The stimulation of human monocytes with interferon-gamma induces the phosphorylation of PU.1 and its binding to gp91phox promoter via activation of a classical protein kinase C

We previously reported that the stimulation of human blood monocytes with IFN-gamma induces the binding of PU.1 to the gp91phox promoter and the consequent expression of gp91phox. In this study, we show that the effect of IFN-gamma is reproduced by the serine phosphatase inhibitor, okadaic acid, and this suggests that serine kinases could be involved in gp91phox expression. We also show that IFN-gamma induces the serine/threonine phosphorylation of PU.1 in cultured monocytes. This phosphorylation, as well as the IFN-gamma-induced PU.1 binding and gp91phox protein synthesis, is slightly affected by the casein kinase II inhibitor, daidzein, but is abrogated by the protein kinase C (PKC) -alpha and -beta inhibitor, Go6976, and by synthetic peptides with sequences based on the endogenous pseudosubstrate region of the classical PKC alpha and beta isoforms. In contrast, peptides reproducing the pseudosubstrate region of PKC beta were without effect. Moreover, we found that the treatment of monocytes with IFN-gamma induces the nuclear translocation and the activation of PKC alpha and betaI, but not of PKC betaII, and that the IFN-gamma-induced phosphorylation of PU.1 was greatly reduced by LY333531, a selective inhibitor of PKC beta isoforms. Finally, nuclear run-on assays demonstrated that while the PKC inhibitors, Go6976 and LY333531, decrease the IFN-gamma-induced gp91phox transcription, the serine phosphatase inhibitor, okadaic acid, enhances the gp91phox gene transcription. Our results indicate that in cultured monocytes, IFN-gamma induces the binding of PU.1 to the gp91phox promoter and the expression of gp91phox by phosphorylation of PU.1 via activation of PKC alpha and/or beta I.