Analysis of gene expression in homocysteine-injured vascular endothelial cells: Demonstration of GRP78/BiP expression, cloning and characterization of a novel reducing agent tunicamycin regulated gene

An elevated plasma level of homocysteine is associated with arteriosclerosi s and thrombosis. The mechanisms by which homocysteine may promote vascular diseases have not yet been elucidated. In the present study, we have appli ed a modified nonradioactive differential display analysis to evaluate chan ges in gene expression induced by homocysteine treatment of cultured human umbilical vein endothelial cells (HUVEC). We identified six upregulated and one downregulated gene. One upregulated gene was GRP78/BiP, a stress prote in, suggesting that unfolded proteins would accumulate in the endoplasmic r eticulum because of redox potential changes caused by homocysteine. Another upregulated gene encoded a bifunctional enzyme with activities of methylen etetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase , which is involved in a homocysteine metabolism. A third upregulated gene encoded activating transcription factor 4. The remaining four were uncharac terized genes. We isolated a full-length cDNA of one of the upregulated gen es from a HUVEC library. It encoded a novel protein with 394 amino acids, w hich was termed reducing-agents and tunicamycin-responsive protein (RTP). N orthern blot analysis revealed that RTP gene expression was induced in HUVE C after 4 h of incubation with homocysteine. RTP mRNA was also observed in unstimulated cells and induced by not only homocysteine but also 2-mercapto ethanol and tunicamycin. The mRNA was ubiquitously expressed in human tissu es. These observations indicate that homocysteine can alter the expressivit y of multiple genes, including a stress protein and several novel genes. Th ese responses may contribute to atherogenesis.