PROTEIN-KINASE-C-ETA MEDIATES LIPOPOLYSACCHARIDE-INDUCED NITRIC-OXIDESYNTHASE EXPRESSION IN PRIMARY ASTROCYTES

The signaling pathway involved in protein kinase C (PRC) activation an d role of PKC isoforms in lipopolysaccharide (LPS)-induced nitric oxid e (NO) release were studied in primary cerebellar astrocytes. LPS caus ed 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 phosp hatidate phosphodrolase inhibitor, propranolol, attenuated the LPS eff ects, whereas the PI-PLC inhibitor, U73122, had no effect. The PKC inh ibitors (staurosporine, Po 31-8220, Go 6976, and calphostin C) also in hibited LPS-induced NO release and iNOS expression. However, long term (24 h) pretreatment of cells with 12-O-tetradecanoyl phorbol-13-aceta te (TPA) did not affect the LPS response. Previous results have shown that TPA-induced translocation, but not down-regulation, of PKC eta oc curs in astrocytes (Chen, C. C., and Chen, W. C. (1996) Glia 17, 63-71 ), suggesting possible involvement of PKC eta in LPS-mediated effects. Treatment with antisense oligonucleotides for PKC eta or delta, anoth er isoform abundantly expressed in astrocytes, demonstrated the involv ement of PKC eta, 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-pro tein complex; this effect was inhibited by genestein, D609, propranolo l, or Po 31-8220 or by PKC eta antisense oligonucleotides, but not by long term TPA treatment. These data suggest that in astrocytes, LPS mi ght activate phosphatidylcholine-phospholipase C and phosphatidylcholi ne-phospholipase D through an upstream protein tyrosine kinase to indu ce PKC activation. Of the PKC isoforms present in these cells, only ac tivation of PKC eta by LPS resulted in the stimulation of NF-kB-specif ic DNA-protein binding and then initiated the iNOS expression and NO r elease. This is further evidence demonstrating that different members of the PKC family within a single cell are involved in specific physio logical responses.