WATANABE Toshio

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Profile and Settings

• Name (Japanese)

  Watanabe

• Name (Kana)

  Toshio

Degree

• Ph D., The University of Tokyo

Research Interests

• ゲノム
• 遺伝子操作マウス
• 生命科学
• 再生医療
• 幹細胞
• 血液
• 免疫
• 生化学
• 分子遺伝学
• 細胞生物学
• 培養細胞
• 細胞分化
• 発生
• がん細胞
• Membrane Traffic
• Gene Manipulated Mice
• Biochemistry
• Molecular Genetics
• Cell Differentiation
• Mouse Development
• Cancer Cells

Research Areas

• Life sciences, Immunology
• Life sciences, Medical biochemistry
• Life sciences, Developmental biology
• Life sciences, Cell biology
• Life sciences, Molecular biology
• Life sciences, Genetics
• Nanotechnology/Materials, Molecular biochemistry

Research Experience

• Apr. 2020, Mar. 2021, 大学院人間文化総合科学研究科, 自然科学専攻, 自然科学専攻長, Japan
• Apr. 2019, Mar. 2021, 奈良女子大学, 理学部長
• Apr. 2006, 9999, Nara Women's University, Faculty, Division of Natural Sciences, Japan
• Apr. 2015, Mar. 2019, 奈良女子大学, 附属中等教育学校長
• 1996, 2006, :東北大学加齢医学研究所・助教授
• 1996, 2006, :Inst. of Dev., Aging & Cancer, Tohoku University ・Associate Professor
• 2006, -:奈良女子大学大学院人間文化研究科・教授
• 2006, -:Graduate School of Humanities and Sciences・Professor
• 1987, 1996, :東京大学応用微生物研究所（現分子細胞生物学研究所）・助手
• 1987, 1996, :Inst. of Applied Microbiol. University of Tokyo・Jyosyu
• 1992, 1994, :マックスプランク免疫生物学研究所・訪問科学者
• 1992, 1994, :Max Planck Inst. of Immunobiol.・Visiting Scientist
• Apr. 2021, Mar. 2023, Nara Women's University, 大学院, 人間文化総合科学研究科 研究科長, Japan

Education

• 1987, The University of Tokyo, 理学系研究科, 生物化学, Japan
• 1987, The University of Tokyo, Graduate School, Division of Science, Biochemistry and Biophysics
• 1982, The University of Tokyo, Faculty of Science, 生物化学, Japan
• 1982, The University of Tokyo, Faculty of Science, Biochemistry and Biophysics

Association Memberships

• 日本生化学学会
• 幹細胞研究会
• 日本分子生物学会
• 日本癌学会
• 日本プロテインホスファターゼ研究会
• 麒麟塾ー血液学若手研究者勉強会ー
• SUMO研究会
• 京都T細胞カンファレンス
• KTCC

Ⅱ．研究活動実績

Published Papers

• Refereed, Mar. 2024
• Refereed, 2024, 693, 149353, 10.1016/j.bbrc.2023.149353.
• Refereed, 10.1002/2211-5463.13775.
• Refereed, 12 2022, 12, 1, 22297
• Refereed, 113, 1613, 1624
• 2022, in press
• Refereed, 2021, 10, 4451, 4464
• 2021
• Refereed, Journal Immunology, Arf1 and Arf6 synergistically maintain survival of T cells during activation., Mami Sumiyoshi; Yui Kotani; Yuki Ikuta; Kazutomo Suzue; Madoka Ozawa; Tomoya Katakai; Taketo Yamada; Yasunori Kanaho; Toshio Watanabe; Satoshi Matsuda, Jan. 2021, 206, 366, 375, Scientific journal

  共同研究・競争的資金等URL
• Refereed, Cancer Science, Wiley, Autophagy regulates levels of tumor suppressor enzyme protein phosphatase 6., Nobuyuki Fujiwara; Shusaku Shibutani; Yusuke Sakai; Toshio Watanabe; Issei Kitabayashi; Hiroko Oshima; Masanobu Oshima; Hisashi Hoshida; Rinji Akada; Tatsuya Usui; Takashi Ohama; Koichi Sato, 2020, Scientific journal, 10.1111/cas.14662

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• Refereed, Nature Communication, Chlorpromazine eliminates acute myeloid leukemia cells by perturbing subcellular localization of FLT3-ITD and KIT-D816V., Shinya Rai; Hirokazu Tanaka; Mai Suzuki; J. Luis Espinoza; Takahiro Kumode; Akira Tanimura; Yasuyoshi Morita; Yoichi Tatsumi; Takafumi Yokota; Kenji Oritani; Toshio Watanabe; Yuzuru Kanakura; Itaru Matsumura, Mutated receptor tyrosine kinases (MT-RTKs) such as internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3 ITD) and a point mutation KIT D816V are driver mutations for acute myeloid leukemia (AML). Clathrin assembly lymphoid myeloid leukemia protein (CALM) regulates intracellular transport of RTKs, however, the precise role for MT-RTKs remains elusive. We here show that CALM knock down leads to severely impaired FLT3 ITD- or KIT D814V-dependent cell growth compared to marginal influence on wild-type FLT3- or KIT-mediated cell growth. An antipsychotic drug chlorpromazine (CPZ) suppresses the growth of primary AML samples, and human CD34+CD38- AML cells including AML initiating cells with MT-RTKs in vitro and in vivo. Mechanistically, CPZ reduces CALM protein at post transcriptional level and perturbs the intracellular localization of MT-RTKs, thereby blocking their signaling. Our study presents a therapeutic strategy for AML with MT-RTKs by altering the intracellular localization of MT-RTKs using CPZ., 2020, 11, 1, 4147, 4147, Scientific journal, True, 10.1038/s41467-020-17666-8

  DOI URL共同研究・競争的資金等URL
• Refereed, Acta Neuropathologica, Picalm reduction exacerbates tau pathology in a murine tauopathy model., Kunie Ando; Robert De Decker; Cristina Vergara; Zehra Yilmaz; Salwa Mansor; Valérie Suain; Kristel Sleegers; Marie-Claude Potier; Charles Duyckaerts; Toshio Watanabe; Luc Buée; Karelle Leroy; Jean-Pierre Brion; the Brainbank; Neuro-CEB Neuropathology Network, 2020, 139, 773, 789, Scientific journal

  共同研究・競争的資金等URL
• Refereed, PLOS ONE, Behavioral and neurological analyses of adult mice carrying null and distinct loss-of-receptor function mutations in protein tyrosine phosphatase receptor type Z (PTPRZ)., Naomi Tanga; Kazuya Kuboyama; Ayako Kishimoto; Miho Kihara; Hiroshi Kiyonari; Toshio Watanabe; Akihiro Fujikawa; Masaharu Noda, Protein tyrosine phosphatase receptor type Z (PTPRZ) is preferentially expressed in the central nervous system as two transmembrane receptor isoforms PTPRZ-A/B and one secretory isoform PTPRZ-S. Ptprz-knockout mice lacking the expression of all three isoforms show behavioral, learning, and neurological abnormalities, including increased exploratory activities to novelty, deficits in spatial and contextual learning, and reduced responses to methamphetamine, relative to wild-type mice. To investigate whether PTPRZ isoforms play distinct physiological roles, we herein performed behavioral studies on two knock-in mouse lines: One expresses the catalytically inactive Cys-1930 to Ser (CS) mutants of PTPRZ-A/B, while the other generated in the present study expresses catalytically active mutants of PTPRZ-A/B lacking the negative regulatory PTP-D2 domain and C-terminal PDZ-binding motif (ΔD2) instead of wild-type PTPRZ-A/-B. In contrast to Ptprz-knockout mice, neither increased responses to novelty in the open field nor memory impairments in the inhibitory-avoidance task were observed in Ptprz-CS or Ptprz-ΔD2 mice. However, the effects of methamphetamine on locomotor activity were significantly weaker in Ptprz-KO mice and CS mutant mice than in wild-type mice, but were normal in ΔD2 mutant mice. Furthermore, microdialysis experiments revealed that methamphetamine-evoked dopamine release in the nucleus accumbens was reduced in Ptprz-KO mice and CS mutant mice. These results suggest that the extracellular region of PTPRZ, including the secretory isoform, is crucial for behavioral responses to novelty and the formation of aversive memories, whereas the PTPase activities of PTPRZ receptor isoforms are involved in regulating the dopaminergic system., Jul. 2019, 14, 6, e0217880, Scientific journal, True, 10.1371/journal.pone.0217880

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• Refereed, GLIA, The PTN-PTPRZ signal activates the AFAP1L2-dependent PI3K-AKT pathway for oligodendrocyte differentiation: Targeted inactivation of PTPRZ activity in mice, Naomi Tanga; Kazuya Kuboyama; Ayako Kishimoto; Hiroshi Kiyonari; Aki Shiraishi; Ryoko Suzuki; Toshio Watanabe; Akihiro Fujikawa; Masaharu Noda, Jan. 2019, 67, 967, 984, Scientific journal
• Refereed, Cancer cell, PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth., Mami Morita; Taku Sato; Miyuki Nomura; Yoshimi Sakamoto; Yui Inoue; Ryota Tanaka; Shigemi Ito; Koreyuki Kurosawa; Kazunori Yamaguchi; Yuki Sugiura; Hiroshi Takizaki; Yoji Yamashita; Ryuichi Katakura; Ikuro Sato; Masaaki Kawai; Yoshinori Okada; Hitomi Watanabe; Gen Kondoh; Shoko Matsumoto; Ayako Kishimoto; Miki Obata; Masaki Matsumoto; Tatsuro Fukuhara; Hozumi Motohashi; Makoto Suematsu; Masaaki Komatsu; Keiichi I Nakayama; Toshio Watanabe; Tomoyoshi Soga; Hiroshi Shima; Makoto Maemondo; Nobuhiro Tanuma, Expression of PKM2, which diverts glucose-derived carbon from catabolic to biosynthetic pathways, is a hallmark of cancer. However, PKM2 function in tumorigenesis remains controversial. Here, we show that, when expressed rather than PKM2, the PKM isoform PKM1 exhibits a tumor-promoting function in KRASG12D-induced or carcinogen-initiated mouse models or in some human cancers. Analysis of Pkm mutant mouse lines expressing specific PKM isoforms established that PKM1 boosts tumor growth cell intrinsically. PKM1 activated glucose catabolism and stimulated autophagy/mitophagy, favoring malignancy. Importantly, we observed that pulmonary neuroendocrine tumors (NETs), including small-cell lung cancer (SCLC), express PKM1, and that PKM1 expression is required for SCLC cell proliferation. Our findings provide a rationale for targeting PKM1 therapeutically in certain cancer subtypes, including pulmonary NETs., 12 Mar. 2018, 33, 3, 355, 367, Scientific journal, True, 10.1016/j.ccell.2018.02.004

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• Not Refereed, Encyclopedia of Signaling Molecules, 2nd Edition, ADP-ribosylation factor 1., Toshio Watanabe; Mami Sumiyoshi, 2018, 412, 414
• Refereed, Cancer Science, Loss of protein phosphatase 6 in mouse keratinocytes enhances K-rasG12D-driven tumor promotion., WATANABE Toshio; Koreyuki; Kurosawa, Yui; Inoue, Yoichiro; Kakugawa; Yoji; Yamashita, Hidekazu; Yamada, Kazuhiro; Kishimoto, Miyuki; Nomura, Yuki; Momoi, Koh; Miura, Ichiro; Sato, Natsuko; Chiba, Mai; Suzuki, Honami; Ogoh, Toshio; Watanabe; Nobuhiro, Tanuma; Masahiro, Tachi; Hiroshi, Shima, 2018, 109, 2178, 2187
• Refereed, Molecular Cancer Research, The BRCA1-interacting protein OLA1 requires the interaction with BARD1 to regulate centrosome number., WATANABE Toshio; uki Yoshino; Huicheng Qi; Hiroki Fujita; Matsuyuki Shirota; Shun Abe; Yuhei Komiyama; Kazuha Shindo; Masahiro Nakayama; Ayako Matsuzawa; Akihiro Kobayashi; Honami Ogoh; Toshio Watanabe; Chikashi Ishioka; Natsuko Chiba, BRCA1 functions as a tumor suppressor in DNA repair and centrosome regulation. Previously, Obg-like ATPase 1 (OLA1) was shown to interact with BARD1, a heterodimer partner of BRCA1. OLA1 binds to BRCA1, BARD1, and γ-tubulin and functions in centrosome regulation. This study determined that overexpression of wild-type OLA1 (OLA1-WT) caused centrosome amplification due to centriole overduplication in mammary tissue-derived cells. Centrosome amplification induced by overexpression of the cancer-derived OLA1 mutant, which is deficient at regulating centrosome number, occurred in significantly fewer cells than in that induced by overexpression of OLA1-WT. Thus, it was hypothesized that overexpression of OLA1 with normal function efficiently induces centrosome amplification, but not that of OLA1 mutants, which are deficient at regulating centrosome number. We analyzed whether overexpression of OLA1 missense mutants of nine candidate phosphorylation residues, three residues modified with acetylation, and two ATP-binding residues caused centrosome amplification and identified five missense mutants that are deficient in the regulation of centrosome number. Three of them did not bind to BARD1. Two phosphomimetic mutations restored the binding to BARD1 and the efficient centrosome amplification by their overexpression. Knockdown and overexpression of BARD1 also caused centrosome amplification. BARD1 mutant reported in cancer failed to bind to OLA1 and rescue the BARD1 knockdown-induced centrosome amplification and reduced its centrosomal localization. Combined, these data reveal that the OLA1-BARD1 interaction is important for the regulation of centrosome number.Implications: Regulation of centrosome number by BRCA1/BARD1 together with OLA1 is important for the genome integrity to prevent tumor development. Mol Cancer Res; 16(10); 1499-511. ©2018 AACR., 2018, 16, 10, 1499, 1511, Scientific journal, True, 10.1158/1541-7786.

  DOI URL共同研究・競争的資金等URL
• Refereed, Scientific reports, Mllt10 knockout mouse model reveals critical role of Af10-dependent H3K79 methylation in midfacial development., Honami Ogoh; Kazutsune Yamagata; Tomomi Nakao; Lisa L Sandell; Ayaka Yamamoto; Aiko Yamashita; Naomi Tanga; Mai Suzuki; Takaya Abe; Issay Kitabayashi; Toshio Watanabe; Daisuke Sakai, Epigenetic regulation is required to ensure the precise spatial and temporal pattern of gene expression that is necessary for embryonic development. Although the roles of some epigenetic modifications in embryonic development have been investigated in depth, the role of methylation at lysine 79 (H3K79me) is poorly understood. Dot1L, a unique methyltransferase for H3K79, forms complexes with distinct sets of co-factors. To further understand the role of H3K79me in embryogenesis, we generated a mouse knockout of Mllt10, the gene encoding Af10, one Dot1L complex co-factor. We find homozygous Mllt10 knockout mutants (Mllt10-KO) exhibit midline facial cleft. The midfacial defects of Mllt10-KO embryos correspond to hyperterolism and are associated with reduced proliferation of mesenchyme in developing nasal processes and adjacent tissue. We demonstrate that H3K79me level is significantly decreased in nasal processes of Mllt10-KO embryos. Importantly, we find that expression of AP2α, a gene critical for midfacial development, is directly regulated by Af10-dependent H3K79me, and expression AP2α is reduced specifically in nasal processes of Mllt10-KO embryos. Suppression of H3K79me completely mimicked the Mllt10-KO phenotype. Together these data are the first to demonstrate that Af10-dependent H3K79me is essential for development of nasal processes and adjacent tissues, and consequent midfacial formation., 20 Sep. 2017, 7, 1, 11922, 11922, True, 10.1038/s41598-017-11745-5

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• Refereed, FEBS LETTERS, WILEY-BLACKWELL, Targeted disruption of the mouse protein phosphatase ppm1l gene leads to structural abnormalities in the brain, Rie Kusano; Kousuke Fujita; Yasuharu Shinoda; Yuko Nagaura; Hiroshi Kiyonari; Takaya Abe; Toshio Watanabe; Yasuhisa Matsui; Masahiro Fukaya; Hiroyuki Sakagami; Tatsuya Sato; Jun-ichi Funahashi; Motoko Ohnishi; Shinri Tamura; Takayasu Kobayashi, PPM1L, a member of the metal-dependent protein phosphatase (PPM) family, is involved in regulating the stress-activated protein kinase pathway and ceramide trafficking. However, the physiological function of PPM1L in the brain is unclear. In this study, we generated and analyzed ppm1l-deficient mice in order to investigate PPM1L functions in the brain. Our results indicate that ppm1l is highly expressed in the central nervous system during mouse development and that ppm1l(Delta/Delta) mice display impaired motor performance and morphological abnormalities in the forebrain. Electron microscopic and immunohistochemical analyses suggest that these abnormalities are due to impaired axonal tract formation. Our novel findings suggest an important role for PPM1L in brain development., Oct. 2016, 590, 20, 3606, 3615, Scientific journal, 10.1002/1873-3468.12429

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• Refereed, HUMAN MOLECULAR GENETICS, OXFORD UNIV PRESS, Partial loss of CALM function reduces A beta 42 production and amyloid deposition in vivo, Kunihiko Kanatsu; Yukiko Hori; Sho Takatori; Toshio Watanabe; Takeshi Iwatsubo; Taisuke Tomita, Aberrant production, clearance and deposition of amyloid-beta protein (A beta) in the human brain have been implicated in the aetiology of Alzheimer disease (AD). gamma-Secretase is the enzyme responsible for generating various Ab species, such as A beta 40 and toxic A beta 42. Recently, genome-wide association studies in late-onset AD patients have identified the endocytosis-related phosphatidylinositol-binding clathrin assembly protein (PICALM) gene as a genetic risk factor for AD. We previously found that the loss of expression of CALM protein encoded by PICALM affects the ratio of production of A beta 42, through the regulation of the clathrin-mediated endocytosis of c-secretase. Here, we show that the binding capacity of the assembly protein 180 N-terminal homology (ANTH) domain of CALM to phosphatidylinositol-4,5-biphosphate, as well as to nicastrin, is critical to the modulation of the internalization of gamma-secretase and to the A beta 42 production ratio. Moreover, reduction of CALM decreases Ab deposition as well as brain levels of insoluble A beta 42 in vivo. These results suggest that CALM expression modifies AD risk by regulating Ab pathology., Sep. 2016, 25, 18, 3988, 3997, Scientific journal, 10.1093/hmg/ddw239

  DOI URL共同研究・競争的資金等URL
• Refereed, MECHANISMS OF DEVELOPMENT, ELSEVIER SCIENCE BV, The protein phosphatase 6 catalytic subunit (Ppp6c) is indispensable for proper post-implantation embryogenesis, Honami Ogoh; Nobuhiro Tanuma; Yasuhisa Matsui; Natsuki Hayakawa; Ayaka Inagaki; Mami Sumiyoshi; Yuki Momoi; Ayako Kishimoto; Mai Suzuki; Nozomi Sasaki; Tsukasa Ohuchi; Miyuki Nomura; Yuriko Teruya; Keiko Yasuda; Toshio Watanabe; Hiroshi Shima, Ppp6c, which encodes the catalytic subunit of phosphoprotein phosphatase 6 (PP6), is conserved among eukaryotes from yeast to humans. In mammalian cells, PP6 targets I kappa B epsilon for degradation, activates DNA-dependent protein kinase to trigger DNA repair, and is reportedly required for normal mitosis. Recently, Ppp6c mutations were identified as candidate drivers of melanoma and skin cancer. Nonetheless, little is known about the physiological role of Ppp6c. To investigate this function in vivo, we established mice lacking the Ppp6c phosphatase domain by crossing heterozygous mutants. No viable homozygous pups were born, indicative of a lethal mutation. Ppp6c homozygous mutant embryos were identified among blastocysts, which exhibited a normal appearance, but embryos degenerated by E7.5 and showed clear developmental defects at E8.5, suggesting that mutant embryos die after implantation. Accordingly, homozygous blastocysts showed significant growth failure of the inner cellmass (ICM) in in vitro blastocyst culture, and primary Ppp6c exon4-deficient MEFs showed greatly reduced proliferation. These results establish for the first time that the Ppp6c phosphatase domain is indispensable for mouse embryogenesis after implantation. (C) 2016 Elsevier Ireland Ltd. All rights reserved., Feb. 2016, 139, 1, 9, Scientific journal, 10.1016/j.mod.2016.02.001

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• Refereed, CANCER LETTERS, ELSEVIER IRELAND LTD, Loss of protein phosphatase 6 in mouse keratinocytes increases susceptibility to ultraviolet-B-induced carcinogenesis, Hiroyuki Kato; Koreyuki Kurosawa; Yui Inoue; Nobuhiro Tanuma; Yuki Momoi; Katsuhisa Hayashi; Honami Ogoh; Miyuki Nomura; Masato Sakayori; Yoichiro Kakugawa; Yoji Yamashita; Koh Miura; Makoto Maemondo; Ryuichi Katakura; Shigemi Ito; Masami Sato; Ikuro Sato; Natsuko Chiba; Toshio Watanabe; Hiroshi Shima, We previously reported that deficiency in the gene encoding the catalytic subunit of protein phosphatase 6 (Ppp6c) predisposes mouse skin tissue to papilloma formation initiated by DMBA. Here, we demonstrate that Ppp6c loss acts as a tumor promoter in UVB-induced squamous cell carcinogenesis. Following UVB irradiation, mice with Ppp6c-deficient keratinocytes showed a higher incidence of skin squamous cell carcinoma than did control mice. Time course experiments showed that following UVB irradiation, Ppp6c-deficient keratinocytes upregulated expression of p53, PUMA, BAX, and cleaved caspase-3 proteins. UVB-induced tumors in Ppp6c-deficient keratinocytes exhibited a high frequency of both p53- and gamma H2AX-positive cells, suggestive of DNA damage. Epidemiological and molecular data strongly suggest that UVB from sunlight induces p53 gene mutations in keratinocytes and is the primary causative agent of human skin cancers. Our analysis suggests that PP6 deficiency underlies molecular events that drive outgrowth of initiated keratinocytes harboring UVB-induced mutated p53. Understanding PP6 function in preventing UV-induced tumorigenesis could suggest strategies to prevent and treat this condition. (C) 2015 Elsevier Ireland Ltd. All rights reserved., Sep. 2015, 365, 2, 223, 228, Scientific journal, 10.1016/j.canlet.2015.05.022

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• Refereed, FEBS LETTERS, ELSEVIER SCIENCE BV, Mice doubly-deficient in the Arf GAPs SMAP1 and SMAP2 exhibit embryonic lethality, Mami Sumiyoshi; Narumi Masuda; Nobuhiro Tanuma; Honami Ogoh; Eri Imai; Mizuki Otsuka; Natsuki Hayakawa; Kinuyo Ohno; Yasuhisa Matsui; Kanae Hara; Risa Gotoh; Mai Suzuki; Shinya Rai; Hirokazu Tanaka; Itaru Matsumura; Hiroshi Shima; Toshio Watanabe, In mammals, the small Arf GTPase-activating protein (SMAP) subfamily of An GTPase-activating proteins consists of closely related members, SMAP1 and SMAP2. These factors reportedly exert distinct functions in membrane trafficking, as manifested by different phenotypes seen in single knockout mice. The present study investigated whether SMAP proteins interact genetically. We report for the first time that simultaneous loss of SMAP1 and SMAP2 promotes apoptosis in the distal region of E7.5 mouse embryos, likely resulting in embryonic lethality. Thus, at least one SMAP gene, either SMAP1 or SMAP2, is required for proper embryogenesis. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved., Sep. 2015, 589, 19, 2754, 2762, Scientific journal, 10.1016/j.febslet.2015.07.050

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• Refereed, EXPERIMENTAL HEMATOLOGY, ELSEVIER SCIENCE INC, TARGETING INTRACELLULAR TRAFFICKING OF FLT3-ITD AND KIT D816V BY CHLORPROMAZINE SHOWS PROMISING PRECLINICAL ACTIVITY AGAINST ACUTE MYELOID LEUKEMIA CELLS, Shinya Rai; Hirokazu Tanaka; Toshio Watanabe; Yuzuru Kanakura; Itaru Matsumura, Sep. 2015, 43, 9, S90, S90, 10.1016/j.exphem.2015.06.233

  DOI URL共同研究・競争的資金等URL
• Refereed, ONCOGENE, NATURE PUBLISHING GROUP, Abrogation of protein phosphatase 6 promotes skin carcinogenesis induced by DMBA, K. Hayashi; Y. Momoi; N. Tanuma; A. Kishimoto; H. Ogoh; H. Kato; M. Suzuki; Y. Sakamoto; Y. Inoue; M. Nomura; H. Kiyonari; M. Sakayori; K. Fukamachi; Y. Kakugawa; Y. Yamashita; S. Ito; I. Sato; A. Suzuki; M. Nishio; M. Suganuma; T. Watanabe; H. Shima, Somatic mutations in the gene encoding the catalytic subunit of protein phosphatase 6 (Ppp6c) have been identified in malignant melanoma and are thought to function as a driver in B-raf-or N-ras-driven tumorigenesis. To assess the role of Ppp6c in carcinogenesis, we generated skin keratinocyte-specific Ppp6c conditional knockout mice and performed two-stage skin carcinogenesis analysis. Ppp6c deficiency induced papilloma formation with 7,12-dimethylbenz (a) anthracene (DMBA) only, and development of those papillomas was significantly accelerated compared with that seen following DMBA/TPA (12-O-tetradecanoylphorbol 13-acetate) treatment of wild-type mice. NF-kappa B activation either by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1 beta was enhanced in Ppp6c-deficient keratinocytes. Overall, we conclude that Ppp6c deficiency predisposes mice to skin carcinogenesis initiated by DMBA. This is the first report showing that such deficiency promotes tumor formation in mice., Aug. 2015, 34, 35, 4647, 4655, Scientific journal, 10.1038/onc.2014.398

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• Not Refereed, 生化学, 日本生化学会, Ppp6c deficiency predisposes mouse skin tissue to carcinogenesis, 渡邊 利雄; 島 礼, 2015, 87, 5, 1, 7

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• Refereed, FEBS Letters, argeted disruption of the mouse protein phosphatase ppm1l gene leads to structural abnormalities in the brain., WATANABE Toshio; Rie Kusano; Kousuke Fujita; Yasuharu Shinoda; Yuko Nagaura; Hiroshi Kiyonari; Takaya Abe; Toshio Watanabe; Yasuhisa Matsui; Masahiro Fukaya; Hiroyuki Sakagami; Tatsuya Sato; Jun-ichi Funahashi; Motoko Ohnishi; Shinri Tamura; Takayasu Kobayashi, 2015, 589, 2754, 2762
• Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, The ADP-ribosylation factor 1 gene is indispensable for mouse embryonic development after implantation, Natsuki Hayakawa; Honami Ogoh; Mami Sumiyoshi; Yasuhisa Matsui; Saori Nishikawa; Kananko Miyamoto; Yuko Maede; Hiroshi Kiyonari; Mai Suzuki; Toshio Watanabe, ADP-ribosylation factor (Arf) 1 is thought to affect the morphologies of organelles, such as the Golgi apparatus, and regulate protein trafficking pathways. Mice have six Arf isoforms. In knockdown experiments with HeLa cells, no single Arf isoform among Arf1-5 is required for organelle morphologies or any membrane trafficking step. This suggests that the cooperation of two or more Arfs is a general feature. Although many cell biological and biochemical analyses have proven the importance of Arf1, the physiological roles of Arfl in mice remain unknown. To investigate the activity of Arfl in vivo, we established Arf1-deficient mice. Arf(-/-) blastocysts were identified at the expected Mendelian ratio. The appearance of these blastocysts was indistinguishable from that of wild-type and Arf(+/-) blastocysts, and they grew normally in an in vitro culture system. However, Arf(-/-) embryos were degenerated at E5.5, and none survived to E12.5, suggesting that they died soon after implantation. These data establish for the first time that the Arf1 gene is indispensable for mouse embryonic development after implantation. (C) 2014 Elsevier Inc. All rights reserved., Oct. 2014, 453, 4, 748, 753, Scientific journal, 10.1016/j.bbrc.2014.10.014

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• Refereed, PLOS ONE, PUBLIC LIBRARY SCIENCE, Clathrin Assembly Protein CALM Plays a Critical Role in KIT Signaling by Regulating Its Cellular Transport from Early to Late Endosomes in Hematopoietic Cells, Shinya Rai; Hirokazu Tanaka; Mai Suzuki; Honami Ogoh; Yasuhiro Taniguchi; Yasuyoshi Morita; Takahiro Shimada; Akira Tanimura; Keiko Matsui; Takafumi Yokota; Kenji Oritani; Kenji Tanabe; Toshio Watanabe; Yuzuru Kanakura; Itaru Matsumura, CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage(-)Sca-1(+)KIT(+) (LSK) cells decreased in the fetal liver of CALM(-/-) mice. Also, colony forming activity was impaired in CALM(-/-) LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM(-/-) LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM(-/-) murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM(-/-) MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM(-/-) MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM(-/-) MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM(-/-) MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM(-/-) KIT+ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy., Oct. 2014, 9, 10, DOI: 10.1371/journal.pone.0109441., Scientific journal, 10.1371/journal.pone.0109441

  DOI URL共同研究・競争的資金等URL
• Refereed, CANCER SCIENCE, WILEY-BLACKWELL, Nuclear export signal within CALM is necessary for CALM-AF10-induced leukemia, Mai Suzuki; Kazutsune Yamagata; Mika Shino; Yukiko Aikawa; Koichi Akashi; Toshio Watanabe; Issay Kitabayashi, The CALM-AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies. Certain HOXA cluster genes and MEIS1 genes are upregulated in patients and mouse models that express CALM-AF10. Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia. To investigate the influence of localization on leukemogenesis involving CALM-AF10, we determined the nuclear export signal (NES) within CALM that is necessary and sufficient for cytoplasmic localization of CALM-AF10. Mutations in the NES eliminated the capacity of CALM-AF10 to immortalize murine bone-marrow cells invitro and to promote development of acute myeloid leukemia in mouse models. Furthermore, a fusion of AF10 with the minimal NES can immortalize bone-marrow cells and induce leukemia in mice. These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm., Mar. 2014, 105, 3, 315, 323, Scientific journal, 10.1111/cas.12347

  DOI URL共同研究・競争的資金等URL
• Refereed, NATURE COMMUNICATIONS, NATURE PUBLISHING GROUP, Decreased CALM expression reduces A beta 42 to total A beta ratio through clathrin-mediated endocytosis of gamma-secretase, Kunihiko Kanatsu; Yuichi Morohashi; Mai Suzuki; Hiromasa Kuroda; Toshio Watanabe; Taisuke Tomita; Takeshi Iwatsubo, A body of evidence suggests that aberrant metabolism of amyloid-beta peptide (A beta) underlies the aetiology of Alzheimer disease (AD). Recently, a single-nucleotide polymorphism in phosphatidylinositol binding clathrin assembly protein (PICALM/CALM) gene, which encodes a protein implicated in the clathrin-mediated endocytosis, was identified as a genetic protective factor for AD, although its mechanistic details have little been explored. Here we show that loss of CALM leads to the selective decrease in the production ratio of the pathogenic A beta species, A beta 42. Active form of gamma-secretase is constitutively endocytosed via the clathrin-mediated pathway in a CALM dependent manner. Alteration in the rate of clathrin-mediated endocytosis of gamma-secretase causes a shift in its steady-state localization, which consequently impacts on the production ratio of A beta 42. Our study identifies CALM as an endogenous modulator of gamma-secretase activity by regulating its endocytosis and also as an excellent target for A beta 42-lowering AD therapeutics., Feb. 2014, 5, DOI. 10.1038/ncomms4386., Scientific journal, 10.1038/ncomms4386

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• Refereed, MOLECULAR BIOLOGY OF THE CELL, AMER SOC CELL BIOLOGY, The Arf GAP SMAP2 is necessary for organized vesicle budding from the trans-Golgi network and subsequent acrosome formation in spermiogenesis, Tomo Funaki; Shunsuke Kon; Kenji Tanabe; Waka Natsume; Sayaka Sato; Tadafumi Shimizu; Naomi Yoshida; Won Fen Wong; Atsuo Ogura; Takehiko Ogawa; Kimiko Inoue; Narumi Ogonuki; Hiromi Miki; Keiji Mochida; Keisuke Endoh; Kentarou Yomogida; Manabu Fukumoto; Reiko Horai; Yoichiro Iwakura; Chizuru Ito; Kiyotaka Toshimori; Toshio Watanabe; Masanobu Satake, The trans-Golgi network (TGN) functions as a hub organelle in the exocytosis of clathrin-coated membrane vesicles, and SMAP2 is an Arf GTPase-activating protein that binds to both clathrin and the clathrin assembly protein (CALM). In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting reveals that SMAP2-deficient male mice are healthy and survive to adulthood but are infertile and exhibit globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increases, TGN structures are distorted, acrosome formation is severely impaired, and reorganization of the nucleus does not proceed properly. CALM functions to regulate vesicle sizes, and this study shows that CALM is not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation. Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation. SMAP2-deficient mice provide a model for globozoospermia in humans., Sep. 2013, 24, 17, 2633, 2644, Scientific journal, 10.1091/mbc.E13-05-0234

  DOI URL共同研究・競争的資金等URL
• Refereed, PLOS ONE, PUBLIC LIBRARY SCIENCE, SMAP2 Regulates Retrograde Transport from Recycling Endosomes to the Golgi, Tatsuyuki Matsudaira; Yasunori Uchida; Kenji Tanabe; Shunsuke Kon; Toshio Watanabe; Tomohiko Taguchi; Hiroyuki Arai, Retrograde transport is where proteins and lipids are transported back from the plasma membrane (PM) and endosomes to the Golgi, and crucial for a diverse range of cellular functions. Recycling endosomes (REs) serve as a sorting station for the retrograde transport and we recently identified evection-2, an RE protein with a pleckstrin homology (PH) domain, as an essential factor of this pathway. How evection-2 regulates retrograde transport from REs to the Golgi is not well understood. Here, we report that evection-2 binds to SMAP2, an Arf GTPase-activating protein. Endogenous SMAP2 localized mostly in REs and to a lesser extent, the trans-Golgi network (TGN). SMAP2 binds evection-2, and the RE localization of SMAP2 was abolished in cells depleted of evection-2. Knockdown of SMAP2, like that of evection-2, impaired the retrograde transport of cholera toxin B subunit (CTxB) from REs. These findings suggest that evection-2 recruits SMAP2 to REs, thereby regulating the retrograde transport of CTxB from REs to the Golgi., Jul. 2013, 8, 7, e69145. doi:10.1371/journal.pone.0069145, Scientific journal, 10.1371/journal.pone.0069145

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• Refereed, JOURNAL OF CLINICAL INVESTIGATION, AMER SOC CLINICAL INVESTIGATION INC, Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia, Shunsuke Kon; Naoko Minegishi; Kenji Tanabe; Toshio Watanabe; Tomo Funaki; Won Fen Wong; Daisuke Sakamoto; Yudai Higuchi; Hiroshi Kiyonari; Katsutoshi Asano; Yoichiro Iwakura; Manabu Fukumoto; Motomi Osato; Masashi Sanada; Seishi Ogawa; Takuro Nakamura; Masanobu Satake, The formation of clathrin-coated vesicles is essential for intracellular membrane trafficking between subcellular compartments and is triggered by the ARF family of small GTPases. We previously identified SMAP1 as an ARF6 GTPase-activating protein that functions in clathrin-dependent endocytosis. Because abnormalities in clathrin-dependent trafficking are often associated with oncogenesis, we targeted Smap1 in mice to examine its physiological and pathological significance. Smap1-deficent mice exhibited healthy growth, but their erythroblasts showed enhanced transferrin endocytosis. In mast cells cultured in SCF, Smap1 deficiency did not affect the internalization of c-KIT but impaired the sorting of internalized c-KIT from multivesicular bodies to lysosomes, resulting in intracellular accumulation of undegraded c-KIT that was accompanied by enhanced activation of ERK and increased cell growth. Interestingly, approximately 50% of aged Smap1-deficient mice developed anemia associated with morphologically dysplastic cells of erythroid-myeloid lineage, which are hematological abnormalities similar to myelodysplastic syndrome (MDS) in humans. Furthermore, some Smap1-deficient mice developed acute myeloid leukemia (AML) of various subtypes. Collectively, to our knowledge these results provide the first evidence in a mouse model that the deregulation of clathrin-dependent membrane trafficking may be involved in the development of MDS and subsequent AML., Mar. 2013, 123, 3, 1123, 1137, Scientific journal, 10.1172/JCI63711

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• Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, Transgene insertion in intronic sequences of Mdga2 gene shows methylation in an imprinted manner in an Acrodysplasia (Adp) mouse line, Mai Suzuki; Davor Solter; Toshio Watanabe, The Acrodysplasia (Adp) mutation arises from the insertion of a transgene containing a mouse metallothionein-promoted bovine papilloma virus and human growth hormone-releasing factor gene. Although the transgene is not expressed, mice that are hemizygous for the transgene show skull and paw deformities when the progeny receive the transgene paternally. To elucidate the molecular mechanisms underlying the mutant phenotype and the modified transmission pattern of the Adp phenotype, a junctional fragment around the transgene integration site was cloned. The transgene was inserted into the intronic sequences between exon 3 and exon 4 of the Mdga2 gene and the degree of methylation of the transgene and the severity of the phenotype were reciprocally related in that the transgene was highly or under methylated in normal and deformed mice, respectively. Thus, methylation of the transgene appears to regulate phenotypic expression and imprinting of Adp. (C) 2012 Elsevier Inc. All rights reserved., Feb. 2012, 418, 3, 439, 444, Scientific journal, 10.1016/j.bbrc.2012.01.032

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• Refereed, PLOS ONE, PUBLIC LIBRARY SCIENCE, The Clathrin Assembly Protein PICALM Is Required for Erythroid Maturation and Transferrin Internalization in Mice, Mai Suzuki; Hirokazu Tanaka; Akira Tanimura; Kenji Tanabe; Natsuko Oe; Shinya Rai; Syunsuke Kon; Manabu Fukumoto; Kohji Takei; Takaya Abe; Itaru Matsumura; Yuzuru Kanakura; Toshio Watanabe, Phosphatidylinositol binding clathrin assembly protein (PICALM), also known as clathrin assembly lymphoid myeloid leukemia protein (CALM), was originally isolated as part of the fusion gene CALM/AF10, which results from the chromosomal translocation t(10;11)(p13;q14). CALM is sufficient to drive clathrin assembly in vitro on lipid monolayers and regulates clathrin-coated budding and the size and shape of the vesicles at the plasma membrane. However, the physiological role of CALM has yet to be elucidated. Here, the role of CALM in vivo was investigated using CALM-deficient mice. CALM-deficient mice exhibited retarded growth in utero and were dwarfed throughout their shortened life-spans. Moreover, CALM-deficient mice suffered from severe anemia, and the maturation and iron content in erythroid precursors were severely impaired. CALM-deficient erythroid cells and embryonic fibroblasts exhibited impaired clathrin-mediated endocytosis of transferrin. These results indicate that CALM is required for erythroid maturation and transferrin internalization in mice., Feb. 2012, 7, 2, 10.1371/journal.pone.0031854, Scientific journal, 10.1371/journal.pone.0031854

  DOI URL共同研究・競争的資金等URL
• Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, Human VAPA and the yeast VAP Scs2p with an altered proline distribution can phenocopy amyotrophic lateral sclerosis-associated VAPB(P56S), Shoko Nakamichi; Kumiko Yamanaka; Mai Suzuki; Toshio Watanabe; Satoshi Kagiwada, A human isoform of the vesicle-associated membrane protein-associated proteins (VAPs), VAPB, causes amyotrophic lateral sclerosis eight due to the missense mutation of Pro-56, whereas human VAPA and the yeast VAR Scs2p proteins are not significantly affected by similar mutations. We have found that VAPA and Scs2p have three prolines present in a conserved region however VAPB has only two prolines in this region. Consequently, this mutation in VAPB (VAPB(P56S)) leaves a single proline in this region whereas other VAPs can retain two proline residues even if the proline equivalent to the Pro-56 is substituted. When Scs2p and VAPA were mutated to be equivalent to VAPB(P56S) in terms of the distribution of proline residues in this region, Scs2p became inactive and aggregated, and VAPA localize to membranous aggregates indistinguishable from those induced by VAPB(P56S). This suggests that the appropriate distribution of three conserved prolines, not the existence of a particular proline, confers VAPA and Scs2p resistance to the Pro-56 mutation and, therefore, is critical for VAR activities. (C) 2010 Elsevier Inc. All rights reserved., Jan. 2011, 404, 2, 605, 609, Scientific journal, 10.1016/j.bbrc.2010.12.011

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• Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, The carboxy-terminal region of SMAP2 directs subcellular localization as well as Arf protein specificity, Ikuko Sakakura; Kenji Tanabe; Natsurni Nouki; Mai Suzuki; Masanobu Satake; Toshio Watanabe, Small G proteins play a central role in the organization of secretory and endocytotic pathways. The recruitment of some effectors, including vesicle coat proteins, is mediated by the ADP-ribosylation factor (Art) family. Arf proteins have distinct subcellular localizations. ArfGAPs (Arf GTPase-activating proteins) regulate Arf GTPase activity. Thus, each ArfGAP is distinctly localized to allow it to maintain a specific interaction with its target Arf(s). However, the domains that regulate the subcellular localization of ArfGAPs and the way in which these subcellular localizations affect the target specificities of ArfGAPs remain unclear. Recently, we identified two novel ArfGAPs, SMAP1 (Small ArfGAP protein 1) and SMAP2. In the current study, we identified sequences in the carboxy-terminal region of SMAP2 that are critical for its specific subcellular localization and its specificity for Arf proteins. (C) 2010 Elsevier Inc. All rights reserved., Jan. 2011, 404, 2, 661, 666, Scientific journal, 10.1016/j.bbrc.2010.12.035

  DOI URL共同研究・競争的資金等URL
• Refereed, CELL STRUCTURE AND FUNCTION, JAPAN SOC CELL BIOLOGY, Localization of SMAP2 to the TGN and its Function in the Regulation of TGN Protein Transport, Tomo Funaki; Shunsuke Kon; Roger E. Ronn; Yuji Henmi; Yuka Kobayashi; Toshio Watanabe; Keiko Nakayama; Kenji Tanabe; Masanobu Satake, SMAP2 is an Arf GTPase-activating protein that is located and functions on early endosome membranes. In the present study, the trans-Golgi network (TGN) was verified as an additional site of SMAP2 localization based on its co-localization with various TGN-marker proteins. Mutation of specific stretches of basic amino acid residues abolished the TGN-localization of SMAP2. Over-expression of wild-type SMAP2, but not of the mutated SMAP2, inhibited the transport of vesicular stomatitis virus-G protein from the TGN to the plasma membrane. In contrast, this transport was enhanced in SMAP2 (-/-) cells characterized by increased levels of the activated form of Arf. SMAP2 therefore belongs to an ArfGAP subtype that resides on the TGN and functions as a negative regulator of vesicle budding from the organelle., 2011, 36, 1, 83, 95, Scientific journal, 10.1247/csf.10022

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• Refereed, JOURNAL OF IMMUNOLOGY, AMER ASSOC IMMUNOLOGISTS, Diminution of the AML1 transcription factor function causes differential effects on the fates of CD4 and CD8 single-positive T cells, K Hayashi; W Natsume; T Watanabe; N Abe; N Iwai; H Okada; Y Ito; M Asano; Y Iwakura; S Habu; Y Takahama; M Satake, In the thymic cortex, T lymphocytes are positively selected to survive and committed either to the CD4 single-positive (SP) or the CD8 SP Lineage. The SP cells then pass through a step of maturation in the medulla and are delivered to peripheral lymphoid tissues, We examined the role of AML1, the gene encoding a transcription factor, in the above processes by using the transgenic mice expressing a dominant interfering form of AML1 as well as mice targeted heterozygously for AML1. One phenotypic change seen in the AML1-diminished mice was the reduction in the numbers of both CD4 SP and CD8 SP thymocytes, reflecting the partial impairment of the transition from the double-positive to SP stage. In addition, distinct from the above abnormality, perturbed were several aspects of SP cells, including the maturation of SP thymocytes, the recent thymic emigration, and the proliferative responsiveness of peripheral T cells to TCR stimulation. Interestingly, the AML1 diminution caused inhibitory and enhancing effects on the CD4 SP and CD8 SP cells, respectively. These differential effects are most likely related to the reduction in the peripheral CD4 SP/CD8 SP ratio observed in the AML1-diminished mice. The AML1 transcription factor thus maintains the homeostasis of each SP subset by functioning at the later stages of T lymphocyte differentiation., Dec. 2000, 165, 12, 6816, 6824, Scientific journal
• Refereed, LEUKEMIA, NATURE PUBLISHING GROUP, The PEBP2 beta/CBF beta-SMMHC chimeric protein is localized both in the cell membrane and nuclear subfractions of leukemic cells carrying chromosomal inversion 16, S Kanto; N Chiba; Y Tanaka; S Fujita; M Endo; N Kamada; K Yoshikawa; A Fukuzaki; S Orikasa; T Watanabe; M Satake, The chromosomal inversion (16)(p13q22), which is associated with the M4-eosinophilia subtype of human acute myeloid leukemia, causes the fusion of two distinct genes. The polypeptide encoded by the chimeric gene, PEBP2 beta/CBF beta-SMMHC, retains the ability to interact with, and dominantly interfere with the function of proteins possessing the Runt homology domain. The Runt protein homologs constitute the DNA binding subunit of the PEBP2/CBF transcription factor. We examined the subcellular localization of PEBP2 beta/CBF beta-SMMHC, as well as that of Runt protein homologs in leukemic cells carrying inversion 16 by immunoblot analysis. A significant amount of the PEBP2 beta/CBF beta-SMMHC protein was recovered from the nuclear fraction along with the Runt protein homologs. Furthermore, some of both polypeptides was retained in the DNA pellet that represents the material remaining after extraction of nuclear fraction with high salt. These observations suggest that the so-called dominant interfering effect of PEBP2 beta/CBF beta-SMMHC on PEBP2/CBF occurs inside the nucleus. In addition, we could detect PEBP2 beta/CBF beta-SMMHC in the cytoplasmic membrane fraction as well. The function of this membrane-located PEBP2 beta/CBF beta-SMMHC, if any, appears to be unrelated to that of Runt protein homologs., Jul. 2000, 14, 7, 1253, 1259, Scientific journal
• Refereed, DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC, The AML1 transcription factor functions to develop and maintain hematogenic precursor cells in the embryonic aorta-gonad-mesonephros region, Y Mukouyama; N Chiba; T Hara; H Okada; Y Ito; R Kanamaru; A Miyajima; M Satake; T Watanabe, We examined the role of the AML1 transcription factor in the development of hematopoiesis in the paraaortic splanchnopleural (P-Sp) and the aorta-gonad-mesonephros (AGM) regions of mouse embryos. The activity of colony-forming units of colonies from the P-Sp/AGM region was reduced severalfold by heterozygous disruption of the AML1 gene, indicating that AML1 functioned in a dosage-dependent manner to generate hematopoietic progenitors. In addition, no hematopoietic progenitor activity was detected in the P-Sp/AGM region of embryos with an AML1 null mutation. Similar results were obtained when a dispersed culture was first prepared from the P-Sp/AGM region before assay of the activity of the colony-forming units. In a culture of cells with the AML1(+)(+) genotype, both hematopoietic and endothelial-like cell types emerged, but in a culture of cells with the AML1(-/-) genotype, only endothelial-like cells emerged. Interestingly, introduction of AML1 cDNA into the P-Sp/AGM culture with the AML1(-/-) genotype partially restored the production of hematopoietic cells. This restoration was observed for cultures prepared from 9.5-day postcoitum (dpc) embryos but not for cultures prepared from 11.5-dpc embryos. Therefore, the population of endothelial-like cells capable of growing in the AML1(-/-) culture would appear to contain inert but nonetheless competent hematogenic precursor cells up until at least the 9.5-dpc period. All these results support the notion that the AML1 transcription factor functions to develop and maintain hematogenic precursor cells in the embryonic P-Sp/AGM region. (C) 2000 Academic Press., Apr. 2000, 220, 1, 27, 36, Scientific journal, 10.1006/dbio.2000.9617

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• Refereed, ONCOGENE, STOCKTON PRESS, Indispensable role of the transcription factor PEBP2/CBF in angiogenic activity of a murine endothelial cell MSS31, K Namba; M Abe; S Saito; M Satake; T Ohmoto; T Watanabe; Y Sato, Mice lacking the AML1/PEBP2 alpha B/CBFa2 gene or PEBP2 beta/CBFb gene exhibit a defect in definitive hematopoiesis and die in utero because of hemorrhage in the central nervous system. Hematopoiesis in the embryo is considered to be tightly associated with vascular development, Here ive examined whether PEBP2/CBF plays any role in angiogenesis besides that in definitive hematopoiesis, We found that AML1/ PEBP2 alpha B/CBFa2, PEBP2 alpha A/CBFa1, and PEBP2 beta/ CBFb were expressed in a murine endothelial cell Line MISS31, The expression of these molecules as cell as the DNA binding activity of PEBP2/CBF were augmented by angiogenic growth factors such as bFGF and VEGF. Moreover, the expression of PEBP2 alpha/CBFa protein in endothelial tells was confirmed at the site of angiogenesis in vivo, To further clarify the role of PEBP2/CF in angiogenesis, we established permanent transfectants of PEBP2 beta-MYH11 gene, one that interacts with the runt domain of the alpha subunit and deregulates PEBP2/CBF in a dominant interfering manner. Proliferation, migration, and tube formation of the PEBP2 beta-MYH11 transfectants were significantly reduced in comparison with those activities of the mock transfectants. These results suggest that transcription factor PEBP2/CBF plays an important role in angiogenesis., Jan. 2000, 19, 1, 106, 114, Scientific journal, 10.1038/sj.onc.1203257

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• Refereed, CURRENT BIOLOGY, CURRENT BIOLOGY LTD, Hematopoietic cells in cultures of the murine embryonic aorta-gonad-mesonephros region are induced by c-Myb, YS Mukouyama; N Chiba; ML Mucenski; M Satake; A Miyajima; T Hara; T Watanabe, Definitive hematopoiesis begins in the para-aortic, splanchnopleural (P-Sp) and aorta-gonad-mesonephros (AGM) regions of mouse embryos and then switches to the fetal liver [1-3]. Gene-targeted mice lacking the c-Myb transcription factor have severe hematopoietic defects in the fetal liver [4], The role of c-Myb, if any, in P-Sp/AGM hematopoiesis has not been examined, however, Recently, we reported that oncostatin M can effectively expand both hematopoietic and endothelial-like cells from in vitro cultures of the AGM region [5]. Using this cell culture system, we examined the involvement of c-Myb in definitive hematopoiesis in the P-Sp and AGM regions. When primary cultures from the P-Sp or AGM regions of wildtype mouse embryos were probed with an anti-c-Myb antibody, hematopoietic cells but not endothelial like cells showed positive staining. In contrast, in the P-Sp/AGM culture from c-myb(-/-) embryos, no hematopoietic cells were generated and endothelial like cells predominated, indicating that the impairment of hematopoiesis in the liver of c-myb(-/-) embryos is actually preceded by a defect in P-Sp/AGM hematopoiesis. Hematogenic precursor cells were, however, still present in an inert but competent form among the endothelial-like, adherent cell population of c-myb(-/-) P-Sp/AGM cultures. When infected with a retrovirus carrying c-myb cDNA, these cultures gave rise to a significant number of hematopoietic cells. The rescued cells, unlike wild-type hematopoietic cells, were negative for c-Kit (a marker of hematopoietic progenitors), but did express other hematopoietic cell surface markers such as Mac-1, Gr-1 (myeloid markers), CD19, B220, Thy-1.2 (lymphoid markers), and Ter119 (an erythroid marker). Thus, c-Myb plays a role in the generation of hematopoietic cells in the embryonic P-Sp and AGM regions., Jul. 1999, 9, 15, 833, 836, Scientific journal, 10.1016/S0960-9822(99)80368-6

