Sep. 2021, 秋山記念生命科学振興財団, 秋山財団賞　　　　　　　　　　　　　　　
松田 正

Sep. 2021, 伊藤医薬学術交流財団, 伊藤太郎学術賞　　　　　　　　　　　　　　　
松田 正

2000, ISI引用最高栄誉賞　　　　　　　　　　　　　　　

2000, ISI Citation Laureate Award　　　　　　　　　　　　　　　

STAP-2 facilitates insulin signaling through binding to CAP/c-Cbl and regulates adipocyte differentiation
Yuichi Sekine, Kazuna Kikkawa, Sachie Honda, Yuto Sasaki, Shoya Kawahara, Akihiro Mizushima, Sumihito Togi, Masahiro Fujimuro, Kenji Oritani, Tadashi Matsuda
Scientific Reports, 14, 1, Springer Science and Business Media LLC, 09 Mar. 2024
Scientific journal, Abstract

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor molecule involved in several cellular signaling cascades. Here, we attempted to identify novel STAP-2 interacting molecules, and identified c-Cbl associated protein (CAP) as a binding protein through the C-terminal proline-rich region of STAP-2. Expression of STAP-2 increased the interaction between CAP and c-Cbl, suggesting that STAP-2 bridges these proteins and enhances complex formation. CAP/c-Cbl complex is known to regulate GLUT4 translocation in insulin signaling. STAP-2 overexpressed human hepatocyte Hep3B cells showed enhanced GLUT4 translocation after insulin treatment. Elevated levels of Stap2 mRNA have been observed in 3T3-L1 cells and mouse embryonic fibroblasts (MEFs) during adipocyte differentiation. The differentiation of 3T3-L1 cells into adipocytes was highly promoted by retroviral overexpression of STAP-2. In contrast, STAP-2 knockout (KO) MEFs exhibited suppressed adipogenesis. The increase in body weight with high-fat diet feeding was significantly decreased in STAP-2 KO mice compared to WT animals. These data suggest that the expression of STAP-2 correlates with adipogenesis. Thus, STAP-2 is a novel regulatory molecule that controls insulin signal transduction by forming a c-Cbl/STAP-2/CAP ternary complex.

Tyrosinase regulates the motility of human melanoma cell line A375 through its hydroxylase enzymatic activity
Sachie Honda, Tadashi Matsuda, Masahiro Fujimuro, Yuichi Sekine
Biochemical and Biophysical Research Communications, 149785, 149785, Elsevier BV, Mar. 2024
Scientific journal

Role of Signal-Transducing Adaptor Protein-1 for T Cell Activation and Pathogenesis of Autoimmune Demyelination and Airway Inflammation
Kota Kagohashi, Yuto Sasaki, Kiyotaka Ozawa, Takuya Tsuchiya, Shoya Kawahara, Kodai Saitoh, Michiko Ichii, Jun Toda, Yasuyo Harada, Masato Kubo, Yuichi Kitai, Ryuta Muromoto, Kenji Oritani, Jun-ichi Kashiwakura, Tadashi Matsuda
The Journal of Immunology, The American Association of Immunologists, 05 Feb. 2024
Scientific journal, Abstract

Signal-transducing adaptor protein (STAP)-1 is an adaptor protein that is widely expressed in T cells. In this article, we show that STAP-1 upregulates TCR-mediated T cell activation and T cell–mediated airway inflammation. Using STAP-1 knockout mice and STAP-1–overexpressing Jurkat cells, we found that STAP-1 enhanced TCR signaling, resulting in increased calcium mobilization, NFAT activity, and IL-2 production. Upon TCR engagement, STAP-1 binding to ITK promoted formation of ITK–LCK and ITK–phospholipase Cγ1 complexes to induce downstream signaling. Consistent with the results, STAP-1 deficiency reduced the severity of symptoms in experimental autoimmune encephalomyelitis. Single-cell RNA-sequencing analysis revealed that STAP-1 is essential for accumulation of T cells and Ifng and Il17 expression in spinal cords after experimental autoimmune encephalomyelitis induction. Th1 and Th17 development was also attenuated in STAP-1 knockout naive T cells. Taken together, STAP-1 enhances TCR signaling and plays a role in T cell–mediated immune disorders.

The Atypical Dual Specificity Phosphatase DUSP15 Regulates Jak1-Mediated STAT3 Activation.
Kazuna Kikkawa, Tadashi Matsuda, Masahiro Fujimuro, Yuichi Sekine
Biological & pharmaceutical bulletin, 47, 9, 1487, 1493, 2024, [Domestic magazines]
English, Scientific journal, The signal transducer and activator of transcription 3 (STAT3) protein is a key regulator of cell differentiation, proliferation, and survival in hematopoiesis, immune responses, and other biological systems. STAT3 transcriptional activity is strictly regulated through various mechanisms, such as phosphorylation and dephosphorylation. In this study, we attempted to identify novel phosphatases which regulate STAT3 activity in response to cytokine stimulations. To this end, leukemia inhibitory factor (LIF)/STAT3 dependent phosphatase induction was evaluated in the mouse hepatoma cell line Hepa1-6. After LIF stimulation, the expression of several atypical dual specific phosphatases (aDUSPs) was upregulated in Hepa1-6 cells. Among the LIF-induced aDUSPs, we focused on DUSP15 and clarified its functions in LIF/STAT3 signaling using RNA interference. DUSP15 knockdown decreased LIF-induced Socs3 mRNA expression and STAT3 translocation. Furthermore, loss of DUSP15 reduced the phosphorylation of STAT3 at Tyr705 and Janus family tyrosine kinase 1 (Jak1) at Tyr1034/1035 in response to LIF. The interaction between Jak1 and DUSP15 was observed in LIF-stimulated Hepa1-6 cells. We also demonstrated the suppression of granulocyte colony-stimulating factor (G-CSF)-mediated gp130/STAT3-dependent cell growth of Ba/F-G133 cells via DUSP15 knockdown. Therefore, DUSP15 functions as a positive feedback regulator in the Jak1/STAT3 signaling cascade.

[Understanding New Regulatory Mechanism of TCR Signal Transduction].
Jun-Ichi Kashiwakura, Tadashi Matsuda
Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan, 144, 5, 497, 501, 2024, [Domestic magazines]
Japanese, Scientific journal, Signal-transducing adaptor protein-2 (STAP-2) is a unique scaffold protein that regulates several immunological signaling pathways, including LIF/LIF receptor and LPS/TLR4 signals. STAP-2 is required for Fas/FasL-dependent T cell apoptosis and SDF-1α-induced T cell migration. Conversely, STAP-2 modulates integrin-mediated T cell adhesion, suggesting that STAP-2 is essential for several negative and positive T cell functions. However, whether STAP-2 is involved in T cell-antigen receptor (TCR)-mediated T cell activation is unknown. STAP-2 deficiency was recently reported to suppress TCR-mediated T cell activation by inhibiting LCK-mediated CD3ζ and ZAP-70 activation. Using STAP-2 deficient mice, it was demonstrated that STAP-2 is required for the pathogenesis of Propionibacterium acnes-induced granuloma formation and experimental autoimmune encephalomyelitis. Here, detailed functions of STAP-2 in TCR-mediated T cell activation, and how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases, are reviewed.

Potential therapeutic applications of targeting signal-transducing adaptor protein-2 in autoimmune diseases
Yuto Sasaki, Shoya Kawahara, Yuichi Sekine, Jun-Ichi Kashiwakura, Kenji Oritani, Tadashi Matsuda
Exploration of Immunology, 3, 6, 604, 612, Open Exploration Publishing, 28 Dec. 2023
Scientific journal, Adaptor proteins are involved in various immune responses via the modulation of many signaling pathways. Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that contains typical domains such as the pleckstrin homology (PH) domain, Src homology domain, and a proline-rich region from the N-terminal region. In T cells, STAP-2 positively regulates T cell receptor (TCR)-mediated signaling by associating with CD3ζ immunoreceptor tyrosine-based activation motifs (ITAMs) and lymphocyte-specific protein tyrosine kinase (LCK). Therefore, a peptide that inhibits the interaction between STAP-2 and CD3ζ ITAMs is likely to suppress TCR-mediated T cell activation, as well as T cell-mediated diseases. As expected, the peptide successfully inhibited the STAP-2/CD3ζ ITAM interaction and suppressed TCR-mediated signaling, cell proliferation, and interleukin (IL)-2 production in human/murine T cells. Furthermore, this inhibitor suppressed the pathogenesis of experimental autoimmune encephalomyelitis (EAE), which is widely recognized as a mouse model of multiple sclerosis, via the downregulation of T cell activation and infiltration of T helper (Th) 1/Th17 cells. These results suggest a new strategy for the treatment of multiple sclerosis and other immune diseases.

STAP-2 negatively regulates BCR-mediated B cell activation by recruiting tyrosine-protein kinase CSK to LYN.
Jun-Ichi Kashiwakura, Shoya Kawahara, Iori Inagaki, Kyosuke Inui, Kodai Saitoh, Kota Kagohashi, Yuto Sasaki, Fuki Kobayashi, Yuichi Kitai, Ryuta Muromoto, Kenji Oritani, Tadashi Matsuda
FEBS letters, 597, 19, 2433, 2445, 05 Sep. 2023, [International Magazine]
English, Scientific journal, Although signal-transducing adaptor protein-2 (STAP-2) acts in certain immune responses, its role in B cell receptor (BCR)-mediated signals remains unknown. In this study, we have revealed that BCR-mediated signals, cytokine production and antibody production were increased in STAP-2 knockout (KO) mice compared with wild-type (WT) mice. Phosphorylation of tyrosine-protein kinase LYN Y508 was reduced in STAP-2 KO B cells after BCR stimulation. Mechanistic analysis revealed that STAP-2 directly binds to LYN, dependently of STAP-2 Y250 phosphorylation by LYN. Furthermore, phosphorylation of STAP-2 enhanced interactions between LYN and tyrosine-protein kinase CSK, resulting in enhanced CSK-mediated LYN Y508 phosphorylation. These results suggest that STAP-2 is crucial for controlling BCR-mediated signals and antibody production by enhanced CSK-mediated feedback regulation of LYN.

STAP-2–Derived Peptide Suppresses TCR-Mediated Signals to Initiate Immune Responses
Yuto Sasaki, Kodai Saitoh, Kota Kagohashi, Toyoyuki Ose, Shoya Kawahara, Yuichi Kitai, Ryuta Muromoto, Yuichi Sekine, Michiko Ichii, Akihiko Yoshimura, Kenji Oritani, Jun-ichi Kashiwakura, Tadashi Matsuda
The Journal of Immunology, The American Association of Immunologists, 07 Jul. 2023
Scientific journal, Abstract

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that contains pleckstrin and Src homology 2–like domains, as well as a proline-rich region in its C-terminal region. Our previous study demonstrated that STAP-2 positively regulates TCR signaling by associating with TCR-proximal CD3ζ ITAMs and the lymphocyte-specific protein tyrosine kinase. In this study, we identify the STAP-2 interacting regions of CD3ζ ITAMs and show that the STAP-2–derived synthetic peptide (iSP2) directly interacts with the ITAM sequence and blocks the interactions between STAP-2 and CD3ζ ITAMs. Cell-penetrating iSP2 was delivered into human and murine T cells. iSP2 suppressed cell proliferation and TCR-induced IL-2 production. Importantly, iSP2 treatment suppressed TCR-mediated activation of naive CD4+ T cells and decreased immune responses in CD4+ T cell–mediated experimental autoimmune encephalomyelitis. It is likely that iSP2 is a novel immunomodulatory tool that modulates STAP-2-mediated activation of TCR signaling and represses the progression of autoimmune diseases.

Signal-transducing adaptor protein-2 modulates T-cell functions
Tadashi Matsuda, Yuto Sasaki, Kota Kagohashi, Kodai Saitoh, Yuichi Sekine, Jun-Ichi Kashiwakura, Kenji Oritani
Exploration of Immunology, 771, 782, Open Exploration Publishing, 27 Dec. 2022
Scientific journal, Immune responses are orchestrated by controlling the initiation, magnitude, and duration of various signaling pathways. Adaptor proteins act as positive or negative regulators by targeting critical molecules of signaling cascades. Signal-transducing adaptor protein-2 (STAP-2) contains typical features of adaptor proteins, like a pleckstrin homology (PH) domain in the N-terminal region and a Src homology 2 (SH2) domain in the central region. STAP-2 binds to a variety of signaling or transcriptional molecules to control multiple steps of inflammatory/immune responses. STAP-2 enhances T-cell receptor (TCR)-mediated signaling via the association with TCR-proximal CD3ζ immunoreceptor tyrosine-based activation motifs (ITAMs) and lymphocyte-specific protein tyrosine kinase (Lck). STAP-2 decreases adherence of T-cells to fibronectin (FN) through an association with focal adhesion kinase (Fak) and Casitas B-lineage Lymphoma (c-Cbl), and increases chemotaxis of T-cells toward stromal cell-derived factor-1α (SDF-1α) through interactions with Vav1 and Ras-related C3 botulinum toxin substrate 1 (Rac1). STAP-2 positively regulates activation-induced cell deathrough the association with Fas and caspase-8. This review describes the current knowledge of the roles of STAP-2 in T-cell-dependent immune responses and the possible clinical utility of STAP-2-targeting therapies.

The Functional Properties and Physiological Roles of Signal-Transducing Adaptor Protein-2 in the Pathogenesis of Inflammatory and Immune Disorders.
Jun-Ichi Kashiwakura, Kenji Oritani, Tadashi Matsuda
Biomedicines, 10, 12, 30 Nov. 2022, [International Magazine]
English, Scientific journal, Adaptor molecules play a crucial role in signal transduction in immune cells. Several adaptor molecules, such as the linker for the activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), are essential for T cell development and activation following T cell receptor (TCR) aggregation, suggesting that adaptor molecules are good therapeutic targets for T cell-mediated immune disorders, such as autoimmune diseases and allergies. Signal-transducing adaptor protein (STAP)-2 is a member of the STAP family of adaptor proteins. STAP-2 functions as a scaffold for various intracellular proteins, including BRK, signal transducer, and activator of transcription (STAT)3, STAT5, and myeloid differentiation primary response protein (MyD88). In T cells, STAP-2 is involved in stromal cell-derived factor (SDF)-1α-induced migration, integrin-dependent cell adhesion, and Fas-mediated apoptosis. We previously reported the critical function of STAP-2 in TCR-mediated T cell activation and T cell-mediated autoimmune diseases. Here, we review how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases in order to develop novel STAP-2-targeting therapeutic strategies.

A peptide derived from adaptor protein STAP-2 inhibits tumor progression by downregulating epidermal growth factor receptor signaling.
Taiga Maemoto, Yuichi Kitai, Runa Takahashi, Haruka Shoji, Shunsuke Yamada, Shiho Takei, Daiki Ito, Ryuta Muromoto, Jun-Ichi Kashiwakura, Haruka Handa, Ari Hashimoto, Shigeru Hashimoto, Toyoyuki Ose, Kenji Oritani, Tadashi Matsuda
The Journal of biological chemistry, 299, 1, 102724, 102724, 18 Nov. 2022, [International Magazine]
English, Scientific journal, Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signals. We previously demonstrated that STAP-2 binds to epidermal growth factor receptor (EGFR) and facilitates its stability and activation of EGFR signaling in prostate cancer cells. Inhibition of this interaction may be a promising direction for cancer treatment. Here, we found that 2D5 peptide, a STAP-2-derived peptide, blocked STAP-2-EGFR interactions and suppressed EGFR-mediated proliferation in several cancer cell lines. 2D5 peptide inhibited tumor growth of human prostate cancer cell line DU145 and human lung cancer cell line A549 in murine xenograft models. Additionally, we determined that EGFR signaling and its stability were decreased by 2D5 peptide treatment during EGF stimulation. In conclusion, our study shows that 2D5 peptide is a novel anti-cancer peptide that inhibits STAP-2-mediated activation of EGFR signaling and suppresses prostate and lung cancer progression.

Central Roles of STAT3-Mediated Signals in Onset and Development of Cancers: Tumorigenesis and Immunosurveillance.
Shigeru Hashimoto, Ari Hashimoto, Ryuta Muromoto, Yuichi Kitai, Kenji Oritani, Tadashi Matsuda
Cells, 11, 16, 22 Aug. 2022, [International Magazine]
English, Scientific journal, Since the time of Rudolf Virchow in the 19th century, it has been well-known that cancer-associated inflammation contributes to tumor initiation and progression. However, it remains unclear whether a collapse of the balance between the antitumor immune response via the immunological surveillance system and protumor immunity due to cancer-related inflammation is responsible for cancer malignancy. The majority of inflammatory signals affect tumorigenesis by activating signal transducer and activation of transcription 3 (STAT3) and nuclear factor-κB. Persistent STAT3 activation in malignant cancer cells mediates extremely widespread functions, including cell growth, survival, angiogenesis, and invasion and contributes to an increase in inflammation-associated tumorigenesis. In addition, intracellular STAT3 activation in immune cells causes suppressive effects on antitumor immunity and leads to the differentiation and mobilization of immature myeloid-derived cells and tumor-associated macrophages. In many cancer types, STAT3 does not directly rely on its activation by oncogenic mutations but has important oncogenic and malignant transformation-associated functions in both cancer and stromal cells in the tumor microenvironment (TME). We have reported a series of studies aiming towards understanding the molecular mechanisms underlying the proliferation of various types of tumors involving signal-transducing adaptor protein-2 as an adaptor molecule that modulates STAT3 activity, and we recently found that AT-rich interactive domain-containing protein 5a functions as an mRNA stabilizer that orchestrates an immunosuppressive TME in malignant mesenchymal tumors. In this review, we summarize recent advances in our understanding of the functional role of STAT3 in tumor progression and introduce novel molecular mechanisms of cancer development and malignant transformation involving STAT3 activation that we have identified to date. Finally, we discuss potential therapeutic strategies for cancer that target the signaling pathway to augment STAT3 activity.

Identification of RPL15 60S Ribosomal Protein as a Novel Topotecan Target Protein That Correlates with DAMP Secretion and Antitumor Immune Activation.
Shunsuke Yamada, Yuichi Kitai, Takashi Tadokoro, Runa Takahashi, Haruka Shoji, Taiga Maemoto, Marie Ishiura, Ryuta Muromoto, Jun-Ichi Kashiwakura, Ken J Ishii, Katsumi Maenaka, Taro Kawai, Tadashi Matsuda
Journal of immunology (Baltimore, Md. : 1950), 209, 1, 171, 179, 20 Jun. 2022, [International Magazine]
English, Scientific journal, Damage-associated molecular patterns (DAMPs) contribute to antitumor immunity during cancer chemotherapy. We previously demonstrated that topotecan (TPT), a topoisomerase I inhibitor, induces DAMP secretion from cancer cells, which activates STING-mediated antitumor immune responses. However, how TPT induces DAMP secretion in cancer cells is yet to be elucidated. Here, we identified RPL15, a 60S ribosomal protein, as a novel TPT target and showed that TPT inhibited preribosomal subunit formation via its binding to RPL15, resulting in the induction of DAMP-mediated antitumor immune activation independent of TOP1. TPT inhibits RPL15-RPL4 interactions and decreases RPL4 stability, which is recovered by CDK12 activity. RPL15 knockdown induced DAMP secretion and increased the CTL population but decreased the regulatory T cell population in a B16-F10 murine melanoma model, which sensitized B16-F10 tumors against PD-1 blockade. Our study identified a novel TPT target protein and showed that ribosomal stress is a trigger of DAMP secretion, which contributes to antitumor immunotherapy.

STAP-2 Is a Novel Positive Regulator of TCR-Proximal Signals.
Kodai Saitoh, Jun-Ichi Kashiwakura, Kota Kagohashi, Yuto Sasaki, Shoya Kawahara, Yuichi Sekine, Yuichi Kitai, Ryuta Muromoto, Michiko Ichii, Hiroko Nakatsukasa, Akihiko Yoshimura, Kenji Oritani, Tadashi Matsuda
Journal of immunology (Baltimore, Md. : 1950), 209, 1, 57, 68, 20 Jun. 2022, [International Magazine]
English, Scientific journal, TCR ligation with an Ag presented on MHC molecules promotes T cell activation, leading to the selection, differentiation, and proliferation of T cells and cytokine production. These immunological events are optimally arranged to provide appropriate responses against a variety of pathogens. We here propose signal-transducing adaptor protein-2 (STAP-2) as a new positive regulator of TCR signaling. STAP-2-deficient T cells showed reduced, whereas STAP-2-overexpressing T cells showed enhanced, TCR-mediated signaling and downstream IL-2 production. For the mechanisms, STAP-2 associated with TCR-proximal CD3ζ immunoreceptor tyrosine activation motifs and phosphorylated LCK, resulting in enhancement of their binding after TCR stimulation. In parallel, STAP-2 expression is required for full activation of downstream TCR signaling. Importantly, STAP-2-deficient mice exhibited slight phenotypes of CD4+ T-cell-mediated inflammatory diseases, such as experimental autoimmune encephalomyelitis, whereas STAP-2-overexpressing transgenic mice showed severe phenotypes of these diseases. Together, STAP-2 is an adaptor protein to enhance TCR signaling; therefore, manipulating STAP-2 will have an ability to improve the treatment of patients with autoimmune diseases as well as the chimeric Ag receptor T cell therapy.

Regulation of NFKBIZ gene promoter activity by STAT3, C/EBPβ, and STAT1
Ryuta Muromoto, Ami Sato, Yuki Komori, Kota Nariya, Yuichi Kitai, Jun-ichi Kashiwakura, Tadashi Matsuda
Biochemical and Biophysical Research Communications, Elsevier BV, May 2022
Scientific journal

A novel intramolecular negative regulation of mouse Jak3 activity by tyrosine 820.
Yuichi Sekine, Kazuna Kikkawa, Bruce A Witthuhn, Jun-Ichi Kashiwakura, Ryuta Muromoto, Yuichi Kitai, Masahiro Fujimuro, Kenji Oritani, Tadashi Matsuda
International immunology, 22 Feb. 2022, [International Magazine]
English, Scientific journal, Jak3, a member of the Janus kinase family, is essential for the cytokine receptor common gamma (γ) chain-mediated signaling. During activation of Jak3, tyrosine residues are phosphorylated and potentially regulate its kinase activity. We identified a novel tyrosine phosphorylation site within mouse Jak3, Y820, which is conserved in human Jak3, Y824. IL-2-induced tyrosine phosphorylation of Jak3 Y824 in human T cell line HuT78 cells was detected by using a phosphospecific, pY824, antibody. Mutation of mouse Jak3 Y820 to alanine (Y820A) showed increased autophosphorylation of Jak3 and enhanced STAT5 tyrosine phosphorylation and transcriptional activation. Stably expressed Jak3 Y820A in F7 cells, an IL-2 responsive mouse pro-B cell line Ba/F3, exhibited enhanced IL-2-dependent cell growth. Mechanistic studies demonstrated that interaction between Jak3 and STAT5 increased in Jak3 Y820A compared to Jak3 WT. These data suggest that Jak3 Y820 plays a role in negative regulation of Jak3-mediated STAT5 signaling cascade upon IL-2-stimulation. We speculate that this occurs through an interaction promoted by the tyrosine phosphorylated Y820 or a conformational change by Y820 mutation with either the STAT directly or with the recruitment of molecules such as phosphatases via a SH2 interaction. Additional studies will focus on these interactions as Jak3 plays a crucial role in disease and health.

Current understanding of the role of tyrosine kinase 2 signaling in immune responses.
Ryuta Muromoto, Kenji Oritani, Tadashi Matsuda
World journal of biological chemistry, 13, 1, 1, 14, 27 Jan. 2022, [International Magazine]
English, Scientific journal, Immune system is a complex network that clears pathogens, toxic substrates, and cancer cells. Distinguishing self-antigens from non-self-antigens is critical for the immune cell-mediated response against foreign antigens. The innate immune system elicits an early-phase response to various stimuli, whereas the adaptive immune response is tailored to previously encountered antigens. During immune responses, B cells differentiate into antibody-secreting cells, while naïve T cells differentiate into functionally specific effector cells [T helper 1 (Th1), Th2, Th17, and regulatory T cells]. However, enhanced or prolonged immune responses can result in autoimmune disorders, which are characterized by lymphocyte-mediated immune responses against self-antigens. Signal transduction of cytokines, which regulate the inflammatory cascades, is dependent on the members of the Janus family of protein kinases. Tyrosine kinase 2 (Tyk2) is associated with receptor subunits of immune-related cytokines, such as type I interferon, interleukin (IL)-6, IL-10, IL-12, and IL-23. Clinical studies on the therapeutic effects and the underlying mechanisms of Tyk2 inhibitors in autoimmune or chronic inflammatory diseases are currently ongoing. This review summarizes the findings of studies examining the role of Tyk2 in immune and/or inflammatory responses using Tyk2-deficient cells and mice.

Signal-transducing adaptor protein-1 and protein-2 in hematopoiesis and diseases
Michiko Ichii, Kenji Oritani, Jun Toda, Naoki Hosen, Tadashi Matsuda, Yuzuru Kanakura
Experimental Hematology, 105, 10, 17, Elsevier BV, Jan. 2022
Scientific journal

Perturbation of IL-17A response by environmental chemicals
MUROMOTO Ryuta, MATSUDA Tadashi
Annual Meeting of the Japanese Society of Toxicology, 49.1, S39-3, The Japanese Society of Toxicology, 2022
Japanese, IL-17A is a cytokine produced by immune cells and acts on non-immune cells such as epithelial cells to stimulate the production of chemokines and antimicrobial peptides. This IL-17A-initiated activation of cells is important for amplifying inflammation and eliminating fungi and bacteria, while an uncontrolled increase in IL-17A action leads to disease, as shown in psoriasis, multiple sclerosis, rheumatoid arthritis patients, and their mouse models. In this study, we report that the IL-17A-induced response is affected by environmental chemicals. We have found IκB-ζ as an IL-17A-induced protein by gene expression analysis using epidermal keratinocytes, and that IκB-ζ plays a role in the expression of other IL-17A-induced genes. The mechanism of IκB-ζ induction by IL-17A was the mRNA stabilization, which is caused by the inactivation of Regnase-1, an mRNA degrading enzyme. The signaling leading to the inactivation of Regnase-1 was inhibited by dimethyl fumarate, a drug used for multiple sclerosis treatment. On the other hand, benzo[a]pyrene, a ligand for aryl hydrocarbon receptor, suppressed the function of Regnase-1 and increased the amount of IκB-ζ mRNA and IL-6 mRNA, which are degradation targets by Regnase-1. In summary, the IL-17A signaling pathway and mRNA stability control mechanisms are positively or negatively affected by various environmental chemicals. These mechanisms may be targets for chemical-induced changes in inflammatory and immune responses.

Signal-transducing adaptor protein-2 has a nonredundant role for IL-33-triggered mast cell activation.
Jun-Ichi Kashiwakura, Nao Koizumi, Kodai Saitoh, Kota Kagohashi, Yuto Sasaki, Fuki Kobayashi, Shoya Kawahara, Yukie Yamauchi, Yuichi Kitai, Ryuta Muromoto, Kenji Oritani, Tadashi Matsuda
Biochemical and biophysical research communications, 572, 80, 85, 01 Oct. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, Signal-transducing adaptor protein (STAP)-2 is one of the STAP family adaptor proteins and ubiquitously expressed in a variety types of cells. Although STAP-2 is required for modification of FcεRI signal transduction in mast cells, other involvement of STAP-2 in mast cell functions is unknown, yet. In the present study, we mainly investigated functional roles of STAP-2 in IL-33-induced mast cell activation. In STAP-2-deficient, but not STAP-1-deficient, mast cells, IL-33-induced IL-6 and TNF-α production was significantly decreased compared with that of wild-type mast cells. In addition, STAP-2-deficiency greatly reduced TLR4-mediated mast cell activation and cytokine production. For the mechanisms, STAP-2 directly binds to IKKα after IL-33 stimulation, leading to elevated NF-κB activity. In conclusion, STAP-2, but not STAP-1, participates in IL-33-induced mast cells activation.

STAP-2 adaptor protein regulates multiple steps of immune and inflammatory responses　　　　　　　　　　　　　　　
Matsuda, T, K.Oritani
Biol. Pharm. Bull., 44, 7, 895, 901, Jul. 2021, [Peer-reviewed]
Scientific journal

Propolis suppresses cytokine production in activated basophils and basophil-mediated skin and intestinal allergic inflammation in mice.
Jun-Ichi Kashiwakura, Mari Yoshihara, Kodai Saitoh, Kota Kagohashi, Yuto Sasaki, Fuki Kobayashi, Iori Inagaki, Yuichi Kitai, Ryuta Muromoto, Tadashi Matsuda
Allergology international : official journal of the Japanese Society of Allergology, 70, 3, 360, 367, Jul. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, BACKGROUND: Propolis is a resinous mixture produced by honey bees that contains cinnamic acid derivatives and flavonoids. Although propolis has been reported to inhibit mast cell functions and mast cell-dependent allergic responses, the effect of propolis on basophil biology remains unknown. This study aimed to investigate the inhibitory effect of propolis on FcεRI-mediated basophil activation. METHODS: To determine the inhibitory effect of propolis on basophil activation in vitro, cytokine production and FcεRI signal transduction were analyzed by ELISA and western blotting, respectively. To investigate the inhibitory effect of propolis in vivo, IgE-CAI and a food allergy mouse model were employed. RESULTS: Propolis treatment resulted in the suppression of IgE/antigen-induced production of IL-4, IL-6 and IL-13 in basophils. Phosphorylation of FcεRI signaling molecules Lyn, Akt and ERK was inhibited in basophils treated with propolis. While propolis did not affect the basophil population in the treated mice, propolis did inhibit IgE-CAI. Finally, ovalbumin-induced intestinal anaphylaxis, which involves basophils and basophil-derived IL-4, was attenuated in mice prophylactically treated with propolis. CONCLUSIONS: Taken together, these results demonstrate the ability of propolis to suppress IgE-dependent basophil activation and basophil-dependent allergic inflammation. Therefore, prophylactic treatment with propolis may be useful for protection against food allergic reactions in sensitive individuals.

Positive interactions between STAP-1 and BCR-ABL influence chronic myeloid leukemia cell proliferation and survival.
Marie Ishiura, Yuichi Kitai, Jun-Ichi Kashiwakura, Ryuta Muromoto, Jun Toda, Michiko Ichii, Kenji Oritani, Tadashi Matsuda
Biochemical and biophysical research communications, 556, 185, 191, 04 Jun. 2021, [International Magazine]
English, Scientific journal, Chronic myeloid leukemia (CML) is a clonal disease characterized by the presence of the Philadelphia chromosome and its oncogenic product, BCR-ABL, which activates multiple pathways involved in cell survival, growth promotion, and disease progression. We recently reported that signal-transducing adaptor protein 1 (STAP-1) is upregulated in CML stem cells (LSCs) and functions to reduce the apoptosis of CML LSCs by upregulating the STAT5-downstream anti-apoptotic genes. In this study, we demonstrate the detailed molecular interactions among BCR-ABL, STAP-1, and signal transducer and activator of transcription 5 (STAT5). Studies with deletion mutants have revealed that STAP-1 interacts with BCR-ABL and STAT5a through its SH2 and PH domains, respectively, suggesting the possible role of STAP-1 as a scaffold protein. Furthermore, the binding of STAP-1 to BCR-ABL stabilizes the BCR-ABL protein in CML cells. Since STAP-1 is highly expressed in CML cells, we also analyzed the STAP-1 promoter activity using a luciferase reporter construct and found that NFATc1 is involved in activating the STAP-1 promoter and inducing STAP-1 mRNA expression. Our results demonstrate that STAP-1 contributes to the BCR-ABL/STAT5 and BCR-ABL/Ca2+/NFAT signals to induce proliferation and STAP-1 mRNA expression in CML cells, respectively.

CD47 promotes T-cell lymphoma metastasis by up-regulating AKAP13-mediated RhoA activation.
Yuichi Kitai, Marie Ishiura, Kodai Saitoh, Naoki Matsumoto, Kimiya Owashi, Shunsuke Yamada, Ryuta Muromoto, Jun-Ichi Kashiwakura, Kenji Oritani, Tadashi Matsuda
International immunology, 33, 5, 273, 280, 22 Apr. 2021, [International Magazine]
English, Scientific journal, CD47, a 50 kDa transmembrane protein, facilitates integrin-mediated cell adhesion and inhibits cell engulfment by phagocytes. Since CD47 blocking promotes engulfment of cancer cells by macrophages, it is important to clarify the mechanism of CD47 signaling in order to develop treatments for diseases involving CD47-overexpressing cancer cells, including breast cancer and lymphoma. Here, we show that CD47 plays an essential role in T-cell lymphoma metastasis by up-regulating basal RhoA activity independent of its anti-phagocytic function. CD47 interacts with AKAP13, a RhoA-specific guanine nucleotide exchange factor (GEF), and facilitates AKAP13-mediated RhoA activation. Our study shows that CD47 has a novel function on the AKAP13-RhoA axis and suggests that CD47-AKAP13 interaction would be a novel target for T-cell lymphoma treatment.

Synthesis of Resolvin E1 and Its Conformationally Restricted Cyclopropane Congeners with Potent Anti-Inflammatory Effect
Kohei Ishimura, Hayato Fukuda, Koichi Fujiwara, Ryuta Muromoto, Koki Hirashima, Yuto Murakami, Mizuki Watanabe, Jun Ishihara, Tadashi Matsuda, Satoshi Shuto
ACS Medicinal Chemistry Letters, 12, 2, 256, 261, American Chemical Society (ACS), 11 Feb. 2021
Scientific journal

Therapeutic Advantage of Tyk2 Inhibition for Treating Autoimmune and Chronic Inflammatory Diseases.
Ryuta Muromoto, Kazuya Shimoda, Kenji Oritani, Tadashi Matsuda
Biological & pharmaceutical bulletin, 44, 11, 1585, 1592, 2021, [Domestic magazines]
English, Scientific journal, Tyrosine kinase 2 (Tyk2) is a member of the Janus family of protein tyrosine kinases (Jaks). Tyk2 associates with interferon (IFN)-α, IFN-β, interleukin (IL)-6, IL-10, IL-12, and IL-23 receptors and mediates their downstream signaling pathways. Based on our data using Tyk2-deficient mice and cells, Tyk2 plays crucial roles in the differentiation, maintenance, and function of T helper 1 (Th1) and Th17 cells, and its dysregulation may promote autoimmune and/or inflammatory diseases. IFN-α-induced growth inhibition of B lymphocyte progenitors is dependent on Tyk2-mediated signals to regulate death-associated protein (Daxx) nuclear localization and Daxx-promyelocytic leukemia protein interactions. Tyk2-deficient mice show impaired constitutive production of type I IFNs by macrophages under steady-state conditions. When heat-killed Cutibacterium acnes is injected intraperitoneally, Tyk2-deficient mice show less granuloma formation through enhanced prostaglandin E2 and protein kinase A activities, leading to high IL-10 production by macrophages. Thus, Tyk2 is widely involved in the immune and inflammatory response at multiple events; therefore, Tyk2 is likely to be a suitable target for treating patients with autoimmune and/or chronic inflammatory diseases. Clinical trials of Tyk2 inhibitors have shown higher response rates and improved tolerability in the treatment of patients with psoriasis and inflammatory bowel diseases. Taken together, Tyk2 inhibition has great potential for clinical application in the management of a variety of diseases.

Pivotal Role of Signal-Transducing Adaptor Protein-2 in Pathogenesis of Autoimmune Hepatitis.
Yuto Sasaki, Kodai Saitoh, Kota Kagohashi, Yuichi Kitai, Ryuta Muromoto, Kenji Oritani, Jun-Ichi Kashiwakura, Tadashi Matsuda
Biological & pharmaceutical bulletin, 44, 12, 1898, 1901, 2021, [Domestic magazines]
English, Scientific journal, Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein involved in inflammatory and immune responses, such as inflammatory bowel disease and allergic responses. In this study, we investigated the role of STAP-2 in the pathogenesis of autoimmune hepatitis. After intravenous injection of concanavalin A (ConA), STAP-2 knock out (KO) mice showed more severe liver necrosis along with substantial lymphocyte infiltration compared to wild type (WT) mice. Serum alanine aminotransferase levels were significantly higher in ConA-injected STAP-2 KO mice than in WT mice. Levels of interferon-γ (IFN-γ), an important factor for liver necrosis, were also significantly increased in sera of STAP-2 KO mice compared to WT mice after ConA injection. Statistically significant upregulation of Fas ligand (FasL) expression was observed in the livers of ConA-injected STAP-2 KO mice compared to WT mice. In accordance with these results, apoptotic signals were facilitated in STAP-2 KO mice compared to WT mice after ConA injection. Correctively, these results suggest that STAP-2 is involved in the pathogenesis of autoimmune hepatitis by regulating the expression of FasL and the production of IFN-γ.

Correction: Expression of signal-transducing adaptor protein-1 attenuates experimental autoimmune hepatitis via down-regulating activation and homeostasis of invariant natural killer T cells.
Jun-Ichi Kashiwakura, Kodai Saitoh, Takeru Ihara, Yuto Sasaki, Kota Kagohashi, Shiyo Enohara, Yuka Morioka, Hiroshi Watarai, Ryuta Muromoto, Yuichi Kitai, Kazuya Iwabuchi, Kenji Oritani, Tadashi Matsuda
PloS one, 16, 4, e0250536, 2021, [International Magazine]
English, [This corrects the article DOI: 10.1371/journal.pone.0241440.].

Implication of NF-κB Activation on Ozone-Induced HO-1 Activation
Sumihito Togi, Misa Togi, Satoshi Nagashima, Yuichi Kitai, Ryuta Muromoto, Jun-ichi Kashiwakura, Toshiaki Miura, Tadashi Matsuda
BPB Reports, 4, 2, 59, 63, Pharmaceutical Society of Japan, 2021
Scientific journal

Graft-versus-host disease develops in mice transplanted with lymphocyte-depleted bone marrow cells from signal-transducing adaptor protein-2 transgenic mice
Hideaki Saito, Michiko Ichii, Jun Toda, Yuichi Kitai, Ryuta Muromoto, Jun-ichi Kashiwakura, Kodai Saitoh, Akira Tanimura, Takafumi Yokota, Hirohiko Shibayama, Tadashi Matsuda, Kenji Oritani, Yuzuru Kanakura, Naoki Hosen
Biochemical and Biophysical Research Communications, 537, 118, 124, Elsevier BV, Jan. 2021
Scientific journal

Expression of signal-transducing adaptor protein-1 attenuates experimental autoimmune hepatitis via down-regulating activation and homeostasis of invariant natural killer T cells
Jun-ichi Kashiwakura, Kodai Saitoh, Takeru Ihara, Yuto Sasaki, Kota Kagohashi, Shiyo Enohara, Yuka Morioka, Hiroshi Watarai, Ryuta Muromoto, Yuichi Kitai, Kazuya Iwabuchi, Kenji Oritani, Tadashi Matsuda
PLOS ONE, 15, 11, e0241440, e0241440, Public Library of Science (PLoS), 11 Nov. 2020
Scientific journal,

Signal-transducing adaptor protein (STAP) family members function as adaptor molecules and are involved in several events during immune responses. Notably however, the biological functions of STAP-1 in other cells are not known. We aimed to investigate the functions of STAP-1 in invariant natural killer T (iNKT) cells and iNKT cell-dependent hepatitis.





We employed concanavalin A (Con A)-induced hepatitis and α-galactosylceramide (α-GalCer)-induced hepatitis mouse models, both are models of iNKT cell-dependent autoimmune hepatitis, and STAP-1 overexpressing 2E10 cells to investigate the role of STAP-1 in iNKT cell activation in vivo an in vitro, respectively.





After Con A- or α-GalCer-injection, hepatocyte necrotic areas and plasma alanine aminotransferase elevation were more severe in STAP-1 knockout (S1KO) mice and milder in lymphocyte-specific STAP-1 transgenic (S1Tg) mice, as compared to wild-type (WT) mice. Two events that may be related to Con A-induced and/or α-GalCer-induced hepatitis were influenced by STAP-1 manipulation. One is that iNKT cell populations in the livers and spleens were increased in S1KO mice and were decreased in S1Tg mice. The other is that Con A-induced interleukin-4 and interferon-γ production was attenuated by STAP-1 overexpression. These effects of STAP-1 were confirmed using 2E10 cells overexpressing STAP-1 that showed impairment of interleukin-4 and interferon-γ production as well as phosphorylation of Akt and mitogen-activated protein kinases in response to Con A stimulation.





These results conclude that STAP-1 regulates iNKT cell maintenance/activation, and is involved in the pathogenesis of autoimmune hepatitis.

Synthesis of Resolvin E3, a Proresolving Lipid Mediator, and Its Deoxy Derivatives: Identification of 18-Deoxy-resolvin E3 as a Potent Anti-Inflammatory Agent
Hayato Fukuda, Hiroyuki Ikeda, Ryuta Muromoto, Koki Hirashima, Kohei Ishimura, Koichi Fujiwara, Haruka Aoki-Saito, Takeshi Hisada, Mizuki Watanabe, Jun Ishihara, Tadashi Matsuda, Satoshi Shuto
The Journal of Organic Chemistry, 85, 21, 14190, 14200, American Chemical Society (ACS), 06 Nov. 2020
Scientific journal

Signal-transducing adapter protein-1 is required for maintenance of leukemic stem cells in CML.
Jun Toda, Michiko Ichii, Kenji Oritani, Hirohiko Shibayama, Akira Tanimura, Hideaki Saito, Takafumi Yokota, Daisuke Motooka, Daisuke Okuzaki, Yuichi Kitai, Ryuta Muromoto, Jun-Ichi Kashiwakura, Tadashi Matsuda, Naoki Hosen, Yuzuru Kanakura
Oncogene, 39, 34, 5601, 5615, 13 Jul. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, The family of signal-transducing adapter proteins (STAPs) has been reported to be involved in a variety of intracellular signaling pathways and implicated as transcriptional factors. We previously cloned STAP-2 as a c-Fms interacting protein and explored its effects on chronic myeloid leukemia (CML) leukemogenesis. STAP-2 binds to BCR-ABL, upregulates BCR-ABL phosphorylation, and activates its downstream molecules. In this study, we evaluated the role of STAP-1, another member of the STAP family, in CML pathogenesis. We found that the expression of STAP-1 is aberrantly upregulated in CML stem cells (LSCs) in patients' bone marrow. Using experimental model mice, deletion of STAP-1 prolonged the survival of CML mice with inducing apoptosis of LSCs. The impaired phosphorylation status of STAT5 by STAP-1 ablation leads to downregulation of antiapoptotic genes, Bcl-2 and Bcl-xL. Interestingly, transcriptome analyses indicated that STAP-1 affects several signaling pathways related to BCR-ABL, JAK2, and PPARγ. This adapter protein directly binds to not only BCR-ABL, but also STAT5 proteins, showing synergistic effects of STAP-1 inhibition and BCR-ABL or JAK2 tyrosine kinase inhibition. Our results identified STAP-1 as a regulator of CML LSCs and suggested it to be a potential therapeutic target for CML.

The mechanism of Tyk2 deficiency-induced immunosuppression in mice involves robust IL-10 production in macrophages
Hirashima K, Muromoto R, Minoguchi H, Matsumoto T, Kitai Y, Kashiwakura JI, Shimoda K, Oritani K, Matsuda T
Cytokine, 130, 155077, 155077, Jun. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, Macrophages are highly plastic in their pro-inflammatory/anti-inflammatory roles. Type I and II interferons (IFNs) are known to modulate macrophage activation. Tyrosine kinase 2 (Tyk2) has an intimate relationship with type I and II IFN signaling. Animal studies have shown that Tyk2 knock-out (KO) in mice is associated with reduced inflammatory responses in various mouse models of diseases. To investigate the role of Tyk2 in inflammation in more detail, we intraperitoneally injected heat-killed Propionibacterium acnes (P. acnes) to Tyk2 KO mice. P. acnes-induced acute peritoneal inflammation, assessed by neutrophil infiltration, was reduced in Tyk2 KO mice. The reduction was accompanied with diminished productions of inflammatory cytokines and an enhanced production of anti-inflammatory IL-10. Unexpectedly, pre-treatment of wild-type mice with the neutralizing antibodies for IFNs did not affect P. acnes-induced neutrophil infiltration. A neutralizing antibody for the IL-10 receptor in Tyk2 KO mice restored P. acnes-induced peritoneal inflammation. Enhanced production of IL-10 from Tyk2 KO peritoneal cells was suppressed by either the cyclooxygenase inhibitor diclofenac or protein kinase A inhibitor H-89. The level of prostaglandin E2 (PGE2) in the steady-state peritoneal cavity in Tyk2 KO mice was higher than that in wild-type mice. Tyk2 KO macrophages showed an enhanced CREB phosphorylation induced by P. acnes plus PGE2. Taken together, these results showed that Tyk2 deficiency potentiates the PGE2-protein kinase A-IL-10 pathway in macrophages, and thereby contributes to potentiation of the immunosuppressive phenotype.

Design and Synthesis of Benzene Congeners of Resolvin E2, a Proresolving Lipid Mediator, as Its Stable Equivalents
Yuto Murakami, Hayato Fukuda, Ryuta Muromoto, Koki Hirashima, Kohei Ishimura, Koichi Fujiwara, Jun Ishihara, Tadashi Matsuda, Mizuki Watanabe, Satoshi Shuto
ACS MEDICINAL CHEMISTRY LETTERS, 11, 4, 479, 484, AMER CHEMICAL SOC, Apr. 2020, [Peer-reviewed]
English, Scientific journal, Resolvins (Rvs) are highly potent anti-inflammatory lipid mediators that are chemically and biologically unstable because of their polyunsaturated structures. To address this issue, we designed benzene congeners of RvE2, i.e., o-, m-, and p-BZ-RvE2s, as stable equivalents of RvE2 by replacing the unstable skipped diene moiety with a benzene ring on the basis of computational conformation studies and synthesized these congeners via a short common route through two Stille couplings. o-BZ-RvE2 exhibited more potent anti-inflammatory activity and much higher metabolic stability than RvE2. Thus, o-BZ-RvE2 was identified as a stable equivalent of RvE2, which is useful as a lead for anti-inflammatory drugs with a new mechanism of action as well as a biotool for investigating RvE2-mediated inflammation resolving pathways.

Tyk2欠損マウスにおける免疫反応抑制にはマクロファージでのIL-10産生増強が関与する　　　　　　　　　　　　　　　
室本 竜太, 平島 洸基, 美濃口 広弥, 松田 正
日本薬学会年会要旨集, 140年会, 26Z, am13, (公社)日本薬学会, Mar. 2020
Japanese

Signal-transducing adaptor protein-2 delays recovery of B lineage lymphocytes during hematopoietic stress.
Michiko Ichii, Kenji Oritani, Jun Toda, Hideaki Saito, Henyun Shi, Hirohiko Shibayama, Daisuke Motooka, Yuichi Kitai, Ryuta Muromoto, Jun-Ichi Kashiwakura, Kodai Saitoh, Daisuke Okuzaki, Tadashi Matsuda, Yuzuru Kanakura
Haematologica, 106, 2, 424, 436, 23 Jan. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, Signal-transducing adaptor protein-2 (STAP-2) was discovered as a C-FMS/M-CSFR interacting protein and subsequently found to function as an adaptor of signaling or transcription factors. These include STAT5, MyD88 and IκB kinase in macrophages, mast cells, and T cells. There is additional information about roles for STAP-2 in several types of malignant diseases including chronic myeloid leukemia, however, none have been reported concerning B lineage lymphocytes. We have now exploited gene targeted and transgenic mice to address this lack of knowledge, and demonstrated that STAP-2 is not required under normal, steady-state conditions. However, recovery of B cells following transplantation was augmented in the absence of STAP-2. This appeared to be restricted to cells of B cell lineage with myeloid rebound noted as unremarkable. Furthermore, all hematological parameters were observed to be normal once recovery from transplantation was complete. Furthermore, overexpression of STAP-2, specifically in lymphoid cells, resulted in reduced numbers of late-stage B cell progenitors within the bone marrow. While numbers of mature peripheral B and T cells were unaffected, recovery from sub-lethal irradiation or transplantation was dramatically reduced. Lipopolysaccharide (LPS) normally suppresses B precursor expansion in response to interleukin 7, however, STAP-2 deficiency made these cells more resistant. Preliminary RNA-Seq analyses indicated multiple signaling pathways in B progenitors as STAP-2-dependent. These findings suggest that STAP-2 modulates formation of B lymphocytes in demand conditions. Further study of this adapter protein could reveal ways to speed recovery of humoral immunity following chemotherapy or transplantation.

Dimethyl fumarate dampens IL-17-ACT1-TBK1 axis-mediated phosphorylation of Regnase-1 and suppresses IL-17-induced IκB-ζ expression.
Ohgakiuchi Y, Saino Y, Muromoto R, Komori Y, Sato A, Hirashima K, Kitai Y, Kashiwakura JI, Oritani K, Matsuda T
Biochemical and biophysical research communications, 521, 4, 957, 963, Nov. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, The signaling elicited by the cytokine interleukin-17A (IL-17) is important for antimicrobial defense responses, whereas excessive IL-17 production leads to autoimmune diseases such as psoriasis and multiple sclerosis. IL-17-induced stabilization of mRNAs has been recognized as a unique and important feature of IL-17 signaling. Previously, we demonstrated that IL-17 signaling protein ACT1 is required to counteract constitutive inhibitor of nuclear factor kappa B zeta (IκB-ζ) mRNA degradation by the ribonuclease Regnase-1. However, information about the mechanism of mRNA stabilization in IL-17-stimulated cells remains insufficient. In the present study, we aimed to clarify the mechanism in more detail and identify an agent that can inhibit IL-17-induced mRNA stabilization. Experiments using small interfering RNA and an inhibitor of TANK-binding kinase 1 (TBK1) revealed that TBK1 was required for IκB-ζ mRNA stabilization through Regnase-1 phosphorylation. Intriguingly, this TBK1-mediated phosphorylation of Regnase-1 was suppressed by the addition of dimethyl fumarate (DMF), an electrophilic small molecule that has been used to treat IL-17-related autoimmune diseases. Confocal microscopic observation of the cellular localization of ACT1 revealed that DMF treatment resulted in the disappearance of ACT1 nuclear dots and perinuclear accumulation of ACT1. These results suggested that DMF is a small molecule that compromises IL-17-induced activation of the ACT1-TBK1 pathway, thereby inhibiting IL-17-induced mRNA stabilization.

The basophil-IL-4-mast cell axis is required for food allergy.
Jun-Ichi Kashiwakura, Tomoaki Ando, Hajime Karasuyama, Masato Kubo, Kenji Matsumoto, Tadashi Matsuda, Toshiaki Kawakami
Allergy, 74, 10, 1992, 1996, Oct. 2019, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

IκB-ζ Expression Requires Both TYK2/STAT3 Activity and IL-17-Regulated mRNA Stabilization.
Muromoto R, Tawa K, Ohgakiuchi Y, Sato A, Saino Y, Hirashima K, Minoguchi H, Kitai Y, Kashiwakura JI, Shimoda K, Oritani K, Matsuda T
ImmunoHorizons, 3, 5, 172, 185, May 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, Cytokine IL-17A (IL-17) acts on various cell types, including epidermal keratinocytes, and induces antimicrobial peptide and chemokine production to elicit antibacterial and antifungal defense responses. Excess IL-17 leads to inflammatory skin diseases such as psoriasis. The IκB family protein IκB-ζ mediates IL-17-induced responses. However, the mechanism controlling IκB-ζ expression in IL-17-stimulated cells remains elusive. In this study, we showed that JAK kinase TYK2 positively regulates IL-17-induced IκB-ζ expression. TYK2-deficient mice showed reduced inflammation and concomitant reduction of IκB-ζ mRNA compared with wild-type mice in imiquimod-induced skin inflammation. The analysis of the IκB-ζ promoter activity using human cell lines (HaCaT and HeLa) revealed that catalytic activity of TYK2 and its substrate transcription factor STAT3, but not IL-17, is required for IκB-ζ promoter activity. In contrast, IL-17-induced signaling, which did not activate STAT3, posttranscriptionally stabilized IκB-ζ mRNA via its 3'-untranslated region. IL-17 signaling protein ACT1 was required to counteract constitutive IκB-ζ mRNA degradation by RNase Regnase-1. These results suggested that transcriptional activation by TYK2-STAT3 pathway and mRNA stabilization by IL-17-mediated signals act separately from each other but complementarily to achieve IκB-ζ induction. Therefore, JAK/TYK2 inhibition might be of significance in regulation of IL-17-induced inflammatory reactions.

STAP-2 positively regulates FcRI-mediated basophil activation and basophil-dependent allergic inflammatory reaction.
Kashiwakura JI, Yamashita S, Yoshihara M, Inui K, Saitoh K, Sekine Y, Muromoto R, Kitai Y, Oritani K, Matsuda T
International immunology, 31, 5, 349, 356, Feb. 2019, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Basophils are an important cell type in the regulation of Th2 immune responses. Recently, we revealed that signal-transducing adaptor protein-2 (STAP-2) negatively regulates mast cell activation via FcεRI. However, the role of STAP-2 in basophil maturation and activation remained unclear. In this study, we demonstrated the normal development of basophils in STAP-2-deficient (STAP-2-/-) mice. We also demonstrated in vitro normal basophil differentiation and FcεRI expression in STAP-2-/- mice, suggesting that STAP-2 is dispensable for basophil maturation. Using bone marrow-derived cultured basophils (BMBs), we showed that degranulation and cytokine production of STAP-2-/- BMBs were lower than those of wild-type (WT) BMBs upon stimulation with IgE/Ag. In accordance with the reduction of degranulation and cytokine production, phosphorylation of several signal molecules such as Lyn, PLC-γ2 and Erk was reduced in STAP-2-/- BMBs after stimulation via FcεRI. Finally, it was observed that IgE-dependent chronic allergic inflammation of STAP-2-/- mice was significantly inhibited compared with WT mice. Taken together, we conclude that STAP-2 is an adaptor molecule that positively regulates FcεRI-mediated basophil activation and basophil-dependent allergic inflammatory reactions.

