Takashi, Kinjyo Ichiko, Yoshimura Akihiko

Immunity 31 （ 1 ） 170 2009年07月

ISSN 1074-7613

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The mTOR pathway is highly activated in diabetic nephropathy and rapamycin has a strong therapeutic potential

Mori Hiroyuki, Inoki Ken, Masutani Kohsuke, Wakabayashi Yu, Komai Kyoko, Nakagawa Ryusuke, Guan Kun Liang, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 384 （ 4 ） 471 - 475 2009年07月

ISSN 0006-291X

Diabetic nephropathy (DN) associated with type 2 diabetes is the most common cause of end-stage renal disease (ESRD) and a serious health issue in the world. Currently, molecular basis for DN has not been established and only limited clinical treatments are effective in abating the progression to ESRD associated with DN. Here we found that diabetic db/db mice which lack the leptin receptor signaling can be used as a model of ESRD associated with DN. We demonstrated that p70S6-kinase was highly activated in mesangial cells in diabetic obese db/db mice. Furthermore, systemic administration of rapamycin, a specific and potent inhibitor of mTOR, markedly ameliorated pathological changes and renal dysfunctions. Moreover, rapamycin treatment shows a significant reduction in fat deposits and attenuates hyperinsulinemia with few side effects. These results indicate that mTOR activation plays a pivotal role in the development of ESRD and that rapamycin could be an effective therapeutic agent for DN. © 2009 Elsevier Inc. All rights reserved.

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Stat1 phosphorylation is a molecular switch of Ras signaling and oncogenesis

Yoshimura Akihiko

Cell Cycle 8 （ 13 ） 1981 - 1982 2009年07月

ISSN 1538-4101

The protein content of an adaptor protein, STAP-2 is controlled by E3 ubiquitin ligase Cbl

Sekine Yuichi, Yamamoto Chikako, Ikeda Osamu, Muromoto Ryuta, Nanbo Asuka, Oritani Kenji, Yoshimura Akihiko, Matsuda Tadashi

Biochemical and Biophysical Research Communications 384 （ 2 ） 187 - 192 2009年06月

ISSN 0006-291X

Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. Our previous study in T cells demonstrated that STAP-2 influences FAK protein levels through recruitment of E3 ubiquitin ligase, Cbl, to FAK. In the present study, we found that Cbl directly controls the protein levels and activity of STAP-2. STAP-2 physically interacted with Cbl through its PH and SH2-like domains. Small-interfering RNA-mediated reduction of endogenous Cbl restored STAP-2 protein levels. In contrast, over-expression of Cbl induced STAP-2 degradation. Importantly, Cbl-mediated regulation of STAP-2 protein levels affected Brk/STAP-2-induced STAT3 activation. These results indicate that Cbl regulates STAP-2 protein levels and Brk/STAP-2-mediated STAT3 activation. © 2009 Elsevier Inc. All rights reserved.

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STAP-2 is phosphorylated at tyrosine-250 by Brk and modulates Brk-mediated STAT3 activation

Ikeda Osamu, Miyasaka Yuto, Sekine Yuichi, Mizushima Akihiro, Muromoto Ryuta, Nanbo Asuka, Yoshimura Akihiko, Matsuda Tadashi

Biochemical and Biophysical Research Communications 384 （ 1 ） 71 - 75 2009年06月

ISSN 0006-291X

Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. STAP-2 is also known as breast tumor kinase (Brk) substrate (BKS). Our previous studies revealed that STAP-2 binds to signal transducer and activator of transcription 3 (STAT3) and STAT5, and regulates the signaling pathways downstream of them. In the present study, we identified tyrosine-250 (Tyr250) in STAP-2 as a major site of phosphorylation by Brk, using a series of STAP-2 YF mutants and anti-phospho-STAP-2 Tyr250 antibody. Furthermore, overexpression of the STAP-2 Y250F mutant protein affected Brk-mediated STAT3 activation. Importantly, small-interfering RNA-mediated reduction of endogenous STAP-2 expression decreased Brk-mediated STAT3 activation. Taken together, our findings demonstrate that STAP-2 is phosphorylated at Tyr250 by Brk, and plays an important role in Brk-mediated STAT3 activation. © 2009 Elsevier Inc. All rights reserved.

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Pulmonary suppressor of cytokine signaling-1 induced by IL-13 regulates allergic asthma phenotype

Fukuyama Satoru, Nakano Takako, Matsumoto Takafumi, Oliver Brian G G, Burgess Janette K., Moriwaki Atsushi, Tanaka Kentaro, Kubo Masato, Hoshino Tomoaki, Tanaka Hiroyuki, McKenzie Andrew N J, Matsumoto Koichiro, Aizawa Hisamichi, Nakanishi Yoichi, Yoshimura Akihiko, Black Judith L., Inoue Hiromasa

American Journal of Respiratory and Critical Care Medicine 179 （ 11 ） 992 - 998 2009年06月

ISSN 1073-449X

Rationale: Th2 cytokines play an important role in allergic diseases. These cytokines activate signal transduction pathways, including Janus kinase/signal transducer and activator of transcription (STAT) signaling. Although the suppressor of cytokine signaling (SOCS) family protein, a negative regulator of the Janus kinase/STAT signaling pathway, contributes to helper T cell differentiation during immune responses, the role of SOCS proteins within the structural cells of a target organ has not been clarified in allergy. Objectives: To study the local function of SOCS in the development of asthma. Methods: We used mouse models of IL-13- and ovalbumin (OVA)-induced allergic airway disease. Airway smooth muscle cells were cultured from patients with asthma. Measurements and Main Results: The administration of IL-13 induced not only airway responses but also SOCS1 expression at the local inflammatory site. The up-regulated SOCS1 markedly suppressed IL-13-dependent STAT6 activation and eotaxin expression and subsequently down-regulated IL-13-induced airway inflammatory responses. The inactivation of SOCS1 induced airway hyperresponsiveness after IL-13 treatment even in hyporesponsive C57BL/6 background mice. In an OVA-induced model of allergic airway disease, allergen exposure up-regulated local SOCS1 expression, and the induction of SOCS1 in the airways attenuated allergen-induced airway responses. Inactivation of IL-13 inhibited SOCS1 induction in a model of allergic airway disease. Interestingly, airway smooth muscle cells from individuals with asthma had impaired upregulation of SOCS1 after IL-13 stimulation. Conclusions: SOCS1 induction by IL-13 in airway structural cells is critical to negatively control allergic airway disease.

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Regulation of cytokine signaling by the SOCS and Spred family proteins

Yoshimura Akihiko

Keio Journal of Medicine 58 （ 2 ） 73 - 83 2009年06月

ISSN 0022-9717

Various cytokines are involved in the regulation of the immune system and of hematopoiesis. Most cytokines utilize the so-called JAK-STAT pathway, but others activate the Ras-ERK pathway, which is more important than the STAT pathway for the proliferation of hematopoietic cells. Dysregulation of cytokine signaling can cause a variety of diseases, including allergy, inflammation, and cancer. We have identified two important regulator families involved in cytokine signaling: the SOCS proteins and the Spred proteins. Suppressors of cytokine signaling (SOCS) proteins bind to JAK and to certain receptors, thereby suppressing further signaling events. Spred family proteins interact with Ras and Raf, thereby suppressing ERK activation. Studies have shown that SOCS and Spred proteins are key physiological regulators of immunity, hematopoiesis, and angiogenesis. Evidence is also emerging for the involvement of these proteins in human diseases.

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A major lipid raft protein raftlin modulates T cell receptor signaling and enhances Th17-mediated autoimmune responses

Saeki Kazuko, Fukuyama Satoru, Ayada Toranoshin, Nakaya Mako, Aki Daisuke, Takaesu Giichi, Hanada Toshikatsu, Matsumura Yumiko, Kobayashi Takashi, Nakagawa Ryusuke, Yoshimura Akihiko

Journal of Immunology 182 （ 10 ） 5929 - 5937 2009年05月

ISSN 0022-1767

The membrane microdomains known as lipid rafts have been shown to act as platforms for the initiation of various receptor signals. Through proteomic analysis, we have identified a novel protein termed Raftlin (raft-linking protein) as a major protein in lipid rafts. To determine the physiological and immunological functions of Raftlin in mammals, we generated Raftlin-deficient mice, as well as Raftlin-transgenic (Tg) mice. Although Raftlin was originally identified in B cells, we observe no severe abnormalities in the B cells of these mice, presumably due to a high expression of Raftlin-homologue (Raftlin-2). T cells, in contrast, expressed a substantial amount of Raftlin but no Raftlin-2. In Raftlin-deficient mice, T cell-dependent Ab production was reduced, and experimental autoimmune encephalomyelitis, a Th17-dependent autoimmune disease model, was ameliorated. In Raftlin-Tg mice, in contrast, Ab production was enhanced and experimental autoimmune encephalomyelitis was more severe. Cytokine production, especially that of IL-17, was reduced in Raftlin-deficient T cells, while it was enhanced in Raftlin-Tg T cells. We found that these changes were associated with the strength of the TCR-mediated signals. Importantly, localization of Lck protein in the lipid rafts was enhanced by Raftlin overexpression and reduced by Raftlin deficiency. These data indicate that Raftlin modulates TCR signals and is necessary for the fine-tuning of T cell-mediated immune responses. Copyright © 2009 by The American Association of Immunologists, Inc.

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Suppression of Sproutys has a therapeutic effect for a mouse model of ischemia by enhancing angiogenesis

Taniguchi Koji, Sasaki Ken Ichiro, Watari Kousuke, Yasukawa Hideo, Imaizumi Tsutomu, Ayada Toranoshin, Okamoto Fuyuki, Ishizaki Takuma, Kato Reiko, Kohno Ri Ichiro, Kimura Hiroshi, Sato Yasufumi, Ono Mayumi, Yonemitsu Yoshikazu, Yoshimura Akihiko

PLoS ONE 4 （ 5 ） 2009年05月

Sprouty proteins (Sproutys) inhibit receptor tyrosine kinase signaling and control various aspects of branching morphogenesis. In this study, we examined the physiological function of Sproutys in angiogenesis, using gene targeting and short-hairpin RNA (shRNA) knockdown strategies. Sprouty2 and Sprouty4 double knockout (KO) (DKO) mice were embryonic-lethal around E12.5 due to cardiovascular defects. The number of peripheral blood vessels, but not that of lymphatic vessels, was increased in Sprouty4 KO mice compared with wild-type (WT) mice. Sprouty4 KO mice were more resistant to hind limb ischemia and soft tissue ischemia than WT mice were, because Sprouty4 deficiency causes accelerated neovascularization. Moreover, suppression of Sprouty2 and Sprouty4 expression in vivo by shRNA targeting accelerated angiogenesis and has a therapeutic effect in a mouse model of hind limb ischemia. These data suggest that Sproutys are physiologically important negative regulators of angiogenesis in vivo and novel therapeutic targets for treating peripheral ischemic diseases. Copyright: © 2009 Taniguchi et al.

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An F-box protein, FBXW5, negatively regulates TAK1 MAP3K in the IL-1β signaling pathway

Minoda Yasumasa, Sakurai Hiroaki, Kobayashi Takashi, Yoshimura Akihiko, Takaesu Giichi

Biochemical and Biophysical Research Communications 381 （ 3 ） 412 - 417 2009年04月

ISSN 0006-291X

TAK1, a member of the MAP3K family, plays an essential role in activation of JNK/p38 MAPKs and IKK in the IL-1β and TNFα signaling pathway. Upon stimulation, TAK1 is rapidly and transiently activated. While the activation mechanism of TAK1 in these signaling pathways is well characterized, how its activity is terminated still remains unclear. To identify the molecule(s) involved in TAK1 regulation, we performed tandem affinity purification (TAP) in HeLa cells stably expressing TAP-tagged TAK1. FBXW5, an F-box family protein, was identified as a previously unknown component of the IL-1β-induced TAK1 complex. FBXW5 associated with endogenous TAK1 in an IL-1β-dependent manner. Overexpression of FBXW5 inhibited IL-1β-induced activation of JNK/p38 MAPKs and NF-κB as well as phosphorylation of TAK1 on Thr187. Conversely, knockdown of FBXW5 resulted in the prolonged activation of TAK1 upon IL-1β stimulation. These results suggest that FBXW5 negatively regulates TAK1 in the IL-1β signaling pathway. © 2009 Elsevier Inc. All rights reserved.

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Cyclic Adenosine Monophosphate Suppresses the Transcription of Proinflammatory Cytokines via the Phosphorylated c-Fos Protein

Koga Keiko, Takaesu Giichi, Yoshida Ryoko, Nakaya Mako, Kobayashi Takashi, Kinjyo Ichiko, Yoshimura Akihiko

Immunity 30 （ 3 ） 372 - 383 2009年03月

ISSN 1074-7613

Intracellular cyclic adenosine monophosphate (cAMP) suppresses innate immunity by inhibiting proinflammatory cytokine production from monocytic cells. Enhanced expression of interleukin-10 (IL-10) has been suggested to be the mechanism of suppression. However, cAMP is still capable of suppressing production of the cytokines TNF-α and IL-12 in IL-10-deficient dendritic cells (DCs). Here, we demonstrated that the transcription factor c-Fos was responsible for the cAMP-mediated suppression of inflammatory cytokine production. c-Fos accumulated at high amounts in response to cAMP and lipopolysaccharide (LPS). Overexpression of c-Fos suppressed LPS-induced cytokine production, whereas cAMP-mediated suppression of TNF-α and IL-12 was impaired in Fos^-/- DCs or in RAW264.7 cells treated with c-Fos siRNA. c-Fos physically interacted with p65 protein and reduced the recruitment of p65 to the Tnf promoter. Multiple sites of c-Fos were phosphorylated by the IKKβ protein. Thus, we propose that c-Fos is a substrate of IKKβ and is responsible for the immunosuppressive effect of cAMP. © 2009 Elsevier Inc. All rights reserved.

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TRAF6 is required for generation of the B-1a B cell compartment as well as T cell-dependent and-independent humoral immune responses

Kobayashi Takashi, Kim Tae Soo, Jacob Anand, Walsh Matthew C., Kadono Yuho, Fuentes-Pananá Ezequiel, Yoshioka Tomoko, Yoshimura Akihiko, Yamamoto Masahiro, Kaisho Tsuneyasu, Akira Shizuo, Monroe John G., Choi Yongwon

PLoS ONE 4 （ 3 ） 2009年03月

TNF receptor superfamily members, such as CD40 and the Toll-like receptors (TLRs), regulate many aspects of B cell differentiation and activation. TRAF6 is an intracellular signaling adaptor molecule for these receptors, but its role in B cells has not been clarified by previous genetic approaches, as the systemic deletion of the TRAF6 gene results in perinatal lethality. Here we show that B cell-specific TRAF6 deficiency results in a reduced number of mature B cells in the bone marrow and spleen. Optimal T cell-dependent (TD) antigen responses, as characterized by isotype switching and long-lived plasma cell generation, are also impaired in B cell-specific TRAF6-deficient mice. B cell-specific TRAF6-deficient mice also exhibit lower levels of serum IgM and IgG2b and defective antigen-specific IgM production in response to T cell-independent (TI) antigens. Unexpectedly, TRAF6-deficient B cell progenitors are unable to generate CD5+ B-1 cells. These results reveal critical roles for TRAF6 in TD and TI humoral immune responses and in inductive fate decisions necessary to generate the B-1 B cell compartment. © 2009 Kobayashi et al.

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PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts

Hikata Tomohiro, Takaishi Hironari, Jiro Takito, Hakozaki Akihiro, Furukawa Mitsuru, Uchikawa Shinichi, Kimura Tokuhiro, Okada Yasunori, Matsumoto Masahito, Yoshimura Akihiko, Nishimura Riko, Reddy Sakamuri V., Asahara Hiroshi, Toyama Yoshiaki

Blood 113 （ 10 ） 2202 - 2212 2009年03月

ISSN 0006-4971

Cytokine signaling via various transcription factors regulates receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation from monocyte/macrophage lineage cells involved in propagation and resolution of inflammatory bone destruction. Protein inhibitor of activated STAT3 (PIAS3) was initially identified as a molecule that inhibits DNA binding of STAT3 and regulates many transcription factors through distinct mechanisms. To analyze PIAS3 function in osteoclasts in vivo, we have generated transgenic mice in which PIAS3 is specifically expressed in the osteoclast lineage using the tartrate-resistant acid phosphatase (TRAP) gene promoter. PIAS3 transgenic mice showed an osteopetrotic phenotype due to impairment of osteoclast differentiation. Overexpression of PIAS3 in RAW264.7 cells suppressed RANKL-induced osteoclastogenesis by inhibiting the expression of c-Fos and NFATc1. Interestingly, PIAS3 inhibits the transcriptional activity of microphthalmia-associated transcription factor (MITF) independent of sumoylation. Down-regulation of PIAS3 markedly enhances RANKL-mediated osteoclastogenesis in RAW264.7 cells. Furthermore, overexpression of PIAS3 in mouse primary osteoblast (POB), down-regulates RANKL expression induced by interleukin-6 (IL-6) cytokine family, and inhibits osteoclast formation from bone marrow macrophages (BMMs) in vitro coculture system. Down-regulation of PIAS3 leads to the accelerated expression of RANKL in POB stimulated with IL-6 and soluble IL-6 receptor (sIL-6R). Taken together, our results clearly indicate that PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts. © 2009 by The American Society of Hematology.

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Sprouty4 negatively regulates protein kinase C activation by inhibiting phosphatidylinositol 4,5-biphosphate hydrolysis

Ayada T., Taniguchi K., Okamoto F., Kato R., Komune S., Takaesu G., Yoshimura A.

Oncogene 28 （ 8 ） 1076 - 1088 2009年02月

ISSN 0950-9232

Sproutys have been shown to negatively regulate growth factor-induced extracellular signal-regulated kinase (ERK) activation, and suggested to be an anti-oncogene. However, molecular mechanism of the suppression has not yet been clarified completely. Sprouty4 inhibits vascular endothelial growth factor (VEGF)-A-induced ERK activation, but not VEGF-C-induced ERK activation. It has been shown that VEGF-A-mediated ERK activation is strongly dependent on protein kinase C (PKC), whereas that by VEGF-C is dependent on Ras. This suggests that Sprouty4 inhibits the PKC pathway more specifically than the Ras pathway. In this study, we confirmed that Sprouty4 suppressed various signals downstream of PKC, such as phosphorylation of MARCKS and protein kinase D (PKD), as well as PKC-dependent nuclear factor (NF)-κB activation. Furthermore, Sprouty4 suppressed upstream signals of PKC, such as Ca²⁺ mobilization, phosphatidylinositol 4,5-biphosphate (PIP₂) breakdown and inositol 1,4,5-triphosphate (IP₃) production in response to VEGF-A. Those effects were dependent on the C-terminal cysteine-rich region, but not on the N-terminal region of Sprouty4, which is critical for the suppression of fibroblast growth factor (FGF)-mediated ERK activation. Sprouty4 overexpression or deletion of the Sprouty4 gene did not affect phospholipase C (PLC) γ-1 activation, which is an enzyme that catalyzes PIP₂ hydrolysis. Moreover, Sprouty4 inhibited not only VEGF-A-mediated PIP₂ hydrolysis but also inhibited the lysophosphatidic acid (LPA)-induced PIP₂ breakdown that is catalyzed by PLCβ/ε activated by G-protein coupled receptor (GPCR). Taken together, Sprouty4 has broader suppression activity for various stimuli than previously thought; it may function as an inhibitor for various types of PLC-dependent signaling as well as for ERK activation. © 2009 Macmillan Publishers Limited All rights reserved.

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Foxp3-Dependent MicroRNA155 Confers Competitive Fitness to Regulatory T Cells by Targeting SOCS1 Protein

Lu Li Fan, Thai To Ha, Calado Dinis Pedro, Chaudhry Ashutosh, Kubo Masato, Tanaka Kentaro, Loeb Gabriel B., Lee Hana, Yoshimura Akihiko, Rajewsky Klaus, Rudensky Alexander Y.

Immunity 30 （ 1 ） 80 - 91 2009年01月

ISSN 1074-7613

Foxp3⁺ regulatory T (Treg) cells limit pathogenic immune responses to self-antigens and foreign antigens. An essential role for microRNA (miRNA) in the maintenance and function of Treg cells, revealed by the Treg cell-specific Dicer ablation, raised a question as to a specific miRNA contribution. We found that Foxp3 controlled the elevated miR155 expression required for maintaining Treg cell proliferative activity and numbers under nonlymphopenic conditions. Moreover, miR155 deficiency in Treg cells resulted in increased suppressor of cytokine signaling 1 (SOCS1) expression accompanied by impaired activation of signal transducer and activator of transcription 5 (STAT5) transcription factor in response to limiting amounts of interleukin-2. Our studies suggest that Foxp3-dependent regulation of miR155 maintains competitive fitness of Treg cell subsets by targeting SOCS1, and they provide experimental support for a proposed role for miRNAs in ensuring the robustness of cellular phenotypes. © 2009 Elsevier Inc. All rights reserved.

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Clinical and mutational spectrum of neurofibromatosis type 1-like syndrome

Messiaen Ludwine, Yao Suxia, Brems Hilde, Callens Tom, Sathienkijkanchai Achara, Denayer Ellen, Spencer Emily, Arn Pamela, Babovic-Vuksanovic Dusica, Bay Carolyn, Bobele Gary, Cohen Bruce H., Escobar Luis, Eunpu Deborah, Grebe Theresa, Greenstein Robert, Hachen Rachel, Irons Mira, Kronn David, Lemire Edmond, Leppig Kathleen, Lim Cynthia, McDonald Marie, Narayanan Vinodh, Pearn Amy, Pedersen Robert, Powell Berkley, Shapiro Lawrence R., Skidmore David, Tegay David, Thiese Heidi, Zackai Elaine H., Vijzelaar Raymon, Taniguchi Koji, Ayada Toranoshin, Okamoto Fuyuki, Yoshimura Akihiko, Parret Annabel, Korf Bruce, Legius Eric

JAMA - Journal of the American Medical Association 302 （ 19 ） 2111 - 2118 2009年

ISSN 0098-7484

Context: Autosomal dominant inactivating sprouty-related EVH1 domain-containing protein 1 (SPRED1) mutations have recently been described in individuals presenting mainly with café au lait macules (CALMs), axillary freckling, and macrocephaly. The extent of the clinical spectrum of this new disorder needs further delineation. Objective: To determine the frequency, mutational spectrum, and phenotype of neurofibromatosis type 1-like syndrome (NFLS) in a large cohort of patients. Design, Setting, and Participants: In a cross-sectional study, 23 unrelated probands carrying a SPRED1 mutation identified through clinical testing participated with their families in a genotype-phenotype study (2007-2008). In a second cross-sectional study, 1318 unrelated anonymous samples collected in 2003-2007 from patients with a broad range of signs typically found in neurofibromatosis type 1 (NF1) but no detectable NF1 germline mutation underwent SPRED1 mutation analysis. Main Outcome Measures: Comparison of aggregated clinical features in patients with or without a SPRED1 or NF1 mutation. Functional assays were used to evaluate the pathogenicity of missense mutations. Results: Among 42 SPRED1-positive individuals from the clinical cohort, 20 (48%; 95% confidence interval [CI], 32%-64%) fulfilled National Institutes of Health (NIH) NF1 diagnostic criteria based on the presence of more than 5 CALMs with or without freckling or an NF1-compatible family history. None of the 42 SPRED1-positive individuals (0%; 95% CI, 0%-7%) had discrete cutaneous or plexiform neurofibromas, typical NF1 osseous lesions, or symptomatic optic pathway gliomas. In the anonymous cohort of 1318 individuals, 34 different SPRED1 mutations in 43 probands were identified: 27 pathogenic mutations in 34 probands and 7 probable nonpathogenic missense mutations in 9 probands. Of 94 probands with familial CALMs with or without freckling and no other NF1 features, 69 (73%; 95% CI, 63%-80%) had an NF1 mutation and 18 (19%; 95% CI, 12%-29%) had a pathogenic SPRED1 mutation. In the anonymous cohort, 1.9% (95% CI, 1.2%-2.9%) of individuals with the clinical diagnosis of NF1 according to the NIH criteria had NFLS. Conclusions: A high SPRED1 mutation detection rate was found in NF1 mutation-negative families with an autosomal dominant phenotype of CALMs with or without freckling and no other NF1 features. Among individuals in this study, NFLS was not associated with the peripheral and central nervous system tumors seen in NF1. ©2009 American Medical Association. All rights reserved.

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Involvement of Th17 cells and the effect of anti-IL-6 therapy in autoimmune uveitis

Yoshimura Takeru, Sonoda Koh Hei, Ohguro Nobuyuki, Ohsugi Yoshiyuki, Ishibashi Tatsuro, Cua Daniel J., Kobayashi Takashi, Yoshida Hiroki, Yoshimura Akihiko

Rheumatology 48 （ 4 ） 347 - 354 2009年

ISSN 1462-0324

Objectives. Human endogenous uveitis is one of the sight-threatening diseases associated with variety of systemic disorders, such as Behcet's disease and sarcoidosis. Recently, biosynthesized antibodies against inflammatory cytokines have been recognized to be useful to control the regional inflammation. In this study, we focused on the possibility of IL-6-based biological therapies for endogenous uveitis. We initially confirmed the significant increase of several inflammatory soluble factors including IL-6 in the vitreous fluids from refractory/chronic engogenous uveitis patients. Methods. To investigate the role of IL-6 in the formation of refractory ocular inflammation, we used the mouse experimental autoimmune uveitis (EAU) model. Both IL-6 and IL-23 are required for the development of IL-17-producing helper T subset (Th17) from naïve CD4⁺T cells. Results. In the EAU model, neither IL-6-deficient mice nor IL-23-deficient mice could induce Th17 cells and the EAU score was decreased in these mice in the entire time course. We also confirmed that systemic administration of anti-IL-6 receptor antibody ameliorates EAU by suppressing both systemic and regional Th17 responses. Conclusions. IL-6 is responsible for causing ocular inflammation, and it is, at least partially, due to IL-6-dependent Th17 differentiation. IL-6 may be a target for therapy of refractory endogenous uveitis in humans. © The Author 2009. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.

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IFN-γ expression in CD8⁺ T cells regulated by IL-6 signal is involved in superantigen-mediated CD4⁺ T cell death

Atsumi Toru, Sato Masae, Kamimura Daisuke, Moroi Arisa, Iwakura Yoichiro, Betz Ulrich A K, Yoshimura Akihiko, Nishihara Mika, Hirano Toshio, Murakami Masaaki

International Immunology 21 （ 1 ） 73 - 80 2009年

ISSN 0953-8178

Infection with pathogens containing superantigens (Sags) can result in massive excessive CD4⁺ T cell activation and death in such conditions as toxic shock, food poisoning and autoimmune diseases. We here showed how enhancement of IL-6 signaling suppresses Sag-mediated activated CD4⁺ T cell death. Sag-induced CD4⁺ T cell death increased in IL-6 knockout (KO) mice, whereas it decreased in mice characterized by enhanced IL-6-gp130-STAT3 signaling. The serum concentration of IFN-γ was inversely correlated with the magnitude of IL-6 signaling, and IFN-γ deficiency inhibited Sag-induced activated CD4⁺ T cell death, suggesting that IL-6 suppresses CD4⁺ T cell death via IFN-γ expression. Interestingly, depletion of activated CD8⁺ T cells inhibited Sag-mediated increases in IFN-γ expression in IL-6 KO mice as well as the augmented CD4⁺ T cell death. The results demonstrate that IL-6-gp130-STAT3 signaling in activated CD8⁺ T cells contributes to Sag-induced CD4⁺ T cell death via IFN-γ expression, highlighting this signaling axis in CD8⁺ T cells as a potential therapeutic target for Sag-related syndromes. © The Japanese Society for Immunology. 2008. All rights reserved.

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Silencing of SOCS1 in macrophages suppresses tumor development by enhancing antitumor inflammation

Hashimoto Masayuki, Ayada Toranoshin, Kinjyo Ichiko, Hiwatashi Kiyokazu, Yoshida Hideyuki, Okada Yasunori, Kobayashi Takashi, Yoshimura Akihiko

Cancer Science 100 （ 4 ） 730 - 736 2009年

ISSN 1347-9032

Inflammation has been shown to contribute to both tumor development and antitumor immunity. However, conditions determining these opposing effects are not well understood. Suppressor of cytokine signaling 1 (SOCS1) has been shown to play an important role in regulating inflammation and tumor development. It has been reported that silencing of SOCS1 gene in dendritic cells potentiates antitumor immunity, while SOCS1-deficiency in whole organs except for T and B cells enhances inflammation-mediated colon tumor development. To determine which types of cells are important for the suppression of tumor development by SOCS1-deficiency, we employed the conditional knockout strategy. SOCS1 gene was deleted in macrophages and neutrophils by crossing SOCS1-flox/ flox mice with LysM-cre mice. Resulting conditional knockout (cKO) mice showed enhanced sensitivity to endotoxin shock. SOCS1-cKO mice survived much longer than wild-type mice after B16 melanoma transplantation. Colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) plus dextran sulfate sodium (DSS) was also reduced in SOCS1-cKO mice. SOCS1-deficiency in monocytic cells enhanced tumor-killing activity of macrophages and tumor-specific cytotoxic T cell activity. These results suggest that inflammation induced by SOCS1-deficiency in monocytes potentiates antitumor immune responses rather than tumor-promoting inflammation. © 2009 Japanese Cancer Association.

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Gfi1 negatively regulates T_h17 differentiation by inhibiting RORγt activity

Ichiyama Kenji, Hashimoto Masayuki, Sekiya Takashi, Nakagawa Ryusuke, Wakabayashi Yu, Sugiyama Yuki, Komai Kyoko, Saba Ingrid, Möröy Tarik, Yoshimura Akihiko

International Immunology 21 （ 7 ） 881 - 889 2009年

ISSN 0953-8178

T_h cells have long been divided into two subsets, T_h1 and T_h2; however, recently, T_h17 and inducible regulatory T (iTreg) cells were identified as new T_hcell subsets. Although T_h1- and T_h 2-polarizing cytokines have been shown to suppress T_h17 and iTreg development, transcriptional regulation of T_h17 and iTreg differentiation by cytokines remains to be clarified. In this study, we found that expression of the growth factor independent 1 (Gfi1) gene, which has been implicated in T_h2 development, was repressed in T_h17 and iTreg cells compared with T_h 1 and T_h2 lineages. Gfi1 expression was enhanced by the IFN-γ/STAT1 and IL-4/STAT6 pathways, whereas it was repressed by the transforming growth factor-β1 stimulation at the promoter level. Over-expression of Gfi1 strongly reduced IL-17A transcription in the EL4 T cell line, as well as in primary T cells. This was due to the blockade of recruitment of retinoid-related orphan receptor γt to the IL-17A promoter. In contrast, IL-17A expression was significantly enhanced in Gfi1-deficient T cells under T_h17-promoting differentiation conditions as compared with wild-type T cells. In contrast, the impacts of Gfi1 in iTregs were not as strong as in T_h17 cells. Taken together, these data strongly suggest that Gfi1 is a negative regulator of T_h17 differentiation, which represents a novel mechanism for the regulation of T_h17 development by cytokines. © The Japanese Society for Immunology. 2009. All rights reserved.

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Spred1 is required for synaptic plasticity and hippocampus-dependent learning

Denayer Ellen, Ahmed Tariq, Brems Hilde, Van Woerden Geeske, Borgesius Nils Zuiderveen, Callaerts-Vegh Zsuzsanna, Yoshimura Akihiko, Hartmann Dieter, Elgersma Ype, D'Hooge Rudi, Legius Eric, Balschun Detlef

Journal of Neuroscience 28 （ 53 ） 14443 - 14449 2008年12月

ISSN 0270-6474

Germline mutations in SPRED1, a negative regulator of Ras, have been described in a neurofibromatosis type 1 (NF1)-like syndrome (NFLS) that included learning difficulties in some affected individuals. NFLS belongs to the group of phenotypically overlapping neurocardio-facial-cutaneous syndromes that are all caused by germ line mutations in genes of the Ras/mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) pathway and that present with some degree of learning difficulties or mental retardation. We investigated hippocampus-dependent learning and memory as well as synaptic plasticity in Spred1^-/- mice, an animal model of this newly discovered human syndrome. Spred1^-/- mice show decreased learning and memory performance in the Morris water maze and visual-discrimination T-maze, but normal basic neuromotor and sensory abilities. Electrophysiological recordings on brain slices from these animals identified defects in short- and long-term synaptic hippocampal plasticity, including a disequilibrium between long-term potentiation (LTP) and long-term depression in CA1 region. Biochemical analysis, 4 h after LTP induction, demonstrated increased ERK-phosphorylation in Spred1^-/- slices compared with those of wild-type littermates. This indicates that deficits in hippocampusdependent learning and synaptic plasticity induced by SPRED1 deficiency are related to hyperactivation of the Ras/ERK pathway. Copyright © 2008 Society for Neuroscience.

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TRAF6 and MEKK1 play a pivotal role in the RIG-I-like helicase antiviral pathway

Yoshida Ryoko, Takaesu Giichi, Yoshida Hideyuki, Okamoto Fuyuki, Yoshioka Tomoko, Choi Yongwon, Akira Shizuo, Kawai Taro, Yoshimura Akihiko, Kobayashi Takashi

Journal of Biological Chemistry 283 （ 52 ） 36211 - 36220 2008年12月

ISSN 0021-9258

Type I interferons (IFN-α/β) are essential for immune defense against viruses and induced through the actions of the cytoplasmic helicases, RIG-I and MDA5, and their downstream adaptor molecule IPS-1. TRAF6 and the downstream kinase TAK1 have been shown to be essential for the production of proinflammatory cytokines through the TLR/MyD88/TRIF pathway. Although binding of TRAF6 with IPS-1 has been demonstrated, the role of the TRAF6 pathway in IFN-α/β production has not been fully understood. Here, we demonstrate that TRAF6 is critical for IFN-α/β induction in response to viral infection and intracellular double-stranded RNA, poly(I:C). Activation of NF-κB, JNK, and p38, but not IRF3, was impaired in TRAF6-deficient mouse embryo fibroblasts in response to vesicular stomatitis virus and poly(I:C). However, TAK1 was not required for IFN-β induction in this process, since normal IFN-α/β production was observed in TAK1-deficient mouse embryo fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN-β promoter in response to poly(I:C). Forced expression of MEKK1 in combination with IRF3 was sufficient for the induction of IFN-β, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN-β by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NF-κB and mitogen-activated protein kinases that are critical for the optimal induction of type I interferons. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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FLN29 deficiency reveals its negative regulatory role in the toll-like receptor (TLR) and retinoic acid-inducible gene I (RIG-I)-like helicase signaling pathway

Sanada Takahito, Takaesu Giichi, Mashima Ryuichi, Yoshida Ryoko, Kobayashi Takashi, Yoshimura Akihiko

Journal of Biological Chemistry 283 （ 49 ） 33858 - 33864 2008年12月

ISSN 0021-9258

FLN29 was identified as an interferon (IFN)-inducible gene, and it has been shown to suppress Toll-like receptor 4-mediated NF-κB activation by binding to TRAF6. To elucidate the physiological roles of FLN29, we generated FLN29-deficient mice. FLN29 deficiency resulted in hyper-response to LPS both in vivo and in vitro, demonstrating the negative regulatory role of FLN29 in TLR4 signaling. Furthermore, we found that FLN29^-/- mice exhibited increased susceptibility to poly(I:C)-induced septic shock compared with WT mice. FLN29^-/- fibroblasts were highly resistant to vesicular stomatitis virus infection, and these cells produced more IFN-β than WT cells did in response to not only intracellular poly(I:C) but also overexpression of IPS-1. Forced expression of FLN29 inhibited the IPS-1-dependent activation of both NF-κB and IRF3. We also found that FLN29 could interact with TRIF, IPS-1, TRAF3, and TRAF6. Together, these results suggest that FLN29, in addition to playing a negative regulatory role in the TLR4 signaling pathway, negatively regulates the RIG-I-like helicase signaling pathway at the level of IPS-1/TRAF6 and IPS-1/TRAF3 complexes. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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FoxO3a regulates hematopoietic homeostasis through a negative feedback pathway in conditions of stress or aging

Miyamoto Kana, Miyamoto Takeshi, Kato Reiko, Yoshimura Akihiko, Motoyama Noboru, Suda Toshio

Blood 112 （ 12 ） 4485 - 4493 2008年12月

ISSN 0006-4971

Stress or aging of tissue-specific stem cells is considered central to the decline of tissue homeostasis in the elderly, although little is known of molecular mechanisms underlying hematopoietic stem cell (HSC) aging and stress resistance. Here, we report that mice lacking the transcription factor forkhead box O3a (FoxO3a) develop neutrophilia associated with inhibition of the up-regulation of negative regulator of cell proliferation, Sprouty-related Ena/VASP homology 1 domain-containing proteins 2 (Spred2) and AKT and ERK activation, in HSCs during hema-topoietic recovery following myelosup-pressive stress conditions. Compared with aged wild-type mice, more severe neutrophilia was also observed in aged Foxo3a-deficient mice. AKT and ERK activation and inhibition of Spred2 were detected in HSCs from aged FoxO3a-deficient mice. Spred2-deficient mice also developed neutrophilia during hematopoietic recovery following myelosuppressive stress, indicating that FoxO3a plays a pivotal role in maintenance, integrity, and stress resistance of HSCs through negative feedback pathways for proliferation. This will provide new insight into the hematopoietic homeostasis in conditions of aging and stress. © 2008 by The American Society of Hematology.

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SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage

Sitko John C., Yeh Brian, Kim Moonhong, Zhou Hong, Takaesu Giichi, Yoshimura Akihiko, McBride William H., Jewett Anahid, Jamieson Christina A M, Cacalano Nicholas A.

Cellular Signalling 20 （ 12 ） 2221 - 2230 2008年12月

ISSN 0898-6568

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.

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Selective inactivation of Socs3 in SF1 neurons improves glucose homeostasis without affecting body weight

Zhang Ren, Dhillon Harveen, Yin Huali, Yoshimura Akihiko, Lowell Bradford B., Maratos-Flier Eleftheria, Flier Jeffrey S.

Endocrinology 149 （ 11 ） 5654 - 5661 2008年11月

ISSN 0013-7227

Suppressor of cytokine signaling 3 (Socs3) has been identified as a mediator of central leptin resistance, but the identity of specific neurons in which Socs3 acts to suppress leptin signaling remains elusive. The ventromedial hypothalamus (VMH) was recently shown to be an important site for leptin action because deleting leptin receptor within VMH neurons causes obesity. To examine the role of VMH Socs3 in leptin resistance and energy homeostasis, we generated mice lacking Socs3 specifically in neurons positive for steroidogenic factor 1 (SF1), which is expressed abundantly in the VMH. These mice had increased phosphorylation of signal transducer and activator of transcription-3 in VMH neurons, suggesting improved leptin signaling, and consistently, food intake and weight-reducing effects of exogenous leptin were enhanced. Furthermore, on either chow or high-fat diets, these mice had reduced food intake. Unexpectedly, energy expenditure was reduced as well. Mice lacking Socs3 in SF1 neurons, despite no change in body weight, had improved glucose homeostasis and were partially protected from hyperglycemia and hyperinsulinemia induced by high-fat diets. These results suggest that Socs3 in SF1 neurons negatively regulates leptin signaling and plays important roles in mediating leptin sensitivity, glucose homeostasis, and energy expenditure. Copyright © 2008 by The Endocrine Society.

