Won Je-Seong, Im Yeong-Bin, Khan Mushfiquddin, Singh Avtar K, Singh Inderjit
Developmental Neurogenetics, Department of Pediatrics, Medical University of South Carolina, Charleston 29425, USA.
J Neurochem. 2004 Feb;88(3):583-93. doi: 10.1046/j.1471-4159.2003.02165.x.
Lipopolysaccharide (LPS) and interferon-gamma (IFN) treatment of C6 rat glioma cells increased the intracellular ceramide level and the expression of the inducible nitric oxide synthase (iNOS) gene. To delineate the possible role of ceramide in the induction of iNOS, we examined the source of intracellular ceramide and associated signal transduction pathway(s) with the use of inhibitors of intracellular ceramide generation. The inhibitor of neutral sphingomyelinase (3-O-methylsphingomyelin, MSM) inhibited the induction of iNOS, whereas inhibitor of acidic sphingomyelinase (SR33557) or that of ceramide de novo synthesis (fumonisin B1) had no effect on the induction of iNOS. MSM-mediated inhibition of iNOS induction was reversed by the supplementation of exogenous C8-ceramide, suggesting that ceramide production by neutral sphingomyelinase (nSMase) is a key mediator in the induction of iNOS. The MSM-mediated inhibition of iNOS gene expression correlated with the decrease in the activity of ras. Inhibition of co-transfected iNOS promoter activity by dominant negative ras supported the role of ras in the nSMase-dependent regulation of iNOS gene. NF-kappaB DNA binding activity and its transactivity were also reduced by MSM pretreatment, and were completely reversed by the supplementation of C8-ceramide. As the dominant negative ras also reduced NF-kappaB transactivity, NF-kappaB activation may be downstream of ras. Our results suggest that ceramide generated by nSMase may be a critical mediator in the regulation of iNOS gene expression via ras-mediated NF-kappaB activation under inflammatory conditions.
用脂多糖（LPS）和干扰素-γ（IFN）处理C6大鼠胶质瘤细胞，可增加细胞内神经酰胺水平以及诱导型一氧化氮合酶（iNOS）基因的表达。为了阐明神经酰胺在诱导iNOS中的可能作用，我们使用细胞内神经酰胺生成抑制剂研究了细胞内神经酰胺的来源及相关信号转导途径。中性鞘磷脂酶抑制剂（3-O-甲基鞘磷脂，MSM）抑制了iNOS的诱导，而酸性鞘磷脂酶抑制剂（SR33557）或神经酰胺从头合成抑制剂（伏马菌素B1）对iNOS的诱导没有影响。补充外源性C8-神经酰胺可逆转MSM介导的iNOS诱导抑制作用，这表明中性鞘磷脂酶（nSMase）产生的神经酰胺是诱导iNOS的关键介质。MSM介导的iNOS基因表达抑制与ras活性降低相关。显性负性ras对共转染的iNOS启动子活性的抑制支持了ras在nSMase依赖性iNOS基因调控中的作用。MSM预处理也降低了NF-κB DNA结合活性及其转录活性，补充C8-神经酰胺可使其完全逆转。由于显性负性ras也降低了NF-κB转录活性，NF-κB激活可能在ras下游。我们的结果表明，在炎症条件下，nSMase产生的神经酰胺可能是通过ras介导的NF-κB激活来调节iNOS基因表达的关键介质。