A Novel α9 Integrin Ligand, XCL1/Lymphotactin,Is Involved in the Development of Murine Models of Autoimmune Diseases
松本, 尚樹, 今, 重之, 中鶴, 拓也, 宮下, 友惠, 乾, 恭輔, 齋藤, 浩大, 鍛代, 悠—, 室本, 竜太, 柏倉, 淳一, 上出, 利光, 松田, 正
Annual report of the Faculty of Pharmacy & Pharmaceutical Sciences, Fukuyama University, 36, 50, 51, 福山大学薬学部, 25 Dec. 2018
Japanese, Research institution

Signal Transducing Adaptor Protein (STAP) Family Accelerates Gut and Thymic Graft-Versus-Host-Disease in Murine Model.　　　　　　　　　　　　　　　
Hideaki Saito, Michiko Ichii, Jun Toda, Yuichi Kitai, Ryuta Muromoto, Jun-ichi Kashiwakura, Kodai Saitoh, Hirohiko Shibayama, Tadashi Matsuda, Kenji Oritani, Yuzuru Kanakura
The American Society of Hematology 60th Annual Meeting, Dec. 2018, [Peer-reviewed]
English

Signal transducing adaptor protein-2(STAP-2)はマウスモデルで慢性GVHDを悪化させる　　　　　　　　　　　　　　　
西東秀晃, 一井倫子, 戸田 淳, 鍛代悠一, 室本竜太, 柏倉淳一, 齋藤浩大, 柴山浩彦, 松田 正, 織谷健司, 金倉 譲
第80回日本血液学会学術集会, Oct. 2018, [Peer-reviewed]
Japanese, Research society

慢性骨髄性白血病におけるSTAPファミリー蛋白の役割　　　　　　　　　　　　　　　
戸田 淳, 一井倫子, 西東秀晃, 鍛代悠一, 室本竜太, 柏倉淳一, 齋藤浩大, 柴山浩彦, 松田 正, 織谷健司, 金倉 譲
第80回日本血液学会学術集会, Oct. 2018, [Peer-reviewed]
Japanese, Research society

骨髄内B細胞分化におけるSTAP-2蛋白の役割　　　　　　　　　　　　　　　
一井倫子, 織谷健司, 柴山浩彦, 戸田 淳, 西東秀晃, 北井勇一, 室本竜太, 柏倉淳一, 齋藤浩大, 松田 正, 金倉 譲
第80回日本血液学会学術集会, Oct. 2018, [Peer-reviewed]
Japanese

慢性骨髄性白血病におけるSTAPファミリー蛋白の役割(Roles of signal trandsducing adaptor proteins in the pathogenesis of chronic myelogenous leukemia)　　　　　　　　　　　　　　　
戸田 淳, 一井 倫子, 西東 秀晃, 鍛代 悠一, 室本 竜太, 柏倉 淳一, 齋藤 浩大, 柴山 浩彦, 松田 正, 織谷 健司, 金倉 譲
臨床血液, 59, 9, 1531, 1531, (一社)日本血液学会-東京事務局, Sep. 2018
English

Signal transducing adaptor protein-2(STAP-2)はマウスモデルで慢性GVHDを悪化させる(Signal transducing adaptor protein-2, STAP-2 accelerates chronic GVHD in murine mouse model)　　　　　　　　　　　　　　　
西東 秀晃, 一井 倫子, 戸田 淳, 鍛代 悠一, 室本 竜太, 柏倉 淳一, 齋藤 浩大, 柴山 浩彦, 松田 正, 織谷 健司, 金倉 譲
臨床血液, 59, 9, 1545, 1545, (一社)日本血液学会-東京事務局, Sep. 2018
English

骨髄内B細胞分化におけるSTAP-2蛋白の役割(Signal-transducing adaptor protein-2 regulates humoral immune reconstitution at pre-B stage)　　　　　　　　　　　　　　　
一井 倫子, 織谷 健司, 柴山 浩彦, 戸田 淳, 西東 秀晃, 北井 勇一, 室本 竜太, 柏倉 淳一, 齋藤 浩大, 松田 正, 金倉 譲
臨床血液, 59, 9, 1695, 1695, (一社)日本血液学会-東京事務局, Sep. 2018
English

Synthesis of Chiral cis-Cyclopropane Bearing Indole and Chromone as Potential TNFα Inhibitors
Ryutaro Kanada, Makoto Tanabe, Ryuta Muromoto, Yukina Sato, Tomoki Kuwahara, Hayato Fukuda, Mitsuhiro Arisawa, Tadashi Matsuda, Mizuki Watanabe, Satoshi Shuto
Journal of Organic Chemistry, 83, 15, 7672, 7682, 03 Aug. 2018, [Peer-reviewed]
Scientific journal, © 2018 American Chemical Society. Conformationally restricted analogues of SPD-304, the first small-molecule TNFα inhibitor, in which two heteroaryl groups, indole and chromone, are connected by chiral methyl- or ethyl-cis-cyclopropane, were designed. Synthesis of these molecules was achieved via Suzuki-Miyaura or Stille coupling reactions with chiral bromomethylenecyclopropane or iodovinyl-cis-cyclopropane as the substrate, both of which were prepared from chiral methylenecyclopropane as a common intermediate, constructing the heteroaryl-methyl or -ethyl-cis-cyclopropane structures as key steps. This study presents an efficient synthesis of a series of chiral cis-cyclopropane conjugates with two heteroaryl groups.

新規α4インテグリンスプライシングバリアントは内在性α4インテグリン抑制分子である　　　　　　　　　　　　　　　
重政 歩美, 乾 恭輔, 本田 真知子, 松田 正, 今 重之
日本薬学会年会要旨集, 138年会, 3, 72, 72, (公社)日本薬学会, Mar. 2018
Japanese

自己免疫疾患における細胞外基質ネフロネクチンの関与　　　　　　　　　　　　　　　
藤岡 和征, 本田 真知子, 松田 正, 今 重之
日本薬学会年会要旨集, 138年会, 3, 72, 72, (公社)日本薬学会, Mar. 2018
Japanese

細胞外基質ネフロネクチンに対するサンドイッチELISAシステムの構築　　　　　　　　　　　　　　　
本田 真知子, 李 順姫, 松田 正, 大槻 剛巳, 今 重之
日本薬学会年会要旨集, 138年会, 3, 73, 73, (公社)日本薬学会, Mar. 2018
Japanese

A new STAT3-binding partner, ARL3, enhances the phosphorylation and nuclear accumulation of STAT3
硎, 澄仁, 室本, 竜太, 平島, 洸基, 鍛代, 悠—, 岡山, 太一郎, 池田, 収, 松本, 尚樹, 今, 重之, 関根, 勇—, 織谷, 健司, 松田, 正
Annual report of the Faculty of Pharmacy & Pharmaceutical Sciences, Fukuyama University, 35, 53, 53, 福山大学薬学部, 25 Dec. 2017
Japanese, Research institution

Anti-IL-17 A blocking antibody reduces cyclosporinA-induced relapse in experimental autoimmune encephalomyelitis mice.
齋藤, 浩大, 今, 重之, 中鶴, 拓也, 乾, 恭輔, 伊原, 建, 松本, 尚樹, 鍛代, 悠—, 室本, 竜太, 松田, 正
Annual report of the Faculty of Pharmacy & Pharmaceutical Sciences, Fukuyama University, 35, 54, 54, 福山大学薬学部, 25 Dec. 2017
Japanese, Research institution

STAP-2 protein promotes prostate cancer growth by enhancing epidermal growth factor receptor stabilization
Yuichi Kitai, Masashi Iwakami, Kodai Saitoh, Sumihito Togi, Serina Isayama, Yuichi Sekine, Ryuta Muromoto, Jun-ichi Kashiwakura, Akihiko Yoshimura, Kenji Oritani, Tadashi Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 292, 47, 19392, 19399, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Nov. 2017, [Peer-reviewed]
English, Scientific journal, Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signaling pathways and promotes tumorigenesis in melanoma and breast cancer cells. However, the contribution of STAP-2 to the behavior of other types of cancer cells is unclear. Here, we show that STAP-2 promotes tumorigenesis of prostate cancer cells through up-regulation of EGF receptor (EGFR) signaling. Tumor growth of a prostate cancer cell line, DU145, was strongly decreased by STAP-2 knockdown. EGF-induced gene expression and phosphorylation of AKT, ERK, and STAT3 were significantly decreased in STAP-2-knockdown DU145 cells. Mechanistically, we found that STAP-2 interacted with EGFR and enhanced its stability by inhibiting c-CBL-mediated EGFR ubiquitination. Our results indicate that STAP-2 promotes prostate cancer progression via facilitating EGFR activation.

Biochanin A enhances ROR gamma activity through STAT3-mediated recruitment of NCOA1
Miki Takahashi, Ryuta Muromoto, Hiroyuki Kojima, Shinji Takeuchi, Yuichi Kitai, Jun-ichi Kashiwakura, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 489, 4, 503, 508, ACADEMIC PRESS INC ELSEVIER SCIENCE, Aug. 2017, [Peer-reviewed]
English, Scientific journal, Interleukin (IL)-17-producing T cells play important roles in autoimmunity, chronic inflammation and host protection against extracellular bacteria and fungi. The retinoic acid receptor-related orphan receptors (ROR) alpha and beta are key regulators of the IL-17-producing phenotype. We previously showed that the isoflavone biochanin A enhanced ROR-mediated transcriptional activity. Here, we investigated the possible mechanisms underlying this ROR activation. Biochanin A-treated murine thymoma EL4 and primary splenocytes demonstrated enhanced induction of IL-17. Biochanin A also induced tyrosine-phosphorylation of signal transducer and activator of transcription 3 (STAT3) in these cells. Stable knockdown of either ROR gamma or STAT3 in EL4 cells canceled biochanin A-induced upregulation of IL-17 expression. Importantly, biochanin A enhanced complex formation between ROR gamma and STAT3 or nuclear-receptor coactivator 1 (NCOA1). Furthermore, the biochanin A-induced ROR gamma-NCOA1 complex was disrupted by a dominant negative mutant of STAT3 or by the STAT3 specific inhibitor Stattic. These results suggest that biochanin A activates ROR gamma-dependent IL-17 transcription through the enhancement of STAT3 phosphorylation and STAT3-mediated recruitment of NCOA1 to ROR gamma. (C) 2017 Elsevier Inc. All rights reserved.

A Novel alpha 9 Integrin Ligand, XCL1/Lymphotactin, Is Involved in the Development of Murine Models of Autoimmune Diseases
Naoki Matsumoto, Shigeyuki Kon, Takuya Nakatsuru, Tomoe Miyashita, Kyosuke Inui, Kodai Saitoh, Yuichi Kitai, Ryuta Muromoto, Jun-ichi Kashiwakura, Toshimitsu Uede, Tadashi Matsuda
JOURNAL OF IMMUNOLOGY, 199, 1, 82, 90, AMER ASSOC IMMUNOLOGISTS, Jul. 2017, [Peer-reviewed]
English, Scientific journal, The integrin alpha 9 beta is a key receptor involved in the development of autoimmune diseases. However, the detailed mechanism for the association of alpha 9 beta 1 integrin with its ligands remains unclear. In this study, we introduce XCL1/lymphotactin, a member of the chemokine family, as a novel ligand for alpha 9 integrin. Using alpha 9 integrin-overexpressing NIH3T3 cells and endogenously alpha 9 integrin-expressing human rhabdomyosarcoma cells, the interaction between XCL1 and alpha 9 integrin was confirmed by pulldown assays. XCL1 enhanced alpha 9 integrin-dependent cell migration of these cells, thus acting on alpha 9 integrin as a chemoattractant. We also analyzed the in vivo function of XCL1 in the development of anti-type II collagen Ab-induced inflammatory arthritis (CAIA) in BALB/c mice and experimental autoimmune encephalomyelitis in C57BL/6 mice, because alpha 9 integrin is involved in these autoimmune disease models. In CAIA, recombinant XCL1 aggravated the disease and this exacerbation was inhibited by an anti-alpha 9 integrin Ab. An XCL1-neutralizing Ab produced in this study also ameliorated CAIA. Furthermore, the XCL1neutralizing Ab abrogated the disease progression in experimental autoimmune encephalomyelitis. Therefore, to our knowledge this study provides the first in vitro and in vivo evidence that the interaction between XCL1 and alpha 9 integrin has an important role for autoimmune diseases.

STAP-2 interacts with Pyk2 and enhances Pyk2 activity in T-cells
Kodai Saitoh, Takuya Tsuchiya, Jun-ichi Kashiwakura, Ryuta Muromoto, Yuichi Kitai, Yuichi Sekine, Kenji Oritani, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 488, 1, 81, 87, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jun. 2017, [Peer-reviewed]
English, Scientific journal, STAP-2 is an adaptor molecule regulating several signaling pathways, including TLRs and cytokine/chemokine receptors in immune cells. We previously reported that STAP-2 enhances SDF-1 a-induced Vavl/Racl-mediated T-cell chemotaxis. However, the detailed mechanisms of STAP-2 involvement in enhancing T-cell chemotaxis remain unknown. In the present study, we demonstrate that STAP-2 directly interacts with Pyk2, which is a key molecule in the regulation of SDF-la/CXCR4-mediated T-cell chemotaxis, and increases phosphorylation of Pyk2. Pyk2 itself can induce STAP-2 Y250 phosphorylation, and this phosphorylation is critical for maximal interactions between STAP-2 and Pyk2. Finally, SDF-1 a induced T-cell chemotaxis is inhibited by treatment with Pyk2 siRNA or AG17, an inhibitor of Pyk2, in Jurkat cells overexpressing STAP-2. Taken together, the Pyk2/STAP-2 interaction is a novel mechanism to regulate SDF-1 alpha-dependent T-cell chemotaxis. (C) 2017 Elsevier Inc. All rights reserved.

Dupuytren拘縮における線維化メカニズムの解析　　　　　　　　　　　　　　　
松井 雄一郎, 今 重之, 船越 忠直, 松田 正, 岩崎 倫政
日本手外科学会雑誌, 34, 1, S207, S207, (一社)日本手外科学会, Apr. 2017
Japanese

Dupuytren拘縮における線維化メカニズムの解析
松井 雄一郎, 船越 忠直, 河村 太介, 亀田 裕亮, 岩崎 倫政, 今 重之, 宮下 友恵, 松田 正
北海道整形災害外科学会雑誌, 58, 2, 302, 302, 北海道整形災害外科学会, Mar. 2017
Japanese

DNA-Containing Exosomes Derived from Cancer Cells Treated with Topotecan Activate a STING- Dependent Pathway and Reinforce Antitumor Immunity
Yuichi Kitai, Takumi Kawasaki, Takuya Sueyoshi, Kouji Kobiyama, Ken J. Ishii, Jian Zou, Shizuo Akira, Tadashi Matsuda, Taro Kawai
JOURNAL OF IMMUNOLOGY, 198, 4, 1649, 1659, AMER ASSOC IMMUNOLOGISTS, Feb. 2017, [Peer-reviewed]
English, Scientific journal, Danger- associated molecular patterns derived from damaged or dying cells elicit inflammation and potentiate antitumor immune responses. In this article, we show that treatment of breast cancer cells with the antitumor agent topotecan (TPT), an inhibitor of topoisomerase I, induces danger-associated molecular pattern secretion that triggers dendritic cell (DC) activation and cytokine production. TPT administration inhibits tumor growth in tumor- bearing mice, which is accompanied by infiltration of activated DCs and CD8 (+) T cells. These effects are abrogated in mice lacking STING, an essential molecule in cytosolic DNA- mediated innate immune responses. Furthermore, TPT- treated cancer cells release exosomes that contain DNA that activate DCs via STING signaling. These findings suggest that a STING- dependent pathway drives antitumor immunity by responding to tumor cell-derived DNA.

Increased expression of v integrin as a regulator of fibrosis in Dupuytren's nodules
Y. Matsui, S. Kon, T. Funakoshi, T. Miyashita, T. Matsuda, N. Iwasaki
JOURNAL OF HAND SURGERY-EUROPEAN VOLUME, 42, 1, 18, 25, SAGE PUBLICATIONS LTD, Jan. 2017, [Peer-reviewed]
English, Scientific journal, Although Dupuytren's contracture is characterized by increased transforming growth factor-1 (TGF-1) and fibrosis in the palmar fascia, the relationship between TGF-1 and integrins, which are considered to be related to fibrosis, remains unclear. We investigated the involvement of TGF-1 and integrins in the pathological palmar fascia of Dupuytren's contracture. Seven patients underwent partial fasciectomy for treatment of this disease. The nodule and cord were isolated from the fascial tissues of the patients. Control fasciae were obtained from seven patients with carpal tunnel syndrome. Immunohistochemical analysis was performed to detect the fibrosis marker -smooth muscle actin and integrins in the fascial tissues. The expression of TGF-1 and integrins was assessed by real-time polymerase chain reaction. The results suggest that nodules may be areas involved in activation of fibrosis in the fascia, associated with increased expression of TGF-1 and v integrin. Thus, v integrin may contribute to fibrosis in Dupuytren's contracture by activating TGF-1. Level of Evidence: IV

Design and Synthesis of Cyclopropane Congeners of Resolvin E2, an Endogenous Proresolving Lipid Mediator, as Its Stable Equivalents
Hayato Fukuda, Ryuta Muromoto, Yuuki Takakura, Kohei Ishimura, Ryutaro Kanada, Daichi Fushihara, Makoto Tanabe, Kotaro Matsubara, Toru Hirao, Koki Hirashima, Hiroshi Abe, Mitsuhiro Arisawa, Tadashi Matsuda, Satoshi Shuto
Organic Letters, 18, 24, 6224, 6227, 16 Dec. 2016, [Peer-reviewed]
Scientific journal, © 2016 American Chemical Society. Lipid chemical mediator resolvins with highly potent anti-inflammatory activity can be leads to develop novel anti-inflammatory drugs; however, they are unstable in oxygen due to their characteristic polyunsaturated structures. To solve the problem, CP-RvE2 has been designed and synthesized in which the cis-olefin of RvE2 was replaced with a cyclopropane. CP-RvE2s were much more stable than RvE2 against autoxidation and equipotent or more potent than RvE2. CP-RvE2s were successfully identified as stable equivalents of RvE2.

Anti-IL-17A blocking antibody reduces cyclosporin A-induced relapse in experimental autoimmune encephalomyelitis mice.
Kodai Saitoh, Shigeyuki Kon, Takuya Nakatsuru, Kyosuke Inui, Takeru Ihara, Naoki Matsumoto, Yuichi Kitai, Ryuta Muromoto, Tadashi Matsuda
Biochemistry and biophysics reports, 8, 139, 145, Dec. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, Cyclosporin A (CsA) is effective at reducing pathogenic immune responses, but upon withdrawal of CsA the immune response often "rebounds" resulting in a relapse or exacerbation of disease. The mechanisms, cells and cytokines involved in the relapse or exacerbation after CsA withdrawal are unknown. We hypothesized that CsA withdrawal induces IL-17 production that could be responsible for relapse, and examined the effect of anti-IL-17A antibody on relapse induced after CsA withdrawal in mouse experimental autoimmune encephalomyelitis (EAE). CsA treatment markedly decreased the EAE disease score during the first episode, but augmented disease severity after CsA withdrawal, compared to untreated mice. After discontinuation of CsA the production of IL-17A was increased and the severity of relapse in EAE was reduced by treatment with anti-IL-17A antibody. These results suggest that the resumption of T cell immune responses after CsA withdrawal leads to a burst of IL-17A production that is at least partially responsible for relapse in EAE mice.

The immunosuppressive effect of domain-deleted dimer of HLA-G2 isoform in collagen-induced arthritis mice
Ami Takahashi, Kimiko Kuroki, Yuki Okabe, Yoshiyuki Kasai, Naoki Matsumoto, Chisato Yamada, Toshiyuki Takai, Toyoyuki Ose, Shigeyuki Kon, Tadashi Matsuda, Katsumi Maenaka
HUMAN IMMUNOLOGY, 77, 9, 754, 759, ELSEVIER SCIENCE INC, Sep. 2016, [Peer-reviewed]
English, Scientific journal, HLA-G is involved in maternal-fetal immune tolerance and is reported to be a natural tolerogenic molecule. Seven-spliced isoforms including dimeric and beta 2m-free forms have been identified. The major isoform, HLA-G1 (and its soluble type HLA-G5), binds to the inhibitory immune receptors, leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2. We previously reported that HLA-G1 also binds to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs, and had a significant immunosuppressive effect in collagen-induced arthritis (CIA) mice. Although HLA-G2 and its soluble form HLA-G6 bind specifically to LILRB2, its functional characteristics are largely unknown. In this study, we report the significant immunosuppressive effect of HLA-G2 dimer in CIA mice. Surface plasmon resonance analysis revealed a specific interaction of HLA-G2 with PIR-B. CIA mice were administered HLA-G2 protein subcutaneously once in the left footpad and clinical severity was evaluated in a double-blind study. A single administration of HLA-G2 maintained a suppressive effect for over 1 month. These results suggested that the HLA-G2 protein might be a useful biopharmaceutical for the treatment of rheumatoid arthritis by binding to inhibitory PIR-B. (C) 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

IL-17A plays a central role in the expression of psoriasis signature genes through the induction of I kappa B-zeta in keratinocytes
Ryuta Muromoto, Toru Hirao, Keisuke Tawa, Koki Hirashima, Shigeyuki Kon, Yuichi Kitai, Tadashi Matsuda
INTERNATIONAL IMMUNOLOGY, 28, 9, 443, 452, OXFORD UNIV PRESS, Sep. 2016, [Peer-reviewed]
English, Scientific journal, In psoriasis lesions, a diverse mixture of cytokines is up-regulated that influence each other generating a complex inflammatory situation. Although this is the case, the inhibition of IL-17A alone showed unprecedented clinical results in patients, indicating that IL-17A is a critical inducer of psoriasis pathogenesis. To elucidate IL-17A-driven keratinocyte-intrinsic signaling pathways, we treated monolayers of normal human epidermal keratinocytes in vitro with a mixture of six cytokines (IL-17A, TNF-alpha, IL-17C, IL-22, IL-36. and IFN-gamma) involved in psoriasis to mimic the inflammatory milieu in psoriasis lesions. Microarray and gene set enrichment analysis revealed that this cytokine mixture induced similar gene expression changes with the previous transcriptome studies using psoriasis lesions. Importantly, we identified a set of IL-17A-regulated genes in keratinocytes, which recapitulate typical psoriasis genes exemplified by DEFB4A, S100A7, IL19 and CSF3, based on the differences in the expression profiles of cells stimulated with six cytokines versus cells stimulated with only five cytokines lacking IL-17A. Furthermore, a specific IL-17A-induced gene, NFKBIZ, which encodes I kappa B-zeta, a transcriptional regulator for NF-kappa B, was demonstrated to have a significant role for IL-17A-induced gene expression. Thus, we present novel in vitro data from normal human keratinocytes that would help elucidating the IL-17A-driven keratinocyte activation in psoriasis.

A New STAT3-binding Partner, ARL3, Enhances the Phosphorylation and Nuclear Accumulation of STAT3.
Sumihito Togi, Ryuta Muromoto, Koki Hirashima, Yuichi Kitai, Taichiro Okayama, Osamu Ikeda, Naoki Matsumoto, Shigeyuki Kon, Yuichi Sekine, Kenji Oritani, Tadashi Matsuda
The Journal of biological chemistry, 291, 21, 11161, 71, 20 May 2016, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Signal transducer and activator of transcription 3 (STAT3) is involved in cell proliferation, differentiation, and cell survival during immune responses, hematopoiesis, neurogenesis, and other biological processes. STAT3 activity is regulated by a variety of mechanisms, including phosphorylation and nuclear translocation. To clarify the molecular mechanisms underlying the regulation of STAT3 activity, we performed yeast two-hybrid screening. We identified ARL3 (ADP-ribosylation factor-like 3) as a novel STAT3-binding partner. ARL3 recognizes the DNA-binding domain as well as the C-terminal region of STAT3 in vivo, and their binding was the strongest when both proteins were activated. Importantly, small interfering RNA-mediated reduction of endogenous ARL3 expression decreased IL-6-induced tyrosine phosphorylation, nuclear accumulation, and transcriptional activity of STAT3. These results indicate that ARL3 interacts with STAT3 and regulates the transcriptional activation of STAT3 by influencing its nuclear accumulation of STAT3.

Dupuytren拘縮におけるインテグリンの関与
松井 雄一郎, 船越 忠直, 瓜田 淳, 河村 太介, 佃 幸憲, 岩崎 倫政, 今 重之, 松田 正
北海道整形災害外科学会雑誌, 57, 2, 288, 288, 北海道整形災害外科学会, Apr. 2016
Japanese

Dupuytren拘縮の最前線 Dupuytren拘縮の進行におけるインテグリンαvの関与　　　　　　　　　　　　　　　
松井 雄一郎, 今 重之, 船越 忠直, 河村 太介, 亀田 裕亮, 宮下 友恵, 松田 正, 岩崎 倫政
日本整形外科学会雑誌, 90, 2, S379, S379, (公社)日本整形外科学会, Mar. 2016
Japanese

Caspase-dependent cleavage regulates protein levels of Epstein-Barr virus-derived latent membrane protein 1
Sumihito Togi, Yosuke Hatano, Ryuta Muromoto, Eri Kawanishi, Osamu Ikeda, Koki Hirashima, Shigeyuki Kon, Yuichi Kitai, Teruhito Yasui, Kenji Oritani, Tadashi Matsuda
FEBS LETTERS, 590, 6, 808, 818, WILEY-BLACKWELL, Mar. 2016, [Peer-reviewed]
English, Scientific journal, Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1) plays pathogenic roles in EBV-related diseases. Thus, host cells employ several mechanisms to regulate LMP1 functions, and we previously reported possible regulation by signal transducing adaptor protein-2 as well as BS69. Here, we found that caspase-3 mainly degraded LMP1 proteins in HeLa cells, leading to decreased NF-kappa B and STAT3 activation. Caspase-3 cleaved the consensus DNTD sequences in the CTAR3 region of LMP1. Of importance, LMP1 expression strongly enhanced caspase-3 activity. Taken together, the reduction of LMP1 protein levels by caspases is likely to be a newly identified host defense against EBV infection.

α9インテグリンバリアントSFα9結合分子に着目した新規自己免疫疾患治療薬の開発　　　　　　　　　　　　　　　
松本 尚樹, 中鶴 拓也, 宮下 友惠, 今 重之, 松田 正
日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [1P0081], [1P0081], (公社)日本生化学会, Dec. 2015
Japanese

β8インテグリンスプライシングバリアントは新規TGF-βシグナル制御分子である　　　　　　　　　　　　　　　
宮下 友惠, 今 重之, 中鶴 拓也, 松本 尚樹, 松井 雄一郎, 岩崎 倫政, 松田 正
日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [1P0082], [1P0082], (公社)日本生化学会, Dec. 2015
Japanese

自己免疫疾患における新規カルシウム結合タンパク質、ネフロネクチンの役割　　　　　　　　　　　　　　　
中鶴 拓也, 今 重之, 宮下 友惠, 松本 尚樹, 乾 恭輔, 石川 清, 瀬川 辰也, 前田 雅弘, 松田 正
日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [1P0084], [1P0084], (公社)日本生化学会, Dec. 2015
Japanese

T細胞活性化におけるSTAP-1及びSTAP-2の役割　　　　　　　　　　　　　　　
齋藤 浩大, 今 重之, 小澤 清貴, 伊原 建, 関根 勇一, 室本 竜太, 鍛代 悠一, 吉村 昭彦, 織谷 健司, 松田 正
日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [3T特, 02(3P1126)], (公社)日本生化学会, Dec. 2015
Japanese

Signal transducer and activator of transcription 3 regulation by novel binding partners.
Tadashi Matsuda, Ryuta Muromoto, Yuichi Sekine, Sumihito Togi, Yuichi Kitai, Shigeyuki Kon, Kenji Oritani
World journal of biological chemistry, 6, 4, 324, 32, 26 Nov. 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal, Signal transducers and activators of transcription (STATs) mediate essential signals for various biological processes, including immune responses, hematopoiesis, and neurogenesis. STAT3, for example, is involved in the pathogenesis of various human diseases, including cancers, autoimmune and inflammatory disorders. STAT3 activation is therefore tightly regulated at multiple levels to prevent these pathological conditions. A number of proteins have been reported to associate with STAT3 and regulate its activity. These STAT3-interacting proteins function to modulate STAT3-mediated signaling at various steps and mediate the crosstalk of STAT3 with other cellular signaling pathways. This article reviews the roles of novel STAT3 binding partners such as DAXX, zipper-interacting protein kinase, Krüppel-associated box-associated protein 1, Y14, PDZ and LIM domain 2 and signal transducing adaptor protein-2, in the regulation of STAT3-mediated signaling.

CCR7 is involved in BCR-ABL/STAP-2-mediated cell growth in hematopoietic Ba/F3 cells
Kaori Kubo, Masashi Iwakami, Ryuta Muromoto, Takuya Inagaki, Yuichi Kitai, Shigeyuki Kon, Yuichi Sekine, Kenji Oritani, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 463, 4, 825, 831, ACADEMIC PRESS INC ELSEVIER SCIENCE, Aug. 2015, [Peer-reviewed]
English, Scientific journal, Chronic myeloid leukemia is a clonal disease characterized by the presence of the Philadelphia chromosome and its oncogenic product, BCR-ABL, which activates multiple pathways involved in cell survival, growth promotion, and disease progression. We previously reported that in murine hematopoietic Ba/F3 cells, signal transducing adaptor protein-2 (STAP-2) binds to BCR-ABL and up-regulates BCR-ABL phosphorylation, leading to enhanced activation of its downstream signaling molecules. The binding of STAP2 to BCR-ABL also influenced the expression levels of chemokine receptors, such as CXCR4 and CCR7. For the induction of CCR7 expression, signals mediated by the MAPK/ERK pathway were critical in Ba/F3 cells expressing BCR-ABL and STAP-2. In addition, STAP-2 cooperated with BCR-ABL to induce the production of CCR7 ligands, CCL19 and CCL21. Our results demonstrate a contribution of CCR7 to STAP-2-dependent enhancement of BCR-ABL-mediated cell growth in Ba/F3 cells. (C) 2015 Elsevier Inc. All rights reserved.

STAP-2 Protein Expression in B16F10 Melanoma Cells Positively Regulates Protein Levels of Tyrosinase, Which Determines Organs to Infiltrate in the Body
Yuichi Sekine, Sumihito Togi, Ryuta Muromoto, Shigeyuki Kon, Yuichi Kitai, Akihiko Yoshimura, Kenji Oritani, Tadashi Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 290, 28, 17462, 17473, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Jul. 2015, [Peer-reviewed]
English, Scientific journal, Melanoma is the most serious type of skin cancer, with a highly metastatic phenotype. In this report, we show that signal transducing adaptor protein 2 (STAP-2) is involved in cell migration, proliferation, and melanogenesis as well as chemokine receptor expression and tumorigenesis in B16F10 melanoma cells. This was evident in mice injected with STAP-2 shRNA (shSTAP-2)-expressing B16F10 cells, which infiltrated organs in a completely different pattern from the original cells, showing massive colonization in the liver, kidney, and neck but not in the lung. The most important finding was that STAP-2 expression determined tyrosinase protein content. STAP-2 colocalized with tyrosinase in lysosomes and protected tyrosinase from protein degradation. It is noteworthy that B16F10 cells with knocked down tyrosinase showed similar cell characteristics as shSTAP-2 cells. These results indicated that tyrosinase contributed to some cellular events beyond melanogenesis. Taken together, one possibility is that STAP-2 positively regulates the protein levels of tyrosinase, which determines tumor invasion via controlling chemokine receptor expression.

Kaposi's sarcoma-associated herpesvirus-encoded LANA associates with glucocorticoid receptor and enhances its transcriptional activities
Sumihito Togi, Misa Nakasuji, Ryuta Muromoto, Osamu Ikeda, Kanako Okabe, Yuichi Kitai, Shigeyuki Kon, Kenji Oritani, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 463, 3, 395, 400, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jul. 2015, [Peer-reviewed]
English, Scientific journal, Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. (C) 2015 Elsevier Inc. All rights reserved.

PML suppresses IL-6-induced STAT3 activation by interfering with STAT3 and HDAC3 interaction
Masaya Kato, Ryuta Muromoto, Sumihito Togi, Masashi Iwakami, Yuichi Kitai, Shigeyuki Kon, Kenji Oritani, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 461, 2, 366, 371, ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2015, [Peer-reviewed]
English, Scientific journal, The promyelocytic leukemia protein PML acts as a tumor suppressor by forming transcription-regulatory complexes with a variety of repressor proteins. In the present study, we found that endogenous PML suppresses interleukin (IL)-6-induced gene expression as well as phosphorylation and transcriptional activation of STAT3 in hepatoma cells. We also found that PML-mediated suppression of IL-6-induced STAT3 activation by disrupting interactions between STAT3 and HDAC3. These results indicate that PML modulates IL-6-induced STAT3 activation and hepatoma cell growth by interacting with HDAC3. (C) 2015 Elsevier Inc. All rights reserved.

新規α9インテグリンリガンドLymphotactinの同定と機能解析　　　　　　　　　　　　　　　
中鶴 拓也, 松本 尚樹, 紅露 ひとみ, 松田 正, 今 重之
日本薬学会年会要旨集, 135年会, 3, 95, 95, (公社)日本薬学会, Mar. 2015
Japanese

T細胞受容体シグナルにおけるSTAP-1及びSTAP-2の役割　　　　　　　　　　　　　　　
小澤 清貴, 齋藤 浩大, 安次富 大, 関根 勇一, 室本 竜太, 今 重之, 松田 正
日本薬学会年会要旨集, 135年会, 3, 96, 96, (公社)日本薬学会, Mar. 2015
Japanese

Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors alpha and gamma
Hiroyuki Kojima, Yukimasa Takeda, Ryuta Muromoto, Miki Takahashi, Toru Hirao, Shinji Takeuchi, Anton M. Jetten, Tadashi Matsuda
TOXICOLOGY, 329, 32, 39, ELSEVIER IRELAND LTD, Mar. 2015, [Peer-reviewed], ［Internationally co-authored］
English, Scientific journal, The retinoic acid receptor-related orphan receptors a and gamma (ROR alpha and ROR gamma), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. In this study, we investigated the effects of isoflavones on ROR alpha/gamma, activity and the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In doxycycline-inducible CHO stable cell lines, we found that four isoflavones, biochanin A (BA), genistein, formononetin, and daidzein, enhanced ROR alpha- or ROR gamma-mediated transcriptional activity in a dose-dependent manner. In an activation assay of the Il17a promoter using Jurkat cells, these compounds enhanced the ROR alpha- or ROR gamma-mediated activation of the Il17a promoter at concentrations of 1 x 10(-6) M to 1 x 10(-5) M. In mammalian two-hybrid assays, the four isoflavones enhanced the interaction between the RORa- or ROR gamma-ligand binding domain and the co-activator LXXLL peptide in a dose-dependent manner. In addition, these isoflavones potently enhanced Il17a mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin, but showed slight enhancement of Il17a gene expression in ROR alpha/gamma-knockdown EL4 cells. Immunoprecipitation and immunoblotting assays also revealed that BA enhanced the interaction between ROR-gamma t and SRC-1, which is a co-activator for nuclear receptors. Taken together, these results suggest that the isoflavones have the ability to enhance IL-17 gene expression by stabilizing the interactions between ROR alpha/gamma, and co-activators. This also provides the first evidence that dietary chemicals can enhance IL-17 gene expression in immune cells. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

Signal-transducing adaptor protein-2 regulates macrophage migration into inflammatory sites during dextran sodium sulfate induced colitis
Natsuko Fujita, Kenji Oritani, Michiko Ichii, Takafumi Yokota, Norimitsu Saitoh, Daisuke Okuzaki, Yuichi Sekine, Shigeyuki Kon, Ryuta Muromoto, Kodai Saitoh, Akihiko Yoshimura, Tadashi Matsuda, Yuzuru Kanakura
EUROPEAN JOURNAL OF IMMUNOLOGY, 44, 6, 1791, 1801, WILEY-BLACKWELL, Jun. 2014, [Peer-reviewed]
English, Scientific journal, Signal-transducing adaptor protein-2 (STAP-2) was cloned as a c-fms/M-CSF receptor interacting protein. STAP-2 is an adaptor protein carrying pleckstrin homology and Src homology 2 like domains, as well as a YXXQ motif. STAP-2 has been indicated to have an ability to bind and modulate a variety of signaling and transcriptional molecules. Especially, our previous in vitro studies showed that STAP-2 is crucial for immune and/or inflammatory responses. Here, we have investigated the role of STAP-2 in intestinal inflammation in vivo. The disruption of STAP-2 attenuates dextran sodium sulfate induced colitis via inhibition of macrophage recruitment. To study whether hematopoietic or epithelial cell derived STAP-2 is required for this phenomenon, we generated BM chimeric mice. STAP-2-deficient macrophages impair the ability of CXCL12-induced migration. Intriguingly, STAP-2 also regulates production of proinflammatory chemokines and cytokines such as CXCL1 and TNF- from intestinal epithelial cells. Therefore, STAP-2 has a potential to regulate plural molecular events during pathological inflammatory responses. Furthermore, our findings not only indicate that STAP-2 is important in regulating intestinal inflammation, but also provide new insights toward the development of novel therapeutic approaches.

The Novel alpha 4B Murine alpha 4 Integrin Protein Splicing Variant Inhibits alpha 4 Protein-dependent Cell Adhesion
Hitomi Kouro, Shigeyuki Kon, Naoki Matsumoto, Tomoe Miyashita, Ayaka Kakuchi, Dai Ashitomi, Kodai Saitoh, Takuya Nakatsuru, Sumihito Togi, Ryuta Muromoto, Tadashi Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 289, 23, 16389, 16398, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Jun. 2014, [Peer-reviewed]
English, Scientific journal, Integrins affect the motility of multiple cell types to control cell survival, growth, or differentiation, which are mediated by cell-cell and cell-extracellular matrix interactions. We reported previously that the alpha 9 integrin splicing variant, SF alpha 9, promotes WT alpha 9 integrin-dependent adhesion. In this study, we introduced a new murine alpha 4 integrin splicing variant, alpha 4B, which has a novel short cytoplasmic tail. In inflamed tissues, the expression of alpha 4B, as well as WT alpha 4 integrin, was up-regulated. Cells expressing alpha 4B specifically bound to VCAM-1 but not other alpha 4 integrin ligands, such as fibronectin CS1 or osteopontin. The binding of cells expressing WT alpha 4 integrin to alpha 4 integrin ligands is inhibited by coexpression of alpha 4B. Knockdown of alpha 4B in metastatic melanoma cell lines results in a significant increase in lung metastasis. Expression levels of WT alpha 4 integrin are unaltered by alpha 4B, with alpha 4B acting as a regulatory subunit for WT alpha 4 integrin by a dominant-negative effect or inhibiting alpha 4 integrin activation.

Tyk2 is a therapeutic target for psoriasis-like skin inflammation
Masayuki Ishizaki, Ryuta Muromoto, Toshihiko Akimoto, Yuichi Sekine, Shigeyuki Kon, Manish Diwan, Hiroaki Maeda, Sumihito Togi, Kazuya Shimoda, Kenji Oritani, Tadashi Matsuda
INTERNATIONAL IMMUNOLOGY, 26, 5, 257, 267, OXFORD UNIV PRESS, May 2014, [Peer-reviewed]
English, Scientific journal, Tyk2 regulates IL-22/IL-23-induced psoriasis.Tyrosine kinase 2 (Tyk2), a member of the Jak kinase family, mediates signals triggered by various cytokines, which are related to the pathogenesis of psoriasis. In this study, we investigated the role of Tyk2 in IL-23-induced psoriasis-like skin inflammation. Tyk2(-/-) mice when injected with IL-23 showed significantly reduced ear skin swelling with epidermal hyperplasia and inflammatory cell infiltration compared with wild-type mice. In addition, Tyk2 deficiency reduced production of pro-inflammatory cytokines and psoriasis-relevant anti-microbial peptides. More noteworthy is that Tyk2 directly regulated IL-22-dependent inflammation and epidermal hyperplasia. Taken together with the inhibition of IL-23-induced inflammation by treatment with neutralizing antibodies against IL-17 or IL-22, Tyk2 participates in both IL-23 and IL-22 signal transduction to mediate psoriasis-like skin inflammation. On the basis of these findings, we demonstrated for the first time that a small-molecule Tyk2 inhibitor significantly inhibited IL-23-induced inflammation and cytokine production in the skin. These observations demonstrate the important role of Tyk2 in experimental skin inflammation and indicate the therapeutic potential of Tyk2 inhibition in human psoriasis.

Promoter Hypermethylation of the Phosphatase DUSP22 Mediates PKA- Dependent TAU Phosphorylation and CREB Activation in Alzheimer's Disease
Jose Vicente Sanchez-Mut, Ester Aso, Holger Heyn, Tadashi Matsuda, Christoph Bock, Isidre Ferrer, Manel Esteller
HIPPOCAMPUS, 24, 4, 363, 368, WILEY-BLACKWELL, Apr. 2014, [Peer-reviewed], ［Internationally co-authored］
English, Scientific journal, Genetic screening in Alzheimer's disease (AD) has identified only a handful of genes that are mutated in the disorder. Thus, for a very large proportion of patients, the biology of their disease is poorly understood. Epigenetic alterations may provide an explanation in these cases. Using DNA methylation profiles of human hippocampus from controls and patients, we have identified the presence of promoter hypermethylation of the dual-specificity phosphatase 22 (DUSP22) gene in AD. DUSP22 is a likely candidate gene for involvement in the pathogenesis of the disorder since, as we demonstrate here, it inhibits PKA activity and thereby determines TAU phosphorylation status and CREB signaling. (c) 2014 The Authors. Hippocampus Published by Wiley Periodicals, Inc.

Signal-Transducing Adaptor Protein-2 Controls the IgE-Mediated, Mast Cell-Mediated Anaphylactic Responses
Yuichi Sekine, Keigo Nishida, Satoru Yamasaki, Ryuta Muromoto, Shigeyuki Kon, Jun-ichi Kashiwakura, Kodai Saitoh, Sumihito Togi, Akihiko Yoshimura, Kenji Oritani, Tadashi Matsuda
JOURNAL OF IMMUNOLOGY, 192, 8, 3488, 3495, AMER ASSOC IMMUNOLOGISTS, Apr. 2014, [Peer-reviewed]
English, Scientific journal, Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that regulates immune and inflammatory responses through interactions with a variety of signaling and transcriptional molecules. In the current study, we clarified the physiological role of STAP-2 in mast cell function, a key mediator of IgE-associated allergic responses. STAP-2 is constitutively expressed in mast cells. STAP-2 deficiency in mast cells greatly enhances Fc epsilon RI-mediated signals, resulting in the increased tyrosine phosphorylation of the phospholipase C-g isoform, calcium mobilization, and degranulation. Of importance, STAP-2-deficient mice challenged with DNP-BSA after passive sensitization with anti-DNP IgE show more severe rectal temperature decrease than do wild-type mice. STAP-2-deficient mice also show increased vascular permeability and more severe cutaneous anaphylaxis after DNP-BSA injection. These regulatory functions performed by STAP-2 indicate that there is an interaction between STAP-2 and Fc epsilon RI. In addition, our previous data indicate that STAP-2 binds to the phospholipase C-gamma isoform and I kappa B kinase-beta. Therefore, our data described in this article strongly suggest that manipulation of STAP-2 expression in mast cells may control the pathogenesis of allergic diseases and have the potential for treating patients with allergy.

Jun Activation Domain-binding Protein 1 (JAB1) Is Required for the Optimal Response to Interferons
Ryuta Muromoto, Maiko Nakajima, Koki Hirashima, Toru Hirao, Shigeyuki Kon, Kazuya Shimoda, Kenji Oritani, Tadashi Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 288, 43, 30969, 30979, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Oct. 2013, [Peer-reviewed]
English, Scientific journal, Degradation of IFN receptor (IFNR) protein is one of the mechanisms to limit the extent of cellular responses to interferons. Tyrosine kinase 2 (TYK2), a JAK family kinase, has been reported to bind to and stabilize IFNR, indicating that TYK2 is a fundamental component of IFNR complex. Herein, we identified Jun activation domain-binding protein 1 (JAB1) as a new TYK2 binding partner and investigated its role in the regulation of IFN responses. siRNA knockdown of JAB1 resulted in suppression of IFN-induced phosphorylation of STAT proteins and their transcriptional activation. Importantly, JAB1 knockdown induced the activation of SCF ubiquitin ligase complex containing Cullin 1 (CUL1), as judged by the enhancement of covalent modification of CUL1 with the ubiquitin-like protein NEDD8, and markedly reduced the basal protein level of IFNR. In contrast, NEDD8 knockdown or inhibition of NEDD8 modification by NEDD8-activating enzyme inhibitor resulted in increased IFNR protein concomitantly with a reduction of NEDD8-modified CUL1. Furthermore, NEDD8-activating enzyme inhibitor treatment enhanced the susceptibility to IFN-alpha in HeLa cells. These data suggest that the NEDD8 modification pathway is involved in the proteolysis of IFNR and that JAB1 acts as a positive regulator of IFN responses by stabilizing IFNR through antagonizing the NEDD8 pathway.

Y14 Positively Regulates TNF-alpha-Induced NF-kappa B Transcriptional Activity via Interacting RIP1 and TRADD Beyond an Exon Junction Complex Protein
Sumihito Togi, Kaname Shiga, Ryuta Muromoto, Masaya Kato, Yuki Souma, Yuichi Sekine, Shigeyuki Kon, Kenji Oritani, Tadashi Matsuda
JOURNAL OF IMMUNOLOGY, 191, 3, 1436, 1444, AMER ASSOC IMMUNOLOGISTS, Aug. 2013, [Peer-reviewed]
English, Scientific journal, Although Y14 is known to be a component of the exon junction complex, we previously reported that Y14 regulates IL-6-induced STAT3 activation. In this study, we showed that endogenous Y14 positively regulated TNF-alpha-induced IL-6 expression in HeLa cells. Small interfering RNA-mediated Y14-knockdown reduced TNF-alpha-induced and NF-kappa B-mediated transcriptional activity, phosphorylation/degradation of I kappa B alpha, and nuclear localization of NF-kappa B/p65. As in the case of IL-6 stimuli, Y14 enhanced TNF-alpha-induced STAT3 phosphorylation, which is important for its nuclear retention. However, our manipulation of Y14 expression indicated that it is involved in TNF-alpha-induced IL-6 expression via both STAT3-dependent and -independent mechanisms. We screened signaling molecules in the TNF-alpha-NF-kappa B pathway and found that Y14 endogenously associated with receptor-interacting protein 1 (RIP1) and TNFR-associated death domain (TRADD). Overexpression of RIP1, but not TRADD, restored TNF-alpha-induced NF-kB activation in Y14-knockdown cells, and Y14 overexpression restored TNF-alpha-induced NF-kB activation in TRADD-knockdown cells, but not in RIP1-knockdown cells, indicating that Y14 lies downstream of TRADD and upstream of RIP1. Of importance, Y14 significantly enhanced the binding between RIP1 and TRADD, and this is a possible new mechanism for Y14-mediated modification of TNF-alpha signals. Although Y14 associates with MAGOH in the exon junction complex, Y14's actions in the TNF-alpha-NF-kappa B pathway are unlikely to require MAGOH. Therefore, Y14 positively regulates signals for TNF-alpha-induced IL-6 production at multiple steps beyond an exon junction complex protein.

Frontiers in Researches of Immune Regulators
Keigo Nishida, Tadashi Matsuda
YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN, 133, 4, 411, 412, PHARMACEUTICAL SOC JAPAN, Apr. 2013, [Peer-reviewed]
Japanese

[Moleculary targeted therapy for JAK/STAT signaling].
Matsuda T, Kon S, Muromoto R
Nihon rinsho. Japanese journal of clinical medicine, 70 Suppl 8, 45, 51, Nov. 2012, [Peer-reviewed]

TET2 is essential for survival and hematopoietic stem cell homeostasis
K. Shide, T. Kameda, H. Shimoda, T. Yamaji, H. Abe, A. Kamiunten, M. Sekine, T. Hidaka, K. Katayose, Y. Kubuki, S. Yamamoto, T. Miike, H. Iwakiri, S. Hasuike, K. Nagata, K. Marutsuka, A. Iwama, T. Matsuda, A. Kitanaka, K. Shimoda
Leukemia, 26, 10, 2216, 2223, Oct. 2012, [Peer-reviewed], ［Internationally co-authored］
English, Scientific journal, Ten-Eleven-Translocation 2 (TET2) is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) and thereby alters the epigenetic state of DNA
somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies. To study the function of TET2 in vivo, we analyzed Ayu17-449 (TET2trap) mice, in which a gene trap insertion in intron 2 of TET2 reduces TET2 mRNA levels to about 20% of that found in wild-type (WT) mice. TET2trap/trap mice were born at Mendelian frequency but died at a high rate by postnatal day 3, indicating the essential role of TET2 for survival. Loss of TET2 results in an increase in the number of hematopoietic stem cells (HSCs)/progenitors in the fetal liver, and TET2trap/trap HSCs exhibit an increased self-renewal ability in vivo. In competitive transplantation assays, TET2 trap/trap HSCs possess a competitive growth advantage over WT HSCs. These data indicate that TET2 has a critical role in survival and HSC homeostasis. © 2012 Macmillan Publishers Limited.

Roles of Cell Signaling Pathways in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission
Asuka Nanbo, Haruna Terada, Kunihiro Kachi, Kenzo Takada, Tadashi Matsuda
JOURNAL OF VIROLOGY, 86, 17, 9285, 9296, AMER SOC MICROBIOLOGY, Sep. 2012, [Peer-reviewed]
English, Scientific journal, Epstein-Barr virus (EBV), a human gamma herpesvirus, establishes a life-long latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence indicate that the efficiency of EBV infection in epithelial cells is accelerated up to 10(4)-fold by coculturing with EBV-infected Burkitt's lymphoma (BL) cells compared to infection with cell-free virions, indicating that EBV infection into epithelial cells is mainly mediated via cell-to-cell contact. However, the molecular mechanisms involved in this pathway are poorly understood. Here, we establish a novel assay to assess cell-to-cell contact-mediated EBV transmission by coculturing an EBV-infected BL cell line with an EBV-negative epithelial cell line under stimulation for lytic cycle induction. By using this assay, we confirmed that EBV was transmitted from BL cells to epithelial cells via cell-to-cell contact but not via cell-to-cell fusion. The inhibitor treatments of extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-kappa B pathways blocked EBV transmission in addition to lytic induction. The blockage of the phosphoinositide 3-kinase (PI3K) pathway impaired EBV transmission coupled with the inhibition of lytic induction. Knockdown of the RelA/p65 subunit of NF-kappa B reduced viral transmission. Moreover, these signaling pathways were activated in cocultured BL cells and in epithelial cells. Finally, we observed that viral replication was induced in cocultured BL cells. Taken together, our data suggest that cell-to-cell contact induces multiple cell signaling pathways in BL cells and epithelial cells, contributing to the induction of the viral lytic cycle in BL cells and the enhancement of viral transmission to epithelial cells.

Signal-Transducing Adaptor Protein-2 Modulates Fas-Mediated T Cell Apoptosis by Interacting with Caspase-8
Yuichi Sekine, Chikako Yamamoto, Michinori Kakisaka, Ryuta Muromoto, Shigeyuki Kon, Dai Ashitomi, Natsuko Fujita, Akihiko Yoshimura, Kenji Oritani, Tadashi Matsuda
JOURNAL OF IMMUNOLOGY, 188, 12, 6194, 6204, AMER ASSOC IMMUNOLOGISTS, Jun. 2012, [Peer-reviewed]
English, Scientific journal, We found that an adaptor protein, signal-transducing adaptor protein (STAP)-2, is a new member of the Fas-death-inducing signaling complex and participates in activation-induced cell death in T cells. STAP-2 enhanced Fas-mediated apoptosis and caspase-8 aggregation and activation in Jurkat T cells. Importantly, STAP-2 directly interacted with caspase-8 and Fas, resulting in enhanced interactions between caspase-8 and FADD in the Fas-death-inducing signaling complex. Moreover, STAP-2 protein has a consensus caspase-8 cleavage sequence, VEAD, in its C-terminal domain, and processing of STAP-2 by caspase-8 was crucial for Fas-induced apoptosis. Physiologic roles of STAP-2 were confirmed by observations that STAP-2-deficient mice displayed impaired activation-induced cell death and superantigen-induced T cell depletion. Therefore, STAP-2 is a novel participant in the regulation of T cell apoptosis after stimulation. The Journal of Immunology, 2012, 188: 6194-6204.