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• Refereed, ONCOGENE, STOCKTON PRESS, AML1(-/-) embryos do not express certain hematopoiesis-related gene transcripts including those of the PU.1 gene, H Okada; T Watanabe; M Niki; H Takano; N Chiba; N Yanai; K Tani; H Hibino; S Asano; ML Mucenski; Y Ito; T Noda; M Satake, The AML1 and PEBP2 beta/CBF beta genes encode the DNA-binding and non-binding subunits, respectively, of the heterodimeric transcription factor, PEBP2/CBF. Targeting each gene results in an almost identical phenotype, namely the complete lack of definitive hematopoiesis in the fetal liver on embryonic day 11.5 (E11.5). We examined and compared the expression levels of various hematopoiesis-related genes in wild type embryos and in embryos mutated for AML1 or PEBP2 beta/CBF beta. The RNAs were prepared from the yolk sacs of E9.5 embryos, from the aorta-gonad- mesonephros regions of E11.5 embryos and from the livers of E11.5 embryos and RT-PCR was performed to detect various gene transcripts. Transcripts were detected for most of the hematopoiesis-related genes that encode transcription factors, cytokines and cytokine receptors, even in tissues from homozygously targeted embryos. On the other hand, PU.1 transcripts were never detected in any tissue of AML1(-/-) or PEBP2 beta/CBF beta(-/-) embryos. In addition, transcripts for the Vav, flk-2/flt-3, M-CSF receptor, G-CSF receptor and c-Myb genes were not detected in certain tissues of the (-/-) embryos. The results suggest that the expression of a particular set of hematopoiesis-related genes is closely correlated with the PEBP2/CBF function., Nov. 1998, 17, 18, 2287, 2293, Scientific journal, 10.1038/sj.onc.1202151

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• Refereed, ONCOGENE, STOCKTON PRESS, Overexpression of AML1 renders a T hybridoma resistant to T cell receptor-mediated apoptosis, M Fujii; K Hayashi; M Niki; N Chiba; K Meguro; K Endo; J Kameoka; S Ito; K Abe; T Watanabe; M Satake, The AML1 gene, which encodes the DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF, is involved in several types of chromosomal translocations associated with human acute myeloid leukemia, and has been shown by gene targeting to be essential for the development of definitive hematopoiesis in the murine fetal liver. In addition, the gene is expressed abundantly in T lymphocytes and has been implicated in T cell specific gene expression. In the present study we examined the function of AML1 in T cell receptor (TCR)-mediated, Fas/Fas-ligand dependent apoptosis of a T hybridoma Line, DO11.10, Several independent cell clones overexpressing the AML1 protein were isolated by transfecting AML1 cDNA into these cells. These clones possessed an increased level of PEBP2/CBF DNA binding activity and were found to be resistant to apoptosis induced by anti-CD3 antibody treatment. Northern blot analysis revealed that induction of the Fas-ligand transcript was markedly suppressed in the anti-CD3 treated clones. Instead, expression of IL-2 receptor alpha subunit (IL-2R alpha), which is a manifestation of proliferative TCR signaling, was induced. This was in contrast to the parental, anti-CD3 treated DO11.10 cells where induction of Fas-ligand but not of IL-2R alpha was observed. Resistance of the AML1 overexpressing cell clones to TCR-mediated apoptosis is most likely attributable to the lack of Fas-ligand induction, since simultaneous treatment with anti-CD3 and anti-Fas antibodies caused apoptosis of the clones. The overall results suggest that the AML1 protein may play a pivotal role in switching TCR signaling between apoptosis and cell proliferation in T lymphocytes., Oct. 1998, 17, 14, 1813, 1820, Scientific journal, 10.1038/sj.onc.1202087

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• Refereed, ONCOGENE, STOCKTON PRESS, The chimeric protein, PEBP2 beta/CBF beta-SMMHC, disorganizes cytoplasmic stress fibers and inhibits transcriptional activation, Y Tanaka; M Fujii; K Hayashi; N Chiba; T Akaishi; R Shineha; T Nishihira; S Satomi; Y Ito; T Watanabe; M Satake, The chromosomal inversion 16(p13;q22) associated with human acute myeloid leukemia generates the chimeric PEBP2 beta/CBF beta-SMMHC gene. The PEBP2 beta/CBF beta portion of the chimeric polypeptide harbors most of the amino acid sequence of the PEBP2 beta/CBF beta protein, the non-DNA. binding subunit of the heterodimeric transcription factor, PEBP2/CBF, whereas the SMMHC portion of the chimera consists of the rod domain of the smooth muscle myosin heavy chain molecule. In this study we examined the subcellular localization of the chimeric protein land its effect both on stress fibers and transcriptional activation by transfecting cDNA into tissue culture cells. The localization of the chimera was investigated by immunocytochemical staining of cells and was found to be both cytoplasmic and nuclear, One aspect of the effect of expression of the chimera was a drastic alteration of cell morphology, The cells appeared elongated and possessed long cytoplasmic processes. Double fluorescent labeling revealed disorganization of the stress fibers and an altered F-actin staining pattern in the transfected cells. Studies using a deletion mutant showed that both the PEBP2 beta/CBF beta and SMMHC domains are necessary for the induction of the morphological alteration, A significant proportion of the chimeric protein was retained in the cytoskeleton after detergent extraction of the cells and could be recuperated as a membrane fraction, suggesting that this is one of the probable sites of action of the PEBP2 beta/CBF beta-SMMHC protein. Another effect of the chimeric protein was inhibition of transcriptional activation dependent on the PEBP2/CBF binding DNA sequence. However, deregulation of PEBP2/CBF site dependent transcription by itself was not sufficient to induce cell morphological changes. Taken together, these results indicate that the PEBP2 beta/CBF beta-SMMHC chimeric protein acts at two levels, at the level of stress fiber organization and at the level of transcriptional activation. We suggest that the action of PEBP2 beta/CBF beta-SMMHC depends to a great extent on whether it is located in the cytoplasm or in the nucleus., Aug. 1998, 17, 6, 699, 708, Scientific journal, 10.1038/sj.onc.1201985

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• Refereed, ONCOGENE, STOCKTON PRESS, The protooncogene product, PEBP2 beta/CBF beta, is mainly located in the cytoplasm and has an affinity with cytoskeletal structures, Y Tanaka; T Watanabe; N Chiba; M Niki; Y Kuroiwa; T Nishihira; S Satomi; Y Ito; M Satake, The Pebp62/Cbfb gene encodes the non-DNA binding beta subunit of the heterodimeric transcription factor, PEBP2/CBF, and has been implicated in a subtype of human acute myeloid leukemia, as well as being indispensable for the development of definitive hematopoiesis in the murine fetal liver, By examining a subcellular localization of the PEBP2 beta/CBF beta protein in tissue culture cells, we could reveal an additional aspect of the protein other than to be a subunit of a transcription factor, Immunoblot and immunocytochemical staining showed that PEBP2 beta/CBF beta was mostly present in the cytoplasm, This PEBP2 beta/CBF beta was free from its DNA-binding partner, the cc subunit of PEBP2/CBF, as judged by the electrophoretic mobility shift assays, Furthermore, a significant amount of PEBP2 beta 1/CBF beta was retained in the cytoskeleton preparation after detergent extraction of the cells and was found by double immunofluorescence to colocalize with the F-actin on stress fibers and the vinculin in membrane processes, Thus, the present study extends PEBP2 beta/CBF beta to be a cytoskeleton-affinitive as well as nuclear protein, The implications of these results are discussed., Aug. 1997, 15, 6, 677, 683, Scientific journal, 10.1038/sj.onc.1201235

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• Refereed, ONCOGENE, STOCKTON PRESS, Differentiation dependent expression and distinct subcellular localization of the protooncogene product, PEBP2 beta/CBF beta, in muscle development, N Chiba; T Watanabe; S Nomura; Y Tanaka; M Minowa; M Niki; R Kanamaru; M Satake, The Pebpb2/Cbfb gene encodes the non-DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF. To examine the expression of the PEBP2 beta/CBF beta protein in vivo, we carried out immunohistochemistry using the tissues from adult mice as well as embryos, Although PEBP2 beta/CBF beta was detected in various tissues to various degrees, interesting features of expression were observed in the skeletal myogenic cells. Here PEBP2 beta/CBF beta was found mainly to occur as cytoplasmic staining and the intensity of this staining increased depending on the differentiation stage of the cells, In the undifferentiated myoblasts PEBP2 beta/CBF beta was undetectable, whereas moderate levels of PEBP2 beta/CBF beta were detected in the elongated and aligned myocytes. PEBP2 beta/CBF beta appeared to accumulate further when the cells fused to each other to become multinucleated myotubes, Once the muscle fibers were established, PEBP2 beta/CBF beta was relocated onto or around the Z-lines. PEBP2 beta/CBF beta was also detected in the cytoplasm of cardiac myocytes and in the smooth muscle cells of the digestive tract. In all the above, the skeletal myotubes were the only case that showed both nuclear and cytoplasmic staining of PEBP2 beta/CBF beta. Thus, we could show differentiation dependent pattern of PEBP2 beta/CBF beta expression in muscle development and establish PEBP2 beta/CBF beta to be a cytoplasmic as well as nuclear protein in vivo., May 1997, 14, 21, 2543, 2552, Scientific journal, 10.1038/sj.onc.1201109

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• Refereed, GENOMICS, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, Assignment of the murine protein kinase gene DLK to chromosome 15 in the vicinity of the bt/Koa locus by genetic linkage analysis, T Watanabe; M Yanagisawa; N Matsubara; M Obinata; Y Matsui, Mar. 1997, 40, 2, 375, 376, Scientific journal, 10.1006/geno.1996.4565

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• Refereed, MOLECULAR REPRODUCTION AND DEVELOPMENT, WILEY-LISS, A novel serine/threonine kinase gene, Gek1, is expressed in meiotic testicular germ cells and primordial germ cells, M Yanagisawa; Y Mukouyama; T Watanabe; M Obinata; Y Matsui, We have isolated a novel serine/threonine kinase gene designated Gek1 from mouse primordial germ cell-derived embryonic germ cell. Gek1 is preferentially expressed in meiotic testicular germ cells and primordial germ cells. Gek1 mRNA is also detected in several other tissues, including hematopoietic organs in adult mice and central nervous system in embryos. The Gek1 cDNA encodes a protein with the consensus sequence of the catalytic domain of protein kinases in its N-terminal region. The deduced amino acid sequence of Gek1 in the kinase domain is related to those encoded by the Saccharomyces cerevisiae STE20, CDC15, and Drosophila melanogaster ninaC. The patterns of expression and the structural features of Gek1 suggest that the gene product is involved in signal transduction or nuclear division of germ cells and other proliferating cells. We also show that Gek1 locates on chromosome 11, near the wr locus, showing neuronal and reproductive defects. (C) 1996 Wiley-Liss, Inc., Dec. 1996, 45, 4, 411, 420, Scientific journal, 10.1002/(SICI)1098-2795(199612)45:4<411::AID-MRD2>3.0.CO;2-U

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• Refereed, DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, A receptor tyrosine kinase, sky, and its ligand gas 6 are expressed in gonads and support primordial germ cell growth or survival in culture, N Matsubara; Y Takahashi; Y Nishina; Y Mukouyama; M Yanagisawa; T Watanabe; T Nakano; K Nomura; H Arita; Y Nishimune; M Obinata; Y Matsui, Although a number of growth factors and their receptors are involved in the proliferation and differentiation of primordial germ cells (PGCs), the only factor that has been shown to be active in vivo is Steel factor, a ligand for c-Kit. To identify new growth factor receptors that may be required for PGCs function in vivo, we used an reverse transcription-polymerase chain reaction-based strategy to screen for protein kinase genes expressed in PGC-derived embryonic germ cells. We report here that one such gene encoding the receptor tyrosine kinase, Sky, is expressed in both PGCs and their supporting cells in male genital ridges after 11.5 dpc. Interestingly, Sky expression was not detected in female genital ridges, although transcripts were detected in supporting cells in the developing ovary at later stages. Gas 6, a ligand for Sky, was also expressed in interstitial cells which surround Sky positive cells in genital ridges, and, in addition, it supported PGC growth or survival in culture. After birth, Sky expression in testis was restricted to Sertoli cells, and Gas 6 was detected around peritubular cells and Leydig cells. These results suggest that Gas 6-Sky signaling plays a role in PGC growth, sexual differentiation, and Sertoli cell functions in vivo. Sky expression in Sertoli cells diminished by 3 weeks of age, when haploid germ cells first appear. On the other hand, the expression in Sertoli cells was markedly upregulated in the testis of germ cell-deficient W/W-V and jsd/jsd mice. The results suggest that signals from differentiated germ cells suppress Sky gene expression in Sertoli cells. High-resolution chromosomal mapping of Sky is also reported. (C) 1996 Academic Press, Inc., Dec. 1996, 180, 2, 499, 510, Scientific journal, 10.1006/dbio.1996.0323

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• Refereed, Cancer Science, Ppp6c haploinsufficiency accelerates UV-induced BRAF(V600E)-initiated melanomagenesis, Kanazawa, Kosuke; Kishimoto, Kazuhiro; Nomura, Miyuki; Kurosawa, Koreyuki; Kato, Hiroyuki; Inoue, Yui; Miura, Koh; Fukui, Katsuya; Yamashita, Yoji; Sato, Ikuro; Tsuji, Hiroyuki; Watanabe, Toshio; Tanaka, Takuji; Yasuda, Jun; Tanuma, Nobuhiro; Shima, Hiroshi, 2021, 112, 2233, 2244, Scientific journal
• Refereed, Cancer Medicine, Ppp6c deficiency accelerates K-rasG12D-induced tongue tumorigenesis., Kazuhiro Kishimoto; Kosuke Kanazawa; Nobuhiro Tanuma; Takuji Tanaka; Miyuki Nomura; Taeko Shigemoto-Kuroda; Katsuya Fukui; Koreyuki Kurosawa; Hiroyuki Kato; Keiko Terasaki; Yoshimi Sakamoto; Yoji Yamashita; Ikuro Sato; Koh Miura; Keiichi Tamai; Issay Kitabayashi; Kazuto Matsuura; Toshio Watanabe; Jun Yasuda; Hiroyuki Tsuji; Hiroshi Shima, BACKGROUND: Effective treatments for cancer harboring mutant RAS are lacking. In Drosophila, it was reported that PP6 suppresses tumorigenicity of mutant RAS. However, the information how PP6 regulates oncogenic RAS in mammals is limited. METHODS: We examined the effects of PP6 gene (Ppp6c) deficiency on tongue tumor development in K (K-rasG12D)- and KP (K-rasG12D + Trp53-deficient)-inducible mice. RESULTS: Mice of K and KP genotypes developed squamous cell carcinoma in situ in the tongue approximately 2 weeks after the induction of Ppp6c deficiency and was euthanized due to 20% loss of body weight. Transcriptome analysis revealed significantly different gene expressions between tissues of Ppp6c-deficient tongues and those of Ppp6c wild type, while Trp53 deficiency had a relatively smaller effect. We then analyzed genes commonly altered by Ppp6c deficiency, with or without Trp53 deficiency, and identified a group concentrated in KEGG database pathways defined as 'Pathways in Cancer' and 'Cytokine-cytokine receptor interaction'. We then evaluated signals downstream of oncogenic RAS and those regulated by PP6 substrates and found that in the presence of K-rasG12D, Ppp6c deletion enhanced the activation of the ERK-ELK1-FOS, AKT-4EBP1, and AKT-FOXO-CyclinD1 axes. Ppp6c deletion combined with K-rasG12D also enhanced DNA double-strand break (DSB) accumulation and activated NFκB signaling, upregulating IL-1β, COX2, and TNF., 2021, 10, 13, 4451, 4464, Scientific journal, True, 10.1002/cam4.3962

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• Refereed, Cancer Science, PP6 deficiency in mice with KRAS mutation and Trp53 loss promotes early death by PDAC with cachexia-like features, Katsuya Fukui; Miyuki Nomura; Kazuhiro Kishimoto; Nobuhiro Tanuma; Koreyuki Kurosawa; Kosuke Kanazawa; Hiroyuki Kato; Tomoki Sato; Shinji Miura; Koh Miura; Ikuro Sato; Hiroyuki Tsuji; Yoji Yamashita; Keiichi Tamai; Toshio Watanabe; Jun Yasuda; Takuji Tanaka; Kennichi Satoh; Toru Furukawa; Keiichi Jingu; Hiroshi Shima, To examine effects of PP6 gene (Ppp6c) deficiency on pancreatic tumor development, we developed pancreas-specific, tamoxifen-inducible Cre-mediated KP (KRAS(G12D) plus Trp53-deficient) mice (cKP mice) and crossed them with Ppp6cflox / flox mice. cKP mice with the homozygous Ppp6c deletion developed pancreatic tumors, became emaciated and required euthanasia within 150 days of mutation induction, phenotypes that were not seen in heterozygous or wild-type (WT) mice. At 30 days, a comparative analysis of genes commonly altered in homozygous versus WT Ppp6c cKP mice revealed enhanced activation of Erk and NFκB pathways in homozygotes. By 80 days, the number and size of tumors and number of precancerous lesions had significantly increased in the pancreas of Ppp6c homozygous relative to heterozygous or WT cKP mice. Ppp6c-/- tumors were pathologically diagnosed as pancreatic ductal adenocarcinoma (PDAC) undergoing the epithelial-mesenchymal transition (EMT), and cancer cells had invaded surrounding tissues in three out of six cases. Transcriptome and metabolome analyses indicated an enhanced cancer-specific glycolytic metabolism in Ppp6c-deficient cKP mice and the increased expression of inflammatory cytokines. Individual Ppp6c-/- cKP mice showed weight loss, decreased skeletal muscle and adipose tissue, and increased circulating tumor necrosis factor (TNF)-α and IL-6 levels, suggestive of systemic inflammation. Overall, Ppp6c deficiency in the presence of K-ras mutations and Trp53 gene deficiency promoted pancreatic tumorigenesis with generalized cachexia and early death. This study provided the first evidence that Ppp6c suppresses mouse pancreatic carcinogenesis and supports the use of Ppp6c-deficient cKP mice as a model for developing treatments for cachexia associated with pancreatic cancer., 2022, 113, 1613, 1624, Scientific journal, True, 10.1111/cas.15315

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• Not Refereed, 生物高分子, 日本生物高分子学会, HDC‐GFP BACトランスジェニックマウスの作製, 箱田温子; 菊田香苗; 丹賀直美; 渡邊利雄; 赤桐里美; 植野洋志, 01 Sep. 2014, 14, 2, 113, 113

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• Not Refereed, 生物高分子, 日本生物高分子学会, ヒスチジンデカルボキシラーゼ(HDC)の生体内局在の探索, 箱田温子; 菊田香苗; 丹賀直美; 渡邊利雄; 赤桐里美; 植野洋志, 01 Oct. 2013, 13, 2, 54, 54

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• Refereed, Scientific Reports, Springer Science and Business Media LLC, Arf1 facilitates mast cell proliferation via the mTORC1 pathway., Yui Kotani; Mami Sumiyoshi; Moe Sasada; Toshio Watanabe; Satoshi Matsuda, Abstract Mast cells are one of major players in allergic responses. Mast cell activation via the high affinity IgE receptor (FcεRI) causes degranulation and release of de novo synthesized proinflammatory cytokines in a process that involves vesicle trafficking. Considering that the GTPase ADP-ribosylation factor 1 (Arf1) orchestrates and maintains membrane traffic and organelle structure, it seems likely that Arf1 contributes to mast cell activation. Actually, it has been reported that pharmaceutical blockade of the Arf1 pathway suppresses cytokine secretion and mast cell degranulation. However, physiological roles of Arf1 in mast cells remain elusive. Here, by using a genetic approach, we demonstrate that Arf1 is required for optimal mTORC1 activation upon IL-3 and facilitates mast cell proliferation. On the other hand, contrary to our expectation, Arf1-deficiency had little impact on FcεRI-induced degranulation nor cytokine secretion. Our findings reveal an unexpected role of Arf1 in mast cell expansion and its potential as a therapeutic target in the mast cell proliferative disorders., Dec. 2022, 12, 1, 22297, Scientific journal, 10.1038/s41598-022-26925-1

  PermalinkURLDOI URL共同研究・競争的資金等URL
• 日本癌学会総会記事, (一社)日本癌学会, Supt6hとFACT complexは白血病関連因子AF10と結合し、MLL融合遺伝子白血病発症に寄与する(AF10 links histone chaperones Supt6h and FACT complex to MLL-fusion leukemia), 山形 和恒; 齋藤 眞理子; 鈴木 舞; 勝本 拓夫; 小河 穂波; 渡邊 利雄; 北林 一生, Sep. 2018, 77回, 1936, 1936
• 日本癌学会総会記事, (一社)日本癌学会, ヒストンシャペロンSupt6hとFACT複合体は、AF10との結合を介してMLL転座関連白血病の発症に寄与する, 山形 和恒; 齋藤 真理子; 鈴木 舞; 小河 穂波; 渡邊 利雄; 北林 一生, Sep. 2017, 76回, P, 2035
• 日本癌学会総会記事, (一社)日本癌学会, MLL融合遺伝子関連白血病発症におけるAF10の役割(The role of AF10 in the development of MLL-fusion leukemia), 山形 和恒; 齋藤 眞理子; 鈴木 麻衣; 相川 祐規子; 勝本 拓夫; 小河 穂波; 渡邊 利雄; 北林 一生, Sep. 2014, 73回, E, 3049
• Refereed, Biochem. Biophys. Res. Commun., Neuron specific loss of Ppp6c induces neonatal death and a decrease in the number of cortical neurons and interneurons., Miki Mtsuoka; Daisuke Sakai; Hiroshi Shima; Toshio Watanabe, Jan. 2024, 693, 149353, Scientific journal

  共同研究・競争的資金等URL
• Refereed, FEBS Open Bio, Wiley, Oncogenic K‐Ras^G12V cannot overcome proliferation failure caused by loss of Ppp6c in mouse embryonic fibroblasts, Mai Ito; Nobuhiro Tanuma; Yui Kotani; Kokoro Murai; Ayumi Kondo; Mami Sumiyoshi; Hiroshi Shima; Satoshi Matsuda; Toshio Watanabe, Protein phosphatase 6 is a Ser/Thr protein phosphatase and its catalytic subunit is Ppp6c. Ppp6c is thought to be indispensable for proper growth of normal cells. On the other hand, loss of Ppp6c accelerates growth of oncogenic Ras‐expressing cells. Although it has been studied in multiple contexts, the role(s) of Ppp6c in cell proliferation remains controversial. It is unclear how oncogenic K‐Ras overcomes cell proliferation failure induced by Ppp6c deficiency; therefore, in this study, we attempted to shed light on how oncogenic K‐Ras modulates tumor cell growth. Contrary to our expectations, loss of Ppp6c decreased proliferation, anchorage‐independent growth in soft agar, and tumor formation of oncogenic Ras‐expressing mouse embryonic fibroblasts (MEFs). These findings show that oncogenic K‐Ras^G12V cannot overcome proliferation failure caused by loss of Ppp6c in MEFs., 06 Feb. 2024, Scientific journal, 10.1002/2211-5463.13775

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• Refereed, Experimental Hematology, Depletion of Ppp6c in hematopoietic and vascular endothelial cells causes embryonic lethality and decreased hematopoietic potential., Ayumi Kondo; Hirokazu Tanaka; Shinya Rai; Hiroshi Shima; Itaru Matsumura; Toshio Watanabe, Mar. 2024, 133, 104205, Scientific journal

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• Refereed, BBA Molecular Basis of Disease, Knockout of Brca1-interacting factor Ola1 in female mice induces tumors with centrosome amplification suppressed by estrogen stimulation, Yuki Yoshino; Honami Ogoh; Yudai Iichi; Tomohiro Sasaki; Takahiro Yoshida; Shiori Ichimura; Masahiro Nakayama; Shi Go; Hiroki Fujita; Megumi Kikuchi; Zhenzhou Fang; Xingming Li; Takaya Abe; Mitsuru Futakuchi; Yasuhiro Nakamura; Toshio Watanabe; Natsuko Chiba, 2024, Scientific journal
• Refereed, FEBS Letters, Increased ROS in Mul1-deficient oocytes results in abnormal embryogenesis before implantation, Ann Nakai; Yuki Fukushima; Mitsuhiro Nishigori; Ayaka Yamamoto; Yuki Amatsu; Yukino Yoshioka; Xiaoyan Chen; Takumi Koshiba; Toshio Watanabe, 2024, Scientific journal

MISC

• 65, 10, 635, 640
• Not Refereed, 細胞工学, 脱リン酸化酵素PP6は皮膚がん抑制遺伝子として働く： 25年目のミッシングピースの発見, 渡邊 利雄; 渡邊利雄; 島礼, 2015, 34, 3, 302, 303
• Not Refereed, BLOOD, AMER SOC HEMATOLOGY, Chlorpromazine, an Inhibitor of Intracellular Trafficking of FLT3-ITD and KIT D816V, Shows Prominent Anti-Leukemic Activities Against AML Cells and AML Stem Cells in Vitro and in Vivo., Shinya Rai; Hirokazu Tanaka; Mai Suzuki; Akira Tanimura; Keiko Matsui; Toshio Watanabe; Yuzuru Kanakura; Itaru Matsumura, Dec. 2014, 124, 21, Summary international conference
• Not Refereed, CANCER RESEARCH, AMER ASSOC CANCER RESEARCH, The nuclear export signal (NES) within CALM is necessary for CALM-AF10-induced leukemia, Mai Suzuki; Kazutsune Yamagata; Yukiko Aikawa; Toshio Watanabe; Issay Kitabayashi, Oct. 2014, 74, 19, Summary international conference, 10.1158/1538-7445.AM2014-1569

