The role of constitutive nitric‐oxide synthase in regulation of IKKα after ultraviolet irradiation

Previously, we reported that UVB irradiation significantly reduces IKKα mRNA levels, while IKKα protein levels remain stable, a phenomenon maintained by constitutive nitric oxide synthase (cNOS) and NF-κB activity. In this study, we systematically investigated the transcriptional regulation of IKKα in response to UVB, with a focus on the role of cNOS. Using a series of luciferase reporter constructs containing deletions and site-specific mutations in the IKKα promoter, we evaluated promoter activity in HEK293 cells (cNOS-null) and HEK293cNOS cells (stably expressing cNOS). Our data identified two regulatory elements critical for UVB-inducible IKKα promoter activity: the second p53-binding site and the Ets-1 site. cNOS overexpression enhanced both basal and UVB-induced promoter activities in a dose-dependent manner. Interestingly, the promoter region spanning -940 to -438 harbors a repressive element that limits IKKα transcription. Although UVB activates the IKKα promoter, as shown by luciferase activity, it simultaneously inhibits transcriptional elongation. This likely explains the paradoxical reduction in endogenous IKKα mRNA levels. The effect is not due to decreased mRNA stability, highlighting transcriptional elongation as a key regulatory bottleneck. In parallel, in vivo studies using SKH-1 mice chronically exposed to solar-simulated UV (sUV) showed that cNOS knockout mice developed more tumors and exhibited significantly reduced IKKα expression compared to wild-type controls. These results demonstrate that cNOS regulates IKKα at multiple levels-promoter activation, transcriptional elongation, and protein stability. This multilayered control enhances our understanding of UV-induced skin pathogenesis and supports cNOS-IKKα signaling as a potential target for therapeutic intervention in skin cancer.