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Amelioration of human lupus-like phenotypes in MRL/lpr mice by overexpression of interleukin 27 receptor α(WSX-1)

Sugiyama N., Nakashima H., Yoshimura T., Sadanaga A., Shimizu S., Masutani K., Igawa T., Akahoshi M., Miyake K., Takeda A., Yoshimura A., Hamano S., Yoshida H.

Annals of the Rheumatic Diseases 67 （ 10 ） 1461 - 1467 2008年10月

ISSN 0003-4967

Objective: In the present work, we investigate the role of interleukin (IL)27/IL27 receptor α (Rα) (WSX-1) in the development of autoimmune disorders in the MRL/lpr mouse, which is considered as an experimental model of systemic lupus erythaematosus (SLE) in humans. Methods: We generated two strains of WSX-1 transgenic mice in the MRL/lpr background with different expression levels of WSX-1, and investigated the effect of WSX-1 overexpression on survival, glomerulonephritis and immunological properties. Results: In comparison with wild type (WT) MRL/lpr and transgenic (Tg) low (TgL) mice, Tg high (TgH) mice exhibited a prolonged lifespan and no apparent development of autoimmune nephritis. Production of anti-dsDNA antibody and total IgG and lgG2a were significantly lower in TgH mice than those of TgL and WT mice. The expressed amounts of interferon (IFN)y and IL4 mRNA by CD4⁺ T cells from Tg mice decreased in a dose-dependent fashion. CD4⁺ splenic lymphocytes in TgH mice were more subject to the IL27-mediated suppression of cytokine production. In vitro stimulation of CD4⁺ T cells by IL27 resulted in over phosphorylation of STAT3 in TgH cells than in WT cells. Conclusion: WSX-1 overexpression in the MRL/lpr background rendered the autoimmune prone mice protected from the development of autoimmune diseases. Our results suggest that IL27 signalling may be a therapeutic target against autoimmune diseases, including human SLE.

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Roles of STAT3/SOCS3 pathway in regulating the visual function and ubiquitin-proteasome-dependent degradation of rhodopsin during retinal inflammation

Ozawa Yoko, Nakao Keiko, Kurihara Toshihide, Shimazaki Takuya, Shimmura Shigeto, Ishida Susumu, Yoshimura Akihiko, Tsubota Kazuo, Okano Hideyuki

Journal of Biological Chemistry 283 （ 36 ） 24561 - 24570 2008年09月

ISSN 0021-9258

Inflammatory cytokines cause tissue dysfunction. We previously reported that retinal inflammation down-regulates rhodopsin expression and impairs visual function by an unknown mechanism. Here, we demonstrate that rhodopsin levels were preserved by suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of STAT3 activation. SOCS3 was expressed mainly in photoreceptor cells in the retina. In the SOCS3-deficient retinas, rhodopsin protein levels dropped sooner, and the reduction was more profound than in the wild type. Visual dysfunction, measured by electroretinogram, was prolonged in retina-specific SOCS3 conditional knockout mice. Visual dysfunction and decreased rhodopsin levels both correlated with increased STAT3 activation enhanced by SOCS3 deficiency. Interleukin 6, one of the inflammatory cytokines found during retinal inflammation, activated STAT3 and decreased rhodopsin protein in adult retinal explants. This was enhanced by inhibiting SOCS3 function in vitro, indicating that rhodopsin reduction was not a secondary effect in the mutant mice. Interestingly, in the inflamed SOCS3-deficient adult retina, rhodopsin decreased post-transcriptionally at least partly through ubiquitin-proteasome-dependent degradation accelerated by STAT3 activation and not transcriptionally as in the developing retina, on which we reported previously. A STAT3-dependent E3 ubiquitin ligase, Ubr1, was responsible for rhodopsin degradation and was up-regulated in the inflamed SOCS3-deficient retinas. These results indicate that in wild-type animals, a decrease in rhodopsin during inflammation is minimized by endogenous SOCS3. However, when STAT3 activation exceeds some threshold beyond the compensatory activity of endogenous SOCS3, rhodopsin levels decrease. These findings suggest SOCS3 as a potential therapeutic target molecule for protecting photoreceptor cell function during inflammation. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Enhanced c-Fms/M-CSF receptor signaling and wound-healing process in bone marrow-derived macrophages of signal-transducing adaptor protein-2 (STAP-2) deficient mice

Ikeda Osamu, Sekine Yuichi, Muromoto Ryuta, Ohbayashi Norihiko, Yoshimura Akihiko, Matsuda Tadashi

Biological and Pharmaceutical Bulletin 31 （ 9 ） 1790 - 1793 2008年09月

ISSN 0918-6158

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein as a c-Fms/M-CSF receptor-interacting protein and constitutively expressed in macrophages. In our previous study, we examined the role of STAP-2 in the c-Fms/M-CSF receptor signaling using a murine macrophage tumor cells line, Raw264.7. Overexpression of STAP-2 in Raw264.7 cells markedly suppressed M-CSF-induced activation of extracellular signal regulated kinase and Akt. In addition, Raw264.7 overexpressing STAP-2 affected cell migration in wound-healing process. These results suggest that STAP-2 deficiency influences endogenous c-Fms/M-CSF receptor signaling. Here we show that loss of STAP-2 expression in knockout mouse macrophages results in marked enhancement of the c-Fms/M-CSF receptor signaling and wound-healing process. We therefore propose that STAP-2 acts as an endogenous regulator in normal macrophages functions. © 2008 Pharmaceutical Society of Japan.

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Interaction and functional interference of glucocorticoid receptor and SOCS1

Haffner Michael C., Jurgeit Andreas, Berlato Chiara, Geley Stephan, Parajuli Nirmala, Yoshimura Akihiko, Doppler Wolfgang

Journal of Biological Chemistry 283 （ 32 ） 22089 - 22096 2008年08月

ISSN 0021-9258

Cytokine and glucocorticoid (GC) hormone signaling act in an integrated fashion to control inflammation and immune response. Here we establish a new mode of interaction of these two pathways and propose Suppressor of Cytokine Signaling (SOCS)-1 as an essential player in mediating cross-talk. We observed that glucocorticoid receptor (GR) and SOCS1 form an intracellular complex through an interaction, which required the SH2 domain of SOCS1 and the ligand binding domain of GR. Furthermore, GC stimulation was found to increase the nuclear level of SOCS1. SOCS1 binding to the GR did not require ligand binding of the receptor; however, it was abolished after long term GC stimulation, suggesting a functional role of the interaction for the early phase of GC action. The interaction between GR and SOCS1 appeared to negatively influence the transcription of the two GR-regulated genes, FKBP5 and MKP1, because the GC-dependent expression of these genes was inhibited by the SOCS1 inducer IFNγ and enhanced in SOCS1-deficient murine embryonic fibroblasts as compared with IFNγ treated wild-type cells. Our results suggest a prominent role of SOCS1 in the early phase of cross-talk between GR and cytokine signaling. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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STAP-2 negatively regulates both canonical and noncanonical NF-κB activation induced by Epstein-Barr virus-derived latent membrane protein

Ikeda Osamu, Sekine Yuichi, Yasui Teruhito, Oritani Kenji, Sugiyma Kenji, Muromoto Ryuta, Ohbayashi Norihiko, Yoshimura Akihiko, Matsuda Tadashi

Molecular and Cellular Biology 28 （ 16 ） 5027 - 5042 2008年08月

ISSN 0270-7306

The signal-transducing adaptor protein 2 (STAP-2) is a recently identified adaptor protein that contains a pleckstrin homology (PH) and Src homology 2 (SH2)-like domains, as well as a proline-rich domain in its C-terminal region. In previous studies, we demonstrated that STAP-2 binds to MyD88 and IKK-α or IKK-β and modulates NF-κB signaling in macrophages. In the present study, we found that ectopic expression of STAP-2 inhibited Epstein-Barr virus (EBV) LMP1-mediated NF-κB signaling and interleukin-6 expression. Indeed, STAP-2 associated with LMP1 through its PH and SH2-like domains, and these proteins interacted with each other in EBV-positive human B cells. We found, furthermore, that STAP-2 regulated LMP1-mediated NF-κB signaling through direct or indirect interactions with the tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) and TNFR-associated death domain (TRADD) proteins. STAP-2 mRNA was induced by the expression of LMP1 in human B cells. Furthermore, transient expression of STAP-2 in EBV-positive human B cells decreased cell growth. Finally, STAP-2 knockout mouse embryonic fibroblasts showed enhanced LMP1-induced cell growth. These results suggest that STAP-2 acts as an endogenous negative regulator of EBV LMP1-mediated signaling through TRAF3 and TRADD. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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SOCS3 negatively regulates the gp130-STAT3 pathway in mouse skin wound healing

Zhu Bing Mei, Ishida Yuko, Robinson Gertraud W., Pacher-Zavisin Margit, Yoshimura Akihiko, Murphy Philip M., Hennighausen Lothar

Journal of Investigative Dermatology 128 （ 7 ） 1821 - 1829 2008年07月

ISSN 0022-202X

Proliferation and differentiation of keratinocytes during wound healing are regulated by cytokines and chemokines, which are secreted by resident and inflammatory cells and activate the transcription factor signal transducer and activator of transcription (STAT)3. However, it is not clear to what extent STAT3 in keratinocytes is activated by gp130-containing receptors. We addressed this question genetically by deleting the suppressor of cytokine signaling (SOCS)3, a negative regulator of gp130-mediated STAT3 activation. Socs3 alleles flanked by loxP sites were deleted in mice with either an MMTV-Cre or K5-Cre transgene. While both transgenes are active in keratinocytes, the MMTV-Cre deletes floxed genes also in immune cells. Deletion of Socs3 using the MMTV-Cre transgene resulted in aberrant STAT3 activation, impaired wound healing, prolonged secretion of chemokines, a hyperproliferative epidermis, and neutrophil infiltration into wounds. Simultaneous deletion of the Socs3 and gp130 genes restored normal wound healing. Moreover, deletion of Socs3 only in keratinocytes caused impaired wound healing. These results demonstrate that wound healing is controlled in keratinocytes by the gp130-SOCS3-STAT3 pathway and an imbalance of this pathway results in delayed wound healing. © 2008 The Society for Investigative Dermatology.

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Foxp3 inhibits RORγt-mediated IL-17A mRNA transcription through direct interaction with RORγt

Ichiyama Kenji, Yoshida Hideyuki, Wakabayashi Yu, Chinen Takatoshi, Saeki Kazuko, Nakaya Mako, Takaesu Giichi, Hori Shohei, Yoshimura Akihiko, Kobayashi Takashi

Journal of Biological Chemistry 283 （ 25 ） 17003 - 17008 2008年06月

ISSN 0021-9258

The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-β1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-β1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor γt (RORγt), was rapidly induced by TGF-β1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Suppressor of cytokine signaling-1 ameliorates dextran sulfate sodium-induced colitis in mice

Horino Jiro, Fujimoto Minoru, Terabe Fumitaka, Serada Satoshi, Takahashi Tsuyoshi, Soma Yoshihito, Tanaka Kentaro, Chinen Takatoshi, Yoshimura Akihiko, Nomura Shintaro, Kawase Ichiro, Hayashi Norio, Kishimoto Tadamitsu, Naka Tetsuji

International Immunology 20 （ 6 ） 753 - 762 2008年06月

ISSN 0953-8178

Inflammatory bowel disease (IBD) is a chronic disorder of the gastrointestinal tract. Although the etiology and pathogenesis of IBD remain unknown, pro-inflammatory cytokines including IFN-γ play an important role in the development of IBD. Suppressor of cytokine signaling-1 (SOCS-1) is a crucial inhibitor of cytokine signaling, particularly of IFN-γ. In this study, we investigated the role of SOCS-1 in the development of murine dextran sulfate sodium (DSS)-induced colitis, a model of colitis resembling human IBD. SOCS-1 heterozygous (SOCS-1^+/-) and wild-type (WT) mice were given 3% DSS dissolved in drinking water for 5 days. Activation and expression of signal transducers and activators of transcription (STAT) in colonic tissues were assessed by western blot analysis. The expression of CD4, IFN-γ, IL-4, IL-17 and Forkhead box P3 (Foxp3) in colonic lamina propria lymphocytes was analyzed by flow cytometry and cytokine concentrations in serum were measured. DSS-treated SOCS^-1+/- mice developed more severe colitis than DSS-treated WT mice. Enhanced activation of STAT1, a higher ratio of CD4⁺ IFN-γ⁺T cells and a lower frequency of Foxp3⁺ regulatory T (Treg) cells, were observed in the colon of DSS-treated SOCS-1^+/- mice compared with DSS-treated WT mice. DSS-treated SOCS-1^+/- mice showed higher levels of IFN-γ in sera than did DSS-treated WT mice. Furthermore, T cell-specific SOCS-1-conditional knockout mice developed more severe colitis than control mice after DSS administration. Our findings suggest that SOCS-1, particularly in T cells, prevents the development of DSS-induced colitis in mice by inhibiting IFN-γ/STAT1 signaling and by subsequently regulating Treg cell development. © The Japanese Society for Immunology. 2008. All rights reserved.

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STAT6 inhibits TGF-β1-mediated Foxp3 induction through direct binding to the Foxp3 promoter, which is reverted by retinoic acid receptor

Takaki Hiromi, Ichiyama Kenji, Koga Keiko, Chinen Takatoshi, Takaesu Giichi, Sugiyama Yuki, Kato Shigeaki, Yoshimura Akihiko, Kobayashi Takashi

Journal of Biological Chemistry 283 （ 22 ） 14955 - 14962 2008年05月

ISSN 0021-9258

It has been shown that transforming growth factor β1 (TGF-β1) is critical in the generation of CD4⁺CD25⁺Foxp3 ⁺-inducible regulatory T cells (iTregs) from naïve CD4 ⁺T cells. However, in contrast to natural Tregs, TGF-β1-induced iTregs rapidly lose both Foxp3 expression and suppression activity. We found that TGF-β1-induced Foxp3 levels were maintained by the addition of the anti-interleukin 4 (IL-4) antibody or by STAT6 gene deletion. Thus, IL-4 is an important suppressor of Foxp3 induction, and T helper 2 development is a major cause for the disappearance of iTreg during long culture. Using promoter analysis in EL4 cells and primary T cells, we identified a silencer region containing a STAT6 binding site. STAT6 binding to this site reduced TGF-β1-mediated Foxp3 promoter activation and chromatin modification. Retinoic acid has also been shown to suppress loss of Foxp3 induced by TGF-β1. Retinoic acid in the presence of TGF-β1 reduced STAT6 binding to the Foxp3 promoter and enhanced histone acetylation, thereby reverting the effect of IL-4. We propose that antagonistic agents for neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and the promotion of tolerance in Th2-dominated diseases such as allergy. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Suppressor of cytokine signaling 1 protects mice against concanavalin A-induced hepatitis by inhibiting apoptosis

Torisu Takehiro, Nakaya Mako, Watanabe Satoko, Hashimoto Masayuki, Yoshida Hideyuki, Chinen Takatoshi, Yoshida Ryoko, Okamoto Fuyuki, Hanada Toshikatsu, Torisu Kumiko, Takaesu Giichi, Kobayashi Takashi, Yasukawa Hideo, Yoshimura Akihiko

Hepatology 47 （ 5 ） 1644 - 1654 2008年05月

ISSN 0270-9139

Acute liver failure is associated with significant mortality. However, the underlying pathophysiological mechanism is not yet fully understood. Suppressor of cytokine signaling-1 (SOCS1), which is a negative-feedback molecule for cytokine signaling, has been shown to be rapidly induced during liver injury. Here, using liver-specific SOCS1-conditional-knockout mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (ConA)-induced hepatitis, which has been shown to be dependent on activated T and natural killer T (NKT) cells. Although serum cytokine level and NKT cell activation were similar in wild-type (WT) and SOCS1-deficient mice after ConA treatment, proapoptotic signals, including signal transducers and activators of transcription 1 (STAT1) and Jun-terminal kinase (JNK) activation, were enhanced in SOCS1-deficient fivers compared with those in WT livers. SOCS1-deficient hepatocytes had higher expression of Fas antigen and were more sensitive to anti-Fas antibody-induced apoptosis than were WT hepatocytes. Furthermore, SOCS1-deficient hepatocytes were more sensitive to tumor necrosis factor (TNF)-α-induced JNK activation and apoptosis. These data indicate that SOCS1 is important to the prevention of hepatocyte apoptosis induced by Fas and TNF-α. In contrast, SOCS1 overexpression in the liver by adenoviral gene transfer prevented ConA-induced fiver injury. Conclusion: These findings indicate that SOCS1 plays important negative roles in fulminant hepatitis and that forced expression of SOCS1 is therapeutic in preventing hepatitis. Copyright © 2008 by the American Association for the Study of Liver Diseases.

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Loss of suppressor of cytokine signaling 1 in helper T cells leads to defective Th17 differentiation by enhancing antagonistic effects of IFN-γ on STAT3 and Smads

Tanaka Kentaro, Ichiyama Kenji, Hashimoto Masayuki, Yoshida Hideyuki, Takimoto Tomohito, Takaesu Giichi, Torisu Takehiro, Hanada Toshikatsu, Yasukawa Hideo, Fukuyama Satoru, Inoue Hiromasa, Nakanishi Yoichi, Kobayashi Takashi, Yoshimura Akihiko

Journal of Immunology 180 （ 6 ） 3746 - 3756 2008年03月

ISSN 0022-1767

Suppressor of cytokine signaling 1 (SOCS1) is an important negative regulator for cytokines; however, the role of SOCS1 in Th17 differentiation has not been clarified. We generated T cell-specific SOCS1-deficient mice and found that these mice were extremely resistant to a Th17-dependent autoimmune disease model, experimental autoimmune encephalomyelitis. SOCS1-deficient naive CD4 ⁺ T cells were predominantly differentiated into Th1 and poorly into Th17 in vitro. These phenotypes were canceled in IFN-γ^-/- background, suggesting that a large amount of IFN-γ in SOCS1-deficient T cells suppressed Th17 differentiation. IL-6 plus TGF-β enhanced retinoic acid receptor-related orphan receptor (ROR)-γt expression and suppressed IFN-γ production in wild-type T cells, whereas these effects were severely impaired in SOCS1-deficient T cells. These phenotypes can be partly explained by STAT3 suppression by enhanced SOCS3 induction through hyper-STAT1 activation in SOCS1-deficient T cells. In addition, SOCS1-deficient T cells were much less sensitive to TGF-β. Suppression of Th1 differentiation by TGF-β was impaired in SOCS1-deficient T cells. TGF-β-mediated Smad transcriptional activity was severely inhibited in SOCS1-deficient cells in the presence of IFN-γ. Such impairment of TGF-γ functions were not observed in SOCS3-overexpressed cells, indicating that suppression of Smads was independent of SOCS3. Therefore, SOCS1 is necessary for Th17 differentiation by suppressing antagonistic effect of IFN-γ on both STAT3 and Smads. Induction of SOCS3 can partly explain IFN-γ-mediated STAT3 suppression, while other mechanism(s) will be involved in IFN-β-mediated Smad suppression. SOCS1-deficient T cells will be very useful to investigate the molecular mechanism for the STAT1-mediated suppression of Th17 development. Copyright © 2008 by The American Association of Immunologists, Inc.

Differential roles for IFN-γ and IL-17 in experimental autoimmune uveoretinitis

Yoshimura Takeru, Sonoda Koh Hei, Miyazaki Yoshiyuki, Iwakura Yoichiro, Ishibashi Tatsuro, Yoshimura Akihiko, Yoshida Hiroki

International Immunology 20 （ 2 ） 209 - 214 2008年02月

ISSN 0953-8178

IL-17-producing CD4⁺ T cells, so called T_h17 cells, constitute a newly identified inflammatogenic cell population, which is critically involved in some inflammatory diseases. To explore the role of T_h17 cells in murine experimental autoimmune uveoretinitis (EAU), a model of human autoimmune uveitis where T_h1 responses predominantly participate in the pathogenesis, IL-17^- mice were immunized with interphotoreceptor retinoid-binding protein peptide 1-20 for disease induction. Funduscopic examination revealed that EAU was induced in IL-17^- mice just like in wild-type (WT) mice at early phases of the disease. However, at later/maintenance phases, the severity was significantly reduced in IL-17^- mice. Expression of IFN-γ and MCP-1 was comparable between WT and IL-17^- mice during the time course. In vivo blockade of IFN-γ and IL-4 resulted in exacerbation of EAU at later phases with augmented IL-17 production. Taken together, our data demonstrated that IL-17/T_h17 participates in the late phases of EAU and also that T_h1 and T_h17 responses are differentially required for EAU. © The Japanese Society for Immunology. 2007. All rights reserved.

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Peptidoglycan and lipopolysaccharide activate PLCγ2, leading to enhanced cytokine production in macrophages and dendritic cells

Aki Daisuke, Minoda Yasumasa, Yoshida Hideyuki, Watanabe Satoko, Yoshida Ryoko, Takaesu Giichi, Chinen Takatoshi, Inaba Toshiya, Hikida Masaki, Kurosaki Tomohiro, Saeki Kazuko, Yoshimura Akihiko

Genes to Cells 13 （ 2 ） 199 - 208 2008年02月

ISSN 1356-9597

In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/ threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cγ-2 (PLCγ2), leading to intracellular free Ca²⁺ mobilization in bone marrow-derived macrophages (BMMφ) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca²⁺ mobilization was not observed in BMDC from PLCγ2 knockout mice. Thus, PLCγ2 is essential for TLR2 and TLR4-mediated Ca²⁺ flux. In PLCγ2-knockdown cells, PGN-induced IκB-α phosphorylation and p38 activation were reduced. Moreover, PLCγ2 was necessary for the full production of TNF-α and IL-6. These data indicate that the PLCγ2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells. Journal compilation © 2008 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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SOCS1 is an inducible host factor during HIV-1 infection and regulates the intracellular trafficking and stability of HIV-1 Gag

Ryo Akihide, Tsurutani Naomi, Ohba Kenji, Kimura Ryuichiro, Komano Jun, Nishi Mayuko, Soeda Hiromi, Hattori Shinichiro, Perrem Kilian, Yamamoto Mikio, Chiba Joe, Mimaya Jun Ichi, Yoshimura Kazuhisa, Matsushita Shuzo, Honda Mitsuo, Yoshimura Akihiko, Sawasaki Tatsuya, Aoki Ichiro, Morikawa Yuko, Yamamoto Naoki

Proceedings of the National Academy of Sciences of the United States of America 105 （ 1 ） 294 - 299 2008年01月

ISSN 0027-8424

Human immunodeficiency virus type 1 (HIV-1) utilizes the macromolecular machinery of the infected host cell to produce progeny virus. The discovery of cellular factors that participate in HIV-1 replication pathways has provided further insight into the molecular basis of virus-host cell interactions. Here, we report that the suppressor of cytokine signaling 1 (SOCS1) is an inducible host factor during HIV-1 infection and regulates the late stages of the HIV-1 replication pathway. SOCS1 can directly bind to the matrix and nucleocapsid regions of the HIV-1 p55 Gag polyprotein and enhance its stability and trafficking, resulting in the efficient production of HIV-1 particles via an IFN signaling-independent mechanism. The depletion of SOCS1 by siRNA reduces both the targeted trafficking and assembly of HIV-1 Gag, resulting in its accumulation as perinuclear solid aggregates that are eventually subjected to lysosomal degradation. These results together indicate that SOCS1 is a crucial host factor that regulates the intracellular dynamism of HIV-1 Gag and could therefore be a potential new therapeutic target for AIDS and its related disorders. © 2008 by The National Academy of Sciences of the USA.

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HTLV-1 Tax-mediated TAK1 activation involves TAB2 adapter protein

Yu Qingsheng, Minoda Yasumasa, Yoshida Ryoko, Yoshida Hideyuki, Iha Hidekatsu, Kobayashi Takashi, Yoshimura Akihiko, Takaesu Giichi

Biochemical and Biophysical Research Communications 365 （ 1 ） 189 - 194 2008年01月

ISSN 0006-291X

Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-κB activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-κB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax. © 2007 Elsevier Inc. All rights reserved.

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Prostaglandin E₂ is a major soluble factor produced by stromal cells for preventing inflammatory cytokine production from dendritic cells

Shiraishi Hiroshi, Yoshida Hideyuki, Saeki Kazuko, Miura Yoshiki, Watanabe Satoko, Ishizaki Takuma, Hashimoto Masayuki, Takaesu Giichi, Kobayashi Takashi, Yoshimura Akihiko

International Immunology 20 （ 9 ） 1219 - 1229 2008年

ISSN 0953-8178

Dendritic cells (DCs) are specialized antigen-presenting cells that play pivotal roles in initiating immune responses. However, DC maturation is usually strongly restricted by the stromal microenvironment, especially in non-lymphoid tissues, such as skin and mucosa. Although suppression of DC maturation by stromal cells has been well documented, the molecular basis of this suppression has not been established. In this study, we examined the role of fibroblasts for DC maturation in vitro. The mouse embryonic fibroblasts (MEFs) strongly suppressed LPS-induced DC maturation. Although suppression of class II MHC and CD40 required DC-MEF contact, soluble factors in the culture supernatant of MEFs were sufficient for the suppression of IL-12 and tumor necrosis factor-α production. Using molecular-size selection and HPLC, we determined that prostaglandin E₂ (PGE₂) is a major soluble inhibitory factor secreted by MEFs. This was confirmed by the fact that cyclooxygenase inhibitors inhibited the production of the suppressive factor by MEFs. These results suggest that PGE₂ is a major soluble factor produced by MEFs for the suppression of inflammatory cytokine production from DCs, while a contact mechanism between MEFs and DCs is required for the suppression to induce T cell-stimulating molecules. © The Japanese Society for Immunology. 2008. All rights reserved.

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SOCS3 Protein Developmentally Regulates the Chemokine Receptor CXCR4-FAK Signaling Pathway during B Lymphopoiesis

Le Yi, Zhu Bing Mei, Harley Brendan, Park Shin Young, Kobayashi Takashi, Manis John P., Luo Hongbo R., Yoshimura Akihiko, Hennighausen Lothar, Silberstein Leslie E.

Immunity 27 （ 5 ） 811 - 823 2007年11月

ISSN 1074-7613

The chemokine CXCL12 induces prolonged focal adhesion kinase (FAK) phosphorylation and sustained proadhesive responses in progenitor bone-marrow (BM) B cells, but not in mature peripheral B cells. Here we demonstrate that suppressor of cytokine signaling 3 (SOCS3) regulated CXCL12-induced FAK phosphorylation through the ubiquitin-proteasome pathway. CXCL12 triggered increased FAK ubiquitination in mature B cells, but not in progenitor B cells. Accordingly, SOCS3 expression was low in progenitor B cells, increased in immature B cells, and highest in mature B cells. SOCS3 overexpression in pro-B cells impaired CXCL12-induced FAK phosphorylation and proadhesive responses. Conversely, SOCS3-deficient mature B cells from Cre^MMTVSocs3^fl/fl mice exhibited prolonged FAK phosphorylation and adhesion to VCAM-1. In contrast to wild-type mice, Cre^MMTVSocs3^fl/fl mice had a 2-fold increase in immature B cells, which were evenly distributed in endosteal and perisinusoidal BM compartments. We propose that the developmental regulation of CXCR4-FAK signaling by SOCS3 is an important mechanism to control the lodgement of B cell precursors in the BM microenvironment. © 2007 Elsevier Inc. All rights reserved.

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Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype

Brems Hilde, Chmara Magdalena, Sahbatou Mourad, Denayer Ellen, Taniguchi Koji, Kato Reiko, Somers Riet, Messiaen Ludwine, De Schepper Sofie, Fryns Jean Pierre, Cools Jan, Marynen Peter, Thomas Gilles, Yoshimura Akihiko, Legius Eric

Nature Genetics 39 （ 9 ） 1120 - 1126 2007年09月

ISSN 1061-4036

We report germline loss-of-function mutations in SPRED1 in a newly identified autosomal dominant human disorder. SPRED1 is a member of the SPROUTY/SPRED family of proteins that act as negative regulators of RAS->RAF interaction and mitogen-activated protein kinase (MAPK) signaling. The clinical features of the reported disorder resemble those of neurofibromatosis type 1 and consist of multiple café-au-lait spots, axillary freckling and macrocephaly. Melanocytes from a café-au-lait spot showed, in addition to the germline SPRED1 mutation, an acquired somatic mutation in the wild-type SPRED1 allele, indicating that complete SPRED1 inactivation is needed to generate a café-au-lait spot in this syndrome. This disorder is yet another member of the recently characterized group of phenotypically overlapping syndromes caused by mutations in the genes encoding key components of the RAS-MAPK pathway. To our knowledge, this is the first report of mutations in the SPRY (SPROUTY)/SPRED family of genes in human disease. © 2007 Nature Publishing Group.

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Signal-transducing adaptor protein-2 regulates integrin-mediated T cell adhesion through protein degradation of focal adhesion kinase

Sekine Yuichi, Tsuji Satoshi, Ikeda Osamu, Sugiyma Kenji, Oritani Kenji, Shimoda Kazuya, Muromoto Ryuta, Ohbayashi Norihiko, Yoshimura Akihiko, Matsuda Tadashi

Journal of Immunology 179 （ 4 ） 2397 - 2407 2007年08月

ISSN 0022-1767

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin homology- and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, we find that STAP-2-deficient splenocytes or T cells exhibit enhanced cell adhesion to fibronectin after PMA treatment, and that STAP-2-deficient T cells contain the increased protein contents of focal adhesion kinase (FAK). Furthermore, overexpression of STAP-2 induces a dramatic decrease in the protein contents of FAK and integrin-mediated T cell adhesion to fibronectin in Jurkat T cells via the degradation of FAK. Regarding the mechanism for this effect, we found that STAP-2 associates with FAK and enhances its degradation, proteasome inhibitors block FAK degradation, and STAP-2 recruits an endogenous E3 ubiquitin ligase, Cbl, to FAK. These results reveal a novel regulation mechanism for integrin-mediated signaling in T cells via STAP-2, which directly interacts with and degrades FAK. Copyright © 2007 by The American Association of Immunologists, Inc.

Selective expansion of Foxp3-positive regulatory T cells and immunosuppression by suppressors of cytokine signaling 3-deficient dendritic cells

Matsumura Yumiko, Kobayashi Takashi, Ichiyama Kenji, Yoshida Ryoko, Hashimoto Masayuki, Takimoto Tomohito, Tanaka Kentaro, Chinen Takatoshi, Shichita Takashi, Wyss-Coray Tony, Sato Katsuaki, Yoshimura Akihiko

Journal of Immunology 179 （ 4 ） 2170 - 2179 2007年08月

ISSN 0022-1767

Dendritic cells (DCs) induce immunity and immunological tolerance as APCs. It has been shown that DCs secreting IL-10 induce IL-10⁺ Tr1-type regulatory T (Treg) cells, whereas Foxp3-positive Treg cells are expanded from naive CD4⁺ T cells by coculturing with mature DCs. However, the regulatory mechanism of expansion of Foxp3⁺ Treg cells by DCs has not been clarified. In this study, we demonstrated that suppressors of cytokine signaling (SOCS)-3-deficient DCs have a strong potential as Foxp3⁺ T cell-inducing tolerogenic DCs. SOCS3^-/- DCs expressed lower levels of class II MHC, CD40, CD86, and IL-12 than wild-type (WT)-DCs both in vitro and in vivo, and showed constitutive activation of STAT3. Foxp3^- effector T cells were predominantly expanded by the priming with WT-DCs, whereas Foxp3⁺ Treg cells were selectively expanded by SOCS3^-/- DCs. Adoptive transfer of SOCS3^-/- DCs reduced the severity of experimental autoimmune encephalomyelitis. Foxp3⁺ T cell expansion was blocked by anti-TGF-β Ab, and SOCS3^-/- DCs produced higher levels of TGF-β than WT-DCs, suggesting that TGF-β plays an essential role in the expansion of Foxp3⁺ Treg cells. These results indicate an important role of SOCS3 in determining on immunity or tolerance by DCs. Copyright © 2007 by The American Association of Immunologists, Inc.

Suppression of SOCS3 expression in the pancreatic β-cell leads to resistance to type 1 diabetes

Mori Hiroyuki, Shichita Takashi, Yu Qingsheng, Yoshida Ryoko, Hashimoto Masayuki, Okamoto Fuyuki, Torisu Takahiro, Nakaya Mako, Kobayashi Takashi, Takaesu Giichi, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 359 （ 4 ） 952 - 958 2007年08月

ISSN 0006-291X

Type 1 diabetes results from the selective destruction of insulin-producing pancreatic β-cells during islet inflammation, which involves inflammatory cytokines and free radicals. However, mechanisms for protecting β-cells from destruction have not been clarified. In this study, we define the role of SOCS3 on β-cell destruction using β-cell-specific SOCS3-conditional knockout (cKO) mice. The β-cell-specific SOCS3-deficient mice were resistant to the development of diabetes caused by streptozotocin (STZ), a genotoxic methylating agent, which has been used to trigger β-cell destruction. The islets from cKO mice demonstrated hyperactivation of STAT3 and higher induction of Bcl-xL than did islets from WT mice, and SOCS3-deficient β-cells were more resistant to apoptosis induced by STZ in vitro than were WT β-cells. These results suggest that enhanced STAT3 signaling protects β-cells from destruction induced by a genotoxic stress and that STAT3/SOCS3 can be a potential therapeutic target for the treatment of type 1 diabetes. © 2007 Elsevier Inc. All rights reserved.

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Ifi202, an IFN-inducible candidate gene for lupus susceptibility in NZB/ W F1 mice, is a positive regulator for NF-κB activation in dendritic cells

Yamauchi Moriyasu, Hashimoto Masayuki, Ichiyama Kenji, Yoshida Ryoko, Hanada Toshikatsu, Muta Tatsushi, Komune Shizuo, Kobayashi Takashi, Yoshimura Akihiko

International Immunology 19 （ 8 ） 935 - 942 2007年08月

ISSN 0953-8178

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies and lupus nephritis. The [New Zealand black (NZB) × New Zealand white (NZW)]F1 (BWF1) mouse has been recognized as an important animal model of human SLE. The T_h1-prone phenotype of BWF1 mice has been shown to contribute to the development of the lupus. However, the molecular basis for T_h1 skewing in BWF1 mice has not been clarified. We noticed that IL-6, IL-12 and other proinflammatory cytokines as well as IκB-ζ induction were higher in mature bone marrow-derived dendritic cells (BMDCs) from NZB and BWF1 mice than those from NZW mice. The expression of an IFN-inducible gene Ifi202, a candidate gene for lupus, was almost undetectable in NZW BMDCs. Thus, we hypothesized that Ifi202 is involved in elevated IL-12 production from BWF1 BMDCs. Overexpression of Ifi202 enhanced the LPS-induced IκB-ζ, IL-12p40 and NF-κB promoter activities, while anti-sense (AS) RNA against Ifi202 strongly suppressed them in a monocytic cell line, RAW 264.7. Furthermore, overexpression of Ifi202 enhanced LPS-induced IL-12p40 and IκB-ζ mRNA induction while Ifi202 AS RNA suppressed these in RAW 264.7 cells. In addition, forced expression of Ifi202 enhanced IL-12p40 mRNA induction in NZW BMDCs. Thus, Ifi202 is an important NF-κB activator in DCs and involved in IL-12 production, which may account for a T_h1-prone phenotype of BWF1 mice. © The Japanese Society for Immunology. 2007. All rights reserved.

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STAP-2 regulates c-Fms/M-CSF receptor signaling in murine macrophage Raw 264.7 cells

Ikeda Osamu, Sekine Yuichi, Kakisaka Michinori, Tsuji Satoshi, Muromoto Ryuta, Ohbayashi Norihiko, Oritani Kenji, Yoshimura Akihiko, Matsuda Tadashi

Biochemical and Biophysical Research Communications 358 （ 3 ） 931 - 937 2007年07月

ISSN 0006-291X

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein as a c-Fms/M-CSF receptor-interacting protein and constitutively expressed in macrophages. Our previous studies also revealed that STAP-2 binds to MyD88 and IKK-α/β, and modulates NF-κB signaling in macrophages. In the present study, we examined physiological roles of the interaction between STAP-2 and c-Fms in Raw 264.7 macrophage cells. Our immunoprecipitation has revealed that c-Fms directly interacts with the PH domain of STAP-2 independently on M-CSF-stimulation. Ectopic expression of STAP-2 markedly suppressed M-CSF-induced tyrosine phosphorylation of c-Fms as well as activation of Akt and extracellular signal regulated kinase. In addition, Raw 264.7 cells over-expressing STAP-2 showed impaired migration in response to M-CSF and wound-healing process. Taken together, our findings demonstrate that STAP-2 directly binds to c-Fms and interferes with the PI3K signaling, which leads to macrophage motility, in Raw 264.7 cells. © 2007 Elsevier Inc. All rights reserved.

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Potentiation of astrogliogenesis by STAT3-mediated activation of bone morphogenetic protein-Smad signaling in neural stem cells

Fukuda Shinji, Abematsu Masahiko, Mori Hiroyuki, Yanagisawa Makoto, Kagawa Tetsushi, Nakashima Kinichi, Yoshimura Akihiko, Taga Tetsuya

Molecular and Cellular Biology 27 （ 13 ） 4931 - 4937 2007年07月

ISSN 0270-7306

Astrocytes play important roles in brain development and injury response. Transcription factors STAT3 and Smadl, activated by leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2), respectively, form a complex with the coactivator p300 to synergistically induce astrocytes from neuroepithelial cells (NECs) (K. Nakashima, M. Yanagisawa, H. Arakawa, N. Kimura, T. Hisatsune, M. Kawabata, K. Miyazono, and T. Taga, Science 284:479-482, 1999). However, the mechanisms that govern astrogliogenesis during the determination of the fate of neural stem cells remain elusive. Here we found that LIF induces expression of BMP2 via STAT3 activation and leads to the consequent activation of Smad1 to efficiently promote astrogliogenic differentiation of NECs. The BMP antagonist Noggin abrogated LIF-induced Smadl activation and astrogliogenesis by inhibiting BMPs produced by NECs. NECs deficient in suppressor of cytokine signaling 3 (SOCS3), a negative regulator of STAT3, readily differentiated into astrocytes upon activation by LIF not only due to sustained activation of STAT3 but also because of the consequent activation of Smadl. Our study suggests a novel LIF-triggered positive regulatory loop that enhances astrogliogenesis. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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Intracellular negative signals and allergy

Yoshimura Akihiko, Tanaka Kentaro, Okamoto Fuyuki, Kobayashi Takashi

Arerugī = [Allergy] 56 （ 6 ） 563 - 569 2007年06月

ISSN 0021-4884

SOCS proteins, cytokine signalling and immune regulation

Yoshimura Akihiko, Naka Tetsuji, Kubo Masato

Nature Reviews Immunology 7 （ 6 ） 454 - 465 2007年06月

ISSN 1474-1733

Suppressor of cytokine signalling (SOCS) proteins are inhibitors of cytokine signalling pathways. Studies have shown that SOCS proteins are key physiological regulators of both innate and adaptive immunity. These molecules positively and negatively regulate macrophage and dendritic-cell activation and are essential for T-cell development and differentiation. Evidence is also emerging of the involvement of SOCS proteins in diseases of the immune system. In this Review we bring together data from recent studies on SOCS proteins and their role in immunity, and propose a cohesive model of how cytokine signalling regulates immune-cell function. © 2007 Nature Publishing Group.

