Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high levels of PG production during inflammation and immune responses. Previous studies with pharmacological inhibitors suggested a role for protein kinase C (PKC) in PG production possibly by regulating COX-2 expression. In this study, we addressed the role of PKC-α in the modulation of COX-2 expression and PGE2 synthesis by the overexpressing of a dominant-negative (DN) mutant of this isoenzyme in the mouse macrophage cell line RAW 264.7. We investigated the effect of various stimuli on COX-2 expression, namely, LPS, IFN-γ, and the intracellular parasite Leishmania donovani. Whereas LPS-induced COX-2 mRNA and protein expression were down-regulated in DN PKC-α-overexpressing clones, IFN-γ-induced COX-2 expression was up-regulated in DN PKC-α-overexpressing clones with respect to normal RAW 264.7 cells. Measurements of PGE2 levels revealed a strong correlation between PGE2 secretion and IFN-γ-induced COX-2 mRNA and protein levels in DN PKC-α-overexpressing clones. Taken together, these results suggest a role for PKC-α in the modulation of LPS- and IFN-γ-induced COX-2 expression, as well as in IFN-γ-induced PGE2 secretion.
Previous studies based on pharmacological evidence suggested a requirement for protein kinase C (PKC) activity in the regulation of IFN-γ-induced MHC class II (MHC-II) expression. In the present study, we investigated the molecular mechanisms by which PKC-α modulates IFN-γ-induced MHC-II expression in the mouse macrophage cell line RAW 264.7. Overexpression of a dominant-negative (DN) mutant of PKC-α inhibited the expression of IFN-γ-induced MHC-II but had no effect on IFN-γ-induced STAT1 nuclear translocation and DNA binding activity, as well as on the expression of inducible NO synthase, IFN consensus sequence binding protein, MHC class I, IFN regulatory factor (IRF)-1, and IFN-γ-inducible protein-10. Further analysis showed that IFN-γ-induced expression of the MHC class II transactivator (CIITA), a transcriptional coactivator essential for MHC-II expression, was inhibited in DN PKC-α-overexpressing cells. Studies with reporter constructs containing the promoter IV region of CIITA revealed that overexpression of a constitutively active mutant of PKC-α enhanced IRF-1, but not IRF-2, transcriptional activity. Furthermore, characterization of IRF-1 from both normal and DN PKC-α-overexpressing cells revealed differences in IRF-1 posttranslational modifications. Collectively, our data suggest a novel regulatory mechanism for IFN-γ-induced MHC-II expression, whereby PKC regulates CIITA expression by selectively modulating the transcriptional activity of IRF-1.
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