Characterization of factors regulating the activity of hepatocyte growth factor which is being developed for a medicine

正在开发用于药物的肝细胞生长因子活性调节因子的表征

• 批准号: 10557017
• 负责人: KITAMURA Naomi
• 金额: $ 7.49万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557017/
• 关键词: hepatocyte growth factor (HGF); liver disease; HGF activator; HGF activator inhibitor; liver injury; protein processing; thrombin; HGFactivator; HGFactivator inhibitor; 血液凝固系

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes, and is being developed for a medicine of liver disease. HGF is activated in the liver by a serine protease, HGF activator (HGFA), following liver injury, and then functions as a liver regeneration factor. In this study, we examined mechanisms for activation and inactivation of HGFA and obtained the following results.1. Analysis of HGFA and its mRNA after liver injury induced by CClィイD24ィエD2 treatment of rat revealed that amount of HGFA mRNA increased, and HGFA, that circulates in the blood as an inactive precursor at normal state, was activated by thrombin following liver injury. These results indicate that HGFA plays an crucial role in regulating the activity of HGF by increase of its amount and proteolytic activation linked to blood coagulation system following liver injury.2. Two types of HGFA inhibitor, HAI-1 and HAI-2, were identified as potent inhibitors of HGFA. Using a monoclonal antibody against HAI-1, we identified a transmembrane form of HA1-1 integrated in the plasma membrane of cultured cells. We also identified several soluble forms of HAI-1 in the conditioned medium of the cells, indicating that multiple sites are present in the transmembrane form of HAI-1 at which proteolytic cleavage releases the extracellular domain. At least two proteases, one of which is a metalloprotease, appear to be responsible for the release. Further, the soluble forms of HAI-1 have different inhibitory activity against HGFA. These findings suggest that proteolytic processing plays important roles in regulation of the inhibitory activity of Hal-1.

肝细胞生长因子（HGF）是一种有效的肝细胞有丝分裂原，目前正被开发用于治疗肝脏疾病。肝损伤后，肝脏中的HGF被丝氨酸蛋白酶激活，HGF激活剂（HGFA），然后作为肝脏再生因子发挥作用。在本研究中，我们研究了HGFA的激活和失活机制，得到了以下结果：对CCl γ γ γ γ - D24 γ γ - D2诱导大鼠肝损伤后HGFA及其mRNA的分析表明，HGFA mRNA数量增加，正常状态下以无活性前体在血液中循环，肝损伤后HGFA被凝血酶激活。这些结果表明，肝损伤后，HGF通过增加HGF的数量和与凝血系统相关的蛋白水解激活，对HGF的活性起着至关重要的调节作用。两种类型的HGFA抑制剂HAI-1和HAI-2被鉴定为HGFA的有效抑制剂。利用一种针对HAI-1的单克隆抗体，我们在培养细胞的质膜中发现了一种跨膜形式的HA1-1。我们还在细胞条件培养基中发现了几种可溶形式的HAI-1，表明在跨膜形式的HAI-1中存在多个位点，在这些位点上蛋白水解裂解释放细胞外结构域。至少有两种蛋白酶，其中一种是金属蛋白酶，似乎与释放有关。此外，可溶形式的HAI-1对HGFA具有不同的抑制活性。这些发现表明，蛋白质水解过程在调节Hal-1的抑制活性中起重要作用。

项目成果

T. Shimomura: "Multiple sites of proteolytic cleavage to release soluble forms of hepatocyte growth factor activator inhibitor type 1 from a transmembrane form"J. Biochem.. 126. 821-828 (1999)

T. Shimomura：“多位点蛋白水解裂解从跨膜形式释放可溶形式的肝细胞生长因子激活剂抑制剂 1 型”J。

Y.Matsubara: "Hepatocyte growth factor activator ; a possible regulator of morphogenesis during the fetal development of rat gastrointestinal tract." Biochem.Biophys.Res.Commun.253. 477-484 (1998)

Y.Matsubara：“肝细胞生长因子激活剂；大鼠胃肠道胎儿发育过程中形态发生的可能调节剂。”

. Qin et al.: "Functional characterization of Kunitz domains in hepatocyte growth factor activator inhibitor type 2"FEBS Letters. 436. 111-114 (1998)

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H.Itoh: "Hepatocyte growth factor activator inhibitor type 2 lacking the first Kunitz-type serine protease inhibitor domain is a predominant product in mouse but not in human"Biochem.Biophys.Res.Commun.. 255. 740-748 (1999)

H.Itoh：“缺乏第一个 Kunitz 型丝氨酸蛋白酶抑制剂结构域的 2 型肝细胞生长因子激活剂抑制剂是小鼠中的主要产物，但在人类中则不然”Biochem.Biophys.Res.Commun.. 255. 740-748 (1999)

H. Itoh: "Up-regulation of hepatocyte growth factor activtor inhibitor type-1 but not type-2 along with regeneration of intestinal mucosa"Am. J. Physiol.. (in press). (2000)

H. Itoh：“随着肠粘膜的再生，1 型肝细胞生长因子激活剂抑制剂上调，但 2 型不上调”Am。