Molecular Mechanism for Coronary Spastic Angina as a Transcriptional Factor-Related Disease

痉挛性心绞痛在日本人中比在高加索人中更常见。提示遗传危险因素参与其发病过程。在此背景下，我们寻找与冠状动脉痉挛性心绞痛相关的内皮型一氧化氮合酶（eNOS）基因多态性。发现eNOS基因第7外显子存在错义突变，5-侧翼区存在单核苷酸多态性，第7外显子T^<-786>→C错义突变不影响eNOS酶活性。而T^<-786>→C则抑制eNOS基因的转录。为了阐明eNOS基因转录减少的分子机制，我们现在已经纯化了一种蛋白质，该蛋白质特异性结合HeLa细胞核提取物中的突变等位基因。纯化的蛋白质与复制蛋白A1（RPA 1）相同，RPA 1是DNA修复所必需的单链DNA结合蛋白。复制和重组。在人脐静脉内皮细胞中，使用反义寡核苷酸抑制RPA 1表达恢复了由突变的启动子序列驱动的转录。而RPA 1的过表达则进一步降低其表达，RPA 1在胎盘组织中的表达也相似。携带T^ →C突变的胎盘中eNOS mRNA水平<-786>显著低于未携带T ^ →C突变的胎盘。携带T^→C突变的个体的血清亚硝酸盐/硝酸盐水平显著低于未携带突变的个体，这一发现揭示了eNOS表达减少的功能重要性<-786>。因此，RPA 1在<-786>与冠状动脉疾病发展相关的eNOS基因转录的T^ →C突变相关的减少中显然起着阻遏蛋白的作用。

Cspastic angina is more frequently observed in Japanese than Caucasians. suggesting genctic risk factor is involved in its pathogenesis. In this context, we searched the polymorphisms in endothelial nitic oxide synthase (eNOS) gene, which are associated with coronary srastic angina. We found a missense mutation in exon 7 and a single nucletide polymorphism in the 5-flanking region of eNOS gene, T^<-786>→C Missense mutation in exon7 did not affect eNOS emzyme activity. but T^<-786>→C reduce eNOS gene transcription. To elucidate the molecular mechanism for the reduced eNOS gene transcription, we have now purified a protein that specifically binds to the mutant allele in nuclear extracts from HeLa cells. The purified protein was identical to replication protein A1 (RPA1), known as a single-stranded-DNA binding protein essential for DNA repair. replication and recombination. In human umbilical vein endothelial cells, inhibition of RPA1 expression using antisense oligonucleotide restored transcription driven by the mutated promoter sequence. while conversely, overexpression of RPA1 further reduced it. RPA1 was similarly detected in placenta. and eNOS mRNA levels in placentas carrying the T^<-786>→C mutation were significantly lower than in placentas without it. The functional importance of the diminished eNOS expression was revealed by the finding that serum nitrite/nitrate levels among individuals carrying the T^<-786>→C mutation were significantly lower than among those without the mutation. RPA1 thus apparently functions as a repressor protein in the T^<-786>→C mutation-related reduction of eNOS gene transcription associated with the development of coronary artery disease.