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

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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.