Hypermethylation of the inducible nitric-oxide synthase gene promoter inhibits its transcription
Hypermethylation of the inducible nitric-oxide synthase gene promoter inhibits its transcription
复制标题
DOI：
10.1074/jbc.m407192200
复制
发表时间：
2004-11-05
影响因子：
4.8
通讯作者：
Kone, BC
中科院分区：
文献类型：
--
作者：
Yu, ZY;Kone, BC
诱导型一氧化氮合酶（inducible nitric-oxide synthase，iNOS）大量产生的一氧化氮（nitric oxide，NO）可在肾小球肾炎和其他炎症性疾病中对宿主细胞造成非预期的损伤。虽然对iNOS的诱导机制了解较多，但对iNOS的转录抑制机制研究较少，本文探讨了胞嘧啶甲基化在iNOS转录调控中的作用。用DNA甲基化抑制剂治疗系膜细胞增强了内源性NO产生和iNOS蛋白水平以及iNOS启动子活性的细胞因子诱导。以相应的方式，在体外甲基化的小鼠iNOS启动子是足以沉默其在系膜细胞的活性。相反，反义敲低DNA甲基转移酶-3b的表达和活性增加iNOS启动子的活性和亚硝酸盐的产生。亚硫酸氢盐处理和iNOS启动子的测序分析鉴定了在-879/-871处形成增强子元件的胞嘧啶的甲基化。体外甲基化抑制NF κ B p50与该元件的结合，并且该元件的缺失导致转录抑制的缓解。这些结果为iNOS基因表达的转录调控提供了独特的分子机制。
Exuberant generation of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) can cause unintended injury to host cells during glomerulonephritis and other inflammatory diseases. Although much is known about the mechanisms of iNOS induction, few transcriptional repression mechanisms have been found. We explored the role of cytosine methylation in the regulation of iNOS transcription. Treatment of mesangial cells with DNA methylation inhibitors augmented cytokine induction of endogenous NO production and iNOS protein levels, as well as iNOS promoter activity. In a corresponding manner, in vitro methylation of the murine iNOS promoter was sufficient to silence its activity in mesangial cells. In contrast, antisense knockdown of DNA methyltransferase-3b expression and activity increased iNOS promoter activity and nitrite production. Bisulfite treatment and sequencing analysis of the iNOS promoter identified methylation of cytosines framing an enhancer element at -879/-871. In vitro methylation inhibited binding of NFkappaB p50 to this element, and deletion of the element resulted in relief of transcriptional repression. These results provide evidence for a unique molecular mechanism involved in transcriptional regulation of iNOS gene expression.