Functional characterization of HGF activator inhibitors which can mediate the regulation of apoptotic pathway via Fas.

• Project/Area Number: 15570112
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Functional biochemistry
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: DENDA Kimitoshi Tokyo Inst.Technol., Dept.Bioloicacl Sciences, Assistant Prof., 大学院・生命理工学研究科, 助手 (50212064)
• Co-Investigator(Kenkyū-buntansha): SHIMOMURA Takeshi Mitsubishi Pharma Corp., Discovery Technology Laboratory, Senior Researcher, 創薬基盤研究所, 主任研究員
• Project Period (FY): 2003 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥3,300,000 (Direct Cost: ¥3,300,000); Fiscal Year 2005: ¥800,000 (Direct Cost: ¥800,000); Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000); Fiscal Year 2003: ¥1,600,000 (Direct Cost: ¥1,600,000)
• Keywords: hepatocyte growth factor (HGF) / HGF activator / serine protease / blood coagulation / apoptosis / Kunitz domain / セリンプロテアーゼカスケード

Research Abstract

Hepatocyte growth factor activator inhibitors type 1 (HAI-1) and type 2 (HAI-2) are Kunitz-type serine protease inhibitors, identified as potent inhibitors toward hepatocyte growth factor (HGF) activator. To clarify the physiological role of these regulatory factors on cellular apoptosis, we have performed the functional analyses of HAI-1 and HAI-2.
1. To investigate the regulatory role of HAIs on cell proliferation, we examined inhibitory spectrum on individual functional domain in HAI molecules. Kunitz I in each HAI molecule appears to be the functional domain for inhibiting the HGF-converting activity of HGF activator. Furthermore, the presence of two Kunitz domains affected the inhibitory activity of HAI-1 against HGF activator. Thus serine protease binding sites of Kunitz I and Kunitz II are located close to each other, and proteolytic processing to generate HAI-1 with only one Kunitz domain regulates the activity of HAI-1. Identification of cognated serine proteases against Kunitz II will reveal the novel biological function of HAIs.
2. In order to identify HAI secretases, we examined protein-protein interaction in the cytoplasmic regions of HAIs using a yeast two-hybrid screening system. Several cytoplasmic proteins, including cytosleletal proteins and intracelluiar enzymes were obtained. One of them was known to be involved in suppression on apoptosis, suggesting that HAI may play important role on the regulation of Fas-mediated death signaling. Analyses of this cellular event is now in progress.
3. To clarify the physiological role of HAI molecules in male germ cells and tissue, we assessed these expression patterns in testicular tissues during sperm formation. We prepared the specific monoclonal antibodies of HAIs. Immunohistochemical analysis reveals that HAI-1 labeled by the monoclonal antibody were localized only in earlier stages in the three developmental phases, suggesting that HAI-1 appears in the morphologic change phase from spermatogonium to round spermatid, whereas HAI-2 was localized along almost entire surface of the cells in the tissues. Similarly, HGF activator was expressed extensively in the seminiferous tubule. HAI-1 and HAI-2 in testis might thus play an important role in the different stages of cellular differentiation, morphologic change or migration during spermatogenesis.
Further analyses of the functional regulation by HAIs in the process of cellular growth will provide a novel mechanism of the regulation of Fas-meidated apoptosis.

Research Products

• [Journal Article] Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is required for branching morphogenesis in the chorioallantoic placenta. (2005)
  • Author(s): Tanaka, H., et al.
  • Journal Title: Mol. Cell. Biol 13 Pages: 5687-5698
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is required for branching morphogenesis in the chorioallantoic placenta. (2005)
  • Author(s): Tanaka H, Nagaike K, Takeda N, Itoh H, Kohama K, Fukushima T, Miyata S, Uchiyaam S, Uchinokura S, Shimomura T, Miyazawa K, Kitamura N, Yamada G, Kataoka H.
  • Journal Title: Mol.Cell.Biol. 13 Pages: 5687-5698
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is required for branching morphogenesis in the chorioallantoic placenta. (2004)
  • Author(s): Tanaka H., et al.
  • Journal Title: Gastroenterology 127 Pages: 1423-1435
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Regeneration of injured intestinal mucosa is impaired in hepatocyte growth factor activator-deficient mice. (2004)
  • Author(s): Itoh H, Naganuma S, Takeda N, Miyata S, Uchinokura S, Fukushima T, Uchiyama S, Tanaka H, Nagaike K, Shimomura T, Miyazawa K, Yamada G, Kitamura N, Koono M, Kataoka H.
  • Journal Title: Gastroenterology 127 Pages: 1423-1435
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Regeneration of injured intestinal mucosa is impaired in hepatocyte growth factor activator-deficient mice. (2004)
  • Author(s): Itoh, H. et al.
  • Journal Title: Gastroenterology 127 Pages: 1423-1435