Evaluation of the role of mitogen-activated protein kinases in the expression of inducible nitric oxide synthase by IFN-gamma and TNF-alpha in mouse macrophages

The expression of inducible nitric oxide synthase (iNOS) by macrophages is stimulated by coexposure to IFN-gamma and a number of stimuli, including TN P-alpha. Recent work has shown that TNF-alpha activates members of the mito gen-activated protein kinase family that subsequently trans-activate transc ription factors implicated in the regulation of MOS expression. The objecti ve of this study was to systematically evaluate the role of: 1) p42(mapk/er k2), 2) p46 c-Jun NH2-terminal kinase/stress-activated protein kinase (p46 JNK/SAPK), and 3) p38(mapk) in the induction of iNOS expression during cost imulation of mouse macrophages with IFN-gamma and TNF-alpha. All three kina ses were activated during costimulation with IFN-gamma and TNF-alpha. Howev er, specific antagonism of the p42(map/erk2) and p38(mapk) with PD98059 and SKF86002, respectively, had no effect on the induction of iNOS expression. In contrast, blockade of all three kinases with N-acetylcysteine completel y blocked the induction of iNOS expression. In addition, specific antagonis m of the JNK/SAPK upstream kinases MEKK (mitogen-activated protein kinase/e xtracellular signal-regulated kinase kinase kinase) and MKK4 (mitogen-activ ated protein kinase kinase 4) with dominant inhibitory mutants blocked tran scriptional activation of the iNOS promoter in response to costimulation,vi th IFN-gamma and TNF-alpha. Collectively, these findings support the involv ement of p46 JNK/SAPK and its upstream kinases in regulating the induction of iNOS following ligation of the TNF-alpha receptor CD120a (p55) in the pr esence of IFN-gamma.