Roles of integral-membrane serine proteinase inhibitors in proliferation, migration and invasion of epithelial cells

整合膜丝氨酸蛋白酶抑制剂在上皮细胞增殖、迁移和侵袭中的作用

基本信息

批准号：
12670209
负责人：
KATAOKA Hiroaki
金额：
$ 1.34万
依托单位：
Miyazaki Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Cell surface proteolysis is an important mechanism for the generation of biologically active proteins that mediates a diverse range of cellular functions. Therefore the interactions between proteinases and cellular surface inhibitors must have important regulatory roles in cellular functions. There is a unique class of SPIs that is synthesized as a transmembrane glycoprotein. These SPIs have one or two extracellular Kunitz-type serine proteinase inhibitor domains, a presumed transmembrane domain near the carboxyl-terminus and a short intracytoplasmic domain. This unique group of SPIs consists of 4 distinct proteins at present. These are APP, APP-like protein 2 (APLP2), HAI-1 and HAI-2. In this project, the functions of these membrane-type inhibitors were extensively analyzed. Following are the results of the project (April, 2000 - March, 2002).(1) Roles of HAI-1 in the regulation of pericellular activation of HGFActivation of hepatocyte growth factor/scatter factor (HGF/SF) is a critic … More al limiting step in the HGF/SF-induced signaling pathway mediated by MET receptor tyrosine kinase. Although HGF/SF-MET signaling could have potentially important roles in the tissue regeneration and tumor progression, little is known about the regulation of HGF/SF activation. In this project, we have shown that HGF activator (HGFA) a recently identified factor XII-like serine proteinase, is critically involved in this process. Furthermore, we also showed that HGF activator inhibitor type 1 (HAI-1) should have an important regulatory role in the pericellular activation of HGF/SF having diverse roles acting as a cell surface specific inhibitor of active HGFA and a reservoir of this enzyme on the cell surface. The latter property might paradoxically ensure the concentrated pericellular HGFA activity in certain cellular conditions in which shedding of HAI-1/HGFA complex from the plasma membrane is upregulated.(2) APP is involved in the growth of colon carcinoma cells in vitro and in vivoIn a panel of human colon carcinoma cell lines, sAPP/PN-II was a major SPI secreted by all the cell lines examined. In this project we found that the downregulation of APP mRNA by an antisense mRNA strategy resulted in significantly reduced cellular proliferation of a colon carcinoma cell line in vitro, indicating that APP is somehow involved in the regulation of cellular proliferation. Of importance was the observation that reduced cellular growth was observed not only in vitro but also in vivo when the antisense colon carcinoma clones were transplanted into nude mice.(3) Generation of HGFA knock-out mouse and HAI-1 knock-out mouse.(4) Identification of a novel nuclear localization signal protein namely HAI-2-related small peptide (H2RSP). Less

细胞表面蛋白水解是产生介导多种细胞功能的生物活性蛋白的重要机制，因此蛋白酶和细胞表面抑制剂之间的相互作用必定在细胞功能中具有重要的调节作用。这些 SPI 具有一个或两个胞外 Kunitz 型丝氨酸蛋白酶抑制剂结构域、一个靠近羧基末端的假定跨膜结构域和一个短结构域。目前，这组独特的 SPI 由 4 种不同的蛋白组成，它们是 APP、APP 样蛋白 2 (APLP2)、HAI-1 和 HAI-2。以下是该项目的结果（2000年4月-2002年3月）。（1）HAI-1在调节HGF激活的细胞周激活中的作用肝细胞生长因子/分散因子 (HGF/SF) 是 MET 受体酪氨酸激酶介导的 HGF/SF 诱导信号通路中的一个关键限制步骤，尽管 HGF/SF-MET 信号传导可能在该过程中发挥潜在的重要作用。在组织再生和肿瘤进展中，人们对 HGF/SF 激活的调节知之甚少。在这个项目中，我们发现 HGF 激活剂 (HGFA) 是一种最近发现的 XII 因子样丝氨酸蛋白酶，在这一过程中发挥着重要作用。此外，我们还表明，HGF 激活剂抑制剂 1 型 (HAI-1) 在 HGF/SF 的细胞周激活中应具有重要的调节作用，具有多种作用，作为活性 HGFA 的细胞表面特异性抑制剂和该酶的储存库。后一种特性可能矛盾地确保了在某些细胞条件下细胞周 HGFA 活性的集中，在这种条件下，HAI-1/HGFA 复合物从质膜上的脱落被上调。(2) APP 参与结肠癌的生长。在一组人结肠癌细胞系中，sAPP/PN-II 是所有检查的细胞系分泌的主要 SPI。在这个项目中，我们发现反义 mRNA 策略导致 APP mRNA 的下调。体外结肠癌细胞系的细胞增殖显着降低，表明APP在某种程度上参与了细胞增殖的调节，重要的是，当反义结肠癌在体外和体内均观察到细胞生长降低。克隆被移植到(3)HGFA敲除小鼠和HAI-1敲除小鼠的产生。(4)新型核定位信号蛋白HAI-2相关小肽(H2RSP)的鉴定。