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，具有丝裂原、形态原和运动原的作用。HGF是由基质细胞作为非活性前体形式分泌的，并被HGF激活剂（HGFA）特异性激活为活性形式。HGF的活性受最近发现的两种特异性抑制剂的调控，即HGF激活因子抑制剂1型（HAI-I）和2型（HAI-2），它们都有两个kunitz型丝氨酸蛋白酶抑制剂结构域。尽管在Met受体酪氨酸激酶介导的HGF/SF诱导的信号通路中，HGF/SF的激活是一个关键的限制步骤，但关于HGF在各组织中的激活调控却知之甚少。在本研究中，我们构建了这些HGF调节分子的敲除小鼠，并在体内检测了它们的功能。首先，我们从小鼠BAC文库中克隆鉴定了小鼠HGFA、HAI-1和HAI-2对应部分的编码基因，然后通过插入阳性/阴性选择制备了靶向载体。通过电穿孔将靶向载体转染到胚胎干细胞中，选择阳性克隆注入C57BL/6雌性小鼠囊胚中。获得了一些嵌合小鼠并进行了交配，产生了杂合突变小鼠。现在，我们有一些杂合子老鼠，它们交配得到同合子突变老鼠。我们还报道了其他一些相关研究结果如下：1)随着肠粘膜的再生，HAI-1上调，而HAI-2不上调；2)HAI-2以第一个Kunitz结构域为主要产物，在小鼠中上调，而在人中不上调；3)在结直肠癌中，HAI-1和HGFA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在细胞表面的特异性细胞表面结合蛋白，是该酶的储存库。这些结果表明，在再生上皮细胞中，HAI-1是活化的HGFA的细胞表面受体，在细胞表面发挥作用，将活化的HGFA定位到即将进入修复过程的细胞上。高浓度的HGF - fa活性可保证受损胃肠道黏膜中HGF/SF的高效细胞周活化，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