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

肝细胞生长因子（HGF）是一种促进肝脏再生的有效因子，目前正在开发用于治疗肝脏疾病的药物。HGF的活性受细胞外因子和细胞内因子的调控。在本研究中，我们分析了这些因子在调节HGF活性中的作用，得到了以下结果。(1) HGF激活剂（HGF activator， HGFA）是一种参与肝损伤后HGF激活的丝氨酸蛋白酶。肝损伤后HGFA分析显示，肝损伤后HGFA mRNA数量增加，HGFA被激活。(2)两种类型的HGFA抑制剂（HAIs）被鉴定为HGFA的有效抑制剂。cDNA克隆结果显示，这两种HAIs均为具有Kunitz结构域的丝氨酸蛋白酶抑制剂。Kunitz结构域突变的引入显著降低了HAIs的活性，表明该结构域对HAIs的活性至关重要。免疫印迹分析表明，HAIs最初以跨膜形式产生，然后由产生细胞通过蛋白水解加工分泌。(3) Hrs及其结合蛋白（Hbp）是调节HGF活性的胞内分子。Hrs/Hbp复合物的功能表征表明，该复合物定位于早期内体，并在内化后调节HGF及其受体的降解中发挥重要作用。此外，去泛素化酶UBPY作为Hbp的SH3结构域的结合伙伴被分离出来，这表明UBPY调节Hrs/Hbp复合物的功能。

Hepatocyte growth factor (HGF) is a potent factor responsible for liver regeneration, and is being developed for a medicine of liver disease. The activity of HGF is regulated by extracellular and intracellular factors. In this study, we analyzed the function of these factors in regulating the activity of HGF and obtained the following results.(1) HGF activator (HGFA) was identified as a serine protease responsible for activation of HGF after liver injury. Analysis of HGFA after liver injury revealed that amount of HGFA mRNA increased, and HGFA was activated following liver injury.(2) Two types of HGFA inhibitors (HAIs) were identified as potent inhibitors of HGFA.cDNA cloning revealed that both HAIs are serine protease inhibitors with Kunitz domains. Introduction of mutations in the Kunitz domains markedly reduced the activity of HAIs, indicating that the domains are essential for the activity. Immunoblotting analysis demonstrated that HAIs are initially produced in transmembrane forms, and then secreted by the producing cells by proteolytic processing.(3) Hrs and its binding protein (Hbp) were identified as intracellular molecules regulating the activity of HGF.Functional characterization of Hrs/Hbp complexes revealed that the complexes are localized to early endosomes and play an important role in regulating the degradation of HGF and its receptor after internalization. Further, deubiquitinating enzyme UBPY was isolated as a binding partner of the SH3 domain of Hbp, suggesting that UBPY regulates the function of the Hrs/Hbp complexes.