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Spreds are essential for embryonic lymphangiogenesis by regulating vascular endothelial growth factor receptor 3 signaling

Taniguchi Koji, Kohno Ri Ichiro, Ayada Toranoshin, Kato Reiko, Ichiyama Kenji, Morisada Tohru, Oike Yuichi, Yonemitsu Yoshikazu, Maehara Yoshihiko, Yoshimura Akihiko

Molecular and Cellular Biology 27 （ 12 ） 4541 - 4550 2007年06月

ISSN 0270-7306

Spred/Sprouty family proteins negatively regulate growth factor-induced ERK activation. Although the individual physiological roles of Spred-1 and Spred-2 have been investigated using gene-disrupted mice, the overlapping functions of Spred-1 and Spred-2 have not been clarified. Here, we demonstrate that the deletion of both Spred-1 and Spred-2 resulted in embryonic lethality at embryonic days 12.5 to 15.5 with marked subcutaneous hemorrhage, edema, and dilated lymphatic vessels filled with erythrocytes. This phenotype resembled that of Syk^-/- and SLP-76^-/- mice with defects in the separation of lymphatic vessels from blood vessels. The number of LYVE-1-positive lymphatic vessels and lymphatic endothelial cells increased markedly in Spred-1/2-deficient embryos compared with WT embryos, while the number of blood vessels was not different. Ex vivo colony assay revealed that Spred-1/2 suppressed lymphatic endothelial cell proliferation and/or differentiation. In cultured cells, the overexpression of Spred-1 or Spred-2 strongly suppressed vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3-mediated ERK activation, while Spred-1/2-deficient cells were extremely sensitive to VEGFR-3 signaling. These data suggest that Spreds play an important role in lymphatic vessel development by negatively regulating VEGF-C/VEGFR-3 signaling. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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Robustness of the signal transduction system of the mammalian JAK/STAT pathway and dimerization steps

Shudo Emi, Yang Jin, Yoshimura Akihiko, Iwasa Yoh

Journal of Theoretical Biology 246 （ 1 ） 1 - 9 2007年05月

ISSN 0022-5193

In the interferon-γ (IFNγ)-activated Janus Kinase (JAK)/signal transducer and activator of transcription 1 (STAT1) pathway, multiple steps of STAT1 dimerization are required prior to gene expression that produce antiviral molecules. By interpreting experimental results, an existing mathematical model suggested that only phosphorylated STAT1 dimers could translocate to the nucleus and activate gene transcription. In this paper, we examine the role of STAT1 dimerization steps by studying the dynamic behaviors of four alternative models. By analyzing several system properties at low input IFNγ signal including the steady-state antiviral molecule production, to the input, the delay of responses triggered by input, and the parameter sensitivity, we found that the mice JAK/STAT1 system identified by experiments (1) suppresses antiviral molecule production at low input signal, (2) has slow kinetics of antiviral molecule production and (3) has low parameter sensitivity of antiviral molecule production at steady state. We conclude that the observed structure of the JAK/STAT1 pathway is responsible for the robust system behavior. © 2007 Elsevier Ltd. All rights reserved.

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Leukemia inhibitory factor-induced phosphorylation of STAP-2 on tyrosine-250 is involved in its STAT3-enhancing activity

Sekine Yuichi, Tsuji Satoshi, Ikeda Osamu, Kakisaka Michinori, Sugiyama Kenji, Yoshimura Akihiko, Matsuda Tadashi

Biochemical and Biophysical Research Communications 356 （ 2 ） 517 - 522 2007年05月

ISSN 0006-291X

Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 binds to signal transducer and activator of transcription 3 (STAT3) and STAT5, and regulates their signaling pathways. In the present study, we identified tyrosine-250 (Tyr250) in STAP-2 as a major site of phosphorylation by v-src and Jak2, using a phospho-specific antibody against STAP-2 phosphorylated at Tyr250. Mutational analyses revealed that Tyr250 was involved in the STAT3-enhancing activity of STAP-2. We further found that leukemia inhibitory factor (LIF) stimulated STAP-2 Tyr250 phosphorylation in 293T and Hep3B cells. Moreover, endogenous STAP-2 was phosphorylated at Tyr250 following LIF stimulation of murine M1 cell line. Taken together, our findings demonstrate that endogenous STAP-2 is phosphorylated at Tyr250 and that this phosphorylation is involved in its function. © 2007 Elsevier Inc. All rights reserved.

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An RNA-binding protein αCP-1 is involved in the STAT3-mediated suppression of NF-κB transcriptional activity

Nishinakamura Hitomi, Minoda Yasumasa, Saeki Kazuko, Koga Keiko, Takaesu Giichi, Onodera Masafumi, Yoshimura Akihiko, Kobayashi Takashi

International Immunology 19 （ 5 ） 609 - 619 2007年05月

ISSN 0953-8178

Signal transducer and activator of transcription 3 (STAT3) has been shown to mediate the anti-inflammatory effect of IL-10. Activated STAT3 suppresses LPS-induced IL-6, tumor necrosis factor-α and IL-12 gene expression in macrophages and dendritic cells. However, the mechanism of Toll-like receptor (TLR) signal suppression by STAT3 has not been clarified. In this study, we investigated the effect of constitutively activated STAT3 (STAT3C) on LPS-induced nuclear factor-κB (NF-κB) activation. The forced expression of STAT3C in HEK293/TLR4 cells, but neither wild-type STAT3 nor dominant-negative form of STAT3, suppressed LPS-TLR4-mediated NF-κB reporter activation. The over-expression of STAT3C did not affect the signal transduction of TLR4, such as the phosphorylation of inhibitory nuclear factor-κBα and mitogen-activated protein kinases and the DNA-binding activity of NF-κB. Thus, STAT3C could suppress the transcriptional and/or translational activity of NF-κB. To define the molecular mechanism, we searched STAT3C-binding proteins by using a proteomic approach and found that a novel RNA-binding protein, αCP-1, interacted with STAT3C. αCP-1 is a K-homology domain-containing RNA-binding protein with specificity for C-rich pyrimidine tracts. Such proteins play pivotal roles in a broad-spectrum of transcriptional and translational events. The over-expression of αCP-1 augmented the suppressive effect of STAT3C on NF-κB activation in HEK293/TLR4 cells. Furthermore, the forced expression of αCP-1 enhanced the antagonistic effect of IL-10 on IL-6 production in RAW264.7 cells, while small interfering RNA against αCP-1 reduced it. These data suggest that αCP-1 is involved in the STAT3-mediated suppression of NF-κB activity. © The Japanese Society for Immunology. 2007. All rights reserved.

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Adaptor protein SH2-B linking receptor-tyrosine kinase and Akt promotes adipocyte differentiation by regulating peroxisome proliferator-activated receptor γ messenger ribonucleic acid levels

Yoshiga Daigo, Sato Naoichi, Torisu Takehiro, Mori Hiroyuki, Yoshida Ryoko, Nakamura Seiji, Takaesu Giichi, Kobayashi Takashi, Yoshimura Akihiko

Molecular Endocrinology 21 （ 5 ） 1120 - 1131 2007年05月

ISSN 0888-8809

Adipocyte differentiation is regulated by insulin and IGF-I, which transmit signals by activating their receptor tyrosine kinase. SH2-B is an adaptor protein containing pleckstrin homology and Src homology 2 (SH2) domains that have been implicated in insulin and IGF-I receptor signaling. In this study, we found a strong link between SH2-B levels and adipogenesis. The fat mass and expression of adipogenic genes including peroxisome proliferator-activated receptor γ (PPARγ) were reduced in white adipose tissue of SH2-B^-/- mice. Reduced adipocyte differentiation of SH2-B-deficient mouse embryonic fibroblasts (MEFs) was observed in response to insulin and dexamethasone, whereas retroviral SH2-B overexpression enhanced differentiation of 3T3-L1 preadipocytes to adipocytes. SH2-B overexpression enhanced mRNA level of PPARγ in 3T3-L1 cells, whereas PPARγ levels were reduced in SH2-B-deficient MEFs in response to insulin. SH2-B-mediated up-regulation of PPARγ mRNA was blocked by a phosphatidylinositol 3-kinase inhibitor, but not by a MAPK kinase inhibitor. Insulin-induced Akt activation and the phosphorylation of forkhead transcription factor (FKHR/Foxo1), a negative regulator of PPARγ transcription, were up-regulated by SH2-B overexpression, but reduced in SH2-B-deficeint MEFs. These data indicate that SH2-B is a key regulator of adipogenesis both in vivo and in vitro by regulating the insulin/IGF-I receptor-Akt-Foxo1-PPARγ pathway. Copyright © 2007 by The Endocrine Society.

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Reciprocal regulation of SOCS1 and SOCS3 enhances resistance to ionizing radiation in glioblastoma multiforme

Zhou Hong, Miki Rika, Eeva Mervi, Fike Francesca M., Seligson David, Yang Lu, Yoshimura Akihiko, Teitell Michael A., Jamieson Christina A M, Cacalano Nicholas A.

Clinical Cancer Research 13 （ 8 ） 2344 - 2353 2007年04月

ISSN 1078-0432

Purpose: The expression of suppressors of cytokine signaling 1 (SOCS1) and SOCS3 genes is dysregulated in several solid tumors, causing aberrant activation of cell growth and survival signaling pathways. In this study, we analyzed SOCS1 and SOCS3 gene expression in glioblastoma multiforme (GBM) and studied the role of each protein in GBM cell signaling and radiation resistance. Experimental Design: SOCS1 and SOCS3 gene expression was analyzed in 10 GBM cell lines by reverse transcription-PCR and Western blotting. SOCS3 expression was also studied in 12 primary GBM tissues by immunohistochemistry. The methylation status of the SOCS1 and SOCS3 loci was determined by methylation-specific PCR. Extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) activation in GBM cell lines overexpressing SOCS1 or lacking SOCS3 was determined by phosphorylated-specific Western blotting. Radiation responses in SOCS1-positive and SOCS3-deficient GBM cell lines and fibroblasts from wild-type and SOCS1 or SOCS3 knockout mice were studied in a clonogenic survival assay. Results: All GBM cell lines tested lacked SOCS1 expression, whereas GBM cell lines and primary GBM tumor samples constitutively expressed SOCS3. SOCS1 gene repression was linked to hypermethylation of the SOCS1 genetic locus in GBM cells. Reintroduction of SOCS1 or blocking SOCS3 expression sensitized cells to radiation and decreased the levels of activated ERK MAPKs in GBM cells. Conclusions: SOCS1 and SOCS3 are aberrantly expressed in GBM cell lines and primary tissues. Altered SOCS gene expression leads to increased cell signaling through the ERK-MAPK pathway and may play a role in disease pathogenesis by enhancing GBM radioresistance. © 2007 American Association for Cancer Research.

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SOCS3 is required to temporally fine-tune photoreceptor cell differentiation

Ozawa Yoko, Nakao Keiko, Shimazaki Takuya, Shimmura Shigeto, Kurihara Toshihide, Ishida Susumu, Yoshimura Akihiko, Tsubota Kazuo, Okano Hideyuki

Developmental Biology 303 （ 2 ） 591 - 600 2007年03月

ISSN 0012-1606

Suppressor of cytokine signaling 3 (SOCS3) is an intracellular, ligand-induced negative feedback modulator of STAT3 activation that acts during inflammation. Here, we demonstrate that SOCS3 expression is important for normal retinal development in the perinatal period. STAT3 is highly activated in the late-embryonic retina, then downregulated at postnatal day 0 (P0), presumably by the depletion of upstream ligands. We found that SOCS3 was required after P0 to shut down the residual STAT3 activation; this loss of activated STAT3 leads to Rhodopsin expression and rod photoreceptor cell differentiation. SOCS3 deficiency failed to terminate STAT3 activation, thereby delaying expression of Rhodopsin and its upstream transcription factor, crx. Development subsequently continued, but its course was temporally erratic, probably because of faulty compensation. Interestingly, SOCS3 protein expression was first detected postnatally, after STAT3 activation was mostly downregulated. It initially appeared in some of the presumptive photoreceptor cells and gradually spread. SOCS3 mRNA level was constant from the late-embryonic to early-postnatal period. Post-transcriptional inhibition of SOCS3 protein expression maintains a high STAT3 activation during late embryogenesis, and after P0, releasing from the inhibition promptly terminates STAT3 activation. Thus, SOCS3 can act as a temporal fine-tuner of STAT3 activation during photoreceptor cell differentiation. © 2006 Elsevier Inc. All rights reserved.

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Socs 3 modulates the activity of the transcription factor Stat3 in mammary tissue and controls alveolar homeostasis

Robinson Gertraud W., Pacher-Zavisin Margit, Zhu Bing Mei, Yoshimura Akihiko, Hennighausen Lothar

Developmental Dynamics 236 （ 3 ） 654 - 661 2007年03月

ISSN 1058-8388

Signal transducer and activator of transcription 5 and 3 (Stat5 and Stat3) control pregnancy-mediated mammary development and involution-dependent remodeling, respectively. Suppressor of cytokine signaling 3 (Socs3) has been implicated in the modulation of both Stat3 and Stat5 activity. To explore the biology of Socs3 in mammary tissue, the gene was deleted using Cre-mediated recombination. Deletion of the Socs3 gene from mammary stem or early progenitor cells did not grossly alter pregnancy-mediated mammary development but resulted in impaired lactation due to attenuated proliferation. Loss of Socs3 from differentiated luminal cells did not interfere with glandular function during lactation, but resulted in accelerated tissue remodeling upon weaning. Loss of Socs3 led to enhanced and precocious Stat3 activation. Thus, Socs3 serves as a modulator of Stat3 activity to ensure controlled proliferation and apoptosis in pregnancy and involution, respectively.

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Corrigendum to "A novel zinc finger protein, ZCCHC11, interacts with TIFA and modulates TLR signaling" [Biochem. Biophys. Res. Commun. 344 (2006) 1023-1030] (DOI

Minoda Yasumasa, Saeki Kazuko, Aki Daisuke, Takaki Hiromi, Sanada Takahito, Koga Keiko, Kobayashi Takashi, Takaesu Giichi, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 353 （ 4 ） 1121 2007年02月

ISSN 0006-291X

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The dual function of hepatic SOCS3 in insulin resistance in vivo

Torisu Takehiro, Sato Naoichi, Yoshiga Daigo, Kobayashi Takashi, Yoshioka Tomoko, Mori Hiroyuki, Iida Mitsuo, Yoshimura Akihiko

Genes to Cells 12 （ 2 ） 143 - 154 2007年02月

ISSN 1356-9597

Inflammation associates with insulin resistance, which dysregulates nutrient homeostasis and leads to diabetes. The suppressor of cytokine signaling 3 (SOCS3), which is induced by pro-inflammatory cytokines, such as TNFα and IL-6, has been implicated in inflammation-mediated insulin resistance in the liver and adipocytes. However, no genetic evidence has been provided for the involvement of SOCS3 on insulin resistance. Here, we generated hepatocyte-specific SOCS3-deficient (L-SOCS3 cKO) mice and examined insulin sensitivity. Being consistent with a previous idea, the loss of SOCS3 in the liver apparently improved insulin sensitivity. However, unexpectedly, L-SOCS3 cKO mice exhibited obesity and systemic insulin resistance with age. Insulin signaling was rather suppressed in muscles, suggesting that deletion of the SOCS3 gene in the liver modulates insulin sensitivity in other organs. Anti-inflammatory reagent, sodium salicylate, partial improved insulin resistance of aged L-SOCS3 cKO mice, suggesting that enhanced inflammatory status is associated with the phenotype of these mice. STAT3 was hyperactivated and acute-phase proteins were elevated in L-SOCS3 cKO mice liver, which were reduced by sodium salicylate treatment. We conclude that hepatic SOCS3 is a mediator of insulin resistance in the liver; however, lack of SOCS3 in the liver promotes systemic insulin resistance by mimicking chronic inflammation. © 2007 The Authors Journal compilation © 2007 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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Sprouty2 and Sprouty4 are essential for embryonic morphogenesis and regulation of FGF signaling

Taniguchi Koji, Ayada Toranoshin, Ichiyama Kenji, Kohno Ri ichiro, Yonemitsu Yoshikazu, Minami Yasuhiro, Kikuchi Akira, Maehara Yoshihiko, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 352 （ 4 ） 896 - 902 2007年01月

ISSN 0006-291X

Sprouty genes encode cytoplasmic membrane-associated proteins that inhibit receptor tyrosine kinase signaling. Four orthologs of Drosophila Sprouty (dSpry) (Sprouty1-4) have been identified in mammals. Physiological function of Sprouty1 and Sprouty2 has been investigated using gene targeting approaches, however to date detailed examination of Sprouty4 knockout (KO) mice has not been reported. In this study, Sprouty4 KO mice were generated and characterized. Although a significant fraction of Sprouty4 KO mice died shortly after birth due to mandible defects, the remainder were viable and fertile. Growth retardation was observed for most Sprouty4-deficient mice, with nearly all Sprouty4 KO mice having polysyndactyly. ERK activation was sustained in Sprouty4 KO mouse embryonic fibroblasts (MEFs) in response to FGF, but not to EGF. Sprouty2 and Sprouty4 double KO (DKO) mice were embryonic lethal and showed severe defects in craniofacial, limb, and lung morphogenesis. These findings suggest both redundant and non-redundant functions for Sprouty2 and Sprouty4 on embryonic development and FGF signaling. © 2006 Elsevier Inc. All rights reserved.

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Role of endogenous inhibitors of cytokine signaling in allergic asthma

Inoue Hiromasa, Fukuyama Satoru, Matsumoto Koichiro, Kubo Masato, Yoshimura Akihiko

Current Medicinal Chemistry 14 （ 2 ） 181 - 189 2007年01月

ISSN 0929-8673

T helper 2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, play an important role in allergic immune disorders, such as bronchial asthma. These cytokines regulate diverse biological functions by binding to receptors at the cell surface to activate complex signal transduction pathways, including the Janus kinase-signal transducer and activator of transcription (JAK-STAT) and the Ras-extracellular signal-regulated kinase (ERK) signaling pathways. The suppressor of cytokine signaling (SOCS) family proteins has been shown to regulate the JAK-STAT pathway, and the Sprouty-related EVH1-domain-containing protein (SPRED) family proteins regulate the Ras-ERK pathway. SOCS3 and SOCS5 are predominantly expressed in Th2 and Th1 cells, respectively, and they reciprocally inhibit the Th1 and Th2 differentiation processes. SOCS3 also has a role in Th3 differentiation. SPRED-1 is expressed in hematopoietic cells, including eosinophils, and negatively controls the eosinophil numbers and functions by modulating IL-5 signaling. Here, we discuss the role of SOCS and SPRED proteins in allergic asthma and explore the potential of these proteins as targets for therapeutic strategies in allergic asthma. © 2007 Bentham Science Publishers Ltd.

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Positive and negative roles of IL-6, STAT3, and SOCS3 in inflammatory arthritis

Kinjyo Ichiko, Ohishi Masanobu, Shouda Takanori, Kobayashi Takashi, Yoshimura Akihiko

113 - 124 2007年

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Loss of SOCS3 gene expression converts STAT3 function from anti-apoptotic to pro-apoptotic

Lu Yang, Fukuyama Satoru, Yoshida Ryoko, Kobayashi Takashi, Saeki Kazuko, Shiraishi Hiroshi, Yoshimura Akihiko, Takaesu Giichi

Journal of Biological Chemistry 281 （ 48 ） 36683 - 36690 2006年12月

ISSN 0021-9258

The transcription factor STAT3 is activated by interleukin-6-related cytokines and has been implicated as an oncogene; it promotes cell proliferation and is anti-apoptotic. However, in some cases, STAT3 has been shown to be pro-apoptotic, especially in mammary epithelial cells. In this report, we generated SOCS3-deficient murine embryonic fibroblasts (MEFs), in which STAT3 activation is extremely enhanced and prolonged. We found that LIF induces caspase-3 activation and apoptosis of SOCS3^-/- MEFs. Exogenous expression of the dominant negative form of STAT3 but not STAT1 suppressed LIF-induced apoptosis of SOCS3^-/- MEFs, indicating that STAT3 plays a critical role in apoptosis induction. As shown in mammary gland epithelial cells, expression of the phosphatidylinositol 3-kinase regulatory subunits p50α and p55α was induced in response to LIF in SOCS3^-/- MEFs but not in wild-type MEFs, and Akt/protein kinase B activity was substantially reduced in SOCS3^-/- MEFs. Furthermore, we found that some of the STAT3 target genes related to apoptosis and proliferation, such as Bcl-2 and cyclin D1, were repressed upon LIF treatment in SOCS3^-/- cells. Not only the up-regulation of p50α and p55α but also the repression of cyclin D1 and Bcl-2 in SOCS3^-/- MEFs was inhibited by dominant negative STAT3. These data suggest that prolonged activation of STAT3 could induce apoptosis/growth arrest rather than anti-apoptosis and proliferation in certain cases, and SOCS3 is a critical regulator of this balance. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Innate defense mechanism against virus infection within the cardiac myocyte requiring gp130-STAT3 signaling

Yajima Toshitaka, Yasukawa Hideo, Jeon Eun Seok, Xiong Dingding, Dorner Andrea, Iwatate Mitsuo, Nara Miwako, Zhou Hanbing, Summers-Torres Daphne, Hoshijima Masahiko, Chien Kenneth R., Yoshimura Akihiko, Knowlton Kirk U.

Circulation 114 （ 22 ） 2364 - 2373 2006年11月

ISSN 0009-7322

BACKGROUND - Little is known about innate immune mechanisms within the cardiac myocyte that determine susceptibility to enterovirus infection, an important cause of myocarditis and subsequent heart failure. Although interferon (IFN) generally plays a key role in innate immunity, ablation of IFN receptors has little or no effect on acute coxsackievirus B3 infection in the heart. Interestingly, gp130-cytokine-mediated stimulation of neonatal ventricular myocytes has a cytoprotective effect against virus infection in culture that can be inhibited by suppressors of cytokine signaling (SOCS)-3, a physiological inhibitor of gp130 signaling that does not affect IFN signaling. Therefore, we hypothesized that inhibition of gp130 signaling by SOCS3 would change cardiac myocyte susceptibility to virus infection without affecting IFN signaling. METHODS AND RESULTS - We generated cardiac-specific SOCS3 transgenic mice. Despite an intact IFN-mediated antiviral response in adult transgenic myocytes, there was a marked increase in susceptibility to viral infection in the SOCS3 transgenic mouse hearts. This indicated the presence of IFN-independent innate defense mechanisms within the cardiac myocyte. Subsequently, we demonstrated that cardiac-specific gp130-knockout mice also had increased susceptibility to viral infection. Furthermore, we demonstrated that the gp130-mediated increase in survival of infected myocytes occurred through a signal transducers and activators of transcription-3-dependent mechanism that did not affect viral replication. This was accompanied by a persistent expression of full-length dystrophin after coxsackievirus B3 infection. In addition, we found that both SOCS3 transgenic and gp130-deficient mice had a decrease in α-sarcoglycan. CONCLUSIONS - SOCS3-mediated regulation of gp130 signaling can affect susceptibility to viral infection in the heart. Increased cardiac cell survival through gp130-signal transducers and activators of transcription-3 signaling appears to play an important role in preserving nondividing cardiac myocytes until specific immune responses begin to clear the virus. © 2006 American Heart Association, Inc.

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Two-sided roles of IL-27

Yoshimura Takeru, Takeda Atsunobu, Hamano Shinjiro, Miyazaki Yoshiyuki, Kinjyo Ichiko, Ishibashi Tatsuro, Yoshimura Akihiko, Yoshida Hiroki

Journal of Immunology 177 （ 8 ） 5377 - 5385 2006年10月

ISSN 0022-1767

Recent lines of evidence have demonstrated that IL-27, a newly identified IL-12-related cytokine, has two apparently conflicting roles in immune responses: one as an initiator of Th1 responses and the other as an attenuator of inflammatory cytokine production. Although the BL-27-mediated Th1 initiation mechanism has been elucidated, little is known about the molecular basis for the suppression of cytokine production. In the present study, we demonstrated that IL-27 suppressed the production of various proinflammatory cytokines by fully activated CD4⁺ T cells while it had no effect on the cytokine production by CD4⁺ T cells at early phases of activation. IL-27 also suppressed IL-17 production by activated CD4⁺ T cells, thereby counteracting IL-23, another IL-12-related cytokine with proinflammatory effects. In fully activated CD4⁺ T cells, STAT3 was preferentially activated by IL-27 stimulation, whereas both STAT1 and 3 were activated by IL-27 in early activated CD4⁺ T cells. Lack of STAT3 in fully activated cells impaired the suppressive effects of IL-27. These data indicated that the preferential activation of STAT3 in fully activated CD4⁺ T cells plays an important role in the cytokine suppression by IL-27/WSX-1. Copyright © 2006 by The American Association of Immunologists, Inc.

Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors

Yoshida T., Hisamoto T., Akiba J., Koga H., Nakamura K., Tokunaga Y., Hanada S., Kumemura H., Maeyama M., Harada M., Ogata H., Yano H., Kojiro M., Ueno T., Yoshimura A., Sata M.

Oncogene 25 （ 45 ） 6056 - 6066 2006年10月

ISSN 0950-9232

Aberrant activation of the Ras/Raf-1/extracellular-regulated kinase (ERK) pathway has been shown to be involved in the progression of human hepatocellular carcinoma (HCC). However, the mechanism of dysregulation of ERK activation is poorly understood. Recently, we identified Sprouty-related protein with Ena/vasodilator-stimulated phosphoprotein homology-1 domain (Spred) as a physiological inhibitor of the Ras/Raf-1/ERK pathway. In this study, we found that the expression levels of Spred-1 and -2 in human HCC tissue were frequently decreased, comparing with those in adjacent non-tumorous tissue. Moreover, Spred expression levels in HCC tissue were inversely correlated with the incidence of tumor invasion and metastasis. Forced expression of Spred-1 inhibited HCC cell proliferation in vitro and in vivo, which was associated with reduced ERK activation. Spred-1 overexpression also reduced the secretion of matrix metalloproteinase-9 (MMP-9) and MMP-2, which play important roles in tumor invasion and metastasis. In addition, Spred-1 inhibited growth factor-mediated HCC cell motility. These data indicate that the reduction of Spred expression in HCC is one of the causes of the acquisition of malignant features. Thus, Spred could be not only a novel prognostic factor but also a new therapeutic target for human HCC. © 2006 Nature Publishing Group. All rights reserved.

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ER stress-induced apoptosis and caspase-12 activation occurs downstream of mitochondrial apoptosis involving Apaf-1

Shiraishi Hiroshi, Okamoto Hideaki, Yoshimura Akihiko, Yoshida Hiroki

Journal of Cell Science 119 （ 19 ） 3958 - 3966 2006年10月

ISSN 0021-9533

Accumulation of unfolded proteins induces endoplasmic reticulum (ER) stress. Excessive and prolonged stresses lead cells to apoptosis. However, the precise molecular mechanisms of ER stress-induced apoptosis have not been fully elucidated. We investigated the involvement of the apoptosome in ER stress-induced cell death pathway using mouse embryonic fibroblasts (MEFs) and mice deficient for Apaf-1. Apaf-1-deficient MEFs showed ore resistance to ER stress-inducing reagents as compared with wild type cells. Despite comparable induction of ER stress in both wild type and Apaf-1-deficient cells, activation of caspase-3 was only observed in wild type, but not Apaf-1-deficient, MEFs. Under ER stress conditions, BAX translocated to mitochondria and cytochrome c was released from mitochondria. We also demonstrated that caspase-12 was processed downstream of Apaf-1 and caspase-3, and neither overexpression nor knockdown of caspase-12 affected susceptibility of the cells to ER stress-induced cell death. Furthermore, in the kidneys of Apaf-1-deficient mice, apoptosis induced by in vivo administration of tunicamycin was remarkably suppressed as compared with wild type mice. These data collectively demonstrated that Apaf-1 and the mitochondrial pathway of apoptosis play significant roles in ER stress-induced apoptosis.

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Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system

Stumhofer Jason S., Laurence Arian, Wilson Emma H., Huang Elaine, Tato Cristina M., Johnson Leanne M., Villarino Alejandro V., Huang Qiulong, Yoshimura Akihiko, Sehy David, Saris Christiaan J M, O'Shea John J., Hennighausen Lothar, Ernst Matthias, Hunter Christopher A.

Nature Immunology 7 （ 9 ） 937 - 945 2006年09月

ISSN 1529-2908

Studies have focused on the events that influence the development of interleukin 17 (IL-17)-producing T helper cells (T_H-17 cells) associated with autoimmunity, such as experimental autoimmune encephalitis, but relatively little is known about the cytokines that antagonize T_H-17 cell effector responses. Here we show that IL-27 receptor-deficient mice chronically infected with Toxoplasma gondii developed severe neuroinflammation that was CD4⁺ T cell dependent and was associated with a prominent IL-17 response. In vitro, treatment of naive primary T cells with IL-27 suppressed the development T_H-17 cells induced by IL-6 and transforming growth factor-β, which was dependent on the intracellular signaling molecule STAT1 but was independent of inhibition of IL-6 signaling mediated by the suppressor protein SOCS3. Thus IL-27, a potent inhibitor of T_H-17 cell development, may be a useful target for treating inflammatory diseases mediated by these cells.

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TGF-β1 suppresses IFN-γ-induced NO production in macrophages by suppressing STAT1 activation and accelerating iNOS protein degradation

Takaki Hiromi, Minoda Yasumasa, Koga Keiko, Takaesu Giichi, Yoshimura Akihiko, Kobayashi Takashi

Genes to Cells 11 （ 8 ） 871 - 882 2006年08月

ISSN 1356-9597

TGF-β1 is a well-known immunosuppressive cytokine; however, little is known of the effect of TGF-β1 on antigen-presenting cells (APCs). In this report, we investigated the molecular mechanisms of the suppressive effects of TGF-β1 on APCs including dendritic cells and macrophages. Although TGF-β1 did not greatly affect the activation of APCs, as assessed by the induction of IL-12 or the upregulation of CD40 in response to LPS, it strongly inhibited IFN-γ-induced nitric oxide (NO) production from macrophages and dendritic cells. Using murine macrophage-like cell line RAW 264.7, we demonstrated that TGF-β1 not only reduced the inducible NO synthase (iNOS) protein stability but also suppressed the iNOS gene transcription. We also found that TGF-β1 directly inhibited IFN-γ-induced STAT1 activation by reducing STAT1 tyrosine phosphorylation. The IFN-γ Type I receptor (IFNGR1) was found to be associated with the TGF-β1 Type I receptor (TGF-βRI) and was phosphorylated by the TGF-βRI. Reduced activation of STAT1 by TGF-β1 was abrogated by the mutation in the IFNGR1 in which the serine residues of potential sites of phosphorylation by TGF-βRI were replaced by alanine residues. Thus, multiple mechanisms are present for the TGF-β1-mediated reduction of iNOS production, and we propose a novel mechanism for regulating inflammatory cytokine by an anti-inflammatory cytokine, TGF-β1; i.e. suppression of IFN-γ-induced STAT1 activation by an association of the IFNGR1 with the TGF-βRI. © 2006 The Authors Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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Enhanced leptin sensitivity and improved glucose homeostasis in mice lacking suppressor of cytokine signaling-3 in POMC-expressing cells

Kievit Paul, Howard Jane K., Badman Michael K., Balthasar Nina, Coppari Roberto, Mori Hiroyuki, Lee Charlotte E., Elmquist Joel K., Yoshimura Akihiko, Flier Jeffrey S.

Cell Metabolism 4 （ 2 ） 123 - 132 2006年08月

ISSN 1550-4131

Suppressor of cytokine signaling-3 (Socs-3) negatively regulates the action of various cytokines, as well as the metabolic hormones leptin and insulin. Mice with haploinsufficiency of Socs-3, or those with neuronal deletion of Socs-3, are lean and more leptin and insulin sensitive. To examine the role of Socs-3 within specific neurons critical to energy balance, we created mice with selective deletion of Socs-3 within pro-opiomelanocortin (POMC)-expressing cells. These mice had enhanced leptin sensitivity, measured by weight loss and food intake after leptin infusion. On chow diet, glucose homeostasis was improved despite normal weight gain. On a high-fat diet, the rate of weight gain was reduced, due to increased energy expenditure rather than decreased food intake; glucose homeostasis and insulin sensitivity were substantially improved. These studies demonstrate that Socs-3 within POMC neurons regulates leptin sensitivity and glucose homeostasis, and plays a key role in linking high-fat diet to disordered metabolism. © 2006 Elsevier Inc. All rights reserved.

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Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen species-mediated vascular endothelial growth factor expression

Yamagishi Sho Ichi, Nakamura Kazuo, Matsui Takanori, Inagaki Yosuke, Takenaka Katsuhiko, Jinnouchi Yuko, Yoshida Yumiko, Matsuura Tetsuro, Narama Isao, Motomiya Yoshihiro, Takeuchi Masayoshi, Inoue Hiroyoshi, Yoshimura Akihiko, Bucala Richard, Imaizumi Tsutomu

Journal of Biological Chemistry 281 （ 29 ） 20213 - 20220 2006年07月

ISSN 0021-9258

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22^phox and gp91^phox, membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8- hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22^PHOX and gp91 ^PHOX. This led to blockade of the AGE-elicited Ras activation and NF-κB-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidase-mediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Deletion of the SOCS3 Gene in Liver Parenchymal Cells Promotes Hepatitis-Induced Hepatocarcinogenesis

Ogata Hisanobu, Kobayashi Takashi, Chinen Takatoshi, Takaki Hiromi, Sanada Takahito, Minoda Yasumasa, Koga Keiko, Takaesu Giichi, Maehara Yoshihiko, Iida Mitsuo, Yoshimura Akihiko

Gastroenterology 131 （ 1 ） 179 - 193 2006年07月

ISSN 0016-5085

Background & Aims: A recent study has suggested that the methylation silencing of the suppressor of cytokine signaling-3 (SOCS3), a negative regulator of interleukin-6-related cytokines, could be involved in hepatocellular carcinoma (HCC). However, the roles of SOCS3 in hepatocellular carcinogenesis and hepatitis have not been established. We investigated the effect of deleting the SOCS3 gene on the development of hepatitis and HCC in hepatitis C virus-infected patients and mouse models. Methods: The expression of SOCS genes in HCC and non-HCC regions of patient samples was determined by real-time reverse-transcription polymerase chain reaction and immunoblotting. The conditional knockout approach in mice was used to determine the hepatocyte-specific roles of SOCS3. To generate a liver-specific deletion, floxed SOCS3 (SOCS3^fl/fl) mice were crossed with albumin-Cre transgenic mice. Hepatitis and HCC were induced by administering concanavalin A and diethylnitrosamine, respectively. Results: SOCS3 expression was reduced in the HCC regions compared with the non-HCC regions. Carcinogen-induced hepatic tumor development was enhanced by deletion of the SOCS3 gene, which was associated with higher levels of the targets of signal transducers and activators of transcription (ie, B-cell lymphoma-XL, B-cell lymphoma-2, C-myelocytomatosis, cyclin D1, and vascular endothelial growth factor). In the concanavalin A-mediated hepatitis model, deletion of the SOCS3 gene in the hepatocytes protected against liver injury through suppression of interferon-γ signaling and induction of the antiapoptotic protein Bcl-XL. Conclusions: Deletion of the SOCS3 gene in hepatocytes promotes the activation of STAT3, resistance to apoptosis, and an acceleration of proliferation, resulting in enhanced hepatitis-induced hepatocarcinogenesis. © 2006 American Gastroenterological Association Institute.

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Conditional ablation of Stat3 or Socs3 discloses a dual role for reactive astrocytes after spinal cord injury

Okada Seiji, Nakamura Masaya, Katoh Hiroyuki, Miyao Tamaki, Shimazaki Takuya, Ishii Ken, Yamane Junichi, Yoshimura Akihiko, Iwamoto Yukihide, Toyama Yoshiaki, Okano Hideyuki

Nature Medicine 12 （ 7 ） 829 - 834 2006年07月

ISSN 1078-8956

In the injured central nervous system (CNS), reactive astrocytes form a glial scar and are considered to be detrimental for axonal regeneration, but their function remains elusive. Here we show that reactive astrocytes have a crucial role in wound healing and functional recovery by using mice with a selective deletion of the protein signal transducer and activator of transcription 3 (Stat3) or the protein suppressor of cytokine signaling 3 (Socs3) under the control of the Nes promoter-enhancer (Nes-Stat3^-/-, Nes-Socs3^-/-). Reactive astrocytes in Nes-Stat3^-/- mice showed limited migration and resulted in markedly widespread infiltration of inflammatory cells, neural disruption and demyelination with severe motor deficits after contusive spinal cord injury (SCI). On the contrary, we observed rapid migration of reactive astrocytes to seclude inflammatory cells, enhanced contraction of lesion area and notable improvement in functional recovery in Nes-Socs3^-/- mice. These results suggest that Stat3 is a key regulator of reactive astrocytes in the healing process after SCI, providing a potential target for intervention in the treatment of CNS injury. © 2006 Nature Publishing Group.

