Protein Kinase C η Mediates Lipopolysaccharide-induced Nitric-oxide Synthase Expression in Primary Astrocytes

• Published in: The Journal of biological chemistry 1998-07, Vol.273 (31), p.19424-19430
• Main Authors: Chen, Ching-Chow, Wang, Jia-Kae, Chen, Wei-Chyuan, Lin, Shwu-Bin
• Format: Article
• Language: English
• Subjects: Animals; Astrocytes - enzymology; Brain - enzymology; Enzyme Activation - physiology; Enzyme Inhibitors - pharmacology; Gene Expression Regulation, Enzymologic - drug effects; Isoenzymes - physiology; Lipopolysaccharides - pharmacology; Nitric Oxide Synthase - metabolism; Nitric Oxide Synthase Type II; Oligonucleotides, Antisense - pharmacology; Phospholipase D - metabolism; Protein Kinase C - physiology; Rats; Rats, Wistar; Signal Transduction - physiology; Tetradecanoylphorbol Acetate - pharmacology; Type C Phospholipases - metabolism
• ISSN: 0021-9258; 1083-351X
• 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 PKCη occurs in astrocytes (Chen, C. C., and Chen, W. C. (1996) Glia 17, 63–71), suggesting possible involvement of PKCη in LPS-mediated effects. Treatment with antisense oligonucleotides for PKCη or δ, another isoform abundantly expressed in astrocytes, demonstrated the involvement of PKCη, but not δ, 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 PKCη 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 PKCη 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.