Abstract
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.
Highlights
Nitric oxide (NO),1 a bioactive free radical, is involved in various physiological and pathological processes in many sys
The results show that LPS might activate phosphatidylcholine-phospholipases C and D (PC-PLC and PC-PLD) via tyrosine phosphorylation to produce protein kinase C (PKC) and nuclear factor (NF)-kB activation, inducible NO synthase (iNOS) expression, and, NO release
When cells were pretreated for 30 min with 30 M genestein, LPSinduced nitrite production was inhibited by 35% and iNOS expression decreased (Fig. 2A)
Summary
Materials—Affinity-purified rabbit polyclonal antibody to iNOS was obtained from Transduction Laboratories (Lexington, KY). Synthesis of PKC and ␦ Antisense Oligonucleotides and Treatment of Cells with Oligonucleotides—Phosphorothioate oligodeoxynucleotides were synthesized in trityl-on mode using an Applied Biosystems model 391 DNA synthesizer, as described previously [19], using A, G, C, and T phosphoramidites, controlled pore glass supports, and sulfuring reagent purchased from Glen Research Corp. Following attachment on day 1 of culture, cells were treated with PKC or ␦ antisense oligonucleotides or control oligonucleotides (10 M) for 9 days (the medium was changed once) before challenge with 1 g/ml LPS for 24 h, and the protein levels of PKC and ␦ were determined by Western blotting using antibodies specific for PKC or ␦. Preparation of Nuclear Extracts and the Electrophoretic Mobility Shift Assay (EMSA)—Control cells or cells pretreated with genestein, D609, propranolol or Ro 31– 8220 for 30 min, TPA for 24 h, or antisense oligonucleotides for 9 days were treated with 1 g/ml LPS for 1 h. Cells were washed with ice-cold phosphate-buffered saline
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