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• Not Refereed, 日本生化学会大会(Web), ヒスタミン合成酵素ヒスチジンデカルボキシラーゼ(HDC)の活性阻害物質の探索とHDC/GFPノックインマウス作製に関する取組み, 箱田温子; 平野真実子; 新田陽子; 菊田香苗; 丹賀直美; 赤桐里美; 渡邊利雄; 植野洋志, 2013, 86th, 3P-387 (WEB ONLY)

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• Not Refereed, 細胞工学, 精子頭部形成を支配するGTPase活性化遺伝子SMAP2：輸送小胞形成不全は受精できない丸頭精子を生み出す。, 渡邊 利雄; 船木智; 昆俊介; 田邊 賢司; 佐竹正延, 2013, 32, 12, 1260, 1261
• Not Refereed, 日本分子生物学会年会プログラム・要旨集(Web), クラスリン小胞形成因子SMAP1の欠損は細胞内小胞輸送の異常をきたし,骨髄異型性症候群を誘引する, 昆俊亮; 峯岸直子; 田邊賢司; 渡邊利雄; 船木智; 坂元大輔; 樋口雄大; 清成寛; 浅野克敏; 福本学; 真田昌; 小川誠司; 中村卓郎; 佐竹正延, 2012, 35th, WEB ONLY 1W2II-4
• Not Refereed, 細胞工学, クラスリン集合因子CALMはトランスフェリンの取り込みに必要で，遺伝子欠損はマウスに貧血を引き起こす：培養細胞研究での定説を覆す, 渡邊 利雄; 鈴木麻衣, 2012, 31, 7, 808, 809
• Not Refereed, 細胞工学, 気づいた時が変わるとき－チロシンホスファターゼ研究との出会い－, 渡邊 利雄, 2011
• Not Refereed, IMMUNOLOGY, WILEY-BLACKWELL, Over-expression of Runx1 transcription factor impairs the development of thymocytes from the double-negative to double-positive stages, Won F. Wong; Megumi Nakazato; Toshio Watanabe; Kazuyoshi Kohu; Takehiro Ogata; Naomi Yoshida; Yusuke Sotomaru; Mamoru Ito; Kimi Araki; Janice Telfer; Manabu Fukumoto; Daisuke Suzuki; Takehito Sato; Katsuto Hozumi; Sonoko Habu; Masanobu Satake, P>Runx1 transcription factor is highly expressed at a CD4/CD8-double-negative (DN) stage of thymocyte development but is down-regulated when cells proceed to the double-positive (DP) stage. In the present study, we examined whether the down-regulation of Runx1 is necessary for thymocyte differentiation from the DN to DP stage. When Runx1 was artificially over-expressed in thymocytes by Lck-driven Cre, the DN3 population was unaffected, as exemplified by proper pre-T-cell receptor expression, whereas the DN4 population was perturbed as shown by the decrease in the CD27hi sub-fraction. In parallel, the growth rate of DN4 cells was reduced by half, as measured by bromodeoxyuridine incorporation. These events impaired the transition of DN4 cells to the DP stage, resulting in the drastic reduction of the number of DP thymocytes. The Runx1 gene has two promoters, a proximal and a distal promoter; and, in thymocytes, endogenous Runx1 was mainly transcribed from the distal promoter. Interestingly, only distal, but not proximal, Runx1 over-expression exhibited an inhibitory effect on thymocyte differentiation, suggesting that the distal Runx1 protein may fulfil a unique function. Our collective results indicate that production of the distal Runx1 protein must be adequately down-regulated for thymocytes to transit from the DN to the DP stage, a critical step in the massive expansion of the T-cell lineage., Jun. 2010, 130, 2, 243, 253, 10.1111/j.1365-2567.2009.03230.x

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• Not Refereed, IMMUNOLOGY, WILEY-BLACKWELL, Over-expression of Runx1 transcription factor impairs the development of thymocytes from the double-negative to double-positive stages, Won F. Wong; Megumi Nakazato; Toshio Watanabe; Kazuyoshi Kohu; Takehiro Ogata; Naomi Yoshida; Yusuke Sotomaru; Mamoru Ito; Kimi Araki; Janice Telfer; Manabu Fukumoto; Daisuke Suzuki; Takehito Sato; Katsuto Hozumi; Sonoko Habu; Masanobu Satake, P>Runx1 transcription factor is highly expressed at a CD4/CD8-double-negative (DN) stage of thymocyte development but is down-regulated when cells proceed to the double-positive (DP) stage. In the present study, we examined whether the down-regulation of Runx1 is necessary for thymocyte differentiation from the DN to DP stage. When Runx1 was artificially over-expressed in thymocytes by Lck-driven Cre, the DN3 population was unaffected, as exemplified by proper pre-T-cell receptor expression, whereas the DN4 population was perturbed as shown by the decrease in the CD27hi sub-fraction. In parallel, the growth rate of DN4 cells was reduced by half, as measured by bromodeoxyuridine incorporation. These events impaired the transition of DN4 cells to the DP stage, resulting in the drastic reduction of the number of DP thymocytes. The Runx1 gene has two promoters, a proximal and a distal promoter; and, in thymocytes, endogenous Runx1 was mainly transcribed from the distal promoter. Interestingly, only distal, but not proximal, Runx1 over-expression exhibited an inhibitory effect on thymocyte differentiation, suggesting that the distal Runx1 protein may fulfil a unique function. Our collective results indicate that production of the distal Runx1 protein must be adequately down-regulated for thymocytes to transit from the DN to the DP stage, a critical step in the massive expansion of the T-cell lineage., Jun. 2010, 130, 2, 243, 253, 10.1111/j.1365-2567.2009.03230.x

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• Not Refereed, 日本細胞生物学会大会講演要旨集, (一社)日本細胞生物学会, メンブレントラフィック クラスリン小胞輸送の機能障害は細胞増殖異常をきたす(Membrane traffic Dysregulation of clathrin-dependent traffic causes neoplasia), 昆 俊亮; 坂元 大輔; 樋口 祐大; 田邊 賢司; 峯岸 直子; 渡邊 利雄; 中村 卓郎; 佐竹 正延, May 2010, 62回, 116, 116
• Not Refereed, BIOCHEMICAL JOURNAL, PORTLAND PRESS LTD, Filamin associates with stress signalling kinases MKK7 and MKK4 and regulates JNK activation, Kentaro Nakagawa; Misato Sugahara; Tokiwa Yamasaki; Hiroaki Kajiho; Shinya Takahashi; Jun Hirayama; Yasuhiro Minami; Yasutaka Ohta; Toshio Watanabe; Yutaka Hata; Toshiaki Katada; Hiroshi Nishina, SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) belongs to the MAPK (mitogen-activated protein kinase) family and is important in many biological contexts. JNK activation is regulated by phosphorylation of specific tyrosine and threonine residues sequentially catalysed by MKK4 and MKK7, which are both dual-specificity MAPKKs (MAPK kinases). Previously, we reported that tyrosine-phosphorylation of JNK by MKK4 precedes threonine-phosphorylation by MKK7, and that both are required for synergistic JNK activation. In the present study, we identify the actin-binding protein-280 (Filamin A) as a presumed 'binder' protein that can bind to MKK7, as well as to MKK4, connecting them in close proximity. We show that Filamin family members A, B and C interact with MKK4 and MKK7, but not with JNK. Filamin A binds to an N-terminal region (residues 31-60) present in the MKK7 gamma and MKK7 beta splice isoforms, but cannot bind to MKK7 alpha which lacks these amino acids. This same N-terminal region is crucial for the intracellular co-localization of MKK7 gamma with actin stress fibres and Filamin A. Experiments using Filamin-A-deletion mutants revealed that the MKK7-binding region of Filamin A differs from its MKK4-binding region, and that MKK7 gamma (but not MKK7 alpha) can form a complex with Filamin A and MKK4. Finally, we used Filamin-A-deficient cells to show that Filamin A enhances MKK7 activation and is important for synergistic stress-induced JNK activation in vivo. Thus Filamin A is a novel member of the group of scaffold proteins whose function is to link two MAPKKs together and promote JNK activation., Apr. 2010, 427, 2, 237, 245, 10.1042/BJ20091011

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• Not Refereed, BIOCHEMICAL JOURNAL, PORTLAND PRESS LTD, Filamin associates with stress signalling kinases MKK7 and MKK4 and regulates JNK activation, Kentaro Nakagawa; Misato Sugahara; Tokiwa Yamasaki; Hiroaki Kajiho; Shinya Takahashi; Jun Hirayama; Yasuhiro Minami; Yasutaka Ohta; Toshio Watanabe; Yutaka Hata; Toshiaki Katada; Hiroshi Nishina, SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) belongs to the MAPK (mitogen-activated protein kinase) family and is important in many biological contexts. JNK activation is regulated by phosphorylation of specific tyrosine and threonine residues sequentially catalysed by MKK4 and MKK7, which are both dual-specificity MAPKKs (MAPK kinases). Previously, we reported that tyrosine-phosphorylation of JNK by MKK4 precedes threonine-phosphorylation by MKK7, and that both are required for synergistic JNK activation. In the present study, we identify the actin-binding protein-280 (Filamin A) as a presumed 'binder' protein that can bind to MKK7, as well as to MKK4, connecting them in close proximity. We show that Filamin family members A, B and C interact with MKK4 and MKK7, but not with JNK. Filamin A binds to an N-terminal region (residues 31-60) present in the MKK7 gamma and MKK7 beta splice isoforms, but cannot bind to MKK7 alpha which lacks these amino acids. This same N-terminal region is crucial for the intracellular co-localization of MKK7 gamma with actin stress fibres and Filamin A. Experiments using Filamin-A-deletion mutants revealed that the MKK7-binding region of Filamin A differs from its MKK4-binding region, and that MKK7 gamma (but not MKK7 alpha) can form a complex with Filamin A and MKK4. Finally, we used Filamin-A-deficient cells to show that Filamin A enhances MKK7 activation and is important for synergistic stress-induced JNK activation in vivo. Thus Filamin A is a novel member of the group of scaffold proteins whose function is to link two MAPKKs together and promote JNK activation., Apr. 2010, 427, 2, 237, 245, 10.1042/BJ20091011

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• Not Refereed, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, NATL ACAD SCIENCES, Identification and functional characterization of paxillin as a target of protein tyrosine phosphatase receptor T, Yiqing Zhao; Xiaodong Zhang; Kishore Guda; Earl Lawrence; Qun Sun; Toshio Watanabe; Yoichiro Iwakura; Masahide Asano; Lanlan Wei; Zhirong Yang; Weiping Zheng; Dawn Dawson; Joseph Willis; Sanford D. Markowitz; Masanobu Satake; Zhenghe Wang, Protein tyrosine phosphatase receptor-type T (PTPRT) is the most frequently mutated tyrosine phosphatase in human cancers. However, the cell signaling pathways regulated by PTPRT largely remain to be elucidated. Here, we show that paxillin is a direct substrate of PTPRT and that PTPRT specifically regulates paxillin phosphorylation at tyrosine residue 88 (Y88) in colorectal cancer (CRC) cells. We engineered CRC cells homozygous for a paxillin Y88F knock-in mutant and found that these cells exhibit significantly reduced cell migration and impaired anchorage-independent growth, fail to form xenograft tumors in nude mice, and have decreased phosphorylation of p130CAS, SHP2, and AKT. PTPRT knockout mice that we generated exhibit increased levels of colonic paxillin phosphorylation at residue Y88 and are highly susceptible to carcinogen azoxymethane-induced colon tumor, providing critical in vivo evidence that PTPRT normally functions as a tumor suppressor. Moreover, similarly increased paxillin pY88 is also found as a common feature of human colon cancers. These studies reveal an important signaling pathway that plays a critical role in colorectal tumorigenesis., Feb. 2010, 107, 6, 2592, 2597, 10.1073/pnas.0914884107

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• Not Refereed, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, NATL ACAD SCIENCES, Identification and functional characterization of paxillin as a target of protein tyrosine phosphatase receptor T, Yiqing Zhao; Xiaodong Zhang; Kishore Guda; Earl Lawrence; Qun Sun; Toshio Watanabe; Yoichiro Iwakura; Masahide Asano; Lanlan Wei; Zhirong Yang; Weiping Zheng; Dawn Dawson; Joseph Willis; Sanford D. Markowitz; Masanobu Satake; Zhenghe Wang, Protein tyrosine phosphatase receptor-type T (PTPRT) is the most frequently mutated tyrosine phosphatase in human cancers. However, the cell signaling pathways regulated by PTPRT largely remain to be elucidated. Here, we show that paxillin is a direct substrate of PTPRT and that PTPRT specifically regulates paxillin phosphorylation at tyrosine residue 88 (Y88) in colorectal cancer (CRC) cells. We engineered CRC cells homozygous for a paxillin Y88F knock-in mutant and found that these cells exhibit significantly reduced cell migration and impaired anchorage-independent growth, fail to form xenograft tumors in nude mice, and have decreased phosphorylation of p130CAS, SHP2, and AKT. PTPRT knockout mice that we generated exhibit increased levels of colonic paxillin phosphorylation at residue Y88 and are highly susceptible to carcinogen azoxymethane-induced colon tumor, providing critical in vivo evidence that PTPRT normally functions as a tumor suppressor. Moreover, similarly increased paxillin pY88 is also found as a common feature of human colon cancers. These studies reveal an important signaling pathway that plays a critical role in colorectal tumorigenesis., Feb. 2010, 107, 6, 2592, 2597, 10.1073/pnas.0914884107

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• Not Refereed, 細胞工学, パキシリンはがん遺伝子でチロシンホスファターゼPTPRTはがん抑制遺伝子である, 渡邊 利雄, 2010, 29, 7, 704, 705
• Not Refereed, 細胞工学, コミュニティ―を感じさせるノックアウトマウスの成果－未だ見知らぬ未来の研究仲間へのエール, 渡邊 利雄, 2010, 29, 8, 824, 825
• Not Refereed, 日本細胞生物学会大会講演要旨集, (一社)日本細胞生物学会, クラスリン小胞輸送の機能障害は細胞増殖異常をきたす(Impairment of clathrin-dependent traffic causes neoplasia), 昆 俊亮; 田邊 賢司; 峯岸 直子; 渡邊 利雄; 佐竹 正延, May 2009, 61回, 195, 195
• Not Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, Hematopoiesis-dependent expression of CD44 in murine hepatic progenitor cells, Shinya Ohata; Makiko Nawa; Takeshi Kasama; Tokiwa Yamasaki; Kenji Sawanobori; Shoji Hata; Takashi Nakamura; Yoichi Asaoka; Toshio Watanabe; Hitoshi Okamoto; Takahiko Hara; Shuji Terai; Isao Sakaida; Toshiaki Katada; Hiroshi Nishina, The fetal liver serves as the predominant hematopoietic organ until birth. However, the mechanisms underlying this link between hematopoiesis and hepatogenesis are unclear. Previously, we reported the isolation of a monoclonal antibody (anti-Liv8) that specifically recognizes an antigen (Liv8) present in murine fetal livers at embryonic day 11.5 (E11.5). Liv8 is a cell surface molecule expressed by hematopoietic cells in both fetal liver and adult mouse bone marrow. Here, we report that Liv8 is also transiently expressed by hepatoblasts at E11.5. Using protein purification and mass spectrometry, we have identified Liv8 as the CD44 protein. Interestingly, the expression of Liv8/CD44 in fetal liver was completely lost in AML1(-/-) murine embryos, which lack definitive hematopoiesis. These results show that hepatoblasts change from Liv8/CD44-negative to Liv8/CD44-positive status in a hematopoiesis-dependent manner by E11.5, and indicate that Liv8/CD44 expression is an important link between hematopoiesis and hepatogenesis during fetal liver development. (C) 2008 Elsevier Inc. All rights reserved., Feb. 2009, 379, 4, 817, 823, 10.1016/j.bbrc.2008.12.149

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• Not Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, Hematopoiesis-dependent expression of CD44 in murine hepatic progenitor cells, Shinya Ohata; Makiko Nawa; Takeshi Kasama; Tokiwa Yamasaki; Kenji Sawanobori; Shoji Hata; Takashi Nakamura; Yoichi Asaoka; Toshio Watanabe; Hitoshi Okamoto; Takahiko Hara; Shuji Terai; Isao Sakaida; Toshiaki Katada; Hiroshi Nishina, The fetal liver serves as the predominant hematopoietic organ until birth. However, the mechanisms underlying this link between hematopoiesis and hepatogenesis are unclear. Previously, we reported the isolation of a monoclonal antibody (anti-Liv8) that specifically recognizes an antigen (Liv8) present in murine fetal livers at embryonic day 11.5 (E11.5). Liv8 is a cell surface molecule expressed by hematopoietic cells in both fetal liver and adult mouse bone marrow. Here, we report that Liv8 is also transiently expressed by hepatoblasts at E11.5. Using protein purification and mass spectrometry, we have identified Liv8 as the CD44 protein. Interestingly, the expression of Liv8/CD44 in fetal liver was completely lost in AML1(-/-) murine embryos, which lack definitive hematopoiesis. These results show that hepatoblasts change from Liv8/CD44-negative to Liv8/CD44-positive status in a hematopoiesis-dependent manner by E11.5, and indicate that Liv8/CD44 expression is an important link between hematopoiesis and hepatogenesis during fetal liver development. (C) 2008 Elsevier Inc. All rights reserved., Feb. 2009, 379, 4, 817, 823, 10.1016/j.bbrc.2008.12.149

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• Not Refereed, Experimental Cell Research, Academic Press Inc., Clathrin dependent endocytosis of E-cadherin is regulated by the Arf6GAP isoform SMAP1, Shunsuke Kon; Kenji Tanabe; Toshio Watanabe; Hisataka Sabe; Masanobu Satake, E-cadherin is a central component of the adherens junction in epithelial cells and continuously undergoes endocytosis via clathrin-coated vesicles and/or caveolae depending on the cell type. In this study, we examined the role of SMAP1, a clathrin-interacting GTPase-activating protein (GAP) for the ADP-ribosylation factor 6 (Arf6) GTPase, in E-cadherin endocytosis. Mardin-Darby canine kidney (MDCK) epithelial cells were used as a model, and SMAP1 localized in the cytoplasm and along the adherens junction where E-cadherin was present. Next, activity of SMAP1 was compared with that of other Arf6GAPs (and/or an effector of Arf6-GTP), namely GIT1 and AMAP2/DDEF2. Overexpression of SMAP1 but not GIT1 nor AMAP2/DDEF2 strongly inhibited basal, as well as phorbolester-induced, internalization of E-cadherin. Notably, AMAP2/DDEF2 rather enhanced the caveolae-mediated incorporation of a membrane protein other than E-cadherin. Thus, in MDCK cells, E-cadherin appeared to be endocytosed solely through SMAP1-regulated clathrin-coated vesicles. Furthermore, MDCK cells overexpressing SMAP1 showed a reduced degree of cell migration compared to untransfected cells, as assessed by wound healing and Transwell assays, and this reduction in migration appeared to be due to the accumulation of E-cadherin at the adherens junction in cells overexpressing SMAP1. Collectively, SMAP1 likely represents a key Arf6GAP in clathrin dependent endocytosis of E-cadherin in MDCK cells. This activity of SMAP1 in E-cadherin turnover may be involved in epithelial organization and/or epithelial-mesenchymal transition. © 2007 Elsevier Inc. All rights reserved., 15 Apr. 2008, 314, 7, 1415, 1428, 10.1016/j.yexcr.2007.11.006

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• Not Refereed, EXPERIMENTAL CELL RESEARCH, ELSEVIER INC, Clathrin dependent endocytosis of E-cadherin is regulated by the Arf6GAP isoform SMAP1, Shunsuke Kon; Kenji Tanabe; Toshio Watanabe; Hisataka Sabe; Masanobu Satake, E-cadherin is a central component of the adherens junction in epithelial cells and continuously undergoes endocytosis via clathrin-coated vesicles and/or caveolae depending on the cell type. In this study, we examined the role of SMAP1, a clathrin-interacting GTPase-activating protein (GAP) for the ADP-ribosylation factor 6 (Arf6) GTPase, in E-cadherin endocytosis. Mardin-Darby canine kidney (MDCK) epithelial cells were used as a model, and SMAP1 localized in the cytoplasm and along the adherens junction where E-cadherin was present. Next, activity of SMAP1 was compared with that of other Arf6GAPs (and/or an effector of Arf6-GTP), namely GIT1 and AMAP2/DDEF2. Overexpression of SMAP1 but not GIT1 nor AMAP2/DDEF2 strongly inhibited basal, as well as phorbolester-induced, internalization of E-cadherin. Notably, AMAP2/DDEF2 rather enhanced the caveolae-mediated incorporation of a membrane protein other than E-cadherin. Thus, in MDCK cells, E-cadherin appeared to be endocytosed solely through SMAP1-regulated clathrin-coated vesicles. Furthermore, MDCK cells overexpressing SMAP1 showed a reduced degree of cell migration compared to untransfected cells, as assessed by wound healing and Transwell assays, and this reduction in migration appeared to be due to the accumulation of E-cadherin at the adherens junction in cells overexpressing SMAP1. Collectively, SMAP1 likely represents a key Arf6GAP in clathrin dependent endocytosis of E-cadherin in MDCK cells. This activity of SMAP1 in E-cadherin turnover may be involved in epithelial organization and/or epithelial-mesenchymal transition. (C) 2007 Elsevier Inc. All rights reserved., Apr. 2008, 314, 7, 1415, 1428, 10.1016/j.yexcr.2007.11.006

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• Not Refereed, SMALL GTPASES IN DISEASE, PART A, ELSEVIER ACADEMIC PRESS INC, A SMAP gene family encoding ARF GTPase-activating proteins and its implication in membrane trafficking, Kenji Tanabe; Shunsuke Kon; Nobuyuki Ichijo; Tomo Funaki; Waka Natsume; Toshio Watanabe; Masanobu Satake, SMAP1 and SMAP2 proteins constitute a subfamily of the Arf-specific GTPase-activating proteins. Both SMAP proteins bind to clathrin heavy chains and are involved in the trafficking of clathrin-coated vesicles. In cells, SMAP1 regulates Arf6-dependent endocytosis of transferrin receptors from the coated pits of the plasma membrane, whereas SMAP2 regulates Arf1-dependent retrograde transport of TGN38 from the early endosome to the trans-Golgi network. The common and distinct features of SMAP1 and SMAP2 activity provide a valuable opportunity to examine the differential regulation of membrane trafficking by these two proteins. In this chapter, we describe several basic experimental procedures that have been used to study the regulation of membrane trafficking using SMAP proteins, including a GAP assay as well as procedures to study the transport of transferrin receptors and TGN38. In addition, a yeast two-hybrid system is described because of its utility in identifying novel molecules that interact with SMAP., 2008, 438, 155, 170, 10.1016/S0076-6879(07)38011-7

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• Not Refereed, SMALL GTPASES IN DISEASE, PART A, ELSEVIER ACADEMIC PRESS INC, A SMAP gene family encoding ARF GTPase-activating proteins and its implication in membrane trafficking, Kenji Tanabe; Shunsuke Kon; Nobuyuki Ichijo; Tomo Funaki; Waka Natsume; Toshio Watanabe; Masanobu Satake, SMAP1 and SMAP2 proteins constitute a subfamily of the Arf-specific GTPase-activating proteins. Both SMAP proteins bind to clathrin heavy chains and are involved in the trafficking of clathrin-coated vesicles. In cells, SMAP1 regulates Arf6-dependent endocytosis of transferrin receptors from the coated pits of the plasma membrane, whereas SMAP2 regulates Arf1-dependent retrograde transport of TGN38 from the early endosome to the trans-Golgi network. The common and distinct features of SMAP1 and SMAP2 activity provide a valuable opportunity to examine the differential regulation of membrane trafficking by these two proteins. In this chapter, we describe several basic experimental procedures that have been used to study the regulation of membrane trafficking using SMAP proteins, including a GAP assay as well as procedures to study the transport of transferrin receptors and TGN38. In addition, a yeast two-hybrid system is described because of its utility in identifying novel molecules that interact with SMAP., 2008, 438, 155, 170, 10.1016/S0076-6879(07)38011-7

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• Not Refereed, CANCER SCIENCE, BLACKWELL PUBLISHING, Involvement of a novel ADP-ribosylation factor GTPase-activating protein, SMAP, in membrane trafficking: Implications in cancer cell biology, Kenji Tanabe; Shunsuke Kon; Waka Natsume; Tetsuo Torii; Toshio Watanabe; Masanobu Satake, The endocytosis of cell membrane proteins is initiated by the binding of activated Arf6, a member of Ras-related GTPases, to the PM. A GAP specific for Arf6 triggers the budding of endocytotic vesicles from the PM by inactivating GTP-bound Arf6. We recently identified the SMAP gene that encodes an ArfGAP and is involved in the endocytosis of TfnR and possibly E-cadherin. In this review, we summarize the process of intracellular membrane trafficking, highlighting the roles played by the SMAP gene. Progression of cancer to malignancy occurs in parallel with the disappearance of E-cadherin, a central component of the adherens junction in epithelial cells. Therefore, elucidation of the molecular mechanism of E-cadherin endocytosis should be one of the key elements in tumor cell biology., Sep. 2006, 97, 9, 801, 806, Book review, 10.1111/j.1349-7006.2006.00251.x

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• Not Refereed, CANCER SCIENCE, BLACKWELL PUBLISHING, Involvement of a novel ADP-ribosylation factor GTPase-activating protein, SMAP, in membrane trafficking: Implications in cancer cell biology, Kenji Tanabe; Shunsuke Kon; Waka Natsume; Tetsuo Torii; Toshio Watanabe; Masanobu Satake, The endocytosis of cell membrane proteins is initiated by the binding of activated Arf6, a member of Ras-related GTPases, to the PM. A GAP specific for Arf6 triggers the budding of endocytotic vesicles from the PM by inactivating GTP-bound Arf6. We recently identified the SMAP gene that encodes an ArfGAP and is involved in the endocytosis of TfnR and possibly E-cadherin. In this review, we summarize the process of intracellular membrane trafficking, highlighting the roles played by the SMAP gene. Progression of cancer to malignancy occurs in parallel with the disappearance of E-cadherin, a central component of the adherens junction in epithelial cells. Therefore, elucidation of the molecular mechanism of E-cadherin endocytosis should be one of the key elements in tumor cell biology., Sep. 2006, 97, 9, 801, 806, Book review, 10.1111/j.1349-7006.2006.00251.x