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SOCS1-negative feedback of STAT1 activation is a key pathway in the dsRNA-induced innate immune response of human keratinocytes

Dai Xiuju, Sayama Koji, Yamasaki Kenshi, Tohyama Mikiko, Shirakata Yuji, Hanakawa Yasushi, Tokumaru Sho, Yahata Yoko, Yang Lujun, Yoshimura Akihiko, Hashimoto Koji

Journal of Investigative Dermatology 126 （ 7 ） 1574 - 1581 2006年07月

ISSN 0022-202X

Toll-like receptor (TLR)3 is a receptor for virus-associated double-stranded RNA, and triggers antiviral immune responses during viral infection. Epidermal keratinocytes express TLR3 and provide an innate immune defense against viral infection. Since the intracellular regulatory mechanism is unknown, we hypothesized that the signal transducers and activators of transcription (STAT)-suppressors of cytokine signaling (SOCS) system regulates the innate immune response of keratinocytes. Treatment with polyinosinic- polycytidylic acid (poly(I:C)) resulted in the rapid translocation of IFN regulatory factor (IRF)-3 into the nucleus, followed by phosphorylation of STAT1 and STAT3. The activation of STATs by poly(I:C) probably occurs in an indirect fashion, through poly(I:C)-induced IFN. We infected cells with the dominant-negative forms of STAT1 (STAT1F), STAT3 (STAT3F), and SOCS1 using adenovirus vectors. Infection with STAT1F suppressed the induction of macrophage inflammatory protein (MIP)-1α by poly(I:C), whereas STAT3F had a minimal effect, which indicates that STAT1 mediates MIP-1α induction. SOCS1, which is a negative feedback regulator of STAT1 signaling, was induced by treatment with poly(I:C). SOCS1 infection inhibited the phosphorylation of STAT1 and significantly reduced poly(I:C)-induced MIP-1α production. Furthermore, STAT1-SOCS1 regulated poly(I:C)-induced TLR3 and IRF-7 expression. However, SOCS1 did not affect NF-κB signaling. Thus, the STAT1-SOCS1 pathway regulates the innate immune response via TLR3 signaling in epidermal keratinocytes. © 2006 The Society for Investigative Dermatology.

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IFNγ-dependent, spontaneous development of colorectal carcinomas in SOCS1-deficient mice

Hanada Toshikatsu, Kobayashi Takashi, Chinen Takatoshi, Saeki Kazuko, Takaki Hiromi, Koga Keiko, Minoda Yasumasa, Sanada Takahito, Yoshioka Tomoko, Mimata Hiromitsu, Kato Seiya, Yoshimura Akihiko

Journal of Experimental Medicine 203 （ 6 ） 1391 - 1397 2006年06月

ISSN 0022-1007

Approximately 20% of human cancers are estimated to develop from chronic inflammation. Recently, the NF-κB pathway was shown to play an essential role in promoting inflammation-associated cancer, but the role of the JAK/STAT pathway, another important signaling pathway of proinflammatory cytokines, remains to be investigated. Suppressor of cytokine signaling-1 (SOCS1) acts as an important physiological regulator of cytokine responses, and silencing of the SOCS1 gene by DNA methylation has been found in several human cancers. Here, we demonstrated that SOCS1-deficient mice (SOCS1^-/-Tg mice), in which SOCS1 expression was restored in T and B cells on a SOCS1^-/- background, spontaneously developed colorectal carcinomas carrying nuclear β-catenin accumulation and p53 mutations at 6 months of age. However, interferon (IFN)γ^-/-SOCS1^-/- mice and SOCS1 ^-/-Tg mice treated with anti-IFNγ antibody did not develop such tumors. STAT3 and NF-κB activation was evident in SOCS1^-/-Tg mice, but these were not sufficient for tumor development because these are also activated in IFNγ^-/-SOCS1^-/- mice. However, colons of SOCS1^-/-Tg mice, but not IFNγ^-/-SOCS1 ^-/- mice, showed hyperactivation of STAT1, which resulted in the induction of carcinogenesis-related enzymes, cyclooxygenase-2 and inducible nitric oxide synthase. These data strongly suggest that SOCS1 is a unique antioncogene which prevents chronic inflammation-mediated carcinogenesis by regulation of the IFNγ/STAT1 pathways. JEM © The Rockefeller University Press.

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A novel Zinc finger protein, ZCCHC11, interacts with TIFA and modulates TLR signaling

Minoda Yasumasa, Saeki Kazuko, Aki Daisuke, Takaki Hiromi, Sanada Takahito, Koga Keiko, Kobayashi Takashi, Takaesu Giichi, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 344 （ 3 ） 1023 - 1030 2006年06月

ISSN 0006-291X

Toll-like receptors (TLRs) play an important role as a sensor of microbial pathogens in the innate immune response. TLRs transmit signals through the recruitment of adaptor proteins including tumor necrosis factor-associated factor 6 (TRAF6), which mediates the activation of IκB kinase (IKK). TIFA (TRAF-interacting protein with a forkhead-associated (FHA) domain) has been shown to bind to TRAF6 and activate IKK by promoting the oligomerization and ubiquitin-ligase activity of TRAF6. FHA domains preferentially bind to phospho-threonine residues in their targets. Here, we identified a novel zinc finger protein, ZCCHC11, that interacts with TIFA from phosphoproteins of a macrophage cell line, RAW 264.7, by using affinity purification with GST-TIFA and mass spectrometric analysis. By a search of the EST database, we found a 200 kDa full-length form (ZCCHC11L). ZCCHC11L was mostly located to the nucleus, but translocated into the cytoplasm in response to LPS and bound to TIFA. Overexpression and knockdown by siRNA indicated that ZCCHC11 functions as a negative regulator of TLR-mediated NF-κB activation. The N-terminal region (ZCCHC11S) including CCHC-type Zn-finger motif was sufficient for suppression of NF-κB. We propose that ZCCHC11 is a unique TLR signal regulator, which interacts with TIFA after LPS treatment and suppresses the TRAF6-dependent activation of NF-κB. © 2006 Elsevier Inc. All rights reserved.

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Signal transduction of inflammatory cytokines and tumor development

Yoshimura Akihiko

Cancer Science 97 （ 6 ） 439 - 447 2006年06月

ISSN 1347-9032

It has been estimated that >20% of all malignancies are initiated or exacerbated by inflammation. Until recently, the molecular basis of this process has not been clarified. However, recent studies have uncovered the molecular mechanism of intracellular signaling pathways of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-6. Three major transcription factors including NF-κB, STAT1 and STAT3 have been shown to play major roles in transmitting inflammatory cytokine signals to the nucleus. One function of NF-κB and STAT3 in tumor cells is the promotion of cell growth and cell survival through the induction of target genes, whose products promote cell division and inhibit apoptosis. In addition, NF-κB and STAT1 are important transcription factors that induce inflammatory mediators from inflammatory cells, especially macrophages, while STAT3 often antagonizes this process. STAT1 is generally believed to be an anti-oncogene because it promotes apoptosis through p53, but it could promote inflammation-mediated tumor development by enhancing tissue injury, remodeling, fibrosis and inflammation. Hence, the inhibition of NF-κB and STATs offers a strategy for treatment of a variety of malignancies and can convert inflammation-induced tumor growth into inflammation-induced tumor regression. © 2006 Japanese Cancer Association.

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Selective regulatory function of Socs3 in the formation of IL-17-secreting T cells

Chen Zhi, Laurence Arian, Kanno Yuka, Pacher-Zavisin Margit, Zhu Bing Mei, Tato Cristina, Yoshimura Akihiko, Hennighausen Lothar, O'Shea John J.

Proceedings of the National Academy of Sciences of the United States of America 103 （ 21 ） 8137 - 8142 2006年05月

ISSN 0027-8424

Suppressor of cytokine signaling (Socs) 3 is a cytokine-inducible inhibitor with critical but selective cell-specific effects. We show that deficiency of Socs3 in T cells had minimal effects on differentiation of T cells to the T helper (Th) 1 or Th2 subsets; accordingly, Socs3 had no effect on IL-12-dependent signal transducer and activator of transcription (Stat) 4 phosphorylation or IL-4-dependent Stat6 phosphorylation. By contrast, Socs3 was found to be a major regulator of IL-23-mediated Stat3 phosphorylation and Th17 generation, and Stat3 directly binds to the IL-17A and IL-17F promoters. We conclude that Socs3 is an essential negative regulator of IL-23 signaling, inhibition of which constrains the generation of Th17 differentiation. © 2006 by The National Academy of Sciences of the USA.

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The neuropeptide neuromedin U activates eosinophils and is involved in allergen-induced eosinophilia

Moriyama Maiko, Fukuyama Satoru, Inoue Hiromasa, Matsumoto Takafumi, Sato Takahiro, Tanaka Kentaro, Kinjyo Ichiko, Kano Tatsuhiko, Yoshimura Akihiko, Kojima Masayasu

American Journal of Physiology - Lung Cellular and Molecular Physiology 290 （ 5 ） 2006年05月

ISSN 1040-0605

Neuromedin U (NMU) is a neuropeptide expressed not only in the central nervous system but also in various organs, including the gastrointestinal tract and lungs. NMU interacts with two G protein-coupled receptors, NMU-R1 and NMU-R2. Although NMU-R2 is expressed in a specific region of the brain, NMU-R1 is expressed in various peripheral tissues, including immune and hematopoietic cells. Our recent study demonstrated an important role of NMU in mast cell-mediated inflammation. In this study, we showed that airway eosinophilia was reduced in NMU-deficient mice in an allergen-induced asthma model. There were no differences in the antigen-induced Th2 responses between wild-type and NMU knockout mice. NMU-R1 was highly expressed in the eosinophil cell line, and NMU directly induced Ca²⁺ mobilization and extracellular/signal- regulated kinase phosphorylation. NMU also induced cell adhesion to components of the extracellular matrix (fibronectin and collagen type I), and chemotaxis in vitro. Furthermore, NMU-R1 was also expressed in human peripheral blood eosinophils, and NMU induced cell adhesion in a dose-dependent manner. These data indicate that NMU promotes eosinophil infiltration into inflammatory sites by directly activating eosinophils. Our study suggests that NMU receptor antagonists could be novel targets for pharmacological inhibition of allergic inflammatory diseases, including asthma. Copyright © 2006 the American Physiological Society.

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Loss of SOCS3 in the liver promotes fibrosis by enhancing STAT3-mediated TGF-β1 production

Ogata H., Chinen T., Yoshida T., Kinjyo I., Takaesu G., Shiraishi H., Iida M., Kobayashi T., Yoshimura A.

Oncogene 25 （ 17 ） 2520 - 2530 2006年04月

ISSN 0950-9232

Recently, DNA methylation and reduced expression of the suppressor of the cytokine signaling-3 (SOCS3) gene in human hepatocellular carcinoma (HCC) patients have been reported. However, the roles of SOCS3 in HCC development in vivo have not been clarified. Using RT-PCR analysis and Western blotting, we confirmed that SOCS3 expression was reduced in HCC patients. However, reduced expression of SOCS3 occurred not only in HCC but also in nontumor regions, and this reduction was stronger as the fibrosis grade increased. Furthermore, SOCS3 levels were inversely correlated with signal transducers and activators of transcription-3 (STAT3) activation as well as transforming growth factor (TGF)-β1 levels in the non-HCC region. To define the molecular consequences of SOCS3 silencing/STAT3 hyperactivation and liver fibrosis, we examined liver-specific SOCS3-deficient mice. We demonstrated that SOCS3 deletion in the liver resulted in hyperactivation of STAT3 and promoted ConA- and chemical-induced liver fibrosis. The expression of TGF-β1, a mediator of fibrosis, was enhanced by SOCS3 gene deletion, but suppressed by the overexpression of a dominant-negative STAT3 or SOCS3 both in vivo and in vitro. These data suggest that TGF-β1 is a target gene of STAT3 and could be one of the mechanisms for enhanced fibrosis in SOCS3-deficient mice. Thus, our present study provides a novel role of SOCS3 and STAT3 in HCC development: in addition to the previously characterized oncogenic potentials, STAT3 enhances hepatic fibrosis through the upregulation of TGF-β1 expression, and SOCS3 prevents this process. © 2006 Nature Publishing Group All rights reserved.

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Loss of SOCS3 in T helper cells resulted in reduced immune responses and hyperproduction of interleukin 10 and transforming growth factor-β1

Kinjyo Ichiko, Inoue Hiromasa, Hamano Shinjiro, Fukuyama Satoru, Yoshimura Takeru, Koga Keiko, Takaki Hiromi, Himeno Kunisuke, Takaesu Giichi, Kobayashi Takashi, Yoshimura Akihiko

Journal of Experimental Medicine 203 （ 4 ） 1021 - 1031 2006年04月

ISSN 0022-1007

Suppressor of cytokine signaling (SOCS)3 is a major negative feedback regulator of signal transducer and activator of transcription (STAT)3-activating cytokines. Transgenic mouse studies indicate that high levels of SOCS3 in T cells result in type 2 T helper cell (Th2) skewing and lead to hypersensitivity to allergic diseases. To define the physiological roles of SOCS3 in T cells, we generated T cell-specific SOCS3 conditional knockout mice. We found that the mice lacking SOCS3 in T cells showed reduced immune responses not only to ovalbumin-induced airway hyperresponsiveness but also to Leishmania major infection. In vitro, SOCS3-deficient CD4⁺ T cells produced more transforming growth factor (TGF)-β1 and interleukin (IL)-10, but less IL-4 than control T cells, suggesting preferential Th3-like differentiation. We found that STAT3 positively regulates TGF-β1 promoter activity depending on the potential STAT3 binding sites. Furthermore, chromatin immunoprecipitation assay revealed that more STAT3 was recruited to the TGF-β1 promoter in SOCS3-deficient T cells than in control T cells. The activated STAT3 enhanced TGF-β1 and IL-10 expression in T cells, whereas the dominant-negative form of STAT3 suppressed these. From these findings, we propose that SOCS3 regulates the production of the immunoregulatory cytokines TGF-β1 and IL-10 through modulating STAT3 activation. © The Rockefeller University Press.

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The neuropeptide neuromedin U promotes IL-6 production from macrophages and endotoxin shock

Moriyama Maiko, Matsukawa Akihiro, Kudoh Shinji, Takahashi Tomoko, Sato Takahiro, Kano Tatsuhiko, Yoshimura Akihiko, Kojima Masayasu

Biochemical and Biophysical Research Communications 341 （ 4 ） 1149 - 1154 2006年03月

ISSN 0006-291X

Neuromedin U (NMU) is a neuropeptide involved in appetite, circadian rhythm, and pronociception. However, the NMU receptor NMU-R1 has been shown to be expressed in immune cells and NMU promotes mast cell-dependent inflammation. In this study, we demonstrated that NMU plays an important role in IL-6 production in macrophages. NMU-deficient mice were resistant against cecal ligation puncture- as well as LPS-induced septic shock. IL-6 but not TNF-α levels were markedly reduced in LPS-treated NMU-deficient mice compared with wild type mice. Both NMU and NMU-R1 were expressed in wild type peritoneal macrophages, and treatment with LPS resulted in up-regulation of NMU but down-regulation of NMU-R1 expression, however, no down-regulation of NMU-R1 was observed in NMU-deficient macrophages where LPS-induced IL-6 production was severely reduced. These data suggest that LPS-induced IL-6 expression is partly dependent on autocrine/paracrine activation of the NMU-NMU-R1 signals in macrophages. © 2006 Elsevier Inc. All rights reserved.

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Divergent mechanisms utilized by SOCS3 to mediate interleukin-10 inhibition of tumor necrosis factor α and nitric oxide production by macrophages

Qasimi Pooran, Ming-Lum Andrew, Ghanipour Ali, Ong Christopher J., Cox Michael E., Ihle James, Cacalano Nicolas, Yoshimura Akihiko, Mui Alice L F

Journal of Biological Chemistry 281 （ 10 ） 6316 - 6324 2006年03月

ISSN 0021-9258

The cytokine interleukin-10 (IL-10) potently inhibits macrophage function through activation of the transcription factor STAT3. The expression of SOCS3 (suppressor of cytokine signaling-3) has been shown to be induced by IL-10 in a STAT3-dependent manner. However, the relevance of SOCS3 expression to the anti-inflammatory effect of IL-10 on macrophages has been controversial. Through kinetic analysis of the requirement for SOCS3 in IL-10 inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α (TNFα) transcription and translation, SOCS3 was found to be necessary for TNFα expression during the early phase, but not the late phase of IL-10 action. SOCS3 was essential for IL-10 inhibition of LPS-stimulated production of iNOS (inducible nitricoxide synthase) protein and nitric oxide (NO). To determine the domains of SOCS3 protein important in mediating these effects, SOCS3 ^-/- macrophages were reconstituted with SOCS3 mutated for the SH2, KIR, SOCS box domains, and tyrosines 204 (Tyr²⁰⁴) and 221 (Tyr ²²¹). The SH2 domain, SOCS box, and both Tyr²⁰⁴ and Tyr²²¹ were required for IL-10 inhibition of TNFα mRNA and protein expression, but interestingly the KIR domain was necessary only for IL-10 inhibition of TNFα protein expression. In contrast, Tyr ²⁰⁴ and Tyr²²¹ were the only structural features of SOCS3 that were necessary in mediating IL-10 inhibition of iNOS protein expression and NO production. These data define SOCS3 as an important mediator of IL-10 inhibition of macrophage activation and that SOCS3 interferes with distinct LPS-stimulated signal transduction events through differing mechanisms. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Involvement of a novel Q-SNARE, D12, in quality control of the endomembrane system

Okumura Akiko Joo, Hatsuzawa Kiyotaka, Tamura Taku, Nagaya Hisao, Saeki Kazuko, Okumura Fumihiko, Nagao Kenji, Nishikawa Mitsuo, Yoshimura Akihiko, Wada Ikuo

Journal of Biological Chemistry 281 （ 7 ） 4495 - 4506 2006年02月

ISSN 0021-9258

The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to α-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomallysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Suppressor of cytokine signaling-1 regulates inflammatory bowel disease in which both IFNγ and IL-4 are involved

Chinen Takatoshi, Kobayashi Takashi, Ogata Hisanobu, Takaesu Giichi, Takaki Hiromi, Hashimoto Masayuki, Yagita Hideo, Nawata Hajime, Yoshimura Akihiko

Gastroenterology 130 （ 2 ） 373 - 388 2006年02月

ISSN 0016-5085

Background & Aims: The suppressor of cytokine signaling-1 (SOCS1) is a potent negative regulator of various cytokines and it has been implicated in the regulation of immune responses. However, the role of SOCS1 in inflammatory bowel diseases (IBDs) has not been clarified. To determine the role of SOCS1 in colitis, we generated SOCS1/T-cell receptor α (TCRα) double knockout (DKO) mice. Methods: The depletion of interferon γ (IFNγ) and IL-4 was achieved by crossing the DKO mice with IFNγ knockout (KO) mice and by the administration of anti-IL-4 antibody, respectively. The activation of cytokine-induced transcription factors was determined by Western blotting with phosphorylation-specific antibodies, and the induction of inflammatory factors was measured by reverse-transcription polymerase chain reaction. Results: Much more severe colitis developed in 100% of the DKO mice within 9 weeks of age than in TCRα-KO mice. Although the proportion and the activation status of CD4⁺ TCRα^-β⁺ T cells in DKO mice were similar to those in TCRα-KO mice, signal transducer and activator of transcription 1, nuclear factor κB, and their target genes were hyperactivated in infiltrated mononuclear cells and colonic epithelial cells in DKO mice. Cytokine-depletion experiments showed that exacerbated colitis in the DKO mice was dependent on both IFNγ and IL-4. SOCS1-deficient cells were hypersensitive to IFNγ, IL-4, and lipopolysaccharides, depending on the target genes. Conclusions: SOCS1 plays an important role in preventing murine colitis by restricting the cytokine signals. SOCS1/TCRα DKO mice could be a useful model for investigating human IBD. © 2006 by the American Gastroenterological Association.

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Mal-function of TLRs by SOCS

Kobayashi Takashi, Takaesu Giichi, Yoshimura Akihiko

Nature Immunology 7 （ 2 ） 123 - 124 2006年02月

ISSN 1529-2908

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Suppressor of cytokine signalling 1 in lymphocytes regulates the development of intestinal inflammation in mice

Inagaki-Ohara K., Sasaki A., Matsuzaki G., Ikeda T., Hotokezaka M., Chijiiwa K., Kubo M., Yoshida H., Nawa Y., Yoshimura A.

Gut 55 （ 2 ） 212 - 219 2006年02月

ISSN 0017-5749

Background and aims: Imbalance between pro- and anti-inflammatory cytokines produced by intestinal T cells induces inflammatory bowel diseases (IBD). However, the importance of regulation of cytokine signalling in IBD has not been fully clarified. We have demonstrated that suppressor of cytokine signalling 1 (SOCS1) is expressed in inflamed tissues in an experimental colitis model. In the present study, we investigated the role of SOCS1 in colitis models to clarify the mechanism of IBD development. Methods: Intestinal T cells in transgenic mice expressing high levels of SOCS1 in lymphocytes (SOCS1Tg mice) were characterised by flow cytometric analysis and cytokine production from intestinal T cells was determined by ELISA. 2,4,6-Trinitrobenzene sulphonic acid (TNBS) induced colitis was induced in SOCS1Tg mice and severity was compared with control littermates by measurement of survival rates, Intracellular signalling was assessed by western blotting analysis. Results: SOCS1Tg mice developed colitis spontaneously with age. Young SOCS1Tg mice less than 15 weeks of age, before the onset of colitis, were susceptible to TNBS induced colitis. Intestinal T cells of SOCS1Tg mice showed increased interferon γ and tumour necrosis factor a production and decreased transforming growth factor γ production. Expression of cytotoxic T lymphocyte associated antigen 4 (CTLA-4), a negative regulator of T cell activation, in SOCS1Tg mice was severely impaired at the protein level although mRNA levels of CTLA-4 in SOCS1Tg mice were comparable with those in control mice. Conclusions: Our data suggest that SOCS1 plays an important role in the regulation of colitis by controlling intestinal T cell activation mediated through CTLA-4 expression.

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Computer simulation of the role of SOCS family protein in helper T cell differentiation

Yamada Satoshi, Tsukada Jun, Yoshimura Akihiko, Kubo Masato

International Immunology 18 （ 2 ） 335 - 345 2006年02月

ISSN 0953-8178

The T_h1/T_h2 balance determines the nature of an immune response, and particular cytokines, IL-4 and IL-12, determine the direction at the initial stage of activation through TCRs. To investigate how cytokine networks and related signaling pathways impact upon the T_h1/T_h2 balance, we have developed a computer model for the simulation of T_h differentiation. The model includes the IL-4, IL-12 and IFN-γ signal transduction pathways, a positive and negative feedback mechanism for cytokine signaling and cytokine-induced negative regulators such as suppressors of cytokine signaling (SOCS)1, SOCS3 and SOCS5. In the present study, we propose a 'T_h0 model', in which naive T cells differentiate neither into T_h1 nor into T_h2 states in unskewed cytokine conditions. The model was found to be consistent with experimental results in BALB/c mice. The results of in silico analysis in the condition with SOCS- and signal transducer and activator of transcription (STAT) family-deficient and transgenic states were well fitted to ex vivo experimental results for T_h1 and T_h2 differentiation profiles in the deficient and transgenic mice. The T_h0 model suggested the possibility that dominant T_h1 differentiation in STAT4/STAT6 double-deficient mice may be due to a positive feedback effect of initial IFN-γ production from T cells. The in silico assessment of beneficial effects of inhibitory drugs by simulation analysis with our T_h0 model indicated that Janus kinase 3-specific inhibitors might be suitable candidates for the modification of T_h2-dominant immune responses. Our results demonstrate that models for the simulation of signaling network, such as our T_h0 model, are useful tools for the in silico evaluation of novel drug candidates. © The Author Society for Immunology. 2006. All rights reserved.

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Association study of polymorphisms in SOCS family genes with type 1 diabetes mellitus

Ni R., Ihara K., Miyako K., Takemoto M., Ishimura M., Kohno H., Matsuura N., Yoshimura A., Hara T.

International Journal of Immunogenetics 33 （ 1 ） 7 - 10 2006年02月

ISSN 1744-3121

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Suppression of IL-6 production and proliferation by blocking STAT3 activation in malignant soft tissue tumor cells

Shouda Takanori, Hiraoka Koji, Komiya Setsuro, Hamada Tetsuya, Zenmyo Michihisa, Iwasaki Hiroshi, Isayama Teruto, Fukushima Nobuhiro, Nagata Kensei, Yoshimura Akihiko

Cancer Letters 231 （ 2 ） 176 - 184 2006年01月

ISSN 0304-3835

There is no established optimum treatment for malignant fibrous histiocytoma (MFH) at present, and few MFH cell lines are established. In the present study, we established new MFH cell lines, KHZ-MFH and SFT85-03, and investigated the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway. We found that MFH cells secreted high levels of IL-6 and that STAT3 was constitutively activated in these cells. The JAK2 kinase inhibitor, tyrphostin AG490, suppressed the growth of MFH cells and inhibited the secretion of IL-6. Furthermore, blockade of activated STAT3 by forced expression of a cytokine signaling repressor, SOCS3 gene as well as a dominant-negative STAT3 in these cells significantly suppressed their growth. These results indicated that an autocrine mechanism of the JAK/STAT3 signaling pathway could promote the growth of MFH cells and that this pathway could be a therapeutic target of MFH. © 2005 Elsevier Ireland Ltd. All rights reserved.

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Modulation of TLR4 signaling by a novel adaptor protein signal-transducing adaptor protein-2 in macrophages

Sekine Yuichi, Yumioka Taro, Yamamoto Tetsuya, Muromoto Ryuta, Imoto Seiyu, Sugiyma Kenji, Oritani Kenji, Shimoda Kazuya, Minoguchi Mayu, Akira Shizuo, Yoshimura Akihiko, Matsuda Tadashi

Journal of Immunology 176 （ 1 ） 380 - 389 2006年01月

ISSN 0022-1767

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies have demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, STAP-2 was found to positively regulate LPS/TLR4-mediated signals in macrophages. Disruption of STAP-2 resulted in impaired LPS/TLR4-induced cytokine production and NF-κB activation. Conversely, overexpression of STAP-2 enhanced these LPS/TLR4-induced biological activities. STAP-2, particularly its Src homology 2-like domain, bound to both MyD88 and IκB kinase (IKK)-αβ, but not TNFR-associated factor 6 or IL-1R-associated kinase 1, and formed a functional complex composed of MyD88-STAP-2-IKK-αβ. These interactions augmented MyD88- and/or IKK-αβ-dependent signals, leading to enhancement of the NF-κB activity. These results demonstrate that STAP-2 may constitute an alternative LPS/TLR4 pathway for NF-κB activation instead of the TNFR-associated factor 6-IL-1R-associated kinase 1 pathway. Copyright © 2005 by The American Association of Immunologists, Inc.

FLN29, a novel interferon- and LPS-inducible gene acting as a negative regulator of toll-like receptor signaling

Mashima Ryuichi, Saeki Kazuko, Aki Daisuke, Minoda Yasumasa, Takaki Hiromi, Sanada Takahito, Kobayashi Takashi, Aburatani Hiroyuki, Yamanashi Yuji, Yoshimura Akihiko

Journal of Biological Chemistry 280 （ 50 ） 41289 - 41297 2005年12月

ISSN 0021-9258

Lipopolysaccharide (LPS) activates macrophages through toll-like receptor (TLR) 4. Although the mechanism of the TLR signaling pathway has been well documented, the mechanism of the negative regulation in response to LPS, particularly LPS tolerance, is still poorly understood. In this study we identified and characterized a novel interferon- and LPS-inducible gene, FLN29, which contains a TRAF6-related zinc finger motif and TRAF family member-associated NF-κB activator-related sequences. The induction of FLN29 was dependent on STAT1. The forced expression of FLN29 in macrophage-like RAW cells resulted in the suppression of TLR-mediated NF-κB and mitogen-activated protein kinase activation, while a reduced expression of FLN29 by small interfering RNA partly cancelled the down-regulation of LPS signaling. Furthermore, we demonstrated that NF-κB activation induced by TRAF6 and TAB2 was impaired by co-expression of FLN29, suggesting FLN29 may regulate the downstream of TRAF6. Taken together, FLN29 is a new negative feedback regulator of TLR signaling. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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SOCS genes

Yoshimura Akihiko, Nishinakamura Hitomi, Takaki Hiromi

International Congress Series 1285 121 - 129 2005年11月

ISSN 0531-5131

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins (ILs) and interferons. Many of these cytokines exert their biological functions through Janus tyrosine kinases (JAKs) and STAT transcription factors. The CIS (cytokine-inducible SH2 protein) and SOCS (suppressors of cytokine signaling) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine-mediated homeostasis, including innate and adaptive immunity. We investigated the roles of suppressors of cytokine signaling (SOCSs) in regulating dendritic cell (DC) maturation and function. We showed that SOCS1-deficient DCs induced stronger Th1-type responses both in vitro and in vivo. SOCS1-deficient DCs induced higher interferon-γ (IFNγ) production from naive T cells than wild-type DCs in vitro. Lymph node T cells also produced higher amount of IFNγ when SOCS1-deficient bone marrow-derived (BM) DCs were transferred in vivo. Moreover, SOCS1^-/- BMDCs raised more effective anti-tumor immunity than wild-type BMDCs. On the other hand, SOCS3-deficeint BMDCs expressed lower levels of MHC, co-stimulators and CD40 in response to LPS. SOCS3-deficient DCs induced lower T cell responses. Thus, SOCS1 and SOCS3 reciprocally regulate DC maturation. © 2005.

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Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37

Tokumaru Sho, Sayama Koji, Shirakata Yuji, Komatsuzawa Hitoshi, Ouhara Kazuhisa, Hanakawa Yasushi, Yahata Yoko, Dai Xiuju, Tohyama Mikiko, Nagai Hiroshi, Yang Lujun, Higashiyama Shigeki, Yoshimura Akihiko, Sugai Motoyuki, Hashimoto Koji

Journal of Immunology 175 （ 7 ） 4662 - 4668 2005年10月

ISSN 0022-1767

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinecyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 μg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration. Copyright © 2005 by The American Association of Immunologists, Inc.

The Sprouty-related protein, Spred-1, localizes in a lipid raft/caveola and inhibits ERK activation in collaboration with caveolin-1

Nonami Atsushi, Taketomi Takaharu, Kimura Akiko, Saeki Kazuko, Takaki Hiromi, Sanada Takahito, Taniguchi Koji, Harada Mine, Kato Reiko, Yoshimura Akihiko

Genes to Cells 10 （ 9 ） 887 - 895 2005年09月

ISSN 1356-9597

Caveolin-1 (Cav-1) has been suggested to function as a negative regulator of mitogen-stimulated proliferation and the Ras-p42/44 ERK (MAP kinase) pathway in a variety of cell types. However, the molecular basis of this suppression has not been clarified. Spred/Sprouty family proteins are also negative regulators of the ERK pathway by interacting with Raf-1. The Spred/Sprouty family proteins contain a cysteine-rich (CR) domain at the C-terminus, which is thought to be palmitoylated like Cav-1 and necessary for membrane anchoring. In this study, we demonstrated that Spred-1 localized in cholesterol-rich membrane raft/caveola fractions and interacted with Cav-1. To clarify the biological effect of Cav-1/Spred-1 interaction, we used hematopoietic cells that lacked expression of caveolins but expressed Spred-1. Forced expression of Cav-1 suppressed SCF- and IL-3-induced proliferation and ERK activation. Furthermore, forced expression of exogenous Spred-1 in Cav-1-expressing cells further suppressed proliferation and ERK activation. These data suggest that Spred-1 inhibits ERK activation in collaboration with Cav-1. © Blackwell Publishing Limited.

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The role of suppressor of cytokine signaling 1 as a negative regulator for aberrant expansion of CD8α⁺ dendritic cell subset

Tsukada Jun, Ozaki Akemi, Hanada Toshikatsu, Chinen Takatoshi, Abe Ryo, Yoshimura Akihiko, Kubo Masato

International Immunology 17 （ 9 ） 1167 - 1178 2005年09月

ISSN 0953-8178

The suppressor of cytokine signaling (SOCS) 1 is a negative regulator in multiple cytokine-related aspects to maintain immunological homeostasis. Here, we studied a role of SOCS1 on dendritic cell (DC) maturation in the mice lacking either TCRα chain or CD28 in SOCS1-deficient background, and found that the SOCS1 could restore acute phase of inflammatory response in SOCS1-deficient mice. The CD11c⁺ CD8^- DC population in freshly isolated splenic DCs from normal mice highly expressed SOCS1. However, in SOCS1-deficient environment, the proportion of CD8α⁺ DCs (CD8 DCs) noticeably increased without affecting the cell number of conventional and plasmacytoid DC populations. This population revealed the CD11c^dull CD8α⁺ CD11b^- CD45RA^- B220^- phenotype, which is a minor population in normal mice. Localization of the abnormal CD8 DCs in splenic microenvironments was mainly restricted to deep within red pulp. The CD8 DCs secrete a large amount of IFN-γ, IL-12 and B lymphocyte stimulator/B cell activation factor of the tumor necrosis factor family in response to LPS and CpG stimulation. This is responsible for the development of DC-mediated systemic autoimmunity in the old age of SOCS1-deficient mice. Moreover, the CD8 DC subsets expressed more indoleamine 2,3-dioxygenase and IL-10, and hence inhibit the allogeneic proliferative T cell response and antigen-induced T_h1 responses. Therefore, SOCS1 expression during DC maturation plays a role in surveillance in controlling the aberrant expansion of abnormal DC subset to maintain homeostasis of immune system. © The Japanese Society for Immunology. 2005. All rights reserved.

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Exacerbation of experimental allergic asthma by augmented Th2 responses in WSX-1-deficient mice

Miyazaki Yoshiyuki, Inoue Hiromasa, Matsumura Mikiko, Matsumoto Koichiro, Nakano Takako, Tsuda Miyuki, Hamano Shinjiro, Yoshimura Akihiko, Yoshida Hiroki

Journal of Immunology 175 （ 4 ） 2401 - 2407 2005年08月

ISSN 0022-1767

WSX-1 (IL-27R) is a class I cytokine receptor with homology to gp130 and IL-12 receptors and is typically expressed on CD4⁺ T lymphocytes. Although previous reports have clarified that IL-27/WSX-1 signaling plays critical roles in both Th1 differentiation and attenuation of cell activation and proinflammatory cytokine production during some bacterial or protozoan infections, little is known about the importance of WSX-1 in cytokine-mediated diseases of allergic origin. To this aim, we took advantage of WSX-1-deficient (WSX-1^-/-) mice and induced experimental asthma, in which Th2 cytokines are central modulators of the pathology. OVA-challenged WSX-1 ^-/- mice showed marked enhancement of airway responsiveness with goblet cell hyperplasia, pulmonary eosinophil infiltration, and increased serum IgE levels compared with wild-type mice. Production of Th2 cytokines, which are largely responsible for the pathogenesis of asthma, was augmented in the lung or in the culture supernatants of peribronchial lymph node CD4⁺ T cells from WSX-1^-/- mice compared with those from wild-type mice. Surprisingly, IFN-γ production was also enhanced in WSX-1^-/- mice, albeit at a low concentration. The cytokine overproduction, thus, seems independent from the Th1-promoting property of WSX-1. These results demonstrated that IL-27/WSX-1 also plays an important role in the down-regulation of airway hyper-reactivity and lung inflammation during the development of allergic asthma through its suppressive effect on cytokine production. Copyright © 2005 by The American Association of Immunologists, Inc.

Loss of mammalian Sprouty2 leads to enteric neuronal hyperplasia and esophageal achalasia

Taketomi Takaharu, Yoshiga Daigo, Taniguchi Koji, Kobayashi Takashi, Nonami Atsushi, Kato Reiko, Sasaki Mika, Sasaki Atsuo, Ishibashi Hitoshi, Moriyama Maiko, Nakamura Kei Ichiro, Nishimura Junji, Yoshimura Akihiko

Nature Neuroscience 8 （ 7 ） 855 - 857 2005年07月

ISSN 1097-6256

We report here that loss of the Sprouty2 gene (also known as Spry2) in mice resulted in enteric nerve hyperplasia, which led to esophageal achalasia and intestinal pseudo-obstruction. Glial cell line-derived neurotrophic factor (GDNF) induced hyperactivation of ERK and Akt in enteric nerve cells. Anti-GDNF antibody administration corrected nerve hyperplasia in Sprouty2-deficient mice. We show Sprouty2 to be a negative regulator of GDNF for the neonatal development or survival of enteric nerve cells. © 2005 Nature Publishing Group.

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The neuropeptide neuromedin U promotes inflammation by direct activation of mast cells

Moriyama Maiko, Sato Takahiro, Inoue Hiromasa, Fukuyama Satoru, Teranishi Hitoshi, Kangawa Kenji, Kano Tatsuhiko, Yoshimura Akihiko, Kojima Masayasu

Journal of Experimental Medicine 202 （ 2 ） 217 - 224 2005年07月

ISSN 0022-1007

Neuromedin U (NMU) is a neuropeptide that is expressed in the gastrointestinal tract and central nervous system. NMU interacts with two G protein-coupled receptors, NMU-R1 and NMU-R2. Whereas NMU-R2 localizes predominantly to nerve cells, NMU-R1 is expressed in peripheral tissues including lymphocytes and monocytes, suggesting a role of NMU in immunoregulation. However, the functions of NMU in peripheral tissues have not been clarified. In this study, using NMU-deficient mice, we first demonstrated that NMU plays an important role in mast cell-mediated inflammation. Complete Freund's adjuvant-induced mast cell degranulation as well as edema and neutrophil infiltration, which occurred weakly in mast cell-deficient WBB6F ₁ - W/W^v mice, did not occur in NMU-deficient mice. Moreover, intraplantar injection of NMU into paws induced early inflammatory responses such as mast cell degranulation, vasodilation, and plasma extravasation in WT mice but not in WBB6F₁ - W/W^v mice. NMU-R1 was highly expressed in primary mast cells, and NMU induced Ca ²⁺ mobilization and degranulation in peritoneal mast cells. These data indicate that NMU promotes mast cell-mediated inflammation; therefore, NMU receptor antagonists could be a novel target for pharmacological inhibition of mast cell-mediated inflammatory diseases. © The Rockefeller University Press.

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WSX-1 over-expression in CD4⁺ T cells leads to hyperproliferation and cytokine hyperproduction in response to TCR stimulation

Takeda Atsunobu, Hamano Shinjiro, Shiraishi Hiroshi, Yoshimura Takeru, Ogata Hisanobu, Ishii Kazunari, Ishibashi Tatsuro, Yoshimura Akihiko, Yoshida Hiroki

International Immunology 17 （ 7 ） 889 - 897 2005年07月

ISSN 0953-8178

WSX-1 is a component of the IL-27R. Analyses of WSX-1 knockout (WSX-1^-/-) mice have shown that IL-27/WSX-1 signaling is essential for the proper development of T_h1 responses and that WSX-1 can suppress cellular activation and pro-inflammatory cytokine production. We have generated transgenic mouse lines over-expressing the WSX-1 gene under the control of the T cell-specific CD2 promoter (WSX-1 Tg mice). Unexpectedly, like activated CD4⁺ T cells from WSX-1^-/- mice, activated CD4⁺ T cells from WSX-1 Tg mice showed increased proliferation, augmented IL-2 production and up-regulated surface expression of activation markers. IL-27-mediated tyrosine phosphorylation of STAT1 was also enhanced in WSX-1 Tg CD4⁺ T cells, but STAT3 activation was normal. Exogenous IL-27 supported the proliferation of wild-type CD4⁺ T cells but suppressed that of WSX-1 Tg cells. WSX-1 over-expression increased IFN-γ production in T_h1-polarized CD4⁺ T cells, but also promoted T_h2 cytokine production under T_h 1-polarizing conditions. Importantly, WSX-1 over-expression failed to suppress T_h2 cytokine production under T_h 2-polarizing conditions. Cytokine hyperproduction was also observed in vivo in WSX-1 Tg mice injected with Con A. Our data suggest that WSX-1 plays a pivotal role in regulating T cell responsiveness to TCR stimulation and that the correct balance of STAT1/STAT3 activation downstream of IL-27R engagement is crucial for the physiological function of IL-27. © The Japanese Society for Immunology 2005. All rights reserved.

