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 TNFα 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 TNFα 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 κ B binding activity in treated macrophages which coincided temporally with the increased transcriptional activity of the two cytokine genes. The polypeptide composition of κB motif binding complexes from LPS- or OA-treated macrophages was similar. However, OA-induced expression of the TNFα 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 TNFα and IP-10 mRNA levels; TNFα 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 TNFα 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.