Hypermethylation of the inducible nitric-oxide synthase gene promoter inhibits its transcription

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.