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Negative regulation of cytokine signaling and immune responses by SOCS proteins

Yoshimura Akihiko, Nishinakamura Hitomi, Matsumura Yumiko, Hanada Toshikatsu

Arthritis Research and Therapy 7 （ 3 ） 100 - 110 2005年06月

ISSN 1478-6354

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins and interferons. Many of these cytokines exert their biological functions through JAKs (Janus tyrosine kinases) and STAT (signal transduction and activators of transcription) transcription factors. CIS (cytokine-inducible SH2 (Src homology 2) protein) and SOCS (suppressor of cytokine signaling) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine-mediated homeostasis, including innate and adaptive immunity. In this review we focus on the molecular mechanism of the action of CIS/SOCS family proteins and their roles in immune regulation and inflammatory diseases including rheumatoid arthritis. © 2005 BioMed Central Ltd.

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Negative regulation of cytokine signaling

Yoshimura Akihiko

Clinical Reviews in Allergy and Immunology 28 （ 3 ） 205 - 220 2005年06月

ISSN 1080-0549

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins and interferons. Many of these cytokines exert their biological functions through Janus kinases and signal transducers and activators of transcription factors. The cytokine-inducible SH2 protein (CIS) and suppressors of cytokine signaling (SOCS) are members of a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine-mediated homeostasis, including innate and adaptive immunity. This article focuses on the molecular mechanism of the action of CIS/SOCS family proteins and their roles in immune regulation and inflammatory diseases. © Copyright 2005 by Humana Press Inc.

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Induction of hyper Th1 cell-type immune responses by dendritic cells lacking the suppressor of cytokine signaling-1 gene

Hanada Toshikatsu, Tanaka Kentaro, Matsumura Yumiko, Yamauchi Moriyasu, Nishinakamura Hitomi, Aburatani Hiroyuki, Mashima Ryuichi, Kubo Masato, Kobayashi Takashi, Yoshimura Akihiko

Journal of Immunology 174 （ 7 ） 4325 - 4332 2005年04月

ISSN 0022-1767

Suppressor of cytokine signaling (SOCS1/JAB) has been shown to play an important role in regulating dendritic cell (DC) function and suppressing inlammatory diseases and systemic autoimmunity. However, role of SOCS1 in DCs for the initiation of Th cell response has not been clarified. Here we demonstrate that SOCS1-deficient DCs induce stronger Th1-type responses both in vitro and in vivo. SOCS1-deficient DCs induced higher IFN-γ production from naive T cells than wild-type (WT) DCs in vitro. Lymph node T cells also produced a higher amount of IFN-γ when SOCS1-deficient bone marrow-derived DCs (BMDCs) were transferred in vivo. Moreover, SOCS1^-/- BMDCs raised more effective anti-tumor immunity than WT BMDCs. Microarray analysis revealed that IFN-inducible genes were highly expressed in SOCS1-deficient DCs without IFN stimulation, suggesting hyper STAT1 activation in SOCS1 ^-/- DCs. These phenotypes of SOCS1-deicient DCs were similar to those of CD8α⁺ DCs, and in the WT spleen, SOCS1 is expressed at higher levels in the Th2-inducing CD4⁺ DC subset, relative to the Th1-inducing CD8α⁺ DC subset. We propose that reduction of the SOCS1 gene expression in DCs leads to CD8α⁺ DC-like phenotype which promotes Th1-type hyperresponses. Copyright © 2005 by The American Association of Immunologists, Inc.

Stat5a inhibits IL-12-induced Th1 cell differentiation through the induction of suppressor of cytokine signaling expression

Takatori Hiroaki, Nakajima Hiroshi, Kagami Shin Ichiro, Hirose Koichi, Suto Akira, Suzuki Kotaro, Kubo Masato, Yoshimura Akihiko, Saito Yasushi, Iwamoto Itsuo

Journal of Immunology 174 （ 7 ） 4105 - 4112 2005年04月

ISSN 0022-1767

In previous studies, we have shown that Th2 cell differentiation is diminished but Th1 cell differentiation is increased in Stat5a-deficient (Stat5a^-/-) CD4⁺ T cells. In the present study, we clarified the molecular mechanisms of Stat5a-mediated Th cell differentiation. We found that enhanced Th1 cell differentiation and the resultant IFN-γ production played a dominant inhibitory role in the down-regulation of IL-4-induced Th2 cell differentiation of Stat5a^-/- CD4⁺ T cells. We also found that IL-12-induced Stat4 phosphorylation and Th1 cell differentiation were augmented in Stat5a^-/- CB4⁺ T cells. Importantly, the expression of suppressor of cytokine signaling (SOCS)3, a potent inhibitor of IL-12-induced Stat4 activation, was decreased in Stat5a ^-/- CD4⁺ T cells. Moreover, a reporter assay showed that a constitutively active form of Stat5a but not Stat6 activated the SOCS3 promoter. Furthermore, chromatin immunoprecipitation assays revealed that Stat5a binds to the SOCS3 promoter in CD4⁺ T cells. Finally, the retrovirus-mediated expression of SOCS3 restored the impaired Th cell differentiation of Stat5a^-/- CD4⁺ T cells. These results suggest that Stat5a forces the Th1/Th2 balance toward a Th2-type by preventing IL-12-induced Th1 cell differentiation through the induction of SOCS3. Copyright © 2005 by The American Association of Immunologists, Inc.

Modulation of TLR signalling by the C-terminal Src kinase (Csk) in macrophages

Aki Daisuke, Mashima Ryuichi, Saeki Kazuko, Minoda Yasumasa, Yamauchi Moriyasu, Yoshimura Akihiko

Genes to Cells 10 （ 4 ） 357 - 368 2005年04月

ISSN 1356-9597

In macrophages and monocytes, lipopolysaccharide (LPS) triggers the production of pro-inflammatory cytokine through Toll-like receptor (TLR) 4. Although major TLR signalling pathways are mediated by serine or threonine kinases including IKK, TAK1, p38 and JNKs, a number of reports suggested that tyrosine phosphorylation of intracellular proteins is involved in LPS signalling. Here, we identified several tyrosine-phosphorylated proteins using mass spectrometric analysis in response to LPS stimulation. Among these proteins, we characterized C-terminal Src kinase (Csk), which negatively regulates Src-like kinases in RAW 264.7 cells using RNAi knockdown technology. Unexpectedly, LPS-induced CD40 activation and the secretion of pro-inflammatory cytokine such as IL-6 and TNF-α, was down-regulated in Csk knockdown cells. Furthermore, overall cellular tyrosine phosphorylation and TLR4-mediated activation of IκB-α, Erk and p38 but not of JNK, were also down-regulated in Csk knockdown cells. The protein expression levels of a tyrosine kinase, Fgr, were reduced in Csk knockdown cells, suggesting that Csk is a critical regulator of TLR4-mediated signalling by modifying the levels of Src-like kinases. © Blackwell Publishing Limited.

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Regulation of dendritic cell activation and autoimmunity by SOCS1

Hanada Toshikatsu, Yoshimura Akihiko

Nishinihon Journal of Urology 67 （ 4 ） 168 - 175 2005年04月

ISSN 0029-0726

The suppressor of cytokine signaling-1 (SOCS1/JAB) negatively regulates not only the cytokine-signaling pathway, but also lipopolysaccharide (LPS)-induced macrophage activation. We found that SOCS1-deficient dendritic cells (DCs) were also hyperresponsive to interferon-γ (IFNγ) and interleukin-4 (IL-4). To define the role of SOCS1-deficient DCs in vivo, we generated mice in which the SOCS1 expression was restored in T and B cells under a SOCS1 ^-/- background. In these mice, DCs were accumulated in the thymus and spleen and produced high levels of BAFF/BLyS, resulting in the aberrant expansion of B cells and autoreactive antibody production. SOCS1-deficient DCs efficiently stimulated B cell proliferation in vitro and auto-antibody production in vivo. These results indicate that SOCS1 plays an essential role in the normal DC functions and in the suppression of systemic autoimmunity.

Keeping DCs awake by putting SOCS1 to sleep

Kobayashi Takashi, Yoshimura Akihiko

Trends in Immunology 26 （ 4 ） 177 - 179 2005年04月

ISSN 1471-4906

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Physical and functional interactions between STAP-2/BKS and STAT5

Sekine Yuichi, Yamamoto Tetsuya, Yumioka Taro, Sugiyama Kenji, Tsuji Satoshi, Oritani Kenji, Shimoda Kazuya, Minoguchi Mayu, Yoshimura Akihiko, Matsuda Tadashi

Journal of Biological Chemistry 280 （ 9 ） 8188 - 8196 2005年03月

ISSN 0021-9258

Signal-transducing adaptor protein family of proteins (STAPs), which currently contains two members, are proposed to be adaptor molecules because of their pleckstrin homology (PH) and Src-homology 2 (SH2)-like domains. STAP-1 has been shown to interact with STAT5 and the tyrosine kinase Tec. With regard to STAP-2/BKS functions, immunoprecipitation experiments and intracellular stainings revealed STAP-2/BKS binds STAT5 in several types of cells. Mutational studies revealed that the PH- and SH2-like domains of STAP-2/BKS interacted with the C-terminal region of STAT5. STAP-2/BKS and STAT5 were found to constitutively co-localize in the cytoplasm of resting cells, but STAP-2/BKS was found to dissociate upon STAT5 phosphorylation, suggesting a role in regulating signaling of STAT5. The physiological role of these interactions is not fully understood, but in studies of overexpression of STAP-2/BKS, cytokine-induced tyrosine phosphorylation and transcriptional activation of STAT5 was diminished. In addition, thymocytes from STAP-2/BKS-deficient mice showed the enhanced interleukin-2-dependent cell growth. Taken together, STAP-2/BKS is an additional modulator of STAT5-mediated signaling. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Suppressors of cytokine signaling-1 and -3 regulate osteoclastogenesis in the presence of inflammatory cytokines

Ohishi Masanobu, Matsumura Yumiko, Aki Daisuke, Mashima Ryuichi, Taniguchi Koji, Kobayashi Takashi, Kukita Toshio, Iwamoto Yukihide, Yoshimura Akihiko

Journal of Immunology 174 （ 5 ） 3024 - 3031 2005年03月

ISSN 0022-1767

Bone metabolism and the immune system have a correlative relationship, and both are controlled by various common cytokines, such as IFNs and ILs, produced in the bone microenvironments. The suppressor of cytokine signaling-1 (SOCS1) and SOCS3 are negative regulators of such cytokines. Although SOCSs are shown to be induced during osteoclast differentiation, their physiological roles in osteoclast differentiation and function have not been clarified. Thus, we examined the roles of SOCS1 and SOCS3 in Osteoclastogenesis using SOCS1- and SOCS3-deficient mice. IFN-γ-mediated inhibition of osteoclast differentiation from bone marrow-derived monocytes (BMMs) was strongly enhanced in SOCS1-deflcient BMMs, but was diminished in SOCS1-overexpressing BMMs. Moreover, LPS-induced Osteoclastogenesis and bone destruction in vivo were suppressed in SOCS1^+/- mice compared with those in wild-type mice, suggesting that SOCS1 antagonizes the inhibitory effect of IFN-γ on Osteoclastogenesis. SOCS3 did not alter the inhibitory effect of IFNs in Osteoclastogenesis in both gain and loss of functional assays; however, the suppressive effect of IL-6 on osteoclast differentiation was greater in SOCS3-deficient BMMs than in wild-type BMMs in vitro. In addition, IL-6 significantly prevented LPS-induced bone destruction in SOCS3-deficient mice, although it failed in wild-type mice in vivo. In SOCS3-deficient BMMs, expression levels of TNF-receptor-associated factor-6 and IκB were drastically reduced and receptor activator of the NF-κB ligand-induced IκB phospliorylation was severely impaired in the presence of IL-6. These data suggest that both SOCS1 and SOCS3 regulate osteoclastogenesis by blocking the inhibitory effect of inflammatory cytokines on receptor activator of the NF-κB ligand-mediated osteoclast differentiation signals. Selective suppression of SOCS1 and SOCS3 in osteoclast precursors may be a possible therapeutic strategy for inflammatory bone destruction.

SOCS3/CIS3 negative regulation of STAT3 in HGF-induced keratinocyte migration

Tokumaru Sho, Sayama Koji, Yamasaki Kenshi, Shirakata Yuji, Hanakawa Yasushi, Yahata Yoko, Dai Xiuju, Tohyama Mikiko, Yang Lujun, Yoshimura Akihiko, Hashimoto Koji

Biochemical and Biophysical Research Communications 327 （ 1 ） 100 - 105 2005年02月

ISSN 0006-291X

Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes. Because HGF has strong effects on the motility of keratinocytes and is produced by fibroblasts, HGF is thought to regulate keratinocyte migration during wound healing. However, the intracellular signaling mechanism of HGF-induced keratinocyte migration is poorly understood. In this report, we clarify the roles of STAT3 and SOCS/CIS family in HGF-induced keratinocyte migration. HGF activated STAT3 and strongly induced keratinocyte migration. Transfection with the dominant-negative mutant of STAT3 almost completely abolished HGF-induced keratinocyte migration and STAT3 phosphorylation. Next, we studied the mechanisms that regulate STAT3 phosphorylation. HGF enhanced the expression of SOCS3/CIS3 by sixfold within 1 h, but had minimum effect on SOCS1/JAB expression. Transfection with SOCS3/CIS3 almost completely abolished HGF-induced STAT3 phosphorylation and keratinocyte migration, indicating that SOCS3/CIS3 acts as a negative regulator of HGF-induced keratinocyte migration. In conclusion, SOCS3/CIS3 regulates HGF-induced keratinocyte migration by inhibiting STAT3 phosphorylation. © 2004 Elsevier Inc. All rights reserved.

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Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness

Inoue Hiromasa, Kato Reiko, Fukuyama Satoru, Nonami Atsushi, Taniguchi Kouji, Matsumoto Koichiro, Nakano Takako, Tsuda Miyuki, Matsumura Mikiko, Kubo Masato, Ishikawa Fumihiko, Moon Byoung Gon, Takatsu Kiyoshi, Nakanishi Yoichi, Yoshimura Akihiko

Journal of Experimental Medicine 201 （ 1 ） 73 - 82 2005年01月

ISSN 0022-1007

T helper 2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, play a critical role in allergic asthma. These cytokines transmit signals through the Janus kinase/signal transducer and activator of transcription (STAT) and the Ras-extracellular signal-regulated kinase (ERK) signaling pathways. Although the suppressor of cytokine signaling (SOCS) family proteins have been shown to regulate the STAT pathway, the mechanism regulating the ERK pathway has not been clarified. The Sprouty-related Ena/VASP homology 1-domain-containing protein (Spred)-1 has recently been identified as a negative regulator of growth factor-mediated, Ras-dependent ERK activation. Here, using Spred-1 -deficient mice, we demonstrated that Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness, without affecting helper T cell differentiation. Biochemical assays indicate that Spred-1 suppresses IL-5-dependent cell proliferation and ERK activation. These data indicate that Spred-1 negatively controls eosinophil numbers and functions by modulating IL-5 signaling in allergic asthma.

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Hypertrophic responses to cardiotrophin-1 are not mediated by STAT3, but via a MEK5-ERK5 pathway in cultured cardiomyocytes

Takahashi Nobuki, Saito Yoshihiko, Kuwahara Koichiro, Harada Masaki, Tanimoto Keiji, Nakagawa Yasuaki, Kawakami Rika, Nakanishi Michio, Yasuno Shinji, Usami Satoru, Yoshimura Akihiko, Nakao Kazuwa

Journal of Molecular and Cellular Cardiology 38 （ 1 ） 185 - 192 2005年01月

ISSN 0022-2828

gp130-dependent signaling is known to play a critical role in the onset of heart failure. In that regard, cardiotrophin-1 (CT-1) activates several signaling pathways via gp130, and induces hypertrophy in neonatal rat cardiomyocytes. Among the mediators activated by CT-1, STAT3 is thought to be important for induction of cell hypertrophy, though its precise function in the CT-1 signaling pathway is not fully understood. In the present study, therefore, to better understand the significance of STAT3 activity in CT-1 signaling, we infected cultured cardiomyocytes with adenoviral vectors harboring a dominant-negative STAT3 mutant or one of two endogenous negative regulators of cytokine signaling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways [suppressor of cytokine signaling (SOCS) 1 and 3] and then examined their effects on three indexes of CT-1-induced cell hypertrophy: protein synthesis, secretion of brain natriuretic peptide and changes in cell surface area. In control cells, CT-1-induced both STAT3 phosphorylation and cell hypertrophy. Overexpression of dominant-negative STAT3 mutant suppressed CT-1-induced STAT3 phosphorylation, but did not affect cell hypertrophy. On the other hand overexpression of SOCS1 or SOCS3 inhibited both CT-1-induced STAT3 phosphorylation and cell hypertrophy. CT-1 also induced phosphorylations of ERK1/2 and ERK5 in cardiomyocytes, and those, too, were suppressed by overexpression of SOCSs. CT-1-induced cell hypertrophy was suppressed by overexpression of a dominant-negative MEK5 mutant, and not by overexpression of a dominant-negative MEK1 mutant. These findings indicate that the major pathway responsible for the hypertrophic responses to CT-1 is not JAK-STAT3 pathway nor MEK1-ERK1/2 pathway, but MEK5-ERK5 pathway. © 2004 Elsevier Ltd. All rights reserved.

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SOCS1/JAB likely mediates the protective effect of cardiotrophin-1 against lipopolysaccharide-induced left ventricular dysfunction in vivo

Tanimoto Keiji, Saito Yoshihiko, Hamanaka Ichiro, Kuwahara Koichiro, Harada Masaki, Takahashi Nobuki, Kawakami Rika, Nakagawa Yasuaki, Nakanishi Michio, Adachi Yuichiro, Shirakami Gotaro, Fukuda Kazuhiko, Yoshimura Akihiko, Nakao Kazuwa

Circulation Journal 69 （ 11 ） 1412 - 1417 2005年

ISSN 1346-9843

Background: Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of cytokine signaling whose expression is induced in the rat heart by cardiotrophin-1 (CT-1). Sepsis-induced myocardial depression results from the expression of inducible nitric oxide synthase (iNOS) evoked by inflammatory cytokines. Methods and Results: The effect of CT-1 on lipopolysaccharide (LPS)-induced cardiac dysfunction was examined in a rat model of sepsis. In the absence of CT-1, LPS (1 mg/kg ip) elicited a reduction of systolic function and dilation of the ventricular cavity within 3-6 h after administration. These physiological effects were accompanied by increased ventricular phosphorylation of signal transducers and activators of transcription (STAT) 1 and STAT3, activation of nuclear factor-^• B^• and expression of iNOS mRNA. Notably, administration of CT-1 (20^• g ^•/kg iv) immediately prior to LPS significantly inhibited all of these LPS-induced changes. To determine whether SOCS1 expression in cardiomyocytes is sufficient to inhibit LPS- and cytokine-induced expression of iNOS mRNA, the effects of forced expression of SOCS1 in cultured neonatal cardiomyocytes were investigated using an adenovirus-mediated transfection system. Forced expression of SOCS1 significantly inhibited iNOS transcription induced by LPS, tumor necrosis factor-^• ^•or interferon-^•.^• Conclusions: CT-1-mediated expression of SOCS1 in cardiomyocytes may be a useful target for preventing sepsis-induced myocardial depression.

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Receptor tyrosine kinases (c-kit, c-fmns, Flt-3, Tie2, etc.)

Yoshimura Akihiko, Sanada Masato, Mashima Ryuichi

Nippon rinsho. Japanese journal of clinical medicine 63 Suppl 4 207 - 214 2005年

ISSN 0047-1852

Negative regulation of cytokine and TLR signalings by SOCS and others

Naka Tetsuji, Fujimoto Minoru, Tsutsui Hiroko, Yoshimura Akihiko

Advances in Immunology 87 61 - 122 2005年

ISSN 0065-2776

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Activation and translocation of PKCδ is necessary for VEGF-induced ERK activation through KDR in HEK293T cells

Kuriyama Masamitsu, Taniguchi Taizo, Shirai Yasuhito, Sasaki Atsuo, Yoshimura Akihiko, Saito Naoaki

Biochemical and Biophysical Research Communications 325 （ 3 ） 843 - 851 2004年12月

ISSN 0006-291X

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-γ-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCδ knock-down using siRNA method showed that PKCδ played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCδ specific inhibitor, or human PKCδ siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCδ-knockdown HEK293T cells, but co-expression of rat PKCδ-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCδ-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCδ-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCδ-knockdown HEK293T cell. These results indicate that PKCδ is involved in VEGF-KDR-induced ERK activation via C1B domain. © 2004 Published by Elsevier Inc.

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Erratum

Kamio Masaki, Yoshida Takafumi, Ogata Hisanobu, Douchi Tsutomu, Nagata Yukihiro, Inoue Makoto, Hasegawa Mamoru, Yonemitsu Yoshikazu, Yoshimura Akihiko

Oncogene 23 （ 58 ） 9449 2004年12月

ISSN 0950-9232

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Spred-1 negatively regulates interleukin-3-mediated ERK/mitogen-activated protein (MAP) kinase activation in hematopoietic cells

Nonami Atsushi, Kato Reiko, Taniguchi Koji, Yoshiga Daigo, Taketomi Takaharu, Fukuyama Satoru, Harada Mine, Sasaki Atsuo, Yoshimura Akihiko

Journal of Biological Chemistry 279 （ 50 ） 52543 - 52551 2004年12月

ISSN 0021-9258

Sprouty/Spred family proteins have been identified as negative regulators of growth factor-induced ERK/mitogen-activated protein (MAP) kinase activation. However, it has not been clarified whether these proteins regulate cytokine-induced ERK activity. We found that Spred-1 is highly expressed in interleukin-3 (IL-3)-dependent hematopoietic cell lines and bone marrow-derived mast cells. To investigate the roles of Spred-1 in hematopoiesis, we expressed wild-type Spred-1 and a dominant negative form, of Spred-1, ΔC-Spred, in IL-3-and stem cell factor (SCF)-dependent cell lines as well as hematopoietic progenitor cells from mouse bone marrow by retrovirus gene transfer. In IL-3-dependent Ba/F3 cells expressing c-kit, forced expression of Spred-1 resulted in a reduced proliferation rate and ERK activation in response to not only SCF but also IL-3. In contrast, ΔC-Spred augmented IL-3-induced cell proliferation and ERK activation. Wild-type Spred-1 inhibited colony formation of bone marrow cells in the presence of cytokines, whereas AC-Spred-1 expression enhanced colony formation. Augmentation of ERK activation and proliferation in response to IL-3 was also observed in Spred-1-deficient bone marrow-derived mast cells. These data suggest that Spred-1 negatively regulates hematopoiesis by suppressing not only SCF-induced but also IL-3-induced ERK activation.

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Minodronate, a newly developed nitrogen-containing bisphosphonate, suppresses melanoma growth and improves survival in nude mice by blocking vascular endothelial growth factor signaling

Yamagishi Sho Ichi, Abe Riichiro, Inagaki Yosuke, Nakamura Kazuo, Sugawara Hiroshi, Inokuma Daisuke, Nakamura Hideki, Shimizu Tadamichi, Takeuchi Masayoshi, Yoshimura Akihiko, Bucala Richard, Shimizu Hiroshi, Imaizumi Tsutomu

American Journal of Pathology 165 （ 6 ） 1865 - 1874 2004年12月

ISSN 0002-9440

Angiogenesis, a process by which new vascular networks are formed from pre-existing capillaries, is required for tumors to grow, invade, and metastasize. Vascular endothelial growth factor (VEGF), a specific mitogen to endothelial cells, is a crucial factor for tumor angiogenesis. In this study, we investigated whether minodronate, a newly developed nitrogen-containing bisphosphonate, could inhibit melanoma growth and improve survival in nude mice by suppressing the VEGF signaling. We found here that minodronate inhibited melanoma growth and improved survival in nude mice by suppressing the tumor-associated angiogenesis and macrophage infiltration. Minodronate completely inhibited the VEGF-induced increase in DNA synthesis and tube formation in endothelial cells by suppressing NADPH oxidase-mediated reactive oxygen species generation and Ras activation. Furthermore, minodronate inhibited the VEGF-induced expression of intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in endothelial cells. Minodronate decreased DNA synthesis and increased apoptotic cell death of cultured melanoma cells as well. Our present study suggests that minodronate might suppress melanoma growth and improve survival in nude mice by two independent mechanisms; one is by blocking the VEGF signaling in endothelial cells, and the other is by inducing apoptotic cell death of melanoma. The present study provides a novel potential therapeutic strategy for the treatment of melanoma.

Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3

Kawaguchi Takumi, Yoshida Takafumi, Harada Masaru, Hisamoto Takao, Nagao Yumiko, Ide Tatsuya, Taniguchi Eitaro, Kumemura Hiroto, Hanada Shinichiro, Maeyama Michiko, Baba Shinji, Koga Hironori, Kumashiro Ryukichi, Ueno Takato, Ogata Hisanobu, Yoshimura Akihiko, Sata Michio

American Journal of Pathology 165 （ 5 ） 1499 - 1508 2004年11月

ISSN 0002-9440

The pathogenesis of hepatitis C virus (HCV)-associated insulin resistance remains unclear. Therefore, we investigated mechanisms for HCV-associated insulin resistance. Homeostasis model assessment for insulin resistance was increased in patients with HCV infection. An increase in fasting insulin levels was associated with the presence of serum HCV core, the severity of hepatic fibrosis and a decrease in expression of insulin receptor substrate (IRS) 1 and IRS2, central molecules of the insulin-signaling cascade, in patients with HCV infection. Down-regulation of IRS1 and IRS2 was also seen in HCV core-transgenic mice livers and HCV core-transfected human hepatoma cells. Carbobenzoxy-L- leucyl-L-leucyl-L-leucinal, a potent proteosomal proteolysis inhibitor, blocked down-regulation of IRS1 and IRS2 in HCV core-transfected hepatoma cells. In human hepatoma cells, HCV core up-regulated suppressor of cytokine signaling (SOCS) 3 and caused ubiquitination of IRS1 and IRS2. HCV core-induced down-regulation of IRS1 and IRS2 was not seen in SOCS3^-/- mouse embryonic fibroblast cells. Furthermore, HCV core suppressed insulin-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase and Akt, activation of 6-phosphofructo-2-kinase, and glucose uptake. In conclusion, HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced SOCS3 promotes proteosomal degradation of IRS1 and IRS2 through ubiquitination.

Pigment epithelium-derived factor inhibits TNF-α-induced interleukin-6 expression in endothelial cells by suppressing NADPH oxidase-mediated reactive oxygen species generation

Yamagishi Sho Ichi, Inagaki Yosuke, Nakamura Kazuo, Abe Riichiro, Shimizu Tadamichi, Yoshimura Akihiko, Imaizumi Tsutomu

Journal of Molecular and Cellular Cardiology 37 （ 2 ） 497 - 506 2004年08月

ISSN 0022-2828

Pigment epithelium-derived factor (PEDF) has recently been shown to be involved in the pathogenesis of proliferative diabetic retinopathy. Atherosclerosis is an inflammatory-fibroproliferative disease as well. Oxidative stress plays a major role in retinopathy and atherosclerosis. Accordingly, we investigated effects of PEDF on reactive oxygen species (ROS) generation, NF-κB activation and interleukin (IL)-6 expression in TNF-α-exposed HUVEC. TNF-α significantly increased intracellular ROS generation, which was completely blocked by PEDF or diphenylene iodonium, an inhibitor of NADPH oxidase. Further, PEDF completely prevented the TNF-α-induced increase in NADPH oxidase activity. PEDF or an antioxidant, N-acetylcysteine, significantly inhibited the TNF-α-induced NF-κB activation. PEDF inhibited TNF-α-induced expression of IL-6 at both mRNA and protein levels. Moreover, TNF-α downregulated PEDF mRNA levels. Ligand blot analysis revealed that HUVEC possessed a membrane protein with binding affinity for PEDF. The results demonstrated that PEDF inhibited TNF-α-induced NF-κB activation and subsequent IL-6 overexpression in HUVEC by suppressing NADPH oxidase-mediated ROS generation. Our present study suggests that PEDF may play an important role in the development and progression of atherosclerosis. © 2004 Elsevier Ltd. All rights reserved.

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The Sprouty-related protein, Spred, inhibits cell motility, metastasis, and Rho-mediated actin reorganization

Miyoshi Kanta, Wakioka Toru, Nishinakamura Hitomi, Kamio Masaki, Yang Lu, Inoue Makoto, Hasegawa Mamoru, Yonemitsu Yoshikazu, Komiya Setsuro, Yoshimura Akihiko

Oncogene 23 （ 33 ） 5567 - 5576 2004年07月

ISSN 0950-9232

Sprouty and the Sprouty-related protein, Spred (Sprouty-related Ena/vasodilator-stimulated phospho-protein homology-1 (EVH1) domain-containing protein), inhibit Ras-dependent extracellular signal-regulated kinase (ERK) signaling induced by a variety of growth factors. Since Sprouty proteins have been shown to inhibit not only ERK activation but also cell migration, we postulated that Spreds also inhibit cellular migration. Using stably highly metastatic LM8 cells infected with the Spred1-Sendai virus vector, we demonstrated that Spred1 inhibits the metastasis of LM8 cells in nude mice. Spred1 overexpression also inhibited migration of cells in vitro in response to chemokines, CCL19 and CCL21. We also found that Spred1 overexpression dissolved actin-stress fibers. Both EVH1 domain and C-terminal Sprouty-related domain were required for actin reassembly. Spred1 and Spred2 suppressed constitutively activated RhoA (V14RhoA)-induced stress fiber formation and serum response factor activation. Spred1 bound to activated RhoA, but not cdc42 and Rac. Spred1 also inhibited chemokine-induced RhoA activation and active RhoA-induced Rho-kinase activation. These data suggest that Spreds are key regulators of RhoA-mediated cell motility and signal transduction. Furthermore, our study suggests that the induction of Spreds could be a novel strategy for preventing cancer cell metastasis.

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Mucosal T cells bearing TCRγδ play a protective role in intestinal inflammation

Inagaki-Ohara Kyoko, Chinen Takatoshi, Matsuzaki Goro, Sasaki Atsuo, Sakamoto Yukiko, Hiromatsu Kenji, Nakamura-Uchiyama Fukumi, Nawa Yukifumi, Yoshimura Akihiko

Journal of Immunology 173 （ 2 ） 1390 - 1398 2004年07月

ISSN 0022-1767

Intestinal intraepithelial lymphocytes (IEL) bearing TCRγδ represent a major T cell population in the murine intestine. However, the role of γδ IEL in inflammatory bowel diseases (IBD) remains controversial. In this study, we show that γδ IEL is an important protective T cell population against IBD. γδ T cell-deficient (Cδ^-/-) mice developed spontaneous colitis with age and showed high susceptibility to Th1-type 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis at a young age. Transfer of γδ IEL to Cδ^-/- mice ameliorated TNBS-induced colitis, which correlated with decrease of IFN-γ and TNF-α production and an increase of TGF-β production by IEL. Furthermore, a high level of IL-15, which inhibits activation-induced cell death to terminate inflammation, was expressed more in intestinal epithelial cells (EC) from TNBS-treated Cδ^-/- mice than in those from wild-type mice. EC from wild-type mice significantly suppressed the IFN-γ production of IEL from TNBS-treated Cδ^-/- mice, whereas EC from TNBS-treated Cδ^-/- mice did not. These data indicate that γδ IEL play important roles in controlling IBD by regulating mucosal T cell activation cooperated with EC function. Our study suggests that enhancement of regulatory γδ T cell activity is a possible new cell therapy for colitis.

Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity

Mori Hiroyuki, Hanada Reiko, Hanada Toshikatsu, Aki Daisuke, Mashima Ryuichi, Nishinakamura Hitomi, Torisu Takehiro, Chien Kenneth R., Yasukawa Hideo, Yoshimura Akihiko

Nature Medicine 10 （ 7 ） 739 - 743 2004年07月

ISSN 1078-8956

Leptin is an adipocyte-derived hormone that plays a key role in energy homeostasis, yet resistance to leptin is a feature of most cases of obesity in humans and rodents. In vitro analysis suggested that the suppressor of cytokine signaling-3 (Socs3) is a negative-feedback regulator of leptin signaling involved in leptin resistance. To determine the functional significance of Socs3 in vivo, we generated neural cell-specific SOCS3 conditional knockout mice using the Cre-IoxP system. Compared to their wild-type littermates, Socs3-deficient mice showed enhanced leptin-induced hypothalamic Stat3 tyrosine phosphorylation as well as pro-opiomelanocortin (POMC) induction, and this resulted in a greater body weight loss and suppression of food intake. Moreover, the Socs3-deficient mice were resistant to high fat diet-induced weight gain and hyperleptinemia, and insulin-sensitivity was retained. These data indicate that Socs3 is a key regulator of diet-induced leptin as well as insulin resistance. Our study demonstrates the negative regulatory role of Socs3 in leptin signaling in vivo, and thus suppression of Socs3 in the brain is a potential therapy for leptin-resistance in obesity.

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SOCS1 is a suppressor of liver fibrosis and hepatitis-induced carcinogenesis

Yoshida Takafumi, Ogata Hisanobu, Kamio Masaki, Joo Akiko, Shiraishi Hiroshi, Tokunaga Yoko, Sata Michio, Nagai Hisaki, Yoshimura Akihiko

Journal of Experimental Medicine 199 （ 12 ） 1701 - 1707 2004年06月

ISSN 0022-1007

Hepatocellular carcinomas (HCCs) mainly develop from liver cirrhosis and severe liver fibrosis that are established with long-lasting inflammation of the liver. Silencing of the suppressor of the cytokine signaling-1 (SOCS1) gene, a negative regulator of cytokine signaling, by DNA methylation has been implicated in development or progress of HCC. However, how SOCS1 contributes to HCC is unknown. We examined SOCS1 gene methylation in >200 patients with chronic liver disease and found that the severity of liver fibrosis is strongly correlated with SOCS1 gene methylation. In murine liver fibrosis models using dimethylnitrosamine, mice with haploinsufficiency of the SOCS1 gene (SOCS1 ^-/+ mice) developed more severe liver fibrosis than did wild-type littermates (SOCS1^+/+ mice). Moreover, carcinogen-induced HCC development was also enhanced by heterozygous deletion of the SO CS1 gene. These findings suggest that SOCS1 contributes to protection against hepatic injury and fibrosis, and may also protect against hepatocarcinogenesis.

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Single-molecule imaging analysis of Ras activation in living cells

Murakoshi Hideji, Iino Ryota, Kobayashi Takeshi, Fujiwara Takahiro, Ohshima Chika, Yoshimura Akihiko, Kusumi Akihiro

Proceedings of the National Academy of Sciences of the United States of America 101 （ 19 ） 7317 - 7322 2004年05月

ISSN 0027-8424

A single-molecule fluorescence resonance energy transfer (FRET) method has been developed to observe the activation of the small G protein Ras at the level of individual molecules. KB cells expressing H- or K-Ras fused with YFP (donor) were microinjected with the fluorescent GTP analogue BodipyTR-GTP (acceptor), and the epidermal growth factor-induced binding of BodipyTR-GTP to YFP-(H or K)-Ras was monitored by single-molecule FRET. On activation, Ras diffusion was greatly suppressed/immobilized, suggesting the formation of large, activated Ras-signaling complexes. These complexes may work as platforms for transducing the Ras signal to effector molecules, further suggesting that Ras signal transduction requires more than simple collisions with effector molecules. GAP334-GFP recruited to the membrane was also stationary, suggesting its binding to the signaling complex. The single-molecules FRET method developed here provides a powerful technique to study the signal-transduction mechanisms of various G proteins.

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SOC1 inhibits HPV-E7-mediated transformation by inducing degradation of E7 protein

Kamio Masaki, Yoshida Takafumi, Ogata Hisanobu, Douchi Tsutomu, Nagata Yukihiro, Inoue Makoto, Hasegawa Mamoru, Yonemitsu Yoshikazu, Yoshimura Akihiko

Oncogene 23 （ 17 ） 3107 - 3115 2004年04月

ISSN 0950-9232

Human papilloma viruses (HPVs) are small double-stranded DNA viruses that infect mucosal and cutaneous epithelium and induce cervical cancer. It has been shown that interferon (IFN)γ suppresses proliferation of HPV-infected cells by suppressing expression of HPV E7. Here, we found that IFNγ induces not only suppression of E7 transcription but also proteasome-dependent degradation. Suppressor of cytokine signaling-1 (SOCS1)/JAB, a suppressor of cytokine signaling, is known to be induced by IFNγ, and functions as an antioncogene against various hematopoietic oncogenic proteins. SOCS1 contains the SOCS-box, which is shown to recruit ubiquitin transferase to the molecules that interact with SOCS1. We found that SOCS1 interacted with HPV E7 protein and induced ubiquitination and degradation of E7 in a SOCS-box-dependent manner. SOCS1 overexpression also increased Rb protein levels and suppressed proliferation of cervical cancer cell lines infected with HPV. Moreover, E7 protein levels were higher and Rb protein levels were lower in SOCS1-deficient fibroblasts infected with retrovirus vector carrying E7 gene than in wild-type fibroblasts. E7 induced anchorage-independent growth in SOCS1-deficient fibroblasts, but not in wild-type cells. These data suggested that SOCS1 plays an important role in regulating the levels of E7 protein and their transforming potential, and could be a new therapeutic tool for HPV-mediated tumors.