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• Not Refereed, MOLECULAR BIOLOGY OF THE CELL, AMER SOC CELL BIOLOGY, SMAP2, a novel ARF GTPase-activating protein, interacts with clathrin and clathrin assembly protein and functions on the AP-1-positive early endosome/trans-Golgi network, W Natsume; K Tanabe; S Kon; N Yoshida; T Watanabe; T Torii; M Satake, We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1-dependent manner. Thus, the SMAP gene family constitutes an important Arf GAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking., Jun. 2006, 17, 6, 2592, 2603, 10.1091/mbc.E05-10-0909

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• Not Refereed, MOLECULAR BIOLOGY OF THE CELL, AMER SOC CELL BIOLOGY, SMAP2, a novel ARF GTPase-activating protein, interacts with clathrin and clathrin assembly protein and functions on the AP-1-positive early endosome/trans-Golgi network, W Natsume; K Tanabe; S Kon; N Yoshida; T Watanabe; T Torii; M Satake, We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1-dependent manner. Thus, the SMAP gene family constitutes an important Arf GAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking., Jun. 2006, 17, 6, 2592, 2603, 10.1091/mbc.E05-10-0909

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• Not Refereed, 血液・腫瘍科, T細胞分化におけるAML1/Runx1の機能, 渡邊 利雄; 渡邊利雄; 佐竹正延, 2006
• Not Refereed, 遺伝子医学MOOK, エンドサイトーシス制御因子Arf6が関与するがんのシグナル伝達, 渡邊 利雄; 渡邊利雄, 2006, 6, 18, 185
• Not Refereed, JOURNAL OF IMMUNOLOGY, AMER ASSOC IMMUNOLOGISTS, Reduction of runx1 transcription factor activity up-regulates Fas and Bim expression and enhances the apoptotic sensitivity of double positive thymocytes, N Abe; K Kohu; H Ohmori; K Hayashi; T Watanabe; K Hozumi; T Sato; S Habu; M Satake, The death or survival of double positive (DP) thymocytes is determined by the strength of their TCR signaling. Of the three Runx family proteins, the DP cells only express the Runx1 transcription factor. We introduced and expressed in murine thymocytes the Runt domain of Runx1, which antagonizes the activity of endogenous Runx1. The Runt transgenic DP thymocytes expressed higher levels of the proapoptotic molecules Fas and Bim compared with the wild-type cells. Furthermore, the Runt transgenic cells were more susceptible to apoptosis induced by the artificial cross-linking of the TCR by the anti-CD3 Ab. This susceptibility was partially abrogated by the lpr/lpr background. In addition, Runx1:HY-TCR-double transgenic DP thymocytes were resistant to the apoptosis induced by the endogenously presented HY Ag. We propose that Runx1 functions to suppress the apoptotic sensitivity of DP thymocytes in the context of TCR signaling., Oct. 2005, 175, 7, 4475, 4482
• Not Refereed, JOURNAL OF IMMUNOLOGY, AMER ASSOC IMMUNOLOGISTS, Reduction of runx1 transcription factor activity up-regulates Fas and Bim expression and enhances the apoptotic sensitivity of double positive thymocytes, N Abe; K Kohu; H Ohmori; K Hayashi; T Watanabe; K Hozumi; T Sato; S Habu; M Satake, The death or survival of double positive (DP) thymocytes is determined by the strength of their TCR signaling. Of the three Runx family proteins, the DP cells only express the Runx1 transcription factor. We introduced and expressed in murine thymocytes the Runt domain of Runx1, which antagonizes the activity of endogenous Runx1. The Runt transgenic DP thymocytes expressed higher levels of the proapoptotic molecules Fas and Bim compared with the wild-type cells. Furthermore, the Runt transgenic cells were more susceptible to apoptosis induced by the artificial cross-linking of the TCR by the anti-CD3 Ab. This susceptibility was partially abrogated by the lpr/lpr background. In addition, Runx1:HY-TCR-double transgenic DP thymocytes were resistant to the apoptosis induced by the endogenously presented HY Ag. We propose that Runx1 functions to suppress the apoptotic sensitivity of DP thymocytes in the context of TCR signaling., Oct. 2005, 175, 7, 4475, 4482
• Not Refereed, MOLECULAR BIOLOGY OF THE CELL, AMER SOC CELL BIOLOGY, A novel GTPase-activating protein for ARF6 directly interacts with clathrin and endocytosis, K Tanabe; T Torii; W Natsume; S Braesch-Andersen; T Watanabe; M Satake, ADP-ribosylation factor 6 (Arf6) is a small-GTPase that regulates the membrane trafficking between the plasma membrane and endosome. It is also involved in the reorganization of the actin cytoskeleton. GTPase-activating protein (GAP) is a critical regulator of Arf function as it inactivates Arf. Here, we identified a novel species of GAP denoted as SMAP1 that preferentially acts on Arf6. Although overexpression of SMAP1 did not alter the subcellular distribution of the actin cytoskeleton, it did block the endocytosis of transferrin receptors. Knock down of endogenous SMAP1 also abolished transferrin internalization, which confirms that SMAP1 is needed for this endocytic process. SMAP1 overexpression had no effect on clathrin-independent endocytosis, however. Intriguingly, SMAP1 binds directly to the clathrin heavy chain via its clathrin-box and mutation studies revealed that its GAP domain and clathrin-box both contribute to the role SMAP1 plays in clathrin-dependent endocytosis. These observations suggest that SMAP1 may be an Arf6GAP that specifically regulates one of the multiple functions of Arf6, namely, clathrin-dependent endocytosis, and that it does so by binding directly to clathrin., Apr. 2005, 16, 4, 1617, 1628, 10.1091/mcb.E04-08-0683

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• Not Refereed, MOLECULAR BIOLOGY OF THE CELL, AMER SOC CELL BIOLOGY, A novel GTPase-activating protein for ARF6 directly interacts with clathrin and endocytosis, K Tanabe; T Torii; W Natsume; S Braesch-Andersen; T Watanabe; M Satake, ADP-ribosylation factor 6 (Arf6) is a small-GTPase that regulates the membrane trafficking between the plasma membrane and endosome. It is also involved in the reorganization of the actin cytoskeleton. GTPase-activating protein (GAP) is a critical regulator of Arf function as it inactivates Arf. Here, we identified a novel species of GAP denoted as SMAP1 that preferentially acts on Arf6. Although overexpression of SMAP1 did not alter the subcellular distribution of the actin cytoskeleton, it did block the endocytosis of transferrin receptors. Knock down of endogenous SMAP1 also abolished transferrin internalization, which confirms that SMAP1 is needed for this endocytic process. SMAP1 overexpression had no effect on clathrin-independent endocytosis, however. Intriguingly, SMAP1 binds directly to the clathrin heavy chain via its clathrin-box and mutation studies revealed that its GAP domain and clathrin-box both contribute to the role SMAP1 plays in clathrin-dependent endocytosis. These observations suggest that SMAP1 may be an Arf6GAP that specifically regulates one of the multiple functions of Arf6, namely, clathrin-dependent endocytosis, and that it does so by binding directly to clathrin., Apr. 2005, 16, 4, 1617, 1628, 10.1091/mcb.E04-08-0683

  DOI URL
• Not Refereed, JOURNAL OF IMMUNOLOGY, AMER ASSOC IMMUNOLOGISTS, Overexpression of the Runx3 transcription factor increases the proportion of mature thymocytes of the CD8 single-positive lineage, K Kohu; T Sato; S Ohno; K Hayashi; R Uchino; N Abe; M Nakazato; N Yoshida; T Kikuchi; Y Iwakura; Y Inoue; T Watanabe; S Hahn; M Satake, The Runx family of transcription factors is thought to regulate the differentiation of thymocytes. Runx3 protein is detected mainly in the CD4(-)8(+) subset of T lymphocytes. In the thymus of Runx3-deficient mice, CD4 expression is de-repressed and CD4(-)8(+) thymocytes do not develop. This clearly implicates Runx3 in CD4 silencing, but does not necessarily prove its role in the differentiation of CD4(-)8(+) thymocytes per se. In the present study, we created transgenic mice that overexpress Runx3 and analyzed the development of thymocytes in these animals. In the Runx3-transgenic thymus, the number of CD4(-)8(+) cells was greatly increased, whereas the numbers of CD4(+)8(+) and CD4(+)8(-) cells were reduced. The CD4(-)8(+) transgenic thymocytes contained mature cells with a TCR(high)HSA(low) phenotype. These cells were released from the thymus and contributed to the elevated level of CD4(-)8(+) cells relative to CD4(+)8(-) cells in the spleen. Runx3 overexpression also increased the number of mature CD4(-)8(+) thymocytes in mice with class II-restricted, transgenic TCR and in mice with a class I-deficient background, both of which are favorable for CD4(+)8(-) lineage selection. Thus, Runx3 can drive thymocytes to select the CD4(-)8(+) lineage. This activity is likely to be due to more than a simple silencing of CD4 gene expression., Mar. 2005, 174, 5, 2627, 2636
• Not Refereed, JOURNAL OF IMMUNOLOGY, AMER ASSOC IMMUNOLOGISTS, Overexpression of the Runx3 transcription factor increases the proportion of mature thymocytes of the CD8 single-positive lineage, K Kohu; T Sato; S Ohno; K Hayashi; R Uchino; N Abe; M Nakazato; N Yoshida; T Kikuchi; Y Iwakura; Y Inoue; T Watanabe; S Hahn; M Satake, The Runx family of transcription factors is thought to regulate the differentiation of thymocytes. Runx3 protein is detected mainly in the CD4(-)8(+) subset of T lymphocytes. In the thymus of Runx3-deficient mice, CD4 expression is de-repressed and CD4(-)8(+) thymocytes do not develop. This clearly implicates Runx3 in CD4 silencing, but does not necessarily prove its role in the differentiation of CD4(-)8(+) thymocytes per se. In the present study, we created transgenic mice that overexpress Runx3 and analyzed the development of thymocytes in these animals. In the Runx3-transgenic thymus, the number of CD4(-)8(+) cells was greatly increased, whereas the numbers of CD4(+)8(+) and CD4(+)8(-) cells were reduced. The CD4(-)8(+) transgenic thymocytes contained mature cells with a TCR(high)HSA(low) phenotype. These cells were released from the thymus and contributed to the elevated level of CD4(-)8(+) cells relative to CD4(+)8(-) cells in the spleen. Runx3 overexpression also increased the number of mature CD4(-)8(+) thymocytes in mice with class II-restricted, transgenic TCR and in mice with a class I-deficient background, both of which are favorable for CD4(+)8(-) lineage selection. Thus, Runx3 can drive thymocytes to select the CD4(-)8(+) lineage. This activity is likely to be due to more than a simple silencing of CD4 gene expression., Mar. 2005, 174, 5, 2627, 2636
• Not Refereed, ONCOGENE, NATURE PUBLISHING GROUP, Runx1 promotes angiogenesis by downregulation of insulin-like growth factor-binding protein-3, K Iwatsuki; K Tanaka; T Kaneko; R Kazama; S Okamoto; Y Nakayama; Y Ito; M Satake; S Takahashi; A Miyajima; T Watanabe; T Hara, Mouse embryos lacking the Runx1 transcription factor exhibit an angiogenic defect accompanied by the absence of hematopoietic stem cells ( HSCs). To ask whether Runx1 plays a direct role in angiogenesis, we established a novel endothelial progenitor cell line, designated AEL-DeltaR1, from the aorta-gonad-mesonephros ( AGM) region of Runx1-null mouse. We introduced Runx1 cDNA into AEL-DeltaR1 cells under the doxycycline-inducible promoter. The ability of AEL-DeltaR1 cells to form vascular networks on matrigel was highly enhanced by the restored expression of Runx1. By molecular comparison of mRNAs in AEL-DeltaR1 cells before and after the induction of Runx1, we found that mRNA expression of insulin-like growth factor-binding protein 3 (IGFBP-3) is downregulated by Runx1. Gel retardation and reporter assays revealed that Runx1 binds to the promoter region of mouse IGFBP-3 gene and represses its transcription. When IGFBP-3 was exogenously added in the matrigel assay, the angiogenesis-enhancing activity of Runx1 was suppressed in a dose-dependent manner. These results demonstrate that Runx1 is directly involved in angiogenesis by repression of IGFBP-3 mRNA expression., Feb. 2005, 24, 7, 1129, 1137, 10.1038/sj.onc.1208287

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• Not Refereed, MOLECULAR AND CELLULAR BIOLOGY, AMER SOC MICROBIOLOGY, Filamin A-bound PEBP2 beta/CBF beta is retained in the cytoplasm and prevented from functioning as a partner of the Runx1 transcription factor, N Yoshida; T Ogata; K Tanabe; SH Li; M Nakazato; K Kohu; T Takafuta; S Shapiro; Y Ohta; M Satake; T Watanabe, The heterodimeric transcription factor PEBP2/CBF is composed of a DNA-binding subunit. called Runx1. and a non-DNA-binding subunit, called PEBP2beta/CBFbeta. The Runx1 protein is detected exclusively in the nuclei of most cells and tissues, whereas PEBP2beta is located in the cytoplasm. We addressed the mechanism by which PEBP2beta localizes to the cytoplasm and found that it is associated with filamin A, an actin-binding protein. Filamin A retains PEBP2beta in the cytoplasm, thereby hindering its engagement as a Run-x1 partner. The interaction with filamin A is mediated by a region within PEBP2beta that includes amino acid residues 68 to 93. The deletion of this region or the repression of filamin A enables PEBP2beta to translocate to the nucleus. Based on these observations, we propose that PEBP2beta has two distinct domains, a newly defined regulatory domain that interacts with filamin A and the previously identified Runx1-binding domain., Feb. 2005, 25, 3, 1003, 1012, 10.1128/MCB.25.3.1003-1012.2005

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• Not Refereed, ONCOGENE, NATURE PUBLISHING GROUP, Runx1 promotes angiogenesis by downregulation of insulin-like growth factor-binding protein-3, K Iwatsuki; K Tanaka; T Kaneko; R Kazama; S Okamoto; Y Nakayama; Y Ito; M Satake; S Takahashi; A Miyajima; T Watanabe; T Hara, Mouse embryos lacking the Runx1 transcription factor exhibit an angiogenic defect accompanied by the absence of hematopoietic stem cells ( HSCs). To ask whether Runx1 plays a direct role in angiogenesis, we established a novel endothelial progenitor cell line, designated AEL-DeltaR1, from the aorta-gonad-mesonephros ( AGM) region of Runx1-null mouse. We introduced Runx1 cDNA into AEL-DeltaR1 cells under the doxycycline-inducible promoter. The ability of AEL-DeltaR1 cells to form vascular networks on matrigel was highly enhanced by the restored expression of Runx1. By molecular comparison of mRNAs in AEL-DeltaR1 cells before and after the induction of Runx1, we found that mRNA expression of insulin-like growth factor-binding protein 3 (IGFBP-3) is downregulated by Runx1. Gel retardation and reporter assays revealed that Runx1 binds to the promoter region of mouse IGFBP-3 gene and represses its transcription. When IGFBP-3 was exogenously added in the matrigel assay, the angiogenesis-enhancing activity of Runx1 was suppressed in a dose-dependent manner. These results demonstrate that Runx1 is directly involved in angiogenesis by repression of IGFBP-3 mRNA expression., Feb. 2005, 24, 7, 1129, 1137, 10.1038/sj.onc.1208287

  DOI URL
• Not Refereed, MOLECULAR AND CELLULAR BIOLOGY, AMER SOC MICROBIOLOGY, Filamin A-bound PEBP2 beta/CBF beta is retained in the cytoplasm and prevented from functioning as a partner of the Runx1 transcription factor, N Yoshida; T Ogata; K Tanabe; SH Li; M Nakazato; K Kohu; T Takafuta; S Shapiro; Y Ohta; M Satake; T Watanabe, The heterodimeric transcription factor PEBP2/CBF is composed of a DNA-binding subunit. called Runx1. and a non-DNA-binding subunit, called PEBP2beta/CBFbeta. The Runx1 protein is detected exclusively in the nuclei of most cells and tissues, whereas PEBP2beta is located in the cytoplasm. We addressed the mechanism by which PEBP2beta localizes to the cytoplasm and found that it is associated with filamin A, an actin-binding protein. Filamin A retains PEBP2beta in the cytoplasm, thereby hindering its engagement as a Run-x1 partner. The interaction with filamin A is mediated by a region within PEBP2beta that includes amino acid residues 68 to 93. The deletion of this region or the repression of filamin A enables PEBP2beta to translocate to the nucleus. Based on these observations, we propose that PEBP2beta has two distinct domains, a newly defined regulatory domain that interacts with filamin A and the previously identified Runx1-binding domain., Feb. 2005, 25, 3, 1003, 1012, 10.1128/MCB.25.3.1003-1012.2005

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• Not Refereed, 細胞工学, 細胞骨格制御因子filamin Aによる転写因子PEBP2/CBFの転写活性調節機構, 渡邊 利雄; 吉田尚美; 佐竹正延; 渡邊利雄, 2005, 24, 256, 257
• Not Refereed, CRITICAL REVIEWS IN EUKARYOTIC GENE EXPRESSION, BEGELL HOUSE INC, Biological implications of filamin A-bound PEBP2 beta/CBF beta retention in the cytoplasm, T Watanabe; N Yoshida; M Satake, Multiple mechanisms regulate dynamic cytoplasmic-to-nuclear transport of transcription factors. However, little is known about the involvement of cytoskeletal proteins in this process. The heterodimeric transcription factor PEBP2/CBF is composed of a DNA-binding subunit, Runx1, and a non-DNA-binding subunit, PEBP2 beta/CBF beta. The Runx1 protein possesses nuclear localization signals and is found exclusively in the nucleus, whereas PEBP2 beta is located in the cytoplasm in most cells and tissues examined thus far. We investigated the mechanism by which PEBP2 beta localizes to the cytoplasm and found that it associates with filamin A, an actin-binding cytoskeletal protein. Filamin A retains PEBP2 beta in the cytoplasm, thereby hindering its engagement as a Runx1 partner. When filamin A is absent, PEBP2 beta moves into the nucleus and enhances Runx1-dependent transcription. These observations highlight the significance of the subcellular localization of PEBP2 beta in regulating its activity as a component of the PEBP2/CBF transcription factor. In humans, PEBP2 beta is frequently targeted in the leukemia-associated chromosomal abnormality, inversion 16 (inv 16). Thus, identifying the factors that mediate the subcellular localization of the PEBP2 beta-derived chimeric transcription factor produced by inv 16 is an important issue that will need to be resolved in order to understand the mechanism(s) involved in inv 16-induced leukemogenesis., 2005, 15, 3, 197, 205
• Not Refereed, Crit. Rev. Eukaryot. Gene Expr., Biological implication of filamin A-bound PEBP2beta/CBFbeta in the cytoplasm, WATANABE Toshio; watanabe T; Yoshida N; Satake M, 2005, 15, 3, 197, 206
• Not Refereed, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, ELSEVIER SCIENCE INC, Transcriptional regulation of angiogenesis-related puromycin-insensitive leucyl-specific aminopeptidase in endothelial cells, O Niizeki; H Miyashita; T Yamazaki; T Akada; M Abe; N Yoshida; T Watanabe; H Yoshimatsu; Y Sato, Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) was expressed in endothelial cells (ECs) and played an important role in angiogenesis. Here, we characterized its transcriptional regulation. Mouse PILSAP gene contained 19 exons and located in the chromosome 13C1-C2. We identified two transcripts; one transcribed from exon 1 and the other from exon 2. Mouse ECs expressed dominantly the one from exon 1. The promoter analysis using 5' upstream region of exon 1 revealed that - 1868 to - 1812 was critical for its transcription in mouse ECs. We identified a motif of the transcription factor PEBP2 in this region, and the deletion or mutation of this motif decreased promoter activity. Protein extracted from mouse ECs bound specifically to this motif. AML1/Runx1/PEBP2alphabeta increased PILSAP mRNA in mouse ECs, whereas dominant interfering chimerical PEBP2beta-MYH11 decreased it. These results indicate that the expression of PILSAP in mouse ECs is regulated, at least in part, by PEBP2. (C) 2004 Elsevier Inc. All rights reserved., Apr. 2004, 424, 1, 63, 71, 10.1016/j.abb.2004.01.022

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• Not Refereed, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, ELSEVIER SCIENCE INC, Transcriptional regulation of angiogenesis-related puromycin-insensitive leucyl-specific aminopeptidase in endothelial cells, O Niizeki; H Miyashita; T Yamazaki; T Akada; M Abe; N Yoshida; T Watanabe; H Yoshimatsu; Y Sato, Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) was expressed in endothelial cells (ECs) and played an important role in angiogenesis. Here, we characterized its transcriptional regulation. Mouse PILSAP gene contained 19 exons and located in the chromosome 13C1-C2. We identified two transcripts; one transcribed from exon 1 and the other from exon 2. Mouse ECs expressed dominantly the one from exon 1. The promoter analysis using 5' upstream region of exon 1 revealed that - 1868 to - 1812 was critical for its transcription in mouse ECs. We identified a motif of the transcription factor PEBP2 in this region, and the deletion or mutation of this motif decreased promoter activity. Protein extracted from mouse ECs bound specifically to this motif. AML1/Runx1/PEBP2alphabeta increased PILSAP mRNA in mouse ECs, whereas dominant interfering chimerical PEBP2beta-MYH11 decreased it. These results indicate that the expression of PILSAP in mouse ECs is regulated, at least in part, by PEBP2. (C) 2004 Elsevier Inc. All rights reserved., Apr. 2004, 424, 1, 63, 71, 10.1016/j.abb.2004.01.022

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• Not Refereed, 加齢医学研究所雑誌, 受容体型チロシンホスファターゼCD148の欠損が引き起こす血管網形成異常 ：大腸癌感受性遺伝子（癌抑制遺伝子）と血管網形成との関係, 渡邊 利雄, 2004, 55, 7, 21
• Not Refereed, 細胞工学, CD148の欠損が引き起こす血管網形成異常：大腸癌感受性遺伝子 （癌抑制遺伝子）と血管網形成の関係, 渡邊 利雄; 渡邊利雄; 高橋孝宗, 2004, 23, 541, 545
• Not Refereed, 加齢医学研究所雑誌, 東北大学加齢医学研究所研究会同窓会, 受容体型チロシンホスファターゼCD148の欠損が引き起こす血管網形成異常 ：大腸癌感受性遺伝子（癌抑制遺伝子）と血管網形成との関係, WATANABE Toshio, 2004, 55, 1, 7, 21

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• Not Refereed, JOURNAL OF IMMUNOLOGY, AMER ASSOC IMMUNOLOGISTS, Morpholino antisense oligonucleotide-mediated gene knockdown during thymocyte development reveals role for Runx3 transcription factor in CD4 silencing during development of CD4(-)/CD8(+) thymocytes, M Ehlers; K Laule-Kilian; M Petter; CJ Aldrian; B Grueter; A Wurch; N Yoshida; T Watanabe; M Satake; Steimle, V, During thymic T cell development, immature CD4(+)/CD8(+) thymocytes develop into either CD4(+)/CD8(-) helper or CD4(-)/CD8(+) CTLs. The molecular mechanisms governing the complex selection and differentiation steps during thymic T cell development are not well understood. Here we developed a novel approach to investigate gene function during thymocyte development. We transfected ex vivo isolated immature thymocytes with gene-specific morpholino antisense oligonucleotides and induced differentiation in cell or organ cultures. A morpholino oligonucleotide specific for CD8alpha strongly reduces CD8 expression. To our knowledge, this is the first demonstrated gene knockdown by morpholino oligonucleotides in primary lymphocytes. Using this approach, we show here that the transcription factor Runx3 is involved in silencing of CD4 expression during CD8 T cell differentiation. Runx3 protein expression appears late in thymocyte differentiation and is confined to mature CD8 single-positive thymocytes, whereas Runx3 mRNA is transcribed in mature CD4 and CD8 thymocytes. Therefore, Runx3 protein expression is regulated at a post-transcriptional level. The knockdown of Runx3 protein expression through morpholino oligonucleotides inhibited the development of CD4(-)/CD8(+) T cells. Instead, mature cells with a CD4(+)/CD8(+) phenotype accumulated. Potential Runx binding sites were identified in the CD4 gene silencer element, which are bound by Runx protein in EMSAs. Mutagenesis of potential Runx binding sites in the CD4 gene silencer abolished silencing activity in a reporter gene assay, indicating that Runx3 is involved in CD4 gene silencing. The experimental approach developed here should be valuable for the functional analysis of other candidate genes in T cell differentiation., Oct. 2003, 171, 7, 3594, 3604
• Not Refereed, JOURNAL OF IMMUNOLOGY, AMER ASSOC IMMUNOLOGISTS, Morpholino antisense oligonucleotide-mediated gene knockdown during thymocyte development reveals role for Runx3 transcription factor in CD4 silencing during development of CD4(-)/CD8(+) thymocytes, M Ehlers; K Laule-Kilian; M Petter; CJ Aldrian; B Grueter; A Wurch; N Yoshida; T Watanabe; M Satake; Steimle, V, During thymic T cell development, immature CD4(+)/CD8(+) thymocytes develop into either CD4(+)/CD8(-) helper or CD4(-)/CD8(+) CTLs. The molecular mechanisms governing the complex selection and differentiation steps during thymic T cell development are not well understood. Here we developed a novel approach to investigate gene function during thymocyte development. We transfected ex vivo isolated immature thymocytes with gene-specific morpholino antisense oligonucleotides and induced differentiation in cell or organ cultures. A morpholino oligonucleotide specific for CD8alpha strongly reduces CD8 expression. To our knowledge, this is the first demonstrated gene knockdown by morpholino oligonucleotides in primary lymphocytes. Using this approach, we show here that the transcription factor Runx3 is involved in silencing of CD4 expression during CD8 T cell differentiation. Runx3 protein expression appears late in thymocyte differentiation and is confined to mature CD8 single-positive thymocytes, whereas Runx3 mRNA is transcribed in mature CD4 and CD8 thymocytes. Therefore, Runx3 protein expression is regulated at a post-transcriptional level. The knockdown of Runx3 protein expression through morpholino oligonucleotides inhibited the development of CD4(-)/CD8(+) T cells. Instead, mature cells with a CD4(+)/CD8(+) phenotype accumulated. Potential Runx binding sites were identified in the CD4 gene silencer element, which are bound by Runx protein in EMSAs. Mutagenesis of potential Runx binding sites in the CD4 gene silencer abolished silencing activity in a reporter gene assay, indicating that Runx3 is involved in CD4 gene silencing. The experimental approach developed here should be valuable for the functional analysis of other candidate genes in T cell differentiation., Oct. 2003, 171, 7, 3594, 3604
• Not Refereed, JOURNAL OF EXPERIMENTAL MEDICINE, ROCKEFELLER UNIV PRESS, The Runx1 transcription factor inhibits the differentiation of naive CD4(+) T cells into the Th2 lineage by repressing GATA3 expression, O Komine; K Hayashi; W Natsume; T Watanabe; Y Seki; N Seki; R Yagi; W Sukzuki; H Tamuchi; K Hozumi; S Habu; M Kubo; M Satake, Differentiation of naive CD4(+) T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation., Jul. 2003, 198, 1, 51, 61, 10.1084/jem.20021200

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• Not Refereed, JOURNAL OF EXPERIMENTAL MEDICINE, ROCKEFELLER UNIV PRESS, The Runx1 transcription factor inhibits the differentiation of naive CD4(+) T cells into the Th2 lineage by repressing GATA3 expression, O Komine; K Hayashi; W Natsume; T Watanabe; Y Seki; N Seki; R Yagi; W Sukzuki; H Tamuchi; K Hozumi; S Habu; M Kubo; M Satake, Differentiation of naive CD4(+) T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation., Jul. 2003, 198, 1, 51, 61, 10.1084/jem.20021200

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• Not Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, Hemocytes of Ciona intestinalis express multiple genes involved in innate immune host defense, K Shida; D Terajima; R Uchino; S Ikawa; M Ikeda; K Asano; T Watanabe; K Azumi; M Nonaka; Y Satou; N Satoh; M Satake; Y Kawazoe; A Kasuya, Ascidians, which are classified as urochordata, appear to employ a primitive system of host defense that is considered to be a prototype of vertebrate innate immunity. We performed a cDNA/EST study to identify the genes expressed in the hemocytes of Ciona intestinalis. We obtained 3357 one-path reads that were then grouped into 1889 independent clusters. Although two thirds of the clusters could not be assigned to any particular gene, the remaining 530 clusters had significant homology to genes with known function. Of these, 62 clusters appeared to be related to host defense mechanisms. These include transcripts whose products are probably involved in cytotoxicity, detoxification, inflammation, and apoptosis. As expected, elements of acquired immunity were not detected. Thus, Ciona hemocytes appear to express a number of host defense-related genes involved in innate immune mechanisms. (C) 2003 Elsevier Science (USA). All rights reserved., Mar. 2003, 302, 2, 207, 218, 10.1016/S0006-291X(03)00113-X

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• Not Refereed, MOLECULAR AND CELLULAR BIOLOGY, AMER SOC MICROBIOLOGY, A mutant receptor tyrosine phosphatase, CD148, causes defects in vascular development, T Takahashi; K Takahashi; PLS John; PA Fleming; T Tomemori; T Watanabe; DR Abrahamson; CJ Drake; T Shirasawa; TO Daniel, Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPeta), participates in developmental vascularization. A mutant allele, CD148(DeltaCyGFP), was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions., Mar. 2003, 23, 5, 1817, 1831, 10.1128/MCB.23.5.1817-1831.2003

  DOI URL
• Not Refereed, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC ELSEVIER SCIENCE, Hemocytes of Ciona intestinalis express multiple genes involved in innate immune host defense, K Shida; D Terajima; R Uchino; S Ikawa; M Ikeda; K Asano; T Watanabe; K Azumi; M Nonaka; Y Satou; N Satoh; M Satake; Y Kawazoe; A Kasuya, Ascidians, which are classified as urochordata, appear to employ a primitive system of host defense that is considered to be a prototype of vertebrate innate immunity. We performed a cDNA/EST study to identify the genes expressed in the hemocytes of Ciona intestinalis. We obtained 3357 one-path reads that were then grouped into 1889 independent clusters. Although two thirds of the clusters could not be assigned to any particular gene, the remaining 530 clusters had significant homology to genes with known function. Of these, 62 clusters appeared to be related to host defense mechanisms. These include transcripts whose products are probably involved in cytotoxicity, detoxification, inflammation, and apoptosis. As expected, elements of acquired immunity were not detected. Thus, Ciona hemocytes appear to express a number of host defense-related genes involved in innate immune mechanisms. (C) 2003 Elsevier Science (USA). All rights reserved., Mar. 2003, 302, 2, 207, 218, 10.1016/S0006-291X(03)00113-X

  DOI URL
• Not Refereed, MOLECULAR AND CELLULAR BIOLOGY, AMER SOC MICROBIOLOGY, A mutant receptor tyrosine phosphatase, CD148, causes defects in vascular development, T Takahashi; K Takahashi; PLS John; PA Fleming; T Tomemori; T Watanabe; DR Abrahamson; CJ Drake; T Shirasawa; TO Daniel, Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPeta), participates in developmental vascularization. A mutant allele, CD148(DeltaCyGFP), was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions., Mar. 2003, 23, 5, 1817, 1831, 10.1128/MCB.23.5.1817-1831.2003

  DOI URL
• Not Refereed, DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC ELSEVIER SCIENCE, SEK1/MKK4-mediated SAPK/JNK signaling participates in embryonic hepatoblast proliferation via a pathway different from NF-kappa B-induced anti-apoptosis, T Watanabe; K Nakagawa; S Ohata; D Kitagawa; G Nishitai; J Seo; S Tanemura; N Shimizu; H Kishimoto; T Wada; J Aoki; H Arai; T Iwatsubo; M Mochita; T Watanabe; M Satake; Y Ito; T Matsuyama; TW Mak; JM Penninger; H Nishina; T Katada, Mice lacking the stress-signaling kinase SEK1 die from embryonic day 10.5 (E10.5) to E12.5. Although a defect in liver formation is accompanied with the embryonic lethality of sek1(-/-) mice, the mechanism of the liver defect has remained unknown. In the present study, we first produced a monoclonal antibody specifically recognizing murine hepatoblasts for the analysis of liver development and further investigated genetic interaction of sek1 with tumor necrosis factor-alpha receptor 1 gene (tnfr1) and protooncogene c-jun, which are also responsible for liver formation and cell apoptosis. The defective liver formation in sek1(-/-) embryos was not protected by additional tnfr1 mutation, which rescues the embryonic lethality of mice lacking NF-kappaB signaling components. There was a progressive increase in the hepatoblast cell numbers of wild-type embryos from E10.5 to E12.5. Instead, impaired hepatoblast proliferation was observed in sek1(-/-) livers from E10.5, though fetal liver-specific gene expression was normal. The impaired phenotype in sek1(-/-) livers was more severe than in c-jun(-/-) embryos, and sek1(-/-) c-jun(-/-) embryos died more rapidly before E8.5. The hepatoblast proliferation required no hematopoiesis, since liver development was not impaired in AML1(-/-) mice that lack hematopoietic functions. Stimulation of stress-activated protein kinase/c-jun N-terminal kinase by hepatocyte growth factor was attenuated in sek1(-/-) livers. Thus, SEK1 appears to play a crucial role in hepatoblast proliferation and survival in a manner apparently different from NF-kappaB or c-jun. (C) 2002 Elsevier Science (USA)., Oct. 2002, 250, 2, 332, 347, 10.1006/dbio.2002.0781

  DOI URL
• Not Refereed, DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS INC ELSEVIER SCIENCE, SEK1/MKK4-mediated SAPK/JNK signaling participates in embryonic hepatoblast proliferation via a pathway different from NF-kappa B-induced anti-apoptosis, T Watanabe; K Nakagawa; S Ohata; D Kitagawa; G Nishitai; J Seo; S Tanemura; N Shimizu; H Kishimoto; T Wada; J Aoki; H Arai; T Iwatsubo; M Mochita; T Watanabe; M Satake; Y Ito; T Matsuyama; TW Mak; JM Penninger; H Nishina; T Katada, Mice lacking the stress-signaling kinase SEK1 die from embryonic day 10.5 (E10.5) to E12.5. Although a defect in liver formation is accompanied with the embryonic lethality of sek1(-/-) mice, the mechanism of the liver defect has remained unknown. In the present study, we first produced a monoclonal antibody specifically recognizing murine hepatoblasts for the analysis of liver development and further investigated genetic interaction of sek1 with tumor necrosis factor-alpha receptor 1 gene (tnfr1) and protooncogene c-jun, which are also responsible for liver formation and cell apoptosis. The defective liver formation in sek1(-/-) embryos was not protected by additional tnfr1 mutation, which rescues the embryonic lethality of mice lacking NF-kappaB signaling components. There was a progressive increase in the hepatoblast cell numbers of wild-type embryos from E10.5 to E12.5. Instead, impaired hepatoblast proliferation was observed in sek1(-/-) livers from E10.5, though fetal liver-specific gene expression was normal. The impaired phenotype in sek1(-/-) livers was more severe than in c-jun(-/-) embryos, and sek1(-/-) c-jun(-/-) embryos died more rapidly before E8.5. The hepatoblast proliferation required no hematopoiesis, since liver development was not impaired in AML1(-/-) mice that lack hematopoietic functions. Stimulation of stress-activated protein kinase/c-jun N-terminal kinase by hepatocyte growth factor was attenuated in sek1(-/-) livers. Thus, SEK1 appears to play a crucial role in hepatoblast proliferation and survival in a manner apparently different from NF-kappaB or c-jun. (C) 2002 Elsevier Science (USA)., Oct. 2002, 250, 2, 332, 347, 10.1006/dbio.2002.0781

  DOI URL
• Not Refereed, JOURNAL OF IMMUNOLOGY, AMER ASSOC IMMUNOLOGISTS, Overexpression of AML1 transcription factor drives thymocytes into the CD8 single-positive lineage, K Hayashi; N Abe; T Watanabe; M Obinata; M Ito; T Sato; S Habu; M Satake, To understand the gene regulation involved in the development of single-positive (SP) thymocytes, we generated transgenic mice in which the AML1 transcription factor is overexpressed. In these mice the number of CD8 SP thymocytes was greatly increased, and this continued to be true even when MHC class I was absent. This promotion to the CDS SP lineage was not, however, observed when both class I and class II were absent. Furthermore, even thymocytes carrying MHC class II-restricted TCR differentiated into the CD8 SP lineage when AML1 was overexpressed. The selected CD8 SP cells were, however, unable to mature, as judged by the expression level of heat-stable Ag. Thus, overexpression of AML1 is able to skew class II-restricted thymocytes into the CD8 SP lineage, but not to drive the maturation of resulting selected CDS SP cells., Nov. 2001, 167, 9, 4957, 4965
• Not Refereed, 日本癌学会総会記事, 日本癌学会, 転写因子c-mybによるAGM造血発生の制御機構, 渡邊 利雄; 佐竹 正延; 小川 峰太郎, Sep. 2001, 60回, 371, 371
• Not Refereed, 日本分子生物学会年会プログラム・講演要旨集, The critical role of AML1, the most frequent target of the leukemia-associated translocations, for embryonic hematopoiesis., 岡田斉; 仁木賢; 高野洋志; 日比野仁; 谷憲三郎; 浅野茂隆; 渡辺利雄; 伊藤嘉明; 野田哲生, Dec. 1997, 20th, 669

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• Not Refereed, ケミカルエンジニアリング, 滅菌不要素早くできる美白因子の快速スクリーニング法開発の試み, 渡邊利雄; 森田詠子, 2020, 65, 10, 635, 640
• 日本生化学会大会(Web), 遺伝学的アルツハイマー病予防因子PICALM/CALMが病態形成に寄与する分子機構の解明, 金津邦彦; 渡邊利雄; 岩坪威; 富田泰輔, 2014, 87th
• 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, アルツハイマー病遺伝学的危険因子PICALM/CALMが病態形成に寄与する分子機構の解明, 金津邦彦; 諸橋雄一; 渡邊利雄; 渡邊利雄; 岩坪威; 富田泰輔, 2013, 12th
• Not Refereed, EXPERIMENTAL HEMATOLOGY, CARDEN JENNINGS PUBL CO LTD, Development of hematopoietic cells in the embryonic aorta gonad-mesonephros region is dependent on the amount of the PEBP2/CBF transcription factor., N Chiba; Y Mukuoyama; T Hara; A Miyajima; M Satake; T Watanabe, Aug. 1998, 26, 8, 716, 716, Summary international conference
• 細胞工学, 秀潤社, AGM造血発生を制御する転写因子 (特集 造血幹細胞の発生起源を求めて) -- (マウス胎仔のPAS,AGM領域における造血発生の解析), 渡辺 利雄; 佐竹 正延, Nov. 1998, 17, 11, 1710, 1715

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• 細胞工学, 秀潤社, 造血幹細胞はどのように発生してくるのか (特集 造血幹細胞の発生起源を求めて), 渡辺 利雄; 佐竹 正延, Nov. 1998, 17, 11, 1666, 1670

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• Tissue culture research communications : the journal of experimental & applied cell culture research = 組織培養研究, 血管新生における転写因子PEBP2/CBFの機能, 難波 克成; 安部 まゆみ; 渡邊 利雄; 佐竹 正延; 佐藤 靖史, 31 Mar. 1999, 18, 1, 55, 55

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• 日本分子生物学会年会プログラム・講演要旨集, モノクローナル抗体を用いたAML1及びc-Myb欠損マウス胎児肝の解析, 渡辺智美; 仁科博史; 餅田みゆき; 佐竹正延; 渡辺利雄; 堅田利明, 2000, 23rd
• Not Refereed, 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, (公社)日本生化学会, ホスファターゼ研究のカティングエッジ メディカルイノベーションへの橋渡しを目指して 脱リン酸化酵素PP6の触媒サブユニットのPpp6cは着床後の胚の正常な発生に不可欠である, 渡邊 利雄; 小河 穂波; 田沼 延公; 早川 夏姫; 住吉 麻実; 岸本 綾子; 松居 靖久; 鈴木 麻衣; 佐々木 希; 大内 司; 野村 美有樹; 照屋 由里子; 安田 恵子; 島 礼, Dec. 2015, 88回・38回, [4W6, p
• CANCER SCIENCE, WILEY, AF10 links histone chaperones Supt6h and FACT complex to MLL-fusion leukemia, Kazutsune Yamagata; Mariko Saito; Mai Suzuki; Takuo Katsumoto; Honami Ogoh; Toshio Watanabe; Issay Kitabayashi, Dec. 2018, 109, 1111, 1111, Summary international conference
• CANCER SCIENCE, WILEY, AF10 links Supt6h and FACT complex to MLL-fusion in leukemia, Kazutsune Yamagata; Mariko Saito; Mai Suzuki; Honami Ogo; Toshio Watanabe; Issay Kitabayashi, Jan. 2018, 109, 589, 589, Summary international conference
• 日本分子生物学会年会プログラム・要旨集(Web), MLL転座関連白血病におけるAF10の役割, 齋藤眞理子; 齋藤眞理子; 山形和恒; 鈴木麻衣; 相川祐規子; 小河穂波; 渡邊利雄; 田代文夫; 北林一生, 2013, 36th
• 日本先天異常学会学術集会プログラム・抄録集, Deregulation of histone methylation is implicated in the etiology of frontonasal dysplasia., OGOH Honami; YAMAGATA Kazutsune; NAKAO Tomomi; SANDELL Lisa L.; YAMAMOTO Ayaka; YAMASHITA Aiko; TANGA Naomi; SUZUKI Mai; ABE Takaya; KITABAYASHI Issay; WATANABE Toshio; SAKAI Daisuke, 2019, 59th
• 日本分子生物学会年会プログラム・要旨集(Web), The defect in differentiation of germinal center B cells in Arf1-BKO mice., 小谷唯; 小谷唯; 住吉麻実; 笹田萌未; 笹田萌未; 小澤まどか; 片貝智哉; 渡邊利雄; 松田達志, 2022, 45th

Books etc

• 史上最強図解 これならわかる！分子生物学, ナツメ社, 2013, Not Refereed, 9784816353499
• バイオイラストレイテッド すくすく育て細胞培養, 秀潤社, 1996, Not Refereed

Presentations

• Poster presentation
• Poster presentation
• Poster presentation
• Poster presentation
• Poster presentation
• Invited oral presentation, 11 Nov. 2022, 15 Nov. 2022
• Poster presentation, 02 Feb. 2023, 03 Feb. 2023
• Poster presentation, 02 Feb. 2023, 03 Feb. 2023
• Poster presentation, 30 Nov. 2022, 02 Dec. 2022
• Poster presentation, 30 Nov. 2022, 02 Dec. 2022
• Poster presentation, 30 Nov. 2022, 02 Dec. 2022
• Poster presentation, 30 Nov. 2022, 02 Dec. 2022
• Poster presentation, 30 Nov. 2022, 02 Dec. 2022
• Poster presentation, 30 Nov. 2022, 02 Dec. 2022
• Oral presentation, 27 May 2022, 28 May 2022
• Oral presentation, 29 May 2021, 29 May 2021
• Poster presentation, 02 Feb. 2022, 03 Feb. 2022
• Poster presentation, 02 Feb. 2022, 03 Feb. 2022
• Poster presentation, 01 Dec. 2021, 03 Dec. 2021
• Poster presentation, 01 Dec. 2021, 03 Dec. 2021
• Poster presentation, 01 Dec. 2021, 03 Dec. 2021
• Poster presentation, 01 Dec. 2021, 03 Dec. 2021
• Poster presentation, 01 Dec. 2021, 03 Dec. 2021
• Oral presentation, 29 Jun. 2021, 02 Jul. 2021
• Invited oral presentation
• Poster presentation
• Poster presentation
• 先端モデル動物支援プラットフォーム 平成29年度若手支援技術講習会, ウイルス感染に対するMul1欠損マウスの応答解析, 2018
• 第91回日本生化学会大会シンポジウム、「リン酸化ー脱リン酸化応答とがん」, 脱リン酸化酵素PP6は、がん抑制遺伝子として働く, 2018
• The 77th Annual Meeting of the Japanese Cancer Association, AF10 links histone chaperones Supt6h and FACT complex to MLL-fusion leukemia, 2018
• Workshop on Frontiers in Phosphatase Research and Drug Discovery, Tissue Specific Deletion of Protein Phosphatase 6 Catalytic Subunit (Ppp6c) Caused Embryonic lethality and postnatal death., 2018
• Workshop on Frontiers in Phosphatase Research and Drug Discovery, Tie2-Cre Induced Tissue Specific Deletion of Ppp6c Caused Abnormal Hematopoiesis during Mouse Embryogenesis., 2018
• Workshop on Frontiers in Phosphatase Research and Drug Discovery, Tumor suppressor activity of protein phosphatase 6., 2018
• 第41回日本分子生物学会年会, B細胞における低分子量Gタンパク質Arf経路の機能解明, 2018
• Analysis of responses to virus infection in Mul1-KO mice, 2018
• The 77th Annual Meeting of the Japanese Cancer Association, AF10 links histone chaperones Supt6h and FACT complex to MLL-fusion leukemia, 2018
• Workshop on Frontiers in Phosphatase Research and Drug Discovery, Tissue Specific Deletion of Protein Phosphatase 6 Catalytic Subunit (Ppp6c) Caused Embryonic lethality and postnatal death., 2018
• Workshop on Frontiers in Phosphatase Research and Drug Discovery, Tie2-Cre Induced Tissue Specific Deletion of Ppp6c Caused Abnormal Hematopoiesis during Mouse Embryogenesis., 2018
• Workshop on Frontiers in Phosphatase Research and Drug Discovery, Tumor suppressor activity of protein phosphatase 6., 2018
• Elucidation of the function of the small G protein Arf family in B cells., 2018
• 第76回日本癌学会学術総会, Ppp6cは、マウス皮膚のがん抑制遺伝子として働く, 2017
• 第76回日本癌学会学術総会, ヒストンシャペロンSupt6hとFACT複合体は、AF10との結合を介してMLL転座関連白血病の発症に寄与する, 2017
• The 3rd Japan-Taiwan Bilateral Conference on Protein Phosphatase., PP6, a target of okadaic acid, is a novel tumor suppressor, 2017
• 2017年度生命科学系学会合同年次大会/第40回日本分子生物学会年会, T細胞における低分子量Gタンパク質Arfファミリーの機能解析, 2017
• 2017年度生命科学系学会合同年次大会/第40回日本分子生物学会年会, BRCA1の新規結合分子Ola1のノックアウトマウスの表現型の解析, 2017
• 2017年度生命科学系学会合同年次大会/第40回日本分子生物学会年会, 細胞膜近傍でのクラスリン集合を担うCALMの欠損は何故MVB/後期エンドソームを介する分解経路の異常を引き起こす原因を探る, 2017
• 2017年度生命科学系学会合同年次大会/第40回日本分子生物学会年会, 制御性T細胞の分化誘導に関わるシグナル伝達経路の解明, 2017
• 2017年度生命科学系学会合同年次大会/第40回日本分子生物学会年会, 小胞輸送を制御するArf GTPase 活性化因子SMAP1とSMAP2の二重欠損胚におけるエンドサイトーシス経路の異常, 2017
• 2017年度生命科学系学会合同年次大会/第40回日本分子生物学会年会, オートファジーによるtype 2A protein phosphataseの制御機構の解明, 2017
• Protein phosphatase 6 functions as a tumor suppressor in mouse skin, 2017
• The 3rd Japan-Taiwan Bilateral Conference on Protein Phosphatase., PP6, a target of okadaic acid, is a novel tumor suppressor, 2017
• Consortium of Biological Sciences 2017, Analysis of how CALM deficiency is involved in the abnormality of the degradation pathway through MVB/Late endosome., 2017
• Consortium of Biological Sciences 2017, The Failure of endocytic pathways in the ArfGAPs, SMAP1 and SMAP2 doubly-deficient mouse embryos., 2017
• 日韓合同癌・老化シンポジウム, Ppp6c deficiency predisposes mouse skin tissue to carcinogenesis initiated by DMBA and UVB., 2016
• 第89回日本生化学会大会, 発がんプロモーターオカダ酸の標的、PP6の皮膚がん抑制遺伝子としての意義, 2016
• 第89回日本生化学会大会, Pyruvate kinase Mの両isoformを欠損するマウスの解析, 2016
• 第89回日本生化学会大会, 遺伝子改変マウスを用いたArf1の胚発生での機能と神経系での機能の解析の試み, 2016
• The 12th International Conference on Protein Phosphatase, Ppp6c deficiency predisposes mouse skin tissue to carcinogenesis., 2016
• The 12th International Conference on Protein Phosphatase, Evidence that Warburg effect functions as anti-cancer barrier., 2016
• The 12th International Conference on Protein Phosphatase, The protein phosphatase 6 catalytic subunit (Ppp6c) is indispensable for proper post-implantation embryogenesis., 2016
• 第75回日本癌学会学術総会, 新規皮膚がん抑制遺伝子Ppp6c変異は、変異型K-RASによる腫瘍発生を強く促進する。, 2016
• 第39回日本分子生物学会年会, 顔面発生におけるH3K79メチル化の機能, 2016
• 第39回日本分子生物学会年会, 小胞輸送を制御するArf GTPase 活性化因子Smap1とSmap2の二重欠損によるマウス胚の表現型の解析, 2016
• 第39回日本分子生物学会年会, クラスリン集合因子CALMはEGF/EGFRシグナル経路に関与する, 2016
• 第39回日本分子生物学会年会, 神経幹細胞特異的にArf1を欠損させたマウスに見られた、誕生後の異常の解析, 2016
• 第39回日本分子生物学会年会, マスト細胞脱顆粒過程におけるPI3K経路の役割解明, 2016
• 13th Korean Japan Joint conference "Cancer and aging", Ppp6c deficiency predisposes mouse skin tissue to carcinogenesis initiated by DMBA and UVB., 2016
• Significance of PP6 as a tumor suppressor of skin, 2016
• Analysis of mice lacking both of pyruvate kinase M (PKM) isoforms, 2016
• An analysis on the physiological roles of Arf1 in embryogenesis and neuronal function, 2016
• The 12th International Conference on Protein Phosphatase, Ppp6c deficiency predisposes mouse skin tissue to carcinogenesis., 2016
• The 12th International Conference on Protein Phosphatase, Evidence that Warburg effect functions as anti-cancer barrier., 2016
• The 12th International Conference on Protein Phosphatase, The protein phosphatase 6 catalytic subunit (Ppp6c) is indispensable for proper post-implantation embryogenesis., 2016
• The 75th Annual Meeting of the Japanese Cancer Association, Loss of protein phosphatase 6 in mouse epidermis enhances K-ras driven tumor promotion., 2016
• The function of H3K79 methylation on craniofacial development, 2016
• The phenotypic analysis of the cause of embryonic lethality, found in mice doubly-deficient in the Arf GAPs Smap1 and Smap2, which regulate vesicular trafficking., 2016
• CALM is involved in EGF/EGFR signaling pathway, 2016
• Neural stem cells specific Arf1 conditional knockout mice shows abnormalities after birth, 2016
• Role of the PI3K signaling pathway in mast cell degranulation, 2016
• 第19回造血器腫瘍研究会, 家族性乳がん原因分子Brca1の新規結合分子Ola1の欠損マウスにおける発がん, 2015
• 日本生化学会東北支部 第81回例会・シンポジウム, ノックアウトマウスを用いたプロテインホスファターゼPPM1Lの新規機能解明, 2015
• 日本生化学会東北支部 第81回例会・シンポジウム, UVB照射により、Ppp6c欠損では、高頻度に皮膚扁平上皮癌が発生する, 2015
• 日本生化学会東北支部 第81回例会・シンポジウム, ワールブルグ効果が腫瘍にもたらすもの, 2015
• 第62回 日本生化学会近畿支部例会, クラスリン集合タンパク質CALM欠損が及ぼすメラニン産生への影響, 2015
• Mechanisms & Models of Cancer 2015, PP6 deficiency potentiates ultraviolet-B-induced skin carcinogenesis, 2015
• 第74回日本癌学会学術総会, Critical roles of c-Myb in c-Kit expression and maintenance of leukemia stem cells, 2015
• 第74回日本癌学会学術総会, Ppp6c欠損皮膚では、UVB誘導発がんへの感受性が高まる。, 2015
• BMB2015, FceRIa-Creノックインマウスを用いマスト細胞機能の解析, 2015
• BMB2015, 脱リン酸化酵素PP6の触媒サブユニットのPpp6cは着床後の胚の正常な発生に不可欠である。, 2015
• BMB2015, 皮膚Ppp6c欠損マウスは、化学発がんおよびUVB誘導発がんに高感受性となる。, 2015
• BMB2015, Arf GTPase活性化因子SMAP2の欠損はEGFの取り込み後の輸送の異常を引き起こすのか, 2015
• BMB2015, メラノーマ細胞でのクラスリン集合因子CALMのノックダウンによるメラニン産生低下の機構を探る, 2015
• BMB2015, クラスリン集合因子CALMのEGFシグナル経路への関与を探る, 2015
• BMB2015, Arf1欠損MEF細胞の作製と引き起こされる異常の解析, 2015
• Mechanisms & Models of Cancer 2015, PP6 deficiency potentiates ultraviolet-B-induced skin carcinogenesis, 2015
• The 74th Annual Meeting of the Japanese Cancer Association, Critical roles of c-Myb in c-Kit expression and maintenance of leukemia stem cells, 2015
• The 74th Annual Meeting of the Japanese Cancer Association, Loss of protein phosphatase 6 in mouse keratinocytes increases susceptibilit y to ultraviolet-B-induced carcinogenesis., 2015
• The protein phosphatase 6 catalytic subunit (Ppp6c) is indispensable for proper post-implantation embryogenesis., 2015
• The protein phosphatase 6 catalytic subunit (Ppp6c) is indispensable for proper post-implantation embryogenesis., 2015
• Does lack of the Arf GAP SMAP2 induces enhanced up-take of EGF or impaired sorting of internalized EGF in MEF cells?, 2015
• Deficiency of CALM accompanies with reduced melanin production in mouse melanoma B16C2M cells., 2015
• An analysis about the roles of CALM on EGF signaling pathway., 2015
• Arf1-deficient MEF cells show prolonged doubling time and increased Arf2 expression., 2015
• 第18回 造血器腫瘍研究会, AF10欠損が引き起こす造血系と血管・リンパ管系の異常の発見, 2014
• 「個体レベルでのがん研究支援活動」ワークショップ, 癌への関与が注目されるPpp6c遺伝子を欠損するマウスの作製と解析, 2014
• 「個体レベルでのがん研究支援活動」ワークショップ, BRCA1の新規結合分Ola1のノックアウトマウスの解析, 2014
• 第6回 日本プロテインホスファターゼ研究会学術集会, 癌と関連するPpp6c遺伝子を欠損するマウスの作製と解析, 2014
• 第6回 日本プロテインホスファターゼ研究会学術集会, GTP脱リン酸化酵素のARFとその活性化因子SMAPの欠損マウス解析, 2014
• 第6回 日本プロテインホスファターゼ研究会学術集会, ノックアウトマウスを用いたPP2Cεの新規機能解明, 2014
• AACR Annual Meeting 2014, Promotion of skin tumorigenesis by deletion of Ppp6c gene, 2014
• AACR Annual Meeting, The nuclear export signal (NES) within CALM is necessary for CALM-AF10-induced leukemia., 2014
• 19th Congress of European Hematology Association, Manipulation of CALM function represents an attractive new strategy for targeting acute myeloid leukemia with mutated RTKs., 2014
• 生化学会 東北支部会, Ppp6c遺伝子欠損はマウス皮膚腫瘍形成を促進する, 2014
• 第11回日本病理学会カンファレンス, 癌への関与が注目されるPpp6c遺伝子を欠損するマウスの作製と解析, 2014
• 第73回日本癌学会, 単一のプルビン酸キナーゼアイソフォームを発現するマウスの解析, 2014
• 第73回日本癌学会, MLL融合遺伝子関連白血病発症におけるAF10の役割, 2014
• 第73回日本癌学会, PP6皮膚特異的欠損マウスは、DMBA誘発皮膚発がんに高感受性をしめす。, 2014
• 11th International Conference on Protein Phosphatase, Ppp6c deficiency leads to embryonic lethality and promotes skin carcinogenesis induced by DMBA, 2014
• 11th International Conference on Protein Phosphatase, Novvel mouse models to dissect isoform-specific functions of pyruvate kinase M., 2014
• 11th International Conference on Protein Phosphatase, Roles of pyruvate kinase M in metabolic rewiring during cellular senescence and transformation., 2014
• 11th International Conference on Protein Phosphatase, Targeted disruption of the mouse protein phosphatase PPM1L gene leads to structural abnormalities in the brain., 2014
• 第37回日本分子生物学会年会, SMAP（Small Arf GAP）1,2両遺伝子欠損胚は致死でアポトーシスを起こしている。, 2014
• 第37回日本分子生物学会年会 ワークショップ, 細胞分化や老化に伴う、ピルビン酸キナーゼMアイソフォームの発現制御, 2014
• 第37回日本分子生物学会年会 ワークショップ, 融合遺伝子による白血病幹細胞の制御機構, 2014
• the 56th ASH Annual Meeting & Exposition,, Chlorpromazine, an Inhibitor of Intracellular Trafficking of FLT3-ITD and KIT D816V, Shows Prominent Anti-Leuke mic Activities Against AML Cells and AML Stem Cells in Vitro and in Vivo., 2014
• AACR Annual Meeting 2014, Promotion of skin tumorigenesis by deletion of Ppp6c gene, 2014
• AACR Annual Meeting, The nuclear export signal (NES) within CALM is necessary for CALM-AF10-induced leukemia., 2014
• 19th Congress of European Hematology Association, Manipulation of CALM function represents an attractive new strategy for targeting acute myeloid leukemia with mutated RTKs., 2014
• Abrogation of protein phosphatase 6 promotes skin carcinogenesis induced by DMBA., 2014
• 11th International Conference on Protein Phosphatase, Ppp6c deficiency leads to embryonic lethality and promotes skin carcinogenesis induced by DMBA, 2014
• 11th International Conference on Protein Phosphatase, Novvel mouse models to dissect isoform-specific functions of pyruvate kinase M., 2014
• 11th International Conference on Protein Phosphatase, Roles of pyruvate kinase M in metabolic rewiring during cellular senescence and transformation., 2014
• 11th International Conference on Protein Phosphatase, Targeted disruption of the mouse protein phosphatase PPM1L gene leads to structural abnormalities in the brain., 2014
• the 56th ASH Annual Meeting & Exposition,, Chlorpromazine, an Inhibitor of Intracellular Trafficking of FLT3-ITD and KIT D816V, Shows Prominent Anti-Leuke mic Activities Against AML Cells and AML Stem Cells in Vitro and in Vivo., 2014
• 第１７回造血器腫瘍研究会, CALM-AF10 のCALM 部分はどのように白血病発症へ関与するか, 2013
• 第１７回造血器腫瘍研究会, AF10 コンディショナルノックアウトマウスの作製, 2013
• 平成24年度「個体レベルでのがん研究支援活動」ワークショップ 個体レベルのがん研究による相乗効果, CALM-AF10融合遺伝子のCALMによる白血病発症と赤血球造血への役割, 2013
• 平成24年度「個体レベルでのがん研究支援活動」ワークショップ 個体レベルのがん研究による相乗効果, Embryonic lethality caused in Mice Lacking the phosphatase domain of the PP6c gene, 2013
• 10th International Conference on Protein Phosphatase, Embryonic lethality caused in Mice Lacking the phosphatase domain of the PP6c gene, 2013
• 第86回日本生化学会大会, PKMアイソフォームに関する生理的、および病態生理的機能解析, 2013
• 第86回日本生化学会大会, ヒスタミン合成酵素ヒスチジンデカルボキシラーゼ（HDC）の活性阻害物質の探索とHCD/GFPノックインマウス作製に関する試み, 2013
• 第72回日本癌学会学術総会, Functional analysis of pyruvate kinase M (PKM) isoforms using knock-in models, 2013
• 第72回日本癌学会学術総会, Nuclear export signal (NES) within CALM is necessary for CALM-AF10-mediated leukemic transformation., 2013
• 第36回日本分子生物学会年会, in vivoにおけるピルビン酸キナーゼMの各スプライシングアイソフォーム特異的な役割精査のためのノックインマウス作製。, 2013
• 第36回日本分子生物学会年会, SMAP（Small Arf GAP）1, 2両遺伝子欠損MEF細胞株の樹立, 2013
• 第36回日本分子生物学会年会, Ppp6c遺伝子の触媒ドメインの欠損胚盤胞は一見正常に増殖できる, 2013
• 第36回日本分子生物学会年会, 小胞形成開始因子のArf1 conditional ノックアウトマウスの作製, 2013
• 第36回日本分子生物学会年会, 小胞形成開始因子Arf1を欠損した胚盤胞のin vitro培養下での増殖解析, 2013
• 第36回日本分子生物学会年会, SMAP2はリサイクリングエンドソームからゴルジ体への物質輸送を制御する, 2013
• 第36回日本分子生物学会年会, 白血病キメラ遺遺伝子の選択的AF10要求性, 2013
• 受容体型プロテインチロシンホスファターゼ研究の新たな方向性 第36回日本分子生物学会年会 公募WS受理番号：WS-065 ワークショップ オーガナイザー, 基質同定とノックアウトマウが明らかにしたチロシンホスファターゼPTPRTのがん抑制機能, 2013
• Embryonic lethality caused in Mice Lacking the phosphatase domain of the PP6c gene, 2013
• 10th International Conference on Protein Phosphatase, Embryonic lethality caused in Mice Lacking the phosphatase domain of the PP6c gene, 2013
• Pathophysiological and isoform-specific functions of pyruvate kinase-M type-1 (PKM1) and type-2 (PKM2), 2013
• The 72nd Annual Meeting of the Japanese Cancer Association, Functional analysis of pyruvate kinase M (PKM) isoforms using knock-in models, 2013
• The 72nd Annual Meeting of the Japanese Cancer Association, Nuclear export signal (NES) within CALM is necessary for CALM-AF10-mediated leukemic transformation., 2013
• 平成24 年度 がん若手研究者ワークショップ, BARD1結合因子BIPのコンディショナルノックアウトマウスの作製, 2012
• 第71回日本癌学会学術集会, PP6 触媒サブユニット遺伝子は、胚の初期発生時期に必須である, 2012
• 第71回日本癌学会学術集会, PKMスイッチ不可能なマウスモデル, 2012
• 第35回日本分子生物学会年会, SMAP（Small Arf GAP）1,2両遺伝子欠損胚は致死である, 2012
• 第35回日本分子生物学会年会, クラスリン集合因子CALMの欠損はトランスフェリンを介した鉄の取り込みを減少させ造血障害を起こす, 2012
• 第35回日本分子生物学会年会, ES細胞におけるピルビン酸キナーゼM（PKM）アイソフォームの機能解析―ノックイン細胞を用いて, 2012
• 第35回日本分子生物学会年会, ワールブルグ効果に関するin vivo解析のためのマウスモデル開発, 2012
• 第85回日本生化学会大会, ピルビン酸キナーゼM（PKM）の発現アイソフォームを固定したマウスの作出, 2012
• 第35回日本分子生物学会年会, mTOR complex 1はB細胞分化に必要である, 2012
• 71th. Annual Meeting of the Japanese Cancer Association, Embryonic lethality caused in mice lacking protein phosphatase 6 catalytic subunit, 2012
• 71th. Annual Meeting of the Japanese Cancer Association, A mouse model incapable of switching PKM isoforms, 2012
• The 35th Annual Meeting of the Molecular Biology Society of Japan, Embryonic Lethality Caused in Mice Lacking both SMAP (Small Arf GAP) 1 and 2., 2012
• The 35th Annual Meeting of the Molecular Biology Society of Japan, Functional analysis of pyruvate kinase M (PKM)-isoforms in self-renewal of mouse ES cell, 2012
• The 35th Annual Meeting of the Molecular Biology Society of Japan, A development of PKM1-knock-in mice lines for analyzing the Warburg effect in vivo., 2012
• The 85th Annual Meeting of the Japanese Biochemical Society, Development of mice in which the switches of pyruvate kinase M (PKM)-isoform s are disabled, 2012
• Toshio Watanabe; Maki Isokawa; Miki Matsuoka; Mai Ito; Ayumi Kondo; Hiroshi Shima, 14th International Conference on Protein Phosphatase., Roles of Ppp6c in neuron, oligodendrocyte, adipose tissue, and the relationship with activated ras in MEF cells., 10 Dec. 2020, 10 Dec. 2020, 12 Dec. 2020
• 吉岡ゆきの; 天津 友貴; 山本 采佳; 安田恵子; 小柴 琢己; 渡邊 利雄, 第43回日本分子生物学会年会, Mul1-deficient mice show infertility, 04 Dec. 2020
• 天津 友貴; 松尾 尚輝; 山本 采佳; 中嵜 詩乃; 小河 穂波; 一戸 猛志; 小柴 琢己; 渡邊 利雄, 第43回日本分子生物学会年会, nalysis of how Mul1-KO MEFs response to virus infection., 04 Dec. 2020
• Toshio Watanabe, Taiwan-Japan Bilateral Conference on Phosphatase., Roles of Ppp6c during mouse early embryonic hematopoiesis., 15 Nov. 2019
• 中井 杏; 吉岡 ゆきの; 安田 恵子; 小柴 琢己; 渡邊 利雄, 【2021年度先端モデル動物支援P】成果発表会, Mul1欠損メスマウスが示す不妊の原因解析, 03 Feb. 2022, 02 Feb. 2022, 03 Feb. 2022
• 松岡 未樹; 酒井大輔; 島礼; 渡邊利雄, 【2021年度先端モデル動物支援P】成果発表会, 神経系における脱リン酸化酵素PP6の機能解析, 03 Feb. 2022, 02 Feb. 2022, 03 Feb. 2022
• 吉岡ゆきの; 中井杏; 小柴琢己; 渡邊利雄, 第44回日本分子生物学会年会202, Mul1遺伝子欠損メスマウスにおける不妊の原因解析, 02 Dec. 2021, 01 Dec. 2021, 03 Dec. 2021
• 中井杏; 吉岡ゆきの; 小柴琢己; 渡邊 利雄, 第44回日本分子生物学会年会, Mul1欠損メスマウスが示す不妊の解析, 01 Dec. 2021, 01 Dec. 2021, 03 Dec. 2021
• 松岡未樹; 酒井大輔; 島礼; 渡邊 利雄, 第44回日本分子生物学会年会, 神経系における脱リン酸化酵素PP6の機能解析, 01 Dec. 2021, 01 Dec. 2021, 03 Dec. 2021
• 小谷 唯; 住吉 麻実; 笹田 萌未; 渡邊 利雄; 松田 達志, 第44回日本分子生物学会年会, 肥満細胞における低分子量Gタンパク質Arf1の機能解明, 01 Dec. 2021, 01 Dec. 2021, 03 Dec. 2021
• 山本 樹; 青木 清乃; 山本 采佳; 山野 荘太郎; 大島 正伸; 八尾 良司; 渡邊 利雄; 田中 知明; 大木 理恵子, 第44回日本分子生物学会年会, p53標的遺伝子p53PAD5は腸管上皮細胞にストレス抵抗性を付与する。, 01 Dec. 2021, 03 Dec. 2021
• 堤 弘次; 長谷川 稜; 戸田 あかり; 堀 羽衣音; 渡邊 利雄; 太田 安隆, 第７３回日本細胞生物学会年会, Arf1 regulates membrane blebbing., 2021, 29 Jun. 2021, 02 Jul. 2021

Works

• 化粧品・医薬部外品の有効性評価及び新規有効成分の探索, Jul. 2010, Jun. 2018
• Ptproのコンディショナルノックイン組換えマウスの作出とその機能解析, Jun. 2014, Mar. 2015
• Ptprj及びPtproのコンディショナルノックイン組換えマウスの作出とその機能解析, Jul. 2013, Mar. 2014
• 改変型Ptprzノックインマウスの作出とその機能解析, Apr. 2012, Mar. 2013
• 新規乳癌関連分子のノックアウトマウスの作成とその解析による新たな発癌機構の解明, Apr. 2012, Mar. 2013
• 新規乳癌関連分子のノックアウトマウスの作成とその解析による新たな発癌機構の解明, Apr. 2011, Mar. 2012
• 改変型Ptprzノックインマウスの作出とその機能解析, Apr. 2011, Mar. 2012
• 改変型Ptprzノックインマウスの作出とその機能解析, Oct. 2010, Mar. 2011

Awards

• Long-Term-Fellowships by Human Frontier Science Program, 1992
• Long-Term-Fellowships by Human Frontier Science Program, 1992
• 加藤記念日本分子生物学会奨励賞, 1987, Japan
• Kato menorial Awords by JMBS, 1987

Research Projects

• 基盤研究(C)（一般）, Apr. 2020, Mar. 2023, Principal investigator, セリン・スレオニン脱リン酸化酵素PP6の「個体レベルでの生理機能の解明」, 渡邊 利雄, 文部科学省, 科学研究費補助金, 奈良女子大学, rm:published_papers;rm:published_papers;rm:published_papers

  対象リソースIRI
• Apr. 2017, Mar. 2020, ミトコンドリア・ダイナミクスの分子機構の解明と高次生理機能の考察, 小柴 琢己, 文部科学省, 科学研究費補助金, 0, 0, 0, Competitive research funding
• Apr. 2016, Mar. 2019, Principal investigator, 組織特異的遺伝子欠損法を用いた神経変性疾患における小胞輸送機構関与の検証, 渡邊 利雄, 文部科学省, 科学研究費補助金, 0, 0, 0, Competitive research funding, rm:published_papers;rm:published_papers

  対象リソースIRI
• Apr. 2016, Mar. 2019, 新規BRCA1結合分子のゲノム安定性維持機構の解明とがん治療法の開発, 千葉奈津子, 文部科学省, 科学研究費補助金, 0, 0, 0, Competitive research funding, rm:published_papers

  対象リソースIRI
• Apr. 2014, Mar. 2017, 哺乳動物におけるミトコンドリア・ダイナミクスの分子基盤解析, 小柴 琢己, 文部科学省, 科学研究費補助金, 0, 0, 0, Competitive research funding
• Apr. 2012, Mar. 2015, Principal investigator, クラスリン依存性小胞輸送の制御機構解明―ノックアウトマウスの利用―, 渡邊 利雄, 文部科学省, 科学研究費補助金, 0, 0, 0, Competitive research funding, rm:published_papers;rm:published_papers;rm:published_papers;rm:published_papers;rm:published_papers;rm:published_papers;rm:published_papers;rm:published_papers

  対象リソースIRI
• Apr. 2012, Mar. 2015, 新規癌抑制分子の細胞分裂制御能の解析による癌抑制機構の解明と癌治療法の開発, 千葉奈津子, 文部科学省, 科学研究費補助金, 0, 0, 0, Competitive research funding
• Apr. 2010, Mar. 2012, キネシンモーターリン酸化の理的意義の解明ー癌、絨毛病への関与ー, 島 礼, 文部科学省, 科学研究費補助金, 0, 0, 0, Competitive research funding
• Apr. 2009, Mar. 2010, 転写制御因子を介した造血幹細胞と白血病幹細胞の制御機構の解析, 北林 一生, 文部科学省, 科学研究費補助金, 0, 0, 0, Competitive research funding
• Apr. 2008, Mar. 2010, Principal investigator, 小胞形成制御因ＳＭＡＰ１，２は細胞外環境の生理機能にどう影響するか, 渡邊 利雄, 文部科学省, 科学研究費補助金, 0, 0, 0, Competitive research funding, rm:published_papers;rm:published_papers;rm:published_papers

  対象リソースIRI
• Apr. 2006, Mar. 2008, Principal investigator, 新規ARF GAPによるARF Gサイクル制御と生理機能の解明, 渡邊 利雄, 文部科学省, 科学研究費補助金, 0, 0, 0, Competitive research funding, rm:published_papers

  対象リソースIRI
• Grant-in-Aid for Challenging Exploratory Research, 01 Apr. 2015, 31 Mar. 2017, 15K15157, Molecular dissection of mast cell function by using newly generated mast cell lineage specific Cre expressing mice, MATSUDA Satoshi; WATANABE Toshio; SUMIYOSHI Mami, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Kansai Medical University, 3770000, 2900000, 870000, To understand mast cell biology, we established mast cell lineage specific Cre expressing mice under the control of FcεRIα locus. Among the signaling molecules functioning downstream of PI3K, we focused Tsc1 and Arf1 along with Arf6. Loss of Tsc1, which causes augmentation of mTORC1 signal, led to marked increase of mast cell number in peritoneal cavity. On the other hand, loss of Arf1 along with Arf6, both of which serve as a vesicle trafficking regulator, impaired mast cell development. These results indicate that mTORC1 signal as well as Arf-mediated vesicle trafficking system play a critical role in mast cell development., kaken

  対象リソースIRI
• 萌芽研究, 2003, 2004, 15659064, 円頭精子症候群の新規原因遺伝子の解析とその修飾遺伝子群を探る試み, 渡邉 利雄, 日本学術振興会, 科学研究費助成事業, 東北大学, 3100000, 3100000, 造血幹細胞発生に必須のRunx1の細胞運命決定への効果を検討するために、Runx1を発生初期の血管内皮細胞に強制発現させたところ、血管網形成が異常となった。この血管網形成異常はこれまでに知られているものと異なっていることから、新しいメカニズムによって血管細胞の運命決定へ影響があったと推察された。現在、造血幹細胞発生が変化していないかを検討中である。一方で、Runx1を造血幹細胞に過剰に発現させた場合には、その個体では外見上異常が見られなかった。現在、造血幹細胞の再建活性能への効果を見ようと準備している。造血幹細胞特異的なRunx1のアイソフォームの特異的Runx1 KIマウスのキメラマウスが得られた。しかし150匹の子供を解析したが生殖系列への伝播がまだ見られてない。一方Runx1の標的遺伝子候補のconditional KOマウスは生殖系列への伝播を確認した。現在順次Creマウスとの交配を行っている。培養T細胞を用いて、100bpのRunx1発現制御領域を同定した。さらに、これまでの一般の予想とは異なり、Runx1が自分自身の発現を負に制御することと、Runxファミリー間での相互抑制機構があることを培養細胞を用いて発見した。内在性のRunx1の発現を再現する発現制御領域検定用のTgマウスは、1系統を樹立したが、培養細胞での解析から決めた発現制御領域では内在性の発現の全ては再現できなかった。現在より広い領域での検討を行うために、組み換え体を作製している。さらに、造血幹細胞細胞の形態変化(細胞骨格の変化)により造血幹細胞発生が制御されている可能性を見いだし、手始めとして、アクチン結合タンパク質のfilamin AによるRunx1の活性制御機構を明らかにして、その成果を論文として公表した。, kaken

  対象リソースIRI
• 特定領域研究, 2003, 2004, 15039201, 転写因子AML1とc-mybとによる造血幹細胞の発生と成立のメカニズム, 渡邉 利雄, 日本学術振興会, 科学研究費助成事業, 東北大学, 5000000, 5000000, 昨年度までに作製した、新規のARF-GAP遺伝子SMAP-2の遺伝子破壊マウスの、詳しい解析を行った。精子の形から、円頭精子症であることを既に明らかにしていたが、これに加えて、精巣の大きさ、精子の数に関しては顕著な異常は見られないものの、ホモのオスが不妊であることを明らかにした。これらは全て、B6/129の混合遺伝背景のもとで解析を行った。解析と平行して、円頭精子症の原因遺伝子を修飾するmodifier遺伝子を探索するために行った戻し交配は、B6マウスとの交配について8代目となったので、現在ホモのオスマウスを作成中である。129系に関しては、交配スペースの問題で未だ解析に至っていない。細胞内局在等を検討するために作製したSMAP-2特異的な抗体は、エスタン解析には使えるが、組織化学的な解析には使用できないことが判明した。このために、培養細胞を用いた遺伝子導入系を用いて、細胞内局在解析を行った。その結果、SMAP-2がTGNとエンドソームに存在している知見を得た。また、CALMなどクラスリンと関係している細胞内小胞輸送系の構成因子とSMAP-2とが会合することも明らかにした。円頭精子症では、ゴルジ体からできることが知られている先体の形成異常が見られることが知られているので、SMAP-2ホモマウスの精子ではどうであるのか今後電子顕微鏡による解析を行う。SMAP-2に加えて、SMAP-2のホモログであるSMAP-1が、クラスリン依存性のエンドサイトーシスに関与していることを明らかにした。通常の遺伝子破壊を行ったところ、ヘテロ欠損体で胎生致死であったので、詳しい解析を行うために、SMAP-1のコンディショナル遺伝子破壊マウスを作製し生殖系列への伝播を確認した。同時に解析に必要なCreマウスのコロニーを作製出来た。, kaken

