Okadaic acid stimulates inflammatory cytokine gene transcription in murine peritoneal macrophages

The involvement of protein phosphorylation in controlling proinflammatory cytokine gene expression by mononuclear phagocytes has been examined using okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A. OA (100 nM) was able to stimulate accumulation of TNF alpha and IP-10 mRNAs in thioglycollate-elicited peritoneal macrophages from C57B1/6 mice. The responses to OA or LPS were similar in several respects. The OA- and LPS-stimulated increases in IP-10 and TNF alpha mRNA were mediated through increases in the transcriptional activity of the genes as measured by nuclear run-on analysis. Both OA- and LPS-stimulated mRNA accumulation were independent of protein synthesis. Furthermore, OA and LPS resulted in elevated kappa B binding activity in treated macrophages which coincided temporally with the increased transcriptional activity of the two cytokine genes. The polypeptide composition of kappa B motif binding complexes from LPS- or OA-treated macrophages was similar. However, OA-induced expression of the TNF alpha and IP-10 genes was markedly delayed (detectable expression was only seen at 4-8 hr after stimulation) relative to that seen in LPS-stimulated macrophages in which both transcription and accumulation of mRNA can be seen within 30 min to 1 hr. Furthermore, the combination of LPS and OA had differential effects on TNF alpha and IP-10 mRNA levels; TNF alpha mRNA expression was prolonged in cells stimulated with LPS plus OA as compared to cells treated with LPS alone. This difference was the result of an increase in the half-life of cytoplasmic TNF alpha mRNA. No similar effect was seen on IP-10 mRNA stability. Thus, although protein phosphorylation appears to be important in regulating cytokine mRNA levels, OA and LPS most likely function via at least partially distinct cellular mechanisms.