Phorbol ester synergistically increases interferon regulatory factor-1 and inducible nitric oxide synthase induction in interferon-gamma-treated RAW 264.7 cells.

The roles of PKC in iNOS induction by IFN-gamma have been shown in some cell types. The effect of a PKC activator, phorbol ester, in iNOS induction is thought to be due to multiple mechanisms, and it is necessary to examine the involvement of phorbol ester on IFN-gamma-induced iNOS in detail. In the present study, we investigated the mechanisms of phorbol ester on IFN-gamma-induced iNOS in RAW 264.7 cells. PMA synergistically increased iNOS activity, protein and mRNA levels in IFN-gamma-treated RAW 264.7 cells. PMA together with IFN-gamma increased iNOS mRNA without affecting the iNOS mRNA degradation, suggesting that the synergistic effect of PMA on IFN-gamma-induced iNOS mRNA production may depend on the elevation of the transcription rate rather than a prolongation of mRNA stability. The DNA binding proteins that are involved in the regulation of iNOS expression are mainly NF-kappa B and IRF-1. IRF-1 transcriptionally regulates many IFN-inducible genes such as iNOS whose promoter contains an IRF-1 binding site. PMA might modulate iNOS induction as a cosignal with IFN-gamma in RAW 264.7 cells because the synergistic effect of PMA was mediated through IRF-1, rather than NF-kappa B. Ro 31-8220, a PKC inhibitor, decreased iNOS activity, protein, mRNA levels and IRF-1 activity, indicating that the effect of PMA on iNOS induction might occur via the PKC pathway. It is evidence that PKC plays an important role in IRF-1 activation and that phorbol ester has a synergistic effect on iNOS induction through IRF-1 activation in IFN-gamma-treated RAW 264.7 cells. The synergistic effect of PMA on IFN-gamma-induced IRF-1 binding activity was observed in macrophage cell line J774 cells as well as RAW 264.7 cells, but not in thioglycollate-elicited peritoneal macrophages.