Induction and stabilization of IκBα by nitric oxide mediates inhibition of NF-κB

To determine the mechanism(s) by which the endogenous mediator nitric oxide (NO) inhibits the activation of transcription factor NF-κB, we stimulated human vascular endothelial cells with tumor necrosis factor-α in the presence of two NO donors, sodium nitroprusside and S-nitrosoglutathione. Electrophoretic mobility shift assays demonstrated that both NO donors inhibited NF-κB activation by tumor necrosis factor-α. This effect was not mediated by guanylyl cyclase activation since the cGMP analogue 8-bromo-cGMP had no similar effect. Inhibition of endogenous constitutive NO production by L-N-monomethylarginine, however, activated NF-κB, suggesting tonic inhibition of NF-κB under basal conditions. NO had little or no effects on other nuclear binding proteins such as AP-1 and GATA. Immunoprecipitation studies showed that NO stabilized the NF-κB inhibitor, IκBα, by preventing its degradation from NF-κB. NO also increased the mRNA expression of IκBα, but not NF-κB subunits, p65 or p50, and transfection experiments with a chloramphenicol acetyltransferase reporter gene linked to the IκBα promoter suggested transcriptional induction of IκBα by NO. We propose that the induction and stabilization of IκBα by NO are important mechanisms by which NO inhibits NF-κB and attenuate atherogenesis.