Studies on genes involved in thrombois on endothelial cells

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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. In the present study, we evaluated changes of gene expression induced by homocysteine treatment of cultured human umbilical vein endothelial cells. We identified six up-regulated and one down-regulated gene. One up-regulated gene was GRP78, a stress protein, suggesting that unfolded proteins would accumulate in the endoplasmic reticulum (ER) because of redox potential changes caused by homocysteine. We isolated cDNA clones of two novel up-regulated genes, RTP (reducing agents and tumicamycin-responsive protein) and Herp (homocysteine inducible ER resident protein). RTP consisted of 394 amino acids. RTP mRNA was induced by not only homocysteine but also 2-mercaptoethanol and tumicamycin. We raised the polyclonal antibody against the recombinant RTP.Immunological analysis revealed that it was a cytosolic protein and was present in the proximal tubule of renal cortex and the microvilli of intestine.RTP was a phosphorylated protein with 8 phosphorylation sites. Herp consisted of 391 amino acids. It was estimated to have a transmembrane domain. Immunological analysis revealed that it was an ER-resident protein. Herp mRNA also induced by reducing agents and tumicamycin. Yeast two-hybrid system revealed that Herp can bind with a novel protein, Herp interacting protein (HIP). HIP was 236 amino acids. HIP showed a sequence homology against an ER-resident protein, neuroendocrine specific protein. Therefore, we speculated that Herp is present in the ER with a complex with an ERresident protein HIP.