Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-κB in human aortic smooth muscle cells

Abstract

Inflammation-induced changes in the activity and expression of NADPH oxidases (Nox) play a key role in atherogenesis. The molecular mechanisms of Nox regulation are scantily elucidated. Since nuclear factor-kappaB (NF-kappaB) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-kappaB signaling in the regulation of Nox1 and Nox4 transcription in human aortic smooth muscle cells (SMCs). Cultured cells were exposed to tumor necrosis factor-alpha (TNFalpha), a potent inducer of both Nox and NF-kappaB, up to 24h. Lucigenin-enhanced chemiluminescence and dichlorofluorescein assays, real-time polymerase chain reaction, and Western blot analysis showed that inhibition of NF-kappaB pathway reduced significantly the TNFalpha-dependent up-regulation of Nox-derived reactive oxygen species production, Nox1 and Nox4 expression. In silico analysis indicated the existence of typical NF-kappaB elements in the promoters of Nox1 and Nox4. Transient overexpression of p65/NF-kappaB significantly increased the promoter activities of both isoforms. Physical interaction of p65/NF-kappaB proteins with the predicted sites was demonstrated by chromatin immunoprecipitation assay. These findings demonstrate that NF-kappaB is an essential regulator of Nox1- and Nox4-containing NADPH oxidase in SMCs. Elucidation of the complex relationships between NF-kappaB and Nox enzymes may lead to a novel pharmacological strategy to reduce both inflammation and oxidative stress in atherosclerosis and its associated complications.