研究者詳細 - 吉村　昭彦

論文 - 吉村　昭彦

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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

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