研究者詳細 - 吉村　昭彦

論文 - 吉村　昭彦

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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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論文 - 吉村　昭彦