上皮・間葉相互作用によるがん悪性化の分子機構とその制御

2000 Fiscal Year Annual Research Report

• Project/Area Number: 12215082
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas (C)
• Research Institution: Osaka University
• Principal Investigator: 中村 敏一 大阪大学, 医学系研究科, 教授 (00049397)
• Co-Investigator(Kenkyū-buntansha): 小清水 右一 大阪大学, 医学系研究科, 助手 (50281126) ; 船越 洋 大阪大学, 医学系研究科, 助手 (40273685) ; 松本 邦夫 大阪大学, 医学系研究科, 助教授 (90201780)
• Keywords: HGF / NK4 / HGFアンタゴニスト / 血管新生 / 浸潤・転移 / クリングルドメイン / LIMキナーゼ / 細胞骨格

Research Abstract

1)NK4による血管新生抑制機構の解明
NK4はN末端ヘアピンドメインと4個のクリングルドメインをもつHGFの分子内断片である。NK4の分子内N末端ヘアピンドメインがc-Metレセプターに結合する上で必須のドメインであり、このドメインを欠く4個のクリングルドメイン(K1-4)はHGFアンタゴニスト活性を失うことが予想された。そこでリコンビナントNK4ならびにK1-4を調製し、両分子のHGFアンタゴニスト活性ならびに血管新生阻害活性を調べた。その結果、NK4はHGFによる肝細胞の増殖促進活性を阻害し、HGFアンタゴニスト活性を示したが、K1-4はHGFアンタゴニスト活性を失っていた。一方、培養ヒト血管内皮細胞に対するNK4ならびにK1-4の作用を調べた結果、K1-4はNK4と同等にbFGF、VEGF、HGFによって促進されるヒト血管内皮細胞の増殖ならびに遊走を阻害した。したがってNK4のHGFアンタゴニスト活性は血管新生阻害作用に関与せず、血管新生阻害作用には分子内クリングルドメインが関与することが明らかになった。
2)LIMキナーゼの活性調節機構および生体機能の解析
LIMキナーゼ(LIMK)はコフィリンを特異的にリン酸化するアクチン細胞骨格の制御因子である。私達はこれまでにLIMK1、LIMK2が異なるRhoファミリー分子の制御下で活性化されること、すなわちLIMK1はRacにより、一方、LIMK2はRhoおよびCdc42により活性化されることを報告しているが、さらに今回、LIMK2はRho依存性キナーゼであるROCKによるリン酸化を受け、活性化されることを明らかにした。またXenopus卵において、LIMKは細胞(減数)分裂の際に重要な役割を担っている微小管形成中心の形成に関与していること、さらにLIMK2ノックアウトマウスではその精子形成に異常が認められることを見い出した。

