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Studies on genes involved in endothelial dysfunction involved in thrombosis and atherosclerosis

血栓形成和动脉粥样硬化内皮功能障碍相关基因的研究

基本信息

批准号：
10044337
负责人：
MIYATA Toshiyuki
金额：
$ 4.16万
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 (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
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 previously isolated two novel cDNA clones, RTP and Herp, both of which have been up-regulated by homocysteine. In the present study, we raised polyclonal anti-serum against recombinant proteins. Western blot analysis showed that RTP was mainly located in the cytoplasm. RTP was partially phosphorylated at seven or more sites. Its phosphorylation was enhanced by the forskolin treatment and inhibited by a protein kinase A inhibitor and a calmodulin kinase inhibitor. Protein kinase A directly phosphorylated recombinant RTP in vitro. The phosphorylated forms were abundant in the cells at the early log phase and then decreased in relation to increased cell density. Immunohistochemistry of mouse tissues demonstrated that RTP was present abundantly in proximal tubule of kidney and in villi of intestine. These data demonstrated that RTP was present abundantly in proximal tubule of kidney and in villi of intestine. These data demonstrated that RTP is the cytoplasmic phosphoprotein and its phosphorylation may be related to cell growth. On the other hand, Western blot analysis of Herp revealed that it was an endoplasmic reticulum (ER)-associated 54-kDa protein, and was strongly induced by perturbation that lead to accumulation of unfolded proteins in the ER. Interestingly, both the N and C termini of Herp were present on the cytoplasmic side of the ER. The human Herp gene consisted of 8 exons spanning 12 kb and localized to chromosome 16q12.2-13. In its promoter region, there was a single 19-bp sequence corresponding to the ER-stress responsive cis-acting element, ERSE. Herp may play an unknown role in the cellular survival response to stress.

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。我们以前分离出两个新的cDNA克隆，RTP和Herp，这两个都已上调同型半胱氨酸。在本研究中，我们提出了针对重组蛋白的多克隆抗血清。Western blot分析显示RTP主要定位于细胞质中。RTP在7个或更多位点被部分磷酸化。它的磷酸化增强毛喉素治疗和抑制蛋白激酶A抑制剂和钙调蛋白激酶抑制剂。蛋白激酶A在体外直接磷酸化重组RTP。磷酸化形式在对数生长早期的细胞中是丰富的，然后随着细胞密度的增加而减少。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。这些结果表明，RTP在肾脏近端小管和小肠绒毛中有丰富的表达。这些数据表明RTP是胞质磷蛋白，其磷酸化可能与细胞生长有关。另一方面，Herp的Western印迹分析显示，它是一种内质网（ER）相关的54-kDa蛋白，并强烈诱导的扰动，导致未折叠的蛋白质在ER中的积累。有趣的是，Herp的N和C末端都存在于ER的胞质侧。人类Herp基因由8个外显子组成，跨越12 kb，定位于染色体16q12.2-13。在其启动子区，有一个19 bp的序列，对应于ER应激反应顺式作用元件ERSE。Herp可能在细胞对应激的生存反应中起着未知的作用。