NO inhibits cytokine-induced iNOS expression and NF-κB activation by interfering with phosphorylation and degradation of IκB-α

Nitric oxide (NO) is known to have antiatherogenic and anti-inflammatory properties, but its effects on the cytokine-induced nuclear factor-kappa B (NF-kappa B) activation pathway in relation to the regulation of inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs) remain elusive. To elucidate the roles of NO in the regulation of cytokine-induced NF-kappa B activation and consequent iNOS gene expression, we studied the effects of NO donors [(+/-)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide (NOR3) and sodium nitroprusside] on interleukin (IL)-1 beta-induced NF-kappa B activation and I kappa B-alpha degradation and subsequent iNOS expression in rat VSMCs. Northern blot and Western blot analyses demonstrated that NO donors decreased IL-1 beta-induced iNOS mRNA and protein expression. Electrophoretic mobility shift assay using synthetic oligonucleotide corresponding to the downstream NF-kappa B site of rat iNOS promoter as a probe showed that NOR3 inhibited IL-beta-induced NF-kappa B activation and its nuclear translocation, as demonstrated with immunocytochemical study. These effects were independent of guanylate cyclase activation; an inhibitor of soluble guanyiate cyclase (1H-oxadiazolo-1,2,4-[4,3-alpha]quinoxaline-1-one) had no effect on NOR3-induced inhibition of NF-kappa B activation or iNOS mRNA expression by IL-1 beta, and a cGMP derivative (8-bromo-cGMP) failed to mimic the effects of NO donors. Western blot analysis using anti-I kappa B-alpha and anti-phospho-I kappa B-alpha antibodies revealed that IL-1 beta induced a transient degradation of I kappa B-alpha preceded by a rapid appearance of phosphorylated I kappa B-alpha, both of which were completely blocked by NOR3, A proteasome inhibitor (MG115) blocked IL-1 beta-induced transient degradation of I kappa B-alpha and stabilized the appearance of phosphorylated I kappa B-alpha stimulated by IL-1 beta. NOR3 inhibited the appearance of IL-1 beta-induced phosphorylated I kappa B-alpha even in the presence of MG115. Our results indicate that an inhibitory action by NO on cytokine-induced NF-kappa B activation and iNOS gene expression is due to its direct blockade on phosphorylation and subsequent degradation of I kappa B-alpha via the cGMP-independent pathway in rat VSMCs.