Research Products

• [Publications] S.Hiscox: "Inhibition of HGF/SF-induced breast cancer cell matility and invasion by the HGF/SF variant, NK4."Breast Cancer Res.& Treat.. 59. 245-254 (2000)
• [Publications] M.Nakano: "Hepatocyte growth factor is essential for amelioration of hyperglycemia in strepto zotocin-induced diabetic mice receiving a marginal mass of intrahepnic islet grafts."Transplantation. 69. 214-221 (2000)
• [Publications] S.Mizuno: "Reciprocal balance of hepatocyte growth factor and transforming growth factor-β1 in renal fibrosis in mice"Kidney Intern.. 57. 937-948 (2000)
• [Publications] K.Beppu: "Elevation of serum hepatocyte growth factor concentration in patients with gastric cancer is mediated by production by tumor tissue."Anticancer Res.. 20. 1263-1268 (2000)
• [Publications] M.Aoki: "Angiogenesis induced by hepatocyte growth factor in non-infarcted myocardium and infarcted myocardium : up-regalation of essential transcription factor for angiogenesis, ets."Gene Therapy. 7. 417-427 (2000)
• [Publications] T.Yamada: "Enhanced expression of hepatocyte growth factor by pulmonary ischemia reperfusion injury in the rat."Am.J.Resp.Crit Care Med.. 162. 707-715 (2000)
• [Publications] Y.Taniyama: "Potential contribution of a novel anti-fibrotic factor, hepatocyte growth factor, to prevention of myocardial fibrosis by angiotensin II blockade in cardiomyopathic hamster."Circulation. 102. 246-252 (2000)
• [Publications] T.Kunisada: "Keratinocyte expression of transgenic hepatocyte growth factor (HGF) affects melanocyte development, leading to dermal melanocytosis."Mech.Dev.. 94. 67-78 (2000)
• [Publications] S.Radaeva: "Unique epithelial cell production of hepatocyte growth factor/scatter factor by putative precancerous intestinal metaplasias and associated "intestinal-type" of biliary cancer chemically-induced in rat liver."Hepatology. 31. 1257-1265 (2000)
• [Publications] J.-M.Otte: "Functional expression of HGF and its receptor in human colorectol Cancer."Digestion. 61. 237-246 (2000)
• [Publications] O.Muto: "HGF/SF-induced spreading of MDCK cells corelates with disappearance of barmotin/7H6, a tight junction-associated protein, from the cell membrane."Cell Biol.Intern.. 24. 439-446 (2000)
• [Publications] T.Kikuchi: "Secretory leukoprotease inhibitor augments hepatocyte growth factor production in human lung fibroblasts."Am.J.Respir.Cell Mol.Biol.. 23. 364-370 (2000)
• [Publications] K.Hayashi: "In vivo transfer of human hepatocyte growth factor gene accelerates re-endothelization and inhibition neointimal formation after balloon injury in rat model."Gene Therapy. 7. 1664-1671 (2000)
• [Publications] H.Nakagami: "Anti-apoptotic action of hepatocyte growth factor through mitogen-activated protein kinase on human aortic endothelial cells."J.Hypertension. 18. 1411-1420 (2000)
• [Publications] T.Nakamura: "Myocardial protection from infarction by endogenous and exogenous hepatocyte growth factor."J.Clin.Invest.. 106. 1511-1519 (2000)
• [Publications] K.Kuba: "HGF/NK4, a four-kringle fragment of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice."Cancer Res.. 60. 6737-6743 (2000)
• [Publications] K.Kuba: "Kringle 1-4 of hepatocyte growth factor inhibits proliferation and migration of human microvascular endothelial cells."Biocem.Biophys.Res.Commun.. 279. 846-852 (2000)
• [Publications] W.Sun: "A sensitive quantification method for evaluating the lebel of hepatocyte growth factor and c-met/HGF receptor mRNAs in the nervous system using competitive RT-PCR."Brain Res.Protoc.. 5. 190-7 (2000)
• [Publications] G.H.Lai: "Unique epithelial cell production of hepatocyte growth factor/scatter factor by putative precancerous intestinal metaplasias and associated"intestinal-type"biliary cancer chemically induced in rat liver."Hepatology. 31. 1257-1265 (2000)
• [Publications] G.Shiota: "In vivo transfer of hepatocyte growth factor gene accelerates proliferation of hepatic oval cells in a 2-acetylaminofluorene/partial hepatectomy model in rats."FEBS Lett.. 470. 325-330 (2000)
• [Publications] K.Matsumoto: "Hepatocyte growth factor and met in tumor invasion-metastasis : Mechanisms related to cancer prevention. In "Cancer metastasis, molecular and cellular biology and clinical aspects""Academic Publishers. 143-193 (2000)
• [Publications] K.Matsumoto: "Hepatocyte growth factor in renal regeneration, renal disease, and potential therapeutics."Hypertension. 9. 395-402 (2000)
• [Publications] T.Takahashi: "Functional involvement of LIM-kinase in Xenopus oocyte maturation."Develop.Biol.. 229. 554-567 (2001)
• [Publications] T.Sumi: "Specific activation of LIM-kinase 2 via threonine-505 phosphorylation by ROCK under the control of Rho."J.Biol.Chem.. 276. 670-676 (2001)
• [Publications] F.Tamura: "FK506 promotes liver regeneration by suppressing natural killer cell activity.".Gastroenterol.and Hepatol.. (in press). (2001)
• [Publications] A.Uchiyama: "Significance of locally-secreted hepatocyte growth factor after thoracic cancer surgery as a stimulator for postoperative cancer invasion."Jpn.J.Clin.Oncol.. (in press). (2001)
• [Publications] H.Azuma: "Hepatocyte growth factor prevents development of chronic allograft nephropathy in an experimental rat transplant model."J.Am.Soc.Nephrol.. (in press). (2001)
• [Publications] S.Mizuno: "Hepatocyte growth factor suppresses interstitial fibrosis in a mouse model of obstructive nephropathy."Kidney Intern.. (in press). (2001)
• [Publications] K.Nakamura: "Molecular cloning and functional characterization of human scavenger receptor with c-type lectin (SRCL), a novel member of scavenger receptor family."Biochem.Biophys.Res.Commun.. (in press). (2001)
• [Publications] N.Maehara: "NK4, a four-kringle antagonist of HGF, inhibits spreading and invasion of human pancreatic cancer cells."Br.J.Cancer. (in press). (2001)
• [Publications] T.Nakamura: "Molecular cloning and characterization of Kremen, a novel Kringle-containing transmembrane protein."Biochem.Biophys.Acta. (in press). (2001)
• [Publications] N.Tsuzuki: "Hepatocyte growth fector reduces the infarct volume after transient focal cerebral ischemia."Neurol.Res.. (in press). (2001)
• [Publications] K.Matsumoto: "Hepatocyte growth factor : renotropic role and potential therapeutics for renal diseases."Kidney Intern.. (in press). (2001)
• [Publications] K.Matsumoto: "Cancer Metastasis: Molecular & Cellular Mechanisms and Clinical Interventions"Kluwer Academic Publisher. 50 (2000)
• [Publications] K.Matsumoto: "Growth Factors and Their Receptors in Cancer Metastasis"Kluwer Academic Publisher (in press). (2001)