Inhibitory effects of azole-type fungicides on interleukin-17 gene expression via retinoic acid receptor-related orphan receptors alpha and gamma
Hiroyuki Kojima, Ryuta Muromoto, Miki Takahashi, Shinji Takeuchi, Yukimasa Takeda, Anton M. Jetten, Tadashi Matsuda
TOXICOLOGY AND APPLIED PHARMACOLOGY, 259, 3, 338, 345, ACADEMIC PRESS INC ELSEVIER SCIENCE, Mar. 2012, [Peer-reviewed]
English, Scientific journal, The retinoic acid receptor-related orphan receptors alpha and gamma (ROR alpha and ROR gamma), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. However, it remains unclear whether environmental chemicals, including pesticides, have agonistic and/or antagonistic activity against ROR alpha/gamma. In this study, we investigated the ROR alpha/gamma activity of several azole-type fungicides, and the effects of these fungicides on the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In the ROR-reporter gene assays, five azole-type fungicides (imibenconazole, trifiumizole, hexaconazole, tetraconazole and imazalil) suppressed ROR alpha- and/or ROR gamma-mediated transcriptional activity as did benzenesulphonamide T0901317, a ROR inverse agonist and a liver X receptor (LXR) agonist. In particular, imibenconazole, triflumizole and hexaconazole showed ROR gamma inverse agonistic activity at concentrations of 10(-6) M. However, unlike T0901317, these fungicides failed to show any LXR alpha/beta agonistic activity. Next, five azole-type fungicides, showing ROR inverse agonist activity, were tested on IL-17 mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin. The quantitative RT-PCR analysis revealed that these fungicides suppressed the expression of IL-17 mRNA without effecting ROR alpha and ROR gamma mRNA levels. In addition, the inhibitory effect of imibenconazole as well as that of T0901317 was absorbed in ROR alpha/gamma-knocked down EL4 cells. Taken together, these results suggest that some azole-type fungicides inhibit IL-17 production via ROR alpha/gamma. This also provides the first evidence that environmental chemicals can act as modulators of IL-17 expression in immune cells. (C) 2012 Elsevier Inc. All rights reserved.

PDLIM2 inhibits T helper 17 cell development and granulomatous inflammation through degradation of STAT3
Takashi Tanaka, Yu Yamamoto, Ryuta Muromoto, Osamu Ikeda, Yuichi Sekine, Michael J. Grusby, Tsuneyasu Kaisho, Tadashi Matsuda
Science Signaling, 4, 202, ra85, 202, 06 Dec. 2011, [Peer-reviewed]
Scientific journal, Granuloma formation is an important host defense mechanism against intracellular bacteria; however, uncontrolled granulomatous inflammation is pathologic. T helper 17 (TH17) cells are thought to have a pathogenic role in autoimmune and inflammatory diseases, including in granulomas. Here, we report that the PDZ-LIM domain protein PDLIM2 inhibited TH17 cell development and granulomatous responses by acting as a nuclear ubiquitin E3 ligase that targeted signal transducer and activator of transcription 3 (STAT3), a transcription factor critical for the commitment of naïve CD4 + T cells to the TH17 lineage. PDLIM2 promoted the polyubiquitination and proteasomal degradation of STAT3, thereby disrupting STAT3-mediated gene activation. Deficiency in PDLIM2 resulted in the accumulation of STAT3 in the nucleus, enhanced the extent of TH17 cell differentiation, and exacerbated granuloma formation. This study delineates an essential role for PDLIM2 in inhibiting TH17 cell-mediated inflammatory responses by suppressing STAT3 signaling and provides a potential therapeutic target for the treatment of autoimmune diseases.

Tyk2 deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice
Masayuki Ishizaki, Ryuta Muromoto, Toshihiko Akimoto, Yuya Ohshiro, Miki Takahashi, Yuichi Sekine, Hiroaki Maeda, Kazuya Shimoda, Kenji Oritani, Tadashi Matsuda
INTERNATIONAL IMMUNOLOGY, 23, 9, 575, 582, OXFORD UNIV PRESS, Sep. 2011, [Peer-reviewed]
English, Scientific journal, Tyrosine kinase-2 (Tyk2) participates in the signaling pathways of multiple cytokines in innate and acquired immunity. In the present study, we investigated the in vivo involvement of Tyk2 in anti-type II collagen antibody-induced arthritis (CAIA) using Tyk2-deficient mice. Hind paws of wild-type mice showed massive swelling and erythema by arthritogenic antibody injection, whereas Tyk2-deficient mice did not show any signs of arthritis. Indeed, neither the infiltration of inflammatory cells nor the fibrillation of articular cartilages was observed in Tyk2-deficient mice. Tyk2 deficiency also reduced the production of T(h)1/T(h)17-related cytokines, the other proinflammatory cytokines and matrix metalloproteases, which are induced in the CAIA paw. Our results demonstrate a critical contribution of Tyk2 in the development of arthritis, and we propose that Tyk2 might be an important candidate for drug development.

Kruppel-Associated Box-Associated Protein 1 Negatively Regulates TNF-alpha-Induced NF-kappa B Transcriptional Activity by Influencing the Interactions among STAT3, p300, and NF-kappa B/p65
Shinya Kamitani, Sumihito Togi, Osamu Ikeda, Misa Nakasuji, Asuka Sakauchi, Yuichi Sekine, Ryuta Muromoto, Kenji Oritani, Tadashi Matsuda
JOURNAL OF IMMUNOLOGY, 187, 5, 2476, 2483, AMER ASSOC IMMUNOLOGISTS, Sep. 2011, [Peer-reviewed]
English, Scientific journal, Kruppel-associated box-associated protein 1 (KAP1) is thought to act mainly as a scaffold for protein complexes, which together silence transcription by triggering the formation of heterochromatin. Using small interfering RNA-mediated KAP1 knockdown, we found that endogenous KAP1 negatively regulated TNF-alpha-induced IL-6 production in HeLa cells. KAP1 is likely to modulate the binding of NF-kappa B to the IL-6 promoter because KAP1 knockdown enhanced TNF-a-induced NF-kappa B-luciferase activity, but not Ik kappa B alpha degradation. Of importance, we found negative regulatory effects of KAP1 on the serine phosphorylation of STAT3, the acetylation of NF-kappa B/p65 by p300, and the nuclear localization of NF-kappa B/p65. In addition, KAP1 associated with NF-kappa B/p65 and inhibited the binding between NF-kappa B/p65 and p300. Thus, KAP1 is likely to negatively control the acetylation of NF-kappa B/p65, which is critical for its nuclear retention. Taken together, KAP1 modulated the acetylation of NF-kappa B/p65 by interfering with the interactions among STAT3, p300, and NF-kappa B/p65, resulting in reduced IL-6 production after TNF-alpha stimulation. Our findings that KAP1 directly interacts with transcriptional factors are new, and will inform further research to elucidate KAP1 function. The Journal of Immunology, 2011, 187: 2476-2483.

Involvement of Tyrosine Kinase-2 in Both the IL-12/Th1 and IL-23/Th17 Axes In Vivo
Masayuki Ishizaki, Toshihiko Akimoto, Ryuta Muromoto, Mika Yokoyama, Yuya Ohshiro, Yuichi Sekine, Hiroaki Maeda, Kazuya Shimoda, Kenji Oritani, Tadashi Matsuda
JOURNAL OF IMMUNOLOGY, 187, 1, 181, 189, AMER ASSOC IMMUNOLOGISTS, Jul. 2011, [Peer-reviewed]
English, Scientific journal, Tyrosine kinase-2 (Tyk2), a member of the Jak family of kinases, mediates the signals triggered by various cytokines, including type I IFNs, IL-12, and IL-23. In the current study, we investigated the in vivo involvement of Tyk2 in several IL-12/Th1- and IL-23/Th17-mediated models of experimental diseases, including methylated BSA injection-induced footpad thickness, imiquimod-induced psoriasis-like skin inflammation, and dextran sulfate sodium-or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. In these disease models, Tyk2 deficiency influenced the phenotypes in immunity and/or inflammation. Our findings demonstrate a somewhat broader contribution of Tyk2 to immune systems than previously expected and suggest that Tyk2 may represent an important candidate for drug development by targeting both the IL-12/Th1 and IL-23/Th17 axes. The Journal of Immunology, 2011, 187: 181-189.

Zipper-interacting Protein Kinase (ZIPK) Modulates Canonical Wnt/beta-Catenin Signaling through Interaction with Nemo-like Kinase and T-cell Factor 4 (NLK/TCF4)
Sumihito Togi, Osamu Ikeda, Shinya Kamitani, Misa Nakasuji, Yuichi Sekine, Ryuta Muromoto, Asuka Nanbo, Kenji Oritani, Taro Kawai, Shizuo Akira, Tadashi Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 286, 21, 19170, 19177, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, May 2011, [Peer-reviewed]
English, Scientific journal, Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in apoptosis and transcriptional regulation. Here, we identified Nemo-like kinase (NLK) as a novel ZIPK-binding partner and found that ZIPK regulates NLK-mediated repression of canonical Wnt/beta-catenin signaling. Indeed, siRNA-mediated reduction of endogenous ZIPK expression reduced Wnt/beta-catenin signaling. Furthermore, ZIPK affected the formation of NLK-T-cell factor 4 (TCF4) complex. Importantly, ZIPK siRNA treatment in human colon carcinoma cells resulted in a reduction of beta-catenin/TCF-mediated gene expression and cell growth. These results indicate that ZIPK may serve as a transcriptional regulator of canonical Wnt/beta-catenin signaling through interaction with NLK/TCF4.

Noncanonical K27-Linked Polyubiquitination of TIEG1 Regulates Foxp3 Expression and Tumor Growth
Dong-Jun Peng, Minghui Zeng, Ryuta Muromoto, Tadashi Matsuda, Kazuya Shimoda, Malayannan Subramaniam, Thomas C. Spelsberg, Wei-Zen Wei, K. Venuprasad
JOURNAL OF IMMUNOLOGY, 186, 10, 5638, 5647, AMER ASSOC IMMUNOLOGISTS, May 2011, [Peer-reviewed]
English, Scientific journal, Earlier, we demonstrated the essential role of Kruppel-like transcription factor, TIEG1, in TGF-beta-induced regulatory T cell (Treg) development. In this article, we demonstrate that IL-6, which promotes Th17 development, abrogated TIEG1 nuclear translocation and inhibited TGF-beta-induced Treg development. Tyrosine kinase Tyk2-mediated phosphorylation of TIEG1 at Tyr179 promoted noncanonical K-27-linked polyubiquitination, which inhibited TIEG1 nuclear translocation. To test the role of TIEG1-regulated Treg/Th17 development in antitumor immunity, we analyzed TRAMP-C2 tumor growth in TIEG1(-/-) mice. The defective Treg development and elevated Th17 response resulted in enhanced immune reactivity in the tumor and inhibition of TRAMP-C2 tumor growth in TIEG1(-/-) mice. Thus, our results uncovered a novel regulatory mechanism that modulates Tregs and may regulate tumor progression. The Journal of Immunology, 2011, 186: 5638-5647.

Involvement of STAP-2 in Brk-mediated phosphorylation and activation of STAT5 in breast cancer cells
Osamu Ikeda, Akihiro Mizushima, Yuichi Sekine, Chikako Yamamoto, Ryuta Muromoto, Asuka Nanbo, Kenji Oritani, Akihiko Yoshimura, Tadashi Matsuda
CANCER SCIENCE, 102, 4, 756, 761, WILEY-BLACKWELL, Apr. 2011, [Peer-reviewed]
English, Scientific journal, Signal-transducing adaptor protein (STAP)-2 is a recently identified adaptor protein that contains Pleckstrin homology and Src homology 2-like domains, and is also known to be a substrate of breast tumor kinase (Brk). In a previous study, we found that STAP-2 upregulated Brk-mediated activation of signal transducer and activator of transcription (STAT) 3 in breast cancer cells. Here, we examined the involvement of STAP-2 in Brk-mediated STAT5 activation in breast cancer cells. Ectopic expression of STAP-2 induced Brk-mediated transcriptional activity of STAT5. Furthermore, STAP-2-knockdown in T47D breast cancer cells induced a marked decrease in proliferation that was as strong as that after Brk- or STAT5b-knockdown. Regarding the mechanism, the Pleckstrin homology domain of STAP-2 is likely to participate in the process by which Brk phosphorylates and activates STAT5. Taken together, our findings provide insights toward the development of novel therapeutic strategies as well as novel prognostic values in breast carcinomas. (Cancer Sci 2011; 102: 756-761)

Interactions of STAP-2 with Brk and STAT3 Participate in Cell Growth of Human Breast Cancer Cells
Osamu Ikeda, Yuichi Sekine, Akihiro Mizushima, Misa Nakasuji, Yuto Miyasaka, Chikako Yamamoto, Ryuta Muromoto, Asuka Nanbo, Kenji Oritani, Akihiko Yoshimura, Tadashi Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 285, 49, 38093, 38103, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Dec. 2010, [Peer-reviewed]
English, Scientific journal, STAP-2 (signal transducing adaptor protein-2) is a recently identified adaptor protein that contains pleckstrin homology (PH) and Src homology 2-like domains, as well as a STAT3-binding motif in its C-terminal region. STAP-2 is also a substrate of breast tumor kinase (Brk). In breast cancers, Brk expression is deregulated and promotes STAT3-dependent cell proliferation. In the present study, manipulated STAP-2 expression demonstrated essential roles of STAP-2 in Brk-mediated STAT3 activation. STAP-2 interacts with both Brk and STAT3. In addition, small interfering RNA-mediated reduction of endogenous STAP-2 expression strongly decreased Brk-mediated STAT3 activation in T47D breast cancer cells. The PH domain of STAP-2 is involved in multiple steps: the binding between Brk and STAP-2, the activation and tyrosine phosphorylation of STAT3, and the activation of Brk. Notably, a STAP-2 PH-Brk fusion protein exhibited robust kinase activity and increased activation and tyrosine phosphorylation of STAT3. Finally, STAP-2 knockdown in T47D cells induced a significant decrease of proliferation, as strong as that of Brk or STAT3 knockdown. Taken together, our findings are likely to inform the development of a novel therapeutic strategy, as well as the determination of novel prognostic values, in breast carcinomas.

Functional involvement of Daxx in gp130-mediated cell growth and survival in BaF3 cells
Ryuta Muromoto, Makoto Kuroda, Sumihito Togi, Yuichi Sekine, Asuka Nanbo, Kazuya Shimoda, Kenji Oritani, Tadashi Matsuda
EUROPEAN JOURNAL OF IMMUNOLOGY, 40, 12, 3570, 3580, WILEY, Dec. 2010, [Peer-reviewed]
English, Scientific journal, Death domain-associated protein (Daxx) is a multifunctional protein that modulates both cell death and transcription. Several recent studies have indicated that Daxx is a mediator of lymphocyte death and/or growth suppression, although the detailed mechanism is unclear. Previously, we reported that Daxx suppresses IL-6 family cytokine-induced gene expression by interacting with STAT3. STAT3 is important for the growth and survival of lymphocytes; therefore, we here examined the role of Daxx in the gp130/STAT3-dependent cell growth/survival signals. We found that Daxx suppresses the gp130/STAT3-dependent cell growth and that Daxx endogenously interacts with STAT3 and inhibits the DNA-binding activity of STAT3. Moreover, small-interfering RNA-mediated knockdown of Daxx enhanced the expression of STAT3-target genes and accelerated the STAT3-mediated cell cycle progression. In addition, knockdown of Daxx-attenuated lactate dehydrogenase leakage from cells, indicating that Daxx positively regulates cell death during gp130/STAT3-mediated cell proliferation. Notably, Daxx specifically suppressed the levels of Bcl2 mRNA and protein, even in cytokine-unstimulated cells, indicating that Daxx regulates Bcl2 expression independently of activated STAT3. These results suggest that Daxx suppresses gp130-mediated cell growth and survival by two independent mechanisms: inhibition of STAT3-induced transcription and down-regulation of Bcl2 expression.

BS69 cooperates with TRAF3 in the regulation of Epstein-Barr virus-derived LMP1/CTAR1-induced NF-kappa B activation
Osamu Ikeda, Yuto Miyasaka, Ryuji Yoshida, Akihiro Mizushima, Kenji Oritani, Yuichi Sekine, Makoto Kuroda, Teruhito Yasui, Masahiro Fujimuro, Ryuta Muromoto, Asuka Nanbo, Tadashi Matsuda
FEBS LETTERS, 584, 5, 865, 872, ELSEVIER SCIENCE BV, Mar. 2010, [Peer-reviewed]
English, Scientific journal, Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-kappa B signaling pathways through two C-terminal regions, CTAR1 and CTAR2. Previous studies have demonstrated that BS69, a multidomain cellular protein, regulates LMP1/CTAR2-mediated NF-kappa B activation by interfering with the complex formation between TRADD and LMP1/CTAR2. Here, we found that BS69 directly interacted with the LMP1/CTAR1 domain and regulated LMP1/CTAR1-mediated NF-kappa B activation and subsequent IL-6 production. Regarding the mechanisms involved, we found that BS69 directly interacted with TRAF3, a negative regulator of NF-kappa B activation. Furthermore, small-interfering RNA-mediated knockdown experiments revealed that TRAF3 was involved in the BS69-mediated suppression of LMP1/CTAR1-induced NF-kappa B activation.
Structured summary:
MINT-7556591: lmp1 (uniprotkb: P03230) physically interacts (MI:0915) with BS69 (uniprotkb:Q15326) by anti tag coimmunoprecipitation (MI: 0007)
MINT-7556646: TRAF6 (uniprotkb:Q9Y4K3) physically interacts (MI: 0915) with BS69 (uniprotkb: Q15326) by anti tag coimmunoprecipitation (MI: 0007)
MINT-7556658, MINT-7556670: TRAF3 (uniprotkb:Q13114) physically interacts (MI: 0915) with BS69 (uniprotkb: Q15326) by anti tag coimmunoprecipitation (MI: 0007)
MINT-7556607: TRAF1 (uniprotkb:Q13077) physically interacts (MI: 0915) with BS69 (uniprotkb: Q15326) by anti tag coimmunoprecipitation (MI: 0007)
MINT-7556634: TRAF5 (uniprotkb:O00463) physically interacts (MI: 0915) with BS69 (uniprotkb: Q15326) by anti tag coimmunoprecipitation (MI: 0007)
MINT-7556622: TRAF2 (uniprotkb:Q12933) physically interacts (MI: 0915) with BS69 (uniprotkb: Q15326) by anti tag coimmunoprecipitation (MI: 0007) (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Signal-Transducing Adaptor Protein-2 Regulates Stromal Cell-Derived Factor-1 alpha-Induced Chemotaxis in T Cells
Yuichi Sekine, Osamu Ikeda, Satoshi Tsuji, Chikako Yamamoto, Ryuta Muromoto, Asuka Nanbo, Kenji Oritani, Akihiko Yoshimura, Tadashi Matsuda
JOURNAL OF IMMUNOLOGY, 183, 12, 7966, 7974, AMER ASSOC IMMUNOLOGISTS, Dec. 2009, [Peer-reviewed]
English, Scientific journal, Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains, as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 regulates integrin-mediated T cell adhesion. In the present study, we find that STAP-2 expression affects Jurkat T cell migration after stromal cell-derived factor-1 alpha (SDF-1 alpha)-treatment. Furthermore, STAP-2-deficient T cells exhibit reduced cell migration after SDF-1 alpha-treatment. Importantly, overexpression of STAP-2 in Jurkat T cells induces activation of small guanine triphosphatases, such as Rac1 and Cdc42. Regarding the mechanism for this effect, we found that STAP-2 associates with Vav1, the guanine-nucleotide exchanging factor for Rac1, and enhances downstream Vav1/Rac1 signaling. These results reveal a novel STAP-2-mediated mechanism for the regulation of SDF-1 alpha-induced chemotaxis of T cells via activation of Vav1/Rac1 signaling. The Journal of Immunology, 2009, 183: 7966-7974.

A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3
Masahiro Yamamoto, Daron M. Standley, Seiji Takashima, Hiroyuki Saiga, Megumi Okuyama, Hisako Kayama, Emi Kubo, Hiroshi Ito, Mutsumi Takaura, Tadashi Matsuda, Dominique Soldati-Favre, Kiyoshi Takeda
JOURNAL OF EXPERIMENTAL MEDICINE, 206, 12, 2747, 2760, ROCKEFELLER UNIV PRESS, Nov. 2009, [Peer-reviewed], ［Internationally co-authored］
English, Scientific journal, Infection by Toxoplasma gondii down-regulates the host innate immune responses, such as proinflammatory cytokine production, in a Stat3-dependent manner. A forward genetic approach recently demonstrated that the type II strain fails to suppress immune responses because of a potential defect in a highly polymorphic parasite-derived kinase, ROP16. We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages. Furthermore, overexpression of ROP16 but not ROP18 in mammalian cells resulted in Stat3 phosphorylation and strong activation of Stat3-dependent promoters. In addition, kinase-inactive ROP16 failed to activate Stat3. Comparison of type I and type II ROP16 revealed that a single amino acid substitution in the kinase domain determined the strain difference in terms of Stat3 activation. Moreover, ROP16 bound Stat3 and directly induced phosphorylation of this transcription factor. These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.

Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor Regulates Enhanced Activation of Signal Transducer and Activator of Transcription 3 by Epstein-Barr Virus-Derived Epstein-Barr Nuclear Antigen 2
Osamu Ikeda, Sumihito Togi, Shinya Kamitani, Ryuta Muromoto, Yuichi Sekine, Asuka Nanbo, Masahiro Fujimuro, Tadashi Matsuda
BIOLOGICAL & PHARMACEUTICAL BULLETIN, 32, 7, 1283, 1285, PHARMACEUTICAL SOC JAPAN, Jul. 2009, [Peer-reviewed]
English, Scientific journal, The Epstein-Barr virus (EBV)-encoded latency protein Epstein-Barr nuclear antigen 2 (EBNA2) is a nuclear transcriptional activator that is essential for EBV-induced cellular transformation. In a previous study, we demonstrated that EBNA2 interacts with signal transducer and activator of transcription 3 (STAT3), a signal transducer for an interleukin (IL)-6 family cytokine, and enhances its transcriptional activity. Here, we show that overexpression of a corepressor, silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), decreases the EBNA2-mediated enhanced STAT3 activation. Furthermore, small-interfering RNA-mediated reduction of endogenous SMRT expression augments the EBNA2-mediated enhanced STAT3 activation. Importantly, EBNA2 reduces interactions between STAT3 and SMRT. These data demonstrate that EBNA2 acts as a transcriptional coactivator of STAT3 by influencing the SMRT corepressor complex.

STAP-2 is phosphorylated at tyrosine-250 by Brk and modulates Brk-mediated STAT3 activation
Osamu Ikeda, Yuto Miyasaka, Yuichi Sekine, Akihiro Mizushima, Ryuta Muromoto, Asuka Nanbo, Akihiko Yoshimura, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 384, 1, 71, 75, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jun. 2009, [Peer-reviewed]
English, Scientific journal, Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. STAP-2 is also known as breast tumor kinase (Brk) substrate (BKS). Our previous studies revealed that STAP-2 binds to signal transducer and activator of transcription 3 (STAT3) and STAT5, and regulates the signaling pal. path-ways downstream of them. In the present study, we identified tyrosine-250 (Tyr250) in STAP-2 as a major site of phosphorylation by Brk, using a series of STAP-2 YF mutants and anti-phospho-STAP-2 Tyr250 antibody. Furthermore, overexpression of the STAP-2 Y250F mutant protein affected Brk-mediated STAT3, activation. importantly, small-interfering RNA-mediated reduction of endogenous STAP-2 expression decreased Brk-mediated STAT3 activation. Taken together, our findings demonstrate that STAP-2 is phosphorylated at Tyr250 by Brk, and plays all important role in Brk-mediated STAT3 activation. (c) 2009 Elsevier Inc. All rights reserved.

The protein content of an adaptor protein, STAP-2 is controlled by E3 ubiquitin ligase Cbl
Yuichi Sekine, Chikako Yamamoto, Osamu Ikeda, Ryuta Muromoto, Asuka Nanbo, Kenji Oritani, Akihiko Yoshimura, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 384, 2, 187, 192, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jun. 2009, [Peer-reviewed]
English, Scientific journal, Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. Our previous Study in T cells demonstrated that STAP-2 influences FAK protein levels through recruitment of E3 ubiquitin ligase, Cbl, to FAK. In the present Study, we found that Cbl directly controls the protein levels and activity of STAP-2. STAP-2 physically interacted with Cbl through its PH and SH2-like domains. Small-interfering RNA-mediated reduction of endogenous Cbl restored STAP-2 protein levels. In contrast, over-expression of Cbl induced STAP-2 degradation. Importantly, Cbl-mediated regulation of STAP-2 protein levels affected Brk/STAP-2-induced STAT3 activation. These results indicate that Cbl regulates STAP-2 protein levels and Brk/STAP-2-mediated STAT3 activation. (C) 2009 Elsevier Inc. All rights reserved.

BS69 negatively regulates the canonical NF-kappa B activation induced by Epstein-Barr virus-derived LMP1
Osamu Ikeda, Yuichi Sekine, Akihiro Mizushima, Kenji Oritani, Teruhito Yasui, Masahiro Fujimuro, Ryuta Muromoto, Asuka Nanbo, Tadashi Matsuda
FEBS LETTERS, 583, 10, 1567, 1574, ELSEVIER SCIENCE BV, May 2009, [Peer-reviewed]
English, Scientific journal, Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates NF-kappa B signaling pathways through the two C-terminal regions, CTAR1 and CTAR2. BS69 has previously been shown to be involved in LMP1-induced c-Jun N-terminal kinase activation through CTAR2 by interacting with tumor necrosis factor (TNFR) receptor-associated factor 6. In the present study, our manipulation of BS69 expression clearly indicates that BS69 negatively regulates LMP1-mediated NF-kappa B activation and up-regulates IL-6 mRNA expression and I kappa B degradation. Our immunoprecipitation experiments suggest that BS69 decreases complex formation between LMP1 and TNFR-associated death domain protein (TRADD).

The exon-junction complex proteins, Y14 and MAGOH regulate STAT3 activation
Ryuta Muromoto, Naohisa Taira, Osamu Ikeda, Kaname Shiga, Shinya Kamitani, Sumihito Togi, Shiho Kawakami, Yuichi Sekine, Asuka Nanbo, Kenji Oritani, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 382, 1, 63, 68, ACADEMIC PRESS INC ELSEVIER SCIENCE, Apr. 2009, [Peer-reviewed]
English, Scientific journal, Signal transducer and activator of transcription 3 (STAT3), which is activated by cytokines and growth factors, mediates biological actions in many physiological processes. In a previous study, we found that Y14, a core component of the exon-junction complex (EJC) bound to STAT3 and upregulated the transcriptional activity of STAT3 by influencing its DNA-binding activity. In the present study, we demonstrate that STAT3 endogenously interacts with Y14. In addition, we found that MAGOH, a Y-14 partner in the EJC, inhibits the STAT3-Y14 complex formation. Furthermore, small-interfering RNA-mediated reduction of MAGOH expression enhanced interleukin-6-induced gene expression. These results indicate that MAGOH regulates the transcriptional activation of STAT3 by interfering complex formation between STAT3 and Y14. (C) 2009 Elsevier Inc. All rights reserved.

HDAC3 influences phosphorylation of STAT3 at serine 727 by interacting with PP2A
Sumihito Togi, Shinya Kamitani, Shiho Kawakami, Osarnu Ikeda, Ryuta Muromoto, Asuka Nanbo, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 379, 2, 616, 620, ACADEMIC PRESS INC ELSEVIER SCIENCE, Feb. 2009, [Peer-reviewed]
English, Scientific journal, Signal transducer and activator of transcription 3 (STAT3), which mediates biological actions in many physiological processes, is activated by cytokines and growth factors, and has been reported to be involved in the pathogenesis of various human diseases. Here, we show that treatment of HeLa cells with a histone deacetylase (HDAC) inhibitor, trichostatin A, or small-interfering RNA (siRNA)-mediated repression of HDAC3, enhances phosphorylation of STAT3 at Ser727. Furthermore, dephosphorylation of STAT3 at Ser727 by protein phosphatase 2A (PP2A) was restored by treatment of cells with HDAC3 siRNA. We further found that formation of a complex between STAT3 and PP2A was enhanced in the presence of HDAC3. Importantly, small-interfering RNA-mediated repression of both HDAC3 and PP2A effectively enhanced leukemia inhibitory factor (LIF)-induced STAT3 activation. These results indicate that HDAC3 may act as a scaffold protein for PP2A to regulate the LIF/STAT3-mediated signaling pathway. (C) 2008 Elsevier Inc. All rights reserved.

Epstein-Barr virus-derived EBNA2 regulates STAT3 activation
Ryuta Murornoto, Osamu Ikeda, Kanako Okabe, Sumihito Togi, Shinya Kamitani, Masahiro Fujimuro, Shizuko Harada, Kenji Oritani, Tadashi Matsud
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 378, 3, 439, 443, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jan. 2009, [Peer-reviewed]
English, Scientific journal, The Epstein-Barr virus (EBV)-encoded latency protein EBNA2 is a nuclear transcriptional activator that is essential for EBV-induced cellular transformation. Here, we show that EBNA2 interacts with STAT3, a signal transducer for an interleukin-6 family cytokine, and enhances the transcriptional activity of STAT3 by influencing its DNA-binding activity. Furthermore, EBNA2 cooperatively acts on STAT3 activation with LMP1, These data demonstrate that EBNA2 acts as a transcriptional coactivator of STAT3. (C) 2008 Elsevier Inc. All rights reserved.

In vitro screening for aryl hydrocarbon receptor agonistic activity in 200 pesticides using a highly sensitive reporter cell line, DR-EcoScreen cells, and in vivo mouse liver cytochrome P450-1A induction by propanil, diuron and linuron
Shinji Takeuchi, Mitsuru Iida, Hisatoshi Yabushita, Tadashi Matsuda, Hiroyuki Kojima
CHEMOSPHERE, 74, 1, 155, 165, PERGAMON-ELSEVIER SCIENCE LTD, Dec. 2008, [Peer-reviewed]
English, Scientific journal, The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates genes involved in xenobiotic metabolism, cellular proliferation and differentiation. In this study, we have developed a highly sensitive AhR-mediated reporter cell line, DR-EcoScreen cells. which are mouse hepatoma Hepa1c1c7 cells stably transfected with a reporter plasmid containing seven copies of dioxin-responsive element. Using these DR-EcoScreen cells, we performed the reporter gene assay and characterized the AhR agonistic activities of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 12 acid amides, 7 triazines, 6 ureas, and 45 others). Eleven of the 200 pesticides (acifluorfen-methyl, bifenox, chlorpyrifos, isoxathion, quinalphos, chlorpropham, diethofencarb, propanil, diuron, linuron, and prochloraz) showed AhR-mediated transcriptional activity. In particular, three herbicides (propanil, diuron, and linuron) have a common chemical structure and showed more potent agonistic activity than other pesticides, To investigate the in vivo effects, we examined the gene expression of AhR-inducible cytochrome P450 1As (CYP1As) in the liver of female C57BL/6 mice intraperitoneally injected with these three herbicides (<= 300 mg kg(-1)) by quantitative RT-PCR, resulting in induction of significant high levels of CYP1A1 and CYP1A2 mRNAs. This indicates that propanil, diuron and linuron possess AhR-mediated transactivation effect in vivo as well as in vitro. Through the present study, we demonstrated that DR-EcoScreen cells are useful for sensitive, rapid and simple identification of AhR agonists among a large number of environmental chemicals. (c) 2008 Elsevier Ltd. All rights reserved.

Enhanced c-Fms/M-CSF receptor signaling and wound-healing process in bone marrow-derived macrophages of signal-transducing adaptor protein-2 (STAP-2) deficient mice
Osamu Ikeda, Yuichi Sekine, Ryuta Muromoto, Norihiko Ohbayashi, Akihiko Yoshimura, Tadashi Matsuda
BIOLOGICAL & PHARMACEUTICAL BULLETIN, 31, 9, 1790, 1793, PHARMACEUTICAL SOC JAPAN, Sep. 2008, [Peer-reviewed]
English, Scientific journal, Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein as a c-Fms/M-CSF receptor-interacting protein and constitutively expressed in macrophages. In our previous study, we examined the role of STAP-2 in the c-Fms/M-CSF receptor signaling using a murine macrophage tumor cells line, Raw264.7. Overexpression of STAP-2 in Raw264.7 cells markedly suppressed M-CSF-induced activation of extracellular signal regulated kinase and Akt. In addition, Raw264.7 overexpressing STAP-2 affected cell migration in wound-healing process. These results suggest that STAP-2 deficiency influences endogenous c-Fms/M-CSF receptor signaling. Here we show that loss of STAP-2 expression in knockout mouse macrophages results in marked enhancement of the c-Fms/M-CSF receptor signaling and wound-healing process. We therefore propose that STAP-2 acts as an endogenous regulator in normal macrophages functions.

An RNA biding protein, Y14 interacts with and modulates STAT3 activation
Norihiko Ohbayashi, Naohisa Taira, Shiho Kawakami, Sumihito Togi, Noriko Sato, Osamu Ikeda, Shinya Kamitani, Ryuta Muromoto, Yuichi Sekine, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 372, 3, 475, 479, ACADEMIC PRESS INC ELSEVIER SCIENCE, Aug. 2008, [Peer-reviewed]
English, Scientific journal, Signal transducer and activator of transcription 3 (STAT3), which mediates biological actions in many physiological processes, is activated by cytokines and growth factors via specific tyrosine-phosphorylation, dimerization, and nuclear translocation. To clarify the molecular mechanisms underlying the regulation of STAT3 activation, we performed yeast two-hybrid screening. We identified Y14, an RNA-binding protein, as a novel STAT3 binding partner. Y14 bound to STAT3 through the C-terminal region of STAT3 in vivo. Importantly, small-interfering RNA-mediated reduction of endogenous Y14 expression decreased IL-6-induced tyrosine-phosphorylation, nuclear accumulation, and DNA-binding activity of STAT3, as well as IL-6/STAT3-dependent gene expression. These results indicate that Y14 interacts with STAT3 and regulates the transcriptional activation of STAT3 by influencing the tyrosine-phosphorylation of STAT3. (C) 2008 Elsevier Inc. All rights reserved.

Physical and functional interactions between ZIP kinase and UbcH5
Norihiko Ohbayashi, Katsuya Okada, Shiho Kawakami, Sumihito Togi, Noriko Sato, Osamu Ikeda, Shinya Kamitani, Ryuta Muromoto, Yuichi Sekine, Taro Kawai, Shizuo Akira, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 372, 4, 708, 712, ACADEMIC PRESS INC ELSEVIER SCIENCE, Aug. 2008, [Peer-reviewed]
English, Scientific journal, Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. In our previous study, we showed that leukemia inhibitory factor stimulated threonine-265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription 3. Here, we identified UbcH5c as a novel ZIPK-binding partner by yeast two-hybrid screening. Importantly, we found that UbcH5c induced ubiquitination of ZIPK. Small-interfering RNA-mediated reduction of endogenous UbcH5 expression decreased ZIPK ubiquitination. Furthermore, coexpression of UbcH5c with ZIPK influenced promyelocytic leukemia protein nuclear body (PML-NB) formation. These results suggest that UbcH5 regulates ZIPK accumulation in PML-NBs by interacting with ZIPK and stimulating its ubiquitination. (c) 2008 Elsevier Inc. All rights reserved

STAP-2 negatively regulates both canonical and noncanonical NF-kappa B activation induced by Epstein-Barr virus-derived latent membrane protein 1
Osamu Ikeda, Yuichi Sekine, Teruhito Yasui, Kenji Oritani, Kenji Sugiyma, Ryuta Muromoto, Norihiko Ohbayashi, Akihiko Yoshimura, Tadashi Matsuda
MOLECULAR AND CELLULAR BIOLOGY, 28, 16, 5027, 5042, AMER SOC MICROBIOLOGY, Aug. 2008, [Peer-reviewed]
English, Scientific journal, The signal-transducing adaptor protein 2 (STAP-2) is a recently identified adaptor protein that contains a pleckstrin homology (PH) and Src homology 2 (SH2)-like domains, as well as a proline-rich domain in its C-terminal region. In previous studies, we demonstrated that STAP-2 binds to MyD88 and IKK-alpha or IKK-beta and modulates NF-kappa B signaling in macrophages. In the present study, we found that ectopic expression of STAP-2 inhibited Epstein-Barr virus (EBV) LMPI-mediated NF-kappa B signaling and interleukin-6 expression. Indeed, STAP-2 associated with LMP1 through its PH and SH2-like domains, and these proteins interacted with each other in EBV-positive human B cells. We found, furthermore, that STAP-2 regulated LMP1-mediated NF-kappa B signaling through direct or indirect interactions with the tumor necrosis factor receptor (TNFR)associated factor 3 (TRAF3) and TNFR-associated death domain (TRADD) proteins. STAP-2 mRNA was induced by the expression of LMP1 in human B cells. Furthermore, transient expression of STAP-2 in EBV-positive human B cells decreased cell growth. Finally, STAP-2 knockout mouse embryonic fibroblasts showed enhanced LMP1-induced cell growth. These results suggest that STAP-2 acts as an endogenous negative regulator of EBV LMPI-mediated signaling through TRAF3 and TRADD.

The IL-6 family of cytokines modulates STAT3 activation by desumoylation of PML through SENP1 induction
Norihiko Ohbayashi, Shiho Kawakami, Ryuta Muromoto, Sumihito Togi, Osamu Ikeda, Shinya Kamitani, Yuichi Sekine, Tsutomu Honjoh, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 371, 4, 823, 828, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jul. 2008, [Peer-reviewed]
English, Scientific journal, Post-translational modification by small ubiquitin-like modifier (SUMO) plays an important role in the regulation of different signaling pathways and is involved in the formation of promyelocytic leukemia (PML) Protein nuclear bodies following sumoylation of PML. In the present study, we found that IL-6 induces desumoylation of PML and dissociation between PML and SUMO1 in hepatoma cells. We also found that IL-6 induces mRNA expression of SENP1, a member of the SUMO-specific protease family. Furthermore, wild-type SENP1 but not an inactive SENP1 mutant restored the PML-mediated suppression of STAT3 activation. These results indicate that the IL-6 family of cytokines modulates STAT3 activation by desumoylation and inactivation PML through SENP1 induction. (c) 2008 Elsevier Inc. All rights reserved.

KAP1 regulates type I interferon/STAT1-mediated IRF-1 gene expression
Shinya Kamitani, Norihiko Ohbayashi, Osamu Ikeda, Sumihito Togi, Ryuta Muromoto, Yuichi Sekine, Kazuhide Ohta, Hironobu Ishiyama, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 370, 2, 366, 370, ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2008, [Peer-reviewed]
English, Scientific journal, Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation, and survival in immune responses, hematopoiesis, neurogenesis, and other biological processes. Recently, we showed that KAP1 is a novel STAT-binding partner that regulates STAT3-mediated transactivation. KAP1 is a universal co-repressor protein for the KRAB zinc finger protein superfamily of transcriptional repressors. In this study, we found KAP1-dependent repression of interferon (IFN)/STAT1-mediated signaling. We also demonstrated that endogenous KAP1 associates with endogenous STAT1 in vivo. importantly, a small-interfering RNA-mediated reduction in KAP1 expression enhanced IFN-induced STAT1-dependent IRF-1 gene expression. These results indicate that KAP1 may act as an endogenous regulator of the IFN/STAT1 signaling pathway. (c) 2008 Elsevier Inc. All rights reserved.

Sumoylation of Smad3 stimulates its nuclear export during PIASy-mediated suppression of TGF-beta signaling
Seiyu Imoto, Norihiko Ohbayashi, Osamu Ikeda, Shinya Kamitani, Ryuta Muromoto, Yuichi Sekine, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 370, 2, 359, 365, ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2008, [Peer-reviewed]
English, Scientific journal, Sma- and MAD-related protein 3 (Smad3) plays crucial roles in the transforming growth factor-beta(TGF-beta)-mediated signaling pathway, which produce a variety of cellular responses, including cell proliferation and differentiation. In our previous study, we demonstrated that protein inhibitor of activated STATy (PIASy) suppresses TGF-beta signaling by interacting with and sumoylating Smad3. In the present study, we examined the molecular mechanisms of Smad3 sumoylation during PIASy-mediated suppression of TGF-beta signaling. We found that small-interfering RNA-mediated reduction of endogenous PIASy expression enhanced TGF-beta-induced gene expression. Importantly, coexpression of Smad3 with PIASy and SUMO1 affected the DNA-binding activity of Smad3. Furthermore, coexpression of Smad3 with PIASy and SUMO1 stimulated the nuclear export of Smad3. Finally, fluorescence resonance energy transfer analyses revealed that Smad3 interacted with SUMO1 in the cytoplasm. These results suggest that PIASy regulates TGF-beta/Smad3-mediated signaling by stimulating sumoylation and nuclear export of Smad3. (c) 2008 Elsevier Inc. All rights reserved.

BART is essential for nuclear retention of STAT3
Ryuta Muromoto, Yuichi Sekine, Seiyu Imoto, Osamu Ikeda, Taichiro Okayama, Noriko Sato, Tadashi Matsuda
INTERNATIONAL IMMUNOLOGY, 20, 3, 395, 403, OXFORD UNIV PRESS, Mar. 2008, [Peer-reviewed]
English, Scientific journal, Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation and survival in immune responses, hematopoiesis, neurogenesis and other biological processes. STAT3, for example, is involved in the epithelial-mesenchymal transition during gastrulation, organogenesis, wound healing and cancer progression. STAT activity is regulated by a variety of mechanisms, including nuclear translocation. To clarify the molecular mechanisms underlying the regulation of STAT activity, we performed yeast two-hybrid screening. Here, we identified binder of ADP-ribosylation factor-like two (BART) as a novel STAT-binding partner. Importantly, we showed that BART is essential for the transcriptional activity and nuclear retention of STAT3. Furthermore, an effector of BART, ADP-ribosylation factor-like 2 (ARL2) was also involved in nuclear retention of STAT3. These results indicate that BART plays an essential role in the nuclear retention of STAT3 through interaction with ARL2.

LIF- and IL-6-Induced acetylation of STAT3 at Lys-685 through PI3K/Akt activation
Norihiko Ohbayashi, Osamu Ikeda, Naohisa Taira, Yu Yamamoto, Ryuta Muromoto, Yalchi Sekine, Kenji Sugiyama, Tsutomu Honjoh, Tadashi Matsuda
BIOLOGICAL & PHARMACEUTICAL BULLETIN, 30, 10, 1860, 1864, PHARMACEUTICAL SOC JAPAN, Oct. 2007, [Peer-reviewed]
English, Scientific journal, Signal transducer and activator of transcription 3 (STAT3), which mediates biological actions in many physiological processes, is activated by cytokines and growth factors via specific tyrosine or serine phosphorylation, dimerization and nuclear translocation. A recent study has demonstrated, by using antibody to acetylated lysine, and a STAT3 mutant with Lys-685-to-Arg substitution, that STAT3 is acetylated at Lys-685 by histone acetyltransferase p300, and that acetylation at Lys-685 is critical for STAT3 activation. In the present study, we created an acetyl-specific antibody against STAT3 acetylated at Lys-685, and found that leukemia inhibitory factor (LIF) or interleukin (IL)-6 induced acetylation of STAT3 at Lys-685 in 293T and Hep3B cells. Moreover, acetylation of STAT3 at Lys-685 was suppressed by PI3K inhibitor LY294002, or a dominant negative Akt. Taken together, our findings demonstrate that endogenous STAT3 is acetylated at Lys-685 by LIF or IL-6 through PI3K/Akt activation.

Chronic thrombopoietin overexpression induces mesangioproliferative glomerulopathy in mice
Haruko K. Shimoda, Masahiro Yamamoto, Kotaro Shide, Kenjirou Kamezaki, Tadashi Matsuda, Katsuhiro Ogawa, Mine Harada, Kazuya Shimoda
AMERICAN JOURNAL OF HEMATOLOGY, 82, 9, 802, 806, WILEY-LISS, Sep. 2007, [Peer-reviewed]
English, Scientific journal, We previously reported that mice transgenic (Tg) for thrombopoietin (TPO) developed progressive fibrosis and osteosclerosis of the bone marrow. Here, we show that TPO-overexpressing mice also exhibited notable histological changes in the kidneys, including an increased number of mesangial cells, expansion of the mesangial matrix in the glomerulus, and atrophy of the renal tubuli. Plasma transforming growth factor (TGF)-beta 1 and platelet-derived growth factor (PDGF)-BB, which could induce mesangioproliferative responses in glomeruli, were both elevated in TPO Tg mice, even though TPO itself has no effect on mesangial cells due to their lack of c-MpI. The mesangial proliferative change in TPO Tg mice was thought to be induced by the elevation of these cytokines. In conclusion, our finding that TPO-overexpressing mice developed mesangioproliferative glomerulopathy might represent an undesirable effect of chronically elevated TPO in vivo, which should be taken into consideration before new TPO-like growth factors become available in clinical practice.

Tyk2 mutation homologous to V617F Jak2 is not found in essential thrombocythaemia, although it induces constitutive signaling and growth factor independence
Kotaro Shide, Kazuya Shimoda, Kenjirou Kamezaki, Haruko Kakumitsu, Takashi Kumano, Akihiko Numata, Fumihiko Ishikawa, Katsuto Takenaka, Ken Yamamoto, Tadashi Matsuda, Mine Harada
LEUKEMIA RESEARCH, 31, 8, 1077, 1084, PERGAMON-ELSEVIER SCIENCE LTD, Aug. 2007, [Peer-reviewed]
English, Scientific journal, A single somatic mutation, V617F, in the pseudokinase domain of the Jak2 is the primary cause of many chronic myeloproliferative diseases. As valine 617 of Jak2 is conserved as valine 678 of Tyk2, we examined the effect of a homologous mutation in Tyk2 (V678F Tyk2) on cell growth. V678F Tyk2 augmented the transcriptional activity of Stat3 and Stat5. The expression of V678F Tyk2 in Ba/F3 cells induced autonomous cell growth and showed hyper-responsiveness to IL-3. Although V678F Tyk2 might cause MPD, no cases of ET patients lacking the V617F Jak2 mutation harbored the Tyk2 mutation. (c) 2006 Elsevier Ltd. All rights reserved.

Signal-transducing adaptor protein-2 regulates integrin-mediated T cell adhesion through protein degradation of focal adhesion kinase
Yuichi Sekine, Satoshi Tsuji, Osamu Ikeda, Kenji Sugiyma, Kenji Oritani, Kazuya Shimoda, Ryuta Muromoto, Norihiko Ohbayashi, Akihiko Yoshimura, Tadashi Matsuda
JOURNAL OF IMMUNOLOGY, 179, 4, 2397, 2407, AMER ASSOC IMMUNOLOGISTS, Aug. 2007, [Peer-reviewed]
English, Scientific journal, Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin homology- and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, we find that STAP-2-deficient splenocytes or T cells exhibit enhanced cell adhesion to fibronectin after PMA treatment, and that STAP-2-deficient T cells contain the increased protein contents of focal adhesion kinase (FAK). Furthermore, overexpression of STAP-2 induces a dramatic decrease in the protein contents of FAK and integrin-mediated T cell adhesion to fibronectin in Jurkat T cells via the degradation of FAK. Regarding the mechanism for this effect, we found that STAP-2 associates with FAK and enhances its degradation, proteasome inhibitors block FAK degradation, and STAP-2 recruits an endogenous E3 ubiquitin ligase, Cbl, to FAK. These results reveal a novel regulation mechanism for integrin-mediated signaling in T cells via STAP-2, which directly interacts with and degrades FAK.

STAP-2 regulates c-Fms/M-CSF receptor signaling in murine macrophage Raw 264.7 cells
Osamu Ikeda, Yuichi Sekine, Michinori Kakisaka, Satoshi Tsuji, Ryuta Muromoto, Norihiko Ohbayashi, Kenji Oritani, Akhko Yoshimura, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 358, 3, 931, 937, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jul. 2007, [Peer-reviewed]
English, Scientific journal, Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein as a c-Fms/M-CSF receptor-interacting protein and constitutively expressed in macrophages. Our previous studies also revealed that STAP-2 binds to MyD88 and IKK-alpha/beta, and modulates NF-kappa B signaling in macrophages. In the present study, we examined physiological roles of the interaction between STAP-2 and c-Fms in Raw 264.7 macrophage cells. Our immunoprecipitation has revealed that c-Fms directly interacts with the PH domain of STAP-2 independently on M-CSF-stimulation. Ectopic expression of STAP-2 markedly suppressed M-CSF-induced tyrosine phosphorylation of c-Fms as well as activation of Akt and extracellular signal regulated kinase. In addition, Raw 264.7 cells over-expressing STAP-2 showed impaired migration in response to M-CSF and wound-healing process. Taken together, our findings demonstrate that STAP-2 directly binds to c-Fms and interferes with the PI3K signaling, which leads to macrophage motility, in Raw 264.7 cells. (C) 2007 Elsevier Inc. All rights reserved.

Leukemia inhibitory factor-induced phosphorylation of STAP-2 on tyrosine-250 is involved in its STAT3-enhancing activity
Yuichi Sekine, Satoshi Tsuji, Osamu Ikeda, Michinori Kakisaka, Kenji Sugiyama, Akihiko Yoshimura, Tadashi Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 356, 2, 517, 522, ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2007, [Peer-reviewed]
English, Scientific journal, Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 binds to signal transducer and activator of transcription 3 (STAT3) and STAT5, and regulates their signaling pathways. In the present study, we identified tyrosine-250 (Tyr250) in STAP-2 as a major site of phosphorylation by v-src and Jak2, using a phospho-specific antibody against STAP-2 phosphorylated at Tyr250. Mutational analyses revealed that Tyr250 was involved in the STAT3-enhancing activity of STAP-2. We further found that leukemia inhibitory factor (LIF) stimulated STAP-2 Tyr250 phosphorylation in 293T and Hep3B cells. Moreover, endogenous STAP-2 was phosphorylated at Tyr250 following LIF stimulation of murine M1 cell line. Taken together, our findings demonstrate that endogenous STAP-2 is phosphorylated at Tyr250 and that this phosphorylation is involved in its function. (c) 2007 Elsevier Inc. All rights reserved.

DUSP22/LMW-DSP2 regulates estrogen receptor-α-mediated signaling through dephosphorylation of Ser-118
Sekine, Y., Ikeda, O., Hayakawa, Y., Tsuji, S., Imoto, S., Aoki, N., Sugiyama, K., Matsuda, T.
Oncogene, 26, 41, 6038, 6049, 2007, [Peer-reviewed]
Scientific journal, In the previous study, we demonstrated the involvement of dual specificity phosphatase 22 (DUSP22/LMW-DSP2) in regulating the leukemia inhibitory factor/interleukin-6/signal transducer and activator of transcription 3-mediated signaling pathway. In this study, we show β-estradiol (E2)-induced DUSP22 mRNA expression in estrogen receptor α (ERα)-positive breast cancer cells, whereas E2-induced phosphorylation and activation of ERα was suppressed by overexpression of DUSP22 but not catalytically inactive mutants. Furthermore, small-interfering RNA-mediated reduction of DUSP22 expression enhanced ERα-mediated transcription and endogenous gene expression. In fact, DUSP22 associated with ERα in vivo and both endogenous proteins interacted in ERα-positive breast cancer T47D cells. These results strongly suggest that DUSP22 acts as a negative regulator of the ERα-mediated signaling pathway. © 2007 Nature Publishing Group All rights reserved.

In vitro screening of 200 pesticides for agonistic activity via mouse peroxisome proliferator-activated receptor (PPAR)alpha and PPAR gamma and quantitative analysis of in vivo induction pathway
Shinji Takeuchi, Tadashi Matsuda, Satoshi Kobayashi, Tetsuo Takahashi, Hiroyuki Kojima
TOXICOLOGY AND APPLIED PHARMACOLOGY, 217, 3, 235, 244, ACADEMIC PRESS INC ELSEVIER SCIENCE, Dec. 2006, [Peer-reviewed]
English, Scientific journal, Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors and key regulators of lipid metabolism and cell differentiation. However, there have been few studies reporting on a variety of environmental chemicals, which may interact with these receptors. In the present study, we characterized mouse PPAR alpha and PPAR gamma agonistic activities of 200 pesticides (29 organochlorines, I I diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, I I acid amides, 7 triazines, 8 ureas and 44 others) by in vitro reporter gene assays using CV-1 monkey kidney cells. Three of the 200 pesticides, diclofop-methyl, pyrethrins and imazalil, which have different chemical structures, showed PPAR alpha-mediated transcriptional activities in a dose-dependent manner. On the other hand, none of the 200 pesticides showed PPAR gamma agonistic activity at concentrations <= 10(-5) M. To investigate the in vivo effects of diclofop-methyl, pyrethrins and imazalil, we examined the gene expression of PPAR alpha-inducible cytochrome P450 4As (CYP4As) in the liver of female mice intraperitoneally injected with these compounds (<= 300 mg/kq). RT-PCR revealed significantly high induction levels of CYP4A10 and CYP4A14 mRNAs in diclofop-methyl- and pyrethrins-treated mice. whereas imazalil induced almost no gene expressions of CYP4As. In particular, diclofop-methyl induced as high levels of CYP4A mRNAs as WY-14643, a potent PPAR alpha agonist. Thus, most of the 200 pesticides tested do not activate PPAR alpha or PPAR gamma in in vitro assays, but only diclofop-methyl and pyrethrins induce PPAR alpha agonistic activity in vivo as well as in vitro. (c) 2006 Elsevier Inc. All rights reserved.

Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2
Megumi Funakoshi-Tago, Stephane Pelletier, Tadashi Matsuda, Evan Parganas, James N. Ihle
EMBO JOURNAL, 25, 20, 4763, 4772, NATURE PUBLISHING GROUP, Oct. 2006, [Peer-reviewed], ［Internationally co-authored］
English, Scientific journal, The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y-119 in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y-119 in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y-119 E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y-119 F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y-119 in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-c.