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Analysis of SOCS-3 Promoter Responses to Interferon yγ

Gatto Luana, Berlato Chiara, Poli Valeria, Tininini Silvia, Kinjyo Ichiko, Yoshimura Akihiko, Cassatella Marco A., Bazzoni Flavia

Journal of Biological Chemistry 279 （ 14 ） 13746 - 13754 2004年04月

ISSN 0021-9258

SOCS-3 (suppressor of cytokine signaling 3) is an intracellular protein that is selectively and rapidly induced by appropriate agonists and that modulates responses of immune cells to cytokines by interfering with the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. On the basis of the observations that interferon γ (IFNγ) up-regulates SOCS-3 gene and protein expression in primary mouse macrophages, J774 macrophage cell line and embryonal fibroblasts, we investigated which sequences of the 5′ SOCS-3 gene are responsive to IFNγ. By promoter deletion analysis we identified a functional IFNγ-responsive element, located at nucleotides -72/-64 upstream from the transcription initiation, whose presence and integrity is necessary to ensure responsiveness to IFNγ. This element contains a STAT consensus binding sequence (SOCS-3/STAT-binding element (SBE)) whose specific mutation totally abolished the responsiveness to IFNγ. In contrast, discrete deletion of other 5' regions of the SOCS-3 promoter did not substantially modify the inducibility by IFNγ. Electromobility shift assay analyses revealed that IFNγ promotes specific DNA binding activities to an oligonucleotide probe containing the SOCS-3/SBE sequence. Even though EFNγ triggered tyrosine phosphorylation of both STAT1 and STAT3 in macrophages and J774 cells, only STAT1 was appropriately activated and thus found to specifically bind to the SOCS-3/SBE oligonucleotide probe. Accordingly, IFNγ-induced SOCS-3 protein expression was not impaired in STAT3-deficient embryonal fibroblasts. Taken together, these results demonstrate that the induction of SOCS-3 by IFNγ depends upon the presence of a STAT-binding element in the SOCS-3 promoter that is specifically activated by STAT1.

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Control of α Subunit of Eukaryotic Translation Initiation Factor 2 (eIF2α) Phosphorylation by the Human Papillomavirus Type 18 E6 Oncoprotein

Kazemi Shirin, Papadopoulou Stavroula, Li Suiyang, Su Qiaozhu, Wang Shuo, Yoshimura Akihiko, Matlashewski Greg, Dever Thomas E., Koromilas Antonis E.

Molecular and Cellular Biology 24 （ 8 ） 3415 - 3429 2004年04月

ISSN 0270-7306

Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) at serine 51 inhibits protein synthesis in cells subjected to various forms of stress including virus infection. The human papillomavirus (HPV) E6 oncoprotein contributes to virus-induced pathogenicity through multiple mechanisms including the inhibition of apoptosis and the blockade of interferon (IFN) action. We have investigated a possible functional relationship between the E6 oncoprotein and eIF2α phosphorylation by an inducible-dimerization form of the IFN-inducible protein kinase PKR. Herein, we demonstrate that HPV type 18 E6 protein synthesis is rapidly repressed upon eIF2α phosphorylation caused by the conditional activation of the kinase. The remainder of E6, however, can rescue cells from PKR-mediated inhibition of protein synthesis and induction of apoptosis. E6 physically associates with GADD34/PP1 holophosphatase complex, which mediates translational recovery, and facilitates eIF2α dephosphorylation. Inhibition of eIF2α phosphorylation by E6 mitigates eIF2α-dependent responses to transcription and translation of proapoptotic genes. These findings demonstrate, for the first time, a role of the oncogenic E6 in apoptotic signaling induced by PKR and eIF2α phosphorylation. The functional interaction between E6 and the eIF2α phosphorylation pathway may have important implications for HPV infection and associated pathogenesis.

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Hyperproduction of Proinflammatory Cytokines by WSX-1-Deficient NKT Cells in Concanavalin A-Induced Hepatitis

Yamanaka Atsushi, Hamano Shinjiro, Miyazaki Yoshiyuki, Ishii Kazunari, Takeda Atsunobu, Mak Tak W., Himeno Kunisuke, Yoshimura Akihiko, Yoshida Hiroki

Journal of Immunology 172 （ 6 ） 3590 - 3596 2004年03月

ISSN 0022-1767

Administration of Con A induces liver injury that is considered to be an experimental model for human autoimmune or viral hepatitis, where immunopathology plays roles mediated by activated lymphocytes, especially NK1.1⁺ CD3⁺ NKT cells, and inflammatory cytokines, including IFN-γ and IL-4. In the present study we investigated the role of WSX-1, a component of IL-27R, in Con A-induced hepatitis by taking advantage of WSX-1 knockout mice. WSX-1-deficient mice were more susceptible to Con A treatment than wild-type mice, showing serum alanine aminotransferase elevation and massive necrosis in the liver. Although the development of NKT cells appeared normal in WSX-1 knockout mice, purified NKT cells from the knockout mice produced more IFN-γ and IL-4 than those from wild-type mice in response to stimulation with Con A both in vitro and in vivo. In addition, hyperproduction of proinflammatory cytokines, including IL-1, IL-6, and TNF-α, was observed in the knockout mice after Con A administration. These data revealed a novel role for WSX-1 as an inhibitory regulator of cytokine production and inflammation in Con A-induced hepatitis.

Spred-2 Suppresses Aorta-Gonad-Mesonephros Hematopoiesis by Inhibiting MAP Kinase Activation

Nobuhisa Ikuo, Kato Reiko, Inoue Hirofumi, Takizawa Makiko, Okita Keisuke, Yoshimura Akihiko, Taga Tetsuya

Journal of Experimental Medicine 199 （ 5 ） 737 - 742 2004年03月

ISSN 0022-1007

In midgestation mouse embryos, the aorta-gonad-mesonephros (AGM) region generates hematopoietic stem cells and definitive hematopoiesis is regulated by cell-cell interaction and signaling molecules. We showed that a Ras/mitogen-activated protein (MAP) kinase signaling-specific inhibitor and a dominant negative mutant Ras blocked the production of CD45⁺ hematopoietic cells in embryonic day 11.5 AGM culture, indicating an essential role for the MAP kinase pathway in AGM hematopoiesis. Overexpression of the Ras/MAP kinase pathway regulator, Spred-2, in the AGM culture significantly reduced the number of CD45⁺ cells. In contrast, production of CD45⁺ cells from the AGM region of Spred-2-null mice was up-regulated as compared with wild-type littermates. Furthermore, Spred-2-deficient mice exhibited elevated hematopoietic colony formation from vascular endothelial-cadherin⁺ cells. These data indicate that Spred-2 functions as a negative regulator of AGM hematopoiesis by inhibiting hematopoietic cytokine signaling.

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The role of the inhibitors of interleukin-6 signal transduction SHP2 and SOCS3 for desensitization of interleukin-6 signalling

Fischer Patrick, Lehmann Ute, Sobota Radoslaw M., Schmitz Jochen, Niemand Claudia, Linnemann Sonja, Haan Serge, Behrmann Iris, Yoshimura Akihiko, Johnston James A., Müller-Newen Gerhard, Heinrich Peter C., Schaper Fred

Biochemical Journal 378 （ 2 ） 449 - 460 2004年03月

ISSN 0264-6021

The immediate early response of cells treated with IL-6 (interleukin-6) is the activation of the signal transducer and activator of transcription (STAT)3. The Src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP2 and the feedback inhibitor SOCS3 (suppressor of cytokine signalling) are potent inhibitors of IL-6 signal transduction. Impaired function of SOCS3 or SHP2 leads to enhanced and prolonged IL-6 signalling. The inhibitory function of both proteins depends on their recruitment to the tyrosine motif 759 within glycoprotein gp130. In contrast to inactivation, desensitization of signal transduction is regarded as impaired responsiveness due to prestimulation. Usually, after activation the sensing receptor becomes inactivated by modifications such as phosphorylation, internalization or degradation. We designed an experimental approach which allows discrimination between desensitization and inactivation of IL-6 signal transduction. We observed that pre-stimulation with IL-6 renders cells less sensitive to further stimulation with IL-6. After several hours, the cells become sensitive again. We show that not only signal transduction through previously activated receptors is affected by desensitization but signalling through receptors which were not targeted by the first stimulation was also attenuated (transdesensitization). Interestingly, in contrast to inhibition, desensitization does not depend on the presence of functional SHP2. Furthermore, cells lacking SOCS3 show constitutive STAT3 activation which is not affected by pre-stimulation with IL-6. All these observations suggest that desensitization and inhibition of signalling are mechanistically distinct.

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Regulation of TLR signaling and inflammation by SOCS family proteins

Yoshimura Akihiko, Ohishi Hiroyuki Mori Masanobu, Aki Daisuke, Hanada Toshikatsu

Journal of Leukocyte Biology 75 （ 3 ） 422 - 427 2004年03月

ISSN 0741-5400

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins and interferons. These cytokines exert their biological functions through Janus tyrosine kinases and signal transducer and activator of transcription factors. The cytokine-inducible Src homology 2 protein (CIS) and suppressors of cytokine signaling (SOCS) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine responses, including those that regulate the inflammatory systems. In this short review, we focused on the molecular mechanism of the action of CIS/SOCS family proteins and their roles in Toll-like receptor signal regulation and inflammatory diseases.

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SOCS3 Is a Physiological Negative Regulator for Granulopoiesis and Granulocyte Colony-stimulating Factor Receptor Signaling

Kimura Akiko, Kinjyo Ichiko, Matsumura Yumiko, Mori Hiroyuki, Mashima Ryuichi, Harada Mine, Chien Kenneth R., Yasukawa Hideo, Yoshimura Akihiko

Journal of Biological Chemistry 279 （ 8 ） 6905 - 6910 2004年02月

ISSN 0021-9258

The suppressor of cytokine signaling-3 (SOCS3/CIS3) has been shown to be an important negative regulator of cytokines, especially cytokines that activate STAT3. To examine the role of SOCS3 in neutrophils and the granulocyte colony-stimulating factor (G-CSF) signaling in vivo, we compared neutrophils from two types of conditional knockout mice, LysM-Cre:SOCS3^fl/fl mice and Tie2-Cre:SOCS3^fl/fl mice, in which the Socs3 gene had been deleted in mature neutrophils and hematopoietic stem cells, respectively. The size of the G-CSF-dependent colonies from Tie2-Cre:SOCS3^fl/fl mouse bone marrow was much larger than that of colonies from control wild-type mice, while the size of interleukin-3-dependent colonies was similar. Moreover, LysM-Cre:SOCS3^fl/fl mice had more neutrophils than SOCS3 ^fl/fl mice, suggesting that SOCS3 is a negative regulator of G-CSF signaling in neutrophils. Consistent with this notion, G-CSF-induced STAT3 as well as mitogen-activated protein kinase activation was much stronger and prolonged in SOCS3-deficient mature neutrophils than in wildtype neutrophils. The preventive effect of G-CSF on apoptosis was more prominent in SOCS3-deficient mature neutrophils than in control neutrophils. These data indicate that SOCS3 negatively regulates granulopoiesis and G-CSF signaling in neutrophils and may contribute to neutrophilia or neutropenia.

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Model analysis of difference between EGF pathway and FGF pathway

Yamada Satoshi, Taketomi Takaharu, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 314 （ 4 ） 1113 - 1120 2004年02月

ISSN 0006-291X

The difference in time course of Ras and mitogen activated protein kinase (MAPK) cascade by different growth factors is considered to be the cause of different cellular responses. We have developed the computer simulation of Ras-MAPK signal transduction pathway containing newly identified negative feedback system, Sprouty, and adaptor molecules. Unexpectedly, negative feedback system did not profoundly affect time course of MAPK activation. We propose the key role of fibroblast growth factor receptor substrate 2 (FRS2) in NGF/FGF pathway for sustained MAPK activation. More Grb2-SOS complexes were recruited to the plasma membrane by binding to membrane-bound FRS2 in FGF pathway than in EGF pathway and caused sustained activation of ERK. The EGF pathway with high concentration of EGF receptor also induced sustained MAPK activation, which is consistent with the results in the PC12 cell overexpressing the EGF receptors. The simulated time courses of FRS2 knock-out cells were consistent with those of the reported experimental results. © 2004 Elsevier Inc. All rights reserved.

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Endoplasmic reticulum stress induces p53 cytoplasmic localization and prevents p53-dependent apoptosis by a pathway involving glycogen synthase kinase-3β

Qu LiKe, Huang Shirley, Baltzis Dionissios, Rivas-Estilla Ana Maria, Pluquet Olivier, Hatzoglou Maria, Koumenis Costas, Taya Yoichi, Yoshimura Akihiko, Koromilas Antonis E.

Genes and Development 18 （ 3 ） 261 - 277 2004年02月

ISSN 0890-9369

The tumor suppressor p53, a sensor of multiple forms of cellular stress, is regulated by post-translational mechanisms to induce cell-cycle arrest, senescence, or apoptosis. We demonstrate that endoplasmic reticulum (ER) stress inhibits p53-mediated apoptosis. The mechanism of inhibition involves the increased cytoplasmic localization of p53 due to phosphorylation at serine 315 and serine 376, which is mediated by glycogen synthase kinase-3 β (GSK-3β). ER stress induces GSK-3β binding to p53 in the nucleus and enhances the cytoplasmic localization of the tumor suppressor. Inhibition of apoptosis caused by ER stress requires GSK-3β and does not occur in cells expressing p53 with mutation(s) of serine 315 and/or serine 376 to alanine(s). As a result of the increased cytoplasmic localization, ER stress prevents p53 stabilization and p53-mediated apoptosis upon DNA damage. It is concluded that inactivation of p53 is a protective mechanism utilized by cells to adapt to ER stress.

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Membrane dynamics and cell signaling as studied by single molecule imaging

Kobayashi Takeshi, Murakami Mizuna, Takeda Yoshie, Kusumi Akihiro, Yoshimura Akihiko

Seikagaku. The Journal of Japanese Biochemical Society 76 （ 2 ） 91 - 100 2004年02月

ISSN 0037-1017

STAT3 and MITF cooperatively induce cellular transformation through upregulation of c-fos expression

Joo Akiko, Aburatani Hiroyuki, Morii Eiichi, Iba Hideo, Yoshimura Akihiko

Oncogene 23 （ 3 ） 726 - 734 2004年01月

ISSN 0950-9232

The signal transducer and activator of transcription (STAT) family proteins are transcription factors critical in mediating cytokine signaling. Among them, STAT3 is frequently activated in a number of human cancers and transformed cell lines and is implicated in tumorigenesis. However, although constitutively activated STAT3 mutant (STAT3C) leads to cellular transformation, its transformation potential such as colony-forming activity in soft-agar is much weaker than that of v-src. To identify tumorigenic factors that cooperatively induce cellular transformation with STAT3C, we screened the retroviral cDNA library. We found that the microphthalmia-associated transcription factor (MITF), an essential transcription factor for melanocyte development and pigmentation, induces anchorage-independent growth of NIH-3T3 cells in cooperation with STAT3C. Microarray analysis revealed that c-fos is highly expressed in transformants expressing STAT3C and MITF. Promoter analysis and chromatin immunoprecipitation assay suggested that both STAT3 and MITF can cooperatively upregulate the c-fos gene. In addition, the transformation of NIH-3T3 cells by both MITF and STAT3C was significantly suppressed by a dominant-negative AP-1 retrovirus. These data indicate that MITF and STAT3 cooperatively induce c-fos, resulting in cellular transformation.

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Regulation of cytokine and toll-like receptor signaling by SOCS family genes

Takagi Hiromi, Sanada Takahito, Minoda Yasumasa, Yoshimura Akihiko

Nippon rinsho. Japanese journal of clinical medicine 62 （ 12 ） 2189 - 2196 2004年

ISSN 0047-1852

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through the Toll-like receptor (TLR) 4. Regulation of TLR signaling is a key step for inflammation, septic shock and innate/adaptive immunity. TLR signaling is shown to be regulated by cytokines, such as interferon-gamma (positive) and interleukin-10 and IL-4 (negative). However, molecular mechanisms of the regulation of LPS signaling by cytokines have not been clarified. Cytokine signaling is regulated by CIS/SOCS family proteins. Both SOCS1 and SOCS3 can inhibit JAK tyrosine kinase activity. We demonstrate that SOCS1 and SOCS3 play an important regulatory role in macrophages and dendritic cells (DCs) by modulating TLR signaling. SOCS1 negatively regulates not only the JAK/STAT pathway, but also the TLR-NF-kappaB pathway. SOCS3 protein was strongly induced by both IL-6 and IL-10 in the presence of LPS, but selectively inhibited IL-6 signaling. Therefore lack of SOCS3 gene in macrophages resulted in suppression of TLR signaling by hyperactivation of STAT3.

Negative regulation of cytokine signaling influences inflammation

Yoshimura Akihiko, Mori Hiroyuki, Ohishi Masanobu, Aki Daisuke, Hanada Toshikatsu

Current Opinion in Immunology 15 （ 6 ） 704 - 708 2003年12月

ISSN 0952-7915

Inflammation is progressed by the action of pro-inflammatory cytokines, including IL-1, TNF, IFN-γ and IL-6, and is resolved by anti-inflammatory cytokines, such as IL-4, IL-10, IL-13, IFN-α and transforming growth factor-β (TGF-β). Intracellular signal transduction pathways initiated by these cytokines have been studied extensively; these pathways ultimately activate transcription factors, such as NF-κB, Smad and STATs. Recently, negative feedback regulation of these pathways, mediated by molecules such as A-20, Smad7 and CIS/SOCS family proteins, has been identified. Negative regulation of cytokine signaling is quite important for inflammation.

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Suppressors of cytokine signaling and immunity

Kubo Masato, Hanada Toshikatu, Yoshimura Akihiko

Nature Immunology 4 （ 12 ） 1169 - 1176 2003年12月

ISSN 1529-2908

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WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production

Hamano Shinjiro, Himeno Kunisuke, Miyazaki Yoshiyuki, Ishii Kazunari, Yamanaka Atsushi, Takeda Atsunobu, Zhang Manxin, Hisaeda Hajime, Mak Tak W., Yoshimura Akihiko, Yoshida Hiroki

Immunity 19 （ 5 ） 657 - 667 2003年11月

ISSN 1074-7613

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors and is essential for resistance to Leishmania major infection. In the present study, we demonstrated that WSX-1 was also required for resistance to Trypanosoma cruzi. WSX-1^-/- mice exhibited prolonged parasitemia, severe liver injury, and increased mortality over wild-type mice. WSX-1 ^-/- splenocytes produced enhanced levels of Th2 cytokines, which were responsible for the prolonged parasitemia. Massive necroinflammatory lesions were observed in the liver of infected WSX-1^-/- mice, and IFN-γ that was overproduced in WSX-1^-/- mice compared with wild-type mice was responsible for the lesions. In addition, vast amounts of various proinflammatory cytokines, including IL-6 and TNF-α, were produced by liver mononuclear cells in WSX-1^-/- mice. Thus, during T. cruzi infection, WSX-1 suppresses liver injury by regulating production of proinflammatory cytokines, while controlling parasitemia by suppression of Th2 responses, demonstrating its novel role as an inhibitory regulator of cytokine production.

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Suppressor of cytokine signaling-1 is essential for suppressing dendritic cell activation and systemic autoimmunity

Hanada Toshikatsu, Yoshida Hiroki, Kato Seiya, Tanaka Kentaro, Masutani Kohsuke, Tsukada Jun, Nomura Yoshio, Mimata Hiromitsu, Kubo Masato, Yoshimura Akihiko

Immunity 19 （ 3 ） 437 - 450 2003年09月

ISSN 1074-7613

Suppressor of cytokine signaling-1 (SOCS1/JAB) negatively regulates not only the cytokine-signaling pathway but also lipopolysaccharide (LPS)-induced macrophage activation. We found that SOCS1-deficient dendritic cells (DCs) were also hyperresponsive to interferon-γ and interleukin-4. To define the role of SOCS1-deficient DCs in vivo, we generated mice in which the SOCS1 expression was restored in T and B cells on a SOCS1^-/- background. In these mice, DCs were accumulated in the thymus and spleen and produced high levels of BAFF/BLyS and APRIL, resulting in the aberrant expansion of B cells and autoreactive antibody production. SOCS1-deficient DCs efficiently stimulated B cell proliferation in vitro and autoantibody production in vivo. These results indicate that SOCS1 plays an essential role in the normal DC functions and suppression of systemic autoimmunity.

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MIP-T3 associates with IL-13Rα1 and suppresses STAT6 activation in response to IL-13 stimulation

Niu Yamei, Murata Takashi, Watanabe Ken, Kawakami Koji, Yoshimura Akihiko, Inoue Jun ichiro, Puri Raj K., Kobayashi Nobuyuki

FEBS Letters 550 （ 1-3 ） 139 - 143 2003年08月

ISSN 0014-5793

To unravel the mechanism of interleukin-13 (IL-13)-specific functions, we sought to identify IL-13 receptor (IL-13R) binding molecules. A novel human IL-13Rα1 binding protein (IL13RBP1) has been identified using yeast tri-hybrid system, which was found to encode the same protein as MIP-T3 (microtubule interacting protein that associates with tumor necrosis factor (TNF) receptor associating factor-3 (TRAF3)). It constitutively associates with IL-13Rα1 and suppresses IL-4/13-induced signal transducer and activator of transcription-6 (STAT6) phosphorylation. IL-13-induced STAT6 activation was also inhibited as determined by dual luciferase assay and electrophoretic mobility shift assay (EMSA). These results suggest that MIP-T3 is a novel inhibitor of IL-13 signaling and may be a useful molecule in ameliorating various conditions in which IL-13 plays a central role. © 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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SOCS-3 regulates onset and maintenance of T_H2-mediated allergic responses

Seki Yoh Ichi, Inoue Hiromasa, Nagata Naoko, Hayashi Katsuhiko, Fukuyama Satoru, Matsumoto Koichiro, Komine Okiru, Hamano Shinjiro, Himeno Kunisuke, Inagaki-Ohara Kyoko, Cacalano Nicholas, O'Garra Anne, Oshida Tadahilo, Saito Hirohisa, Johnston James A., Yoshimura Akihiko, Kubo Masato

Nature Medicine 9 （ 8 ） 1047 - 1054 2003年08月

ISSN 1078-8956

Members of the suppressor of cytokine signaling (SOCS) family are involved in the pathogenesis of many inflammatory diseases. SOCS-3 is predominantly expressed in T-helper type 2 (T_H2) cells, but its role in T _H2-related allergic diseases remains to be investigated. In this study we provide a strong correlation between SOCS-3 expression and the pathology of asthma and atopic dermatitis, as well as serum IgE levels in allergic human patients. SOCS-3 transgenic mice showed increased T_H2 responses and multiple pathological features characteristic of asthma in an airway hypersensitivity model system. In contrast, dominant-negative mutant SOCS-3 transgenic mice, as well as mice with a heterozygous deletion of Socs3, had decreased T_H2 development. These data indicate that SOCS-3 has an important role in regulating the onset and maintenance of T _H2-mediated allergic immune disease, and suggest that SOCS-3 may be a new therapeutic target for the development of antiallergic drugs.

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Negative regulation of cytokine signaling and inflammatory diseases

Inagaki-Ohara Kyoko, Hanada Toshikatsu, Yoshimura Akihiko

Current Opinion in Pharmacology 3 （ 4 ） 435 - 442 2003年08月

ISSN 1471-4892

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins and interferons. These cytokines exert their biological functions through Janus tyrosine kinases and signal transducer and activators of transcription (STATs). The cytokine-inducible SH2 proteins (CIS) and suppressors of cytokine signaling (SOCS) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine responses, including those that regulate the inflammatory system. Several recent advances have been made in this field, including treatment of inflammatory diseases by modulating extracellular and intracellular signaling pathways.

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Regulation of FcεRI-mediated signaling by an adaptor protein STAP-2/BSK in rat basophilic leukemia RBL-2H3 cells

Yamamoto Tetsuya, Yumioka Taro, Sekine Yuichi, Sato Noriko, Minoguchi Mayu, Yoshimura Akihiko, Matsuda Tadashi

Biochemical and Biophysical Research Communications 306 （ 3 ） 767 - 773 2003年07月

ISSN 0006-291X

Crosslinking of multivalent antigen bound IgE transduces FcεRI mediated signaling cascades, which activate nonreceptor-type protein-tyrosine kinases and subsequent tyrosine phosphorylation of cellular proteins, and these are critical elements for degranulation in mast cells. We cloned a novel adaptor molecule, signal transducing adaptor protein (STAP)-2 containing PH and SH2-like domains as a c-fms interacting protein. STAP-2 was identical to a recently cloned adaptor molecule, BKS, a substrate of BRK (breast tumor kinase) tyrosine kinase, although its function is still unknown. To examine a novel function of STAP-2/BSK, we expressed STAP-2/BSK or its mutants in rat basophilic leukemia RBL-2H3 cells. Overexpression of STAP-2/BSK resulted in a suppression of FcεRI-mediated calcium mobilization and degranulation. FcεRI-induced tyrosine phosphorylation of phospholipase C-γ (PLC-γ) but not Syk was significantly suppressed in these cells. Furthermore, STAP-2/BSK associated with PLC-γ in vivo. These data indicate that STAP-2/BSK negatively controls the FcεRI-mediated calcium mobilization and degranulation by direct modulation of tyrosine phosphorylation of PLC-γ. © 2003 Elsevier Science (USA). All rights reserved.

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Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding to Raf1.

Sasaki Atsuo, Taketomi Takaharu, Kato Reiko, Saeki Kazuko, Nonami Atsushi, Sasaki Mika, Kuriyama Masamitsu, Saito Naoaki, Shibuya Masafumi, Yoshimura Akihiko

Cell cycle (Georgetown, Tex.) 2 （ 4 ） 281 - 282 2003年07月

ISSN 1538-4101

The B cell-specific major raft protein, Raftlin, is necessary for the integrity of lipid raft and BCR signal transduction

Saeki Kazuko, Miura Yoshiki, Aki Daisuke, Kurosaki Tomohiro, Yoshimura Akihiko

EMBO Journal 22 （ 12 ） 3015 - 3026 2003年06月

ISSN 0261-4189

Recent evidence indicates that membrane microdomains, termed lipid rafts, have a role in B-cell activation as platforms for B-cell antigen receptor (BCR) signal initiation. To gain an insight into the possible functioning of lipid rafts in B cells, we applied liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodologies to the identification of proteins that co-purified with lipid rafts of Raji cells. Among these raft proteins, we characterized a novel protein termed Raftlin (raft-linking protein). Like the Src family kinase, Raftlin is localized exclusively in lipid rafts by fatty acylation of N-terminal Gly2 and Cys3, and is co-localized with BCR before and after BCR stimulation. Disruption of the Raftlin gene in the DT40 B-cell line resulted in a marked reduction in the quantity of lipid raft components, including Lyn and ganglioside GM1, while overexpression of Raftlin increased the content of raft protein. Moreover, BCR-mediated tyrosine phosphorylation and calcium mobilization were impaired by the lack of Raftlin and actually potentiated by overexpression of Raftlin. These data suggest that Raftlin plays a pivotal role in the formation and/ or maintenance of lipid rafts, therefore regulating BCR-mediated signaling.

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IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages

Yasukawa Hideo, Ohishi Masanobu, Mori Hiroyuki, Murakami Masaaki, Chinen Takatoshi, Aki Daisuke, Hanada Toshikatsu, Takeda Kiyoshi, Akira Shizuo, Hoshijima Masahiko, Hirano Toshio, Chien Kenneth R., Yoshimura Akihiko

Nature Immunology 4 （ 6 ） 551 - 556 2003年06月

ISSN 1529-2908

Whereas interleukin-6 (IL-6) is a proinflammatory cytokine, IL-10 is an anti-inflammatory cytokine. Although signal transducer and activator of transcription 3 (STAT3) is essential for the function of both IL-6 and IL-10, it is unclear how these two cytokines have such opposing functions. Here we show that suppressor of cytokine signaling 3 (SOCS3) is a key regulator of the divergent action of these two cytokines. In macrophages lacking the Socs3 gene or carrying a mutation of the SOCS3-binding site in gp130, the lipopolysaccharide-induced production of tumor necrosis factor (TNF) and IL-12 is suppressed by both IL-10 and IL-6. SOCS3 specifically prevents activation of STAT3 by IL-6 but not IL-10. Taken together, these data indicate that SOCS3 selectively blocks signaling by IL-6, thereby preventing its ability to inhibit LPS signaling.

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Cutting edge

Takeda Atsunobu, Hamano Shinjiro, Yamanaka Atsushi, Hanada Toshikatsu, Ishibashi Tatsuro, Mak Tak W., Yoshimura Akihiko, Yoshida Hiroki

Journal of Immunology 170 （ 10 ） 4886 - 4890 2003年05月

ISSN 0022-1767

WSX-1 is a member of the class I cytokine receptor family with homology to IL-12Rβ2 and is essential for the initial mounting of Th1 responses. STAT1 interacts with tyrosine-phosphorylated WSX-1, and the conserved tyrosine residue of the cytoplasmic domain of WSX-1 is essential for transcriptional activation of STAT1. IL-27 stimulation induced STAT1 phosphorylation in wild-type but not in WSX-1-deficient naive CD4⁺ T cells. Although IL-27 did not directly induce IFN-γ production by wild-type CD4⁺ T cells, IL-12-dependent IFN-γ production was augmented by IL-27 stimulation in wild-type naive CD4⁺ T cells but was impaired in WSX-1-deficient naive CD4⁺ T cells. Additionally, IL-27 stimulation induced T-bet and IL-12Rβ2 expression in wild-type, but not in WSX-1-deficient, CD4⁺ T cells. Thus, during the initiation of Th1 differentiation, the IL-27/WSX-1 signaling system plays a pivotal role by STAT1-mediated T-bet induction before the IL-12R system.

Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding to Raf1

Sasaki Atsuo, Taketomi Takaharu, Kato Reiko, Saeki Kazuko, Nonami Atsushi, Sasaki Mika, Kuriyama Masamitsu, Saito Naoaki, Shibuya Masabumi, Yoshimura Akihiko

Nature Cell Biology 5 （ 5 ） 427 - 432 2003年05月

ISSN 1465-7392

The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation^1-3. Raf is activated through both Ras-dependent and Ras-independent mechanisms^4-6, but the regulatory mechanisms of Raf activation remain unclear^7-9. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified^10-13 and characterized as negative regulators of growth-factor-induced ERK activation^14-25. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling^19,25, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.

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Inhibition of STAT3 prevents neointima formation by inhibiting proliferation and promoting apoptosis of neointimal smooth muscle cells

Shibata Rei, Kai Hisashi, Seki Yukihiko, Kato Seiya, Wada Yoshihiro, Hanakawa Yasushi, Hashimoto Koji, Yoshimura Akihiko, Imaizumi Tsutomu

Human Gene Therapy 14 （ 7 ） 601 - 610 2003年05月

ISSN 1043-0342

In cultured vascular smooth muscle cells (SMCs), STAT3 mediates proliferation signal by directly activating transcription of early growth response genes. Recently, we have found that balloon injury transiently induces JAK2 and STAT3 expressions and activations with a peak at day 7 in rat carotid artery. However, the specific role of STAT3 in neointima formation remains unknown. Adenoviral vector encoding a dominant negative STAT3 (AxCAdnSTAT3) or β-galactosidase (control) was overexpressed in a balloon-injured artery to inhibit endogenous STAT3 activation selectively. In controls, neointima became evident after day 4, and reached a maximum at day 14. The number of bromodeoxyuridine (BrdU)-positive proliferating or TUNEL-positive apoptotic neointimal SMCs peaked at day 7, decreasing to lower levels by day 14. AxCAdnSTAT3 not only abrogated STAT3 phosphorylation but also decreased BrdU labeling index by 60% and increased TUNEL index by 35% at day 7 versus controls, resulting in the 40% reduction in the intima/media area ratio at day 14. At day 7, in controls, vascular injury upregulated antiapoptotic mediator Mcl-1 and Bcl-xL expression by 8-fold to 5-fold, respectively, versus sham, whereas proapoptotic Bax slightly increased by 1.5-fold versus sham. AxCAdnSTAT3 reversed the upregulated Mcl-1 and Bcl-xL levels by 70% and 37%, respectively, while having no affect on Bax expression. In conclusion, the STAT3-mediated pathway plays an important role in neointima formation through enhanced vascular SMC accumulation by promoting cell proliferation and survival.

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Suppressor of cytokine signaling 1/JAB and suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 negatively regulate the signal transducers and activators of transcription signaling pathway in normal human epidermal keratinocytes

Yamasaki Kenshi, Hanakawa Yasushi, Tokumaru Sho, Shirakata Yuji, Sayama Koji, Hanada Toshikatsu, Yoshimura Akihiko, Hashimoto Koji

Journal of Investigative Dermatology 120 （ 4 ） 571 - 580 2003年04月

ISSN 0022-202X

The suppressor of cytokine signaling/cytokine-inducible SH2 containing proteins are cytokine inducible and are negative regulators of the signal transducers and activators of the transcription signaling pathway. We investigated the mechanism regulating signal transducers and activators of transcription and the suppressor of cytokine signaling/cytokine-inducible SH2 containing protein family in keratinocytes, one of the major target cells for cytokines. Suppressor of cytokine signaling 1 mRNA was upregulated 3 h post-interferon γ, and a 8.1-fold increase in the suppressor of cytokine signaling 1 mRNA occurred 48 h post-interferon γ. The suppressor of cytokine signaling 3 mRNA was also upregulated from 1 h post-interferon γ, and a 6.7-fold increase in the suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 mRNA occurred between 6 and 12 h post-interferon γ. Interleukin-6 exposure for 1 h enhanced the expression of the suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 mRNA, but the suppressor of cytokine signaling 1/JAB mRNA was not induced by interleukin-6. Interleukin-4 upregulated the suppressor of cytokine signaling 1/JAB and cytokine-inducible SH2 containing protein 1 mRNA, with 3.4-fold and 5.1-fold increases in mRNA observed at 1 h post-interleukin-4, respectively. In contrast, epidermal growth factor, which phosphorylates signal transducers and activators of transcription 3, did not influence the level of the suppressor of cytokine signaling/cytokine-inducible SH2 containing protein family mRNA expression. Transfection of an adenovirus vector expressing the suppressor of cytokine signaling 1/JAB completely inhibited interferon γ-dependent signal transducers and activators of transcription 1 phosphorylation and interleukin-4-dependent signal transducers and activators of transcription 6 phosphorylation. Transfection of adenovirus vector expressing the suppressor of cytokine signaling 1/JAB did not inhibit interleukin-6-dependent signal transducers and activators of transcription 3 phosphorylation - several reports show that the suppressor of cytokine signaling 1/JAB is a potent inhibitor of signal transducers and activators of transcription 3 signaling in the myeloid leukemia M1 cell. Transfection of the adenovirus vector expressing suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 completely inhibited interleukin-6-dependent signal transducers and activators of transcription 3 phosphorylation and partially inhibited interferon γ-dependent signal transducers and activators of transcription 1 phosphorylation. Transfection of the adenovirus vector expressing suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3, however, did not inhibit interleukin-4-dependent signal transducers and activators of transcription 6 phosphorylation. Transfection of the adenovirus vector expressing cytokine-inducible SH2 containing protein 1 had no effect on signal transducers and activators of transcription 1, 3, and 6 signaling in normal keratinocytes. Therefore, the relationship between signal transducers and activators of transcription and suppressor of cytokine signaling is unique in the keratinocytes, and the suppressor of cytokine signaling regulates cytokine signals in these cells.

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Oncostatin M, an interleukin-6 family cytokine, upregulates matrix metalloproteinase-9 through the mitogen-activated protein kinase kinase-extracellular signal-regulated kinase pathway in cultured smooth muscle cells

Nagata Tsuyoshi, Kai Hisashi, Shibata Rei, Koga Mitsuhisa, Yoshimura Akihiko, Imaizumi Tsutomu

Arteriosclerosis, Thrombosis, and Vascular Biology 23 （ 4 ） 588 - 593 2003年04月

ISSN 1079-5642

Objectives - Matrix metalloproteinase (MMP)-9 is implicated in extracellular matrix (ECM) degradation of atherosclerotic lesions. Oncostatin M (OSM) regulates ECM metabolism in various kinds of cells. Thus, we sought to investigate whether OSM regulates MMP-9 expression in cultured rat aortic smooth muscle cells (SMCs) and, if so, to determine the signaling pathway for MMP-9 induction by OSM. Methods and Results - Competitive reverse transcriptase polymerase chain reaction showed that OSM upregulated MMP-9 mRNA expression, peaking at 4 hours and returning to unstimulated levels by 24 hours. Gelatin zymography revealed that MMP-9 activity was increased in the conditioned medium after the 24-hour OSM treatment. Immunoblot analysis demonstrated that OSM transiently induced extracellular signal-regulated kinase (ERK)1/2 and STAT3 phosphorylations with a peak at 15 and 5 minutes, respectively. A MEK1 inhibitor, PD98059, not only blocked ERK1/2 phosphorylation but also abolished the OSM-induced MMP-9 upregulation, whereas the MMP-9 induction was not affected by overexpressing dominant-negative STAT3. In addition, OSM slightly upregulated MMP-2 and downregulated tissue inhibitors of MMP-1 and -3 through different mechanisms from that in case of MMP-9. Conclusions - OSM upregulates MMP-9 expression in SMCs through the MEK-ERK but not STAT3 pathway.

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STAP-2/BKS, an adaptor/docking protein, modulates STAT3 activation in acute-phase response through its YXXQ motif

Minoguchi Mayu, Minoguchi Shigeru, Aki Daisuke, Joo Akiko, Yamamoto Tetsuya, Yumioka Taro, Matsuda Tadashi, Yoshimura Akihiko

Journal of Biological Chemistry 278 （ 13 ） 11182 - 11189 2003年03月

ISSN 0021-9258

As a c-fms-interacting protein, we cloned a novel adaptor molecule, signal-transducing adaptor protein-2 (STAP-2), which contains pleckstrin homology- and Src homology 2-like (PH and SRC) domains and a proline-rich region. STAP-2 is structurally related to STAP-1/BRDG1 (BCR downstream signaling-1), which we had cloned previously from hematopoietic stem cells. STAP-2 is a murine homologue of a recently identified adaptor molecule, BKS, a substrate of BRK tyrosine kinase. STAP-2 was tyrosine-phosphorylated and translocated to the plasma membrane in response to epidermal growth factor when overexpressed in fibroblastic cells. To define the function of STAP-2, we generated mice lacking the STAP-2 gene. STAP-2 mRNA was strongly induced in the liver in response to lipopolysaccharide and in isolated hepatocytes in response to interleukin-6. In the STAP-2^-/- hepatocytes, the interleukin-6-induced expression of acute-phase (AP) genes and the tyrosine-phosphorylation level of STAT3 were reduced specifically at the late phase (6-24 h) of the response. These data indicate that STAP-2 plays a regulatory role in the AP response in systemic inflammation. STAP-2 contains a YXXQ motif in the C-terminal region that is a potential STAT3-binding site. Overexpression of wild-type STAP-2, but not of mutants lacking this motif, enhanced the AP response element reporter activity and an AP protein production. These data suggest that STAP-2 is a new class of adaptor molecule that modulates STAT3 activity through its YXXQ motif.

