Pahan K, Sheikh F G, Khan M, Namboodiri A M, Singh I
Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 1998 Jan 30;273(5):2591-600. doi: 10.1074/jbc.273.5.2591.
The sphingomyelin signal transduction pathway is known to play a role in mediating the action of various cytokines. Here we examined the possible role of the sphingomyelin signaling pathway on lipopolysaccharide (LPS)- and cytokine-mediated production of NO and the expression of inducible nitric-oxide synthase (iNOS). Sphingomyelinase (SMase) treatment of astrocytes increased the cellular levels of ceramide without the induction of NO production. However, incubation of LPS or cytokine-stimulated astrocytes with SMase or by increasing intracellular ceramide by cell-permeable ceramide analogs (C2- or C6-ceramide) or inhibitor of ceramidase (N-oleoyl ethanolamine) led to a time- and dose-dependent increase in the production of NO. This increase in NO production was accompanied by an increase in iNOS activity, iNOS protein, and iNOS mRNA. Similar to astrocytes, SMase or ceramide analogs also stimulated the LPS- and cytokine-mediated expression of iNOS in the C6 glial cell line. Since activation of NF-kappaB is necessary for the induction of iNOS, we examined the effect of SMase and C2-ceramide on the activation of NF-kappaB. Although SMase or C2-ceramide alone was ineffective in activating NF-kappaB, both stimulated the LPS-mediated activation of NF-kappaB in LPS-activated astrocytes. Inhibition of ceramide and LPS-mediated induction of iNOS by antioxidant inhibitors of NF-kappaB (N-acetylcysteine and pyrrolidine dithiocarbamate) suggest that the stimulatory effect of ceramide on the induction of iNOS is due to the stimulation of NF-kappaB activation and that cellular redox plays a role in the activation of NF-kappaB and induction of iNOS. Inhibition of LPS-mediated as well as LPS and ceramide-mediated induction of iNOS and activation of NF-kappaB by PD98059, a specific inhibitor of activation of mitogen-activated protein (MAP) kinase kinase (MEK), and FPT inhibitor II, a selective inhibitor of Ras farnesyl protein transferase, indicate that the Ras-MAP kinase pathway is involved in LPS-ceramide induced activation of NF-kappaB and induction of iNOS, and that ceramide-mediated signaling events probably converge into the LPS-modulated MAP kinase signaling pathway resulting in greater activation of NF-kappaB and iNOS induction. This study illustrates a novel role of the sphingomyelin-ceramide signaling pathway in stimulating the expression of iNOS via LPS- or cytokine-mediated activation of NF-kappaB in astrocytes.
已知鞘磷脂信号转导途径在介导多种细胞因子的作用中发挥作用。在此，我们研究了鞘磷脂信号通路对脂多糖（LPS）和细胞因子介导的一氧化氮（NO）产生以及诱导型一氧化氮合酶（iNOS）表达的可能作用。用鞘磷脂酶（SMase）处理星形胶质细胞可增加细胞内神经酰胺水平，而不诱导NO产生。然而，用SMase处理LPS或细胞因子刺激的星形胶质细胞，或通过细胞可渗透的神经酰胺类似物（C2 - 或C6 - 神经酰胺）或神经酰胺酶抑制剂（N - 油酰乙醇胺）增加细胞内神经酰胺，会导致NO产生呈时间和剂量依赖性增加。NO产生的这种增加伴随着iNOS活性、iNOS蛋白和iNOS mRNA的增加。与星形胶质细胞类似，SMase或神经酰胺类似物也刺激了C6神经胶质细胞系中LPS和细胞因子介导的iNOS表达。由于NF - κB的激活对于iNOS的诱导是必需的，我们研究了SMase和C2 - 神经酰胺对NF - κB激活的影响。尽管单独的SMase或C2 - 神经酰胺在激活NF - κB方面无效，但两者都刺激了LPS激活的星形胶质细胞中LPS介导的NF - κB激活。NF - κB的抗氧化剂抑制剂（N - 乙酰半胱氨酸和吡咯烷二硫代氨基甲酸盐）对神经酰胺和LPS介导的iNOS诱导的抑制表明，神经酰胺对iNOS诱导的刺激作用是由于对NF - κB激活的刺激，并且细胞氧化还原在NF - κB的激活和iNOS的诱导中起作用。丝裂原活化蛋白（MAP）激酶激酶（MEK）激活的特异性抑制剂PD98059和Ras法尼基蛋白转移酶的选择性抑制剂FPT抑制剂II对LPS介导的以及LPS和神经酰胺介导的iNOS诱导和NF - κB激活的抑制表明，Ras - MAP激酶途径参与LPS - 神经酰胺诱导的NF - κB激活和iNOS诱导，并且神经酰胺介导的信号事件可能汇聚到LPS调节的MAP激酶信号通路中，导致NF - κB的更大激活和iNOS诱导。这项研究说明了鞘磷脂 - 神经酰胺信号通路在通过LPS或细胞因子介导的星形胶质细胞中NF - κB激活来刺激iNOS表达方面的新作用。