Sumoylation of Daxx regulates IFN-induced growth suppression of B lymphocytes and the hormone receptor-mediated transactivation
Ryuta Muromoto, Masato Ishida, Kenji Sugiyama, Yuichi Sekine, Kenji Oritani, Kazuya Shimoda, Tadashi Matsuda
JOURNAL OF IMMUNOLOGY, 177, 2, 1160, 1170, AMER ASSOC IMMUNOLOGISTS, Jul. 2006, [Peer-reviewed]
English, Scientific journal, Daxx has been shown to play an essential role in type I IFN-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the molecular mechanisms of how Daxx acts on growth suppression of B lymphocytes, we examined functions of a sumoylation-defective Daxx KA mutant (Daxx K630/631A), which substituted Lys 630 and Lys 631 to Ala. Importantly, Daxx KA localized in the cytoplasm, whereas wild-type Daxx localized in the nucleus. Murine pro-B cell line Ba/F3 expressing Daxx KA revealed a resistance to the IFN-induced growth suppression. It is noteworthy that treatment with an exportin inhibitor, leptomycin B, resulted in nuclear localization of Daxx KA and recovery of the IFN-induced growth suppression in Ba/F3 cells. Moreover, Daxx KA decreased the binding potential to promyelocytic leukemia protein (PML), and overexpression of PML recruited Daxx KA into PML oncogenic domains. Notably, a Daxx-small ubiquitin-related modifier fusion protein exhibited increased nuclear localization and ability to suppress cell growth in Ba/F3 cells. These results demonstrate that the IFN-induced growth suppression of B lymphocytes requires nuclear localization of Daxx through its sumoylation and proper interactions with PML.

Phosphorylation of threonine-265 in Zipper-interacting protein kinase plays an important role in its activity and is induced by IL-6 family cytokines
N Sato, N Kamada, R Muromoto, T Kawai, K Sugiyama, T Watanabe, S Imoto, Y Sekine, N Ohbayashi, M Ishida, S Akira, T Matsuda
IMMUNOLOGY LETTERS, 103, 2, 127, 134, ELSEVIER SCIENCE BV, Mar. 2006, [Peer-reviewed]
English, Scientific journal, Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. Here, we identified threonine-265 (Thr265) in ZIPK as a major autophosphorylation site. Mutational analyses revealed that autophosphorylation of Thr265 were essential for its full catalytic activity toward an exogenous substrate as well as for cell death induction. Furthermore, leukemia inhibitory factor (LIF) stimulated Thr265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription (STAT3). Taken together, our findings demonstrate that ZIPK is positively regulated through Thr265 phosphorylation and that this phosphorylation is essential for its function. (C) 2005 Elsevier B.V. All rights reserved.

Physical and functional interactions between Daxx and STAT3.
Muromoto R, Nakao K, Watanabe T, Sato N, Sekine Y, Sugiyama K, Oritani K, Shimoda K, Matsuda T
Oncogene, 25, 14, 2131, 2136, Nature Publishing Group, Mar. 2006, [Peer-reviewed]
English, Signal transducer and activator of transcription 3 (STAT3) play key roles in the intracellular signaling pathways of the interleukin (IL)-6 family of cytokines, which exhibit a diverse set of cellular responses, including cell proliferation and differentiation. Dysregulated IL-6/STAT3 signaling is involved in the pathogenesis of several diseases, for example autoimmune diseases and tumors. Type I interferon (IFN) induces the expression of proapoptotic genes and has been used in the clinical treatment of several tumors. In the present study, we found that type I IFN suppressed IL-6/STAT3-mediated transcription and gene expression. Furthermore, a type I IFN-induced protein, Daxx, also suppressed STAT3-mediated transcriptional activation, while overexpression of Daxx inhibited IL-6/STAT3-mediated gene expression. Importantly, small-interfering RNA-mediated reduction of Daxx expression enhanced IL-6/leukemia inhibitory factor (LIF)-induced STAT3-dependent transcription. Co-immunoprecipitation studies revealed a physical interaction between Daxx and STAT3 in transiently transfected 293T cells. We further found that Daxx and STAT3 were co-localized in the nucleus. These results indicate that Daxx may serve as a transcriptional regulator of type I IFN-mediated suppression of the IL-6/STAT3 signaling pathway.

Physical and functional interactions between STAT3 and Kaposi's sarcoma-associated herpesvirus-encoded LANA
R Muromoto, K Okabe, M Fujimuro, K Sugiyama, H Yokosawa, T Seya, T Matsuda
FEBS LETTERS, 580, 1, 93, 98, ELSEVIER SCIENCE BV, Jan. 2006, [Peer-reviewed]
English, Scientific journal, The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) is known to modulate viral and cellular gene expression. We show that LANA directly associates with an interleukin-6 signal transducer, signal transducer and activator of transcription 3 (STAT3) and that LANA enhances the transcriptional activity of STAT3. Coinummoprecipitation studies documented a physical interaction between LANA and STAT3 in transiently transfected 293T cells as well as the KSHV-infected primary effusion lymphoma (PEL) cell line. Furthermore, small-interfering RNA-mediated reduction of LANA expression decreased the STAT3-dependent transcription in KSHV-positive PEL cells, whereas overexpression of LANA enhanced STAT3 activity in KSHV-negative B lymphoma cells. These data demonstrate that LANA is a transcriptional co-activator of STAT3, and may have implications for the pathogenesis of KSHV-associated diseases. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Modulation of TLR4 signaling by a novel adaptor protein signal-transducing adaptor protein-2 in macrophages
Y Sekine, T Yumioka, T Yamamoto, R Muromoto, S Imoto, K Sugiyma, K Oritani, K Shimoda, M Minoguchi, S Akira, A Yoshimura, T Matsuda
JOURNAL OF IMMUNOLOGY, 176, 1, 380, 389, AMER ASSOC IMMUNOLOGISTS, Jan. 2006, [Peer-reviewed]
English, Scientific journal, Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies have demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, STAP-2 was found to positively regulate LPS/TLR4-mediated signals in macrophages. Disruption of STAP-2 resulted in impaired LPS/TLR4-induced cytokine production and NF-kappa beta activation. Conversely, overexpression of STAP-2 enhanced these LPS/TLR4-induced biological activities. STAP-2, particularly its Src homology 2-like domain, bound to both MyD88 and licB kinase (IKK)-alpha beta, but not TNFR-associated factor 6 or IL-1R-associated kinase 1, and formed a functional complex composed of MyD88-STAP-2-IKK-alpha beta. These interactions augmented MyD88- and/or IKK-alpha beta-dependent signals, leading to enhancement of the NF-kappa beta activity. These results demonstrate that STAP-2 may constitute an alternative LPS/TLR4 pathway for NF-kappa beta activation instead of the TNFR-associated factor 6-IL-IRassociated kinase 1 pathway.

Regulation of STAT3-mediated signaling by LMW-DSP2
Sekine, Y., Tsuji, S., Ikeda, O., Sato, N., Aoki, N., Aoyama, K., Sugiyama, K., Matsuda, T.
Oncogene, 25, 42, 5801, 5806, 2006, [Peer-reviewed]
Scientific journal, Signal transducer and activator of transcription 3 (STAT3), which mediates biological actions in many physiological processes, is activated by cytokines and growth factors, and has been reported to be constitutively activated in numerous cancer cells. In this study, we examined whether low molecular weight-dual specificity phosphatase two (LMW-DSP2) is involved in the regulation of the interleukin 6 (IL-6)/leukemia inhibitory factor (LIF)/STAT3-mediated signaling pathway. IL-6/LIF-induced LMW-DSP2 expression in murine testicular or hepatoma cell lines, while LMW-DSP2 overexpression in 293T cells suppressed IL-6-induced phosphorylation and activation of STAT3. Furthermore, LMW-DSP2 suppressed the expression of IL-6-induced endogenous genes. In contrast, small-interfering RNA-mediated reduction of LMW-DSP2 expression enhanced IL-6-induced STAT3-dependent transcription. In fact, LMW-DSP2 interacted with STAT3 in vivo and endogenous LMW-DSP2 bound to STAT3 in murine testicular GC-1 cells. These results strongly suggest that LMW-DSP2 acts as a negative regulator of the IL-6/LIF/STAT3-mediated signaling pathway. © 2006 Nature Publishing Group. All rights reserved.

Physical and functional interactions between STAT3 and ZIP kinase
N Sato, T Kawai, K Sugiyama, R Muromoto, S Imoto, Y Sekine, M Ishida, S Akira, T Matsuda
INTERNATIONAL IMMUNOLOGY, 17, 12, 1543, 1552, OXFORD UNIV PRESS, Dec. 2005, [Peer-reviewed]
English, Scientific journal, Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor that can be activated by cytokines and growth factors. It plays important roles in cell growth, apoptosis and cell transformation, and is constitutively active in a variety of tumor cells. In this study, we provide evidence that zipper-interacting protein kinase (ZIPK) interacts physically with STAT3. ZIPK specifically interacted with STAT3, and did not bind to STAT1, STAT4, STAT5a, STAT5b or STAT6. ZIPK phosphorylated STAT3 on serine 727 (Ser727) and enhanced STAT3 transcriptional activity. Small interfering RNA-mediated reduction of ZIPK expression decreased leukemia inhibitory factor (LIF)- and IL-6-induced STAT3-dependent transcription. Furthermore, LIF- and IL-6-mediated STAT3 activation stimulated ZIPK activity. Taken together, our data suggest that ZIPK interacts with STAT3 within the nucleus to regulate the transcriptional activity of STAT3 via phosphorylation of Ser727.

Nuclear retention of STAT3 through the coiled-coil domain regulates its activity
N Sato, R Tsuruma, S Imoto, Y Sekine, R Muromoto, K Sugiyama, T Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 336, 2, 617, 624, ACADEMIC PRESS INC ELSEVIER SCIENCE, Oct. 2005, [Peer-reviewed]
English, Scientific journal, Signal transducer and activator of transcription 3 (STAT3), which mediates biological actions in many physiological processes, is activated by cytokines and growth factors via specific tyrosine phosphorylation, dimerization, and nuclear translocation. However, the mechanism involved in its nuclear translocation remains unclear. A previous study demonstrated that STAT3 with Arg-214/215 mutations in the coiled-coil domain (R214A/R215A; STAT3 RA) failed to undergo nuclear translocation. Here, we re-examined the nature of the STAT3 RA mutant and found that it showed higher and more extensive tyrosine-phosphorylation as well as much higher STAT3 transcriptional activity in response to stimuli. Furthermore, STAT3 RA showed nuclear translocation and faster nuclear export than wild-type STAT3 after stimulation. Moreover, nuclear retention of STAT3 RA by a chromosomal region maintenance 1 (CRM1) inhibitor, leptomycin B, decreased the enhanced STAT3 activation by stimuli. These data demonstrate that Arg-214/215 are involved in CRM1-mediated STAT3 nuclear export and the regulation of STAT3 activity. (c) 2005 Elsevier Inc. All rights reserved.

A novel mutation in the juxtamembrane intracellular sequence of the granulocyte colony-stimulating factor (G-CSF) receptor gene in a patient with severe congenital neutropenia augments G-CSF proliferation activity but not through the MAP kinase cascade
T Yokoyama, S Okamura, Y Asano, K Kamezaki, A Numata, H Kakumitsu, K Shide, H Nakashima, T Kanaji, Y Sekine, Y Mizuno, J Okamura, T Matsuda, M Harada, Y Niho, K Shimoda
INTERNATIONAL JOURNAL OF HEMATOLOGY, 82, 1, 28, 34, CARDEN JENNINGS PUBL CO LTD, Jul. 2005, [Peer-reviewed]
English, Scientific journal, We analyzed the structure of the granulocyte colony-stimulating factor (G-CSF) receptor gene in a 6-year-old female patient with severe congenital neutropenia (SCN) who experienced severe recurrent infections since I month of age. There is no family history of any similar disease. When the patient was 4 months old, she began receiving treatment with recombinant human G-CSF that resulted in a small increase in the neutrophil count sufficient for the prevention and treatment of bacterial infection. An analysis of complementary DNA for the patient's G-CSF receptor revealed a 3-base pair deletion in the juxtamembrane intracellular sequence. This deletion at the beginning of exon 16 was thought to be caused by alternative splicing; analysis of the DNA revealed a G-to-A point mutation of the final nucleotide of intron 15. To evaluate the functional activity of the G-CSF receptor with this 3-base pair deletion of the juxtamembrane region, we transfected this G-CSF receptor mutant into an interleukin 3-dependent cell line, BAF/3. BAF/3 cells expressing the mutant G-CSF receptor showed augmented proliferation activity in response to G-CSF compared with cells having the wild-type G-CSF receptor. Although the proliferation signal of G-CSF in normal hematopoiesis is transduced through the activation of MAP kinases, this G-CSF receptor mutant showed decreased activation of ERK1/2 in response to G-CSF compared with the wild type, but the transduced signal for Stat3 activation by G-CSF was of the same magnitude as that of the wild-type G-CSF receptor. This result means that the augmented proliferation activity in response to G-CSF that we observed in cells having the G-CSF receptor gene with the 3-base pair deletion is transduced through an intracellular signaling pathway other than MAP kinase. Because SCN patients with a mutation in the G-CSF receptor frequently develop leukemia, this 3-base pair deletion in the juxtamembrane sequence of the G-CSF receptor gene in this patient may be one step in the course of leukemic transformation.

Novel pathway for LPS-induced afferent vagus nerve activation: Possible role of nodose ganglion
Hosoi, T, Okuma, Y, Matsuda, T, Nomura, Y
AUTONOMIC NEUROSCIENCE-BASIC & CLINICAL, 120, 1-2, 104, 107, ELSEVIER SCIENCE BV, 15 Jun. 2005, [Peer-reviewed]
Scientific journal, The afferent vagus nerve has been suggested to be an important component for transmitting peripheral immune signals to the brain. However, there is inconsistent evidence showing that subdiaphragmatic vagotomy did not inhibit the brain mediated behavioral and neural effects induced by the peripheral application of lipopolysaccharide (LPS). LPS triggers innate immune cells through Toll-like receptor 4 (TLR4). In the present study, we found that TLR4 mRNA and protein was expressed in the rat nodose ganglion. Thus, it is suggested that LPS could activate afferent vagus nerve at the level of nodose ganglion, which exists centrally from the subdiaphragmatic level of vagus nerve. The results could provide evidence for the novel pathway of LPS-induced afferent vagus nerve activation. (c) 2005 Elsevier B.V. All rights reserved.

Differential effects of phthalate esters on transcriptional activities via human estrogen receptors alpha and beta, and androgen receptor
S Takeuchi, M Iida, S Kobayashi, K Jin, T Matsuda, H Kojima
TOXICOLOGY, 210, 2-3, 223, 233, ELSEVIER IRELAND LTD, Jun. 2005, [Peer-reviewed]
English, Scientific journal, Some phthallates are suspected to disrupt the endocrine system, especially by mimicking estrogens. In this study, we characterized the activities of human estrogen receptor a (hERalpha), human estrogen receptor beta (hERbeta), and human androgen receptor (hAR) in the presence of 22 phthalates including 3 of their metabolites using highly sensitive reporter gene assays. Of the 22 compounds tested, several phthalate diesters with alkyl chains ranging in length from C-3 to C-6 exhibited not only hERalpha-mediated estrogenic activity, but also hERbeta-mediated antiestrogenic activity in a dose-dependent manner. In addition, we found that some phthalate diesters possess hAR-mediated antiandrogenic activity. However, the phthalates having side chains with very short length (diethyl) or very long length (diheptyl), and three metabolites (monoesters) were found to have no effect on the activities of the three receptors. These results indicate that several phthalate esters simultaneously act as agonists and/or antagonists via one or more hormonal receptors, and interaction of phthalate esters with the estrogen and androgen receptors requires certain size and bulkiness with alkyl groups. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide stimulate interleukin-6 production in prostate cancer cells and prostatic epithelial cells.
Nagakawa O, Junicho A, Akashi T, Koizumi K, Matsuda T, Fuse H, Saiki I
Oncology reports, 13, 6, 1217, 1221, Jun. 2005, [Peer-reviewed]

Roles for lysine residues of the MH2 domain of Smad3 in transforming growth factor-beta e signaling
S Imoto, K Sugiyama, Y Sekine, T Matsuda
FEBS LETTERS, 579, 13, 2853, 2862, ELSEVIER SCIENCE BV, May 2005, [Peer-reviewed]
English, Scientific journal, Sma and MAD-related protein 3 (Smad3) plays a key role in the intracellular signaling of the transforming growth factor-beta (TGF-beta) family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. Smad3 has the N-terminal Mad homology (MH) 1 and the C-terminal MH2 domains. MH2 domain is essential for the TGF-beta-induced transcriptional activation, because the MH2 domain of Smad3 is involved in the interactions with several transcriptional cofactors as well as the type I TGF-beta receptor (T beta R-I). In this study, we examined the roles for four lysine residues (Lys-333, Lys-341, Lvs-378, and Lys-409) in the Smad3 MH2 domain. Mutation of the lysine (K)-378 to arginine (R) (K378R) abolished the interaction with T beta R-I, phosphorylation, transcriptional activation by an active T beta R-I. The K341R mutant also failed to stimulate TGF-beta-induced transcription by resting in the cytoplasm. However, the K409R mutant showed a higher transcriptional activity by stronger interactions with coactivators, such as p300/CBP. Furthermore, both the K341R and K378R mutants act as dominant-negative inhibitors in the TGF-beta-induced target genes of endogenous TGF-beta signal. Thus, the lysine residues of Smad3 MH2 domain play important roles in the transcriptional regulation of TGF-beta signals through T beta R-I. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Physical and functional interactions between STAP-2/BKS and STAT5
Y Sekine, T Yamamoto, T Yumioka, K Sugiyama, S Tsuji, K Oritani, K Shimoda, M Minoguchi, A Yoshimura, T Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 280, 9, 8188, 8196, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Mar. 2005, [Peer-reviewed]
English, Scientific journal, Signal-transducing adaptor protein family of proteins (STAPs), which currently contains two members, are proposed to be adaptor molecules because of their pleckstrin homology (PH) and Src-homology 2 (SH2)-like domains. STAP-1 has been shown to interact with STAT5 and the tyrosine kinase Tec. With regard to STAP-2/BKS functions, immunoprecipitation experiments and intracellular stainings revealed STAP-2/BKS binds STAT5 in several types of cells. Mutational studies revealed that the PH- and SH2-like domains of STAP-2/BKS interacted with the C-terminal region of STAT5. STAP-2/BKS and STAT5 were found to constitutively co-localize in the cytoplasm of resting cells, but STAP-2/BKS was found to dissociate upon STAT5 phosphorylation, suggesting a role in regulating signaling of STAT5. The physiological role of these interactions is not fully understood, but in studies of overexpression of STAP-2/BKS, cytokine-induced tyrosine phosphorylation and transcriptional activation of STAT5 was diminished. In addition, thymocytes from STAP-2/BKS-deficient mice showed the enhanced interleukin-2-dependent cell growth. Taken together, STAP-2/BKS is an additional modulator of STAT5-mediated signaling.

Roles of Stat3 and ERK in G-CSF signaling
K Kamezaki, K Shimoda, A Numata, T Haro, H Kakumitsu, M Yoshie, Y Yamamoto, K Takeda, T Matsuda, M Akira, K Ogawa, O Harada
STEM CELLS, 23, 2, 252, 263, ALPHAMED PRESS, Feb. 2005, [Peer-reviewed]
English, Scientific journal, G-CSF specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil-granulocyte lineage. Although Stat3 was thought to be essential for the transduction of G-CSF-induced cell proliferation and differentiation signals, mice deficient for Stat3 in hematopoietic cells show neutrocytosis and was not induced by G-CSF stimulation. Stat3-null bone marrow cells displayed a significant activation of extracellular regulated kinase I (ERK1)/ERK2 under basal conditions, and the activation of ERK was enhanced and sustained by G-CSF stimulation. Furthermore, the augmented proliferation of Stat3-deficient bone marrow infiltration of cells into the digestive tract. The number of cells in response to G-CSF was dramatically decreased progenitor cells in the neutrophil lineage is not changed, and G-CSF-induced proliferation of progenitor cells and prolonged neutrophil survival were observed in Stat3deficient mice. In hematopoietic cells from Stat3-deficient mice, trace levels of SOCS3, a negative regulator of granulopoiesis, were observed, and SOCS3 expression by addition of a MEK1 inhibitor. These results indicate that Stat3 functions as a negative regulator of G-CSF signaling by inducing SOCS3 expression and that ERK activation is the major factor responsible for inducing the proliferation of hematopoietic cells in response to G-CSF.

Cytokine induction by a bacterial DNA-specific modified base
H Tsuchiya, T Matsuda, H Harashima, H Kamiya
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 326, 4, 777, 781, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jan. 2005, [Peer-reviewed]
English, Scientific journal, Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals. Here we show that N-6-methyladenine (N-6-MeA), a bacterium-specific modified base, also causes cytokine production. An oligodeoxyribonucleotide (ODN) containing N-6-MeA induced cytokines when injected into mice. Co-injection of N-6-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone. Plasmid DNA containing N-6-MeA, complexed with cationic lipids, induced IL-12. These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N-6-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine. (C) 2004 Elsevier Inc. All rights reserved.

Determination of the transphosphorylation sites of Jak2 kinase
T Matsuda, J Feng, BA Witthuhn, Y Sekine, JN Ihle
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 325, 2, 586, 594, ACADEMIC PRESS INC ELSEVIER SCIENCE, Dec. 2004, [Peer-reviewed], ［Internationally co-authored］
English, Scientific journal, Janus kinases are the key enzymes involved in the initial transmission of signals in response to type I and II cytokines. Activation of the signal begins with the transphosphorylation of Jak kinases. Substrates that give rise to downstream events are recruited to the receptor complex in part by interactions with phosphorylated tyrosines. The identity of many of the phosphotyrosines responsible for recruitment has been elucidated as being receptor-based tyrosines. The ability of Jaks to recruit substrates through their own phosphotyrosines has been demonstrated for tyrosines in the kinase activation loop. Recent studies demonstrate that other tyrosines have implications in regulatory roles of Jak kinase activity. In this study, baculovirus-produced Jak2 was utilized to demonstrate that transphosphorylation of Jak kinases occurs on multiple residues throughout the protein. We demonstrate that among the tyrosines phosphorylated, those in the kinase domain occur as expected, but many other sites are also phosphorylated. The tyrosines conserved in the Jak family are the object of this study, although many of them are phosphorylated, many are not. This result suggests that conservation of tyrosines is perhaps as important in maintaining structure of the Jak family. Additionally, non-Jak family conserved tyrosines are phosphorylated suggesting that the individual Jaks ability to phosphorylated specific tyrosines may influence signals emitting from activated Jaks. (C) 2004 Elsevier Inc. All rights reserved.

Molecular dynamics of STAT3 on IL-6 signaling pathway in living cells
K Watanabe, K Saito, M Kinjo, T Matsuda, M Tamura, S Kon, T Miyazaki, T Uede
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 324, 4, 1264, 1273, ACADEMIC PRESS INC ELSEVIER SCIENCE, Nov. 2004, [Peer-reviewed]
English, Scientific journal, Signal transducer and activator of transcription 3 (STAT3) is a critical signal transducer of interleukin-6 (IL-6) signaling. To investigate the mobility and the dynamics of STAT3 complex on IL-6 signaling in living cells, we generated a chimeric gene consisting of STAT3 fused to enhanced green fluorescence protein, STAT3-GFP. STAT3-GFP was expressed in Hep3B cells and the dynamics of this protein were analyzed by fluorescence correlation spectroscopy. After IL-6 stimulation, STAT3 translocated from the cytoplasm to the nucleus, as shown previously. According to the analysis of STAT3 diffusion in stable transformants, the number of STAT3 molecules at the cytoplasmic membrane and in the cytoplasm decreased after IL-6 stimulation. In the nucleus, the diffusion speed of STAT3 complex strongly decreased after IL-6 stimulation. Furthermore, we found that STAT3 existed as a complex whose molecular weight was less than 400 kDa before IL-6 addition. However, IL-6 stimulation induced the formation of STAT3 dimer as a megacomplex form whose molecular weight was more than 1 MDa at the cytoplasm and a very slow diffusion complex in the nucleus. (C) 2004 Elsevier Inc. All rights reserved.

Bacterial endotoxin induces IL-20 expression in the glial cells
T Hosoi, S Wada, S Suzuki, Y Okuma, S Akira, T Matsuda, Y Nomura
MOLECULAR BRAIN RESEARCH, 130, 1-2, 23, 29, ELSEVIER SCIENCE BV, Nov. 2004, [Peer-reviewed]
English, Scientific journal, The regulatory mechanisms leading to IL-20 expression during infection have not been elucidated. In the present study, we found that bacterial lipopolysaccharide (LPS) induced IL-20 expression in the primary cultured glial cells and RAW264.7 macrophage cell line. Pretreatment with protein synthesis inhibitor puromycin or cycloheximide failed to inhibit the expression of IL-20, suggesting that the expression was not dependent on de novo protein synthesis. Myeloid differentiation factor 88 (MyD88) is an important adaptor molecule for Toll-like receptor signaling. We observed complete inhibition of LPS-induced expression of IL-20 in the primary, cultured glial cells prepared from MyD88-deficient mice. Furthermore, a p38 MAP kinase inhibitor, SB203580, inhibited LPS-induced expression of IL-20 mRNA. LPS-induced p38 MAP kinase phosphorylation was delayed in MyD88-deficient glial cells. Therefore, it is suggested that LPS induces IL-20 expression through MyD88-p38-dependent mechanisms. As dexamethasone inhibited LPS-induced IL-20 expression, the expression of IL-20 is regulated by a negative feedback loop mediated through glucocorticoids. Therefore, it is suggested that IL-20 may play a crucial role in inflammatory conditions in the brain. (C) 2004 Elsevier B.V. All rights reserved.

Bacterial endotoxin induces STAT3 activation in the mouse brain
T Hosoi, Y Okuma, T Kawagishi, Qi, X, T Matsuda, Y Nomura
BRAIN RESEARCH, 1023, 1, 48, 53, ELSEVIER SCIENCE BV, Oct. 2004, [Peer-reviewed]
English, Scientific journal, In the present study, we investigated regulatory mechanisms of bacterial endotoxin-induced STAT3 activation in the brain. Intraperitoneal injection of lipopolysaccharide (LPS) dose-dependently (0.5-5000 mug/kg) induced STAT3 phosphorylation in the hypothalamus. LPS-induced STAT3 phosphorylation was peaked at 2-4 h and declined there after. Moreover, intracerebroventricular injection of LPS induced STAT3 phosphorylation in the cortex and the hippocampus, indicating that central as well as peripheral LPS can act in the brain to induce STAT3 activation. Glucocorticoids are known to play a physiological role in the feedback inhibition of immune/inflammatory responses in the endocrine system. Interestingly, we observed no effect of dexamethasone on LPS-induced STAT3 phosphorylation in the hypothalamus. These findings point to the important role of STAT3 in the neuroimmune interaction of inflammation in the brain. (C) 2004 Elsevier B.V All rights reserved.

Differential expression of integrin subunits in DU-145/AR prostate cancer cells
O Nagakawa, T Akashi, Y Hayakawa, A Junicho, K Koizumi, Y Fujiuchi, Y Furuya, T Matsuda, H Fuse, Saiki, I
ONCOLOGY REPORTS, 12, 4, 837, 841, PROFESSOR D A SPANDIDOS, Oct. 2004, [Peer-reviewed]
English, Scientific journal, We have established a clonal DU-145 prostate cancer cell line (DU-145/AR) stably transfected with androgen receptor cDNA. We investigated the expression of integrin subunits, adhesion to extracellular matrices, the invasion of DU-145/AR prostate cancer cells. The expression of various integrin subunits and adhesion to various extracellular matrices in DU-145, DU-145/Neo and DU-145/AR cells were examined. The haptoinvasion and the haptotactic migration of these cells were investigated using a Transwell cell culture chamber assay. DU-145/AR cells exhibited lower expression of alpha6 and beta4 integrin subunits and higher expression of alpha2 and alpha5 than DU-145 cells. DU-145/AR cells showed significantly lower adhesion to fibronectin, laminin-1 and laminin-5 than DU-145/Neo cells, whereas DU-145/AR cells showed higher adhesion to type I and type IV collagen. Haptoinvasion of DU-145/AR cells into Matrigel/fibronectin-coated filter was significantly reduced as compared with DU-145/Neo or DU-145 cells, but there was no significant difference between DU-145/AR and control cells in the haptotactic migration to fibronectin. Dihydrotestosterone (DHT) inhibited the invasive ability of DU-145/AR cells. These results indicate that androgen receptor may play a role in the regulation of adhesion to the extracellular matrices and invasion of prostate cancer cells through influencing the expression of specific integrin subunits.

Hapten-induced contact hypersensitivity is enhanced in Tyk2-deficient mice
M Hosogi, H Tonogaito, A Aioi, K Hamada, K Shimoda, R Muromoto, T Matsuda, Y Miyachi
JOURNAL OF DERMATOLOGICAL SCIENCE, 36, 1, 51, 56, ELSEVIER SCI IRELAND LTD, Oct. 2004, [Peer-reviewed]
English, Scientific journal, Background: Previous studies have shown that Tyk2, a member of the Janus family of protein tyrosine kinases, which are activated by a variety of cytokines, plays a crucial role in interleukin (IL)-12-mediated T-cell functions such as IFN-gamma production. On the other hand, hapten-induced contact hypersensitivity (CHS) is mediated by IFN-gamma producing CD8+ T cells and regulated by CD4+ T cells.
Objective: This study hypothesized that the CHS response might be reduced in Tyk2-deficient mice because of a tack of IFN-gamma production from CD4+ and CD8+ T cells.
Methods: The CHS reaction was evoked in wild-type and Tyk2-deficient mice and the ears of the mice were examined to measure for several cytokines.
Results: Ear swelling during CHS was significantly enhanced in Tyk2-deficient mice compared with the controls. IL-12 and IFN-gamma levels at the reaction sites in Tyk2-deficient mice were significantly lower than in the controls, whereas IL-2 and IL-4 levels were elevated. Furthermore, STAT3- and STAT4-phosphorylation in the draining lymph node cells of Tyk2-deficient mice decreased.
Conclusion: These results suggest that the tack of Tyk2-mediated signal transduction enhances a compensative pathway during CHS. (C) 2004 Japanese Society for Investigative Dermatology.

The role of Tyk2, Stat1 and Stat4 in LPS-induced endotoxin signals
K Kamezaki, K Shimoda, A Numata, T Matsuda, K Nakayama, M Harada
INTERNATIONAL IMMUNOLOGY, 16, 8, 1173, 1179, OXFORD UNIV PRESS, Aug. 2004, [Peer-reviewed]
English, Scientific journal, Mice lacking Tyk2, Stat1 or Stat4, which are members of the Jak-Stat signaling cascade, were resistant to LPS-induced endotoxin shock. Interestingly, Tyk2-deficient mice had higher resistance to LIPS challenge than mice lacking either Stat1 or Stat4. The activation of MAPK and NF-kappaB by LIPS, and the production of TNF-alpha and IL-12 after LPS injection, were not abrogated by the absence of Tyk2, Stat1 or Stat4l. In Stat1-deficient mice, the induction of IFN-beta by LIPS in macrophages was severely reduced, although the serum level of IFN-gamma was elevated after LPS injection. In contrast, in Stat-4 deficient mice, the induction of IFN-beta by LIPS was normal, but the serum level of IFN-gamma remained low after LPS injection. Interestingly, the induction of both IFN-beta and IFN-gamma by LIPS was severely reduced in Tyk2-deficient mice. Therefore, Stat1 and Stat4 independently play substantial roles in the susceptibility to LIPS. Tyk2 is essential for LIPS-induced endotoxin shock, and this signaling pathway is transduced by the activation of Stat1 and Stat4.

Tyrosine kinase 2 interacts with and phosphorylates receptor for-activated c kinase-1, a WD motif-containing protein
T Haro, K Shimoda, H Kakumitsu, K Kamezaki, A Numata, F Ishikawa, Y Sekine, R Muromoto, T Matsuda, M Harada
JOURNAL OF IMMUNOLOGY, 173, 2, 1151, 1157, AMER ASSOC IMMUNOLOGISTS, Jul. 2004, [Peer-reviewed]
English, Scientific journal, Receptor for activated C kinase (Rack)-1 is a protein kinase C-interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase Cgamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alphabeta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudokinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N terminus and one in the middle portion (aa 138-203) of Rack-1. Jak activation causes the phosphorylation of tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.

The RING domain of PIASy is involved in the suppression of bone morphogenetic protein-signaling pathway
S Imoto, K Sugiyama, T Yamamoto, T Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 319, 1, 275, 282, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jun. 2004, [Peer-reviewed]
English, Scientific journal, Bone morphogenetic proteins (BMPs) play central roles in differentiation, development, and physiologic tissue remodeling. Recently, we have demonstrated that a protein inhibitor of activated STAT, PIASy, suppresses TGF-beta signaling by interacting with Sma and MAD-related protein 3 (Smad3). In this study, we examined a PIASy-dependent inhibitory effect on BMP signaling. PIASy expression was induced by BMP-2 stimulation and suppressed BMP-2-dependent Smad activity in hepatoma cells. Furthermore, BMP-2-regulated Smads directly bound to PIASy. We also demonstrated that the RING domain of PIASy played an important role in PIASy-mediated suppression of Smad activity. We here provide evidence that the inhibitory action of PIASy on BMP-regulated Smad activity was due to direct physical interactions between Smads and PIASy through its RING domain. (C) 2004 Elsevier Inc. All rights reserved.

Physical and functional interactions between Daxx and TSG101
R Muromoto, K Sugiyama, T Yamamoto, K Oritani, K Shimoda, T Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 316, 3, 827, 833, ACADEMIC PRESS INC ELSEVIER SCIENCE, Apr. 2004, [Peer-reviewed]
English, Scientific journal, Daxx has been reported to mediate the Fas/JNK-dcpendcnt signals in the cytoplasm. However, several evidences have suggested that Daxx is located mainly in the nucleus and functions as a transcriptional regulator. Recently, we identified DMAP1, a TSG101-interacting protein as a Daxx binding partner by yeast two-hybrid screening. TSG101 has been shown to act as transcriptional corepressor of nuclear hormone receptors. Here we examined whether TSG101 also interacts with Daxx directly. The association of Daxx and TSG101 was confirmed using co-expressed tagged proteins. The interaction regions in both proteins were also mapped, and the cellular localization of the interaction was examined. TSG101 formed a complex with Daxx through its coiled-coil domain and co-localized in the nucleus. Furthermore, TSG101 enhanced Daxx-mediated repression of glucocorticoid receptor transcriptional activity. These results provide the novel molecular interactions between Daxx and TSG101, which establish an efficient repressive transcription complex in the nucleus. (C) 2004 Elsevier Inc. All rights reserved.

Cross-talk between endocrine-disrupting chemicals and cytokine signaling through estrogen receptors
Y Sekine, T Yamamoto, T Yumioka, S Imoto, H Kojima, T Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 315, 3, 692, 698, ACADEMIC PRESS INC ELSEVIER SCIENCE, Mar. 2004, [Peer-reviewed]
English, Scientific journal, STAT3 mainly acts as a signal transducer of IL-6 family cytokines and transcriptionally activates specific target genes. STAT3 has also been demonstrated to mediate cellular transformation and is found in numerous cancers. Endocrine-disrupting chemicals (EDCs) are a diverse group of chemicals that bind to estrogen receptors (ERs), mimic estrogenic actions, and may have adverse effects on human health. In our previous study, we demonstrated that estrogens suppressed the STAT3-mediated transcription activity through ERs. In this study, we examined the effects of EDCs on STAT3-mediated signaling through ERs. Surprisingly, some of EDCs enhanced STAT3-mediated transcription activity through ERs. This finding strongly suggests that EDCs may play an important role in the endocrine functions by mimicking cytokine activity by stimulating STAT3 actions through ERs. (C) 2004 Elsevier Inc. All rights reserved.

Physical and functional interactions between Daxx and DNA methyltransferase 1-associated protein, DMAP1
R Muromoto, K Sugiyama, A Takachi, S Imoto, N Sato, T Yamamoto, K Oritani, K Shimoda, T Matsuda
JOURNAL OF IMMUNOLOGY, 172, 5, 2985, 2993, AMER ASSOC IMMUNOLOGISTS, Mar. 2004, [Peer-reviewed]
English, Scientific journal, Daxx has been shown to play an essential role in type I IFN-alphabeta-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify how Daxx regulates B cell development, we examined Daxx interacting partners by yeast two-hybrid screening. DNA methyltransferase I (DNMT1)-associated protein (DMAP1) was identified and demonstrated to interact with Daxx. The interaction regions in both proteins were mapped, and the cellular localization of the interaction was examined. Both Daxx and DMAP1 formed a complex with DNMT1 and colocalized in the nucleus. DMAP1 enhanced Daxx-mediated repression of glucocorticoid receptor transcriptional activity. Furthermore, Daxx protected protein degradation of DMAP1 in vivo. These results provide the novel molecular link between Daxx and DNMT1, which establishes a repressive transcription complex in the nucleus.

Involvement of NF-kappa B in TGF-beta-mediated suppression of IL-4 signaling
T Yamamoto, S Imoto, Y Sekine, K Sugiyama, T Akimoto, A Muraguchi, T Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 313, 3, 627, 634, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jan. 2004, [Peer-reviewed]
English, Scientific journal, Control of immune response requires the coordinated integration of both stimulatory and inhibitory factors. Therefore, the cross-talk of different signaling pathways is critical in providing an integrated cellular response to multiple external signals. Both interleukin-4 (IL-4) and transforming growth factor (TGF-beta) are pleiotropic cytokines and play critical roles in controlling immune responses. For example, IL-4 mediates important pro-inflammatory functions in asthma including induction of the IgE isotype switch and expression of vascular cell adhesion molecules. Whereas, TGF-beta is secreted from B, T, and dendritic cells as well as macrophages, and negatively regulates their proliferation, differentiation, and activation by other cytokines. In this study, we examined the effect of TGF-beta on IL-4 signaling using B cells as well as embryonic kidney cells. TGF-beta inhibited IL-4-induced IgG1 production and gene expression of germline epsilon transcripts in B cells. In embryonic kidney cells, TGF-beta signals suppressed IL-4-induced transcription, when we monitored using germline epsilon promoter DNA. Furthermore, activation of NF-kappaB resulted in a resistance to TGF-beta-mediated suppression of IL-4 signaling. These results indicate that TGF-beta-mediated regulation of IL-4 signaling may act by targeting NF-kappaB signaling. (C) 2003 Elsevier Inc. All rights reserved.

Regulation of TGF-beta signaling by PIASy
S Imoto, K Sugiyama, T Yamamoto, T Matsuda
IMMUNOLOGY 2004: CYTOKINE NETWORK, REGULATORY CELLS, SIGNALING, AND APOPTOSIS, 253, 256, MEDIMOND PUBLISHING CO, 2004, [Peer-reviewed]
English, International conference proceedings, Signals by transforming growth factor-beta (TGF-beta) superfamily are negatively regulated by inhibitory Smads. Smad7 inhibits signaling by both TGF-beta and bone morphogenetic proteins. In this study, we identified a protein inhibitor of activated STAT (signal transducers and activators of transcription), PIASy, as a novel interaction partner of Smad7. Moreover, we found that PIASy also associated with other Smads including Smad3, and suppressed TGF-beta/Smad3 -mediated signaling. We also demonstrated that TGF-beta-induced endogenous PIASy expression. These findings suggest that PIASy regulates TGF-b signaling using the negative feedback loop.

Limitin, an interferon-like cytokine, transduces inhibitory signals on B-cell growth through activation of Tyk2, but not Stat1, followed by induction and nuclear translocation of Daxx
K Aoki, K Shimoda, K Oritani, T Matsuda, K Kamezaki, R Muromoto, A Numata, S Tamiya, T Haro, F Ishikawa, K Takase, T Yamamoto, T Yumioka, T Miyamoto, K Nagafuji, H Gondo, S Nagafuchi, K Nakayama, M Harada
EXPERIMENTAL HEMATOLOGY, 31, 12, 1317, 1322, ELSEVIER SCIENCE INC, Dec. 2003, [Peer-reviewed]
English, Scientific journal, Objective. Limitin, an interferon-like cytokine, suppresses B lymphopoiesis through ligation of the interferon-alpha/beta (IFN-alpha/beta) receptor. The aim of this study was to examine the intracellular signal transduction pathways activated by limitin.
Materials and Methods. The effects of limitin on cell growth, the activation of Jak kinase and Stat proteins, and the induction of interferon regulatory factor-1 (IRF-1) and Daxx were examined using the mouse pre-B-cell line 18.81, wild-type, and Tyk2-deficient mouse bone marrow cells. In addition, the change of localization of the Daxx protein after limitin treatment in wild-type and Tyk2-deficient mice was examined.
Results. Limitin phosphorylates Tyk2, Jak1, Stat1, and Stat2 and rapidly induces IRF-1 mRNA production. Phosphorylation of Stat1 by limitin is partially dependent on Tyk2. Suppression of B-cell growth by limitin, however, is severely impaired in the absence of Tyk2, whereas it is unaffected by the absence of Stat1. Limitin also induces the expression and nuclear translocation of Daxx, which is essential for IFN-alpha-induced inhibition of B-lymphocyte development. The absence of Tyk2 abrogates this induction of Daxx expression and nuclear translocation.
Conclusions. Limitin suppresses B-cell growth through activation of Tyk2, resulting in the up-regulation and nuclear translocation of Daxx. This limitin-mediated signaling pathway does not require Stat1. 2003 International Society for Experimental Hematology. Published by Elsevier Inc.

Intracellular signal transduction of interferon on the suppression of haematopoietic progenitor cell growth
K Kato, K Kamezaki, K Shimoda, A Numata, T Haro, K Aoki, F Ishikawa, K Takase, H Ariyama, T Matsuda, T Miyamoto, K Nagafuji, H Gondo, KI Nakayama, M Harada
BRITISH JOURNAL OF HAEMATOLOGY, 123, 3, 528, 535, BLACKWELL PUBLISHING LTD, Nov. 2003, [Peer-reviewed]
English, Scientific journal, Interferon (IFN)-alpha and IFN-gamma suppress the growth of haematopoietic progenitor cells. IFN-alpha activates Janus kinase-1 (Jak1) and Tyrosine kinase-2 (Tyk2), followed by the phosphorylation of the signal transducers and activators of transcription, Stat1 and Stat2. IFN-gamma activates Jak1 and Jak2, followed by the activation of Stat1. Activated Stats bind the promoter regions of IFN-inducible genes. We evaluated the role of Tyk2 and Stat1 in the IFN-mediated inhibition of haematopoietic progenitor cell growth. While IFN-alpha (1000 U/ml) suppressed the number of granulocyte-macrophage colony-forming units (CFU-GM) or erythroid burst-forming units (BFU-E) from wild-type mouse bone marrow cells, this suppression was partially inhibited by a deficiency in Tyk2 and completely inhibited by a deficiency in Stat1. High levels of IFN-alpha (10 000 U/ml) suppressed the CFU-GM or BFU-E obtained from Stat1-deficient mice, but did not suppress this growth in cells from Tyk2-deficient mice. Stat1 was phosphorylated by IFN-alpha in Tyk2-deficient cells, although the level of phosphorylation was weaker than that observed in wild type mice. Thus, the inhibitory signal on haematopoietic progenitor cells mediated by IFN-alpha may be transduced by two signalling pathways, one regulated by Tyk2 and the other dependent on Stat1. IFN-gamma also suppressed the number of CFU-GM or BFU-E, and this pathway was mediated by IFN-gamma in a Stat1-dependent manner, independently of Tyk2.

Regulation of transforming growth factor-beta signaling by protein inhibitor of activated STAT, PIASy through Smad3
S Imoto, K Sugiyama, R Muromoto, N Sato, T Yamamoto, T Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 278, 36, 34253, 34258, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Sep. 2003, [Peer-reviewed]
English, Scientific journal, Smads proteins play a key role in the intracellular signaling of the transforming growth factor (TGF)-beta family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. In particular, Smad7 acts as an antagonist of TGF-beta signaling, which could determine the intensity or duration of its signaling cascade. In this study we identified a protein inhibitor of activated STAT ( signal transducers and activators of transcription), PIASy, as a novel interaction partner of Smad7 by yeast two-hybrid screening using the MH2 domain of Smad7 as bait. The association of Smad7 and PIASy was confirmed using co-expressed tagged proteins in 293T cells. Moreover, we found that other Smads including Smad3 also associated with PIASy through its MH2 domain, and PIASy suppressed TGF-beta-mediated activation of Smad3. PIASy also stimulated the sumoylation of Smad3 in vivo. Furthermore, endogenous PIASy expression was induced by TGF-beta in Hep3B cells. These findings provide the first evidence that a PIAS family protein, PIASy, associates with Smads and involves the regulation of TGF-beta signaling using the negative feedback loop.

Regulation of Fc epsilon RI-mediated signaling by an adaptor protein STAP-2/BSK in rat basophilic leukemia RBL-2H3 cells
T Yamamoto, T Yumioka, Y Sekine, N Sato, M Minoguchi, A Yoshimura, T Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 306, 3, 767, 773, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jul. 2003, [Peer-reviewed]
English, Scientific journal, Crosslinking of multivalent antigen bound IgE transduces FcepsilonRI 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 FcepsilonRI-mediated calcium mobilization and degranulation. FcepsilonRI-induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) but not Syk was significantly suppressed in these cells. Furthermore, STAP-2/BSK associated with PLC-gamma in vivo. These data indicate that STAP-2/BSK negatively controls the FcepsilonRI-mediated calcium mobilization and degranulation by direct modulation of tyrosine phosphorylation of PLC-gamma. (C) 2003 Elsevier Science (USA). All rights reserved.

Molecular interactions between STAT3 and protein inhibitor of activated STAT3, and androgen receptor
T Yamamoto, N Sato, Y Sekine, T Yumioka, S Imoto, A Junicho, H Fuse, T Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 306, 2, 610, 615, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jun. 2003, [Peer-reviewed]
English, Scientific journal, STAT3 mainly acts as a signal transducer of IL-6 family cytokines and transcriptionally activates specific target genes. The recently discovered protein inhibitor of activated STAT3 (PIAS3) binds directly to STAT3 and blocks transcriptional activation. In our previous report, we demonstrated that PIAS3 directly interacted with androgen receptor (AR) and affected AR-mediated gene activation. Furthermore, we also showed that AR associated with STAT3 and enhanced its activity. Here, we examined molecular interactions between STAT3, PIAS3, and AR to underline the mechanism of how they regulate each other. AR activation overcame the inhibitory effect on STAT3-mediated transcription by PIAS3. Co-immunoprecipitation experiments revealed that an active form of AR relieved STAT3 from STAT3-PIAS3 complex formation. These results indicate that AR and PIAS3 regulate the STAT3-mediated transcriptional activity by their physical protein-protein competition on STAT3. (C) 2003 Elsevier Science (USA). All rights reserved.

Daxx enhances Fas-mediated apoptosis in a murine pro-B cell line, BAF3
R Muromoto, T Yamamoto, T Yumioka, Y Sekine, K Sugiyama, K Shimoda, K Oritani, T Matsuda
FEBS LETTERS, 540, 1-3, 223, 228, ELSEVIER SCIENCE BV, Apr. 2003, [Peer-reviewed]
English, Scientific journal, Daxx has been shown to play an essential in type I interferon (IFN-alpha/beta)-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and nuclear translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the mechanism of Daxx-mediated apoptosis signaling in B lymphocyte progenitors, here we introduced an efficient suicide switch in a murine pro-B cell line, BAF3, by expressing FK506-binding protein-fused Fas intracellular domain (FKBP-Fas) and Daxx. It allows us to monitor Fas/Daxx-mediated signal by induction of Fas dimerization with the dimerizer drug AP20187. AP20187-mediated Fas dimerization induced not only apoptosis but also Jun N-terminal kinase (JNK) activation. However, AP20187 had no effect on cells expressing either Fas or Daxx only. Furthermore, expression of a JNK inhibitor, the JNK-binding domain of JIP-1, resulted in resistance to AP20187-mediated apoptosis in cells expressing FKBP-Fas and Daxx. These results imply that our novel suicide switch system may provide a powerful tool to delineate or identify the signaling molecules for Daxx-mediated apoptotic machinery in B lymphocyte progenitors through JNK activation. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

STAP-2/BKS, an adaptor/docking protein, modulates STAT3 activation in acute-phase response through its YXXQ motif
M Minoguchi, S Minoguchi, D Aki, A Joo, T Yamamoto, T Yumioka, T Matsuda, A Yoshimura
JOURNAL OF BIOLOGICAL CHEMISTRY, 278, 13, 11182, 11189, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Mar. 2003, [Peer-reviewed]
English, Scientific journal, As a c-fins-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.

Involvement of heat-shock protein 90 in the interleukin-6-mediated signaling pathway through STAT3
N Sato, T Yamamoto, Y Sekine, T Yumioka, A Junicho, H Fuse, T Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 300, 4, 847, 852, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jan. 2003, [Peer-reviewed]
English, Scientific journal, Interleukin-6 (IL-6) is a multifunctional cytokine playing roles in the immune system, hematopoiesis, and acute phase reactions. IL-6 also regulates the growth of various types of human malignant tumors. Here we demonstrate that IL-6-induced gene expression was suppressed by a specific heat-shock protein 90 (Hsp90) inhibitor, geldanamycin (GA) in human hepatoma Hep3B cells. GA also suppressed the IL-6-induced activation of signal transducer and activator of transcription 3 (STAT3) in a human embryonic kidney carcinoma 293T cells. This inhibitory effect of GA on STAT3 activation was reversed by overexpression of Hsp90. Furthermore, Hsp90 directly bound to STAT3 via its N-terminal region, which interacted with GA. We provide evidence that the action of GA on IL-6 functions was due to the inhibition of direct physical interactions between STAT3 and Hsp90, which represents a novel role of Hsp90 in the IL-6 signaling pathways. (C) 2002 Elsevier Science (USA). All rights reserved.

Cutting edge: Tyk2 is required for the induction and nuclear translocation of Daxx which regulates IFN-alpha-induced suppression of B lymphocyte formation
K Shimoda, K Kamesaki, A Numata, K Aoki, T Matsuda, K Oritani, S Tamiya, K Kato, K Takase, R Imamura, T Yamamoto, T Miyamoto, K Nagafuji, H Gondo, S Nagafuchi, KI Nakayama, M Harada
JOURNAL OF IMMUNOLOGY, 169, 9, 4707, 4711, AMER ASSOC IMMUNOLOGISTS, Nov. 2002, [Peer-reviewed]
English, Scientific journal, IFN-alpha inhibits B lymphocyte development, and the nuclear protein Daxx has been reported to be essential for this biological activity. We show in this study that IFN-a inhibits the clonal proliferation of B lymphocyte progenitors in response to IL-7 in wild-type, but not in tyk2-deficient, mice. In addition, the IFN-alpha-induced up-regulation and nuclear translocation of Daxx are completely abrogated in the absence of tyk2. Therefore, tyk2 is directly involved in IFN-a signaling for the induction and translocation of Daxx, which may result in B lymphocyte growth arrest and/or apoptosis.

Mapping of the sites responsible for factor I-cofactor activity for cleavage of C3b and C4b on human C4b-binding protein (C4bp) by deletion mutagenesis
A Fukui, T Yuasa-Nakagawa, Y Murakami, K Funami, N Kishi, T Matsuda, T Fujita, T Seya, S Nagasawa
JOURNAL OF BIOCHEMISTRY, 132, 5, 719, 728, JAPANESE BIOCHEMICAL SOC, Nov. 2002, [Peer-reviewed]
English, Scientific journal, Human C4b-binding protein (C4bp) facilitates the factor I-mediated proteolytic cleavage of the active forms of complement effectors C3b and C4b into their inactive forms. C4bp comprises a disulfide-linked heptamer of alpha-chains with complement (C) regulatory activity and a beta-chain. Each alpha-chain contains 8 short consensus repeat (SCR) domains. Using SCR-deletion mutants of recombinant multimeric C4bp, we identified the domains responsible for the C3b/C4b-binding and C3b/C4b-inactivating cofactor activity. The C4bp mutant with deletion of SCR2 lost the C4b-binding ability, as judged on C3b/C4b-Sepharose binding assaying and ELISA. In contrast, the essential domains for C3b-binding extended more to the C-terminus, exceeding SCR4. Using fluid phase cofactor assaying and deletion mutants of C4bp, SCR2 and 3 were found to be indispensable for C4b cleavage by factor I, and SCR1 contributed to full expression of the factor I-mediated C4b cleaving activity. On the other hand, SCR1, 2, 3, 4, and 5 participated in the factor I-cofactor activity for C3b cleavage, and SCR2, 3, and 4 were absolutely required for C3b inactivation. Thus, different sets of SCRs participate in C3b and C4b inactivation, and the domain repertoire supporting C3b cofactor activity is broader than that supporting C4b inactivation by C4bp and factor I. Furthermore, the domains participating in C3b/C4b binding are not always identical to those responsible for cofactor activity. The necessity of the wide range of SCRs in C3b inactivation compared to C4b inactivation by C4bp and factor I may reflect the physiological properties of C4bp, which is mainly directed to C4b rather than C3b.

Lymphoid enhancer-binding factor-1 binds and activates the recombination-activating gene-2 promoter together with c-Myb and Pax-5 in immature B cells
ZX Jin, H Kishi, XC Wei, T Matsuda, S Saito, A Muraguchi
JOURNAL OF IMMUNOLOGY, 169, 7, 3783, 3792, AMER ASSOC IMMUNOLOGISTS, Oct. 2002, [Peer-reviewed]
English, Scientific journal, The recombination-activating gene (RAG)-1 and RAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of Ag receptor genes. We studied the regulation of murine RAG-2 promoter and revealed that -41/-17 RAG-2 promoter region, which was indispensable for the RAG-2 promoter activity in B cell lines, contained binding sites for lymphoid enhancer-binding factor-1 (LEF-1), c-Myb, and Pax-5. We showed that these three transcription factors bound the promoter region in vitro and in vivo. Cotransfection assays using a human embryonic kidney cell line (293T) showed that LEF-1, c-Myb, and Pax-5 cooperatively activated the RAG-2 promoter, via their synergistic DNA binding. We also showed that LEF-1, c-Myb, and Pax-5 physically interact in the cells. Finally, we demonstrated that a dominant-negative LEF-1 protein, which lacks the binding site for beta-catenin, suppressed the RAG-2 promoter activity as well as the endogenous RAG-2 expression in a pre-B cell line (18.81). These results suggest that LEF-1/beta-catenin complex regulates the RAG-2 promoter activation in concert with c-Myb and Pax-5 in immature B cells. The link between LEF-1/-beta-catenin and Wnt signaling in B lineage cells will be discussed.