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Molecular cloning of mammalian Spred-3 which suppresses tyrosine kinase-mediated Erk activation

Kato Reiko, Nonami Atsushi, Taketomi Takaharu, Wakioka Toru, Kuroiwa Asato, Matsuda Yoichi, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 302 （ 4 ） 767 - 772 2003年03月

ISSN 0006-291X

We have reported on Spred-1 and Spred-2, which inhibit MAP kinase activation by interacting with c-kit and ras/raf. Here, we report the cloning of a third member in this family, Spred-3. Spred-3 is expressed exclusively in the brain and its gene locates in chromosome 19q13.13 in human. Like Spred-1 and -2, Spred-3 contains an EVH1 domain in the N-terminus and a Sprouty-related cysteine-rich region (SPR domain) in the C-terminus that is necessary for membrane localization. However, Spred-3 does not possess a functional c-kit binding domain (KBD), since the critical amino acid Arg residue in this region was replaced with Gly in Spred-3. Although Spred-3 suppressed growth factor-induced MAP kinase (Erk) activation, inhibitory activity of Spred-3 was lower than that of Spred-1 or Spred-2. By the analysis of chimeric molecules between Spred-3 and Spred-1, we found that the SPR domain, rather than KBD, is responsible for efficient Erk suppression. The finding of Spred-3 revealed the presence of a novel family of regulators for the Ras/MAP kinase pathway, each member of which may have different specificities for extracellular signals. © 2003 Elsevier Science (USA). All rights reserved.

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The adapter protein APS associates with the multifunctional docking sites Tyr-568 and Tyr-936 in c-Kit

Wollberg Patrik, Lennartsson Johan, Gottfridsson Eva, Yoshimura Akihiko, Rönnstrand Lars

Biochemical Journal 370 （ 3 ） 1033 - 1038 2003年03月

ISSN 0264-6021

The adapter protein APS has previously been shown to be involved in recruiting the ubiquitin E3 ligase c-Cbl to the insulin receptor, the platelet-derived growth factor β-receptor and the erythropoietin receptor, leading to increased degradation of the receptors and inhibition of mitogenesis. Here we demonstrate, by use of immobilized synthetic phosphopeptides corresponding to various autophosphorylated tyrosine residues in the receptor for stem-cell factor (c-Kit), that APS preferentially associates with phosphorylated Tyr-568 and Tyr-936. Tyr-568 has previously been identified as the binding site of the Src family of tyrosine kinases, the Csk-homologous kinase CHK, and the protein tyrosine phosphatase SHP-2. We have recently demonstrated that Tyr-936 is an autophosphorylation site involved in binding the adapter proteins Grb2 and Grb7. We could further demonstrate that the critical determinant for binding of APS is the presence of either a leucine or an isoleucine residue in the position +3 to the phosphorylated tyrosine. This allowed us to design mutants that selectively failed to associate with APS, while still associating with Src family members, SHP-2 and Grb2, respectively.

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Interaction of the growth hormone receptor with cytokine-induced Src homology domain 2 protein in rat adipocytes

Du Ling, Frick G. Peter, Tai Lih Ruey, Yoshimura Akihiko, Goodman H. Maurice

Endocrinology 144 （ 3 ） 868 - 876 2003年03月

ISSN 0013-7227

GH stimulates the phosphorylation of tyrosine residues in the GH receptor (GHR), Janus kinase 2 (JAK2), and other signaling proteins in a transient manner that subsides within 1 h. To assess the possible roles of cytokine-induced Src homology domain 2 (SH2) (CIS/SOCS) proteins in these transient responses, we studied the expression and disposition of CIS/SOCS proteins in rat adipocytes, a physiological target of GH action. A tyrosine-phosphorylated protein that appears to be the GHR was coprecipitated from extracts of GH-treated adipocytes with α-CIS. In contrast, no tyrosine-phosphorylated adipocyte proteins were recovered after immunoprecipitation with α-SOCS3, although coprecipitation of GHR with SOCS3 was readily detected in extracts of 3T3-F442A fibroblasts. Interaction of GHR with CIS peaked between 2 and 10 min after adipocytes were treated with GH, when tyrosine phosphorylation of the GHR was maximal. By 60 min after GH, tyrosine phosphorylation of the GHR declined to very low levels, and its interaction with CIS was reduced correspondingly. Proteasome inhibitors prevented the decline in tyrosine-phosphorylated GHR and prolonged interaction of GHR and CIS for at least 1 h. These findings demonstrate the interaction of CIS with the GHR in vivo and suggest that CIS may enhance degradation of the receptor by a proteasomal pathway.

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Differential control of the NIMA-related kinases, Nek6 and Nek7, by serum stimulation

Minoguchi Shigeru, Minoguchi Mayu, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 301 （ 4 ） 899 - 906 2003年02月

ISSN 0006-291X

Neks (NIMA-related kinases) are mammalian serine/threonine (Ser/Thr) protein kinases structurally related to Aspergillus NIMA (Never in Mitosis, gene A), which plays essential roles in mitotic signaling. Among these kinases, Nek6 and Nek7 are structurally related and constitute a subfamily in the NIMA/Nek family, although their functions still remain almost elusive. In this report, we studied the enzymatic regulation of Nek6 and Nek7 to gain an insight into their cellular functions. Recombinant Nek7 produced in bacteria was active comparably to Nek6; however, the Nek7 activity in mammalian cells was found to be significantly lower than Nek6. Since Nek6 previously has been reported to in vitro phosphorylate p70 ribosomal S6 kinase at Thr412, we examined if Nek6 and Nek7 activities were controlled by the amino acid supplement, which is known to affect the phosphorylation at Thr412, and did not observe any significant effect. However, we unexpectedly found that Nek7 kinase activity was rapidly and efficiently increased by serum deprivation, while Nek6 activity was decreased. This is well consistent with the lower activity of Nek7 in cells under normal growth conditions. In addition, it was suggested that Nek7 activity would be regulated in a cell cycle-dependent manner, although Nek6 was not. These clear differences in enzymatic control between the highly similar kinases, Nek6 and Nek7, suggest their distinct signaling functions in mammalian cells. © 2003 Elsevier Science (USA). All rights reserved.

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A role of suppressor of cytokine signaling 3 (SOCS3/CIS3/SSI3) in CD28-mediated interleukin 2 production

Matsumoto Akira, Seki Yoh ichi, Watanabe Ryosuke, Hayashi Katsuhiko, Johnston James A., Harada Yohsuke, Abe Ryo, Yoshimura Akihiko, Kubo Masato

Journal of Experimental Medicine 197 （ 4 ） 425 - 436 2003年02月

ISSN 0022-1007

Suppressor of cytokine signaling (SOCS)3 has been characterized as a negative feedback regulator in cytokine-mediated Janus kinase signal transducer and activator of transcription signaling. However, this study shows that T cells from transgenic mice expressing SOCS3 exhibit a significant reduction in interleukin (IL)-2 production induced by T cell receptor cross-linking when T cells are costimulated with CD28. Decreased protein expression in SOCS3^+/- mice enhanced CD28-mediated IL-2 production, clearly indicating the correlation between expression level of SOCS3 and IL-2 production ability. The SOCS3 protein interacted with phosphorylated CD28 through its SH2 domain but not the kinase inhibitory region. In addition, a point mutation in the SOCS3 SH2 domain attenuated the inhibition of CD28 function in IL-2 promoter activation. Committed T helper (Th)2 cells exclusively expressed SOCS3 and production of Th2 cytokines, such as IL-4 and IL-5, was much less dependent on CD28 costimulation compared with interferon γ and IL-2 production in Th1 cells. Consistent with this notion, the expression level of SOCS3 in early T cell activation influenced the ability of IL-2 production induced by CD28 costimulation. Therefore, the SOCS3 may play an alternative role in prohibiting excessive progression of CD28-mediated IL-2 production.

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The suppressor of cytokine signaling-1 (SOCS1) is a novel therapeutic target for enterovirus-induced cardiac injury

Yasukawa Hideo, Yajima Toshitaka, Duplain Hervé, Iwatate Mitsuo, Kido Masakuni, Hoshijima Masahiko, Weitzman Matthew D., Nakamura Tomoyuki, Woodard Sarah, Xiong Dingding, Yoshimura Akihiko, Chien Kenneth R., Knowlton Kirk U.

Journal of Clinical Investigation 111 （ 4 ） 469 - 478 2003年02月

ISSN 0021-9738

Enteroviral infections of the heart are among the most commonly identified causes of acute myocarditis in children and adults and have been implicated in dilated cardiomyopathy. Although there is considerable information regarding the cellular immune response in myocarditis, little is known about innate signaling mechanisms within the infected cardiac myocyte that contribute to the host defense against viral infection. Here we show the essential role of Janus kinase (JAK) signaling in cardiac myocyte antiviral defense and a negative role of an intrinsic JAK inhibitor, the suppressor of cytokine signaling (SOCS), in the early disease process. Cardiac myocyte-specific transgenic expression of SOCS1 inhibited enterovirus-induced signaling of JAK and the signal transducers and activators of transcription (STAT), with accompanying increases in viral replication, cardiomyopathy, and mortality in coxsackievirus-infected mice. Furthermore, the inhibition of SOCS in the cardiac myocyte through adeno-associated virus-mediated (AAV-mediated) expression of a dominant-negative SOCS1 increased the myocyte resistance to the acute cardiac injury caused by enteroviral infection. These results indicate that strategies directed at inhibition of SOCS in the heart and perhaps other organs can augment the host-cell antiviral system, thus preventing viral-mediated endorgan damage during the early stages of infection.

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The N-terminal truncated isoform of SOCS3 translated from an alternative initiation AUG codon under stress conditions is stable due to the lack of a major ubiquitination site, Lys-6

Sasaki Atsuo, Inagaki-Ohara Kyoko, Yoshida Takafumi, Yamanaka Atsushi, Sasaki Mika, Yasukawa Hideo, Koromilas Antonis E., Yoshimura Akihiko

Journal of Biological Chemistry 278 （ 4 ） 2432 - 2436 2003年01月

ISSN 0021-9258

The suppressor of cytokine signaling-3 (SOCS3/CIS-33/SSI-3) is an important negative regulator of cytokine signaling. Here, we show that an N-terminal truncated isoform (AN-SOCS3) translated from the internal AUG codon 12 was profoundly induced by endoplasmic reticulum (ER) stress- or active double-stranded RNA-activated protein kinase PKR, as a result of induction of eukaryotic initiation factor 2α phosphorylation. ΔN-SOCS3 exhibited a stronger cytokine-inhibitory activity and a higher stability than WT-SOCS3 in Ba/F3 hematopoietic cells. A potential ubiquitination residue, Lys-6, at the N terminus is evolutionary conserved among SOCS3 species. The K6Q-SOCS3 mutant showed a much longer half-life than WT-SOCS3 in Ba/F3 cells. Furthermore, inhibition of the 26 S proteasome pathway increased both ubiquitination and protein levels of WT-SOCS3 but had no effect on K6Q-SOCS3. SOCS3 mutant lacking the carboxyl-terminal SOCS-box exhibited the same stability as K6Q-SOCS3. These observations suggest that the short form of SOCS3 is a naturally occurring stabilized inhibitory protein, whereas WT-SOCS3 is a short-lived protein modulated by Lys-6 ubiquitination and proteasome-dependent degradation. Our findings provide strong evidence for the first time that translational control plays an important role in stabilization and function of SOCS3.

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Control mechanism of JAK/STAT signal transduction pathway

Yamada Satoshi, Shiono Satoru, Joo Akiko, Yoshimura Akihiko

FEBS Letters 534 （ 1-3 ） 190 - 196 2003年01月

ISSN 0014-5793

Suppressor of cytokine signaling-1 (SOCS1) was identified as the negative regulator of Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signal transduction pathway. However, the kinetics and control mechanism of the pathway have not yet been fully understood. We have developed the computer simulation of the JAK/STAT pathway. Without nuclear phosphatase, SOCS1's binding to JAK did not cause the decrease in nuclear phosphorylated STAT1. However, without SH2 domain-containing tyrosine phosphatase 2 (SHP-2) or cytoplasmic phosphatase, it did. So nuclear phosphatase is considered to be the most important in this system. By changing parameters of the model, dynamical characteristics and control mechanism were investigated. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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SHP2 and SOCS3 contribute to Tyr-759-dependent attenuation of interleukin-6 signaling through gp130

Lehmann Ute, Schmitz Jochen, Weissenbach Manuela, Sobota Radoslaw M., Hörtner Michael, Friederichs Kerstin, Behrmann Iris, Tsiaris William, Sasaki Atsuo, Schneider-Mergener Jens, Yoshimura Akihiko, Neel Benjamin G., Heinrich Peter C., Schaper Fred

Journal of Biological Chemistry 278 （ 1 ） 661 - 671 2003年01月

ISSN 0021-9258

Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kinase cascade. Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has been identified as being involved in negative regulation of IL-6-induced gene induction and activation of the Jak/STAT pathway. Because this site is known to be a recruitment motif for the protein-tyrosine phosphatase SHP2, it has been suggested that SHP2 is the mediator of tyrosine 759-dependent signal attenuation. We recently observed that the suppressor of cytokine-signaling SOCS3 also acts through the tyrosine motif 759 of gp130. However, the relative contributions of SHP2 and SOCS3 to the repression of IL-6 signaling are not understood. Therefore, we designed experiments allowing the independent recruitment of each of these proteins to the IL-6-receptor complex. We show that receptor- and membrane-targeted SHP2 counteracts IL-6 signaling independent of SOCS3 binding to gp130. On the other hand, SOCS3 inhibits signaling in cells expressing a truncated SHP2 protein, which is not recruited to gp130. These data suggest, that there are two, largely distinct modes of negative regulation of gp130 activity, despite the fact that both SOCS3 and SHP2 are recruited to the same site within gp130.

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CIS1 interacts with the Y532 of the prolactin receptor and suppresses prolactin-dependent STAT5 activation

Endo Takaho, Sasaki Atsuo, Minoguchi Mayu, Joo Akiko, Yoshimura Akihiko

Journal of Biochemistry 133 （ 1 ） 109 - 113 2003年01月

ISSN 0021-924X

Prolactin (PRL) interacts with a single-chain prolactin-specific receptor of the cytokine receptor superfamily. PRL triggers the activation of JAK2 kinase, which phosphorylates the PRL receptor itself, and of STAT5, a member of the family of signal transducers and activators of transcription (STAT). We have shown that the STAT5-dependent immediate early gene, CIS1 (Cytokine-Inducible SH2 domain-containing protein-1), suppresses PRL-induced STAT5 activation in vitro as well as in transgenic mice. To facilitate the study of the interactions between CIS1 and the PRL receptor, we have developed the yeast tri-hybrid system, a modification of the yeast two-hybrid system. We expressed CIS1 fused to the DNA-binding domain and PRL receptor cytoplasmic domain fused to the transcription activation domain in the presence or absence of the tyrosine kinase domain of JAK2 in yeast. CIS1 bound to the PRL receptor cytoplasmic domain in a JAK2-dependent manner. Moreover, we determined that the phosphorylated Y532 of the murine PRL receptor is the binding site for CIS1. Interestingly, Y532 has been shown to be unnecessary for STAT5 activation, although CIS1 overexpression suppressed PRL-induced STAT5 activation. These data suggest that the suppression of STAT5 activation by CIS1 is not due to a simple competition with STAT5 but rather to a modification of the receptor by CIS1 binding.

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Negative regulation of cytokine signaling by CIS/SOCS family proteins and their roles in inflammatory diseases.

Hanada T., Kinjyo I., Inagaki-Ohara K., Yoshimura A.

Reviews of physiology, biochemistry and pharmacology 149 72 - 86 2003年

ISSN 0303-4240

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins (ILs) and interferons. These cytokines exert their biological functions through Janus tyrosine kinases (JAKs) and STAT transcription factors. The CIS (cytokine-inducible SH2 protein) and SOCS (suppressors of cytokine signaling) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine responses, including those that regulate the inflammatory systems. In this review, we focused on the molecular mechanism of the action of CIS/SOCS family proteins and their roles in inflammatory diseases. Furthermore, we illustrate several approaches for treating inflammatory diseases by modulating extracellular and intracellular signaling pathways.

Combined hypermethylation and chromosome loss associated with inactivation of SSI-1/SOCS-1/JAB gene in human hepatocellular carcinomas

Nagai Hisaki, Kim Yong Sung, Konishi Noboru, Baba Masaru, Kubota Takeo, Yoshimura Akihiko, Emi Mitsuru

Cancer Letters 186 （ 1 ） 59 - 65 2002年12月

ISSN 0304-3835

We previously demonstrated using restriction landmark genomic scanning-based 2-dimensional genome electrophoresis method decreased results of 16 primary hepatocellular carcinomas (HCCs) revealed reduction of intensity of 60 NotI-landmark spots, and increase in five spots that were frequently observed in HCCs. Most frequently decreased spot (14/16 HCCs) was identified to it corresponds to a gene encoding SSI-1, a JAK-binding protein (SSI-1/SOCS-1/JAB) that regulated the JAK/STAT signal transduction pathway. This signaling pathway is important for relaying signals from various cytokines outside the cell to the inside. Expression level of SOCS-1 messenger RNA was markedly suppressed in 50% of HCCs (4/8). Loss of heterozygosity at the SSI-1 gene, was found in all cases with aberrant expression. Methylation analysis of the CpG-rich regions of SSI-1 gene revealed hypermethylation of these regions. In an additional series of methylation analysis using 30 HCCs, 16 (53%) showed hypermethylation of the gene. These results indicate that the SSI-1 gene is silenced in a substantial portion of HCC though the combined mechanisms of methylation of either 5′ or exon CpG rich regions and by a chromosomal loss of the remaining allele. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

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SOCS1/JAB is a negative regulator of LPS-induced macrophage activation

Kinjyo Ichiko, Hanada Toshikatsu, Inagaki-Ohara Kyoko, Mori Hiroyuki, Aki Daisuke, Ohishi Masanobu, Yoshida Hiroki, Kubo Masato, Yoshimura Akihiko

Immunity 17 （ 5 ） 583 - 591 2002年11月

ISSN 1074-7613

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor (TLR) 4. We show here that the suppressor of cytokine-signaling-1 (SOCS1/JAB) is rapidly induced by LPS and negatively regulates LPS signaling. SOCS1^+/- mice or SOCS1^-/- mice with interferon-γ (IFNγ)-deficient background were more sensitive to LPS-induced lethal effects than were wild-type littermates. LPS-induced NO₂^- synthesis and TNFα production were augmented in SOCS1^-/- macrophages. Furthermore, LPS tolerance, a protection mechanism against endotoxin shock, was also strikingly reduced in SOCS1^-/- cells. LPS-induced I-κB and p38 phosphorylation was upregulated in SOCS1^-/- macrophages, and forced expression of SOCS1 suppressed LPS-induced NF-κB activation. Thus, SOCS1 directly suppresses TLR4 signaling and modulates innate immunity.

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Expression of the suppressor of cytokine signaling-5 (SOCS5) negatively regulates IL-4-dependent STAT6 activation and Th2 differentiation

Seki Yoh Ichi, Hayashi Katsuhiko, Matsumoto Akira, Seki Noriyasu, Tsukada Jun, Ransom John, Naka Tetsuji, Kishimoto Tadamitsu, Yoshimura Akihiko, Kubo Masato

Proceedings of the National Academy of Sciences of the United States of America 99 （ 20 ） 13003 - 13008 2002年10月

ISSN 0027-8424

The development of helper T (Th) cell subsets, which secrete distinct cytokines, plays an important role in determining the type of immune response. The IL-4-mediated Janus kinase-signal transducer and activator of transcription signaling pathway is crucial for mediating Th2 cell development. Notably, this pathway is selectively impaired in Th1 cells, although the molecular basis of this impairment remains unclear. We show here that during Th1 differentiation a reduction in the association of Janus kinase 1 with the IL-4 receptor (IL-4R) correlated with the appearance of the suppressor of cytokine signaling-5 (SOCS5). SOCS5 protein was preferentially expressed in committed Th1 cells and interacted with the cytoplasmic region of the IL-4Rα chain irrespective of receptor tyrosine phosphorylation. This unconventional interaction of SOCS5 protein with the IL-4R resulted in the inhibition of IL-4-mediated signal transducer and activator of transcription-6 activation. T cells from transgenic mice constitutively expressing SOCS5 exhibited a significant reduction of IL-4-mediated Th2 development. Therefore, the induced SOCS5 protein in Th1 differentiation environment may play an important role by regulating Th1 and Th2 balance.

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Activation of STAT3 by the hepatitis C virus core protein leads to cellular transformation

Yoshida Takafumi, Hanada Toshikatsu, Tokuhisa Takeshi, Kosai Ken Ichiro, Sata Michio, Kohara Michinori, Yoshimura Akihiko

Journal of Experimental Medicine 196 （ 5 ） 641 - 653 2002年09月

ISSN 0022-1007

The signal transducer and activator of transcription (STAT) family proteins are transcription factors critical in mediating cytokine signaling. Among them, STAT3 is often constitutively phosphorylated and activated in human cancers and in transformed cell lines and is implicated in tumorigenesis. However, cause of the persistent activation of STAT3 in human tumor cells is largely unknown. The hepatitis C virus (HCV) is a major etiological agent of non-A and non-B hepatitis, and chronic infection by HCV is associated with development of liver cirrhosis and hepatocellular carcinoma. HCV core protein is proposed to be responsible for the virus-induced transformation. We now report that HCV core protein directly interacts with and activates STAT3 through phosphorylation of the critical tyrosine residue. Activation of STAT3 by the HCV core in NIH-3T3 cells resulted in rapid proliferation and up-regulation of Bcl-XL and cyclin-D1. Additional expression of STAT3 in HCV core-expressing cells resulted in anchorage-independent growth and tumorigenesis. We propose that the HCV core protein cooperates with STAT3, which leads to cellular transformation.

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Regulation of cytokine signaling and inflammation

Hanada Toshitkatsu, Yoshimura Akihiko

Cytokine and Growth Factor Reviews 13 （ 4-5 ） 413 - 421 2002年08月

ISSN 1359-6101

Inflammation progresses by the action of pro-inflammatory cytokines, including interleukin-1 (IL-1), the tumor necrosis factor (TNF), gamma-interferon (IFNγ), IL-12, IL-18, and the granulocyte-macrophage colony-stimulating factor, and is resolved by anti-inflammatory cytokines such as IL-4, IL-10, IL-13, IFNα, and the transforming growth factor (TGF)β. The intracellular signal transduction pathways of these cytokines have been studied extensively, and these pathways ultimately activate transcription factors, such as NF-κB, Smad, and STATs. Recently, the negative -feedback regulation of these pathways has been identified. In this review, we provide examples of the relationship between cytokine signal transduction, negative-signal regulation, and inflammatory disease models. Furthermore, we illustrate several approaches for treating inflammatory diseases by modulating extracellular and intracellular signaling pathways. © 2002 Elsevier Science Ltd. All rights reserved.

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Involvement of suppressor of cytokine signaling-3 as a mediator of the inhibitory effects of IL-10 on lipopolysaccharide-induced macrophage activation

Berlato Chiara, Cassatella Marco A., Kinjyo Ichiko, Gatto Luana, Yoshimura Akihiko, Bazzoni Flavia

Journal of Immunology 168 （ 12 ） 6404 - 6411 2002年06月

ISSN 0022-1767

Previous studies have shown that IL-10 can induce the expression of the suppressor of cytokine signaling 3 (SOCS-3) mRNA in human monocytes and neutrophils, suggesting that the capacity of IL-10 to inhibit the expression of LPS-inducible proinflammatory genes may depend on SOCS-3 induction. However, no direct experimental evidence has been provided to support such hypothesis. Herein, we show that stable transfection of SOCS-3 into the mouse macrophage cell line J774 resulted in an inhibition of NO, TNF-α, IL-6, and GM-CSF secretion in response to LPS at levels similar to those exerted by IL-10 in LPS-stimulated wild-type J774. Constitutive SOCS-3 expression also down-regulated the mRNA expression of inducible NO synthase and IL-6 and impaired the production of TNF-α, mainly at a post-transcriptional level. In addition, SOCS-3-transfected cells displayed a constitutive expression of the IL-1R antagonist gene, consistent with the observation that IL-10 enhances IL-1R antagonist mRNA in LPS-stimulated wild-type cells. Furthermore, in peritoneal macrophages harvested from mice carrying heterozygous disruption of the SOCS-3 gene, IL-10 was less effective in repressing LPS-stimulated TNF-α and NO production. Taken together, our data show that SOCS-3 inhibits LPS-induced macrophage activation, strongly supporting the idea that it plays a role in the molecular mechanism by which IL-10 down-modulates the effector functions of LPS-activated macrophages. Finally, we show that forced expression of SOCS-3 significantly suppresses the ability of IL-10 to trigger tyrosine phosphorylation of STAT3. Therefore, SOCS-3 functions both as an LPS signal inhibitor and as a negative feedback regulator of IL-10/STAT3 signaling.

Stat3β inhibits γ-globin gene expression in erythroid cells

Foley Heather A., Ofori-Acquah Solomon F., Yoshimura Akihiko, Critz Stuart, Surendra Baliga B., Pace Betty S.

Journal of Biological Chemistry 277 （ 18 ） 16211 - 16219 2002年05月

ISSN 0021-9258

We demonstrated previously γ-globin gene inhibition in K562 cells and primary erythroid progenitors treated with interleukin-6. Although several cis-acting elements have been identified in the globin promoters, the precise mechanism for cytokine-mediated globin gene regulation remains to be elucidated. In this report we demonstrate inhibitors of Stat3 phosphorylation abrogate interleukin-6-mediated γ gene silencing in erythroid cells. DNA-protein binding studies established Stat3 interaction in the 5′-untranslated γ-globin promoter region. Furthermore, co-transfection experiments with Stat3β demonstrate γ promoter inhibition in a concentration-dependent manner, which was significantly reversed when the cognate Stat3-binding site in the 5′-untranslated region was mutated. These studies establish a novel mechanism for γ gene silencing through the STAT signal transduction pathway.

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Erratum

Hara Hiromitsu, Takeda Atsunobu, Takeuchi Michiyo, Wakeham Andrew C., Itié Annick, Sasaki Masafumi, Mak Tak W., Yoshimura Akihiko, Nomoto Kikuo, Yoshida Hiroki

Journal of Immunology 168 （ 8 ） 4241 2002年04月

ISSN 0022-1767

The apoptotic protease-activating factor 1-mediated pathway of apoptosis is dispensable for negative selection of thymocytes

Hara Hiromitsu, Takeda Atsunobu, Takeuchi Michiyo, Wakeham Andrew C., Itié Annick, Sasaki Masafumi, Mak Tak W., Yoshimura Akihiko, Nomoto Kikuo, Yoshida Hiroki

Journal of Immunology 168 （ 5 ） 2288 - 2295 2002年03月

ISSN 0022-1767

Negative selection is a process to delete potentially autoreactive clones in developing thymocytes. Programmed cell death or apoptosis is thought to play an important role in this selection process. In this study, we investigated the role of apoptotic protease-activating factor 1 (Apaf1), a mammalian homologue of CED-4, in programmed cell death during the negative selection in thymus. There was no developmental abnormality in thymocytes from newborn Apaf1^-/- mice in terms of CD4 and CD8 expression pattern and thymocyte number. Clonal deletion by endogenous male H-Y Ag of Apaf1-deficient thymocytes with transgenic expression of H-Y Ag-specific TCRs (H-Y Tg/Apaf1^-/- thymocytes) was normally observed in lethally irradiated wild-type mice reconstituted with fetal liver-derived hemopoietic stem cells. Clonal deletion induced in vitro by a bacterial superantigen was also normal in fetal thymic organ culture. Thus, Apaf1-mediated pathway of apoptosis is dispensable for the negative selection of thymocytes. However, H-Y Tg/Apaf1^-/- thymocytes showed partial resistance to H-Y peptide-induced deletion in vitro as compared with H-Y Tg/Apaf1^+/- thymocytes, implicating the Apaf1-mediated apoptotic pathway in the negative selection in a certain situation. In addition, the peptide-induced deletion was still observed in H-Y Tg/Apaf1^-/- thymocytes in the presence of a broad spectrum caspase inhibitor, z-VAD-fmk, suggesting the presence of caspase-independent cell death pathway playing roles during the negative selection. We assume that mechanisms for the negative selection are composed of several cell death pathways to avoid failure of elimination of autoreactive clones.

Induction of SOCS family proteins in inflammatory diseases and suppression of inflammatory arthritis model by the adenovirus gene transfer

Yoshimura Akihiko, Shouda Takanori, Yoshida Takafumi, Hanada Toshikatsu

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 47 （ 16 Suppl ） 2355 - 2361 2002年

ISSN 0039-9450

SH2-B is required for both male and female reproduction

Ohtsuka Satoshi, Takaki Satoshi, Iseki Masanori, Miyoshi Kanta, Nakagata Naomi, Kataoka Yuki, Yoshida Nobuaki, Takatsu Kiyoshi, Yoshimura Akihiko

Molecular and Cellular Biology 22 （ 9 ） 3066 - 3077 2002年

ISSN 0270-7306

Many growth factors and hormones modulate the reproductive status in mammals. Among these, insulin and insulin-like growth factor I (IGF-I) regulate the development of gonadal tissues. SH2-B has been shown to interact with insulin and IGF-I receptors, although the role of SH2-B in these signals has not been clarified. To investigate the role of SH2-B, we generated mice with a targeted disruption of the SH2-B gene. Both male and female SH2-B^-/- mice showed slight retardation in growth and impaired fertility. Female knockout mice possess small, anovulatory ovaries with reduced numbers of follicles and male SH2-B^-/- mice have small testes with a reduced number of sperm. SH2-B^-/-cumulus cells do not respond to either follicle-stimulating hormone or IGF-I. These data suggest that SH2-B plays a critical role in the IGF-I-mediated reproductive pathway in mice.

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Target cell defense prevents the development of diabetes after viral infection

Flodström Malin, Maday Amy, Balakrishna Deepika, Cleary Mary Malo, Yoshimura Akihiko, Sarvetnick Nora

Nature Immunology 3 （ 4 ） 373 - 382 2002年

ISSN 1529-2908

The mechanisms that regulate susceptibility to virus-induced autoimmunity remain undefined. We establish here a fundamental link between the responsiveness of target pancreatic β cells to interferons (IFNs) and prevention of coxsackievirus B4 (CVB4)-induced diabetes. We found that an intact β cell response to IFNs was critical in preventing disease in infected hosts. The antiviral defense, raised by β cells in response to IFNs, resulted in a reduced permissiveness to infection and subsequent natural killer (NK) cell-dependent death. These results show that β cell defenses are critical for β cell survival during CVB4 infection and suggest an important role for IFNs in preserving NK cell tolerance to β cells during viral infection. Thus, alterations in target cell defenses can critically influence susceptibility to disease.

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Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins

Ben-Yair Liora, Slaaby Rita, Herman Asael, Cohen Yael, Biener Eva, Moran Nava, Yoshimura Akihiko, Whittaker Jonathan, De Meyts Pierre, Herman Brian, Gertler Arieh

Protein Expression and Purification 25 （ 3 ） 456 - 464 2002年

ISSN 1046-5928

To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment. © 2002 Elsevier Science (USA). All rights reserved.

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Regulation and function of the cytokine-inducible SH-2 domain proteins, CIS and SOCS3, in mammary epithelial cells

Tonko-Geymayer Sibylle, Goupille Olivier, Tonko Martin, Soratroi Claudia, Yoshimura Akihiko, Streuli Charles, Ziemiecki Andrew, Kofler Reinhard, Doppler Wolfgang

Molecular Endocrinology 16 （ 7 ） 1680 - 1695 2002年

ISSN 0888-8809

The cytokine-inducible src homology 2 (SH-2) proteins, CIS (cytokine inducible SH-2 domain protein) and SOCS3 (suppressor of cytokine signaling 3), are implicated in the negative regulation of prolactin (PRL) receptor-mediated activation of signal transducer and activator of transcription 5 (STAT5). We have studied the expression and function of CIS and SOCS3 proteins in the mouse mammary gland and in HC11 mammary epithelial cells. CIS and SOCS3 were differentially regulated: high expression levels of CIS mRNA were measured during the second half of pregnancy, whereas SOCS3 expression was high during the first 12 d post conceptum. SOCS3 levels increased, whereas CIS levels decreased, in the initial phase of involution. At the beginning of the lactation period both CIS and SOCS3 were high. PRL and epidermal growth factor (EGF) were able to induce CIS and SOCS3, whereas glucocorticoids inhibited their expression in mammary epithelial cells. The effect of EGF was much stronger on SOCS3 than on CIS. Ectopic expression of both SOCS3 and CIS inhibited STAT5 activation. Our data indicate that in the mammary gland CIS and SOCS3 are involved in regulating STAT5 signaling at three different instances: 1) SOCS3 serves as a mediator of the inhibitory EGF effect on PRL-induced STAT5 activation; 2) CIS and SOCS3 play a role as negative feedback inhibitors of PRL action; 3) Inhibition of CIS and SOCS3 expression by glucocorticoids contributes to the positive effect of glucocorticoids on PRL-induced STAT5 activation.

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Interleukin-10 and cAMP-elevating agents cooperate to induce suppressor of cytokine signaling-3 via a protein kinase A-independent signal

Gasperini Sara, Crepaldi Luca, Calzetti Federica, Gatto Luana, Berlato Chiara, Bazzoni Flavia, Yoshimura Akihiko, Cassatella Marco A.

European Cytokine Network 13 （ 1 ） 47 - 53 2002年

ISSN 1148-5493

We have recently shown that IL-10 represents an efficient stimulus for suppressor of cytokine signalling (SOCS)-3 mRNA expression in human neutrophils and PBMC. Herein, we identify cAMP-elevating agents such as prostaglandin E₂ (PGE₂), PGE₁, forskolin, dibutyryl cAMP (dbcAMP) and cholera toxin as a novel class of agonists able to induce SOCS-3 mRNA and protein expression in human leukocytes, cooperating with interleukin-10 (IL-10) in such activities. While PGE₂ or dbcAMP prolonged the stability of SOCS-3 mRNA isolated from IL-10-treated leukocytes, inhibitors of cAMP-dependent protein kinase A (H89, KT5720, and St-Ht31 peptide) did not influence the action of PGE₂/dbcAMP and/or IL-10 on SOCS-3 mRNA and protein expression, implying that their effect are mediated through a PKA-independent pathway. Taken together, our data identify cAMP-elevating substances as a novel class of agonists able to trigger SOCS-3 expression, and suggest that SOCS-3 might be involved in the regulatory effects of cAMP-elevating substances.

Angiogenesis induced by advanced glycation end products and its prevention by cerivastatin.

Okamoto Tamami, Yamagishi Sho ichi, Inagaki Yosuke, Amano Shinjiro, Koga Kohachiro, Abe Riichiro, Takeuchi Masayoshi, Ohno Shigeaki, Yoshimura Akihiko, Makita Zenji

The FASEB journal : official publication of the Federation of American Societies for Experimental Biology 16 （ 14 ） 1928 - 1930 2002年

We previously have found that advanced glycation end products (AGE), senescent macroproteins formed at an accelerated rate in diabetes, arise in vivo not only from glucose but also from reducing sugars. Furthermore, we recently have shown that glyceraldehyde- and glycolaldehyde-derived AGE (glycer- and glycol-AGE) are mainly involved in loss of pericytes, the earliest histopathological hallmark of diabetic retinopathy. However, the effects of these AGE proteins on angiogenesis, another vascular derangement in diabetic retinopathy, remain to be elucidated. In this study, we investigated whether these AGE proteins elicit changes in cultured endothelial cells that are associated with angiogenesis. When human skin microvascular endothelial cells (EC) were cultured with glycer-AGE or glycol-AGE, growth and tube formation of EC, the key steps of angiogenesis, were significantly stimulated. The AGE-induced growth stimulation was significantly enhanced in AGE receptor (RAGE)-overexpressed EC. Furthermore, AGE increased transcriptional activity of nuclear factor-kB (NF-kB) and activator protein-1 (AP-1) and then up-regulated mRNA levels of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) in EC. Cerivastatin, a hydroxymethylglutaryl CoA reductase inhibitor; pyrrolidinedithiocarbamate; or curcumin was found to completely prevent the AGE-induced increase in NF-kB and AP-1 activity, VEGF mRNA up-regulation, and the resultant increase in DNA synthesis in microvascular EC. These results suggest that the AGE-RAGE interaction elicited angiogenesis through the transcriptional activation of the VEGF gene via NF-kB and AP-1 factors. By blocking AGE-RAGE signaling pathways, cerivastatin might be a promising remedy for treating patients with proliferative diabetic retinopathy.

Incadronate disodium inhibits advanced glycation end products-induced angiogenesis in vitro

Okamoto Tamami, Yamagishi Sho ichi, Inagaki Yosuke, Amano Shinjiro, Takeuchi Masayoshi, Kikuchi Seiji, Ohno Shigeaki, Yoshimura Akihiko

Biochemical and Biophysical Research Communications 297 （ 2 ） 419 - 424 2002年

ISSN 0006-291X

We have previously shown that advanced glycation end products (AGE), senescent macroprotein derivatives formed at an accelerated rate in diabetes, induced angiogenesis through overgeneration of autocrine vascular endothelial growth factor (VEGF). In the present study, effects of incadronate disodium, a nitrogen-containing bisphosphonate on AGE-elicited angiogenesis in vitro, were studied. Incadronate disodium was found to completely inhibit AGE-induced increase in DNA synthesis as well as tube formation of human microvascular endothelial cells (EC). Furthermore, incadronate disodium significantly prevented transcriptional activation of nuclear factor-κB and activator protein-1 and the subsequent up-regulation of VEGF mRNA levels in AGE-exposed EC. Farnesyl pyrophosphate, but not geranylgeranyl pyrophosphate, was found to completely restore the anti-angiogenic effects of incadronate disodium on EC. These results suggest that incadronate disodium could block the AGE-signaling pathway in microvascular EC through inhibition of protein farnesylation. Incadronate disodium may be a promising remedy for treatment of patients with proliferative diabetic retinopathy. © 2002 Elsevier Science (USA). All rights reserved.

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Recombinant human CIS2 (SOCS2) protein

Biener Eva, Maurice Sarah, Sandowski Yael, Cohen Yael, Gusakowsky Eugene E., Hooghe Robert, Yoshimura Akihiko, Livnah Oded, Gertler Arieh

Protein Expression and Purification 25 （ 2 ） 305 - 312 2002年

ISSN 1046-5928

The 1x myc-tagged cDNA encoding for human CIS2 protein was subcloned into a pET-29a+ vector in order to express and produce a recombinant S-peptide tagged and 1x myc-tagged protein in Escherichia coli BL21(DE3). The constitutively expressed protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by anion-exchange chromatography on Q-Sepharose. The recombinant form was found to be pure and monomeric as judged by both SDS-PAGE and gel-filtration chromatography and its biological activity was proven by its ability to bind to the tyrosine-phosphorylated cytosolic fragment of human growth hormone receptor fused to glutathione-S-transferase. Recombinant CIS2 was compared by biochemical, immunological, and molecular methods to the CIS2 protein expressed in eukaryotic cells. This report describes the first substantial production of biologically active recombinant human CIS2. © 2002 Elsevier Science (USA). All rights reserved.

