Nonradioactive differential display cloning of genes induced by homocysteine in vascular endothelial cells

Hyperhomocysteinemia is known to be a risk factor for arteriosclerosis and thrombosis. To elucidate the mechanisms by which homocysteine may promote v ascular diseases. we have applied a modified nonradioactive differential di splay analysis that evaluates changes in gene expression induced by homocys teine treatment of cultured human umbilical vein endothelial cells (HUVECs) . We identified six upregulated and one downregulated gene. One upregulated gene was GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chape rone, suggesting that unfolded proteins would accumulate in the ER because of redox potential changes caused by homocysteine. Another upregulated gene encoded a bifunctional enzyme with activities of methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase, which is involv ed in homocysteine metabolism. A third upregulated gene encoded activating transcription factor 4. Homology searches of the remaining four clones fail ed to retrieve any similar sequences with a known function. We isolated a f ull-length cDNA of one of the upregulated genes from a HUVEC library. It en coded a novel protein with 394 amino acids, which was termed RTP (reducing agent and tunicamycin-responsive protein). Northern blot analysis revealed that RTP mRNA expression was induced in HUVECs treated with not only homocy steine but also 2-mercaptoethanol and tunicamycin, both of which are known to induce ER stress. RTP mRNA was ubiquitously expressed in human adult org ans, and seemed to be regulated in mouse embryogenesis. Consequently, our d ifferential display analysis revealed that homocysteine alters the expressi vity of multiple proteins, especially ER stress-responsive ones. This poten tial ability of homocysteine may be involved in atherogenesis, (C) 1998 Aca demic Press.