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Studies on new proteins involved in vascular endothelial injury

参与血管内皮损伤的新蛋白研究

基本信息

批准号：
09680607
负责人：
MIYATA Toshiyuki
金额：
$ 1.86万
依托单位：
National Cardiovascular Center Research Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

An elevated level of homocysteine is associated with arteriosclerosis and thrombosis. The mechanisms by which homocysteine may promote vascular diseases have not been elucidated yet. We identified a novel homocysteine responsive protein, RTP, in human umbilical vein endothelial cells (HUVEC). Its mRNA was increased by a variety of endoplasmic reticulum-stress inducing agents such as tunicamycin, A23187, and thapsigargin. Cytokines had little or no effect on RTP mRNA expression. Western blot analysis using recombinant RTP antiserum showed that protein level of RTP (47 kDa) was detectable in untreated HUVEC and increased in homocysteine- or tunicamycin-treated cells. It was also demonstrated that RTP was partially phosphorylated and contained seven or more phosphorylation sites. Phosphorylation of RTP was enhanced by the forskolin treatment and inhibited by a protein kinase A inhibitor. Furthermore, protein kinase A directly phosphorylated recombinant RTP in vitro. The phosphorylated forms were abundant in the proliferating cells and decreased in relation to increased cell density. Immunocytochemical analysis indicated that RTP was mainly located in the cytoplasm of cultured cells. Immunohistochemistry of mouse tissues demonstrated that RTP was present abundantly in proximal tubule of kidney and in villi of intestine. The present study raises the possibility that cellular functions in patients with hyperhomocysteinemia might be altered through changes in the RTP expression level and its phosphorylation state.

同型半胱氨酸水平升高与动脉硬化和血栓形成有关。同型半胱氨酸可能促进血管疾病的机制尚未阐明。我们在人脐静脉内皮细胞（HUVEC）中发现了一种新的同型半胱氨酸反应蛋白RTP。它的mRNA增加了各种内质网应激诱导剂，如衣霉素，A23187，和毒胡萝卜素。细胞因子对RTP mRNA表达的影响很小或没有影响。使用重组RTP抗血清的蛋白质印迹分析表明，RTP（47 kDa）的蛋白质水平在未处理的HUVEC中是可检测的，并在同型半胱氨酸或衣霉素处理的细胞中增加。RTP被部分磷酸化，含有7个或更多的磷酸化位点。毛喉素处理增强RTP的磷酸化，并抑制蛋白激酶A抑制剂。此外，蛋白激酶A直接磷酸化重组RTP在体外。磷酸化形式在增殖细胞中丰富，并随着细胞密度的增加而减少。免疫细胞化学分析表明，RTP主要定位于培养细胞的胞浆中。免疫组化结果显示，RTP在小鼠肾脏近端小管和小肠绒毛中表达丰富。本研究提出了高同型半胱氨酸血症患者的细胞功能可能通过RTP表达水平及其磷酸化状态的变化而改变的可能性。