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

Inflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF alpha), are known to activate sphingomyelinase (SMase) and nuclear factor-kappaB (NF-kappaB) 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-kappaB activation pathway similar to that of IL-1beta and TNF alpha in cultured rat VSMCs. Neutral SMase, although less potently than IL-1beta 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-1beta, and TNF alpha activated NF-kappaB, as revealed by electrophoretic mobility shift assay, and its nuclear translocation, as demonstrated by immunocytochemical study. Neutral SMase potentiated NOx production, iNOS expression, and NF-kappaB activation stimulated by TNF alpha, but not by IL-1beta. Aldehyde peptide proteasome inhibitors completely blocked NOx production, iNOS expression, NF-kappaB activation, and its nuclear translocation induced by cytokines and neutral SMase. IL-1beta and TNF alpha, but not neutral SMase, caused a transient decrease in IkappaB-alpha protein levels, whereas IkappaB-beta protein expression was not affected by either agent. Proteasome inhibitors prevented cytokine-mediated IkappaB-alpha degradation. Several cell-permeable ceramide analogs (C2, C6, and C8), hydrolysis products of sphingomyelin, activated NF-kappaB less potently than neutral SMase, but had no effect on NOx production. These results demonstrate an essential role of NF-kappaB activation in mediation of neutral SMase-induced iNOS expression, but distinct from the proteasome-mediated IkappaB-alpha degradation by cytokines, suggesting the possible involvement of an additional signaling pathway(s).