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A Mutant Form of JAB/SOCS1 Augments the Cytokine-induced JAK/STAT Pathway by Accelerating Degradation of Wild-type JAB/CIS Family Proteins through the SOCS-box

Hanada Toshikatsu, Yoshida Takafumi, Kinjyo Ichiko, Minoguchi Shigeru, Yasukawa Hideo, Kato Seiya, Mimata Hiromitsu, Nomura Yoshio, Seki Youichi, Kubo Masato, Yoshimura Akihiko

Journal of Biological Chemistry 276 （ 44 ） 40746 - 40754 2001年11月

ISSN 0021-9258

Cytokines exert biological functions by activating Janus tyrosine kinases (JAKs), and JAK inhibitors JAB (also referred to as SOCS1 and SSI1) and CIS3 (SOCS3) play an essential role in the negative regulation of cytokine signaling. We have found that transgenic (Tg) mice expressing a mutant JAB (F59D.JAB) exhibited a more potent STAT3 activation and a more severe colitis than did wild-type littermates after treatment with dextran sulfate sodium. We now find that there is a prolonged activation of JAKs and STATs in response to a number of cytokines in T cells from Tg mice with lck promoter-driven F59D-JAB. Overexpression of F59D-JAB also sustained activation of JAK2 in Ba/F3 cells. These data suggested that F59D-JAB up-regulated STAT activity by sustaining JAK activation. To elucidate molecular mechanisms related to F59D-JAB, we analyzed the effects of F59D-JAB on the JAK/STAT pathway using the 293 cell transient expression system. We found that the C-terminal SOCS-box played an essential role in augmenting cytokine signaling by F59D-JAB. The SOCS-box interacted with the Elongin BC complex, and this interaction stabilized JAB. F59D-JAB induced destabilization of wild-type JAB, whereas overexpression of Elongin BC canceled this effect. Levels of endogenous JAB and CIS3 in T cells from F59D-JAB Tg-mouse were lower than in wild-type mice. We propose that F59D-JAB destabilizes wild-type, endogenous JAB and CIS3 by chelating the Elongin BC complex, thereby sustaining JAK activation.

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Identification of a Dominant Negative Mutant of Sprouty that Potentiates Fibroblast Growth Factor-but Not Epidermal Growth Factor-induced ERK Activation

Sasaki Atsuo, Taketomi Takaharu, Wakioka Toru, Kato Reiko, Yoshimura Akihiko

Journal of Biological Chemistry 276 （ 39 ） 36804 - 36808 2001年09月

ISSN 0021-9258

Various mitogenic stimuli such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and phorbol 12,13-dibutyrate (PDBu) activate the Ras-Raf-MEK-ERK pathway, but the regulatory mechanism of this pathway remains to be investigated. Here we found that in 293 cells, mammalian Sprouty2 and Sprouty4 were rapidly induced by EGF, FGF, and PDBu in an ERK pathway-dependent manner. Forced expression of Sprouty2 and Sprouty4 inhibited FGF-induced ERK activation but did not affect EGF- or PDBu-induced ERK activation. To examine whether endogenous Sproutys were also selective inhibitors, we generated a dominant negative form of Sprouty2 (Y55A) and Sprouty4 (Y53A) in which conserved tyrosine residues were mutated. These mutants reverted the suppressive effect of both Sprouty2 and Sprouty4 but not that of RasGAP or SPRED (Sprouty-related EVH1 domain-containing protein), another Sprouty-related Ras suppressor. Expression of dominant negative Sprouty2 and Sprouty4 enhanced and prolonged FGF- but not EGF-induced ERK activation in 293 cells. In PC12 cells, endogenous Sprouty4 was also induced by FGF. Overexpression of wild-type Sprouty4 blocked FGF-induced differentiation, whereas Y53A-Sprouty4 enhanced it. These observations suggest that endogenous Sprouty2 and Sprouty4 are physiological negative feedback regulators of growth factor-mediated ERK pathway and that there are Sprouty-sensitive and -insensitive ERK activation pathways. Finding a dominant negative form of Sproutys will facilitate the study of the molecular mechanism and physiological function of Sproutys.

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Src-catalyzed Phosphorylation of c-Cbl Leads to the Interdependent Ubiquitination of Both Proteins

Yokouchi Masahiro, Kondo Takeshi, Sanjay Archana, Houghton Adam, Yoshimura Akihiko, Komiya Seturo, Zhang Hui, Baron Roland

Journal of Biological Chemistry 276 （ 37 ） 35185 - 35193 2001年09月

ISSN 0021-9258

The protooncogene c-Cbl has recently emerged as an E3 ubiquitin ligase for activated receptor tyrosine kinases. We report here that c-Cbl also mediates the ubiquitination of another protooncogene, the non-receptor tyrosine kinase c-Src, as well as of itself. The c-Cbl-dependent ubiquitination of Src and c-Cbl requires c-Cbl's RING finger, Src kinase activity, and c-Cbl's tyrosine phosphorylation, probably on Tyr-371. In vitro, c-Cbl forms a stable complex with the ubiquitin-conjugating enzyme UbcH7, but active Src destabilizes this interaction. In contrast, Src inhibition stabilizes the c-Cbl-UbcH7·Src complex. Finally, c-Cbl reduces v-Src protein levels and suppresses v-Src-induced STAT3 activation. Thus, in addition to mediating the ubiquitination of activated receptor tyrosine kinases, c-Cbl also acts as a ubiquitin ligase for the non-receptor tyrosine kinase Src, thereby down-regulating Src.

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Spred is a sprouty-related suppressor of Ras signalling

Wakioka Toru, Sasaki Atsuo, Kato Reiko, Shouda Takanori, Matsumoto Akira, Miyoshi Kanta, Tsuneoka Makoto, Komiya Setsuro, Baron Roland, Yoshimura Akihiko

Nature 412 （ 6847 ） 647 - 651 2001年08月

ISSN 0028-0836

Cellular proliferation, and differentiation of cells in response to extracellular signals, are controlled by the signal transduction pathway of Ras, Raf and MAP (mitogen-activated protein) kinase. The mechanisms that regulate this pathway are not well known. Here we describe two structurally similar tyrosine kinase substrates, Spred-1 and Spred-2. These two proteins contain a cysteine-rich domain related to Sprouty (the SPR domain) at the carboxy terminus. In Drosophila, Sprouty inhibits the signalling by receptors of fibroblast growth factor (FGF) and epidermal growth factor (EGF) by suppressing the MAP kinase pathway. Like Sprouty, Spred inhibited growth-factor-mediated activation of MAP kinase. The Ras-MAP kinase pathway is essential in the differentiation of neuronal cells and myocytes. Expression of a dominant negative form of Spred and Spred-antibody microinjection revealed that endogenous Spred regulates differentiation in these types of cells. Spred constitutively associated with Ras but did not prevent activation of Ras or membrane translocation of Raf. Instead, Spred inhibited the activation of MAP kinase by suppressing phosphorylation and activation of Raf. Spred may represent a class of proteins that modulate Ras-Raf interaction and MAP kinase signalling.

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The SOCS Box of SOCS-1 Accelerates Ubiquitin-dependent Proteolysis of TEL-JAK2

Kamizono Shintaro, Hanada Toshikatsu, Yasukawa Hideo, Minoguchi Shigeru, Kato Reiko, Minoguchi Mayu, Hattori Kimihiko, Hatakeyama Shigetsugu, Yada Masayoshi, Morita Sumiyo, Kitamura Toshio, Kato Hirohisa, Nakayama Kei Ichi, Yoshimura Akihiko

Journal of Biological Chemistry 276 （ 16 ） 12530 - 12538 2001年04月

ISSN 0021-9258

Fusion of the TEL gene on 12p13 to the JAK2 tyrosine kinase gene on 9p24 has been found in human leukemia. TEL-mediated oligomerization of JAK2 results in constitutive activation of the tyrosine kinase (JH1) domain and confers cytokine-independent proliferation on interleukin-3-dependent Ba/F3 cells. Forced expression of the JAK inhibitor gene SOCS1/JAB/SSI-1 induced apoptosis of TEL-JAK2-transformed Ba/F3 cells. This suppression of TEL-JAK2 activity was dependent on SOCS box-mediated proteasomal degradation of TEL-JAK2 rather than on kinase inhibition. Degradation of JAK2 depended on its phosphorylation and its high affinity binding with SOCS1 through the kinase inhibitory region and the SH2 domain. It has been demonstrated that von Hippel-Lindau disease (VHL) tumor-suppressor gene product possesses the SOCS box that forms a complex with Elongin B and C and Cullin-2, and it functions as a ubiquitin ligase. The SOCS box of SOCS1/JAB has also been shown to interact with Elongins; however, ubiquitin ligase activity has not been demonstrated. We found that the SOCS box interacted with Cullin-2 and promoted ubiquitination of TEL-JAK2. Furthermore, overexpression of dominant negative Cullin-2 suppressed SOCS1-dependent TEL-JAK2 degradation. Our study demonstrates the substrate-specific E3 ubiquitin-ligase-like activity of SOCS1 for activated JAK2 and may provide a novel strategy for the suppression of oncogenic tyrosine kinases.

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Induction of JAB/SOCS-1/SSI-1 and CIS3/SOCS-3/SSI-3 is involved in gp130 resistance in cardiovascular system in rat treated with cardiotrophin-1 in vivo

Hamanaka I., Saito Y., Yasukawa H., Kishimoto I., Kuwahara K., Miyamoto Y., Harada M., Ogawa E., Kajiyama N., Takahashi N., Izumi T., Kawakami R., Masuda I., Yoshimura A., Nakao K.

Circulation Research 88 （ 7 ） 727 - 732 2001年04月

ISSN 0009-7330

CIS (cytokine-inducible SH2 protein), SOCS (suppressor of cytokine signaling), or SSI (signal transducers and activators of transcription [STAT]-induced STAT inhibitor) proteins are a family of cytokine-inducible negative regulators of cytokine signaling via Janus kinase (JAK)-STAT pathways. Given the evidence that the JAK-STAT pathway plays a critical role in the cardiovascular system, the primary objective of this study was to assess the effects of the CIS family on JAK-STAT signaling in the cardiovascular system in rats treated with cardiotrophin-1 (CT-1), an interleukin-6 family of cytokines. Intravenous injection of 20 μg/kg body weight of CT-1 induced a transient, marked increase in STAT3 activation in various tissues, including heart and lung, and subsequent upregulation of 2 members of the CIS family, JAK-binding protein (JAB)/SOCS-1/SSI-1 and CIS3/SOCS-3/SSI-3, in the same tissues. It was also observed that CIS3 was directly associated with JAK2 in vivo. Pretreatment with the same dose of CT-1 60 minutes before significantly attenuated the STAT3 activation induced by a second injection of CT-1. We previously reported that intravenous injection of CT-1 results in the nitric oxide (NO)-dependent hypotension accompanied by the induction of inducible NO synthase mRNA. In rats pretreated with CT-1, the induction of inducible NO synthase mRNA or hypotension by subsequent CT-1 injection was not observed. Forced expression of JAB or CIS3, bur not other CISs, directly blocked CT-1-induced STAT3 activation in 293 cells. These results suggest that JAB and CIS3 serve as endogenous inhibitors of CT-1-mediated JAK-STAT signaling in the cardiovascular system in vivo.

Hematopoietic cell-specific adapter proteins, SLP-76 and BLNK, effectively activate NF-AT as well as NF-κB by Syk and Tec PTKs in non-lymphoid cell lines

Yamamoto Tetsuya, Matsuda Tadashi, Junicho Akira, Kishi Hiroyuki, Yoshimura Akihiko, Muraguchi Atsushi

FEBS Letters 491 （ 3 ） 272 - 278 2001年03月

ISSN 0014-5793

To investigate the roles of various hematopoietic cell-specific adapter proteins in T cell receptor (TCR)-signaling leading to nuclear factor of activated T cell (NF-AT) and nuclear factor of κB (NF-κB) activation, we reconstituted TCR-signaling with CD8/ζ, various protein tyrosine kinases (PTKs), and adapter proteins in a non-lymphoid cell line, 293T. We show that SLP-76 and BLNK, but not LAT, effectively co-operated with Syk and Tec family PTKs to activate NF-AT and NF-κB. We also show that Tec family PTKs enhanced endogenous phospholipase C (PLC)-γ1 phosphorylation induced by CD8/ζ and Syk in 293T cells. These results imply that PLC-γ1 may play a critical role in a hematopoietic cell-specific adapter protein-mediated NF-AT and NF-κB activation in a non-lymphoid cell. © 2001 Federation of European Biochemical Societies.

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CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation

Suzuki Asuka, Hanada Toshikatsu, Mitsuyama Keiichi, Yoshida Takafumi, Kamizono Shintaro, Hoshino Tomoaki, Kubo Masato, Yamashita Atsuko, Okabe Masaru, Takeda Kiyoshi, Akira Shizuo, Matsumoto Satoshi, Toyonaga Atsushi, Sata Michio, Yoshimura Akihiko

Journal of Experimental Medicine 193 （ 4 ） 471 - 481 2001年02月

ISSN 0022-1007

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins (ILs) and interferons. These cytokines exert their biological functions through Janus tyrosine kinases and signal transducer and activator of transcription (STAT) transcription factors. We recently identified two intrinsic Janus kinase (JAK) inhibitors, JAK binding protein (JAB; also referred to as suppressor of cytokine signaling [SOCS1]/STAT-induced STAT inhibitor [SSI1]) and cytokine-inducible SH2 protein (CIS)3 (or SOCS3/SSI3), which play an essential role in the negative regulation of cytokine signaling. We have investigated the role of STATs and these JAK inhibitors in intestinal inflammation. Among STAT family members, STAT3 was most strongly tyrosine phosphorylated in human ulcerative colitis and Crohn's disease patients as well as in dextran sulfate sodium (DSS)-induced colitis in mice. Development of colitis as well as STAT3 activation was significantly reduced in IL-6-deficient mice treated with DSS, suggesting that STAT3 plays an important role in the perpetuation of colitis. CIS3, but not JAB, was highly expressed in the colon of DSS-treated mice as well as several T cell-dependent colitis models. To define the physiological role of CIS3 induction in colitis, we developed a JAB mutant (F59D-JAB) that overcame the inhibitory effect of both JAB and CIS3 and created transgenic mice. DSS induced stronger STAT3 activation and more severe colitis in F59D-JAB transgenic mice than in their wild-type littermates. These data suggest that hyperactivation of STAT3 results in severe colitis and that CIS3 plays a negative regulatory role in intestinal inflammation by downregulating STAT3 activity.

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JAB/SOCS1/SSI-1 is an interleukin-2-induced inhibitor of IL-2 signaling

Sporri Bernhard, Kovanen Panu E., Sasaki Atsuo, Yoshimura Akihiko, Leonard Warren J.

Blood 97 （ 1 ） 221 - 226 2001年01月

ISSN 0006-4971

JAB/suppressor of cytokine signaling 1 (SOCS1) STAT-induced STAT inhibitor-1 (SSI-1) (JAB/SOCS1/SSI-1) is an SH2-domain-containing protein that is induced by and negatively regulates signaling by a number of cytokines including interleukin-4 (IL-4), IL-6, interferon (IFN)-γ, prolactin, growth hormone, and erythropoietin. The role of JAB/SOCS1/SSI-1 in IL-2 signaling has been analyzed. JAB/SOCS1/SSI-1 is strongly induced by IL-2 in peripheral blood T cells, and JAB/SOCS1/SSI-1 overexpression strongly inhibits IL-2-induced signal transducer and activator of transcription-5 (Stat5) phosphorylation and transcriptional activity. In cotransfection experiments, JAB/SOCS1/SSI-1 associates with both Jak1 and Jak3; however, JAB/SOCS1/SSI-1 had a greater effect on Jak1 tyrosine phosphorylation and kinase activity. JAB/SOCS1/SSI-1 also interacts with IL-2Rβ, and this interaction requires the A region (residues 313-382) of IL-2Rβ. However, this interaction was not essential for the inhibitory action of JAB. Thus, JAB/SOCS1/SSI-1 is an IL-2-induced inhibitor of IL-2 signaling that functions by inhibiting Jak kinase activity. This suggests an important role for JAB/SOCS1/SSI-1 in regulating T-cell responses. © 2001 by The American Society of Hematology.

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CIS1, a cytokine-inducible SH2 protein, suppresses BCR/ABL-mediated transformation

Tauchi Tetsuzo, Yoshimura Akihiko, Ohyashiki Kazuma

Experimental Hematology 29 （ 3 ） 356 - 361 2001年

ISSN 0301-472X

Objective. BCR/ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive leukemia. A general understanding of BCR/ABL signaling events is emerging, but little is known about the endogenous inhibitors of p210 BCR/ABL. The present study focused attention on CIS1, a cytokine-inducible SH2 protein, as a potential physiologic antagonist for BCR/ABL. Materials and Methods. The murine hematopoietic cell line NSF/N1.H7 stably transfected with BCR/ABL was compared to the parental counterparts for induction of CIS1 by immunoblotting and immunoprecipitation. Cells were treated with a proteasome inhibitor to examine the effect of a proteasome inhibitor on CIS1 protein expression. To determine the effect of CIS1 on BCR/ABL-mediated transformation, we generated Rat-1 fibroblasts transfected with either a control vector, CIS1, BCR/ABL p210, or CIS1 plus BCR/ABL p210.ResultsThree forms of CIS1 with molecular masses of 32, 37, and 47 kDa were detected in BCR/ABL-transformed cells. The 47-kDa protein was a ubiquitinated protein. The proteasome inhibitor increased the formation of complexes between CIS1 and BCR/ABL. Transformation of p210 BCR/ABL was significantly suppressed in cells overexpressing CIS1. Conclusion. The results suggest that CIS1 is an endogenous inhibitor of p210 BCR/ABL and is likely to be important in the pathogenesis of Ph-positive leukemia. Copyright © 2001 International Society for Experimental Hematology.

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Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis

Shouda Takanori, Yoshida Takafumi, Hanada Toshikatsu, Wakioka Toru, Oishi Masanobu, Miyoshi Kanta, Komiya Setsuro, Kosai Ken Ichiro, Hanakawa Yasushi, Hashimoto Koji, Nagata Kensei, Yoshimura Akihiko

Journal of Clinical Investigation 108 （ 12 ） 1781 - 1788 2001年

ISSN 0021-9738

Immune and inflammatory systems are controlled by multiple cytokines, including ILs and INFs. These cytokines exert their biological functions through Janus tyrosine kinases and STAT transcription factors. One such cytokine, IL-6, has been proposed to contribute to the development of rheumatoid arthritis (RA). We found that STAT3 was strongly tyrosine phosphorylated in synovial tissue of RA patients, but not those with osteoarthritis. Blockade of the IL-6-gp130-JAK-STAT3-signaling pathway might therefore be beneficial in the treatment of RA. We show here that the mRNA for the endogenous cytokine signaling repressor CIS3/SOCS3 is abundantly expressed in RA patients. To determine whether CIS3 is effective in treating experimental arthritis, a recombinant adenovirus carrying the CIS3 cDNA was injected periarticularly into the ankle joints of mice with antigeninduced arthritis or collagen-induced arthritis (CIA). Periarticular injection of CIS3 adenovirus drastically reduced the severity of arthritis and joint swelling compared with control groups. CIS3 was more effective than a dominant-negative form of STAT3 in the CIA model. Thus, induction of CIS3 could represent a new approach for effective treatment of RA.

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Suppressor of cytokine signaling-3 is a biomechanical stress-inducible gene that suppresses gp130-mediated cardiac myocyte hypertrophy and survival pathways

Yasukawa Hideo, Hoshijima Masahiko, Gu Yusu, Nakamura Tomoyuki, Pradervand Sylvain, Hanada Toshikatsu, Hanakawa Yasushi, Yoshimura Akihiko, Ross John, Chien Kenneth R.

Journal of Clinical Investigation 108 （ 10 ） 1459 - 1467 2001年

ISSN 0021-9738

The gp130 cytokine receptor activates a cardiomyocyte survival pathway during the transition to heart failure following the biomechanical stress of pressure overload. Although gp130 activation is observed transiently during transverse aortic constriction (TAC), its mechanism of inactivation is largely unknown in cardiomyocytes. We show here that suppressor of cytokine signaling 3 (SOCS3), an intrinsic inhibitor of JAK, shows biphasic induction in response to TAC. The induction of SOCS3 was closely correlated with STAT3 phosphorylation, as well as the activation of an embryonic gene program, suggesting that cardiac gp130-JAK signaling is precisely controlled by this endogenous suppressor. In addition to its cytoprotective action, gp130-dependent signaling induces cardiomyocyte hypertrophy. Adenovirus-mediated gene transfer of SOCS3 to ventricular cardiomyocytes completely suppressed both hypertrophy and antiapoptotic phenotypes induced by leukemia inhibitory factor (LIF). To our knowledge, this is the first clear evidence that these two separate cardiomyocyte phenotypes induced by gp130 activation lie downstream of JAK. Three independent signaling pathways, STAT3, MEK1-ERK1/2, and AKT activation, that are coinduced by LIF stimulation were completely suppressed by SOCS3 overexpression. We conclude that SOCS3 is a mechanical stress-inducible gene in cardiac muscle cells and that it directly modulates stress-induced gp130 cytokine receptor signaling as the key molecular switch for a negative feedback circuit for both myocyte hypertrophy and survival.

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Mitogen-activated protein kinase plays an essential role in the erythropoietin-dependent proliferation of CTLL-2 cells

Sakamoto H., Kitamura T., Yoshimura A.

Journal of Biological Chemistry 275 （ 46 ） 35857 - 35862 2000年11月

ISSN 0021-9258

Erythropoietin (EPO) and its receptor (EPOR) are required for development of erythrocytes. It has been shown that the ectopic expression of EPOR confers EPO-dependent proliferation on an interleukin 3 (IL3)-dependent cell line, Ba/F3, whereas the IL2-dependent T cell line, CTLL-2 expressing the EPOR (T-ER), fails to proliferate in response to EPO. However, the molecular basis of the EPO unresponsiveness in CTLL-2 has not been clarified. We found that the expression level of JAK2 in T-ER cells was much lower than that in Ba/F3 cells. Therefore, we examined the effects of forced expression of JAK2 in T-ER cells. In T-ER transformants expressing JAK2 (T-JER), EPO induced tyrosine phosphorylation of the EPOR, JAK2, and STAT5, and consequently STAT5-responsive genes including bcl-X and cis1 were normally induced. Furthermore, T-JER cells were resistant to apoptosis until at least 72 h after switching from IL2 to EPO. Although T-JER cells could not continuously proliferate in the presence of EPO, additional expression of JAK2 in T-JER (T-JJER) to a level similar to that in Ba/F3 cells supported long term proliferation in response to EPO. JAK2 was equally co-immunoprecipitated with the EPOR among T-JER, T-JJER, and Ba/F3 cells expressing the EPOR (BF-ER). However, EPO-dependent mitogen-activated protein (MAP) kinase activation was observed in T-JJER and BF-ER cells but not in T-JER cells. EPO-dependent long term proliferation of T-JER cells was conferred by expression of the constitutively activated form of MEK1. Our results suggest that MAP kinase activation is, at least in part, an important component for mitotic signal from the EPOR, and CTLL-2 cells probably lack signaling molecule(s) in JAK2 and the Ras-MAP kinase pathway.

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The JAK-inhibitor, JAB/SOCS-1 selectively inhibits, cytokine-induced, but not v-Src induced JAK-STAT activation

Iwamoto Takashi, Senga Takeshi, Naito Yuko, Matsuda Satoru, Miyake Yozo, Yoshimura Akihiko, Hamaguchi Michinari

Oncogene 19 （ 41 ） 4795 - 4801 2000年09月

ISSN 0950-9232

Recently, constitutive activation of JAK kinases (JAKs) and/or signal transducers and activators of transcription (STATs) has been reported in growing numbers of human cancer cells as well as oncogene-transformed cells. JAB/SOCS-1 has been shown to be an intrinsic JAK tyrosine kinase inhibitor and to suppress the cytokine-dependent JAK-STAT pathway. In this report, we investigated the effect of ectopic expression of JAB on v-Src-induced JAK-STAT activation. Forced expression of JAB in v-Src-transformed NIH3T3 cells neither suppressed phosphorylation of STAT3 and JAK1/JAK2 nor blocked STAT3-reporter gene activation. Colony forming assay also showed that JAB did not suppress v-Src-lnduced transformation of NIH3T3 cells, while dominant negative STAT3 suppressed it. In contrast, JAB could downregulate pbospborylation of STAT1 and STAT3 induced by interferon gamma (IFN(γ)) and interleukin-6 (IL-6) plus soluble IL6 receptor (sIL-6R), respectively. Furthermore, in vitro kinase assay indicated that JAB suppressed byperactivation of JAK1/JAK2 and JAK1 induced by INF(γ) and IL-6 plus sIL-6R respectively, but not v-Src-induced basal JAKI/JAK2 activity. Nevertheless, both JAK1/JAK2 activated by v-Src and that activated by IL-6 plus slL-6R could similarly bind JAB. These results clearly demonstrate that JAB distinguishes cytokine-induced JAK-STAT signaling from v-Src-induced one and can not suppress the transformation with v-Src.

CIS3/SOCS-3 suppresses erythropoietin (EPO) signaling by binding the EPO receptor and JAK2

Sasaki Atsuo, Yasukawa Hideo, Shouda Takanori, Kitamura Toshio, Dikic Ivan, Yoshimura Akihiko

Journal of Biological Chemistry 275 （ 38 ） 29338 - 29347 2000年09月

ISSN 0021-9258

The cytokine-inducible SH2 protein-3 (CIS3/SOCS-3/SSI-3) has been shown to inhibit the JAK/STAT pathway and act as a negative regulator of fetal liver erythropoiesis. Here, we studied the molecular mechanisms by which CIS3 regulates the erythropoietin (EPO) receptor (EPOR) signaling in erythroid progenitors and Ba/F3 cells expressing the EPOR (BF-ER). CIS3 binds directly to the EPOR as well as JAK2 and inhibits EPO-dependent proliferation and STAT5 activation. We have identified the region containing Tyr⁴⁰¹ in the cytoplasmic domain of the EPOR as a direct binding site for CIS3. Deletion of the Tyr⁴⁰¹ region of the EPOR reduced the inhibitory effect of CIS3, suggesting that binding of CIS3 to the EPOR augmented the negative effect of CIS3. Both N- and C-terminal regions adjacent to the SH2 domain of CIS3 were necessary for binding to EPOR and JAK2. In the N-terminal region of CIS3, the amino acid Gly⁴⁵ was critical for binding to the EPOR but not to JAK2, while Leu²² was critical for binding to JAK2. The mutation of G45A partially reduced ability of CIS3 to inhibit EPO-dependent proliferation and STAT5 activation, while L22D mutant CIS3 was completely unable to suppress EPOR signaling. Moreover, overexpression of STAT5, which also binds to Tyr⁴⁰¹, reduced the binding of CIS3 to the EPOR, and the inhibitory effect of CIS3 against EPO signaling, while it did not affect JAB/SOCS-1/SSI-1. These data demonstrate that binding of CIS3 to the EPOR augments the inhibitory effect of CIS3. CIS3 binding to both EPOR and JAK2 may explain a specific regulatory role of CIS3 in erythropoiesis.

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Intracellular retention and degradation of the epidermal growth factor receptor, Two distinct processes mediated by benzoquinone ansamycins

Supino-Rosin Lia, Yoshimura Akihiko, Yarden Yossef, Elazar Zvulun, Neumann Drorit

Journal of Biological Chemistry 275 （ 29 ） 21850 - 21855 2000年07月

ISSN 0021-9258

Epidermal growth factor (EGF) stimulates the growth of various types of cells via its cell surface tyrosine kinase receptor. The EGF receptor (EGF-R) has an on-cogenic potential when overexpressed in a wide range of tumor cells. Geldanamycin (GA) and herbimycin (HA), specific inhibitors of the cytosolic chaperone HSP 90 and its endoplasmic reticulum homologue GRP 94, were shown to accelerate degradation of the EGF-R and of its homologue p185(c)^-(erbB)^-2. Here we compared the effects of GA and HA on intracellular degradation and maturation of EGF-R. By using an inhibitor of proteasomal degradation, we learned that GA, but not HA, blocks processing of newly synthesized EGF-R. The effects of GA and HA on receptor degradation are mediated by the cytosolic portion of EGF-R and could be conferred to the erythropoietin receptor (EPO-R), by employing the respective chimera. Neither HA nor GA affected stability of newly synthesized EGF-R lacking the cytosolic domain (Ex EGF-R), but GA caused intracellular retention of this mutant. Taken together, our results imply that GA has two distinct targets of action on the EGF-R, one for promoting its degradation and another for mediating its intracellular retention. Apparently, degradation of the EGF-R mediated by GA or HA requires the presence of the EGF-R cytosolic domain, whereas intracellular retention in the presence of GA is coupled to the extracellular domain of the EGF-R.

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Role of the JAK/STAT pathway in rat carotid artery remodeling after vascular injury

Seki Yukihiko, Kai Hisashi, Shibata Rei, Nagata Tsuyoshi, Yasukawa Hideo, Yoshimura Akihiko, Imaizumi Tsutomu

Circulation Research 87 （ 1 ） 12 - 18 2000年07月

ISSN 0009-7330

In cultured vascular smooth muscle cells (VSMCs), Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) are expressed constitutively and play a role in angiotensin II (Ang II)-induced intracellular signaling and proliferation. However, little is known regarding the relevance of these proteins to the process of vascular remodeling. The role of JAK and STAT proteins in vascular remodeling and their functional coupling with Ang II were examined in balloon-injured rat carotid artery. Immunoreactive Jak2, Tyk2, Stat1, and Stat3 were not detected in the intact artery. Immunohistostaining showed transient expressions of these JAKs and STATs in medial and neointimal VSMCs at days 2 and 5, respectively, with a peak at day 7 in both layers. The expressions declined to insignificant levels by day 14. Ang II type 1 receptors (AT₁s) were coexpressed in the medial and neointimal VSMCs expressing Jak2 and Stat3. The Jak2 and Stat3 inductions in the injured artery were accompanied by constitutive Jak2 and Stat3 phosphorylations, which were enhanced by ex vivo Ang II stimulation via AT₁. Additionally, a Jak2 inhibitor, AG490, blocked the Ang II-induced Stat3 phosphorylation. Furthermore, local treatment with AG490 inhibited constitutive Stat3 phosphorylation and neointimal VSMC replication and subsequently reduced neointima formation in the injured artery. In conclusion, JAK and STAT proteins were inducible in medial and neointimal VSMCs after vascular injury and were functionally coupled to AT₁. The inductions of JAKs and STATs would be involved in the mechanisms of neointima formation after vascular injury.

SOCS-1 can suppress CD3ζ- and Syk-mediated NF-AT activation in a non-lymphoid cell line

Matsuda Tadashi, Yamamoto Tetsuya, Kishi Hiroyuki, Yoshimura Akihiko, Muraguchi Atsushi

FEBS Letters 472 （ 2-3 ） 235 - 240 2000年04月

ISSN 0014-5793

To elucidate T cell antigen receptor (TCR) signaling leading to activation nuclear factor of activated T cells (NF-AT), we reconstituted TCR signaling to activate NF-AT in a non-lymphoid cell line, 293T. We demonstrated that co-expression of CD8/ζ and Syk were necessary for NF-AT activation in 293T. This NF-AT response was completely inhibited by the addition of cyclosporin A or FK506, but markedly enhanced by the additional expression of Tec protein tyrosine kinase. We also show that the cytokine signaling suppressor, suppressor of cytokine signaling 1, potently inhibited this response by interacting with Syk and immunoreceptor tyrosine-based activation motifs in CD8/ζ. These results imply that this novel system may provide a useful tool to delineate or identify the regulatory molecules for CD3ζ/Syk-mediated NF-AT activation. Copyright (C) 2000 Federation of European Biochemical Societies.

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Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains

Masuhara Masaaki, Nagao Kenji, Nishikawa Mitsuo, Sasaki Mika, Yoshimura Akihiko, Osawa Masatake

Biochemical and Biophysical Research Communications 268 （ 3 ） 697 - 703 2000年02月

ISSN 0006-291X

To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-low/negative, Sca-1-positive, c-kit-positive, and lineage marker-negative (CD34(low/-)Sca-1⁺c-kit⁺Lin^-) cells were sorted by a fluorescence-activated cell sorter from mouse bone marrow cells and a yeast two-hybrid cDNA library was constructed. By screening with c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, the Src homology 2 (SH2) domain, and a number of tyrosine phosphorylation sites. RT-PCR analysis revealed that STAP-1 expression is restricted in the bone marrow cell fraction expressing c-kit. The highest expression was observed in the CD34(low/-)Sca-1⁺c-kit⁺Lin^- stem cell-enriched fraction. The murine myeloid cell line, M1, expressed a high level of STAP-1. However, the expression was strongly repressed in response to leukemia inhibitory factor (LIF) which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associated with the undifferentiated cell type. A two-hybrid assay indicated that STAP-1 bound not only to c-kit but also to c-fms but not to JAK2 or Pyk2. In 293 cells, STAP-1 was tyrosine-phosphorylated by activated c-kit. An in vitro binding assay suggested that the STAP-1 SH2 domain interacted with several tyrosine-phosphorylated proteins including c-kit and STAT5. These suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells. (C) 2000 Academic Press.

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The Jak-Stat pathway in normal and perturbed hematopoiesis

Ward Alister C., Touw Ivo, Yoshimura Akihiko

Blood 95 （ 1 ） 19 - 29 2000年01月

ISSN 0006-4971

Signal transduction of cytokine receptors and its regulation

Yoshimura A.

Seikagaku 72 （ 2 ） 75 - 90 2000年

ISSN 0037-1017

IL-5-induced JAB-JAK2 interaction

Zahn Stefan, Godillot Paul, Yoshimura Akihiko, Chaiken Irwin

Cytokine 12 （ 9 ） 1299 - 1306 2000年

ISSN 1043-4666

Receptor activation by the haematopoietic growth factor proteins interleukin 5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) leads to phosphorylation of JAK2 as a key trigger of signal transduction. JAB has recently been identified as a regulator of JAK2 phosphorylation and activity by binding phosphorylated JAK2 and inducing its degradation. As part of our effort to define molecular recognition networks that lead to signalling, we investigated the effect of JAB on both JAK2 phosphorylation and JAK2 interaction state that ensue upon IL-5 stimulation in recombinant 293T cells cotransfected 293T cells with IL-5Rα, βc and hJAK2 either with or without JAB. Without JAB, stimulation with wild-type and re-engineered single chain (sc) IL-5 induced a time-dependent phosphorylation of JAK2. In the presence of JAB cotransfection, no phospho-JAK2 was observed, and JAB was observed co-immunoprecipitated with non-phosphorylated JAK2. The time dependence of JAB co-immunoprecipitation correlated with the time dependence of JAK2 phosphorylation when JAB was absent. Since JAB has already been shown to bind JAK2 via a phosphorylated tyrosine, the current data suggest that JAB binds to phosphorylated JAK2, enhances JAK2 dephosphorylation and remains associated in a complex, with dephosphorylated JAK2, that may be a precursor leading to irreversible JAK2 degradation. (C) 2000 Academic Press.

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Molecular cloning and characterization of interleukin 13 receptor binding protein

Murata Takashi, Kurokawa Kenji, Sasaki Atsuo, Yoshimura Akihiko, Puri Raj K.

Blood 96 （ 11 PART II ） 2000年

ISSN 0006-4971

Interleukin 13 (IL-13) has been shown to play a critical role in the allergic diseases and bronchial asthma. However, signal transduction mechanisms for IL-13 receptor complex is still completely not clear. To investigate its signal transduction pathways, we have cloned new IL-13Ralpha' binding protein by yeast tri-hybrid system and designated it interleukin 13 receptor binding protein 1 (IL-I3RBP1). IL-13RBP1 cDNA is 2370 bp in length with open reading frame predicted to encode a protein of 625 amino acids. MOTIF search analysis revealed that IL-13RBP1 cDNA contains a myosin heavy chain like sequence in the N terminal end, nuclear localization signal in the middle part and coiled-coil region in C terminal end. We next examined mRNA expression in human tissues by Northern analysis and found that two different sizes (4.4 and 2.4 kb) of IL13RBP1 transcripts were present in most tissues. In addition, testis contained an additional 3 types of transcripts of -2.7 kb. Database searches utilizing BLAST and FASTA programs failed to identify any mammalian proteins having significant sequence similarities to IL13RBP1. However, C. elegans and D. melanogaster proteins of unknown function share -20% similarity to IL-13RBP1. IL13RBP1 may provide a novel mechanism in IL-13R signal transduction pathways.

Negative regulation of cytokine signaling pathways

Yasukawa Hideo, Sasaki Atsuo, Yoshimura Akihiko

Annual Review of Immunology 18 143 - 164 2000年

ISSN 0732-0582

The Janus family of protein tyrosine kinases (JAKs) and STAT transcription factors regulate cellular processes involved in cell growth, differentiation, and transformation through their association with cytokine receptors. The CIS family of proteins (also referred to as the SOCS or SSI family) has been implicated in the regulation of signal transduction by a variety of cytokines. Most of them appear to be induced after stimulation with several different cytokines, and at least three of them (CIS 1, CIS3/SOCS3, and JAB/SOCS 1) negatively regulate cytokine signal transduction by various means: CIS 1 inhibits STAT5 activation by binding to cytokine receptors that recruit STAT5, whereas JAB/SOCS-1 and CIS3/SOCS-3 directly bind to the kinase domain of JAKs, thereby inhibiting tyrosine-kinase activity. Therefore, these CIS family members seem to function in a classical negative feedback loop of cytokine signaling. Biochemical characterization as well as gene disruption studies indicate that JAB/SOCS-1/SSI-1 is an important negative regulator of interferon γ signaling. The mechanisms by which these inhibitors of cytokine signal transduction exert their effects have been extensively studied and will provide useful information for regulating tyrosine-kinase activity.

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The janus kinase inhibitor, JAB/SOCS-1, is an interferon-γ inducible gene and determines the sensitivity to interferons

Sakamoto Hiroshi, Kinjyo Ichiko, Yoshimura Akihiko

Leukemia and Lymphoma 38 （ 1-2 ） 49 - 58 2000年

ISSN 1042-8194

The Janus family of protein tyrosine kinases (JAKs) and STAT transcription factors regulate cellular processes involved in cell growth, differentiation, and transformation through their association with cytokine receptors. The CIS family of proteins (also referred as the SOCS or SSI family) has been implicated in the regulation of signal transduction by a variety of cytokines. Among them, we have shown that JAB/SOCS-1 is strongly induced by interferon-γ and forced expression of JAB/SOCS-1 conferred cells interferon resitance. This resistance was caused by inhibition of JAK1 and JAK2 activation in response to lFNγ. Moreover, recent detailed analysis of JAB/SOCS-1 knockout mice revealed that JAB/SOCS-1 is indeed a 'negative feedback regulator' that determine the sensitivity of cells to IFNγ. Using in vitro mutagensis, we defined a functional structure of JAB/SOCS-1 and proposed a mechanism for how JAB inhibits JAK kinase activity.

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吉村 昭彦

論文 - 吉村 昭彦

吉村　昭彦

論文 - 吉村　昭彦