The nuclear isoform of protein-tyrosine phosphatase TC-PTP regulates interleukin-6-mediated signaling pathway through STAT3 dephosphorylation
T Yamamoto, Y Sekine, K Kashima, A Kubota, N Sato, N Aoki, T Matsuda
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 297, 4, 811, 817, ACADEMIC PRESS INC ELSEVIER SCIENCE, Oct. 2002, [Peer-reviewed]
English, Scientific journal, In the previous study, we demonstrated that the nuclear isoform of T-cell protein-tyrosine phosphatase (TC-PTP) dephosphorylated and deactivated signal transducer and activator of transcription 5a (STAT5a) and STAT5b, thereby negatively regulating prolactin (PRL)-mediated signaling pathway. In this study, we examined the involvement of the nuclear isoform of TC-PTP in interleukin-6 (IL-6)-mediated signaling pathway. IL-6 is a multifunctional cytokine that plays important roles in the immune system, hematopoiesis, and acute phase reactions, and has also implicated in IL-6-related diseases. Here, we demonstrate that IL-6-induced tyro sine-phosphorylation and activation of STAT3 were suppressed by overexpression of the nuclear isoform of TC-PTP in 293T cells. Tyrosine-phosphorylated STAT3 directly interacted with a substrate-trapping mutant of TC-PTP, Furthermore, retrovirus-mediated overexpression of the nuclear isoform of TC-PTP suppressed the IL-6-induced growth arrest of myeloid leukemia M1 cells. Endogenous TC-PTP complexed with STAT3 in the nucleus of M1 cells. These results strongly suggest that the nuclear isoform of TC-PTP may serve as a negative regulator of IL-6-mediated signaling pathway. (C) 2002 Elsevier Science (USA). All rights reserved.

Cross-talk between bone morphogenic proteins and estrogen receptor signaling
T Yamamoto, F Saatcioglu, T Matsuda
ENDOCRINOLOGY, 143, 7, 2635, 2642, ENDOCRINE SOC, Jul. 2002, [Peer-reviewed]
English, Scientific journal, Bone morphogenic proteins (BMPs) play central roles in differentiation, development, and physiological tissue remodeling. Estrogens have key roles in a variety of biological events, such as the development and maintenance of numerous target tissues. Previous studies demonstrated that estrogens suppress BMP functions by repressing BMP gene expression. Here we present a novel mechanism for the inhibitory effect of estrogens on BMP function. BMP-2-induced activation of Sma and Mad (mothers against decapentaplegic)-related protein (Smad) activity and BMP-2-mediated gene expression were suppressed by 17beta-E2 in breast cancer cells and mesangial cells. E2-mediated inhibition of Smad activation was reversed by tamoxifen, an ER antagonist. We provide evidence that the inhibitory action of ER on Smad activity was due to direct physical interactions between Smads and ER, which represents a novel mechanism for the cross-talk between BMP and ER signaling pathways.

Characterization of chromatin structure and enhancer elements for murine recombination activating gene-2
XC Wei, H Kishi, ZX Jin, WP Zhao, S Kondo, T Matsuda, S Saito, A Muraguchi
JOURNAL OF IMMUNOLOGY, 169, 2, 873, 881, AMER ASSOC IMMUNOLOGISTS, Jul. 2002, [Peer-reviewed]
English, Scientific journal, Recombination-activating genes (RAGs) play a critical role in V(D)J recombination machinery and their expression is specifically regulated during lymphocyte ontogeny. To elucidate the molecular mechanisms regulating murine RAG-2 expression, we examined a chromatin structure of 25-kb DNA segment adjacent to murine RAG-2 by analyzing DNase I hypersensitive (HS) sites. In a RAG-2-expressing murine pre-B cell line, three lymphoid cell-specific HS sites (HS1, HS2, and HS3) were identified. Among these HS sites, one HS site (HS3) that locates in the RAG-2 promoter was associated only with RAG-2-expressing cell lines. Using the transient enhanced green fluorescence protein reporter gene assays, we identified two enhancer elements in the 5'-upstream region of RAG-2 that corresponded to HS1 and HS2. One of the enhancer elements (D3) exhibited enhancer activity only in the lymphoid cell lines. Analysis of the transgenic mice carrying the enhanced green fluorescence protein-reporter gene linked with D3 revealed that D3 activated the reporter gene-expression in the primary lymphoid tissues, but not in the secondary lymphoid tissues or nonlymphoid tissues. D3 was active in CD4(-)CD8(-), but not in CD4(+)CD8(+) or CD4(+)CD8(-) thymocytes in the thymus, and also active in B220(+)IgM(-), but not in B220(+)IgM(+), cells in the bone marrow. Finally, our data suggested that C/EBP may bind to the D3 enhancer and function as one of the transcription factor(s) responsible for the enhancer activity. These results show that the tissue- and stage-specific expression of murine RAG-2 is regulated by alteration of the chromatin structure as well as cis-regulatory enhancer elements.

Partial impairment of interleukin-12 (IL-12) and IL-18 signaling in Tyk2-deficient mice
K Shimoda, H Tsutsui, K Aoki, K Kato, T Matsuda, A Numata, K Takase, T Yamamoto, H Nukina, T Hoshino, Y Asano, H Gondo, T Okamura, S Okamura, KI Nakayama, K Nakanishi, Y Niho, M Harada
BLOOD, 99, 6, 2094, 2099, AMER SOC HEMATOLOGY, Mar. 2002, [Peer-reviewed]
English, Scientific journal, Tyk2 is activated in response to interleukin-12 (IL-12) and is essential for IL-12-induced T-cell function, including interferon-gamma (IFN-gamma) production and Th1 cell differentiation. Because IL-12 is a stimulatory factor for natural killer (NK) cell-mediated cytotoxicity, we examined whether tyk2 is required for IL-12-induced NK cell activity. IL-12-induced NK cell activity in cells from tyk2-deficient mice was drastically reduced compared to that in cells from wild-type mice. IL-18 shares its biologic functions with IL-12. However, the molecular mechanism of IL-18 signaling, which activates an IL-1 receptor-associated kinase and nuclear translocation of nuclear factor-kappaB, is different from that of IL-12. We next examined whether biologic functions induced by IL-18 are affected by the absence of tyk2. NK cell activity and IFN-gamma production induced by IL-18 were reduced by the absence of tyk2. Moreover, the synergistic effect of IL-12 and IL-18 for the production of IFN-gamma was also abrogated by the absence of tyk2. This was partially due to the absence of any up-regulation of the IL-18 receptor treated with IL-12, and it might suggest the presence of the cross-talk between Jak-Stat and mitogen-activated protein kinase pathways In cytokine signaling.

Vasoactive intestinal peptide (VIP) enhances the cell motility of androgen receptor-transfected DU-145 prostate cancer cells (DU-145/AR)
O Nagakawa, J Murata, A Junicho, T Matsuda, Y Fujiuchi, H Fuse, Saiki, I
CANCER LETTERS, 176, 1, 93, 99, ELSEVIER SCI IRELAND LTD, Feb. 2002, [Peer-reviewed]
English, Scientific journal, We established a clonal DU-145 prostate cancer cell line (DU-145/AR) stably transfected with androgen receptor (AR) cDNA and investigated the expression of type I vasoactive intestinal peptide (VIP) receptor (VIP1R) and type 2 VIP receptor (VIP2R) mRNA in these cells by reverse transcriptase-polymerase chain reaction analysis and the effect of VIP on the invasion and the haptotactic migration of these cells. DU-145/AR cells constitutively expressed both VIP1R and VIP2R mRNA, but the parent DU-145 cells did not. VIP increased the invasive capacity of DU-145/AR cells. VIP also enhanced the haptotactic migration of these cells to fibronectin. However, the growth of these tumor cells was not affected by VIP at any concentrations used in this study. These results indicate that VIP may play a role in the regulation of the invasion of prostate cancer. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Cooperative binding of c-Myb and Pax-5 activates the RAG-2 promoter in immature B cells
H Kishi, ZX Jin, XC Wei, T Nagata, T Matsuda, S Saito, A Muraguchi
BLOOD, 99, 2, 576, 583, AMER SOC HEMATOLOGY, Jan. 2002, [Peer-reviewed]
English, Scientific journal, The recombination activating gene-1 (RAG-1)and RAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of antigen receptor genes. The regulation of murine RAG-2 promoter was studied and it was revealed that the -41/-17 RAG-2 promoter region, which is conserved between humans and mice, was indispensable for the RAG-2 promoter activity in B-cell lines. The region contained 2 cis elements that bound c-Myb and Pax-5. Mutation in the c-Myb-binding site in the promoter reduced the promoter activity in B-cell lines. Cooperative activation of the RAG-2 promoter was seen by a combination of c-Myb and Pax-5 in a human embryonic kidney cell line (293T), via their synergistic DNA-binding. Deletion experiments showed that the C-terminus of c-Myb was responsible for their interaction. Furthermore, the dominant-negative c-Myb mutant suppressed the activation of the RAG-2 promoter in a pre-B-cell line as well as In 293T cells. These results suggest that cooperative binding of c-Myb and Pax-5 to the RAG-2 promoter Is one of the mechanisms to direct the restricted expression of the RAG-2 In Immature B cells. (Blood. 2002;99:576-583) (C) 2002 by The American Society of Hematology.

Cross-talk between transforming growth factor-beta and estrogen receptor signaling through Smad3
T Matsuda, T Yamamoto, A Muraguchi, F Saatcioglu
JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 46, 42908, 42914, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Nov. 2001, [Peer-reviewed], ［Internationally co-authored］
English, Scientific journal, Transforming growth factor-beta (TGF-beta) plays central roles in embryonic development, organogenesis, and physiologic connective tissue remodeling during wound healing and tissue repair as well as in carcinogenesis. Estrogens have key roles in a variety of biological events, such as the development and maintenance of female reproductive organs and bone and lipid metabolism. Previous studies demonstrated that estrogens suppress TGF-beta -induced gene expression, such as type IV collagen in kidney mesangial cells. However, the molecular mechanisms that mediate this antagonistic effect are unknown. To elucidate the mechanisms of cross-talk between TGF-beta and estrogen receptor (ER) signaling pathways, we reconstituted TGF-beta and ER signaling in human kidney carcinoma cells. Here we demonstrate that TGF-beta -induced activation of Sma and MAD-related protein 3 (Smad3) activity, one of the major intracellular transducers of TGF-beta signaling, was suppressed by ER, whereas ER-mediated transcriptional activation was enhanced by TGF-beta signaling. We provide evidence that this two-way cross-talk between the estrogen and TGF-beta signaling pathways was the result of direct physical interactions between Smad3 and ER. These findings have implications for a variety of disease states, such as the pathophysiology of kidney function, atherosclerosis, and breast cancer.

The role of interleukin 6 in plasmacytomagenesis.
T. Hirano, S. Suematsu, T. Matsusaka, T. Matsuda, T. Kishimoto
Ciba Foundation symposium, 167, 188, 196200, 1992
English, Scientific journal, Interleukin 6 (IL-6) is a polyfunctional cytokine which regulates the immune response, the acute-phase reaction and haemopoiesis. IL-6 plays a critical role in differentiation of B cells into plasma cells, and is a potent growth factor for plasmacytomas and myelomas. A relationship between IL-6 and polyclonal plasma cell abnormalities has been demonstrated. Abnormal production of IL-6 was first suggested to be related to hypergammaglobulinaemia with autoantibody production in patients with cardiac myxoma. A role of IL-6 in the generation of plasmacytoma has also been indicated. In support of these clinical and experimental observations, we demonstrated that transgenic C57BL/6 mice carrying the human IL-6 gene showed a massive polyclonal plasmacytosis with production of autoantibodies. However, the tumour was not transplantable to syngeneic animals. Susceptibility to pristane-induced plasmacytomagenesis is genetically determined--pristane can induce plasmacytomas in BALB/c but not in C57BL/6 mice. IL-6 transgenic C57BL/6 mice were backcrossed to BALB/c mice to elucidate the genetic influence on plasmacytomagenesis. Transplantable monoclonal plasmacytoma with a t(12
15) chromosomal translocation was generated in some of the backcrossed mice, indicating that IL-6 plays a key role in the multistep oncogenesis of plasma cell neoplasia.

INTERLEUKIN-6 (IL-6) AND ITS RECEPTOR (IL-6R) IN MYELOMA PLASMACYTOMA
S SUEMATSU, M HIBI, T SUGITA, M SAITO, M MURAKAMI, T MATSUSAKA, T MATSUDA, T HIRANO, T TAGA, T KISHIMOTO
MECHANISMS IN B-CELL NEOPLASIA 1990, 13, 22, SPRINGER-VERLAG BERLIN, 1990, [Peer-reviewed]
English, International conference proceedings

IL-6 BSF2 IN NORMAL AND ABNORMAL REGULATION OF IMMUNE-RESPONSES
T MATSUDA, S SUEMATSU, M KAWANO, K YOSHIZAKI, B TANG, O TANABE, T NAKAJIMA, S AKIRA, T HIRANO, T KISHIMOTO
REGULATION OF THE ACUTE PHASE AND IMMUNE RESPONSES : INTERLEUKIN-6, 557, 466, 477, NEW YORK ACAD SCIENCES, 1989, [Peer-reviewed]
English, International conference proceedings

MOLECULAR-CLONING OF THE CDNAS FOR INTERLEUKIN-6 B-CELL STIMULATORY FACTOR-II AND ITS RECEPTOR
T HIRANO, T TAGA, K YAMASAKI, T MATSUDA, K YASUKAWA, Y HIRATA, H YAWATA, O TANABE, S AKIRA, T KISHIMOTO
REGULATION OF THE ACUTE PHASE AND IMMUNE RESPONSES : INTERLEUKIN-6, 557, 167, 180, NEW YORK ACAD SCIENCES, 1989, [Peer-reviewed]
English, International conference proceedings

INTERLEUKIN-6 AND ITS RECEPTOR IN IMMUNE REGULATION
T KISHIMOTO, T TAGA, T MATSUDA, M HIBI, S SUEMATSU, B TANG, H YAWATA, Y HIRATA, K YAMASAKI, T HIRANO
PROGRESS IN IMMUNOLOGY, VOL 7, 633, 640, SPRINGER-VERLAG BERLIN, 1989, [Peer-reviewed]
English, International conference proceedings

Pathogenic significance of BSF-2/IL-6 in castleman's disease
K. Yoshizaki, T. Matsuda, N. Nishimoto, T. Kuritani, L. Taeho, K. Aozasa, T. Nakahata, H. Tagoh, T. Komori, S. Kishimoto, T. Hirano, T. Kishimoto
International Journal of Immunopharmacology, 10, 1, 13, 1988, [Peer-reviewed]
English, International conference proceedings

B cell stimulatory factor 2(BSF2/IL-6) and rheumatoid arthritis.
HIRANO Toshio, MATSUDA Tadashi, TURNER Martin, FELDMANN Marc, TANG Bo, MIYASAKA Nobuyuki, SHIMIZU Masatoshi, KISHIMOTO Tadamitsu
Proceedings of the Japan Academy, 63, 7, 281, 283, The Japan Academy, 1987
English

Analysis of the relationship between protein NEDDylation mechanism and Regnase-1 activity
YU Tingchi, 小森雄喜, 室本竜太, 松田正, 日本免疫毒性学会学術年会講演要旨集, 30th, 2023

Effect of Benzo[a]pyrene on IL-17 mediated signaling
成家康太, 室本竜太, 松田正, 衛生薬学・環境トキシコロジー講演要旨集, 2023, 2023

Role of Tyk2 in immune and inflammatory responses
室本竜太, 松田正, 日本薬学会年会要旨集(Web), 143rd, 2023

Neddylation inhibition inactivates Regnase-1 via MALT1-mediated cleavage
YU Tingchi, 小森雄喜, 室本竜太, 松田正, 日本薬学会年会要旨集(Web), 143rd, 2023

Upregulated Glo1 gene expression in Tyk2-deficient macrophages
阿部丈一朗, 宮下清隆, 美濃口広弥, 室本竜太, 松田正, 日本薬学会年会要旨集(Web), 143rd, 2023

Role of Tyk2 in LPS-induced acute lung injury model in mice
金光聡馬, 大久保優菜, 室本竜太, 松田正, 日本薬学会年会要旨集(Web), 143rd, 2023

X線小角散乱(SEC-SAXS)による,Breast tumor kinaseの活性化機構の解明
宇賀神魁, 武井梓穂, 松田正, 尾瀬農之, 日本蛋白質科学会年会プログラム・要旨集, 23rd (CD-ROM), 2023

【IL-6阻害療法の基礎と臨床】IL-6とそのシグナル伝達系の分子基盤　　　　　　　　　　　　　　　
松田 正, 室本 竜太, 鍛代 悠一, 織谷 健司, リウマチ科, 69, 1, 1, 12, Jan. 2023
(有)科学評論社, Japanese

Benzo[a]pyrene enhances IL-17-mediated responses by suppressing expression of Regnase-1 through AhR
成家康太, 室本竜太, 松田正, 日本薬学会年会要旨集(Web), 142nd, 2022

抗炎症脂質レゾルビンE2の1,4,7-トリエン-ベンゼン置換による安定等価体の創製研究
田中伸英, 村上侑斗, 平島洸基, 福田隼, 藤原広一, 渡邉瑞貴, 室本竜太, 松田正, 周東智, 反応と合成の進歩シンポジウム講演要旨集, 48th, 2022

Benzo[a]pyrene enhances IL-17 response via AhR-mediated Regnase-1 suppression
室本竜太, 成家康太, 斎野由佳, 松田正, 日本免疫毒性学会学術年会講演要旨集, 28th, 2021

STAT1 negatively regulates IκB-ζ gene transcription
室本竜太, 佐藤亜美, 松田正, 日本薬学会年会要旨集(Web), 141st, 2021

Role of the adaptor molecule, Signal-transducing adaptor protein-2, for regulatory mechanisms of allergic responses
柏倉淳一, 松田正, 月刊アレルギーの臨床, 553, 2021

Tyk2欠損マウスにおける免疫反応抑制にはマクロファージでのIL-10産生増強が関与する　　　　　　　　　　　　　　　
室本 竜太, 平島 洸基, 美濃口 広弥, 松田 正, 日本薬学会年会要旨集, 140年会, 26Z, am13, Mar. 2020
(公社)日本薬学会, Japanese

Signal Transducing Adaptor Protein(STAP)-2はGVHD増悪に関与する
西東秀晃, 一井倫子, 戸田淳, 鍛代悠一, 室本竜太, 柏倉淳一, 斎藤宏大, 横田貴史, 柴山浩彦, 松田正, 織谷健司, 金倉譲, 金倉譲, 保仙直毅, 日本血液学会学術集会抄録(Web), 82nd, 2020

炎症収束脂質レゾルビンE2のスキップジエン構造をベンゼン環で置換した安定等価体創製研究　　　　　　　　　　　　　　　
村上 侑斗, 福田 隼, 石村 航平, 平島 洸基, 室本 竜太, 渡邉 瑞貴, 松田 正, 周東 智, 日本薬学会年会要旨集, 139年会, 2, 96, 96, Mar. 2019
(公社)日本薬学会, Japanese

IL-17誘導性IκB-ζmRNA安定化へのTBK1の関与　　　　　　　　　　　　　　　
斎野 由佳, 大垣内 優衣, 室本 竜太, 松田 正, 日本薬学会年会要旨集, 139年会, 3, 64, 64, Mar. 2019
(公社)日本薬学会, Japanese

トポテカンの新規標的である60SリボソームタンパクRPLAはDAMPsの放出およびがん免疫の活性化に関与する
山田駿介, 鍛代悠一, 室本竜太, 柏倉淳一, 松田正, 日本分子生物学会年会プログラム・要旨集(Web), 42nd, 2019

Role of Signal Transducing Adaptor Protein-1 (STAP-1) in Chronic Myelogenous Leukemia Stem Cells
Jun Toda, Michiko Ichii, Hirohiko Shibayama, Hideaki Saito, Yuichi Kitai, Ryuta Muromoto, Jun-ichi Kashiwakura, Kodai Saitoh, Tadashi Matsuda, Kenji Oritani, Yuzuru Kanakura, BLOOD, 132, Nov. 2018
0, AMER SOC HEMATOLOGY, English, Summary international conference

角化細胞におけるIL‐17誘導性IκB‐ζ発現機構の解析
室本竜太, 大垣内優衣, 斎野由佳, 佐藤亜美, 多和佳佑, 松田正, 日本免疫毒性学会学術年会講演要旨集, 25th, 66, Aug. 2018
Japanese

新規α4インテグリンスプライシングバリアントは内在性α4インテグリン抑制分子である
重政歩美, 乾恭輔, 本田真知子, 松田正, 今重之, 日本薬学会年会要旨集(CD-ROM), 138th, 2018

自己免疫疾患における細胞外基質ネフロネクチンの関与
藤岡和征, 本田真知子, 松田正, 今重之, 日本薬学会年会要旨集(CD-ROM), 138th, 2018

細胞外基質ネフロネクチンに対するサンドイッチELISAシステムの構築
本田真知子, 李順姫, 松田正, 大槻剛巳, 今重之, 日本薬学会年会要旨集(CD-ROM), 138th, 2018

Role of Signal Transducing Adaptor Protein (STAP) Family in Chronic Myelogenous Leukemia
Jun Toda, Michiko Ichii, Hirohiko Shibayama, Hideaki Saito, Yuichi Kitai, Ryuta Muromoto, Jun-ichi Kashiwakura, Kodai Saitoh, Tadashi Matsuda, Kenji Oritani, Yuzuru Kanakura, BLOOD, 130, Dec. 2017
0, AMER SOC HEMATOLOGY, English, Summary international conference

TYK2-mediated basal STAT3 activity and IL-17-induced mRNA stabilization coordinately dictate the expression level of I kappa B-zeta in keratinocytes
Ryuta Muromoto, Keisuke Tawa, Yui Ohgakiuchi, Tadashi Matsuda, CYTOKINE, 100, 90, 91, Dec. 2017
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, English, Summary international conference

P.acnes誘導性炎症・免疫応答の解析
松田正, 平島洸基, 齋藤浩大, 鍛代悠一, 柏倉淳一, 室本竜太, 微生物シンポジウム講演要旨集, 29th, 60‐61, 29 Aug. 2017
Japanese

Biochanin Aによる核内受容体RORγ活性化機構の解析
室本竜太, 高橋美妃, 小島弘幸, 武内伸治, 松田正, 衛生薬学・環境トキシコロジー講演要旨集, 2017, 237, 18 Aug. 2017
Japanese

JAKファミリーキナーゼ阻害が角化細胞のIL‐17応答に及ぼす影響
室本竜太, 多和佳佑, 大垣内優衣, 松田正, 日本免疫毒性学会学術年会講演要旨集, 24th, 70, Aug. 2017
Japanese

SIGNAL TRANSDUCING ADAPTOR PROTEIN-1 (STAP-1) MAINTAINS CHRONIC MYELOID LEUKEMIC STEM CELLS
J. Toda, M. Ichii, K. Oritani, H. Saito, Y. Kitai, R. Muromoto, J. -I. Kashiwakura, K. Saitoh, T. Matsuda, Y. Kanakura, HAEMATOLOGICA, 102, 231, 231, Jun. 2017
FERRATA STORTI FOUNDATION, English, Summary international conference

炎症収束性脂質レゾルビンE1配座制限誘導体の設計と合成
石村航平, 福田隼, 室本竜太, 平島洸基, 渡邉瑞貴, 松田正, 周東智, 次世代を担う有機化学シンポジウム講演要旨集, 15th, 26‐27, 19 May 2017
Japanese

STAP-2によるT細胞活性化制御機構の解析
齋藤浩大, 今重之, 柏倉淳一, 室本竜太, 鍛代悠一, 関根勇一, 吉村昭彦, 織谷健司, 織谷健司, 松田正, 次世代を担う若手ファーマ・バイオフォーラム講演要旨集, 16th, 2017

Concanavalin-A誘導性肝炎におけるSTAP-1の機能解析
伊原建, 齋藤浩大, 柏倉淳一, 今重之, 室本竜太, 鍛代悠一, 松田正, 次世代を担う若手ファーマ・バイオフォーラム講演要旨集, 16th, 2017

シクロプロパン構造特性に基づくレゾルビンE2の安定等価体の創製研究
松原光太郎, 池田紘之, 石村航平, 平島洸基, 室本竜太, 松田正, 福田隼, 渡邉瑞貴, 周東智, 日本薬学会年会要旨集(CD-ROM), 137th, ROMBUNNO.27R‐am10S, 2017
Japanese

トポテカンによる細胞死を介したDNA内包エクソソームの放出とSTING依存的がん免疫誘導機構の解明
鍛代悠一, 鍛代悠一, 川崎拓実, 末吉拓也, 小檜山康司, 石井健, 雛健, 審良静男, 松田正, 河合太郎, 日本薬学会年会要旨集(CD-ROM), 137th, ROMBUNNO.26PB‐pm021, 2017
Japanese

IL‐17誘導性IκB‐ζ発現におけるTyk2‐STAT3経路の役割
室本竜太, 多和佳佑, 松田正, 日本薬学会年会要旨集(CD-ROM), 137th, ROMBUNNO.26PB‐am058, 2017
Japanese

内因性炎症収束脂質レゾルビン類の創薬化学研究
池田紘之, 福田隼, 高倉夕季, 石村航平, 平島洸基, 平尾徹, 室本竜太, 渡邉瑞貴, 松田正, 周東智, メディシナルケミストリーシンポジウム講演要旨集, 34th, 143, 11 Nov. 2016
Japanese

Dupuytren拘縮における線維化メカニズムの解析
松井雄一郎, 今重之, 船越忠直, 河村太介, 亀田裕亮, 宮下友恵, 松田正, 岩崎倫政, 北海道整形災害外科学会, 130th, 2016

Dupuytren拘縮の進行におけるインテグリンαvの関与
松井雄一郎, 今重之, 船越忠直, 河村太介, 亀田裕亮, 宮下友恵, 松田正, 岩崎倫政, 日本整形外科学会雑誌, 90, 2, 2016

T細胞活性化及び免疫応答におけるSTAP-2の機能解析
齋藤浩大, 今重之, 今重之, 小澤清貴, 伊原建, 関根勇一, 室本竜太, 鍛代悠一, 吉村昭彦, 織谷健司, 松田正, 日本分子生物学会年会プログラム・要旨集(Web), 39th, 2016

α9インテグリンの新規リガンドXCL1/Lymphotactinは自己免疫疾患増悪化に関与する
松本尚樹, 中鶴拓也, 宮下友惠, 乾恭輔, 蝦名佳貴, 今重之, 今重之, 松田正, 日本分子生物学会年会プログラム・要旨集(Web), 39th, 2016

悪性黒色腫の発症•進行•転移メカニズム　　　　　　　　　　　　　　　
松田 正, 関根勇一, 鍛代悠一, 織谷健司, 次世代のがん治療薬・診断のための研究開発, 3, 10, 2016
Japanese

自然免疫による癌細胞DNAの認識と抗腫瘍免疫発動機構の解析
鍛代悠一, 雛健, 川崎拓実, 末吉拓也, 小檜山康司, 石井健, 審良静男, 松田正, 河合太郎, 日本Cell Death学会学術集会プログラム抄録集, 25th, 64, 2016
Japanese

Arl5BによるMDA5依存的な抗ウイルス自然免疫応答の抑制機構の解明
鍛代悠一, 織大祐, 末吉拓也, 川崎拓実, 竹内理, 松田正, 審良静男, 河合太郎, 日本薬学会年会要旨集(CD-ROM), 136th, ROMBUNNO.29AB‐AM249, 2016
Japanese

ケラチノサイトのIL‐17応答におけるTYK2による間接的制御
室本竜太, 多和佳佑, 松田正, 日本薬学会年会要旨集(CD-ROM), 136th, ROMBUNNO.29AB‐AM142, 2016
Japanese

炎症収束性脂質・レゾルビンE2の安定等価体の創製
石村航平, 福田隼, 高倉夕季, 金田龍太郎, 平尾徹, 平島洸基, 室本竜太, 松田正, 阿部洋, 周東智, メディシナルケミストリーシンポジウム講演要旨集, 33rd, 78, 01 Nov. 2015
Japanese

内因性炎症収束脂質レゾルビンE2安定等価体の創製
福田隼, 高倉夕季, 石村航平, 金田龍太郎, 平尾徹, 平島洸基, 室本竜太, 松田正, 阿部洋, 有澤光弘, 周東智, 反応と合成の進歩シンポジウム講演要旨集, 41st, 37, 09 Oct. 2015
Japanese

STAT3活性化を介したIL‐17A遺伝子発現に及ぼすバイオカニンAの影響
KOJIMA HIROYUKI, MUROMOTO RYUTA, TAKAHASHI MIKI, HIRAO TOORU, MATSUDA TADASHI, 日本免疫毒性学会学術年会講演要旨集, 22nd, 80, Aug. 2015
Japanese

細胞表面へのホスホセリン提示可能なホスファチジルセリン二量体の設計と合成
佐藤耀, 川村周平, 平尾徹, 室本竜太, 福田隼, 阿部洋, 松田正, 周東智, 万有生命科学振興国際交流財団札幌シンポジウム, 27th, 42, 04 Jul. 2015
Japanese

Dupuytren拘縮におけるインテグリンの関与
松井雄一郎, 今重之, 船越忠直, 瓜田淳, 河村太介, 佃幸憲, 松田正, 岩崎倫政, 日本整形外科学会雑誌, 89, 2, 2015

α4インテグリンスプライシングバリアントによるインテグリン機能調節機構の解明と創薬への発展
今重之, 紅露ひとみ, 中鶴拓也, 宮下友惠, 松本尚樹, 松田正, 日本薬学会年会要旨集(CD-ROM), 135th, 2015

T細胞受容体シグナルにおけるSTAP-1及びSTAP-2の役割
小澤清貴, 齋藤浩大, 安次富大, 関根勇一, 室本竜太, 今重之, 松田正, 日本薬学会年会要旨集(CD-ROM), 135th, 2015

新規α9インテグリンリガンドLymphotactinの同定と機能解析
中鶴拓也, 松本尚樹, 紅露ひとみ, 松田正, 今重之, 日本薬学会年会要旨集(CD-ROM), 135th, 2015

Dupuytren拘縮におけるインテグリンの関与
松井雄一郎, 船越忠直, 瓜田淳, 河村太介, 佃幸憲, 岩崎倫政, 今重之, 松田正, 北海道整形災害外科学会, 128th, 2015

α9インテグリンバリアントSFα9結合分子に着目した新規自己免疫疾患治療薬の開発
松本尚樹, 中鶴拓也, 宮下友惠, 今重之, 松田正, 日本生化学会大会(Web), 88th, 2015

自己免疫疾患における新規カルシウム結合タンパク質,ネフロネクチンの役割
中鶴拓也, 今重之, 宮下友惠, 松本尚樹, 乾恭輔, 石川清, 瀬川辰也, 前田雅弘, 松田正, 日本生化学会大会(Web), 88th, 2015

β8インテグリンスプライシングバリアントは新規TGF-βシグナル制御分子である
宮下友惠, 今重之, 中鶴拓也, 松本尚樹, 松井雄一郎, 岩崎倫政, 松田正, 日本生化学会大会(Web), 88th, 2015

T細胞活性化におけるSTAP-1及びSTAP-2の役割
齋藤浩大, 今重之, 小澤清貴, 伊原建, 関根勇一, 室本竜太, 鍛代悠一, 吉村昭彦, 織谷健司, 松田正, 日本生化学会大会(Web), 88th, 2015

IL−６とがん IL-6から見える景色　　　　　　　　　　　　　　　
松田 正, Keynote R•A 先端医学社, 3, 3, 2015
Japanese, Introduction scientific journal

ファゴサイトーシス誘導性ホスファチジルセリン二量体類縁化合物の創製研究
SATO YO, KAWAMURA SHUHEI, HIRAO TOORU, MUROMOTO RYUTA, FUKUDA HAYATO, ABE HIROSHI, ABE HIROSHI, MATSUDA TADASHI, SHUTO SATOSHI, 日本薬学会年会要旨集(CD-ROM), 135th, ROMBUNNO.28T-AM12S, 2015
Japanese

ケラチノサイトのIL‐17応答調節におけるJAKファミリーチロシンキナーゼの役割
TAWA KEISUKE, MUROMOTO RYUTA, MATSUDA TADASHI, 日本薬学会年会要旨集(CD-ROM), 135th, ROMBUNNO.28PB-PM107, 2015
Japanese

マクロファージの脂質代謝系制御におけるTyrosine kinase2(TYK2)の関与
HIRASHIMA KOKI, MUROMOTO RYUTA, MATSUDA TADASHI, 日本薬学会年会要旨集(CD-ROM), 135th, ROMBUNNO.27W-PM10, 2015
Japanese

The Role of Signal-Transducing Adaptor Protein-2 in Early T Lymphopoiesis in Thymus
Michiko Ichii, Natsuko Fujita, Daisuke Okuzaki, Tetsuo Maeda, Yuichi Sekine, Shigeyuki Kon, Ryuta Muromoto, Kenji Oritani, Tadashi Matsuda, Yuzuru Kanakura, BLOOD, 124, 21, Dec. 2014
AMER SOC HEMATOLOGY, English, Summary international conference

REGULATION OF B LYMPHOPOIESIS BY SIGNAL-TRANSDUCING ADAPTOR PROTEIN-2, STAP-2
M. Ichii, K. Oritani, N. Fujita, N. Saitoh, Y. Sekine, R. Muromoto, S. Kon, K. Saitoh, T. Matsuda, Y. Kanakura, HAEMATOLOGICA, 99, 175, 175, Jun. 2014
FERRATA STORTI FOUNDATION, English, Summary international conference

Y14 positively regulates TNF-alpha induced NF-kappa B transcriptional activity via interacting RIP1 and TRADD beyond an exon junction complex protein
Tadashi Matsuda, Sumihito Togi, Shigeyuki Kon, Yuichi Sekine, Ryuta Muromoto, FASEB JOURNAL, 28, 1, Apr. 2014
FEDERATION AMER SOC EXP BIOL, English, Summary international conference

乾癬の発症とTYK2
ISHIZAKI MASAYUKI, MUROMOTO RYUTA, KON SHIGEYUKI, ORITANI KENJI, MATSUDA TADASHI, 月刊臨床免疫・アレルギー科, 61, 2, 214, 220, 25 Feb. 2014
科学評論社, Japanese

Dupuytren拘縮におけるインテグリンの関与
松井雄一郎, 今重之, 船越忠直, 瓜田淳, 河村太介, 佃幸憲, 松田正, 岩崎倫政, 日本整形外科学会雑誌, 88, 8, 2014

Dupuytren拘縮におけるインテグリンの関与
松井雄一郎, 今重之, 船越忠直, 瓜田淳, 河村太介, 佃幸憲, 松田正, 岩崎倫政, 日本関節病学会誌, 33, 3, 2014

上皮がんにおけるBRKとSTAP-2の機能解析
岩上昌史, 久保果央莉, 室本竜太, 今重之, 松田正, 日本分子生物学会年会プログラム・要旨集(Web), 37th, 2014

STAP-2の翻訳後修飾によるT細胞活性化調節機構の解析
安次富大, 今重之, 齋藤浩大, 関根勇一, 室本竜太, 織谷健司, 松田正, 日本分子生物学会年会プログラム・要旨集(Web), 37th, 2014

白血病細胞増殖におけるアダプター分子STAP-2制御性ケモカインレセプターの機能解析
久保果央莉, 岩上昌史, 室本竜太, 今重之, 関根勇一, 松田正, 日本分子生物学会年会プログラム・要旨集(Web), 37th, 2014

A novel cryptic binding motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved osteopontin as a novel ligand for α9β1 integrin is involved in the anti-type II collagen antibody-induced arthritis.
Shigeyuki Kon, Yosuke Nakayama, Naoki Matsumoto, Koyu Ito, Masashi Kanayama, Chiemi Kimura, Hitomi Kouro, Dai Ashitomi, Tadashi Matsuda, Toshimitsu Uede, PloS one, 9, 12, e116210, 2014, [International Magazine]
Osteopontin (OPN) is a multifunctional protein that has been linked to various intractable inflammatory diseases. One way by which OPN induces inflammation is the production of various functional fragments by enzyme cleavage. It has been well appreciated that OPN is cleaved by thrombin, and/or matrix metalloproteinase-3 and -7 (MMP-3/7). Although the function of thrombin-cleaved OPN is well characterized, little is known about the function of MMP-3/7-cleaved OPN. In this study, we found a novel motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved mouse OPN binds to α9β1 integrin. Importantly, this novel motif is involved in the development of anti-type II collagen antibody-induced arthritis (CAIA). This study provides the first in vitro and in vivo evidence that OPN cleavage by MMP-3/7 is an important regulatory mechanism for CAIA., English

イソフラボン類によるSTAT3とRORγを介したIL‐17産生増強作用
小島弘幸, 室本竜太, 高橋美妃, 武内伸治, 松田正, 日本免疫毒性学会講演抄録集, 20th, 59, Aug. 2013
Japanese

奨励賞受賞 関根勇一氏の業績
松田 正, ファルマシア, 49, 6, 01 Jun. 2013
Japanese

An RNA binding protein, Y14 regulates TNF-alpha-induced NF-kappa B activation and IL-6 expression
Sumihito Togi, Ryuta Muromoto, Shigeyuki Kon, Yuichi Sekine, Tadashi Matsuda, JOURNAL OF IMMUNOLOGY, 190, May 2013
AMER ASSOC IMMUNOLOGISTS, English, Summary international conference

微量オゾンによる炎症関連転写因子群のクロストークの解明
けい澄仁, 今重之, 室本竜太, 松田正, 日本医療・環境オゾン学会研究講演会要旨集, 18th, 2013

アダプタータンパク質STAP-2によるBRK活性化機構の解析
岩上昌史, 久保果央莉, 室本竜太, 今重之, 関根勇一, 松田正, 日本薬学会年会要旨集(CD-ROM), 133rd, 2013

T細胞受容体シグナルにおけるアダプタータンパク質STAP-2の役割
齋藤浩大, 赤坂大地, 関根勇一, 室本竜太, 今重之, 松田正, 日本薬学会年会要旨集(CD-ROM), 133rd, 2013

細胞内配列の異なる新規変異型α4インテグリンの機能解析
紅露ひとみ, 今重之, 松田正, 日本薬学会年会要旨集(CD-ROM), 133rd, 2013

アダプター分子STAP-2によるBCR-ABLチロシンキナーゼの制御機構
久保果央莉, 岩上昌史, 室本竜太, 今重之, 関根勇一, 松田正, 日本薬学会年会要旨集(CD-ROM), 133rd, 2013

TNF-α/NF-κBシグナル経路におけるSTAT3とNF-κBのクロストークの解析
加藤聖弥, 研澄仁, 室本竜太, 今重之, 松田正, 日本薬学会年会要旨集(CD-ROM), 133rd, 2013

微量オゾンによる炎症関連転写因子群のクロストークの解明
とぎ澄仁, 坂内明日香, 今重之, 室本竜太, 松田正, 日本医療・環境オゾン学会会報, 20, 3, 2013

アダプター分子STAP-2によるアポトーシス制御機構の解析
安次富大, 赤坂大地, 角地彩花, 関根勇一, 室本竜太, 今重之, 松田正, 日本薬学会年会要旨集(CD-ROM), 133rd, 2013

細胞内配列の異なる新規変異型α4インテグリンはα4インテグリン依存性細胞接着を抑制する
紅露ひとみ, 松本尚樹, 今重之, 松田正, 日本分子生物学会年会プログラム・要旨集(Web), 36th, 2013

アダプター分子STAP-2による白血病細胞の増殖制御機構の解析
久保果央莉, 岩上昌史, 室本竜太, 今重之, 関根勇一, 松田正, 日本分子生物学会年会プログラム・要旨集(Web), 36th, 2013

T細胞活性化におけるSTAP-2の機能解析
安次富大, 今重之, 斉藤浩大, 関根勇一, 室本竜太, 織谷健司, 松田正, 日本分子生物学会年会プログラム・要旨集(Web), 36th, 2013

結晶構造解析に向けた,乳がん特異的キナーゼBrkとアダプタータンパク質STAP‐2の機能解析
神田諒, 関根勇一, 前仲勝実, 松田正, 尾瀬農之, 日本分子生物学会年会プログラム・要旨集(Web), 36th, 2P-0302 (WEB ONLY), 2013
Japanese

核内受容体RORα/γ依存的IL‐17産生に及ぼすイソフラボン類の影響
小島弘幸, 高橋美妃, 武田行正, 室本竜太, 武内伸治, ANTON M. Jetten, 松田正, 日本薬学会年会要旨集(CD-ROM), 133rd, ROMBUNNO.29AME-024, 2013
Japanese

RNA結合タンパクY14によるTNF‐α/NF‐κBシグナル制御機構の解析
研澄仁, 志賀要, 室本竜太, 今重之, 松田正, 日本薬学会年会要旨集(CD-ROM), 133rd, ROMBUNNO.28S-AM21S, 2013
Japanese

ケモカイン産生におけるTyk2の役割の解析
相馬悠樹, 大城裕也, 室本竜太, 松田正, 日本薬学会年会要旨集(CD-ROM), 133rd, ROMBUNNO.28S-PM22S, 2013
Japanese

イソフラボン類のIL-17産生増強作用
小島弘幸, 室本竜太, 高橋美妃, 武内伸治, 松田正, ImmunoTox Letter（日本免疫毒性学会誌）, 18, 2, 12, 14, 2013, [Invited]
Japanese, Report scientific journal

ダイオキシン受容体を介したIL‐17遺伝子発現に及ぼす環境化学物質の影響
小島弘幸, 室本竜太, 高橋美妃, 武内伸治, 松田正, 日本内分泌かく乱化学物質学会研究発表会要旨集, 15th, 45, 18 Dec. 2012
Japanese

分子標的薬の作用機序・薬理作用 がん関連標的分子・標的経路 JAK/STATシグナル伝達経路を標的とした分子標的薬
MATSUDA TADASHI, KON SHIGEYUKI, MUROMOTO RYUTA, 日本臨床, 70, 45, 51, 20 Nov. 2012
Japanese

マウスEL4細胞のIL‐17及びIL‐22遺伝子発現に及ぼすAhRアゴニストの影響
小島弘幸, 室本竜太, 高橋美妃, 武内伸治, 松田正, 日本免疫毒性学会講演抄録集, 19th, 65, Aug. 2012
Japanese

A novel murine alpha 4 integrin splicing variant is a specific receptor for VCAM-1
Shigeyuki Kon, Hitomi Kouro, Tadashi Matsuda, JOURNAL OF IMMUNOLOGY, 188, May 2012
AMER ASSOC IMMUNOLOGISTS, English, Summary international conference

ZIPK modulates canonical Wnt/beta-catenin signaling through interaction with NLK/TCF4
Sumihito Togi, Ryuta Muromoto, Tadashi Matsuda, FASEB JOURNAL, 26, Apr. 2012
FEDERATION AMER SOC EXP BIOL, English, Summary international conference

P.acnes死菌誘導性実験的腹膜炎におけるTyk2の関与
大城裕也, 室本竜太, 松田正, 日本薬学会年会要旨集, 132nd, 3, 97, 05 Mar. 2012
Japanese

炎症性サイトカインIL‐17産生に及ぼすBiochanin Aの影響に関する研究
高橋美妃, 室本竜太, 小島弘幸, 武内伸治, 松田正, 日本薬学会年会要旨集, 132nd, 3, 98, 05 Mar. 2012
Japanese

環境化学物質における核内受容体RORα/γ活性の探索とIL‐17遺伝子発現への影響
小島弘幸, 室本竜太, 高橋美妃, 武内伸治, 松田正, 日本薬学会年会要旨集, 132nd, 3, 268, 05 Mar. 2012
Japanese

メラノーマがん細胞転移におけるアダプター分子STAP‐2の役割
角地彩花, 赤坂大地, 関根勇一, 今重之, 室本竜太, 松田正, 日本薬学会年会要旨集, 132nd, 3, 113, 05 Mar. 2012
Japanese

KAP1/STAT3によるTNF‐α誘導性IL‐6発現経路の制御機構
硎 澄仁, 室本竜太, 松田正, 日本薬学会年会要旨集, 132nd, 3, 135, Mar. 2012
Japanese

STAP-2によるT細胞抗原受容体シグナル伝達制御機構の解析
赤坂大地, 角地彩花, 今重之, 関根勇一, 松田正, 日本薬学会年会要旨集, 132nd, 3, 2012

T細胞受容体シグナルにおけるアダプタータンパク質STAP-2の役割
今重之, 赤坂大地, 関根勇一, 室本竜太, 吉村昭彦, 松田正, 日本分子生物学会年会プログラム・要旨集(Web), 35th, 2012

RORα/γ依存的なIL‐17産生に及ぼすBiochanin Aの影響
小島弘幸, 室本竜太, 高橋美妃, 武内伸治, 松田正, 日本内分泌かく乱化学物質学会研究発表会要旨集, 14th, 40, 01 Dec. 2011
Japanese

アダプター分子STAP‐2よる活性化誘導性細胞死(AICD)制御機構の解析
山本千香子, 関根勇一, 室本竜太, 松田正, 次世代を担う若手ファーマ・バイオフォーラム講演要旨集, 10th, 30, Sep. 2011
Japanese

新規乳癌治療標的としてのチロシンキナーゼBRKの機能解析
水嶋明宏, 池田収, 関根勇一, 関根勇一, 室本竜太, 室本竜太, 松田正, 松田正, 次世代を担う若手ファーマ・バイオフォーラム講演要旨集, 10th, 75, Sep. 2011
Japanese

RORγt依存的なIL‐17遺伝子発現誘導に対するイソフラボン類の影響
小島弘幸, 室本竜太, 高橋美妃, 武内伸治, 松田正, 日本免疫毒性学会講演抄録集, 18th, 73, Aug. 2011
Japanese

Adaptor protein, signal-transducing adaptor protein-2 regulates T-cell functions
関根 勇一, 松田 正, Clinical immunology & allergology, 55, 5, 507, 514, May 2011
科学評論社, Japanese

TYK2とJun activation domain‐binding protein 1(JAB1)の機能的相互作用の解析
室本竜太, 高橋美妃, 坪内真知子, 関根勇一, 南保明日香, 松田正, 日本薬学会年会要旨集, 131st, 3, 102, 05 Mar. 2011
Japanese

核内コリプレッサーKAP1によるSTAT3を介したTNF‐α/NF‐κBシグナル制御機構の解析
硎澄仁, 上谷晋也, 池田収, 川上志保, 関根勇一, 室本竜太, 南保明日香, 松田正, 日本薬学会年会要旨集, 131st, 3, 136, Mar. 2011
Japanese

Regulatory proteins of IL-6/STAT3 signaling pathway: Daxx, KAP1 and STAP-2
松田 正, 室本 竜太, 関根 勇一, 医学のあゆみ, 234, 5, 401, 407, 31 Jul. 2010
医歯薬出版, Japanese

IN VIVO FUNCTION OF A NOVEL ADAPTOR PROTEIN STAP2 IN THE PROGRESS OF INFLAMMATION
N. Fujita, K. Oritani, T. Yokota, N. Saitoh, T. Sudo, Y. Sekina, T. Matsuda, Y. Kanakura, HAEMATOLOGICA-THE HEMATOLOGY JOURNAL, 95, 667, 667, Jun. 2010
FERRATA STORTI FOUNDATION, English, Summary international conference

KAP1 regulates TNF-Induced NF-kappaB transcriptional activity by influencing the interactions between p300 and NF-kappaB
Sumihito Togi, Shinya Kamitani, Shiho Kawakami, Osamu Ikeda, Ryuta Muromoto, Yuichi Sekine, Asuka Nanbo, Tadashi Matsuda, FASEB JOURNAL, 24, Apr. 2010
FEDERATION AMER SOC EXP BIOL, English, Summary international conference

BS69 negatively regulates the canonical NF-kappaB activation induced by Epstein-Barr virus-derived LMP1
Osamu Ikeda, Yuichi Sekine, Ryuta Muromoto, Asuka Nanbo, Tadashi Matsuda, FASEB JOURNAL, 24, Apr. 2010
FEDERATION AMER SOC EXP BIOL, English, Summary international conference

EJC構成タンパク質Y14,MAGOHによるSTAT3活性の制御
志賀要, 室本竜太, 松田正, 日本薬学会年会要旨集, 130th, 3, 74, 05 Mar. 2010
Japanese

P.acnes誘導性肉芽腫形成におけるTyk2の役割
黒田誠, 室本竜太, 松田正, 日本薬学会年会要旨集, 130th, 3, 80, 05 Mar. 2010
Japanese

STAP-2 regulates integrin-mediated T-cell adhesion through protein degradation of FAK
Tadashi Matsuda, Osamu Ikeda, Yuichi Sekine, FASEB JOURNAL, 22, Apr. 2008
FEDERATION AMER SOC EXP BIOL, English, Summary international conference

サイトカインのシグナル伝達系制御機構の解析
松田 正, 三共生命科学研究振興財団研究報告集, 24, 0, 177, 186, 2008
三共生命科学研究振興財団, Japanese

インターフェロンによるB細胞の増殖制御
室本竜太, 織谷健司, 下田和哉, 松田正, 月刊臨床免疫・アレルギー科, 47, 6, 735-744, 744, 25 Jun. 2007
科学評論社, Japanese

インターフェロン誘導性タンパク質Daxxによる転写因子STAT3の機能制御
室本竜太, 北本竜生, 中嶋麻衣子, 杉山憲司, 石田雅人, 松田正, 日本薬学会年会要旨集, 127th, 2, 17, 05 Mar. 2007
Japanese

Regulation of the Epstein-Barr virus LMP1-mediated NF-kappaB signaling pathway by a novel adaptor protein STAP-2
Osamu Ikeda, Yuichi Sekine, Teruhito Yasui, Suita Kenji Sugiyma, Kenji Oritani, Suita Akihiko Yoshimura, Tadashi Matsuda, CLINICAL IMMUNOLOGY, 123, S33, S34, 2007
ACADEMIC PRESS INC ELSEVIER SCIENCE, English, Summary international conference

Regulation of the STAT3-mediated signaling by Daxx
Kenji Sugiyama, Ryuta Muromoto, Masato Ishida, Yuichi Sekine, Kenji Oritani, Tadashi Matsuda, CLINICAL IMMUNOLOGY, 123, S159, S159, 2007
ACADEMIC PRESS INC ELSEVIER SCIENCE, English, Summary international conference

ウイルス感染と宿主応答・宿主側の要因 新規アダプター分子STAP-2とEBウイルス産物LMP1の機能的相互作用の解析　　　　　　　　　　　　　　　
池田 収, 関根 勇一, 辻 暁司, 安居 輝人, 杉山 憲司, 吉村 昭彦, 松田 正, 日本免疫学会総会・学術集会記録, 36, 206, 206, Nov. 2006
(NPO)日本免疫学会, Japanese

KSHV遺伝子産物LANAによるSTAT3の活性化
室本竜太, 岡部可菜子, 杉山憲司, 松田正, 日本薬学会年会要旨集, 126th, 3, 81, 06 Mar. 2006
Japanese

ZIPKとNLKの機能的相互作用の解析
渡部匡史, 室本竜太, 佐藤紀子, 杉山憲司, 松田正, 日本薬学会年会要旨集, 126th, 3, 81, 06 Mar. 2006
Japanese

Modulation of toll-like receptor 4 signaling by a novel adaptor protein STAP-2 in macrophages
Y Sekine, S Tsuji, O Ikeda, K Sugiyama, S Akira, A Yoshimiura, T Matsuda, FASEB JOURNAL, 20, 4, A114, A114, Mar. 2006
FEDERATION AMER SOC EXP BIOL, English, Summary international conference

Physical and functional interactions between Daxx and STAT3
T Matsuda, R Muromoto, K Nakao, T Watanabe, N Sato, Y Sekine, K Sugiyma, FASEB JOURNAL, 20, 4, A533, A533, Mar. 2006
FEDERATION AMER SOC EXP BIOL, English, Summary international conference

DaxxによるSTAT3転写活性化抑制機構の解析
中尾圭, 室本竜太, 杉山憲司, 渡部匡史, 織谷健司, 下田和哉, 松田正, 日本免疫学会総会・学術集会記録, 35, 285, 15 Nov. 2005
Japanese

アダプター分子STAP‐2によるSTAT蛋白の機能調節
SEKINE YUICHI, TSUJI SATOSHI, IKEDA OSAMU, MATSUDA TADASHI, 臨床免疫, 44, 3, 339, 344, 25 Sep. 2005
Japanese

