研究者詳細 - 吉村　昭彦

論文 - 吉村　昭彦

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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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吉村 昭彦

論文 - 吉村 昭彦

吉村　昭彦

論文 - 吉村　昭彦