Katsuyama K, Shichiri M, Marumo F, Hirata Y
Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
Endocrinology. 1998 Nov;139(11):4506-12. doi: 10.1210/endo.139.11.6309.
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).
已知炎性细胞因子，如白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNFα），可在某些细胞类型中激活鞘磷脂酶（SMase）和核因子-κB（NF-κB），这也会刺激血管平滑肌细胞（VSMC）中的诱导型一氧化氮合酶（iNOS）基因。然而，SMase途径是否参与VSMC中iNOS基因的表达仍不清楚。因此，本研究旨在检测在培养的大鼠VSMC中，SMase是否通过与IL-1β和TNFα类似的NF-κB激活途径诱导iNOS基因表达。中性SMase虽然效力低于IL-1β和TNFα，但可刺激亚硝酸盐/硝酸盐（NOx）生成以及iNOS信使核糖核酸和蛋白质表达，分别通过Northern印迹分析和Western印迹分析进行评估。电泳迁移率变动分析显示，中性SMase、IL-1β和TNFα可激活NF-κB，免疫细胞化学研究表明其可发生核转位。中性SMase可增强TNFα刺激的NOx生成、iNOS表达和NF-κB激活，但对IL-1β刺激的无此作用。醛肽蛋白酶体抑制剂可完全阻断细胞因子和中性SMase诱导的NOx生成、iNOS表达、NF-κB激活及其核转位。IL-1β和TNFα可使IκB-α蛋白水平短暂降低，但中性SMase无此作用，而IκB-β蛋白表达不受任何一种试剂影响。蛋白酶体抑制剂可阻止细胞因子介导的IκB-α降解。几种细胞可渗透的神经酰胺类似物（C2、C6和C8），即鞘磷脂的水解产物，激活NF-κB的效力低于中性SMase，但对NOx生成无影响。这些结果表明，NF-κB激活在介导中性SMase诱导的iNOS表达中起重要作用，但不同于细胞因子介导的蛋白酶体介导的IκB-α降解，提示可能涉及其他信号通路。