A novel mutation in the juxtamembrane intracellular sequence of the granulocyte colony-stimulating factor (G-CSF) receptor gene in a patient with severe congenital neutropenia augments G-CSF proliferation activity but not through the MAP kinase cascade
T Yokoyama, S Okamura, Y Asano, K Kamezaki, A Numata, H Kakumitsu, K Shide, H Nakashima, T Kanaji, Y Sekine, Y Mizuno, J Okamura, T Matsuda, M Harada, Y Niho, K Shimoda, INTERNATIONAL JOURNAL OF HEMATOLOGY, 82, 1, 28, 34, Jul. 2005
We analyzed the structure of the granulocyte colony-stimulating factor (G-CSF) receptor gene in a 6-year-old female patient with severe congenital neutropenia (SCN) who experienced severe recurrent infections since I month of age. There is no family history of any similar disease. When the patient was 4 months old, she began receiving treatment with recombinant human G-CSF that resulted in a small increase in the neutrophil count sufficient for the prevention and treatment of bacterial infection. An analysis of complementary DNA for the patient's G-CSF receptor revealed a 3-base pair deletion in the juxtamembrane intracellular sequence. This deletion at the beginning of exon 16 was thought to be caused by alternative splicing; analysis of the DNA revealed a G-to-A point mutation of the final nucleotide of intron 15. To evaluate the functional activity of the G-CSF receptor with this 3-base pair deletion of the juxtamembrane region, we transfected this G-CSF receptor mutant into an interleukin 3-dependent cell line, BAF/3. BAF/3 cells expressing the mutant G-CSF receptor showed augmented proliferation activity in response to G-CSF compared with cells having the wild-type G-CSF receptor. Although the proliferation signal of G-CSF in normal hematopoiesis is transduced through the activation of MAP kinases, this G-CSF receptor mutant showed decreased activation of ERK1/2 in response to G-CSF compared with the wild type, but the transduced signal for Stat3 activation by G-CSF was of the same magnitude as that of the wild-type G-CSF receptor. This result means that the augmented proliferation activity in response to G-CSF that we observed in cells having the G-CSF receptor gene with the 3-base pair deletion is transduced through an intracellular signaling pathway other than MAP kinase. Because SCN patients with a mutation in the G-CSF receptor frequently develop leukemia, this 3-base pair deletion in the juxtamembrane sequence of the G-CSF receptor gene in this patient may be one step in the course of leukemic transformation., CARDEN JENNINGS PUBL CO LTD, English

Transgenic mice overexpressing murine thrombopoietin develop myelofibrosis and osteosclerosis
B Kakumitsu, B Kamezaki, B Shimoda, K Karube, T Haro, A Numata, K Shide, T Matsuda, K Oshima, M Harada, LEUKEMIA RESEARCH, 29, 7, 761, 769, Jul. 2005
Thrombopoietin (TPO) regulates megakaryocytopoiesis and platelet production in vivo and in vitro. Exogenous overexpression of TPO in vivo by viral-mediated gene transfer induced bone marrow (BM) fibrosis and osteosclerosis. On the other hand, transgenic mice (Tg) overexpressing TPO using a liver-specific apolipoprotein E (Apo-E) promoter did not exhibit myelofibrosis or osteosclerosis. These discrepancies in phenotype are not fully understood. Then we have investigated the consequences of long-term in vivo overexpression of TPO in a mouse model. Murine TPO Tg mice driven by the IgH promoter were generated. The number of platelets and neutrophils in peripheral blood, and the number of megakaryocytes and granulocytic immature cells in the BM was elevated, together with the number of progenitor cells for megakaryocyte and myeloid cells. TPO Tg mice demonstrated anemia but the number of progenitor cells for the erythrocyte was increased. TPO Tg mice developed myelofibrosis and osteosclerosis as they aged with extramedullary hematopoiesis in the spleen. As plasma transforming growth factors (TGF)-beta 1 and osteoprotegerin (OPG) levels were higher in TPO Tg mice than in wild-type mice, the development of myelofibrosis and osteosclerosis depends on local TPO levels in BM and might be due to elevated TGF-beta 1 and OPG. (c) 2005 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD, English

Differential effects of phthalate esters on transcriptional activities via human estrogen receptors alpha and beta, and androgen receptor
S Takeuchi, M Iida, S Kobayashi, K Jin, T Matsuda, H Kojima, TOXICOLOGY, 210, 2-3, 223, 233, Jun. 2005
Some phthallates are suspected to disrupt the endocrine system, especially by mimicking estrogens. In this study, we characterized the activities of human estrogen receptor a (hERalpha), human estrogen receptor beta (hERbeta), and human androgen receptor (hAR) in the presence of 22 phthalates including 3 of their metabolites using highly sensitive reporter gene assays. Of the 22 compounds tested, several phthalate diesters with alkyl chains ranging in length from C-3 to C-6 exhibited not only hERalpha-mediated estrogenic activity, but also hERbeta-mediated antiestrogenic activity in a dose-dependent manner. In addition, we found that some phthalate diesters possess hAR-mediated antiandrogenic activity. However, the phthalates having side chains with very short length (diethyl) or very long length (diheptyl), and three metabolites (monoesters) were found to have no effect on the activities of the three receptors. These results indicate that several phthalate esters simultaneously act as agonists and/or antagonists via one or more hormonal receptors, and interaction of phthalate esters with the estrogen and androgen receptors requires certain size and bulkiness with alkyl groups. (c) 2005 Elsevier Ireland Ltd. All rights reserved., ELSEVIER IRELAND LTD, English

Roles for lysine residues of the MH2 domain of Smad3 in transforming growth factor-beta e signaling
S Imoto, K Sugiyama, Y Sekine, T Matsuda, FEBS LETTERS, 579, 13, 2853, 2862, May 2005
Sma and MAD-related protein 3 (Smad3) plays a key role in the intracellular signaling of the transforming growth factor-beta (TGF-beta) family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. Smad3 has the N-terminal Mad homology (MH) 1 and the C-terminal MH2 domains. MH2 domain is essential for the TGF-beta-induced transcriptional activation, because the MH2 domain of Smad3 is involved in the interactions with several transcriptional cofactors as well as the type I TGF-beta receptor (T beta R-I). In this study, we examined the roles for four lysine residues (Lys-333, Lys-341, Lvs-378, and Lys-409) in the Smad3 MH2 domain. Mutation of the lysine (K)-378 to arginine (R) (K378R) abolished the interaction with T beta R-I, phosphorylation, transcriptional activation by an active T beta R-I. The K341R mutant also failed to stimulate TGF-beta-induced transcription by resting in the cytoplasm. However, the K409R mutant showed a higher transcriptional activity by stronger interactions with coactivators, such as p300/CBP. Furthermore, both the K341R and K378R mutants act as dominant-negative inhibitors in the TGF-beta-induced target genes of endogenous TGF-beta signal. Thus, the lysine residues of Smad3 MH2 domain play important roles in the transcriptional regulation of TGF-beta signals through T beta R-I. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV, English

Physical and functional interactions between STAP-2/BKS and STAT5
Y Sekine, T Yamamoto, T Yumioka, K Sugiyama, S Tsuji, K Oritani, K Shimoda, M Minoguchi, A Yoshimura, T Matsuda, JOURNAL OF BIOLOGICAL CHEMISTRY, 280, 9, 8188, 8196, Mar. 2005
Signal-transducing adaptor protein family of proteins (STAPs), which currently contains two members, are proposed to be adaptor molecules because of their pleckstrin homology (PH) and Src-homology 2 (SH2)-like domains. STAP-1 has been shown to interact with STAT5 and the tyrosine kinase Tec. With regard to STAP-2/BKS functions, immunoprecipitation experiments and intracellular stainings revealed STAP-2/BKS binds STAT5 in several types of cells. Mutational studies revealed that the PH- and SH2-like domains of STAP-2/BKS interacted with the C-terminal region of STAT5. STAP-2/BKS and STAT5 were found to constitutively co-localize in the cytoplasm of resting cells, but STAP-2/BKS was found to dissociate upon STAT5 phosphorylation, suggesting a role in regulating signaling of STAT5. The physiological role of these interactions is not fully understood, but in studies of overexpression of STAP-2/BKS, cytokine-induced tyrosine phosphorylation and transcriptional activation of STAT5 was diminished. In addition, thymocytes from STAP-2/BKS-deficient mice showed the enhanced interleukin-2-dependent cell growth. Taken together, STAP-2/BKS is an additional modulator of STAT5-mediated signaling., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, English

Roles of a STAT3 kinase in STAT3-mediated signaling
N Sato, Y Kamata, K Sugiyama, Y Sekine, T Matsuda, FASEB JOURNAL, 19, 5, A1419, A1419, Mar. 2005
FEDERATION AMER SOC EXP BIOL, English, Summary international conference

Roles of a STAT binding protein in cytokine signaling
T Okayama, N Sato, K Sugiyama, T Matsuda, FASEB JOURNAL, 19, 5, A1420, A1420, Mar. 2005
FEDERATION AMER SOC EXP BIOL, English, Summary international conference

Roles of Stat3 and ERK in G-CSF signaling
K Kamezaki, K Shimoda, A Numata, T Haro, H Kakumitsu, M Yoshie, Y Yamamoto, K Takeda, T Matsuda, M Akira, K Ogawa, O Harada, STEM CELLS, 23, 2, 252, 263, Feb. 2005
G-CSF specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil-granulocyte lineage. Although Stat3 was thought to be essential for the transduction of G-CSF-induced cell proliferation and differentiation signals, mice deficient for Stat3 in hematopoietic cells show neutrocytosis and was not induced by G-CSF stimulation. Stat3-null bone marrow cells displayed a significant activation of extracellular regulated kinase I (ERK1)/ERK2 under basal conditions, and the activation of ERK was enhanced and sustained by G-CSF stimulation. Furthermore, the augmented proliferation of Stat3-deficient bone marrow infiltration of cells into the digestive tract. The number of cells in response to G-CSF was dramatically decreased progenitor cells in the neutrophil lineage is not changed, and G-CSF-induced proliferation of progenitor cells and prolonged neutrophil survival were observed in Stat3deficient mice. In hematopoietic cells from Stat3-deficient mice, trace levels of SOCS3, a negative regulator of granulopoiesis, were observed, and SOCS3 expression by addition of a MEK1 inhibitor. These results indicate that Stat3 functions as a negative regulator of G-CSF signaling by inducing SOCS3 expression and that ERK activation is the major factor responsible for inducing the proliferation of hematopoietic cells in response to G-CSF., ALPHAMED PRESS, English

サイトカインシグナル伝達系とその制御―シグナル伝達分子STAT3を中心に―
SATO NORIKO, SEKINE YUICHI, MATSUDA TADASHI, 日本応用酵素協会誌, 39, 11, 17, 01 Feb. 2005
Japanese

Cytokine induction by a bacterial DNA-specific modified base
Biochem. Biophys. Res. Commun., 326:777-781, 2005

KSHV遺伝子産物LANAによるSTAT3の活性化
岡部可菜子, 室本竜太, 杉山憲司, 松田正, 日本免疫学会総会・学術集会記録, 34, 183, 05 Nov. 2004
Japanese

Daxxの変異体によるインターフェロンのBリンパ球前駆細胞増殖抑制活性の阻害
室本竜太, 杉山憲司, 織谷健司, 下田和哉, 松田正, 日本免疫学会総会・学術集会記録, 34, 191, 05 Nov. 2004
Japanese

Bacterial endotoxin induces IL-20 expression in the glial cells
T Hosoi, S Wada, S Suzuki, Y Okuma, S Akira, T Matsuda, Y Nomura, MOLECULAR BRAIN RESEARCH, 130, 1-2, 23, 29, Nov. 2004
The regulatory mechanisms leading to IL-20 expression during infection have not been elucidated. In the present study, we found that bacterial lipopolysaccharide (LPS) induced IL-20 expression in the primary cultured glial cells and RAW264.7 macrophage cell line. Pretreatment with protein synthesis inhibitor puromycin or cycloheximide failed to inhibit the expression of IL-20, suggesting that the expression was not dependent on de novo protein synthesis. Myeloid differentiation factor 88 (MyD88) is an important adaptor molecule for Toll-like receptor signaling. We observed complete inhibition of LPS-induced expression of IL-20 in the primary, cultured glial cells prepared from MyD88-deficient mice. Furthermore, a p38 MAP kinase inhibitor, SB203580, inhibited LPS-induced expression of IL-20 mRNA. LPS-induced p38 MAP kinase phosphorylation was delayed in MyD88-deficient glial cells. Therefore, it is suggested that LPS induces IL-20 expression through MyD88-p38-dependent mechanisms. As dexamethasone inhibited LPS-induced IL-20 expression, the expression of IL-20 is regulated by a negative feedback loop mediated through glucocorticoids. Therefore, it is suggested that IL-20 may play a crucial role in inflammatory conditions in the brain. (C) 2004 Elsevier B.V. All rights reserved., ELSEVIER SCIENCE BV, English

The roles of stat3 and ERK in G-CSF signaling.
K Kamezaki, K Shimoda, A Numata, M Yoshie, M Yamamoto, K Takeda, T Matsuda, S Akira, K Ogawa, M Harada, BLOOD, 104, 11, 597A, 597A, Nov. 2004
AMER SOC HEMATOLOGY, English, Summary international conference

タイプIインターフェロンシグナル伝達系の新たな機能分子Daxx
室本 竜太, 松田 正, 臨床免疫, 42, 5, 570, 575, Nov. 2004
科学評論社, Japanese

Differential expression of integrin subunits in DU-145/AR prostate cancer cells
O Nagakawa, T Akashi, Y Hayakawa, A Junicho, K Koizumi, Y Fujiuchi, Y Furuya, T Matsuda, H Fuse, Saiki, I, ONCOLOGY REPORTS, 12, 4, 837, 841, Oct. 2004
We have established a clonal DU-145 prostate cancer cell line (DU-145/AR) stably transfected with androgen receptor cDNA. We investigated the expression of integrin subunits, adhesion to extracellular matrices, the invasion of DU-145/AR prostate cancer cells. The expression of various integrin subunits and adhesion to various extracellular matrices in DU-145, DU-145/Neo and DU-145/AR cells were examined. The haptoinvasion and the haptotactic migration of these cells were investigated using a Transwell cell culture chamber assay. DU-145/AR cells exhibited lower expression of alpha6 and beta4 integrin subunits and higher expression of alpha2 and alpha5 than DU-145 cells. DU-145/AR cells showed significantly lower adhesion to fibronectin, laminin-1 and laminin-5 than DU-145/Neo cells, whereas DU-145/AR cells showed higher adhesion to type I and type IV collagen. Haptoinvasion of DU-145/AR cells into Matrigel/fibronectin-coated filter was significantly reduced as compared with DU-145/Neo or DU-145 cells, but there was no significant difference between DU-145/AR and control cells in the haptotactic migration to fibronectin. Dihydrotestosterone (DHT) inhibited the invasive ability of DU-145/AR cells. These results indicate that androgen receptor may play a role in the regulation of adhesion to the extracellular matrices and invasion of prostate cancer cells through influencing the expression of specific integrin subunits., PROFESSOR D A SPANDIDOS, English

Hapten-induced contact hypersensitivity is enhanced in Tyk2-deficient mice
M Hosogi, H Tonogaito, A Aioi, K Hamada, K Shimoda, R Muromoto, T Matsuda, Y Miyachi, JOURNAL OF DERMATOLOGICAL SCIENCE, 36, 1, 51, 56, Oct. 2004
Background: Previous studies have shown that Tyk2, a member of the Janus family of protein tyrosine kinases, which are activated by a variety of cytokines, plays a crucial role in interleukin (IL)-12-mediated T-cell functions such as IFN-gamma production. On the other hand, hapten-induced contact hypersensitivity (CHS) is mediated by IFN-gamma producing CD8+ T cells and regulated by CD4+ T cells.
Objective: This study hypothesized that the CHS response might be reduced in Tyk2-deficient mice because of a tack of IFN-gamma production from CD4+ and CD8+ T cells.
Methods: The CHS reaction was evoked in wild-type and Tyk2-deficient mice and the ears of the mice were examined to measure for several cytokines.
Results: Ear swelling during CHS was significantly enhanced in Tyk2-deficient mice compared with the controls. IL-12 and IFN-gamma levels at the reaction sites in Tyk2-deficient mice were significantly lower than in the controls, whereas IL-2 and IL-4 levels were elevated. Furthermore, STAT3- and STAT4-phosphorylation in the draining lymph node cells of Tyk2-deficient mice decreased.
Conclusion: These results suggest that the tack of Tyk2-mediated signal transduction enhances a compensative pathway during CHS. (C) 2004 Japanese Society for Investigative Dermatology., ELSEVIER SCI IRELAND LTD, English

Bacterial endotoxin induces STAT3 activation in the mouse brain
T Hosoi, Y Okuma, T Kawagishi, Qi, X, T Matsuda, Y Nomura, BRAIN RESEARCH, 1023, 1, 48, 53, Oct. 2004
In the present study, we investigated regulatory mechanisms of bacterial endotoxin-induced STAT3 activation in the brain. Intraperitoneal injection of lipopolysaccharide (LPS) dose-dependently (0.5-5000 mug/kg) induced STAT3 phosphorylation in the hypothalamus. LPS-induced STAT3 phosphorylation was peaked at 2-4 h and declined there after. Moreover, intracerebroventricular injection of LPS induced STAT3 phosphorylation in the cortex and the hippocampus, indicating that central as well as peripheral LPS can act in the brain to induce STAT3 activation. Glucocorticoids are known to play a physiological role in the feedback inhibition of immune/inflammatory responses in the endocrine system. Interestingly, we observed no effect of dexamethasone on LPS-induced STAT3 phosphorylation in the hypothalamus. These findings point to the important role of STAT3 in the neuroimmune interaction of inflammation in the brain. (C) 2004 Elsevier B.V All rights reserved., ELSEVIER SCIENCE BV, English

The role of Tyk2, Stat1 and Stat4 in LPS-induced endotoxin signals
K Kamezaki, K Shimoda, A Numata, T Matsuda, K Nakayama, M Harada, INTERNATIONAL IMMUNOLOGY, 16, 8, 1173, 1179, Aug. 2004
Mice lacking Tyk2, Stat1 or Stat4, which are members of the Jak-Stat signaling cascade, were resistant to LPS-induced endotoxin shock. Interestingly, Tyk2-deficient mice had higher resistance to LIPS challenge than mice lacking either Stat1 or Stat4. The activation of MAPK and NF-kappaB by LIPS, and the production of TNF-alpha and IL-12 after LPS injection, were not abrogated by the absence of Tyk2, Stat1 or Stat4l. In Stat1-deficient mice, the induction of IFN-beta by LIPS in macrophages was severely reduced, although the serum level of IFN-gamma was elevated after LPS injection. In contrast, in Stat-4 deficient mice, the induction of IFN-beta by LIPS was normal, but the serum level of IFN-gamma remained low after LPS injection. Interestingly, the induction of both IFN-beta and IFN-gamma by LIPS was severely reduced in Tyk2-deficient mice. Therefore, Stat1 and Stat4 independently play substantial roles in the susceptibility to LIPS. Tyk2 is essential for LIPS-induced endotoxin shock, and this signaling pathway is transduced by the activation of Stat1 and Stat4., OXFORD UNIV PRESS, English

Influence of endoplasmic reticulum stress on iNOS induction in glial cells
T Hosoi, Y Okuma, M Takakuwa, T Matsuda, Y Nomura, NITRIC OXIDE-BIOLOGY AND CHEMISTRY, 11, 1, 109, 109, Aug. 2004
ACADEMIC PRESS INC ELSEVIER SCIENCE, English, Summary international conference

Tyrosine kinase 2 interacts with and phosphorylates receptor for-activated c kinase-1, a WD motif-containing protein
T Haro, K Shimoda, H Kakumitsu, K Kamezaki, A Numata, F Ishikawa, Y Sekine, R Muromoto, T Matsuda, M Harada, JOURNAL OF IMMUNOLOGY, 173, 2, 1151, 1157, Jul. 2004
Receptor for activated C kinase (Rack)-1 is a protein kinase C-interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase Cgamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alphabeta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudokinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N terminus and one in the middle portion (aa 138-203) of Rack-1. Jak activation causes the phosphorylation of tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades., AMER ASSOC IMMUNOLOGISTS, English

The RING domain of PIASy is involved in the suppression of bone morphogenetic protein-signaling pathway
S Imoto, K Sugiyama, T Yamamoto, T Matsuda, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 319, 1, 275, 282, Jun. 2004
Bone morphogenetic proteins (BMPs) play central roles in differentiation, development, and physiologic tissue remodeling. Recently, we have demonstrated that a protein inhibitor of activated STAT, PIASy, suppresses TGF-beta signaling by interacting with Sma and MAD-related protein 3 (Smad3). In this study, we examined a PIASy-dependent inhibitory effect on BMP signaling. PIASy expression was induced by BMP-2 stimulation and suppressed BMP-2-dependent Smad activity in hepatoma cells. Furthermore, BMP-2-regulated Smads directly bound to PIASy. We also demonstrated that the RING domain of PIASy played an important role in PIASy-mediated suppression of Smad activity. We here provide evidence that the inhibitory action of PIASy on BMP-regulated Smad activity was due to direct physical interactions between Smads and PIASy through its RING domain. (C) 2004 Elsevier Inc. All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE, English

Physical and functional interactions between Daxx and TSG101
R Muromoto, K Sugiyama, T Yamamoto, K Oritani, K Shimoda, T Matsuda, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 316, 3, 827, 833, Apr. 2004
Daxx has been reported to mediate the Fas/JNK-dcpendcnt signals in the cytoplasm. However, several evidences have suggested that Daxx is located mainly in the nucleus and functions as a transcriptional regulator. Recently, we identified DMAP1, a TSG101-interacting protein as a Daxx binding partner by yeast two-hybrid screening. TSG101 has been shown to act as transcriptional corepressor of nuclear hormone receptors. Here we examined whether TSG101 also interacts with Daxx directly. The association of Daxx and TSG101 was confirmed using co-expressed tagged proteins. The interaction regions in both proteins were also mapped, and the cellular localization of the interaction was examined. TSG101 formed a complex with Daxx through its coiled-coil domain and co-localized in the nucleus. Furthermore, TSG101 enhanced Daxx-mediated repression of glucocorticoid receptor transcriptional activity. These results provide the novel molecular interactions between Daxx and TSG101, which establish an efficient repressive transcription complex in the nucleus. (C) 2004 Elsevier Inc. All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE, English

Cross-talk between endocrine-disrupting chemicals and cytokine signaling through estrogen receptors
Y Sekine, T Yamamoto, T Yumioka, S Imoto, H Kojima, T Matsuda, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 315, 3, 692, 698, Mar. 2004
STAT3 mainly acts as a signal transducer of IL-6 family cytokines and transcriptionally activates specific target genes. STAT3 has also been demonstrated to mediate cellular transformation and is found in numerous cancers. Endocrine-disrupting chemicals (EDCs) are a diverse group of chemicals that bind to estrogen receptors (ERs), mimic estrogenic actions, and may have adverse effects on human health. In our previous study, we demonstrated that estrogens suppressed the STAT3-mediated transcription activity through ERs. In this study, we examined the effects of EDCs on STAT3-mediated signaling through ERs. Surprisingly, some of EDCs enhanced STAT3-mediated transcription activity through ERs. This finding strongly suggests that EDCs may play an important role in the endocrine functions by mimicking cytokine activity by stimulating STAT3 actions through ERs. (C) 2004 Elsevier Inc. All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE, English

Physical and functional interactions between Daxx and DNA methyltransferase 1-associated protein, DMAP1
R Muromoto, K Sugiyama, A Takachi, S Imoto, N Sato, T Yamamoto, K Oritani, K Shimoda, T Matsuda, JOURNAL OF IMMUNOLOGY, 172, 5, 2985, 2993, Mar. 2004
Daxx has been shown to play an essential role in type I IFN-alphabeta-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify how Daxx regulates B cell development, we examined Daxx interacting partners by yeast two-hybrid screening. DNA methyltransferase I (DNMT1)-associated protein (DMAP1) was identified and demonstrated to interact with Daxx. The interaction regions in both proteins were mapped, and the cellular localization of the interaction was examined. Both Daxx and DMAP1 formed a complex with DNMT1 and colocalized in the nucleus. DMAP1 enhanced Daxx-mediated repression of glucocorticoid receptor transcriptional activity. Furthermore, Daxx protected protein degradation of DMAP1 in vivo. These results provide the novel molecular link between Daxx and DNMT1, which establishes a repressive transcription complex in the nucleus., AMER ASSOC IMMUNOLOGISTS, English

TEL-Syk fusion constitutively activates PI3-K/Akt, MAPK and JAK2-independent STAT5 signal pathways
T Kanie, A Abe, T Matsuda, Y Kuno, M Towatari, T Yamamoto, H Saito, N Emi, T Naoe, LEUKEMIA, 18, 3, 548, 555, Mar. 2004
We previously reported the fusion of the TEL gene to the Syk gene in myelodysplastic syndrome with t(9;12)(q22;p12). TEL-Syk fusion transformed interleukin-3 (IL-3)-dependent murine hematopoietic cell line BaF3 to growth factor independence. Here, we investigate the intracellular signal transduction of the stable transfectants. TEL-Syk fusion protein was associated with the p85 subunit of phosphatidyl inositol 3 kinase (PI3-K) followed by the activation of Akt in the absence of IL-3. Vav, phospholipase C-gamma2 and mitogen-activated protein kinase (MAPK) were also constitutively activated. TEL-Syk also activated the signal transducer and activator of transcription 5 (STAT5) in the absence of Janus kinase 2 activation. None of these kinases were phosphorylated in the BaF3 cells transfected with TELDeltaPNT-Syk in which the oligomerization domain of TEL was deleted. Inhibitor analysis showed that the MAPK pathway was important in TEL-Syk-mediated cell proliferation. The immunofluorescence technique revealed that the TEL-Syk fusion protein was located in the cytoplasm. These data suggest that TEL-Syk fusion protein in the cytoplasm leads to the constitutive activation of PI3-K/Akt, MAPK and STAT5 signal pathways, which are closely involved in IL-3-independent cell proliferation of BaF3 cells., NATURE PUBLISHING GROUP, English

Molecular dynamics of STAT3 on IL-6 signaling pathway in living cells
Biochem. Biophys. Res. Commun., 324, 1264-1273, 2004

Involvement of NF-kappa B in TGF-beta-mediated suppression of IL-4 signaling
T Yamamoto, S Imoto, Y Sekine, K Sugiyama, T Akimoto, A Muraguchi, T Matsuda, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 313, 3, 627, 634, Jan. 2004
Control of immune response requires the coordinated integration of both stimulatory and inhibitory factors. Therefore, the cross-talk of different signaling pathways is critical in providing an integrated cellular response to multiple external signals. Both interleukin-4 (IL-4) and transforming growth factor (TGF-beta) are pleiotropic cytokines and play critical roles in controlling immune responses. For example, IL-4 mediates important pro-inflammatory functions in asthma including induction of the IgE isotype switch and expression of vascular cell adhesion molecules. Whereas, TGF-beta is secreted from B, T, and dendritic cells as well as macrophages, and negatively regulates their proliferation, differentiation, and activation by other cytokines. In this study, we examined the effect of TGF-beta on IL-4 signaling using B cells as well as embryonic kidney cells. TGF-beta inhibited IL-4-induced IgG1 production and gene expression of germline epsilon transcripts in B cells. In embryonic kidney cells, TGF-beta signals suppressed IL-4-induced transcription, when we monitored using germline epsilon promoter DNA. Furthermore, activation of NF-kappaB resulted in a resistance to TGF-beta-mediated suppression of IL-4 signaling. These results indicate that TGF-beta-mediated regulation of IL-4 signaling may act by targeting NF-kappaB signaling. (C) 2003 Elsevier Inc. All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE, English

The mechanisms of bacterial endotoxin-induced endoplasmic reticulum stress response
T Hosoi, Y Okuma, M Takakuwa, T Matsuda, Y Nomura, JOURNAL OF PHARMACOLOGICAL SCIENCES, 94, 299P, 299P, 2004
JAPANESE PHARMACOLOGICAL SOC, English, Summary international conference

Limitin, an interferon-like cytokine, transduces inhibitory signals on B-cell growth through activation of Tyk2, but not Stat1, followed by induction and nuclear translocation of Daxx
K Aoki, K Shimoda, K Oritani, T Matsuda, K Kamezaki, R Muromoto, A Numata, S Tamiya, T Haro, F Ishikawa, K Takase, T Yamamoto, T Yumioka, T Miyamoto, K Nagafuji, H Gondo, S Nagafuchi, K Nakayama, M Harada, EXPERIMENTAL HEMATOLOGY, 31, 12, 1317, 1322, Dec. 2003
Objective. Limitin, an interferon-like cytokine, suppresses B lymphopoiesis through ligation of the interferon-alpha/beta (IFN-alpha/beta) receptor. The aim of this study was to examine the intracellular signal transduction pathways activated by limitin.
Materials and Methods. The effects of limitin on cell growth, the activation of Jak kinase and Stat proteins, and the induction of interferon regulatory factor-1 (IRF-1) and Daxx were examined using the mouse pre-B-cell line 18.81, wild-type, and Tyk2-deficient mouse bone marrow cells. In addition, the change of localization of the Daxx protein after limitin treatment in wild-type and Tyk2-deficient mice was examined.
Results. Limitin phosphorylates Tyk2, Jak1, Stat1, and Stat2 and rapidly induces IRF-1 mRNA production. Phosphorylation of Stat1 by limitin is partially dependent on Tyk2. Suppression of B-cell growth by limitin, however, is severely impaired in the absence of Tyk2, whereas it is unaffected by the absence of Stat1. Limitin also induces the expression and nuclear translocation of Daxx, which is essential for IFN-alpha-induced inhibition of B-lymphocyte development. The absence of Tyk2 abrogates this induction of Daxx expression and nuclear translocation.
Conclusions. Limitin suppresses B-cell growth through activation of Tyk2, resulting in the up-regulation and nuclear translocation of Daxx. This limitin-mediated signaling pathway does not require Stat1. 2003 International Society for Experimental Hematology. Published by Elsevier Inc., ELSEVIER SCIENCE INC, English

サイトカインシグナル伝達分子STAT3に会合する新規分子の同定
佐藤紀子, 杉山憲司, 石黒真琴, 井本世祐, 室本竜太, 山本哲也, 松田正, 日本免疫学会総会・学術集会記録, 33, 257, 05 Nov. 2003
Japanese

Bリンパ球前駆細胞の増殖抑制に関与するDaxxの新規結合分子同定と解析
室本竜太, 杉山憲司, 井本世祐, 佐藤紀子, 山本哲也, 織谷健司, 下田和哉, 松田正, 日本免疫学会総会・学術集会記録, 33, 264, 05 Nov. 2003
Japanese

Intracellular signal transduction of interferon on the suppression of hematopoietic progenitor cell growth.
K Kamezaki, K Kato, K Shimoda, A Numata, T Haro, K Aoki, F Ishikawa, K Takase, H Ariyama, T Matsuda, T Miyamoto, K Nagafuji, H Gondo, K Nakayama, M Harada, BLOOD, 102, 11, 167B, 167B, Nov. 2003
AMER SOC HEMATOLOGY, English, Summary international conference

Regulation of transforming growth factor-beta signaling by protein inhibitor of activated STAT, PIASy through Smad3
S Imoto, K Sugiyama, R Muromoto, N Sato, T Yamamoto, T Matsuda, JOURNAL OF BIOLOGICAL CHEMISTRY, 278, 36, 34253, 34258, Sep. 2003
Smads proteins play a key role in the intracellular signaling of the transforming growth factor (TGF)-beta family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. In particular, Smad7 acts as an antagonist of TGF-beta signaling, which could determine the intensity or duration of its signaling cascade. In this study we identified a protein inhibitor of activated STAT ( signal transducers and activators of transcription), PIASy, as a novel interaction partner of Smad7 by yeast two-hybrid screening using the MH2 domain of Smad7 as bait. The association of Smad7 and PIASy was confirmed using co-expressed tagged proteins in 293T cells. Moreover, we found that other Smads including Smad3 also associated with PIASy through its MH2 domain, and PIASy suppressed TGF-beta-mediated activation of Smad3. PIASy also stimulated the sumoylation of Smad3 in vivo. Furthermore, endogenous PIASy expression was induced by TGF-beta in Hep3B cells. These findings provide the first evidence that a PIAS family protein, PIASy, associates with Smads and involves the regulation of TGF-beta signaling using the negative feedback loop., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, English

Regulation of transforming growth factor-beta signaling by protein inhibitor of activated STAT, PIASy through Smad3
S Imoto, K Sugiyama, R Muromoto, N Sato, T Yamamoto, T Matsuda, JOURNAL OF BIOLOGICAL CHEMISTRY, 278, 36, 34253, 34258, Sep. 2003
Smads proteins play a key role in the intracellular signaling of the transforming growth factor (TGF)-beta family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. In particular, Smad7 acts as an antagonist of TGF-beta signaling, which could determine the intensity or duration of its signaling cascade. In this study we identified a protein inhibitor of activated STAT ( signal transducers and activators of transcription), PIASy, as a novel interaction partner of Smad7 by yeast two-hybrid screening using the MH2 domain of Smad7 as bait. The association of Smad7 and PIASy was confirmed using co-expressed tagged proteins in 293T cells. Moreover, we found that other Smads including Smad3 also associated with PIASy through its MH2 domain, and PIASy suppressed TGF-beta-mediated activation of Smad3. PIASy also stimulated the sumoylation of Smad3 in vivo. Furthermore, endogenous PIASy expression was induced by TGF-beta in Hep3B cells. These findings provide the first evidence that a PIAS family protein, PIASy, associates with Smads and involves the regulation of TGF-beta signaling using the negative feedback loop., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, English

Regulation of Fc epsilon RI-mediated signaling by an adaptor protein STAP-2/BSK in rat basophilic leukemia RBL-2H3 cells
T Yamamoto, T Yumioka, Y Sekine, N Sato, M Minoguchi, A Yoshimura, T Matsuda, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 306, 3, 767, 773, Jul. 2003
Crosslinking of multivalent antigen bound IgE transduces FcepsilonRI 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 FcepsilonRI-mediated calcium mobilization and degranulation. FcepsilonRI-induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) but not Syk was significantly suppressed in these cells. Furthermore, STAP-2/BSK associated with PLC-gamma in vivo. These data indicate that STAP-2/BSK negatively controls the FcepsilonRI-mediated calcium mobilization and degranulation by direct modulation of tyrosine phosphorylation of PLC-gamma. (C) 2003 Elsevier Science (USA). All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE, English

Regulation of Fc epsilon RI-mediated signaling by an adaptor protein STAP-2/BSK in rat basophilic leukemia RBL-2H3 cells
T Yamamoto, T Yumioka, Y Sekine, N Sato, M Minoguchi, A Yoshimura, T Matsuda, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 306, 3, 767, 773, Jul. 2003
Crosslinking of multivalent antigen bound IgE transduces FcepsilonRI 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 FcepsilonRI-mediated calcium mobilization and degranulation. FcepsilonRI-induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) but not Syk was significantly suppressed in these cells. Furthermore, STAP-2/BSK associated with PLC-gamma in vivo. These data indicate that STAP-2/BSK negatively controls the FcepsilonRI-mediated calcium mobilization and degranulation by direct modulation of tyrosine phosphorylation of PLC-gamma. (C) 2003 Elsevier Science (USA). All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE, English

Molecular interactions between STAT3 and protein inhibitor of activated STAT3, and androgen receptor
T Yamamoto, N Sato, Y Sekine, T Yumioka, S Imoto, A Junicho, H Fuse, T Matsuda, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 306, 2, 610, 615, Jun. 2003
STAT3 mainly acts as a signal transducer of IL-6 family cytokines and transcriptionally activates specific target genes. The recently discovered protein inhibitor of activated STAT3 (PIAS3) binds directly to STAT3 and blocks transcriptional activation. In our previous report, we demonstrated that PIAS3 directly interacted with androgen receptor (AR) and affected AR-mediated gene activation. Furthermore, we also showed that AR associated with STAT3 and enhanced its activity. Here, we examined molecular interactions between STAT3, PIAS3, and AR to underline the mechanism of how they regulate each other. AR activation overcame the inhibitory effect on STAT3-mediated transcription by PIAS3. Co-immunoprecipitation experiments revealed that an active form of AR relieved STAT3 from STAT3-PIAS3 complex formation. These results indicate that AR and PIAS3 regulate the STAT3-mediated transcriptional activity by their physical protein-protein competition on STAT3. (C) 2003 Elsevier Science (USA). All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE, English

Daxx enhances Fas-mediated apoptosis in a murine pro-B cell line, BAF3
R Muromoto, T Yamamoto, T Yumioka, Y Sekine, K Sugiyama, K Shimoda, K Oritani, T Matsuda, FEBS LETTERS, 540, 1-3, 223, 228, Apr. 2003
Daxx has been shown to play an essential in type I interferon (IFN-alpha/beta)-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and nuclear translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the mechanism of Daxx-mediated apoptosis signaling in B lymphocyte progenitors, here we introduced an efficient suicide switch in a murine pro-B cell line, BAF3, by expressing FK506-binding protein-fused Fas intracellular domain (FKBP-Fas) and Daxx. It allows us to monitor Fas/Daxx-mediated signal by induction of Fas dimerization with the dimerizer drug AP20187. AP20187-mediated Fas dimerization induced not only apoptosis but also Jun N-terminal kinase (JNK) activation. However, AP20187 had no effect on cells expressing either Fas or Daxx only. Furthermore, expression of a JNK inhibitor, the JNK-binding domain of JIP-1, resulted in resistance to AP20187-mediated apoptosis in cells expressing FKBP-Fas and Daxx. These results imply that our novel suicide switch system may provide a powerful tool to delineate or identify the signaling molecules for Daxx-mediated apoptotic machinery in B lymphocyte progenitors through JNK activation. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies., ELSEVIER SCIENCE BV, English

STAP-2/BKS, an adaptor/docking protein, modulates STAT3 activation in acute-phase response through its YXXQ motif
M Minoguchi, S Minoguchi, D Aki, A Joo, T Yamamoto, T Yumioka, T Matsuda, A Yoshimura, JOURNAL OF BIOLOGICAL CHEMISTRY, 278, 13, 11182, 11189, Mar. 2003
As a c-fins-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., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, English

STAP-2/BKS, an adaptor/docking protein, modulates STAT3 activation in acute-phase response through its YXXQ motif
M Minoguchi, S Minoguchi, D Aki, A Joo, T Yamamoto, T Yumioka, T Matsuda, A Yoshimura, JOURNAL OF BIOLOGICAL CHEMISTRY, 278, 13, 11182, 11189, Mar. 2003
As a c-fins-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., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, English

Molecular interactions between STAT3 and protein inhibitor of activated STAT3, and androgen receptor　　　　　　　　　　　　　　　
Biochem.Biophys.Ress.Commun, 360, 767, 773, 2003

Involvement of heat-shock protein 90 in the interleukin-6-mediated signaling pathway through STAT3
N Sato, T Yamamoto, Y Sekine, T Yumioka, A Junicho, H Fuse, T Matsuda, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 300, 4, 847, 852, Jan. 2003
Interleukin-6 (IL-6) is a multifunctional cytokine playing roles in the immune system, hematopoiesis, and acute phase reactions. IL-6 also regulates the growth of various types of human malignant tumors. Here we demonstrate that IL-6-induced gene expression was suppressed by a specific heat-shock protein 90 (Hsp90) inhibitor, geldanamycin (GA) in human hepatoma Hep3B cells. GA also suppressed the IL-6-induced activation of signal transducer and activator of transcription 3 (STAT3) in a human embryonic kidney carcinoma 293T cells. This inhibitory effect of GA on STAT3 activation was reversed by overexpression of Hsp90. Furthermore, Hsp90 directly bound to STAT3 via its N-terminal region, which interacted with GA. We provide evidence that the action of GA on IL-6 functions was due to the inhibition of direct physical interactions between STAT3 and Hsp90, which represents a novel role of Hsp90 in the IL-6 signaling pathways. (C) 2002 Elsevier Science (USA). All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE, English

Mapping of the sites responsible for factor I-cofactor activity for cleavage of C3b and C4b on human C4b-binding protein (C4bp) by deletion mutagenesis
A Fukui, T Yuasa-Nakagawa, Y Murakami, K Funami, N Kishi, T Matsuda, T Fujita, T Seya, S Nagasawa, JOURNAL OF BIOCHEMISTRY, 132, 5, 719, 728, Nov. 2002
Human C4b-binding protein (C4bp) facilitates the factor I-mediated proteolytic cleavage of the active forms of complement effectors C3b and C4b into their inactive forms. C4bp comprises a disulfide-linked heptamer of alpha-chains with complement (C) regulatory activity and a beta-chain. Each alpha-chain contains 8 short consensus repeat (SCR) domains. Using SCR-deletion mutants of recombinant multimeric C4bp, we identified the domains responsible for the C3b/C4b-binding and C3b/C4b-inactivating cofactor activity. The C4bp mutant with deletion of SCR2 lost the C4b-binding ability, as judged on C3b/C4b-Sepharose binding assaying and ELISA. In contrast, the essential domains for C3b-binding extended more to the C-terminus, exceeding SCR4. Using fluid phase cofactor assaying and deletion mutants of C4bp, SCR2 and 3 were found to be indispensable for C4b cleavage by factor I, and SCR1 contributed to full expression of the factor I-mediated C4b cleaving activity. On the other hand, SCR1, 2, 3, 4, and 5 participated in the factor I-cofactor activity for C3b cleavage, and SCR2, 3, and 4 were absolutely required for C3b inactivation. Thus, different sets of SCRs participate in C3b and C4b inactivation, and the domain repertoire supporting C3b cofactor activity is broader than that supporting C4b inactivation by C4bp and factor I. Furthermore, the domains participating in C3b/C4b binding are not always identical to those responsible for cofactor activity. The necessity of the wide range of SCRs in C3b inactivation compared to C4b inactivation by C4bp and factor I may reflect the physiological properties of C4bp, which is mainly directed to C4b rather than C3b., JAPANESE BIOCHEMICAL SOC, English

Tyk2 is required for the induction and nuclear translocation of Daxx which regulates interferon-alpha-induced suppression of B lymphocyte formation.
K Shimoda, K Kamesaki, A Numata, K Aoki, T Matsuda, K Oritani, K Kato, K Takase, R Imamura, T Miyamoto, K Nagafuji, H Gondo, KI Nakayama, M Harada, BLOOD, 100, 11, 190A, 190A, Nov. 2002
AMER SOC HEMATOLOGY, English, Summary international conference

AML1/MTG8 leukemic protein induces activation of STAT3
K Aoki, K Shimoda, T Matsuda, A Numata, K Kamezaki, K Takase, T Haro, T Miyamoto, K Nagafuji, H Gondo, M Harada, BLOOD, 100, 11, 209B, 209B, Nov. 2002
AMER SOC HEMATOLOGY, English, Summary international conference

リミチンによるBリンパ球前駆細胞アポトーシスの誘導とDaxxの関与
松田正, 山本哲也, 室本竜太, 織谷健司, 下田和哉, 日本免疫学会総会・学術集会記録, 32, 173, 31 Oct. 2002
Japanese

Lymphoid enhancer-binding factor-1 binds and activates the recombination-activating gene-2 promoter together with c-Myb and Pax-5 in immature B cells
ZX Jin, H Kishi, XC Wei, T Matsuda, S Saito, A Muraguchi, JOURNAL OF IMMUNOLOGY, 169, 7, 3783, 3792, Oct. 2002
The recombination-activating gene (RAG)-1 and RAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of Ag receptor genes. We studied the regulation of murine RAG-2 promoter and revealed that -41/-17 RAG-2 promoter region, which was indispensable for the RAG-2 promoter activity in B cell lines, contained binding sites for lymphoid enhancer-binding factor-1 (LEF-1), c-Myb, and Pax-5. We showed that these three transcription factors bound the promoter region in vitro and in vivo. Cotransfection assays using a human embryonic kidney cell line (293T) showed that LEF-1, c-Myb, and Pax-5 cooperatively activated the RAG-2 promoter, via their synergistic DNA binding. We also showed that LEF-1, c-Myb, and Pax-5 physically interact in the cells. Finally, we demonstrated that a dominant-negative LEF-1 protein, which lacks the binding site for beta-catenin, suppressed the RAG-2 promoter activity as well as the endogenous RAG-2 expression in a pre-B cell line (18.81). These results suggest that LEF-1/beta-catenin complex regulates the RAG-2 promoter activation in concert with c-Myb and Pax-5 in immature B cells. The link between LEF-1/-beta-catenin and Wnt signaling in B lineage cells will be discussed., AMER ASSOC IMMUNOLOGISTS, English

The nuclear isoform of protein-tyrosine phosphatase TC-PTP regulates interleukin-6-mediated signaling pathway through STAT3 dephosphorylation
T Yamamoto, Y Sekine, K Kashima, A Kubota, N Sato, N Aoki, T Matsuda, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 297, 4, 811, 817, Oct. 2002
In the previous study, we demonstrated that the nuclear isoform of T-cell protein-tyrosine phosphatase (TC-PTP) dephosphorylated and deactivated signal transducer and activator of transcription 5a (STAT5a) and STAT5b, thereby negatively regulating prolactin (PRL)-mediated signaling pathway. In this study, we examined the involvement of the nuclear isoform of TC-PTP in interleukin-6 (IL-6)-mediated signaling pathway. IL-6 is a multifunctional cytokine that plays important roles in the immune system, hematopoiesis, and acute phase reactions, and has also implicated in IL-6-related diseases. Here, we demonstrate that IL-6-induced tyro sine-phosphorylation and activation of STAT3 were suppressed by overexpression of the nuclear isoform of TC-PTP in 293T cells. Tyrosine-phosphorylated STAT3 directly interacted with a substrate-trapping mutant of TC-PTP, Furthermore, retrovirus-mediated overexpression of the nuclear isoform of TC-PTP suppressed the IL-6-induced growth arrest of myeloid leukemia M1 cells. Endogenous TC-PTP complexed with STAT3 in the nucleus of M1 cells. These results strongly suggest that the nuclear isoform of TC-PTP may serve as a negative regulator of IL-6-mediated signaling pathway. (C) 2002 Elsevier Science (USA). All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE, English

樹状細胞におけるCD40発現の選択的制御　　　　　　　　　　　　　　　
飯島 則文, 柳川 芳毅, 松田 正, 小野江 和則, 日本免疫学会総会・学術集会記録, 32, 54, 54, Oct. 2002
(NPO)日本免疫学会, Japanese

Characterization of chromatin structure and enhancer elements for murine recombination activating gene-2
XC Wei, H Kishi, ZX Jin, WP Zhao, S Kondo, T Matsuda, S Saito, A Muraguchi, JOURNAL OF IMMUNOLOGY, 169, 2, 873, 881, Jul. 2002
Recombination-activating genes (RAGs) play a critical role in V(D)J recombination machinery and their expression is specifically regulated during lymphocyte ontogeny. To elucidate the molecular mechanisms regulating murine RAG-2 expression, we examined a chromatin structure of 25-kb DNA segment adjacent to murine RAG-2 by analyzing DNase I hypersensitive (HS) sites. In a RAG-2-expressing murine pre-B cell line, three lymphoid cell-specific HS sites (HS1, HS2, and HS3) were identified. Among these HS sites, one HS site (HS3) that locates in the RAG-2 promoter was associated only with RAG-2-expressing cell lines. Using the transient enhanced green fluorescence protein reporter gene assays, we identified two enhancer elements in the 5'-upstream region of RAG-2 that corresponded to HS1 and HS2. One of the enhancer elements (D3) exhibited enhancer activity only in the lymphoid cell lines. Analysis of the transgenic mice carrying the enhanced green fluorescence protein-reporter gene linked with D3 revealed that D3 activated the reporter gene-expression in the primary lymphoid tissues, but not in the secondary lymphoid tissues or nonlymphoid tissues. D3 was active in CD4(-)CD8(-), but not in CD4(+)CD8(+) or CD4(+)CD8(-) thymocytes in the thymus, and also active in B220(+)IgM(-), but not in B220(+)IgM(+), cells in the bone marrow. Finally, our data suggested that C/EBP may bind to the D3 enhancer and function as one of the transcription factor(s) responsible for the enhancer activity. These results show that the tissue- and stage-specific expression of murine RAG-2 is regulated by alteration of the chromatin structure as well as cis-regulatory enhancer elements., AMER ASSOC IMMUNOLOGISTS, English

Cross-talk between bone morphogenic proteins and estrogen receptor signaling
T Yamamoto, F Saatcioglu, T Matsuda, ENDOCRINOLOGY, 143, 7, 2635, 2642, Jul. 2002
Bone morphogenic proteins (BMPs) play central roles in differentiation, development, and physiological tissue remodeling. Estrogens have key roles in a variety of biological events, such as the development and maintenance of numerous target tissues. Previous studies demonstrated that estrogens suppress BMP functions by repressing BMP gene expression. Here we present a novel mechanism for the inhibitory effect of estrogens on BMP function. BMP-2-induced activation of Sma and Mad (mothers against decapentaplegic)-related protein (Smad) activity and BMP-2-mediated gene expression were suppressed by 17beta-E2 in breast cancer cells and mesangial cells. E2-mediated inhibition of Smad activation was reversed by tamoxifen, an ER antagonist. We provide evidence that the inhibitory action of ER on Smad activity was due to direct physical interactions between Smads and ER, which represents a novel mechanism for the cross-talk between BMP and ER signaling pathways., ENDOCRINE SOC, English

The regulation of DNAse activities in subcellular compartments of activated thymocytes
T Nagata, H Kishi, QL Liu, T Matsuda, T Imanaka, K Tsukada, D Kang, A Muraguchi, IMMUNOLOGY, 105, 4, 399, 406, Apr. 2002
Thymocytes expressing self-reactive T-cell receptors (TCR) are eliminated in the thymus through a TCR-mediated signal. This cell death signal (negative selection) generates nuclear morphological change and DNA fragmentation in thymocytes. However, the pathway leading to DNA fragmentation of thymocytes following TCR engagement remains obscure. In this study, we investigated the localization and function of caspase-activated DNAse (CAD) and its inhibitor (ICAD) in thymocytes prior to or after in vivo TCR stimulation. We showed that CAD and ICAD are co-localized in microsome, nuclei and cytosol in unstimulated thymocytes. Following in vivo TCR engagement, ICAD located in cytosol and microsome was degraded and the resulting activated CAD induced chromosomal DNA fragmentation. CAD present in cytosol and microsome of unstimulated thymocytes was activated by recombinant caspase-3, and microsomal CAD was released to the cytosol. These results demonstrate that TCR engagement of thymocytes induces caspase-3-dependent activation of CAD localized in both cytosol and microsome, leading to DNA fragmentation in harmony., BLACKWELL PUBLISHING LTD, English

Partial impairment of interleukin-12 (IL-12) and IL-18 signaling in Tyk2-deficient mice
K Shimoda, H Tsutsui, K Aoki, K Kato, T Matsuda, A Numata, K Takase, T Yamamoto, H Nukina, T Hoshino, Y Asano, H Gondo, T Okamura, S Okamura, KI Nakayama, K Nakanishi, Y Niho, M Harada, BLOOD, 99, 6, 2094, 2099, Mar. 2002
Tyk2 is activated in response to interleukin-12 (IL-12) and is essential for IL-12-induced T-cell function, including interferon-gamma (IFN-gamma) production and Th1 cell differentiation. Because IL-12 is a stimulatory factor for natural killer (NK) cell-mediated cytotoxicity, we examined whether tyk2 is required for IL-12-induced NK cell activity. IL-12-induced NK cell activity in cells from tyk2-deficient mice was drastically reduced compared to that in cells from wild-type mice. IL-18 shares its biologic functions with IL-12. However, the molecular mechanism of IL-18 signaling, which activates an IL-1 receptor-associated kinase and nuclear translocation of nuclear factor-kappaB, is different from that of IL-12. We next examined whether biologic functions induced by IL-18 are affected by the absence of tyk2. NK cell activity and IFN-gamma production induced by IL-18 were reduced by the absence of tyk2. Moreover, the synergistic effect of IL-12 and IL-18 for the production of IFN-gamma was also abrogated by the absence of tyk2. This was partially due to the absence of any up-regulation of the IL-18 receptor treated with IL-12, and it might suggest the presence of the cross-talk between Jak-Stat and mitogen-activated protein kinase pathways In cytokine signaling., AMER SOC HEMATOLOGY, English

Vasoactive intestinal peptide (VIP) enhances the cell motility of androgen receptor-transfected DU-145 prostate cancer cells (DU-145/AR)
O Nagakawa, J Murata, A Junicho, T Matsuda, Y Fujiuchi, H Fuse, Saiki, I, CANCER LETTERS, 176, 1, 93, 99, Feb. 2002
We established a clonal DU-145 prostate cancer cell line (DU-145/AR) stably transfected with androgen receptor (AR) cDNA and investigated the expression of type I vasoactive intestinal peptide (VIP) receptor (VIP1R) and type 2 VIP receptor (VIP2R) mRNA in these cells by reverse transcriptase-polymerase chain reaction analysis and the effect of VIP on the invasion and the haptotactic migration of these cells. DU-145/AR cells constitutively expressed both VIP1R and VIP2R mRNA, but the parent DU-145 cells did not. VIP increased the invasive capacity of DU-145/AR cells. VIP also enhanced the haptotactic migration of these cells to fibronectin. However, the growth of these tumor cells was not affected by VIP at any concentrations used in this study. These results indicate that VIP may play a role in the regulation of the invasion of prostate cancer. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved., ELSEVIER SCI IRELAND LTD, English

LEF-1 binds and activates the recombination-activating gene-2 promoter together with c-Myb and Pax-5 in immature B cells.　　　　　　　　　　　　　　　
J. Immunol., 169,3783-3792, 2002

Cooperative binding of c-Myb and Pax-5 activates the RAG-2 promoter in immature B cells
H Kishi, ZX Jin, XC Wei, T Nagata, T Matsuda, S Saito, A Muraguchi, BLOOD, 99, 2, 576, 583, Jan. 2002
The recombination activating gene-1 (RAG-1)and RAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of antigen receptor genes. The regulation of murine RAG-2 promoter was studied and it was revealed that the -41/-17 RAG-2 promoter region, which is conserved between humans and mice, was indispensable for the RAG-2 promoter activity in B-cell lines. The region contained 2 cis elements that bound c-Myb and Pax-5. Mutation in the c-Myb-binding site in the promoter reduced the promoter activity in B-cell lines. Cooperative activation of the RAG-2 promoter was seen by a combination of c-Myb and Pax-5 in a human embryonic kidney cell line (293T), via their synergistic DNA-binding. Deletion experiments showed that the C-terminus of c-Myb was responsible for their interaction. Furthermore, the dominant-negative c-Myb mutant suppressed the activation of the RAG-2 promoter in a pre-B-cell line as well as In 293T cells. These results suggest that cooperative binding of c-Myb and Pax-5 to the RAG-2 promoter Is one of the mechanisms to direct the restricted expression of the RAG-2 In Immature B cells. (Blood. 2002;99:576-583) (C) 2002 by The American Society of Hematology., AMER SOC HEMATOLOGY, English