  対象リソースIRI
• 特定領域研究, 2003, 2003, 15019009, 転写因子AML1によるT細胞のTh1/Th2系列への振り分け制御機構の解明, 渡邊 利雄, 日本学術振興会, 科学研究費助成事業, 東北大学, 3300000, 3300000, 昨年度までに、Runx1がGATA-3の発現を抑制することを通してTh2細胞分化を抑制すること(小峰らJ.Exp.Men.,2003)、Runx3がCD4T細胞の発生を抑制すること(EhlersらJ.Immunol.,2003)を、in vitroの実験から明らかにした。これらの成果から、転写因子Runx1,3が、感染応答に重要なCD4SPT細胞の発生と、Th1・Th2細胞分化の制御因子である可能性を示した。今年度はRunx1,3がCD4,8細胞分化や、Th1、Th2細胞分化を如何にコントロールしているのかを、遺伝子導入マウスを用いて明らかにし、CD4SP T細胞の発生や、Th1、Th2細胞分化をコントロールするための基礎となる成果を得ることを目的とした。lck-Runx3-Tgマウスを作製したところ、CD4細胞の激減とCD8細胞の激増が見られた。これより、Runx3はCD4サイレンサーとして働きCD4細胞分化を制御していることに加えて、細胞性免疫を担うCD8細胞の分化に促進的に働く可能性が示された。一方Runx1に関しては、我々や海外のグループの解析結果から、T細胞ではこれまで主に解析されてきたN末端の短いp-Runx1ではなくN末端の長いd-Runx1が主として発現していることが最近判明した。そこでT細胞分化における2つのアイソフォームの機能を解析するためにcre-loxPシステムを用いてconditionalに各アイソフォームを発現するTgマウスを作成した。lck-creマウスを用いた解析から、d-Runx1-TgマウスではDP細胞が激減しCD4、CD8細胞分化が抑制された。一方p-Runx1-Tgマウスでは、DP細胞の激減は見られなかったが、CD4細胞の減少とCD8細胞の増加が見られた。, kaken

  対象リソースIRI
• 特定領域研究(C), 2001, 2001, 13216009, 転写因子AML1とチロシンホスファターゼPTPβ2から探る血管新生制御の分子機構, 渡邊 利雄, 日本学術振興会, 科学研究費助成事業, 東北大学, 3800000, 3800000, 我々は、白血病染色体転座部位に存在する癌関連遺伝子として単離された転写因子であるAML1が、胎児期における造血幹細胞の発生と特定の領域での血管新生に不可欠であることを明らかにした。また、白血病細胞株の脱癌化(赤血球分化)を誘導するがん抑制遺伝子、チロシンホスファターゼPTPβ2/CD148のKOマウスでは、血管新生異常があり、マウスは貧血状態に見えること観察した。胎生期における血管新生と血球発生の各々の、そして相互の関係のシナリオを理解するためには、今後これらの分子メカニズムを明らかにすることが大きく寄与すると考えられる。一方、癌細胞の増殖に伴った血管新生の機構も胎生期と同様のシナリオで進むものと考えられているが、こちらも未だ不明の点が多い。この癌細胞の増殖に伴った血管新生の機構の解明を目指して、AML1転写因子とチロシンホスファターゼPTPβ2/CD148のKOマウスを用いて、血管新生と血球発生のメカニズムについて解析を加えることにした。本研究では、血球発生時、血管新生時にAML1転写因子の転写活性可能がどのような機構で制御され、作用しているのかについてと、チロシンホスファターゼPTPβ2/CD148の血管形成、血球発生における役割はいったい何なのかを明らかにすることを目的とした。その結果、AML1の転写活性を抑制する細胞骨格制御タンパク質の存在を明らかにした。またPTPβ2/CD148欠損マウスでは、血球発生能は異常が見られないが、確かに血管新生の異常による血管網形成の異常がみられることを明らかにした。さらに、少なくともin vitroの系では、血管形成能には異常が見られないことから、PTPβ2/CD148は血管を誘導する環境側の細胞で機能している可能性が示唆された。さらに、この機能を担うPTPβ2/CD148の細胞内情報伝達系の一端を明らかにした。, kaken

  対象リソースIRI
• 特定領域研究(A), 2001, 2001, 13016201, 転写因子とチロシンホスファターゼから探る造血発生と血管新生制御の分子機構, 渡邊 利雄, 日本学術振興会, 科学研究費助成事業, 東北大学, 1800000, 1800000, 胎生期における血管新生と血球発生の各々の、そして相互の関係のシナリオの解明を目指して、各々の作用機序が大きく異なる転写因子とチロシンホスファターゼのKOマウスを用いて、転写制御と細胞内情報伝違系の側面から血管新生と血球発生に解析を加えた。今回はAML1転写因子とチロシンホスファターゼPTPβ2/CD148のKOマウスを用いて、血管新生と血球発生のメカニズムについて解析を加えることにした。本研究で取り上げた具体的なテーマは、血球発生時、血管新生時にAML1転写因子の転写活性化能がどのような機構で制御されているのかについて明らかにすることと、チロシンホスファターゼPTPβ2/CD148の血管形成、血球発生における役割はいったい何なのかを明らかにすることである。研究の結果、AML1の転写活性を抑制する細胞骨格制御タンパク質の存在を明らかにし、血球発生時には、これまでに知られていなかった機構でAML1の転写活性が制御されている可能性があることを明らかにし得た。またPTPβ2/CD148欠損マウスのホモ欠損体では、血球発生能は異常が見られないが、確かに血管網形成の異常がみられることを明らかにした。予想外のことに、少なくともin vitroのP-Sp器官倍養系では、成体型の血球発生能にも、血管網形成能にも異常が見られなかった。このことからPTPβ2/CD148は血管内皮細胞ではなく、血管を誘導する環境側の細胞で機能している可能性が示唆された。さらに、この機能を担うPTPβ2/CD148の細胞内情報伝達系の一端を明らかにするために、PTPβ2/CD148の会合因子の候補を得た。, kaken

  対象リソースIRI
• 特定領域研究(C), 2000, 2000, 12215012, AML1による胎児期血管新生、造血発生制御の分子機構の解明, 渡邊 利雄, 日本学術振興会, 科学研究費助成事業, 東北大学, 3000000, 3000000, 互いに密接な関係にあると推定されている造血発生と血管新生の両者に共通する機構の実体は、依然として不明のままである。そこでこの問題を解明するために、一つの転写因子AML1を欠損したマウスに見られる胎児期血管新生と造血発生の二つの以上に関わる、共通の分子機構を明らかにすることを目的とした。血管発生をin vitroで見ることができるP-Sp領域の器官培養系ではAML1を欠損したマウスから成体型の造血発生が起きないと同時に血管新生が一切見られないことを明らかにした。次にこの血管新生の異常はAngiopoietin-1の添加で回復することを見出した。さらに、血管新生と造血発生との相互作用があることを想定し、AML1欠損マウスのP-Sp領域の器官培養系に造血幹細胞を添加したところAngiopoietin-1と同様に血管新生が回復された。これに加えて、造血幹細胞でのみAngiopoietin-1が発現していること、造血幹細胞を移植すると生体内で異所的に血管新生が起こることを示した。以上のことから、発生のある時期の特定の領域では造血幹細胞から供給されるAngiopoietin-1によって血管新生が起きているという、造血幹細胞の新しい機能を提唱した。この結果は高い評価を受けた。一方培養細胞における血管新生のモデルシステムを用い、転写因子AML1の機能阻害はこの細胞系の血管新生に対して抑制的に働くことを明らかにした。また、cDNA subtraction、cDNA Tipを用いた包括的解析によりAML1の標的遺伝子検索を行い、現在候補遺伝子を得つつある。, kaken

  対象リソースIRI
• 特定領域研究(A), 1998, 1998, 10181201, c-myb,PEBP2欠損マウスのAGM領域での造血発生の変化とその機構の解析, 渡邊 利雄; 佐竹 正延, 日本学術振興会, 科学研究費助成事業, 東北大学, 3000000, 3000000, c-myb,PEBP2(=AMLl)遺伝子を欠損するマウスでは、卵黄嚢におけるプリミティブな造血能は傷害を受けないが、胎仔肝におけるデフィニティブな造血能が全く発生せず、胎令12.5日で死亡することが、我々を含めた幾つかの研究グループにより示されている。一方近年、デフィニティブな造血前駆細胞は卵黄嚢よりは遅く、しかし肝よりは早く、AGM(aorta,gonad and mesonephros)領域に発生することが判明した。9.5,11.5日令のマウス胎仔のAGM領域を細胞培養することにより、血球細胞をin vitroで発生・増殖できる系を遺伝子破壊マウスに適用し、次の2点を明らかにした。即ち、1)c-myb(-/-)またはAML1(-/-)胎仔肝におけるデフィニティブな造血の欠如は、本来なら肝に先行してAGM領域に局在するはずの、造血前駆細胞の傷害によるものである。2)AGMにおいて造血前駆細胞は、PEBP2(=AML1)転写因子の量に依存して発生・増殖する。, kaken

  対象リソースIRI
• Grant-in-Aid for Scientific Research (C), 1997, 1998, 09680684, Functions of the PEBP2beta protooncogene product in muscle cell differentiation., WATANABE Toshio, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Institute of Development, Aging and Cancer, Tohoku University, 2600000, 2600000, The PEBP2beta gene encodes a non-DNA binding subunit of a heterodimeric transcription factor, PEBP2, in the chromosomal inversion 16 associated with human acute myeloid leukemia (AML), the PEBP2beta and smooth musclemysin heavy chain (SMMHC) genes fuse each other and the chimeric PEBP2beta-SMMHC gene is generated. We analyzed functions of cytoplasmic-located PEBP2beta and PEBP2beta-SMMHC proteins, which are distinct from their functios as a subunit of transcription factor. The results are as follows. 1)Differentiation dependent expression and distinct subcellular localization of the protooncogene product, PEBP2beta/CBFbeta, in muscle development. 2)The protooncogene product, PEBP2beta/CBFfbeta, is mainly located in the cytoplasm and has an affinity with cytoskeletalstructures. 3)The chimeric protein, PEBP2beta-SMMHC, disorganizes cytoplasmic stress fibers and inhibits transcriptionalactivation., kaken

  対象リソースIRI
• Grant-in-Aid for international Scientific Research, 1997, 1998, 09044249, Functions of the runt gene family in development, SATAKE Masanobu; CHIBA Natsuko; NIKI Masaru; WATANABE Toshio; JIM Jaynes, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Institute of Development, Aging and Cancer, Tohoku University, 4600000, 4600000, The PEBP2 transcription factor functions as a heterodimer composed of the alpha and beta subunits. The alpha subunit is responsible for the DNA binding activity of PEBP2, which is furthermore enhanced by interacting with the beta subunit. The AML1 gene, encoding the alpha subunit of PEBP2, corresponds to the mammalian homologue of Drosophila runt gene, a gene involved in Segmentation, neural development and sex determination in fly. The mammalian PEBP2beta gene encoding the beta subunit of PEBP2 represents a runt-partner gene. Both the AMI.I and PEBP2beta genes are involved in chromosomal rearrangements associated which human acute myeloid leukemia. As a result of translocation and/or inversion, the chimeric genes are generated. The study was carried out to reveal the functions of mammalian runt and runt-partner genes in cell differentiation, transformation and development. The results are summarized as follows. l)The PEBP2 transcription factor is indispensable and functions in a dose dependent manner for the hematopoietic development in the embryonic aorta-gonad-mesonephros region. 2)The product of the chimeric gene associate with human AML and involving the PEBP2beta gene impairs the architecture of cytoplasmic cytoskeleton. 3)A dose of AML1 transcription factor is critical in deternuning the quality of T cell receptor-mediated signal to cell survival or cell death., kaken

  対象リソースIRI
• Grant-in-Aid for Scientific Research (A), 1996, 1998, 08308035, Physiological significance of protein phosphatases on cellular signal transduction, TAKEDA Masao; YAKURA Hidetaka; KASUGA Masato; MIYAMOTO Eishichi; TAMURA Shinri; KIKUCHI Kunimi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, HIROSHIMA UNIVERSITY, 18100000, 18100000, (Takeda) Human erythrocyte PP2A (CAB") was phosphorylated in vitro by A-kinase at Ser-60, -75 and -573 of B" without dissociation of B" from CA, and was stimulated in the activity toward P-histones ; (Kikuchi) Activities and amounts of nuclear type I protein phosphatase (PP1) were elevated in human HepG2 and rat AHI3 hepatoma cells compared with primary cultured hepatocytes. The nuclear PP1 activities were more elevated at the (Gl/S transition in hepatoma cells, the extent of the elevation being much less in hepatoma cells than in hepatocytes ; (Kuno) Disruption of the PP2B gene in fission yeast resulted in a drastic chloride ion-sensitive growth defect. High copy of two novel genes pmpl+ and pekl+ suppress this defect. pmpl+ encodes a MAP kinase phosphatase which negatively regulates Pmkl, the third MAP kinase in fission yeast pekl+ encodes a novel MAPKK member, which phosphorylates and activates the Pmkl MAPK ; (Tamura) Protein phosphatase 2Calpha (PP2Calpha) or PP2Cbeta-l expressed in COS7 cells suppressed anisomycin- and NaCI-enhanced phosphorylations of p38 coexpressed in the cells. PP2Calpha or PP2Cbeta-l expression also suppressed both basal and stress-enhanced phosphorylations of MKK3b, MKK6b and MKK4 but not those of MKKla. A dominant negative form of PP2Cbeta-l enhanced farther the NaCl-stimulated phosphorylations of MKK3b, MKK4 and MKK6b. These results suggest that PP2C inhibits selectively the SAPK pathways through suppression of MKK3b, MKK4, and MKK6b activities in mammalian cells ; (Miyamoto) l)Protein phosphatases which are responsible for the phosphorylation sites by proline-directed protein kinases were investigated. In this connection, protein phosphatases for phosphorylated tau in neurofibrillary tangles Alzheimer's brain was studied. 2) Decreases in protein phosphatase activities during induction of long-term potentiation in hippocampus was studied ; (Fujiki, ) Inhibitors of PP-l and PP-2A, okadaic acid and microcystin are potent tumor promoters of skin, glandular stomach and liver. However, tautomycin, an inhibitor of PP-1 and PP-2A, did not show any tumor promoting activity in mouse skin and rat glandular stomach. Okadaic acid and microcystin induced tumor necrosis factor-alpha (TNF-alpha) gene expression in their target organs, but tautomycin did not. Effects of tautomycin on JNK activation snd cell cycle regulation are different from those of okadaic acid ; (Kasuga) SHP substrate-l (SHIPS-1) with Ig-like domains in its extracellular region and four YXX(L/ V/I) motifs in its cytoplasmic region was identified. Various mitogens induced tyrosine phosphorylation of SHPS-i and its subsequent association with SAP-2/SHP-2. Thus, SHPS-l may be a direct substrate for both tyrosine kinases and SAP-2/SAP-2 and also be a specific docking protein for SH2 domain-containing PTPases ; (Hinoda) Tyrosine phosphorylation of SHP-2 was involved in the signal transduction pathways by IL-2 stimulation. Since this SHP-2 activation is irrelevant to, kaken

  対象リソースIRI
• 重点領域研究, 1991, 1991, 03252105, 正常造血幹細胞,造血因子と造血器腫瘍との比較と相互関係, 穂積 本男; 白川 茂; 平井 久丸; 斎藤 政樹; 帯刀 益夫; 渡辺 利雄, 日本学術振興会, 科学研究費助成事業, 埼玉県立がんセンター, 30000000, 30000000, 1.骨髄性白血病M1細胞の分化誘導因子(D因子)は、テラトカルシノ-マF9細胞の分化誘導抑制作用および骨芽細胞の骨吸収作用のあることを見出した。また、インタ-フェロンβ,ILー4、ILー6は単球性白血病細胞の増殖と分化を著しく修飾することを認めた(穂積)。 2.フレンド白血病細胞の分化誘導に関与するチロシンキナ-ゼはEGF受容体チロンキナ-ゼ様であることを明らかにし、燐酸化チロシンホスファタ-ゼ活性の低下と分化誘導能とが密接に関連していることを明らかにした(大石)。 3.cーmyc遺伝子がフレンド白血病細胞の転写因子として機能していることを証明し、cーmycが細胞増殖、分化の転換を制御していることを示唆した(帯刀)。 4.シアロ糖脂質ガングリオシド(GM_3、2‐3sialosylparagloboside(SPG)の各種ヒト白血病細胞に対する分化誘導と増殖抑制効果を明らかにし、その作用機序を検討した(斎藤)。 5.ヒト胎盤cDNAライブラリ-より、正常ヒトltk遺伝子を単離してその構造を解析し、ltk遺伝子の翻訳開始点、遺伝子産物、コ-ディング領域を明らかにした(平井)。 6.白血病細胞の表現型、遺伝子型、染色体、増血因子に対する反応性などの解析から幹細胞性白血病が従来の想定より高頻度に存在しており、特定の形質発現の異常から診断可能であることを明らかにした。また、白血病の細胞種、白血病細胞の分化段階の差違によってプロティンキナ-ゼCアイソザイムの発現が異なることを見出した(白川)。, kaken

  対象リソースIRI
• 重点領域研究, 1990, 1990, 02256106, 正常造血幹細胞,造血因子と造血器腫瘍との比較と相互関係, 穂積 本男; 白川 茂; 平井 久丸; 斎藤 政樹; 帯刀 益夫; 渡辺 利雄, 日本学術振興会, 科学研究費助成事業, 埼玉県立がんセンター, 30000000, 30000000, 1.マウス骨髄性白血病M1細胞の分化誘導因子(D因子)は、1)各種サイトカインとの併用処理でM1細胞の分化誘導を修飾すること、2)ブタ腎上皮LLCーPK_1細胞の分化を抑制することを明らかにした。また、各種サイトカインはレチノイン酸、ビタミンD_3活性体によるヒト骨髄性白血病細胞の分化誘導と増殖抑制を促進することを見出した(穂積)。2.フレンド白血病細胞の3種類の細胞内分化因子(DIFーI、DIFーII、DIFーIII)の中で、DIFーIIIはDIFーIのリン酸化チロシンのDIFーIIによる脱リン酸化反応によって生成することを明らかにした(渡辺)。3.cーmyc遺伝子によるフレンド白血病細胞の分化誘導抑制機序を解析し、cーmycは分化の決定の過程と分化特異的遺伝子発現の過程とを抑制していることを明らかにした。また、cーmyc蛋白質の機能領域を解析し、癌原性と分化抑制を関与する領域は同一であるが、分化特異的遺伝子発現を抑制する領域とは異なることを明らかにした(帯刀)。4.シアロコレステロ-ルとシアロジグリセリドにヒト前骨髄球性白血病HLー60細胞の分化誘導と増殖抑制効果のあることを見出した。また、分化誘導性ガングリオシドはインシュリン受容体βサブユニットの自己リン酸化反応の特異的な抑制作用を介してHLー60細胞を分化させることを示唆した。(斎藤)。5.造血細胞に特異的に発現し、造血細胞の増殖に関与すると推察される新たなチロシンキナ-ゼ型受容体遺伝子1tkをヒト白血病K562細胞のcDNAライブラリ-から単離し、構造を明らかにした(平井)。6.リンパ球、骨髄球両者の性質を示すヒト白血病細胞は多能性幹細胞に由来し、特定の染色体相互転座発生は細胞の分化と密接に関連していることを明らかにした。また。白血病細胞型および分化段階によってプロティンキナ-ゼC亜型に差違があることを明らかにした(白川)。, kaken

  対象リソースIRI
• がん特別研究, 1989, 1989, 01015024, マウス白血病細胞分化の分子機構, 大石 道夫; 渡辺 利雄; 野村 慎太郎; 鮎沢 大, 日本学術振興会, 科学研究費助成事業, 東京大学, 6700000, 6700000, 赤芽球性白血病細胞マウス・フレンド細胞の分化を引き起こす3種の細胞内因子(DIFーI、II、III)の相互作用を解析した結果、DIFーIIIはDIFーIとDIFーIIとの脱リン酸化を含む共役反応により生成され、DIFーIIはDIFーIに作用する脱リン酸化酵素であることが推定された。また反応の解析の結果から、フレンド細胞分化の誘導(分化決定因子DIFーIIIの生成)にはリン酸化チロシン脱リン酸化反応が関与していると考えられる。この結果より、チロシンキナーゼ阻害剤を用いてマウスフレンド細胞の分化が誘導されるかについて検討した。その結果、herbimycin Aは単独で、genisteinとST638とはDIFーIの誘導剤マイトマイシンCと相乗的に、フレンド細胞の分化を誘導し、分化の決定にリン酸化チロシンの脱リン酸化が関与していることを強く示唆した。さらにherbimycin Aは、マウス胚性腫瘍細胞(F9細胞)のparietal endodermへの分化を引き起こすことから、初期発生のモデルであるF9細胞においてもフレンド細胞におけるのと同様な機構が存在している可能性が示唆される。又、分化誘導初期におけるリン酸化チロシンの含量を調べたところ、フレンド細胞、F9細胞のいずれも低下していることが判明した。一方herbimycin AによるF9細胞分化の機構を別の方面から探るため、herbimycin A処理後短時間で顕著に発現がみられる遺伝子のクローニングを行い、1つのクローンを得た。この遺伝子の発現誘導には___ー novoの蛋白質合成が関与していないと思われることから、herbimycin Aによるタンパク質のリン酸化の阻害の直接の結果として発現が誘導されていると考えられる。この遺伝子はレチノイン酸のみでは誘導されないが、レチノイン酸とdibutyryl CAMPとの共存で誘導されることから、F9細胞のparietal endoderm細胞への分化に関与していると思われる。さらに塩基配列の解析から、この遺伝子はheat shockタンパク質の1つ(hsp86)であることが明らかとなった。, kaken

  対象リソースIRI
• Grant-in-Aid for Scientific Research (C), 01 Apr. 2013, 31 Mar. 2016, 25460602, The role of mTORC1 in dendritic cells in adaptive immune response, OHTANI Masashi; NAGAI Shigenori; WATANABE Toshio, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, 5070000, 3900000, 1170000, We have previously demonstrated that, in dendritic cells (DC), mTORC1 regulates the production of IL-12 as well as the T helper (Th) cell differentiation. In this study, we investigated the roles of mTORC1 in DC in T cell mediated-humoral immunity using DC-specific mTORC1 function-deleted (KO) mice. In KO mice, bovine serum albumin (BSA)-alum induced the production of antigen-specific IgG1 was decreased when compared with control mice. We also found the reduction of the proportion of Th2 cells in the spleen of immunized KO mice, suggesting that the mTORC1 in DC influences the antigen-specific antibody production through the regulation of Th cell differentiation., kaken

  対象リソースIRI
• Grant-in-Aid for General Scientific Research (B), 1988, 1989, 63480509, Studies on intracellular factors involved in cellular differentiation of mouse and human cells., OISHI Michio; WATANABE Toshio; NOMURA Shintaro; AYUSAWA Dai, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Inst. of Applied Microbiol. University of Tokyo, 7000000, 7000000, We have found that herbimycin A is an effective inducing agent capable of triggering differentiation in two typical mouse in vitro differentiation systems, which have been considered to be quite different in their mechanism of induction: endoderm differentiation of embryonal carcinoma (F9) cells and terminal erythroid differentiation of (MEL) cells. The results suggest that there is a common step in the intracellular differentiation cascade which is, directly or indirectly, associated with phosphorylation at specific (tyrosine) residues of cellular proteins. An erythroid-inducing activity was detected in cell-free extracts from MEL cells which had been incubated with dimethyl sulfoxide or hexamehylenebisacetamide for a prolonged period of time. The newly detected activity was apparently different from the previously reported differentiation-inducing factors and seemed to be derived from a proeinacious factor in the cytoplasm. The timing of the appearance of the activity in the extracts coincided with that of cellular commitment to differentiation. The activity was not induced in a differentiation defective MEL cell line and the induction of the activity was inhabited by PMA, a specific inhibitor for MEL cell differentiation., kaken

  対象リソースIRI
• がん特別研究, 1988, 1988, 63015019, マウス白血病細胞分化の分子機構, 大石 道夫; 渡辺 利雄; 西森 克彦, 日本学術振興会, 科学研究費助成事業, 東京大学, 7100000, 7100000, 従来の研究によりマウスフレンド細胞の分化に関する3種のタンパク質性因子(DIF-I,DIF-II,DIF-III)の関与が証明された。最近それぞれ部分精製したDIF-IとDIF-IIからDIF-IIIがin vitroで生成する再構成系が確立された。この系では反応終了後、DIF-IIIの生成と同時にDIF-Iが消失した。このことはDIF-IとDIF-IIとの相互作用によりDIF-IIIが生成し、このDIF-IIIが究極的にフレンド細胞の分化の決定に関与していることを強く示唆している。さらにDIF-IIIが生成する際にDIF-Iが検出されなくなることから、DIF-IIによりDIF-Iが修飾を受けDIF-IIIに変換するのではないかと考えられた。上述した試験管内でDIF-IIIを生成するin vitroの反応は脱燐酸化阻害剤Na_3VO_4やZnCl_2によって阻害される。また、DIF-Iをアルカリ.ホスファターゼ、ウシ心筋プロテインホスファターゼで処理してもDIF-IIの関与なしにDIF-IIIが生成した。これらのことから、DIF-IとDIF-IIとの共役反応には脱燐酸化反応が含まれることが推定される。そこでこの仮説が正しいかについて、細胞を用いても検討を行った。in vitro反応を阻害したNa_3VO_4やZnCl_2はDMSOによるフレンド細胞の分化誘導を阻害した。Na_3VO_4は細胞内でもDIF-I,IIの生成は阻害しなかったが両因子からのDIF-IIIの生成を阻害した。以上の結果は、DIF-Iが燐酸化タンパク質であることを示唆している。Na_3VO_4やZnCl_2は、燐酸化チロシンホスファターゼの阻害剤として知られていることから、最近見い出されたチロシンキナーゼ阻害剤、herbimycin A,genistern,ST638を用いた解析を行った。その結果、genistern,ST638はDIF-Iの誘導剤マイトマイシンCと相乗的にフレンド細胞の分化を引き起こし、細胞内にはDIF-IIIが誘導されていた。又、herbimycin Aは単独でフレンド細胞の分化を誘導した。又これらの物質はフレンド細胞のみならず、胚性腫瘍細胞(F9)の分化も誘導した。これらの事実から脱癌-再分化のプロセスには蛋白質のチロシン残基の脱燐酸化が関与していると云う仮説を提唱したい。, kaken

  対象リソースIRI

Ⅲ．社会連携活動実績

１．公的団体の委員等（審議会、国家試験委員、他大学評価委員，科研費審査委員等）

• Jul. 2006, Jul. 2007, Society
• Jun. 2007, Society
• Apr. 2000, Society