Studies on new proteins involved in vascular endothelial injury

参与血管内皮损伤的新蛋白研究

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680607/
• 关键词: homocysteine; arteriosclerosis; thrombosis; vascular disease; endothelial cells; phosphorylation; hyperhomocysteinemia; kidney; ホモシステイン; 小胞体; 内皮傷害; 分子シャペロン; 血栓症; 高ホモシステイン血症; 動脈硬化症; RTP; homocysteine; arteriosclerosis; thrombosis; vascular disease; endothelial cells; phosphorylation; hyperhomocysteinemia; kidney; ホモシステイン; 小胞体; 内皮傷害; 分子シャペロン; 血栓症; 高ホモシステイン血症; 動脈硬化症; RTP

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粥样硬化和血栓形成有关。尚未阐明同型半胱氨酸可能促进血管疾病的机制。我们在人脐静脉内皮细胞（HUVEC）中鉴定了一种新型的同型半胱氨酸反应蛋白RTP。多种内质网应力诱导剂，例如衣霉素，A23187和Thapsigargin增加了它的mRNA。细胞因子对RTP mRNA表达几乎没有影响。使用重组RTP抗血清的蛋白质印迹分析表明，在未处理的HUVEC中可以检测到RTP的蛋白质水平（47 kDa），并在同型半胱氨酸或女霉素处理的细胞中增加。还证明RTP被部分磷酸化并包含七个或更多的磷酸化位点。 RTP的磷酸化通过Forskolin治疗增强，并被蛋白激酶A抑制剂抑制。此外，蛋白激酶在体外直接磷酸化的重组RTP。磷酸化形式在增殖细胞中丰富，并且与细胞密度增加有关。免疫细胞化学分析表明，RTP主要位于培养细胞的细胞质中。小鼠组织的免疫组织化学表明，RTP大量存在于肾脏近端小管中，在肠道的绒毛中。本研究提高了高脑结膜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。