Protein kinase C eta mediates lipopolysaccharide-induced nitric-oxide synthase expression in primary astrocytes.
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.
Institutes of Pharmacology, College of Medicine, National Taiwan University, No.1, Jen-Ai Road, 1st Section, Taipei 10018, Taiwan. cchen@ha.mc.ntu.edu.tw
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D000818:Animals; D001253:Astrocytes / Q000201:enzymology*; D001921:Brain / Q000201:enzymology; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007527:Isoenzymes / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008070:Lipopolysaccharides; D016376:Oligonucleotides, Antisense; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D011493:Protein Kinase C; D010738:Type C Phospholipases; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate