Miyata T, Kokame K, Agarwala K L, Kato H
National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Semin Thromb Hemost. 1998;24(3):285-91. doi: 10.1055/s-2007-995856.
An elevated plasma level of homocysteine is associated with arteriosclerosis and thrombosis. The mechanisms by which homocysteine may promote vascular diseases have not yet been elucidated. In the present study, we have applied a modified nonradioactive differential display analysis to evaluate changes 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 protein, suggesting that unfolded proteins would accumulate in the endoplasmic reticulum 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 involved in a homocysteine metabolism. A third upregulated gene encoded activating transcription factor 4. The remaining four were uncharacterized genes. We isolated a full-length cDNA of one of the upregulated genes from a HUVEC library. It encoded a novel protein with 394 amino acids, which was termed reducing-agents and tunicamycin-responsive protein (RTP). Northern blot analysis revealed that RTP gene expression was induced in HUVEC after 4 h of incubation with homocysteine. RTP mRNA was also observed in unstimulated cells and induced by not only homocysteine but also 2-mercaptoethanol and tunicamycin. The mRNA was ubiquitously expressed in human tissues. These observations indicate that homocysteine can alter the expressivity of multiple genes, including a stress protein and several novel genes. These responses may contribute to atherogenesis.
血浆同型半胱氨酸水平升高与动脉硬化和血栓形成有关。同型半胱氨酸促进血管疾病的机制尚未阐明。在本研究中，我们应用改良的非放射性差异显示分析法，评估同型半胱氨酸处理培养的人脐静脉内皮细胞（HUVEC）诱导的基因表达变化。我们鉴定出六个上调基因和一个下调基因。一个上调基因是GRP78/BiP，一种应激蛋白，这表明由于同型半胱氨酸引起的氧化还原电位变化，未折叠蛋白会在内质网中积累。另一个上调基因编码一种具有亚甲基四氢叶酸脱氢酶和亚甲基四氢叶酸环水解酶活性的双功能酶，其参与同型半胱氨酸代谢。第三个上调基因编码激活转录因子4。其余四个是未鉴定的基因。我们从HUVEC文库中分离出一个上调基因的全长cDNA。它编码一种含有394个氨基酸的新型蛋白质，被称为还原剂和衣霉素反应蛋白（RTP）。Northern印迹分析显示，同型半胱氨酸孵育4小时后，HUVEC中RTP基因表达被诱导。在未刺激的细胞中也观察到RTP mRNA，它不仅由同型半胱氨酸诱导，还由2-巯基乙醇和衣霉素诱导。该mRNA在人体组织中广泛表达。这些观察结果表明，同型半胱氨酸可改变多个基因的表达，包括一种应激蛋白和几个新基因。这些反应可能有助于动脉粥样硬化的发生。
