Role of nuclear factor-κB activation in cytokine- and sphingomyelinase-stimulated inducible nitric oxide synthase gene expression in vascular smooth muscle cells

Inflammatory cytokines, such as interleulrin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha), are known to activate sphingomyelinase (SMase) and nuclear factor-kappa B (NF-kappa B) in certain cell types, which also stimulate inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs). However, it remains unknown whether the SMase pathway is involved in iNOS gene expression in VSMCs. Therefore, the present study was designed to examine whether SMase induces iNOS gene expression via the NF-kappa B activation pathway similar to that of IL-1 beta and TNF alpha in cultured rat VSMCs. Neutral SMase, although less potently than IL-1 beta and TNF alpha, stimulated nitrite/nitrate (NOx) production, and iNOS messenger RNA and protein expression, as assessed by Northern and Western blot analyses, respectively. Neutral SMase, IL-1 beta, and TNF alpha activated NF-kappa B, as revealed by electrophoretic mobility shift assay, and its nuclear translocation, as demonstrated by immunocytochemical study. Neutral SMase potentiated NOx production, MOS expression, and NF-kappa B activation stimulated by TNF alpha, but not by IL-1 beta. Aldehyde peptide proteasome inhibitors completely blocked NOx production, iNOS expression, NF-kappa B activation, and its nuclear translocation induced by cytokines and neutral SMase. IL-1 beta and TNF alpha, but not neutral SMase, caused a transient decrease in I kappa B-alpha protein levels, whereas I kappa B-beta protein expression was not affected by either agent. Proteasome inhibitors prevented cytokine-mediated I kappa B-alpha degradation. Several cell-permeable ceramide analogs (CZ, C6, and C8), hydrolysis products of sphingomyelin, activated NF-kappa B less potently than neutral SMase, but had no effect on NOx production. These results demonstrate an essential role of NF-kappa B activation in mediation of neutral SMase-induced MOS expression, but distinct from the proteasome-mediated I kappa B-alpha degradation by cytokines, suggesting the possible involvement of an additional signaling pathway(s).