Constitutive STAT5 activation by the TEL-SYK fusion gene from a patient with myelodysplastic syndrome.
T Kanie, A Abe, T Matsuda, Y Kuno, M Towatari, N Emi, H Saito, BLOOD, 98, 11, 563A, 563A, Nov. 2001
AMER SOC HEMATOLOGY, English, Summary international conference

Cross-talk between signal transducer and activator of transcription 3 and androgen receptor signaling in prostate carcinoma cells
T Matsuda, A Junicho, T Yamamoto, H Kishi, K Korkmaz, F Saatcioglu, H Fuse, A Muraguchi, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 283, 1, 179, 187, Apr. 2001, ［Internationally co-authored］
Interleukin 6 (IL-6) plays important roles in the immune system, hematopoiesis, as well as the growth of various tumors. Androgens are important in the initiation and progression of prostate cancer and their effects are mediated by androgen receptor (AR). Here we present a molecular mechanism for the effects of IL-6 on prostate cancer cells through a cross-talk between IL-6 and AR signaling pathways. IL-6-induced activation of signal transducer and activator of transcription 3 (STAT3) was augmented by AR in the presence of dihydrotestosterone (DHT). In addition, DHT treatment augmented endogenous STAT3-mediated gene expression by IL-6. Conversely, DHT-induced AR activity was increased by IL-6, and a dominant negative form of STAT3 inhibited AR activation. In contrast, DHT-mediated enhancement of STAT3 activation was inhibited by flutamide, an AR antagonist. We provide evidence that these activities are due to direct physical interactions between STAT3 and AR in prostate cancer cells. (C) 2001 Academic Press., ACADEMIC PRESS INC, English

Cross-talk between IL-6 and TGF-beta signaling in hepatoma cells
T Yamamoto, T Matsuda, A Muraguchi, K Miyazono, M Kawabata, FEBS LETTERS, 492, 3, 247, 253, Mar. 2001
Interleukin-6 (IL-6) is a multifunctional cytokine that plays important roles in the immune system, hematopoiesis, and acute phase reactions. Transforming growth factor-beta (TGF-beta) also has pleiotropy including the production of acute phase proteins in hepatocytes, To elucidate the cross-talk between IL-6 and TGF-beta signaling pathways in hepatic cells, we investigated the effects of TGF-beta on IL-6-induced signal transducer and activator of transcription-3 (STAT3) activation in a human hepatoma cell line, Hep3B, IL-6-induccd activation of STAT3 activity and STAT3-mediated gene expression were augmented by TGF-beta in Hep3B cells, We provide evidence that these activities were due to physical interactions between STAT3 and Sma- and MAD-related protein-3, bridged by p300. These results demonstrate a molecular mechanism of a cross-talk between STAT3 and TGF-beta signaling pathways in hepatocytes, (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved., ELSEVIER SCIENCE BV, English

Cross-talk between IL-6 and TGF-beta signaling in hepatoma cells
T Yamamoto, T Matsuda, A Muraguchi, K Miyazono, M Kawabata, FEBS LETTERS, 492, 3, 247, 253, Mar. 2001
Interleukin-6 (IL-6) is a multifunctional cytokine that plays important roles in the immune system, hematopoiesis, and acute phase reactions. Transforming growth factor-beta (TGF-beta) also has pleiotropy including the production of acute phase proteins in hepatocytes, To elucidate the cross-talk between IL-6 and TGF-beta signaling pathways in hepatic cells, we investigated the effects of TGF-beta on IL-6-induced signal transducer and activator of transcription-3 (STAT3) activation in a human hepatoma cell line, Hep3B, IL-6-induccd activation of STAT3 activity and STAT3-mediated gene expression were augmented by TGF-beta in Hep3B cells, We provide evidence that these activities were due to physical interactions between STAT3 and Sma- and MAD-related protein-3, bridged by p300. These results demonstrate a molecular mechanism of a cross-talk between STAT3 and TGF-beta signaling pathways in hepatocytes, (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved., ELSEVIER SCIENCE BV, English

Hematopoietic cell-specific adapter proteins, SLP-76 and BLNK, effectively activate NF-AT as well as NF-kappa B by Syk and Tec PTKs in non-lymphoid cell lines
T Yamamoto, T Matsuda, A Junicho, H Kishi, A Yoshimura, A Muraguchi, FEBS LETTERS, 491, 3, 272, 278, Mar. 2001
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 KB (NF-kappaB) activation, we reconstituted TCR-signaling with CD8/zeta, 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-kappaB. We also show that Tec family PTKs enhanced endogenous phospholipase C (PLC)-gamma1 phosphorylation induced by CD8/zeta and Syk in 293T cells. These results imply that PLC-yl may play a critical role in a hematopoietic cell-specific adapter protein-mediated NF-AT and NF-kappaB activation in a non-lymphoid cell. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved., ELSEVIER SCIENCE BV, English

前立腺癌細胞におけるProtein inhibitor of activated STAT3(PIAS3)によるアンドロゲン受容体の負の制御機構
十二町 明, 松田 正, 永川 修, 村口 篤, 布施 秀樹, 日本泌尿器科學會雜誌, 92, 2, 267, 267, 20 Feb. 2001
社団法人日本泌尿器科学会, Japanese

Cross-talk between TGF-beta and estrogen receptor signaling through Smad3.　　　　　　　　　　　　　　　
J. Biol. Chem., 276: 42907-42914, 2001

Cross-talk between TGF-beta and estrogen receptor signaling through Smad3.　　　　　　　　　　　　　　　
J. Biol. Chem., 276: 42907-42914, 2001

Cross-talk between signal transducer and activator of transcription 3 and estrogen receptor signaling
T Yamamoto, T Matsuda, A Junicho, H Kishi, F Saatcioglu, A Muraguchi, FEBS LETTERS, 486, 2, 143, 148, Dec. 2000, ［Internationally co-authored］, [International Magazine]
Interleukin-6 (IL-6) is a multifunctional cytokine that plays important roles in the immune system, hematopoiesis, and acute phase reactions. Estrogens have significant roles in a variety of biological events, such as the development and maintenance of female reproductive organs, and bone and lipid metabolism. Previous studies demonstrated that estrogens suppress IL-6-induced osteoporosis and the growth of multiple myeloma cells by repressing IL-6 and IL-6 receptor gene expression. Here we present a novel mechanism for the inhibitory effect of estrogens on IL-6 function. IL-6-induced activation of signal transducer and activator of transcription 3 (STAT3) activity and STAT3-mediated gene expression were suppressed by 17 beta -estradiol (E2) in breast cancer cells. E2-mediated inhibition of STAT3 activation was reversed by tamoxifen, an estrogen receptor (ER) antagonist. We provide evidence that the inhibitory action of ER on STAT3 activity was due to direct physical interactions between STAT3 and ER which represents a novel form of cross-talk between STAT3 and ER signaling pathways. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved., ELSEVIER SCIENCE BV, English

Protein inhibitor of activated STAT3 regulates androgen receptor signaling in prostate carcinoma cells
A Junicho, T Matsuda, T Yamamoto, H Kishi, K Korkmaz, F Saatcioglu, H Fuse, A Muraguchi, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 278, 1, 9, 13, Nov. 2000, ［Internationally co-authored］, [International Magazine]
Protein inhibitor of activated STAT3 (PIAS3) is a specific inhibitor of signal transducer and activator of transcription 3 (STAT3). PIAS3 binds to STAT3 and inhibits its DNA-binding activity, and thereby STAT3-mediated gene activation. PIAS1, another member of the PIAS family, was recently shown to interact with the androgen receptor (AR), a nuclear hormone receptor that has an important role in both physiological and pathological processes, and acts as a cofactor for AR. Here we demonstrate that PIAS3 is expressed in prostate cancer cells and its expression is induced in response to dihydrotestosterone (DHT) treatment. Ectopic overexpression of PIAS3 suppressed AR-mediated gene activation induced by DHT-stimulation in LNCaP cells. We provide evidence that these activities were due to direct physical interactions between PIAS3 and AR. These results indicate that PIAS3 acts as a coregulator of AR signaling pathway in prostate cancer cells, (C) 2000 Academic Press., ACADEMIC PRESS INC, English

Tyk2 plays a restricted role in IFN alpha signaling, although it is required for IL-12-mediated T cell function
K Shimoda, K Kato, K Aoki, T Matsuda, A Miyamoto, M Shibamori, M Yamashita, A Numata, K Takase, S Kobayashi, S Shibata, Y Asano, H Gondo, K Sekiguchi, K Nakayama, T Nakayama, T Okamura, S Okamura, Y Niho, K Nakayama, IMMUNITY, 13, 4, 561, 571, Oct. 2000
Janus kinases (Jaks) play an important role in signal transduction via cytokine receptors. Tyk2 is a Janus kinase, and we developed tyk2-deficient mice to study the requirement for tyk2 in vivo. Tyk2-deficient mice show no overt developmental abnormalities; however, they display a lack of responsiveness to a small amount of IFN alpha, although a high concentration of IFN alpha can fully transduce its signal even in the absence of tyk2. Furthermore, IL-12-induced T cell function is defective in these mice. In contrast, these mice respond normally to IL-6 and IL-10, both of which activate tyk2 in vitro. These observations demonstrate that tyk2 plays only a restricted role in mediating IFN alpha -dependent signaling while being required in mediating IL-12-dependent biological responses., CELL PRESS, English

Possible involvement of cyclophilin B and caspase-activated deoxyribonuclease in the induction of chromosomal DNA degradation in TCR-stimulated thymocytes
T Nagata, H Kishi, QL Lin, T Yoshino, T Matsuda, ZX Jin, K Murayama, K Tsukada, A Muraguchi, JOURNAL OF IMMUNOLOGY, 165, 8, 4281, 4289, Oct. 2000
TCR engagement of immature CD4(+)CD8(+) thymocytes induces clonal maturation (positive selection) as well as clonal deletion (negative selection) in the thymus, However, the cell death execution events of thymocytes during the negative selection process remain obscure. Using a cell-free system, we identified two different DNase activities in the cytosol of in vivo anti-TCR-stimulated murine thymocytes: one that induced chromosomal DNA fragmentation, which was inhibited by an inhibitor of caspase-activated DNase, and another that induced plasmid DNA degradation, which was not inhibited by an inhibitor of caspase-activated DNase, We purified the protein to homogeneity that induced plasmid DNA degradation from the cytosol of anti-CD3-stimulated thymocytes and found that it is identical with cyclophilin B (Cyp B), which was reported to locate in endoplasmic reticulum, Ab against Cyp B specifically inhibited the DNA degradation activity in the cytosol of anti-CD3-stimulated thymocytes, Furthermore, recombinant Cyp B induced DNA degradation of naked nuclei, but did not induce internucleosomal DNA fragmentation, Finally, we demonstrated that TCR engagement of a murine T cell line (EL4) with anti-CD3/CD28 resulted in the release of Cyp B from the microsome fraction to the cytosol/nuclear fraction. Our data strongly suggest that both active caspase-activated DNase and Cyp B may participate in the induction of chromosomal DNA degradation during cell death execution of TCR-stimulated thymocytes., AMER ASSOC IMMUNOLOGISTS, English

Lineage-specific regulation of the murine RAG-2 promoter: GATA-3 in T cells and Pax-5 in B cells
H Kishi, XC Wei, ZX Jin, Y Fujishiro, T Nagata, T Matsuda, A Muraguchi, BLOOD, 95, 12, 3845, 3852, Jun. 2000
Recombination activating gene-1 (RAG-1) and RAGE are expressed in lymphoid cells undergoing the antigen receptor gene rearrangement. A study of the regulation of the mouse RAG-2 promoter showed that the lymphocyte-specific promoter activity is conferred 80 nucleotide (nt) upstream of RAG-2. Using an electrophoretic mobility shift assay, it was shown that a B-cell-specific transcription protein, Pax-5, and a T-cell-specific transcription protein, GATA-3, bind to the -80 to -17 nt region in B cells and T cells, respectively, Mutation of the RAG-2 promoter for Pax-5- and GATA-3-binding sites results in the reduction of promoter activity in B cells and T cells, These results indicate that distinct DNA binding proteins, Pax-5 and GATA-3, may regulate the murine RAG-2 promoter in B and T lineage cells, respectively. (C) 2000 by The American Society of Hematology., AMER SOC HEMATOLOGY, English

SOCS-1 can suppress CD3 zeta- and Syk-mediated NF-AT activation in a non-lymphoid cell line
T Matsuda, T Yamamoto, H Kishi, A Yoshimura, A Muraguchi, FEBS LETTERS, 472, 2-3, 235, 240, Apr. 2000
To elucidate T cell antigen receptor (TCR) signaling leading to activation nuclear factor of activated T cells (NF-AIT), we reconstituted TCR signaling to activate NF-AT in a non-lymphoid cell line, 293T, We demonstrated that co-expression of CD8/zeta and Syk were neccesary for NF-AT activation in 293T, This NF-AT response was completely inhibited hy the addition of cyclosporin A or FK506, but markedly enhanced by the additional expression of Tee protein tyrosine kinase, We also show that the cytokine signaling suppressor, suppressor of cytokine signaling 1, potently inhibited this response by interacting with Syk and immunoreceptor tyrosine-based activation motifs in CD8/zeta, These results imply that this novel system may provide a useful tool to delineate or identify the regulatory molecules for CD3 zeta/Syk-mediated NF-AT activation. (C) 2000 Federation of European Biochemical Societies., ELSEVIER SCIENCE BV, English

Apoptosis-inducing activity in a microsomal fraction of TCR-stimulated mouse thymocytes
T Nagata, H Kishi, KL Lyu, T Yoshino, T Matsuda, A Muraguchi, FASEB JOURNAL, 14, 6, A923, A923, Apr. 2000
FEDERATION AMER SOC EXP BIOL, English, Summary international conference

サイトカインシグナル伝達系の研究
松田 正, 癌と人, 27, 38, 39, 31 Mar. 2000
大阪癌研究会, Japanese

IL-6シグナル伝達系とアンドロゲン受容体のクロストーク
十二町 明, 松田 正, 村口 篤, 永川 修, 布施 秀樹, 日本泌尿器科學會雜誌, 91, 3, 346, 346, 20 Mar. 2000
社団法人日本泌尿器科学会, Japanese

Lactate dehydrogenase A-dependent surface expression of immature thymocyte antigen-1: an implication for a novel trafficking function of lactate dehydrogenase-A during T cell development
Y Fujishiro, H Kishi, T Matsuda, H Fuse, A Muraguchi, EUROPEAN JOURNAL OF IMMUNOLOGY, 30, 2, 516, 524, Feb. 2000
A possible involvement of lactate dehydrogenase A (LDHA) in the translocation of a thymic differentiation antigen, immature thymocyte antigen-1 (IMT-1), from cytoplasm to cell surface membrane during thymocyte differentiation is described. Transfection of cDNA far LDHA, but not LDHB, into EL4 cells, which expressed IMT-1 in the cytoplasm but not on the cell surface, induced the expression of IMT-1 on the cell surface. This translocation process seemed to be dependent on the translation of LDHA cDNA in EL4 cells, as well as the native structure of LDHA composing of coenzyme binding domain, catalysis domain, and subunit contact domain. Immunoprecipitation analysis revealed that LDHA could be co-precipitated with IMT-1 from the cell surface of EL4 cells that had been transfected with LDHA cDNA, suggesting that some of LDHA is associated with cell surface IMT-1 on the transfectants. Flow cytometry analysis of thymocyte subpopulations showed that some thymocytes at the CD4(-)CD8(-) double negative stage express both IMT-1 and LDHA on their surface; These data indicate that LDHA, in addition to its function in the metabolism in the glycolytic pathway, may have a novel function in the expression of a cell surface antigen during T cell development., WILEY-V C H VERLAG GMBH, English

胸腺T細胞のアポトーシス誘導とその制御　　　　　　　　　　　　　　　
臨床免疫, 34, 424-433, 2000

Distinct factors, GATA-3 and Pax-5, may reciprocally regulate the murine RAG2 promoter in T-and B-lineage cells.　　　　　　　　　　　　　　　
Blood., 95: 3845-3852, 2000

Distinct factors, GATA-3 and Pax-5, may reciprocally regulate the murine RAG2 promoter in T-and B-lineage cells.　　　　　　　　　　　　　　　
Blood., 95: 3845-3852, 2000

A bone marrow-derived stroma cell line, ST2, can support the differentiation of fetal thymocytes from the CD4(-) CD8(-) double negative to the CD4(+) CD8(+) double positive differentiation stage in vitro
JJ Tong, H Kishi, T Matsuda, A Muraguchi, IMMUNOLOGY, 97, 4, 672, 678, Aug. 1999
T-cell precursors differentiate into mature T cells predominantly in the thymus. However, it has also been reported that T-cell precursors mature in extrathymic organs such as the liver, bone marrow, or intestines. In order to investigate the nature of the extrathymic microenvironment that supports T-cell maturation, we examined the effect of a bone marrow-derived stroma cell line, ST2, on T-cell precursors by using a reaggregate thymic organ culture (RTOC) system. We found that ST2 cells supported the differentiation of fetal thymocytes at day 14.5 of gestation from a CD4(-) CD8(-) double negative (DN) to a CD4(+) CD8(+) double positive (DP) differentiation stage in a manner similar to that observed in thymus. Anti-interleukin-7 receptor (IL-7R) and anti-c-kit antibodies blocked the growth of thymocytes in RTOC with ST2 cells, but did not inhibit the generation of DP thymocytes. These data indicate that a bone marrow-derived stroma cell, ST2, which supports B-cell differentiation, is also able to support T-cell development and may constitute one of the microenvironmental components for extrathymic T;cell development., BLACKWELL SCIENCE LTD, English

The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop
H Yasukawa, H Misawa, H Sakamoto, M Masuhara, A Sasaki, T Wakioka, S Ohtsuka, T Imaizumi, T Matsuda, JN Ihle, A Yoshimura, EMBO JOURNAL, 18, 5, 1309, 1320, Mar. 1999, ［Internationally co-authored］
The Janus family of protein tyrosine kinases (JAKs) regulate cellular processes involved in cell growth, differentiation and transformation through their association with cytokine receptors, However, compared with other kinases, little is known about cellular regulators of the JAKs, We have recently identified a JAK-binding protein (JAB) that inhibits JAK signaling in cells. In the studies presented here we demonstrate that JAB specifically binds to the tyrosine residue (Y1007) in the activation loop of JAK2, whose phosphorylation is required for activation of kinase activity. Binding to the phosphorylated activation loop requires the JAB SH2 domain and an additional N-terminal 12 amino acids (extended SH2 subdomain) containing two residues (Ile68 and Leu75) that are conserved in JAB-related proteins. An additional N-terminal 12-amino-acid region (kinase inhibitory region) of JAB also contributes to high-affinity binding to the JAK2 tyrosine kinase domain and is required for inhibition of JAK2 signaling and kinase activity. Our studies define a novel type of regulation of tyrosine kinases and might provide a basis for the design of specific tyrosine kinase inhibitors., OXFORD UNIV PRESS, English

Activation of Jak2 catalytic activity requires phosphorylation of Y-1007 in the kinase activation loop
J Feng, BA Witthuhn, T Matsuda, F Kohlhuber, IM Kerr, JN Ihle, MOLECULAR AND CELLULAR BIOLOGY, 17, 5, 2497, 2501, May 1997, ［Internationally co-authored］, [International Magazine]
The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes, The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks, In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain, Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y-1007 and Y-1008, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y-1007 and Y-1008 are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions, Mutation of Y-1007, or both Y-1007 and Y-1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y-1008 to phenylalanine had no detectable effect on kinase activity, The mutants were also examined for the ability to reconstitute erythropoietin signaling in gamma 2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y-1007 to phenylalanine eliminated the ability to restore signaling, Moreover, phosphorylation of a kinase-inactive mutant ((KE)-E-882) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y-1007 in Jak2 regulation and function., AMER SOC MICROBIOLOGY, English

Phosphorylation of tyrosine 1007 is required for the activation of Jak2.
J Feng, BA Witthuhn, T Matsuda, IM Kerr, JN Ihle, BLOOD, 88, 10, 1777, 1777, Nov. 1996
AMER SOC HEMATOLOGY, English, Summary international conference

ACTIVATION OF FES TYROSINE KINASE BY GP130, AN INTERLEUKIN-6 FAMILY CYTOKINE SIGNAL TRANSDUCER, AND THEIR ASSOCIATION
T MATSUDA, T FUKADA, M TAKAHASHITEZUKA, Y OKUYAMA, Y FUJITANI, Y HANAZONO, H HIRAI, T HIRANO, JOURNAL OF BIOLOGICAL CHEMISTRY, 270, 19, 11037, 11039, May 1995
gp130 is a signal-transducing subunit of receptors for the interleukin-6 (IL-6)-related cytokine subfamily including IL-6, leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotrophic factor, indicating that gp130-mediated signals are involved in the immune response, hematopoiesis, inflammation, and endocrine and nervous system activity. We previously showed that gp130 stimulation rapidly activates Jak, Btk, and Tec tyrosine kinases, all of which constitutively associate with gp130, To further elucidate intracellular signal transduction through gp130, we examined the possible involvement of another nonreceptor tyrosine kinase, p92(c-fes) (Fes). We showed that gp130 stimulation rapidly induced tyrosine phosphorylation of Fes and actually activated its kinase activity in hematopoietic lineage cells. Furthermore, Fes associated with gp130 independently of ligand stimulation like Jak, Btk, and Tec tyrosine kinases. These results indicate that multiple nonreceptor tyrosine kinases are involved in the gp130-mediated signal transduction pathway. Because both gp130 and Fes are expressed not only in hematopoietic lineage cells but also in heart and nerve cells, Fes may play a role in signal transduction through gp130 in these tissues., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, English, Introduction scientific journal

ASSOCIATION AND ACTIVATION OF BTK AND TEC TYROSINE KINASES BY GP130, A SIGNAL TRANSDUCER OF THE INTERLEUKIN-6 FAMILY OF CYTOKINES
T MATSUDA, M TAKAHASHITEZUKA, T FUKADA, Y OKUYAMA, Y FUJITANI, S TSUKADA, H MANE, H HIRAI, ON WITTE, T HIRANO, BLOOD, 85, 3, 627, 633, Feb. 1995, ［Internationally co-authored］, [International Magazine]
Interleukin-8 (IL-6), leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotrophic factor constitute the IL-6 family of cytokines and play important roles in hematopoiesis, immune response, and nervous system. The receptors for the IL-6 family of cytokines share gp130 through which signals are generated, although the cytoplasmic region of gp130 does not contain any catalytic domain. In this study we show that in addition to Jak family tyrosine kinase, the stimulation of gp130 by IL-6 plus soluble IL-6 receptor cu induced the activation of Btk and Tec tyrosine kinases, whereas IL-3 and granulocyte colony-stimulating factor activated Tec but not Btk in a pro-B cell line. Furthermore, both Btk and Tec kinases were associated with gp130 without the ligand stimulation. Because Btk is a critical tyrosine kinase for B lymphopoiesis and Tec is considered to be involved in hematopoiesis. the results suggest the involvement of gp130-Btk-Tec signal pathway in early lymphohematopoiesis. (C) 1995 by The American Society of Hematology., W B SAUNDERS CO, English, Introduction scientific journal

サイトカインとそのレセプター インターロイキン6(IL-6)　　　　　　　　　　　　　　　
臨床免疫, 27(Suppl. 16), 64-72, 1995

Signal transduction through IL-6 receptor: Involvement of multiple protein kinases, stat factors, and a novel H7-sensitive pathway
K Nakajima, T Matsuda, Y Fujitani, H Kojima, Y Yamanaka, K Nakae, T Takeda, T Hirano, INTERLEUKIN-6 TYPE CYTOKINES, 762, 55, 70, 1995
NEW YORK ACAD SCIENCES, English

ESTABLISHMENT OF THE ELISA FOR MURINE SOLUBLE GP130, A SIGNAL TRANSDUCER FOR THE IL-6 FAMILY CYTOKINE, AND ITS DETECTION IN THE ASCITIC FLUIDS OF TUMOR-BEARING MICE
T MATSUDA, T HIRANO, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 202, 1, 637, 642, Jul. 1994
Interleukin-6 (IL-6) signal is transduced through a membrane glycoprotein gp130, which associates with the IL-6 receptor a chain (IL-6R alpha) in the presence of IL-6. We prepared monoclonal antibodies (mAbs) specific to murine gp130 by immunizing rats and hamsters with a chimeric molecule consisting of the extracellular domain of murine gp130 and human immunoglobulin (Ig) G(1) Fc (m130Ig). Furthermore, we developed a sandwich ELISA for detection of murine soluble gp130 (sgp130) and showed that sgp130 was present in the ascitic fluids of tumor-bearing mice. Because sgp130 can inhibit the biological activities of the IL-6-related cytokine subfamily that can enhance anti-tumor immune response and also has both growth inducing and growth inhibitory activities on various tumors, the results suggest that sgp130 in tumor-bearing host modulates tumor progression. (C) 1994 Academic Press, Inc., ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, English

SIGNAL-TRANSDUCTION THROUGH GP130 THAT IS SHARED AMONG THE RECEPTORS FOR THE INTERLEUKIN-6 RELATED CYTOKINE SUBFAMILY
T HIRANO, T MATSUDA, K NAKAJIMA, STEM CELLS, 12, 3, 262, 277, May 1994
Interleukin 6 (IL-6) and related cytokines, such as leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and IL-11 exhibit multiple functions and redundancy in biological activities and play important roles in the immune response, hematopoiesis, the nervous system and acute phase reactions. These IL-6 family cytokines exhibit a similar helical structure, and their receptors are structurally similar and constitute a cytokine receptor super family. In addition, a receptor subunit is shared among these IL-6 related cytokine subfamily receptors, contributing to one of the mechanisms of functional redundancy of cytokine activities and suggesting the presence of a common signal transduction pathway among these receptors. In this review, we describe the structure of the receptors for IL-6 and its related cytokine subfamily members. Furthermore, we propose a novel mechanism for the generation of cytokine diversity, i.e. the complex of a cytokine and one of its receptor subunits act as a novel cytokine on the cells that express the other receptor subunit(s) capable of acting as a receptor for the complex. Finally, we describe a Ras-independent novel signal transduction pathway that utilizes Jak tyrosine kinase family, Stat protein family and yet unidentified H-7-sensitive pathway. This signal transduction pathway is commonly generated through the receptors for a wide range of cytokines and growth factors., ALPHAMED PRESS, English, Book review

INTERLEUKIN-6-INDUCED TYROSINE PHOSPHORYLATION OF MULTIPLE PROTEINS IN MURINE HEMATOPOIETIC LINEAGE CELLS
T MATSUDA, Y YAMANAKA, T HIRANO, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 200, 2, 821, 828, Apr. 1994
Interleukin-6 (IL-6) is a multifunctional cytokine playing various roles in the immune system, hematopoiesis and acute phase reactions. To elucidate the intracellular signal transduction mechanism through the IL-6 receptor, we investigated IL-6-induced protein tyrosine phosphorylation in murine hematopoietic cell lines, BAFm130 and Y6. IL-6 stimulated tyrosine phosphorylation of multiple cellular proteins, such as gp130, an IL-6 signal transducer; Jak family of the cytoplasmic tyrosine kinases; and the latent cytoplasmic signal transducer and activator of transcription. We showed that the pattern of these tyrosine-phosphorylated molecules was different between BAFm130 and Y6 cells on which IL-6 exhibited different biological activities. (C) 1994 Academic Press, Inc., ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, English

シグナル伝達機構の人為的再構築と修飾　　　　　　　　　　　　　　　
免疫薬理, 12(4), 446-452, 1994

Interleukin 6 receptor and signal transduction, Advances in Cell and Molecular.　　　　　　　　　　　　　　　
Biology of Membranes and Organelles., 3: 65-92, 1994

Association of p72 tyrosine kinase with Stat factors and its activation by IL-3, IL-6 and G-CSF.　　　　　　　　　　　　　　　
Blood, 83: 3457-3461, 1994

Interleukin 6 receptor and signal transduction, Advances in Cell and Molecular.　　　　　　　　　　　　　　　
Biology of Membranes and Organelles., 3: 65-92, 1994

Association of p72 tyrosine kinase with Stat factors and its activation by IL-3, IL-6 and G-CSF.　　　　　　　　　　　　　　　
Blood, 83: 3457-3461, 1994

URINARY LEVELS OF IL-6 IN PATIENTS WITH ACTIVE LUPUS NEPHRITIS
M IWANO, K DOHI, E HIRATA, N KURUMATANI, Y HORII, H SHIIKI, A FUKATSU, T MATSUDA, T HIRANO, T KISHIMOTO, H ISHIKAWA, CLINICAL NEPHROLOGY, 40, 1, 16, 21, Jul. 1993
Using the IL-6 dependent hybridoma, MH60.BSF2, we measured urinary levels of interleukin 6 (IL-6) in 29 patients' with active lupus nephritis. We detected IL-6 activity in the urine of 24 (83%) of 29 patients before the initiation of therapy. The median value of urinary IL-6 levels in patients with a histologic diagnosis of WHO class IV on renal biopsy was significantly higher than that in patients with other classes (p<0.01). After treatment, urinary levels of IL-6 decreased significantly (p<0.001). These data suggest that urinary levels of IL-6 may be a valuable tool for monitoring the progression of lupus nephritis., DUSTRI-VERLAG DR KARL FEISTLE, English

インターロイキン6　　　　　　　　　　　　　　　
外科と代謝・栄養, 27(5), 341-356, 1993

インターロイキン6受容体に連鎖するチロシンキナーゼの解析　　　　　　　　　　　　　　　
成人病の研究, 12, 3-4, 1993

脳神経系と免疫系をめぐるサイトカインシグナル　　　　　　　　　　　　　　　
免疫薬理, 11(2), 125-136, 1993

INTERLEUKIN-6 IN MYELOMA PLASMACYTOMA
S SUEMATSU, T MATSUSAKA, T MATSUDA, T HIRANO, T KISHIMOTO, INTERNATIONAL REVIEW OF EXPERIMENTAL PATHOLOGY, 34, 91, 98, 1993
ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, English, Book review

ABNORMAL DISTRIBUTION OF IL-6 RECEPTOR IN AGED MRL/LPR MICE - ELEVATED EXPRESSION ON B-CELLS AND ABSENCE ON CD4+ CELLS
KOBAYASHI, I, T MATSUDA, T SAITO, K YASUKAWA, H KIKUTANI, T HIRANO, T TAGA, T KISHIMOTO, INTERNATIONAL IMMUNOLOGY, 4, 12, 1407, 1412, Dec. 1992
A mAb against murine IL-6 receptor (IL-6R), KMH7, was obtained by immunization of hamster with recombinant soluble murine IL-6R. Flow cytometry analysis of IL-6R distribution on lymphocytes in BALB/c showed that IL-6R was expressed on peripheral lymph node (LN) T cells of either CD4+ or CD8+ phenotype, and Peyer's patch IgA+ B cells, but not on splenic B cells and thymocytes. A similar distribution was observed in 5 week old MRL/Ipr and 16-week-old MRL/n mice. In contrast, in 16 week old MRL/Ipr mice of both sexes, IL-6R was expressed on splenic IgM+ cells. Peripheral LN CD4+ T cells in 16 week old female MRL/Ipr mice did not express IL-6R. Thymocytes in any population with a phenotype of CD4+ or CD8+, double negative, and double positive were not stained with KMH7 in both BALB/c and MRL/Ipr mice. In both strains, IL-6R was induced in CD4+ or CD8+ thymocytes after 2 days of culture, suggesting that CD4+ thymocytes in MRL/Ipr have a potential to express IL-6R. Our results suggest that overexpression of IL-6R on B cells and absence of IL-6R on peripheral CD4+ cells are concurrent with, or may contribute to, B cell hyperreactivity and T cell abnormality in this strain., OXFORD UNIV PRESS UNITED KINGDOM, English

CHARACTERIZATION OF GROWTH INHIBITORY FACTORS FOR MOUSE MONOCYTIC LEUKEMIA-CELLS
T KASUKABE, J OKABEKADO, Y HONMA, M HOZUMI, T MATSUDA, T HIRANO, LEUKEMIA RESEARCH, 16, 2, 139, 144, Feb. 1992
Growth inhibitory (GI) factors for mouse monocytic leukemia cells were previously found in conditioned medium (CM) of a clone of mouse myeloblastic leukemia MI cells (R1-GI factor) and in CM of mouse lung tissue (L-GI factor). In the present study, the effects of these GI factors on the growth of variant cell lines (Mm-A, Mm-P and Mm-S2) of mouse monocytic line Mm-1 cells were examined. Mm-A are highly leukemogenic to syngeneic SL mice, Mm-P cells are moderately leukemogenic, while Mm-S2 cells have little or no leukemogenicity. The R1-GI factor markedly suppressed the growth of Mm-A cells, whereas it inhibited the growth of Mm-P and Mm-S2 cells only moderately. Recombinant mouse interferon-beta (IFN-beta) had similar target specificities to those of the R1-GI factor on these variant cells. Moreover, anti-mouse IFN-beta antibody completely neutralized the GI activity of the R1-GI factor on Mm-A cells. These results show that the R1-GI factor and mouse IFN-beta are very similar and probably identical proteins. The L-GI factor had different target specificities from the R1-GI factor: it showed strongest GI activity on Mm-P cells, moderate GI activity on Mm-S2 cells and weak GI activity on Mm-A cells. Recombinant human interleukin 6 (IL-6) had similar target specificities to the L-G1 factor on these Mm-1 variant cells. Furthermore, the L-GI factor couLd support the proliferation of IL-6-dependent MH60.BSF2 cells. Anti-mouse IL-6 antiserum neutralized the GI actIvity of the L-GI factor on Mm-P cells. Thus the L-GI factor and mouse IL-6 seem to be closely related and possibly to be identical proteins., PERGAMON-ELSEVIER SCIENCE LTD, English

Interleukin 6.　　　　　　　　　　　　　　　
Encyclopedia of Immunology, 2: 917-919, 1992

Interleukin 6.　　　　　　　　　　　　　　　
Encyclopedia of Immunology, 2: 917-919, 1992

Generation of plasmacytomas with the chromosomal translocation t(12;15) in interleukin 6 transgenic mice
Sachiko Suematsu, Taiji Matsusaka, Tadashi Matsuda, Shinsuke Ohno, Jun-Ichi Miyazaki, Ken-Ichi Yamamura, Toshio Hirano, Tadamitsu Kishimoto, Proceedings of the National Academy of Sciences of the United States of America, 89, 1, 232, 235, 1992
The mechanisms through which pristane or mineral oil can induce plasmacytomas in BALB/c or NZB mice are not fully understood, but involvement of interleukin 6 (IL-6), a growth factor for plasmacytomas and myelomas, has been strongly suggested. To clarify the role of IL-6 in plasmacytomagenesis, a human IL-6 cDNA was introduced into mouse germ lines under the transcriptional control of the murine major histocompatibility complex class I (H-2Ld) promoter. IL-6 transgenic mice of C57BL/6 origin developed a massive plasmacytosis but not plasmacytomas. However, introduction of BALB/c genetic background into IL-6 transgenic mice could generate monoclonal transplantable plasmacytomas with the chromosomal translocation t(12
15). These results provide firm evidence of the critical role of IL-6 in the plasmacytoma development., National Academy of Sciences, English

DISTRIBUTION OF INTERLEUKIN-6 IN NORMAL AND DISEASED HUMAN KIDNEY
A FUKATSU, S MATSUO, H TAMAI, N SAKAMOTO, T MATSUDA, T HIRANO, LABORATORY INVESTIGATION, 65, 1, 61, 66, Jul. 1991
Distribution of interleukin 6 (IL-6) in normal and diseased kidneys was examined by an immunohistochemical method. Four specimens of normal kidney tissue and 47 specimens obtained from diseased kidneys by biopsy were studied by indirect immunofluorescence microscopy using specific monoclonal antibody to human IL-6. In the normal kidney, IL-6 was distributed in the glomerular mesangial area and in vascular walls. In the diseased kidneys, IL-6 was observed in the glomerulus in various patterns. The extent of the distribution of IL-6 correlated with the average number of glomerular cells. When sclerosis appeared, the expression of IL-6 decreased. Occasionally, IL-6 was found in the area of synechiae or crescents as well as in the parietal epithelial cells of the glomerulus with synechiae and/or crescents. In the interstitium, IL-6 was seen in atrophic tubules, and the distribution correlated with the extent of tubular atrophy. The binding of anti-IL-6 monoclonal antibody to the renal sections was completely inhibited by preincubation of the antibody with recombinant IL-6.
These results suggests that IL-6 expression is a good marker for glomerular cell proliferation and that IL-6 may be involved in the formation of synechiae or crescents and in tubulointerstitial injury., NATURE PUBLISHING GROUP, English

Cytokine and hemopoietic factors.Interleukin.6.(IL-6).
FUJIBAYASHI MARI, MATSUDA TADASHI, 臨床検査, 35, 5, 481, 484, May 1991
Japanese

AGE-ASSOCIATED INCREASE IN INTERLEUKIN-6 IN MRL LPR MICE
B TANG, T MATSUDA, S AKIRA, N NAGATA, S IKEHARA, T HIRANO, T KISHIMOTO, INTERNATIONAL IMMUNOLOGY, 3, 3, 273, 278, Mar. 1991
It has been demonstrated that abnormal expression of interleukin 6 (IL-6) may be involved in the pathogenesis of a variety of autoimmune diseases and glomerulonephritis. In this study, we demonstrate an age-associated increase in IL-6 in the sera of MRL/lpr mice but not in control congenic MRL/+ mice, normal strains of BALB/c, C3H/HeN mice, and other autoimmune prone mice, such as (NZB x NZW)F1, (NZB x BXSB)F1 mice. IL-6 activity was detected in the sera of MRL/lpr mice by 3 weeks of age and it was neutralized by anti-mouse IL-6 antibody. The serum IL-6 level was gradually increased with age and reached up to 410 pg/ml around 30 weeks of age. The expression of IL-6 mRNA was detected in the spleen and lymph nodes from 8 weeks of age. Expression of IL-6 mRNA was also detected in C57BL/6-lpr mice, indicating the possible linkage of abnormal expression of the IL-6 gene and the lpr gene. Southern blot analysis demonstrated that both the promoter region and 3' untranslated region of the IL-6 gene were apparently normal, suggesting that deregulated production of IL-6 may be due to abnormal transcriptional control. The data suggest that abnormal expression of the IL-6 gene may play a key role in the development of polyclonal B cell activation and glomerulonephritis in MRL/lpr mice., OXFORD UNIV PRESS UNITED KINGDOM, English

Interleukin 6　　　　　　　　　　　　　　　
現代医療, 23(11), 3077-3087, 1991

インターロイキン6―ベッドサイドから分子生物学へ　　　　　　　　　　　　　　　
侵襲と免疫, 1, 3-14, 1991

サイトカインレセプターファミリー　　　　　　　　　　　　　　　
治療学, 25, 1428-1529, 1991

脳と免疫ーサイトカインの関わり（Ⅱ）ー　　　　　　　　　　　　　　　
ブレインサイエンス, 2: p607-611, 1991

脳と免疫ーサイトカインの関わり（Ⅰ）ー　　　　　　　　　　　　　　　
ブレインサイエンス, 2: p491-495, 1991

インターロイキン6（IL-6）
臨床検査, 35(5), 481-484, 1991

インターロイキン6とそのレセプター　　　　　　　　　　　　　　　
蛋白質・核酸・酵素, 36(7), 1184-1194, 1991

IL-6と免疫応答　　　　　　　　　　　　　　　
造血因子, 2(1), 14-24, 1991

Special issue : from IL-6 to IL-11, SCF.1 IL-6 and immune response.
ORITANI KENJI, MATSUDA TADASHI, HIRANO TOSHIO, 造血因子, 2, 1, 14, 24, Jan. 1991
Japanese

IL-6 IS PRODUCED BY OSTEOBLASTS AND INDUCES BONE-RESORPTION
Y ISHIMI, C MIYAURA, CH JIN, T AKATSU, E ABE, Y NAKAMURA, A YAMAGUCHI, S YOSHIKI, T MATSUDA, T HIRANO, T KISHIMOTO, T SUDA, JOURNAL OF IMMUNOLOGY, 145, 10, 3297, 3303, Nov. 1990
AMER ASSOC IMMUNOLOGISTS, English

PURIFICATION AND CHARACTERIZATION OF SOLUBLE HUMAN IL-6 RECEPTOR EXPRESSED IN CHO CELLS
K YASUKAWA, T SAITO, T FUKUNAGA, Y SEKIMORI, Y KOISHIHARA, H FUKUI, Y OHSUGI, T MATSUDA, H YAWATA, T HIRANO, T TAGA, T KISHIMOTO, JOURNAL OF BIOCHEMISTRY, 108, 4, 673, 676, Oct. 1990
JAPAN BIOCHEMICAL SOC, English

Production of autoimmune diseases by the transfer of IL-6 transgenic bone marrow.
松阪泰二, 末松佐知子, 審良静男, 松田正, 平野俊夫, 青笹克之, 宮崎純一, 山村研一, 岸本忠三, 日本免疫学会総会・学術集会記録, 20, 154, Oct. 1990
Japanese

IL-6 and diseases.
MATSUDA TADASHI, HIRANO TOSHIO, 代謝, 27, 10, 897, 907, Oct. 1990
Japanese

Structure and function of cytokine and receptor.11 Relevance between diversity of a function of IL-6 and disease.
MATSUDA TADASHI, 臨床科学, 26, 9, 1115, 1122, Sep. 1990
Japanese

Interleukin-6(IL-6/BSF-2).
MATSUDA TADASHI, HIRANO TOSHIO, 日本臨床, 48, ’90 Zokan Jo Rinsho Meneki, 292, 297, 11 Jul. 1990
Japanese

PRODUCTION OF INTERLEUKIN-6 BY NORMAL HUMAN TROPHOBLAST
T KAMEDA, N MATSUZAKI, K SAWAI, T OKADA, F SAJI, T MATSUDA, T HIRANO, T KISHIMOTO, O TANIZAWA, PLACENTA, 11, 3, 205, 213, May 1990
W B SAUNDERS CO LTD, English

Lymphokine determination methods. 7. Determination of interleukin-6 (IL-6).
MATSUDA TADASHI, Pure Chem Daiichi, 21, 1, 20, 24, Mar. 1990
Japanese

サイトカインレセプターとシグナル伝達　　　　　　　　　　　　　　　
感染・炎症・免疫, 20(4), 257-275, 1990

IL-6／IL-6受容体の構造と機能および異常発現と疾患との関わり　　　　　　　　　　　　　　　
代謝, 27(10), 897-907, 1990

サイトカインとレセプターの構造と機能IL-6の機能多様化と疾患とのかかわり　　　　　　　　　　　　　　　
臨床科学, 26(9), 1115-1122, 1990

サイトカイン各論 インターロイキン－6（IL-6/BSF-2）　　　　　　　　　　　　　　　
日本臨床, 48(増刊 臨床免疫 上), 292-297, 1990

インターロイキン6（IL-6）と自己免疫疾患　　　　　　　　　　　　　　　
日本臨床, 48(増刊 臨床免疫 上), 785-790, 1990

インターロイキン6産生腫瘍　　　　　　　　　　　　　　　
BIO medica, 5(3), 258-261, 1990

Interleukin 6 (IL-6).　　　　　　　　　　　　　　　
Biotherapy, 2: 363-373, 1990

Interleukin 6 (IL-6).　　　　　　　　　　　　　　　
Biotherapy, 2: 363-373, 1990

INTERLEUKIN-6 AND ITS RECEPTOR IN THE IMMUNE-RESPONSE AND HEMATOPOIESIS
T HIRANO, T TAGA, T MATSUDA, M HIBI, S SUEMATSU, B TANG, M MURAKAMI, T KISHIMOTO, INTERNATIONAL JOURNAL OF CELL CLONING, 8, 155, 167, Jan. 1990
ALPHAMED PRESS, English

Special issue. Anti-receptor antibodies and disorders. Autoimmune disorders and IL-6. For receptor treatment.
HIBI MASAHIKO, SUEMATSU SACHIKO, HIRATA YUICHI, MATSUDA TADASHI, 免疫薬理, 8, 1, 51, 58, Jan. 1990
Japanese

INVOLVEMENT OF IL-6 IN MESANGIAL PROLIFERATIVE GLOMERULONEPHRITIS
Y HORII, A MURAGUCHI, M IWANO, T MATSUDA, T HIRAYAMA, H YAMADA, Y FUJII, K DOHI, H ISHIKAWA, Y OHMOTO, K YOSHIZAKI, T HIRANO, T KISHIMOTO, JOURNAL OF IMMUNOLOGY, 143, 12, 3949, 3955, Dec. 1989
AMER ASSOC IMMUNOLOGISTS, English

Introductory lectures to clinical aspects of allergy. 62. Interleukin 6 (IL-6). Part 2.
ISSHIKI HIROSHI, MATSUDA TADASHI, 月刊アレルギーの臨床, 114, 965, 967, Dec. 1989
Japanese

IGG1 PLASMACYTOSIS IN INTERLEUKIN-6 TRANSGENIC MICE
S SUEMATSU, T MATSUDA, K AOZASA, S AKIRA, N NAKANO, S OHNO, J MIYAZAKI, K YAMAMURA, T HIRANO, T KISHIMOTO, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 86, 19, 7547, 7551, Oct. 1989
NATL ACAD SCIENCES, English

IL-6 and its clinical aspects.
IWANO MASAYUKI, MATSUDA TADASHI, HORII YASUHIRO, HIRANO TOSHIO, Med Immunol, 18, 4, 568, 574, Oct. 1989
Japanese

Interleukin 6(IL-6). 1.
ISSHIKI HIROSHI, MATSUDA TADASHI, 月刊アレルギーの臨床, 112, 819, 822, Oct. 1989
Japanese

Plasmacytosis in IL-6 transgenic mice.
末松佐知子, 松田正, 審良静男, 青笹克之, 中野直子, 宮崎純一, 山村研一, 平野俊夫, 岸本忠三, 日本免疫学会総会・学術集会記録, 19, 105, Oct. 1989
Japanese

PATHOGENIC SIGNIFICANCE OF INTERLEUKIN-6 (IL-6/BSF-2) IN CASTLEMANS DISEASE
K YOSHIZAKI, T MATSUDA, N NISHIMOTO, T KURITANI, L TAEHO, K AOZASA, T NAKAHATA, H KAWAI, H TAGOH, T KOMORI, S KISHIMOTO, T HIRANO, T KISHIMOTO, BLOOD, 74, 4, 1360, 1367, Sep. 1989
W B SAUNDERS CO, English

INTERLEUKIN-6 TRIGGERS THE ASSOCIATION OF ITS RECEPTOR WITH A POSSIBLE SIGNAL TRANSDUCER, GP130
T TAGA, M HIBI, Y HIRATA, K YAMASAKI, K YASUKAWA, T MATSUDA, T HIRANO, T KISHIMOTO, CELL, 58, 3, 573, 581, Aug. 1989
CELL PRESS, English

CONSTITUTIVE PRODUCTION OF INTERLEUKIN 6/B CELL STIMULATORY FACTOR-II FROM INFLAMMATORY SYNOVIUM
N MIYASAKA, K SATO, J HASHIMOTO, H KOHSAKA, K YAMAMOTO, M GOTO, K INOUE, T MATSUDA, T HIRANO, T KISHIMOTO, K NISHIOKA, CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY, 52, 2, 238, 247, Aug. 1989
ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, English

HUMAN INTERLEUKIN-6 AND TUMOR NECROSIS FACTOR-ALPHA PRODUCTION STUDIED AT A SINGLE-CELL LEVEL
U ANDERSSON, T MATSUDA, EUROPEAN JOURNAL OF IMMUNOLOGY, 19, 6, 1157, 1160, Jun. 1989
VCH PUBLISHERS INC, English, Introduction scientific journal

IL-6 BSF2 IN NORMAL AND ABNORMAL REGULATION OF IMMUNE-RESPONSES
T MATSUDA, S SUEMATSU, M KAWANO, K YOSHIZAKI, B TANG, O TANABE, T NAKAJIMA, S AKIRA, T HIRANO, T KISHIMOTO, ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, 557, 466, 477, Jun. 1989
NEW YORK ACAD SCIENCES, English

MOLECULAR-CLONING OF THE CDNAS FOR INTERLEUKIN-6 B-CELL STIMULATORY FACTOR-II AND ITS RECEPTOR
T HIRANO, T TAGA, K YAMASAKI, T MATSUDA, K YASUKAWA, Y HIRATA, H YAWATA, O TANABE, S AKIRA, T KISHIMOTO, ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, 557, 167, 180, Jun. 1989
NEW YORK ACAD SCIENCES, English

PRODUCTION OF INTERLEUKIN-6 AND ITS RELATION TO THE MACROPHAGE DIFFERENTIATION OF MOUSE MYELOID-LEUKEMIA CELLS (M1) TREATED WITH DIFFERENTIATION-INDUCING FACTOR AND 1-ALPHA,25-DIHYDROXYVITAMIN-D3
C MIYAURA, CH JIN, Y YAMAGUCHI, M TOMIDA, M HOZUMI, T MATSUDA, T HIRANO, T KISHIMOTO, T SUDA, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 158, 3, 660, 666, Feb. 1989
ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, English

インターロイキン6（IL-6）　　　　　　　　　　　　　　　
内科, 63(6), 1193-1194, 1989

インターロイキン6　　　　　　　　　　　　　　　
BIOTHERAPY, 3(6), 1472-1486, 1989

サイトカインと自己免疫疾患 特にインターロイキン6を例として　　　　　　　　　　　　　　　
細胞工学, 8(11), 949-957, 1989

免疫グロブリンのクラス決定とB細胞刺激因子
松田 正, 平野 俊夫, 臨床免疫, 21(4), 588-602, 4, p588, 602, 1989
科学評論社, Japanese

脳と免疫 生化学的立場から　　　　　　　　　　　　　　　
代謝, 26(臨時増刊), 45-54, 1989

IL-6の構造と機能　　　　　　　　　　　　　　　
Medical Immunology, 17(1), 6-8, 1989

IL-6の構造・機能・レセプター　　　　　　　　　　　　　　　
実験医学, 7(1), 13-20, 1989

インターロイキン6（その2）　　　　　　　　　　　　　　　
アレルギーの臨床, 9(13), 965-967, 1989

インターロイキン6（その1）　　　　　　　　　　　　　　　
アレルギーの臨床, 9(11), 819-822, 1989

B細胞刺激因子　　　　　　　　　　　　　　　
MEDICO, 21(1), 23-28, 1989

Current concepts of b cell modulation
Tadashi Matsuda, Katsuhiko Yamasaki, Tetsuya Taga, Toshio Hirano, Tadamitsu Kishimoto, International Reviews of Immunology, 5, 2, 97, 109, 1989
Three interleukms with distinct functions, IL-4, IL-5 and IL-6, are involved in the regulation of B cell response into antibody producing cells. The studies with recombinant interleukins, however, demonstrated that the activities of these interleukins were not restricted to B lineage cells but showed a wide variety of biological functions on various tissues and cells. One of the most typical example of multifunctional interleukins is IL-6. It acts not only on B cells as B cell differentiation factor but also on T cells, hematopoietic stem cells, hepatocytes, nerve cells and myeloma cells. Deregulation of the expression of these interleukins was shown to be responsible for various diseases, such as i) IL-4 vs. immediate type hypersensitivity and ii) IL-6 vs. autoimmunity and multiple myelomas. © 1989 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted., Informa Healthcare, English

Interleukin 6 and its receptor: Pathological role in autoimmunity and lymphoid malignancy.　　　　　　　　　　　　　　　
Advances in Immunopharmacology, 4: 161-168, 1989

A MULTIFUNCTIONAL CYTOKINE (IL-6 BSF-2) AND ITS RECEPTOR
T HIRANO, T TAGA, K YAMASAKI, T MATSUDA, B TANG, A MURAGUCHI, Y HORII, S SUEMATSU, Y HIRATA, H YAWATA, M SHIMIZU, M KAWANO, T KISHIMOTO, INTERNATIONAL ARCHIVES OF ALLERGY AND APPLIED IMMUNOLOGY, 88, 1-2, 29, 33, Jan. 1989
KARGER, English

IgGl plasmacytosis in interleukin 6 transgenic mice.
Proc. Natl. Acad. Sci. USA., 86: 7547-7551, 1989

Interleukin 6 and its receptor: Pathological role in autoimmunity and lymphoid malignancy.　　　　　　　　　　　　　　　
Advances in Immunopharmacology, 4: 161-168, 1989

IDENTIFICATION OF ALPHA-2-MACROGLOBULIN AS A CARRIER PROTEIN FOR IL-6
T MATSUDA, T HIRANO, S NAGASAWA, T KISHIMOTO, JOURNAL OF IMMUNOLOGY, 142, 1, 148, 152, Jan. 1989
AMER ASSOC IMMUNOLOGISTS, English

Interleukin-6. Structure, functions and receptors of IL-6.
MATSUDA TADASHI, TAGA TETSUYA, YAMASAKI KATSUHIKO, 実験医学, 7, 1, 13, 20, Jan. 1989
Japanese

EXCESSIVE PRODUCTION OF INTERLEUKIN-6/B CELL STIMULATORY FACTOR-II IN RHEUMATOID-ARTHRITIS
T HIRANO, T MATSUDA, M TURNER, N MIYASAKA, G BUCHAN, B TANG, K SATO, M SHIMIZU, R MAINI, M FELDMAN, T KISHIMOTO, EUROPEAN JOURNAL OF IMMUNOLOGY, 18, 11, 1797, 1801, Nov. 1988, ［Internationally co-authored］, [International Magazine]
VCH PUBLISHERS INC, English

