Regulation of growth factor activity by Kunitz-type serine proteinase inhibitors

肝细胞生长因子（HGF）是一种多功能生长因子，具有促细胞分裂、促细胞形态形成和促细胞运动等作用。HGF由基质细胞分泌为非活性前体形式，并被HGF激活剂（HGFA）特异性激活为活性形式。HGFA的活性受两种最近鉴定的特异性抑制剂调节，即HGF激活物抑制剂1型（HAI-I）和2型（HAI-2），这两种抑制剂都具有两个Kunitz型丝氨酸蛋白酶抑制剂结构域。虽然HGF/SF的激活是由Met受体酪氨酸激酶介导的HGF/SF诱导的信号通路中的关键限制步骤，但是关于HGF在各种组织中的激活的调节知之甚少。在这项研究中，我们产生了这些HGF调节分子的敲除小鼠，并检查它们在体内的功能。首先，我们从小鼠BAC文库中克隆了编码小鼠HGFA、HAI-1和HAI-2的基因，并对其进行了鉴定，然后通过正/负选择插入法构建了靶向载体。 ...更多信息 n个标记。电穿孔法将靶向载体转染ES细胞，筛选阳性克隆并注射入C57 BL/6小鼠囊胚内。获得了一些嵌合小鼠并进行交配以产生杂合突变小鼠。现在，我们有一些杂合子小鼠，它们正在交配以获得纯合子突变小鼠。我们还报告了其他一些相关研究结果如下：1）HAI-1而非HAI-2的上调沿着肠粘膜的再生，2）具有第一Kunitz结构域的HAI-2是小鼠而非人中的主要产物，3）结肠直肠癌中HAI-1和HGFA的上调，4）HAI-1而不是HAI-2不仅是活性HGFA的抑制剂，而且是活性HGFA的特异性细胞表面结合蛋白，在细胞表面上充当该酶的储库。这些结果表明，HAI-1是再生上皮细胞中活化的HGFA的细胞表面受体，在细胞表面上起作用以将活性HGFA定位在将要进入修复过程的细胞上。高浓度的HGFA活性可确保损伤胃肠道粘膜中HGF/SF的有效细胞周活化，促进胃肠道上皮细胞的增殖和迁移。少

Hepatocyte growth factor (HGF) is a multifunctinal growth factor that acts as mitogen, morphogen, and motogen. HGF is secreted by stromal cells as an inactive precursor form and is specifically activated by HGF activator (HGFA) to the active form. Activity of HGFA is regulated by two recently identified specific inhibitors, namely HGF activator inhibitor type 1 (HAI-I) and type 2 (HAI-2), both of which have two Kunitz-type serine proteinase inhibitor domains. Although the activation of HGF/SF is a critical limiting step in HGF/SF-induced signaling pathway mediated by Met receptor tyrosine kinase, little is known concerning the regulation of HGF activation in various tissues. In this study, we generated the knock-out mice of these HGF regulatory molecules and examine their function in vivo. First, we cloned characterized the genes coding for mouse counter parts of HGFA, HAI-1 and HAI-2 from mouse BAC library, and then made the targeting vectors by insertion of positive/negative selectio … More n markers. Targeting vecters were transfected into ES cells by electroporation and positive clones were selected and injected into blastcysts from C57BL/6 female mice. Some chimeric mice were obtained and mated for generating heterozygous mutant mice. Now, we have some heterozygous mice and they are mating to obtain homo zygous mutant mice. We also reported some other results of relevant studies as follows ; 1) Upregulation of HAI-1 but not HAI-2 along with regeneration of intestinal mucosa, 2) HAI-2 taking the first Kunitz domain is a major product in mouse but not in human, 3) upregulation of HAI-1 and HGFA in colorectal carcinomas, 4) HAI-1 but not HAI-2 is not only an inhibitor but also a specific cell surface binding protein of active HGFA acting a reservoir of this enzyme on the cell surface. These results suggest that HAI-1 is a cell surface acceptor of activated HGFA in regenerative epithelial cells, functioning on the cellular surface to localize the active HGFA on the cells which are going to enter the repair process. The concentrated HGFA activity would ensure the efficient pericellular activation of HGF/SF in the damaged gastrointestinal mucosa and promote proliferation and migration of gastrointestinal epithelial cells. Less