Chen C C, Wang J K, Chen W C, Lin S B
Institutes of Pharmacology, College of Medicine, National Taiwan University, No.1, Jen-Ai Road, 1st Section, Taipei 10018, Taiwan.
J Biol Chem. 1998 Jul 31;273(31):19424-30. doi: 10.1074/jbc.273.31.19424.
The signaling pathway involved in protein kinase C (PKC) activation and role of PKC isoforms in lipopolysaccharide (LPS)-induced nitric oxide (NO) release were studied in primary cerebellar astrocytes. LPS caused a dose- and time-dependent increase in NO release and inducible NO synthase (iNOS) expression. The tyrosine kinase inhibitor, genestein, the phosphatidylcholine-phospholipase C inhibitor, D609, and the phosphatidate phosphodrolase inhibitor, propranolol, attenuated the LPS effects, whereas the PI-PLC inhibitor, U73122, had no effect. The PKC inhibitors (staurosporine, Ro 31-8220, Go 6976, and calphostin C) also inhibited LPS-induced NO release and iNOS expression. However, long term (24 h) pretreatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) did not affect the LPS response. Previous results have shown that TPA-induced translocation, but not down-regulation, of PKCeta occurs in astrocytes (Chen, C. C., and Chen, W. C. (1996) Glia 17, 63-71), suggesting possible involvement of PKCeta in LPS-mediated effects. Treatment with antisense oligonucleotides for PKCeta or delta, another isoform abundantly expressed in astrocytes, demonstrated the involvement of PKCeta, but not delta, in LPS-mediated effects. Stimulation of cells for 1 h with LPS caused activation of nuclear factor (NF)-kB in the nuclei as detected by the formation of a NF-kB-specific DNA-protein complex; this effect was inhibited by genestein, D609, propranolol, or Ro 31-8220 or by PKCeta antisense oligonucleotides, but not by long term TPA treatment. These data suggest that in astrocytes, LPS might activate phosphatidylcholine-phospholipase C and phosphatidylcholine-phospholipase D through an upstream protein tyrosine kinase to induce PKC activation. Of the PKC isoforms present in these cells, only activation of PKCeta by LPS resulted in the stimulation of NF-kB-specific DNA-protein binding and then initiated the iNOS expression and NO release. This is further evidence demonstrating that different members of the PKC family within a single cell are involved in specific physiological responses.
在原代小脑星形胶质细胞中研究了蛋白激酶C（PKC）激活所涉及的信号通路以及PKC亚型在脂多糖（LPS）诱导的一氧化氮（NO）释放中的作用。LPS导致NO释放和诱导型NO合酶（iNOS）表达呈剂量和时间依赖性增加。酪氨酸激酶抑制剂染料木黄酮、磷脂酰胆碱 - 磷脂酶C抑制剂D609以及磷脂酸磷酸二酯酶抑制剂普萘洛尔减弱了LPS的作用，而磷脂酰肌醇 - 磷脂酶C抑制剂U73122则无作用。PKC抑制剂（星形孢菌素、Ro 31 - 8220、Go 6976和钙泊三醇C）也抑制LPS诱导的NO释放和iNOS表达。然而，用12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）对细胞进行长期（24小时）预处理并不影响LPS反应。先前的结果表明，TPA诱导的PKCeta转位而非下调发生在星形胶质细胞中（Chen，C.C.，和Chen，W.C.（1996）Glia 17，63 - 71），提示PKCeta可能参与LPS介导的效应。用针对PKCeta或delta（星形胶质细胞中大量表达的另一种亚型）的反义寡核苷酸处理表明，PKCeta而非delta参与LPS介导的效应中的作用。用LPS刺激细胞1小时导致细胞核中核因子（NF）-kB激活，通过形成NF - kB特异性DNA - 蛋白质复合物检测到；这种效应被染料木黄酮、D609、普萘洛尔或Ro 31 - 8220或PKCeta反义寡核苷酸抑制，但不受长期TPA处理抑制。这些数据表明，在星形胶质细胞中，LPS可能通过上游蛋白酪氨酸激酶激活磷脂酰胆碱 - 磷脂酶C和磷脂酰胆碱 - 磷脂酶D以诱导PKC激活。在这些细胞中存在的PKC亚型中，只有LPS激活PKCeta导致NF - kB特异性DNA - 蛋白质结合的刺激，然后启动iNOS表达和NO释放。这是进一步的证据表明单个细胞内PKC家族的不同成员参与特定的生理反应。