INTERLEUKIN-6 (B-CELL STIMULATORY FACTOR 2)-DEPENDENT GROWTH OF A LENNERT LYMPHOMA-DERIVED T-CELL LINE (KT-3)
S SHIMIZU, T HIRANO, R YOSHIOKA, S SUGAI, T MATSUDA, T TAGA, T KISHIMOTO, S KONDA, BLOOD, 72, 5, 1826, 1828, Nov. 1988
W B SAUNDERS CO, English

IL-6 BSF-2 FUNCTIONS AS A KILLER HELPER FACTOR IN THE INVITRO INDUCTION OF CYTO-TOXIC T-CELLS
M OKADA, M KITAHARA, S KISHIMOTO, T MATSUDA, T HIRANO, T KISHIMOTO, JOURNAL OF IMMUNOLOGY, 141, 5, 1543, 1549, Sep. 1988
AMER ASSOC IMMUNOLOGISTS, English

REGULATION OF BSF-2 IL-6 PRODUCTION BY HUMAN MONONUCLEAR-CELLS - MACROPHAGE-DEPENDENT SYNTHESIS OF BSF-2 IL-6 BY T-CELLS
Y HORII, A MURAGUCHI, S SUEMATSU, T MATSUDA, K YOSHIZAKI, T HIRANO, T KISHIMOTO, JOURNAL OF IMMUNOLOGY, 141, 5, 1529, 1535, Sep. 1988
AMER ASSOC IMMUNOLOGISTS, English

Monoclonal antibody. 2. Techniques for making monoclonal antibody. 6. Effective techniques for making hybridoma using IL-6.
MATSUDA TADASHI, HIRANO TOSHIO, 実験医学, 6, 10, 920, 924, Sep. 1988
Japanese

最新のバイオテクノロジ--21-細胞分子と遺伝子--インタ-ロイキン6とそのレセプタ-
松田 正, 代謝, 25, 9, p859, 869, Sep. 1988
中山書店, Japanese

INDUCTION OF NEURONAL DIFFERENTIATION IN PC12 CELLS BY B-CELL STIMULATORY FACTOR-II INTERLEUKIN-6
T SATOH, S NAKAMURA, T TAGA, T MATSUDA, T HIRANO, T KISHIMOTO, Y KAZIRO, MOLECULAR AND CELLULAR BIOLOGY, 8, 8, 3546, 3549, Aug. 1988
AMER SOC MICROBIOLOGY, English, Introduction scientific journal

Pleiotropic functions of BSF-2/IL-6.
MATSUDA TADASHI, HIRANO TOSHIO, 蛋白質 核酸 酵素, 33, 8, 1317, 1322, Jul. 1988
Japanese

免疫研究の最前線-15-BSF-2/IL-6の生物学的機能の多様性
松田 正, 平野 俊夫, 蛋白質核酸酵素, 33, 8, p1317, 1322, Jul. 1988
共立出版, Japanese

ESTABLISHMENT OF AN INTERLEUKIN 6 (IL 6)/B-CELL STIMULATORY FACTOR 2-DEPENDENT CELL-LINE AND PREPARATION OF ANTI-IL-6 MONOCLONAL-ANTIBODIES
T MATSUDA, T HIRANO, T KISHIMOTO, EUROPEAN JOURNAL OF IMMUNOLOGY, 18, 6, 951, 956, Jun. 1988
VCH PUBLISHERS INC, English

Functional diversity and abnormal expression of BSF-2/IL-6.
HIRANO TOSHIO, TAGA TETSUYA, MATSUDA TADASHI, HORII YASUHIRO, TANG B, KAWANISHI YOSHIKAZU, SUEMATSU SACHIKO, KISHIMOTO TADAMITSU, 日本臨床, 46, 5, 1029, 1035, May 1988
Japanese

INTERLEUKIN-6 (BSF2/IL-6) IS AN AUTOCRINE GROWTH-FACTOR FOR HUMAN MULTIPLE MYELOMAS
M KAWANO, T HIRANO, T MATSUDA, T TAGA, Y HORII, K IWATO, H ASAOKU, B TANG, O TANABE, H TANAKA, A KURAMOTO, T KISHIMOTO, PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES, 64, 3, 68, 71, Mar. 1988
JAPAN ACAD, English

AUTOCRINE GENERATION AND REQUIREMENT OF BSF-2/IL-6 FOR HUMAN MULTIPLE MYELOMAS
M KAWANO, T HIRANO, T MATSUDA, T TAGA, Y HORII, K IWATO, H ASAOKU, B TANG, O TANABE, H TANAKA, A KURAMOTO, T KISHIMOTO, NATURE, 332, 6159, 83, 85, Mar. 1988
MACMILLAN MAGAZINES LTD, English

THE ESSENTIAL ROLE OF B-CELL STIMULATORY FACTOR-II (BSF-2/IL-6) FOR THE TERMINAL DIFFERENTIATION OF B-CELLS
A MURAGUCHI, T HIRANO, B TANG, T MATSUDA, Y HORII, K NAKAJIMA, T KISHIMOTO, JOURNAL OF EXPERIMENTAL MEDICINE, 167, 2, 332, 344, Feb. 1988
ROCKEFELLER UNIV PRESS, English

インターロイキン6とそのレセプター　　　　　　　　　　　　　　　
代謝, 25(9), 859-869, 1988

IL-6とリウマチ疾患　　　　　　　　　　　　　　　
生体防御, 5(2), 211-216, 1988

BSF-2／IL-6の生物学的機能の多様性　　　　　　　　　　　　　　　
蛋白質・核酸・酵素, 33(8), 1317-1322, 1988

BSF-2／IL-6の機能多様性と異常発現　　　　　　　　　　　　　　　
日本臨床, 46(5), 1029-1035, 1988

〔自己免疫病 基礎･臨床研究の新しい動向〕B細胞刺激因子（BSF-2）と自己免疫疾患　　　　　　　　　　　　　　　
日本臨床, 46(4), 749-754, 1988

IL-6を用いた効率的ハイブリドーマ作成法　　　　　　　　　　　　　　　
実験医学増刊, 6(10), 66-71, 1988

B細胞の増殖・分化因子とその異常　　　　　　　　　　　　　　　
癌・免疫・栄養, 2(4), 1-5, 1988

ABSENCE OF ANTIVIRAL ACTIVITY IN RECOMBINANT B-CELL STIMULATORY FACTOR-II (BSF-2)
T HIRANO, T MATSUDA, K HOSOI, A OKANO, H MATSUI, T KISHIMOTO, IMMUNOLOGY LETTERS, 17, 1, 41, 45, Jan. 1988
ELSEVIER SCIENCE BV, English

STRUCTURE AND EXPRESSION OF HUMAN B-CELL STIMULATORY FACTOR-II (BSF-2/IL-6) GENE
K YASUKAWA, T HIRANO, Y WATANABE, K MURATANI, T MATSUDA, S NAKAI, T KISHIMOTO, EMBO JOURNAL, 6, 10, 2939, 2945, Oct. 1987
OXFORD UNIV PRESS UNITED KINGDOM, English

B細胞刺激因子とB細胞の分化　　　　　　　　　　　　　　　
Medical Immunology, 14(5), 645-651, 1987

COMPLEMENTARY-DNA FOR A NOVEL HUMAN INTERLEUKIN (BSF-2) THAT INDUCES LYMPHOCYTES-B TO PRODUCE IMMUNOGLOBULIN
T HIRANO, K YASUKAWA, H HARADA, T TAGA, Y WATANABE, T MATSUDA, S KASHIWAMURA, K NAKAJIMA, K KOYAMA, A IWAMATSU, S TSUNASAWA, F SAKIYAMA, H MATSUI, Y TAKAHARA, T TANIGUCHI, T KISHIMOTO, NATURE, 324, 6092, 73, 76, Nov. 1986
MACMILLAN MAGAZINES LTD, English

LIMITED PROTEOLYSIS OF A CHEMICALLY MODIFIED 3RD COMPONENT OF HUMAN-COMPLEMENT, C-3, BY CATHEPSIN-G OF HUMAN-LEUKOCYTES
T MATSUDA, S NAGASAWA, T KOIDE, J KOYAMA, JOURNAL OF BIOCHEMISTRY, 98, 1, 229, 236, 1985
JAPANESE BIOCHEMICAL SOC, English

LOCATION OF THE INTER-CHAIN DISULFIDE BONDS OF THE 3RD COMPONENT OF HUMAN-COMPLEMENT
T MATSUDA, T SEYA, S NAGASAWA, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 127, 1, 264, 269, 1985
ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, English

Interleukin-6 (IL-6).　　　　　　　　　　　　　　　
Cytokine Reference Academic Press., 2000

Interleukin-6 (IL-6).　　　　　　　　　　　　　　　
Cytokine Reference Academic Press., 2000

IL-6の遺伝子発現機構　　　　　　　　　　　　　　　
Annual Review免疫 1995, 1995

サイトカインシグナル　　　　　　　　　　　　　　　
中外医学社, 1994

GM-CSF受容体　　　　　　　　　　　　　　　
広川タンパク質化学 第2巻 受容体タンパク質Ⅰ, 1993

インターロイキン6受容体　　　　　　　　　　　　　　　
広川タンパク質化学 第2巻 受容体タンパク質Ⅰ, 1993

サイトカインレセプターとシグナル伝達　　　　　　　　　　　　　　　
Annual Review 免疫 1992, 1992

IL-6　　　　　　　　　　　　　　　
サイトカイン療法 南江堂, 1992

インターロイキン6　　　　　　　　　　　　　　　
医学のあゆみ 別冊巻 サイトカイン 基礎から臨床応用まで, 1992

B細胞の増殖・分化　　　　　　　　　　　　　　　
免疫・炎症・膠原病 メデイカル葵出版, 1991

IL-6とそのレセプター　　　　　　　　　　　　　　　
Annual Review 細胞生物学 1991, 1991

インターロイキン6(IL-6)の機能多様性とB細胞異常 改訂　　　　　　　　　　　　　　　
がん遺伝子研究最近の進歩, 1991

サイトカインと疾患　　　　　　　　　　　　　　　
小児医学の進歩’91A 中山書店, 1991

インターロイキン6　　　　　　　　　　　　　　　
新生化学実験講座12 分子免疫学Ⅰ 東京化学同人, 1989

増殖分化因子遺伝子の単離　　　　　　　　　　　　　　　
新生化学実験 講座 7 増殖分化因子とその受容体 東京化学同人, 1989

Interleukin 6 and its receptor in immune regulation.　　　　　　　　　　　　　　　
Progress in Immunology., 1989

Interleukin-6 and its receptor.　　　　　　　　　　　　　　　
Lymphokine Receptor Interations. Eds D. Fradelizi, J. Bertoglio. Colloque INSERM/John Libbey Eurotext Ltd., 1989

Differentiation and regulation of immunocompetent cells: interleukin 6 in immune regulation.　　　　　　　　　　　　　　　
Progress in Allergy and Clinical Immunology. Editors W. J. Pichler, B. M. Stadler, C. A. Dahinden, A. T. P'ecoud, P. Frei, C. H. Schneider, A. L.. de Weck. Hogrefe and Huber Publeshers, Toronto., 1989

Normal and abnormal regulation of human B cell differentiation by a new cytokine, BSF-2/IL-6.　　　　　　　　　　　　　　　
Mechanisms of Lymphocyte Activation and Immune Regulation, vol 2 edited by S. Gupta, and W. E. Paul., Plenum Publishing Corporation., 1989

Interleukin 6 and its receptor.　　　　　　　　　　　　　　　
Proceedings of the Mochida Memorial Symposium on Recent Progress in Cytokine Research. Modical View Co. Ltd, Tokyo,, 1989

Interleukin 6 and its receptor in immune regulation.　　　　　　　　　　　　　　　
Progress in Immunology., 1989

Interleukin-6 and its receptor.　　　　　　　　　　　　　　　
Lymphokine Receptor Interations. Eds D. Fradelizi, J. Bertoglio. Colloque INSERM/John Libbey Eurotext Ltd., 1989

Differentiation and regulation of immunocompetent cells: interleukin 6 in immune regulation.　　　　　　　　　　　　　　　
Progress in Allergy and Clinical Immunology. Editors W. J. Pichler, B. M. Stadler, C. A. Dahinden, A. T. P'ecoud, P. Frei, C. H. Schneider, A. L.. de Weck. Hogrefe and Huber Publeshers, Toronto., 1989

Normal and abnormal regulation of human B cell differentiation by a new cytokine, BSF-2/IL-6.　　　　　　　　　　　　　　　
Mechanisms of Lymphocyte Activation and Immune Regulation, vol 2 edited by S. Gupta, and W. E. Paul., Plenum Publishing Corporation., 1989

Interleukin 6 and its receptor.　　　　　　　　　　　　　　　
Proceedings of the Mochida Memorial Symposium on Recent Progress in Cytokine Research. Modical View Co. Ltd, Tokyo,, 1989

A new cytokine, BSF-2/IL-6 and its receptor.　　　　　　　　　　　　　　　
B cell development, edited by O. N. Witte, N. Klinman and M C. Howard. Alan R. Liss Inc., New York., 1988

A new cytokine, BSF-2/IL-6 and its receptor.　　　　　　　　　　　　　　　
B cell development, edited by O. N. Witte, N. Klinman and M C. Howard. Alan R. Liss Inc., New York.., 1988

Molecular structure and immunological function of human B cell differentiation factor (BSF2).　　　　　　　　　　　　　　　
Molecular basis of lymphokine action, edited by D. R. Webb, C. W. Pierce, and S. Cohen, The Human Press Inc., 1987

Molecular structure and immunological function of human B cell differentiation factor (BSF2).　　　　　　　　　　　　　　　
Molecular basis of lymphokine action, edited by D. R. Webb, C. W. Pierce, and S. Cohen, The Human Press Inc., 1987

生命薬学特論　　　　　　　　　　　　　　　
北海道大学生命科学院

生命医薬科学概論　　　　　　　　　　　　　　　
北海道大学生命科学院

薬局実習　　　　　　　　　　　　　　　
北海道大学薬学部

薬学概論　　　　　　　　　　　　　　　
北海道大学薬学部

教養科目　　　　　　　　　　　　　　　
北海道大学

公衆衛生学　　　　　　　　　　　　　　　
北海道大学薬学部

衛生化学　　　　　　　　　　　　　　　
北海道大学薬学部

免疫学　　　　　　　　　　　　　　　
北海道大学薬学部

ポストコロナの感染症・がん予防に向けての貪食促進型赤血球ワクチンの創製
科学研究費助成事業
30 Jun. 2023 - 31 Mar. 2025
松田 正, 半田 悠, 室本 竜太, 鍛代 悠一
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 23K18170

Study of signaling pathways for treatment of immune/allergic diseases
Grants-in-Aid for Scientific Research
01 Apr. 2019 - 31 Mar. 2022
Matsuda Tadashi
Cytokines control the proliferation and differentiation as well as effecter functions of immune cells to eliminate invasive pathogens; however, they sometimes involve in the onset and development of immune/allergic diseases. We identified new regulatory molecules in the cytkine signaling and analyzed their functions. Among them, we focused on STAT3 / NF-κB-related signals, which are important for controlling the function of cytokines responsible for immune / allergic responses. However, the details of the involvement of these molecules in the development of immune / allergic diseases are not yet clear. In this study, we further examined functions of these STAT3 / NF-κB-related signaling molecules and showed new mechanisms in the onset of diseases such as allergies / immune diseases.This study will provide clues toward development of novel drugs for immune/allergic diseases in the near future.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 19H03364

Design and synthesis of phosphatidylserine dimers with antitumor effect via the phagocytosis-inducing property
Grants-in-Aid for Scientific Research
01 Apr. 2013 - 31 Mar. 2015
SHUTO satoshi, FUKUDA Hayato, ASAI Akira, MATSUDA Tadashi
We designed and synthesized dimer derivatives of phosphatidylserine as phagocytosis inducer via exposure of phosphor-serine polar-head moieties on cell surface, which are potentially used in immune cancer therapy. We successfully synthesized these dimer derivatives. We demonstrated that the liposomes containing the synthesized dimer derivatives of phosphatidylserine actually exposed the phosphor-serine polar-head moieties on cell surface in the evaluation with tumor cells.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 25670048

Effects of environmental chemicals on Th17 cells differenciation via aryl hydrocarbon receptor
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2012 - 31 Mar. 2015
KOJIMA HIROYUKI, TAKEUCHI Shinji, MUROMOTO Ryuta, MATSUDA Tadashi
The retinoic acid receptor-related orphan receptor (ROR) and aryl hydrocarbon receptor (AhR) are key regulators of helper T (Th)17 cell differentiation. However, it remains unclear whether environmental chemicals, including dioxins, affect Th17 cell functions. In this study, we examined the AhR activity of 200 environmental chemicals using DR-EcoScreen assay, and found that several pesticides and isoflavones enhanced AhR-mediated transcriptional activity. These compounds increased the expression of IL-17A mRNA without effecting ROR mRNA levels in mouse T lymphoma EL4 cells. On the other hand, these chemicals did not affect the IL-17A gene expression in ROR- or AhR-knockdown EL4 cells. Taken together, these results suggest that several AhR agonists might enhance IL-17 production via ROR and AhR. This study also provides the evidence that environmental chemicals can act as modulators of IL-17 gene expression in immune cells.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido Institute of Public Health, 24590773

CMLがん細胞増殖におけるSTAP－２蛋白の役割
科学研究費助成事業
01 Apr. 2013 - 31 Mar. 2014
松田 正
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 25670017

P.acnes由来肉芽腫形成因子の同定とワクチンへの応用
科学研究費助成事業 新学術領域研究(研究領域提案型)
01 Apr. 2012 - 31 Mar. 2014
松田 正
肉芽腫形成は自然炎症反応による病変のひとつであり、細菌感染など病原体の侵入に対して、病原体を隔離する生体防御のひとつと考えられている。興味深いことにシグナル伝達分子Tyk2欠損マウスにおいてP.acnes誘導性の肉芽腫形成反応が抑制されることを見出しており、Tyk2欠損マウスを利用しつつ、肉芽腫形成を担うP.acnes由来因子を同定し、その特異的モノクローナル抗体の獲得と、特異的モノクローナル抗体による肉芽腫形成の阻害、さらにP.acnes由来因子による肉芽腫形成の分子機構の解明するため本年度は以下の研究を行なった。1.肉芽腫形成応答におけるケモカイン群の解析 P.acnes誘導性の肉芽腫形成反応に対応して種々のサイトカインやケモカインの産生が誘導されるが、Tyk2KOマウスにおいてはそれらの産生低下が顕著である。特にP.acnesによりマクロファージにより誘導されるケモカイン群発現に注目し、その分子機構を検討し、Tyk2下流のSTAT3の活性化が重要であることが明らかとなっている。2. P.acnes菌体からのP.acnes由来肉芽腫形成因子の精製とP.acnes由来肉芽腫形成因子に対する抗体の作製 現在、P.acnes由来蛋白に対するポリクローナル、モノクローナル抗体を作成しており、それらの抗体を用いた肉芽腫形成や免疫活性化効果への影響を検討する予定である。3.活性型Tyk2変異体発現トランスジェニックマウスでのP.acnes誘導肉芽腫形成の解析 すでに種々のTyk2点変異体の作成により活性増強されたTyk2変異体を発見しており、より効率的P.acnes誘導性の肉芽腫形成反応検出のための活性型Tyk2Tgマウスを作製する予定である。さらにこれらの研究過程でTyk2が炎症応答を惹起するサイトカインIL-23による病気発症に重要であることも明らかにした（論文投稿中）。
日本学術振興会, 新学術領域研究(研究領域提案型), 北海道大学, 24117701

癌・免疫疾患発症とシグナル異常：STAT3とNFーκBを中心として
科学研究費助成事業 基盤研究(B)
01 Apr. 2011 - 31 Mar. 2014
松田 正, 室本 竜太, 今 重之, 関根 勇一, 南保 明日香
生体の恒常性の維持にはサイトカインの存在が不可欠であり、中でも炎症性サイトカイン、インターロイキン6(IL-6)の異常産生やシグナル異常は癌、自己免疫疾患の発症に深く関与している。申請者はIL-6のシグナル分子STAT3の活性化及び活性化制御に関与する多くの分子を同定し、機能解析を行ってきた。本研究ではSTAT3と炎症応答を制御するNF-kappaBとの機能的相互作用の解析を中心に生体内STAT3制御法の確立を目指し、本年度は以下の研究を行った。1.STAT3結合蛋白のSTAT3機能への影響解析 これまでSTAT3制御因子としてY14を同定したが、Y14がNF-kappaBの活性制御にも関与する新規機能分子であることを明らかにした（論文投稿中）。2.STAT3とNF-kappaBとのクロストークを指標にしたSTAT3制御機構の解析 これまで核内蛋白KAP1がSTAT3、NF-kappaB、p300等と相互作用し、TNF/NF-kappaB依存的な炎症性サイトカインIL-6の遺伝子発現を制御することを見い出し報告したが、今回STAT3によるNF-kappaB活性化調節にSTAT3のセリンリン酸化が関与することを明らかにした。3.STAT3結合蛋白の新規機能解析STAT3の活性化を制御するSTAT3結合蛋白STAP-2が免疫系T細胞の活性化細胞死やCML慢性骨髄性白血病の原因蛋白BCR-ABLの活性化に関与することも明らかにした。さらに植物性イソフラボンが免疫系細胞においてSTAT3の活性化を誘導し、炎症性サイトカイン産生を増強することも世界で初めて明らかにした。
日本学術振興会, 基盤研究(B), 北海道大学, 23390015

疾患関連シグナルにおける低分子量二重特異性ホスファターゼの機能解析
科学研究費助成事業 挑戦的萌芽研究
2009 - 2010
松田 正, 室本 竜太, 関根 勇一
近年の自己免疫疾患やがんの疾患発症の分子メカニズムの研究により明らかになり始めた『シグナル伝達系の制御』白群の代表であるプロテインホスファターゼのうち、近年その生理的機能の一部が明らかになり、注目されるユニー群、低分子量二重特異性ホスファターゼを中心に、ホスファターゼによる疾患関連シグナルの制御機構の解析を行っ
1.種々の組織・細胞株でのDUSP遺伝子発現とサトカイン、増殖因子、ステロイドホルモン等の各種シグナルによ誘導の解析
サイトカイン、ステロイドホルモン刺激による各種DUSPのmRNA発現の誘導をRT-PCRにより、評価し、それらの刺激に導されるDUSPを同定した。
2.疾患シグナルにおけるDUSP遺伝子ノックダウンによる影響を解析
申請者らが確立したサイトカイン、増殖因子、ステロイドホルモンレセプターシグナルをモニターしうるレポーター法を用いて、細胞株で種々のDUSP遺伝子をsiRNAノックダウンすることにより、当該DUSPのシグナルへの影響を評価し連サイトカインシグナルを正に制御するDUSPを同定し、現在論文投稿準備中である。
3.ホスファターゼによる転写活性化複合体の制御機構の解析
疾患シグナルを制御する転写活性化制御複合体の重要な構成蛋白であるKAP1が疾患関連シグナル伝達分子STAT3のリン酸化、脱リン酸化に影響することによって転写活性化制御を担うことを明かにし、現在論文投稿中である。
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 21659014

Activation and regulation of the disease-related IL-6/STAT3 signaling
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
2008 - 2010
MATSUDA Tadashi, MUROMOTO Ryuta, SEKINE Yuichi, OHBAYASHI Norihiko, NANBO Asuka
Since we cloned interleukin-6 (IL-6) as B cell stimulatory factor 2 in 1986, IL-6 has been reported to influence cell proliferation, differentiation, and survival as well as mature cell functions. Signal transducer and activator of transcription 3 (STAT3) is a key molecule to mediate IL-6-induced cellular events. Indeed, STAT3 is activated in many tumor tissues, and the dimmer of STAT3 promotes cellular transformation, indicating that STAT3 itself can act as a proto-oncogene. In addition to oncogenesis, IL-6/STAT3 axis influences host immune systems. For example, IL-6/STAT3 axis is involved in the Toll-like receptor signaling pathways, the development and maturation of dendritic cells, and the differentiation of Th17 T-cell subset. These facts are likely to suggest that manipulation of IL-6/STAT3 signals could be suitable target for a variety of disease such as cancer and autoimmune diseases. We have energetically examined regulatory mechanisms of IL-6/STAT3-mediated signal transduction for the development of novel therapeutic strategies.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 20390017

Molecular mechanisms of STAT3 activation and their therapeutic applications
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
2006 - 2007
MATSUDA Tadashi, OHBAYASHI Norihiko, SEKINE Yuichi
STAT3 protein is mainly activated by IL-6 family of cytokines, epidermal growth factor, and leptin. Like other members of STAT family, STAT3 is tyrosine-phosphorylated by Jak kinases, then forms a dimer and translocates into the nucleus to activate target genes. It has been shown that the activated STAT3 alone can mediate cellular transformation. We cloned IL-6 as B cell stimularory factor 2 in 1986, and found its multiple functions and the relationship with several autoimmune diseases and cancers. After these outstanding works, we have continued the research concerning the mechanisms of IL-6 signal transduction. Especially, we have focused on STAT3 molecule in the IL-6/STAT3-mediated signaling pathway and reported several molecules, which regulate STAT3 positively or negatively. We also identified novel STAT3 binding proteins by using yeast two-hybrid system. Among them, one is ZIPK, a Ser/Thr kinase which phosphorylates STAT3 directly. We have also reported other STAT3 regulators, STAP-2, Daxx and LMW-DSP2 (DUSP22). In our research, we expect that STAT3 regulators, which directly interact with STAT3, may have critical roles in the regulation of STAT3 activation. More detailed understanding of interactions between these molecules and STAT3 is therefore important as new information may provide novel therapeutic approaches for the pathological conditions, including cancer and autoimmune diseases.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 18390017

エピジェニック効果を指標とした新規臨床検査法の確立
科学研究費助成事業 萌芽研究
2006 - 2007
松田 正, 大林 典彦
免疫疾患や癌は、さらに増加傾向の一途を辿っている。加えて、その重症化も進み、日常生活に著しい支障を慢性的にきたすことから、国民の健康上重大な問題となっている。さらに患者のQOLの向上を図るためにも、疾患の状況を把握しうる確実なる診断法開発も必要である。本研究においては、近年の疾患発症の分子メカニズムの研究により明らかになり始めている核タンパク質のエピジェニック効果にスポットを当て、疾患における核タンパク質の異常なタンパク質修飾を特異的に、かつ迅速に検出しうる診断法の開発を目指すものである。実際、本研究において免疫疾患や癌等の疾患関連核タンパク質を標的としてその核タンパク質修飾を検出しうる測定系を樹立することにより、診断をより容易なものとし、最終的にはタンパク質修飾を標的とした特異的な治療薬開発を目指す。国民の保険医療において、安価で簡便な診断、副作用の少ない特異的治療薬の開発は医療費削減にも重要なポイントであ。本研究においては核タンパク質のエピジェニック効果に伴うタンパク質修飾を特異的に検出する抗体の作製により新規診断法の確立を行う。本年度はタンパク質のリン酸化部位あるいはアセチル化部位の同定とその特異的抗体の作成により、生体内での特異的修飾を検出できた。しかしながら抗体の力価さらにあげ、大量に産生しうるモノクローナル抗体の作成を進める必要があり、現在進めている。また、それを用いてのELISA測定法の予備実験にも成功しており、さらに産業化に向けて研究を進める。
日本学術振興会, 萌芽研究, 北海道大学, 18659036

Supression of the expression of cyclooxygenase-2 by ozonides of oleate ester
Grants-in-Aid for Scientific Research
2004 - 2005
MIURA Toshiaki, MATSUDA Tadashi, TAMOTO Kouichi
Ozonized olive oil has been reported to exert anti-inflammatory effect. However, the details of chemical structure of ozonized olive oil and its action mechanism have long been unclear. We have recently demonstrated that the major component of the ozonized olive oil is triozonide of triolein. In this study, we identified several minor components of ozonized olive oil, including "cross ozonides" of low and high molecular weight. In addition, to elucidate the anti-inflammatory action mechanism of ozonized olive oil, we investigated the effects of ozonized olive oil and methyl oleate ozonides on the generation of PGE_2 and the expression of Cyclooxygenase-2 (COX-2) in Lipopolysaccharide (LPS)-stimulated macrophage-like THP-1 cells. Olive oil, methyl oleate and their ozonides did not affect cell viability and morphology within a concentration used. LPS-induced COX-2 expression and PGE_2 generation in macrophage-like THP-1 cells were not affected by olive oil itself. However, ozonized olive oil inhibited both reactions in a concentration-dependent manner. Methyl oleate ozonides also inhibited COX-2 expression and PGE_2 generation whereas oleic acid and methyl oleate did not show such inhibitory effects. Such actions of ozonized olive oil and methyl oleate ozonide were shown to be due to their inhibitory effect on lкB phosphorylation which is required for Nuclear factor-kappa B (NFкB) activation and subsequent COX-2 gene transcription in LPS-stimulated macrophages. The inhibitory effect was also observed with ozonide having no functional group except for ozonide ring. In addition, ozonized olive oil and methyl oleate ozonide showed no effect on the phosphorylations of p38 and JNK, which are involved in the activation of AP-1. These results demonstrate that ozonized olive oil and methyl oleate ozonides suppress LPS-induced PGE_2 generation in macrophage-like THP-1 cells through inhibition of COX-2 gene transcription by suppressing the lкBαNFкB pathway.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), HOKKAIDO UNIVERSITY, 16590080

サイトカインのシグナル伝達　　　　　　　　　　　　　　　
ライフサイエンス基礎科学研究
1995 - 2005
Competitive research funding

サイトカインシグナル伝達系におけるSTAT3制御蛋白の役割
科学研究費助成事業 特定領域研究
2004 - 2004
松田 正, 平 敬宏, 奈良 敏文
申請者らが世界に先駆けクローニングし、機能を明らかにしたIL-6の研究を元として、IL-6の下流のシグナル伝達分子STAT3結合蛋白を網羅的にクローニングし、解析を行なった。すでに申請者らはSTAT3の機能を修飾する蛋白としてステロイドレセプターやHSP90,チロシンホスファターゼTC-PTPに関する論文を報告し、IL-6/STAT3のシグナル伝達系の解析法を確立した。さらに最近STAT3に結合するアダプター蛋白STAP-2を同定し、そのノックアウトマウスにおいてLPS,IL-6刺激によるSTAT3の活性化、急性期蛋白誘導が抑制されることを明らかにした。また、このSTAP-2がSTAT5とも機能的相互作用することも明らかにした。
特に、本申請研究においてはSTAT3に特異的に結合するキナーゼとSTAT3の核移行に重要な新規トランスロケーターを同定し、その機能解析を行い、現在論文投稿中である。STAT3キナーゼはSTAT3のSer727をリン酸化し、STAT3の活性化を正に制御する。STAT3トランスロケーターはSTAT3の核移行に必須である。今後、STAT3キナーゼやトランスロケーター分子のSTAT3を介した自己免疫疾患の発症や細胞の癌化における役割を明らかにし、疾患におけるこのキナーゼ、トランスロケーターの生理的意義を明らかなものとする。
日本学術振興会, 特定領域研究, 北海道大学, 16043201

The mechanism of granulopoiesis and leukemogenesis by the activation of Stat3
Grants-in-Aid for Scientific Research
2003 - 2004
SHIMODA Kazuya, MATSUDA Tadashi, HARADA Mine, NAGAFUJI Kouji, MIYAMOTO Toshihiro
The Jak-Stat pathway plays an essential role in cytokine signaling. G-CSF promotes granulopoiesis, and Stat3 is the principle Stat protein activated by G-CSF. Upon treatment with G-CSF, the IL-3 dependent cell line 32D clone 3(32Dcl3) differentiates into neutrophils, and 32Dcl3 cells expressing dominant-negative Stat3 (32Dcl3/DNStat3) proliferate in G-CSF without differentiation. Gene expression profile of G-CSF-stimulated cell lines revealed that the expression of C/EBP_α was up-regulated by the activation of Stat3. And activated Stat3 bound to C/EBP_α, leading to the enhancement of the transcription activity of C/EBP_α. Conditional expression of C/EBP_α in 32Dcl3/DNStat3 cells after G-CSF stimulation abolishes the G-CSF dependent cell proliferation and induces granulocytic differentiation. These results show that one of the major roles of Stat3 in G-CSF signaling pathway is to augment the function of C/EBP_α.
The function of Stat3 in vivo was examined using the Stat3 conditional deficient mice. Mice deficient for Stat3 in hematopoietic cells show neutrocytosis, and G-CSF-induced proliferation of progenitor cells was observed in Stat3-deficient mice. In hematopoietic cells from Stat3-deficient mice, trace levels of SOCS3, a negative regulator of granulopoiesis, were observed, and SOCS3 expression was not induced by G-CSF stimulation. Stat3 null bone marrow cells displayed a significant activation of ERK1/2 under basal conditions, and it was enhanced and sustained by G-CSF stimulation. Furthermore, the augmented proliferation of Stat3-deficient bone marrow cells in response to G-CSF was dramatically decreased by addition of a MEK1 inhibitor. Stat3 functions in vivo as a negative regulator of G-CSF signaling by inducing SOCS3 expression, and ERK activation is the major factor responsible for inducing the proliferation of hematopoietic cells in response to G-CSF.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), KYUSHU UNIVERSITY, 15390302

環境ホルモンによるステロイド、サイトカインおよび増殖因子のクロストークへの影響
科学研究費助成事業 特定領域研究
2002 - 2003
松田 正
本研究ではヒト胎性腎癌細胞株293T細胞にエストロゲンレセプターやそのレポーター遺伝子を導入することによってエストロゲンレセプターシグナルモデル系を、さらにサイトカインのシグナル伝達分子STATあるいはTGF-βの活性をモニターしうるレポーター遺伝子を導入することによってサイトカインあるいは増殖因子のシグナルモデルを再構築した。エストロゲンレセプターの活性化は17β-エストラジオール(E2)によりSTAT (STAT3)の活性化は293T細胞をIL-6やLIF (Leukemia inhibitory factor)で刺激することにより行ない、Smadの活性化は活性化タイプI型TGF-βレセプターを発現させることにより行なった。これらの刺激を組み合わせることによって互いのクロストークを観察することにより、エストロゲンレセプターがSTAT3と相互作用し、IL-6あるいはLIFの作用を抑制することを明らかにした。さらに増殖因子TGF-βやBMPのシグナル伝達分子であるSmad蛋白がエストロゲンレセプターと相互作用することにより、TGF-βやBMPの作用を制御し、エストゲンレセプター活性を増強することを293T細胞での再構築系やヒト乳癌細胞株MCF-7を用いて明らかにした。本研究においては、これらエストロゲンレセプターおよび増殖因子、サイトカインのシグナル伝達系を用いることにより、種々の環境ホルモンのこれらシグナル系への影響を検討した。エストロゲンレセプターを刺激するE2同様に種々の環境ホルモンによるエストロゲンレセプターの活性化は増殖因子TGF-βの刺激、すなわちTGF-βレセプターの活性化によって増強された。すなわち環境ホルモンもE2同様に増殖因子のシグナル伝達系とクロストークすることが観察された。また、E2によって増殖因子、サイトカインのシグナル系が抑制されることを明らかにしたが、検討した環境ホルモンのなかにはサイトカインによるSTAT3の活性化を逆に増強するものが存在した。その増強効果はドミナントネガテイブSTAT3やアンチエストロゲン、タモキシフェンによって抑制が認められた。また、増強効果を示す環境ホルモンによってMAPキナーゼの活性増強も観察された。
日本学術振興会, 特定領域研究, 北海道大学, 14042216

TNFファミリー分子及びサイトカインを介するアポトーシス増殖シグナル伝達機構解明
科学研究費助成事業 特定領域研究
2002 - 2002
宮崎 忠昭, 松田 正
(1)細胞接着を阻害し、integrin receptor等の接着分子からのシグナルを抑制することにより、アポトーシス(接着依存性アポトーシス:anoikis)が誘導される。癌細胞の増殖および転移においてanoikisのメカニズムを解明することは癌治療のために重要であり、我々はこのanoikis誘導の機構においてDAP3 (death associated protein 3)が機能を有していることを見い出した。さらにこのDAP3の機能の制御はそのリン酸化によって行われていることを確認し、そのリン酸化を行うkinaseについても同定と解析を行った。
(2)癌の発症、増殖および浸潤とDAP3との関連性を検討するため、胃癌患者の癌組織部においてDAP3の発現をwestern blottingおよび組織切片の免疫染色を行って検討した。その結果、胃癌の中では印環細胞癌、低分化腺癌、腺扁平上皮癌においてDAP3の発現が著しく亢進している例を認めたため、現在、その発現が亢進しているDAP3のアミノ酸変異の有無について解析を行っている。また、胃癌患者由来の組織を用いて作成したcDNAを使用して、DAP3のシークエンスをPCRにより確認したところ、アミノ酸置換が考えられるDNAの変異を確認した。
(3)新たなアポトーシスを誘導する分子としてクローニングされたDeath Receptor 6 (DR6)のシグナル伝達機構を解明するため、DR6の細胞内領域に物理的に会合する分子を酵母two hybrid法によりスクリーニングした結果、その候補として18個の遺伝子産物をクローニングした。これらのうちクローニングされた頻度の高かった2個の遺伝子産物について哺乳類細胞内での会合を検討した結果、DR6との会合を確認した。
(4)TDAG8 (T cell death associated gene 8)遺伝子の発現誘導は、グルココルチコイドによる胸腺細胞株のアポトーシス誘導と相関していた。TDAG8のトランスジェニックマウスを作製し解析した結果、TDAG8はcaspaseの活性化に関与していることが明らかとなった。
日本学術振興会, 特定領域研究, 北海道大学, 14028002

Tyk2ノックアウトマウスを用いたIL-12のシグナル伝達の解析
科学研究費助成事業
2001 - 2001
松田 正, 下田 和哉
サイトカインのシグナル伝達におけるJakファミリーキナーゼの重要性は十分理解されているが、その中でTyk2キナーゼは、当初IFNαのシグナル伝達に必須のキナーゼとして同定された。In vivoでのTyk2の役割を明確にするために我々はTyk2欠損マウスを作成し、Tyk2はIFNαの大部分のシグナル伝達に必須ではないことを明らかにした。さらにIFNα以外にその刺激によりTyk2を活性化する抗癌サイトカインとして知られるIL-12のシグナル伝達にTyk2が重要であることも示したが、今回さらにIL-12とならび生体内の抗癌作用に重要なlFNY誘導サイトカインであるIL-18についてTyk2欠損マウスを用いて検討した。IL-18は、Jak-STAT経路ではなく、NF-κBを介する経路でそのシグナルを伝達するが、Tyk2欠損マウス由来脾細胞では、野生型マウスと比べIL-12刺激時に約1/6、IL-18刺激時に約1/3とNK細胞活性が減少していた。また、IL-12,IL-18刺激によるT細胞の増殖にはTyk2の欠損は影響を与えなかったが、IL-12,IL-18刺激によるIFNγの産生は著明に低下していた。野生型マウスでIL-12刺激時にみられるT, NK細胞におけるIL-18レセプターの発現亢進が、Tyk2欠損マウス由来T, NK細胞では見られておらず、このことがJak-STAT経路ではなく、MAPキナーゼ、NF-κBを介する経路をシグナル伝達に用いるIL-18の作用がTyk2の欠損により傷害される機序の一つとして考えられる。
現在、これらサイトカインによる抗癌メカニズム解明に向けてTyk2ノックアウトマウス及び野生型マウス由来脾細胞をIL-12及びIL-18刺激により新たに誘導される遺伝子発現をMicroarray法により解析中であり、同定された新たな遺伝子の解析も進める予定である。
日本学術振興会, 特定領域研究(C), 富山医科薬科大学, 13214037

ABNORMAL RECOMBINATION ACTIVATING GENE (RAG) AND IMMUNO DEFICIENCY
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B).
1999 - 2000
MURAGUCHI Atsushi, NAGATA Takuya, MATSUDA Tadashi, KISHI Hiroyuki
1. Human Rag genomic locus ; Using a fragment of human RAG-1 cDNA, we isolated genomic DNA clones that contained either first or second exon of human RAG-1. We determined its exon/intron organization and the transcriptional start site. Similarly, we isolated genomic DNA clones that contains first or second exon of human RAG-2 and determined its exon/intron structure and the transcription initiation site for RAG-2. These led us to reveal, for the first time, the organization of human genomic RAG-1 and RAG-2 locus.
2. Regulation of human RAG transcription ; We characterized promoter regions for human RAG-1 and RAG-2, and revealed that the 5' promoter region of RAG-lwas indispensable for its basal promoter activity. On the contrary, the sequences between -63 to -107 of RAG-2 were shown to be essential for its promoter acticity, suggesting the regulation of RAG-1 and RAG-2 transcription may be controled by different transcriptional mechanisms.
3. Regulation of murine RAG-2 ; We found that the regulation of the mouse RAG-2 promoter activity is confered to 80 nucleotide upstream of RAG-2. We also found that a B cell-specific transcription protein, Pax-5, and a T cell-specific transcription protein, GATA-3, bind to the -80 to -17 nt region in B cells and T cells, respectively. These results indicate that distinct DNA binding proteins, Pax-5 and GATA-3, may regulate the murine RAG-2 promoter in B and T lineage cells, respectively.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B)., TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY, 11470169

造血系構築とくにマクロファージ分化におけるIL-6シグナルの解析
科学研究費助成事業 重点領域研究
1994 - 1994
松田 正, 平野 俊夫
IL-6は造血系において、IL-6は、IL-3とともに多能性幹細胞コロニー形成を誘導し、巨核球に対してはその成熟化を誘導する。また、IL-6はマウスの骨髄性白血病細胞株M1のマクロファージへの分化を誘導する因子として注目されている。IL-6受容体は分子量80kDaのIL-6結合分子とそのシグナル伝達分子であるgp130により構成される。gp130分子の細胞内ドメインにはチロシンキナーゼ等のエフェクター分子に類似した構造は認められず、gp130分子を介するシグナル伝達系を明らかにするためにはgp130会合分子の同定は必須である。申請者らは非受容体型チロシンキナーゼであるJAKファミリーキナーゼならびにその下流に存在すると考えられるSTAT分子に注目し、それらに対する特異的抗体を用いた免疫沈降法にてIL-6刺激によるチロシンリン酸化の有無を検討した。JAKキナーゼ及びSTAT蛋白に対する抗体は合成ペプチドをウサギに免疫することにより得た。マウスgp130分子を高発現させたマウスプロB細胞株BAFm130細胞あるいはIL-6によってマクロファージへの分化が誘導されるY6細胞をIL-6と可溶性IL-6レセプターa鎖で刺激し、NP-40溶液で可溶化後、抗ホスホチロシン抗体あるいは抗JAK抗体、抗STAT抗体で免疫沈降したのち抗ホスホチロシン抗体によるウエスタンブロットを行った。BAFm130においたはgp130分子を刺激することにより種々の蛋白のチロシンリン酸化が誘導された(BBRC200:821,1994)。また、gp130刺激でJAKキナーゼとSTAT分子のチロシンリン酸化が観察された。さらに、JAKキナーゼは構成的にgp130分子に会合していることが明らかとなった。加えて、抗STAT抗体による免疫沈降物中には分子量72kDaの新たなチロシンキナーゼ(p72^)(p72STAT-associatcd tyrosinc kinasc)が含まれることを明らかにした。また、p72^のチロシンキナーゼ活性はgp130刺激で上昇することを明らかにした(Blood83:3457,1994)。さらに、p72^チロシンキナーゼのチロシンリン酸化はIL-6によってマクロファージへの分化が誘導されるY6細胞においてもチロシンリン酸化が誘導されることや他の造血系サイトカインであるIL-3やG-CSFによっても誘導されることから造血系サイトカインレセプター下流に共通に存在するチロシンキナーゼであると考えられる。現在、このp72^のチロシンキナーゼの単離、精製を試みている。
日本学術振興会, 重点領域研究, 大阪大学, 06277211

IL-6による発癌機構の解析
科学研究費助成事業
1993 - 1993
平野 俊夫, 松田 正, 中嶋 弘一
インターロイキン6(IL-6)受容体を介するシグナル伝達経路を明かにした。すなわち、初期応答遺伝子の一つであるjunB遺伝子のIL-6応答領域(JRE-IL6)に至るシグナル伝達経路には、Jakチロシンキナーゼと、未知の分子量7万のチロシンキナーゼが関与していることと、シグナル伝達性転写因子であるStatファミリー蛋白が関与していることを明かにした。Stat蛋白がDNA結合能を獲得するためには、チロシンキナーゼによるStat蛋白のチロシンリン酸化が必須である。さらに、H7感受性のプロセスがStat蛋白が転写活性を獲得するために必須であることを明かにした。このシグナル伝達経路は、Ras,Raf,Cキナーゼ,Ca/CM依存性キナーゼを使用しておらず、未知の経路であることを明かにした。さらにJRE-IL6活性化に至るシグナル伝達経路は、アデノウイルスのE1A蛋白によって抑制されること、この抑制は、E1AのCR1領域を含む部分が必要であることを明かにした。今後、IL-6で活性化される分子量7万のチロシンキナーゼの同定、及びH7感受性プロセスを明かにすると共に、E1A分子の標的分子を同定することによって、この新しいRas非依存性のシグナル伝達経路の全貌を明かにしたい。
日本学術振興会, がん特別研究, 大阪大学, 05152073

自己免疫疾患の発症機序に関する分子生物学的研究
科学研究費助成事業 一般研究(B)
1993 - 1993
平野 俊夫, 松田 正
慢性関節リウマチ(RA)患者の骨髄ストローマ細胞株を樹立し、正常人由来骨髄ストローマ細胞株と、プレB細胞増殖支持能を比較したところ、患者由来細胞株は、高い支持能を有していた。この支持能亢進は、未知の細胞膜分子によることを明らかにした。さらにこの未知の膜分子を同定するために、RA由来骨髄ストローマ細胞株に対するモノクローナル抗体、RF3を作製した。ひきつづきRF3が認識する膜蛋白(BST-1と命名)をコードするcDNAをクローニングした。BST-1はGPIアンカー型の新しい膜蛋白で、CD38と約30%のホモロジーを有していた。さらに、BALB3T3細胞に発現させることによって、プレB細胞に対する増殖支持能を示した。また、BST-1の染色体遺伝子は、第14番染色体のq32.3に位置していることを示した。BST-1分子は、RA由来骨髄ストローマ細胞株では、その発現が亢進していた。この事実は、RA骨髄には、なんらかの異常が存在し、その結果BST-1遺伝子の発現が亢進していると考えられる。IL-6の遺伝子異常発現の分子機序と共に、RAの原因を追及するための有用なアプローチとなると考えられる。
日本学術振興会, 一般研究(B), 大阪大学, 05454208

IL-6による発癌機構の解析
科学研究費助成事業
1992 - 1992
平野 俊夫, 改正 恒康, 松田 正, 中嶋 弘一
1.IL-6シグナル伝達機構の解析
前年までに、IL-6によって誘導される初期応答遺伝子の一つであるJunB遺伝子プロモーターのIL-6応答領域(JRE-IL6)を決定し、JRE-IL6がEts結合部位とCREB/ATF結合領域を含んでいることを明かにした。本年度は、引き続きJRE-IL6活性化に至るIL-6シグナル伝達経路を詳細に解析した結果、このシグナル伝達経路には、C-キナーゼ、A-キナーゼ、Ras、Rafが全く関与せず未知のシグナル経路であることを明かにした。又、Ets結合部位に結合可能なDNA結合蛋白をコードするcDNA結合蛋白をコードするcDNAをIL-6反応細胞よりクローニングした。引き続き、このEts結合蛋白がIL-6シグナルによって実際に活性化されるか否かを検討中である。
2.IL-6受容体会合分子の解析
IL-6受容体は、分子量8万のα鎖と、シグナル伝達分子であるgp130によって構成されているが、我々は第三の分子が会合している可能性を示す結果を得た。引き続き、この第三分子の機能を解析中である。
3.IL-6遺伝子異常発現機構の解析
多発性骨髄腫患者より樹立した骨髄ストローマ細胞は、IL-6プロモーターをトランスに活性化しうる何らかの因子を有している。この因子をクローニングするためのモデル実験として、HTLV-1のTaxが実際にIL-6プロモーターをNF-κB結合部位を介して活性化することを明かにした。又、Tax遺伝子を100倍に希釈しても、IL-6プロモーター支配下のルシフェラーゼ遺伝子の発現が誘導されることを明かにした。現在、引き続きストローマ細胞由来トランスアクチベーター因子のクローニングを行っている。
日本学術振興会, がん特別研究, 大阪大学, 04152076

Investigation of the molecular mechanisms of immunological disorders with abnormal expression of the interleukin 6 gene.
Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (A)
1990 - 1992
HIRANO Toshio, KAISHO Tsuneyasu, MATSUDA Tadashi
To elucidate the molecular mechanism(s) of immunological disorders where abnormal expression of the interleukin 6(IL-6) gene is considered to play some roles, we first made a working hypothesis of the pathogenesis of autoimmune disease(s) such as rheumatoid arthritis (RA). We postulate the involvement of endogenous retrovirus and that virus-derived transactivator, such as HTLV-1 p40tax would induce the expression of a variety of genes including the IL-6 gene of which expression is essential for disease onset. On the basis of this hypothesis, we established the cloning system by which we can ultimately clone the cDNA encoding p40tax like molecule present in the synovial tissue of RA patient. We further demonstrated that HTLV-1 p40tax can induce IL-60 gene expression through NF-kB binding site. Furthermore, to develop efficient inhibitors of IL-6 actions, we investigated the IL-6 signal transduction. We identified a novel IL-6 responsive element of the junB gene (JRE-IL6) and demonstrated that the signalings activating the JRE-IL6 does not contain PKC,PKA,ras,and raf activation, demonstrating the presence of a novel signal transduction pathway.
Japan Society for the Promotion of Science, Grant-in-Aid for General Scientific Research (A), Osaka University, 02404032

ILー6による発癌機構の解析
科学研究費助成事業
1990 - 1990
平野 俊夫, 改正 恒康, 松田 正, 中嶋 弘一
1.ILー6シグナル伝達機構の解析
ILー6によって誘導される初期応答遺伝子の一つであるjunB遺伝子プロモ-タ-のILー6応答領域を決定した。ILー6応答領域は、5'上流ー149〜ー125塩基対に位置し、ETS結合領域とCREB/ATF結合領域を含んでいることが明らかになった。ILー6応答には両部位の存在が必須であり、両結合部位に各々ETSファミリ-とCREB/ATFファミリ-に属するDNA結合蛋白が結合しうることを明らかにした。さらに、Etsー1,CREーBP1、及びjunBが協調的にILー6応答領域に作用しうることを直接証明した。
2.多発性骨髄腫ストロ-マ細胞の機能異常の解析
多発性骨髄腫の発症が骨髄ストロ-マ細胞の異常に基づく可能性を明らかにする目的で、患者より骨髄ストロ-マ細胞株を樹立した。B細胞前駆細胞の増殖支持能を検討したところ、正常骨髄由来ストロ-マ細胞と比較して、3〜10倍強い活性があることを明らかにした。又、この増殖支持能は、何らかの未知の接着因子によることを示した。
3.ILー6遺伝子異常発現機構の解析
多発性骨髄腫患者より樹立した骨髄ストロ-マ細胞に、ILー6プロモ-タ-とCATを結合した遺伝子を導入することにより、患者ストロ-マ細胞には、ILー6プロモ-タ-をトランスに活性化しうる何らかのトランスアクチベ-タ-が存在することを明らかにした。
日本学術振興会, がん特別研究, 大阪大学, 03152078

形質細胞腫/骨髄腫発生機構におけるインタ-ロイキン6とその受容体発現異常の解析
科学研究費助成事業 がん特別研究
1990 - 1990
平野 俊夫, 改正 恒康, 松田 正
我々は、インタ-ロイキン6(ILー6)遺伝子の異常発現がミエロ-マ形質細胞腫の発生に重要な役割を果たしていることを報告してきた。
C57BL/6(B6)マウスを使用して、ヒトILー6遺伝子を構成的に発現するトランスジェニックマウス(B6Tg)を作成したところ、著明な形質細胞の増加を認めることを以前に報告した。しかしながら、これらの形質細胞は、同系マウスに移植できなかった。
今回、プリスティンの投与にて形質細胞腫が容易に発生するBALB/cマウスとB6Tgの交配実験を行なったところ、t(12;15)のクロモゾ-ム転座を伴い、かつヌ-ドマウスに移植可能な形質細胞腫が発生することが明らかとなった。又免疫グロブリン遺伝子は、モノクロ-ナルな再構成パタ-ンを示していた。以上の事実より、ILー6遺伝子の異常発現とcーmyc遺伝子等の癌遺伝子の異常発現により、形質細胞腫が発生することが明かとなった。
本研究によって、少なくともマウスにおいては、ILー6遺伝子の異常発現によって、最終的に、形質細胞腫が発生しうること、又このためには、他の癌遺伝子の異常発現が必要であることが明かとなった。ILー6は、ヒトのミエロ-マ/形質細胞腫の増殖因子であり、臨床的に癌が増加する時期には、これら患者血清中にILー6が増加することが知られている。これらの事実は、ILー6遺伝子の異常発現がヒトミエロ-マ/形質細胞腫の発生にも重要な役割を演じていることを示唆している。今後解明されなければならない重要な問題は、ミエロ-マ患者におけるILー6遺伝子の異常発現を誘導している分子機構の解明であると考えられる。
日本学術振興会, がん特別研究, 大阪大学, 02152069

免疫異常症の病態および病因におけるIL-61BSF-2の免疫学的分子生物学的解析
科学研究費助成事業 奨励研究(A)
1989 - 1989
松田 正
日本学術振興会, 奨励研究(A), 大阪大学, 01790523