Studies on new proteins involved in vascular endothelial injury

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。多种内质网应激诱导剂如衣霉素、A23187和thapsigargin可使其mRNA水平升高。细胞因子对RTP基因的表达影响很小或没有影响。用重组RTP抗血清进行Western印迹分析表明，在未处理的HUVEC中检测到RTP(47 KDa)的蛋白水平，而在同型半胱氨酸或衣霉素处理的细胞中检测到RTP蛋白水平的增加。研究还表明，RTP被部分磷酸化，并含有7个或更多的磷酸化位点。Forsklin处理可促进RTP的磷酸化，而蛋白激酶A抑制剂则抑制RTP的磷酸化。此外，蛋白激酶A在体外可直接磷酸化重组RTP。在增殖的细胞中，磷酸化形式丰富，并且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。免疫组织化学结果显示，RTP在小鼠肾近端小管和肠绒毛中大量表达。目前的研究提出，高同型半胱氨酸血症患者的细胞功能可能通过改变RTP的表达水平及其磷酸化状态而改变。

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.