ROLE OF NUCLEAR FACTOR-KAPPA-B ACTIVATION IN CYTOKINE-STIMULATED 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 tu mor necrosis factor-alpha (TNF alpha), are known to activate sphingomy elinase (SMase) and nuclear factor-kappa B (NF-kappa B) in certain cel l types, which also stimulate inducible nitric oxide synthase (iNOS) g ene in vascular smooth muscle cells (VSMCs). However, it remains unkno wn whether the SMase pathway is involved in iNOS gene expression in VS MCs. Therefore, the present study was designed to examine whether SMas e induces iNOS gene expression via the NF-kappa B activation pathway s imilar to that of IL-1 beta and TNF alpha in cultured rat VSMCs. Neutr al SMase, although less potently than IL-1 beta and TNF alpha, stimula ted nitrite/nitrate (NOx) production, and iNOS messenger RNA and prote in expression, as assessed by Northern and Western blot analyses, resp ectively. 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 S Mase potentiated NOx production, MOS expression, and NF-kappa B activa tion stimulated by TNF alpha, but not by IL-1 beta. Aldehyde peptide p roteasome inhibitors completely blocked NOx production, iNOS expressio n, NF-kappa B activation, and its nuclear translocation induced by cyt okines and neutral SMase. IL-1 beta and TNF alpha, but not neutral SMa se, caused a transient decrease in I kappa B-alpha protein levels, whe reas I kappa B-beta protein expression was not affected by either agen t. Proteasome inhibitors prevented cytokine-mediated I kappa B-alpha d egradation. Several cell-permeable ceramide analogs (CZ, C6, and C8), hydrolysis products of sphingomyelin, activated NF-kappa B less potent ly than neutral SMase, but had no effect on NOx production. These resu lts demonstrate an essential role of NF-kappa B activation in mediatio n of neutral SMase-induced MOS expression, but distinct from the prote asome-mediated I kappa B-alpha degradation by cytokines, suggesting th e possible involvement of an additional signaling pathway(s).