Role of protein kinase C activity in tumor necrosis factor-alpha gene expression. Involvement at the transcriptional...

Primary rat astrocytes express TNF-alpha protein in response to various stimuli including a combined treatment with IFN-gamma and LPS, or IFN-gamma and IL-1 beta. This study was undertaken to further elucidate the mechanisms underlying TNF-alpha gene expression in the astrocyte, and to determine the intracellular signaling pathways involved in IFN-gamma/LPS and/or IFN-gamma/IL-1 beta induction of the TNF-alpha gene. We demonstrate that TNF-alpha mRNA is rapidly induced, and mRNA levels peak after 2 h of stimulation. De novo protein synthesis is not required for TNF-alpha expression because the inclusion of cycloheximide does not prevent expression of the gene and acts to superinduce TNF-alpha mRNA levels. IFN-gamma/LPS induces transcriptional activation of the TNF-alpha gene as assessed by nuclear run-on experiments. Cycloheximide acts to increase both transcription of the TNF-alpha gene and stability of TNF-alpha mRNA thereby resulting in increased TNF-alpha steady state mRNA levels. Two protein kinase C (PKC) inhibitors, H7 and staurosporine, abrogate IFN-gamma/LPS- and IFN-gamma/IL-1 beta-induced TNF-alpha expression in a dose-dependent manner. PKC activity is required for transcription of the TNF-alpha gene, and does not appear to be involved in TNF-alpha mRNA stabilization. Taken together, these data demonstrate that TNF-alpha gene expression in primary rat astrocytes is induced in a PKC-dependent manner.