AbstractThe expression of inducible nitric oxide synthase (iNOS) by macrophages is stimulated by coexposure to IFN-γ and a number of stimuli, including TNF-α. Recent work has shown that TNF-α activates members of the mitogen-activated protein kinase family that subsequently trans-activate transcription factors implicated in the regulation of iNOS expression. The objective of this study was to systematically evaluate the role of: 1) p42mapk/erk2, 2) p46 c-Jun NH2-terminal kinase/stress-activated protein kinase (p46 JNK/SAPK), and 3) p38mapk in the induction of iNOS expression during costimulation of mouse macrophages with IFN-γ and TNF-α. All three kinases were activated during costimulation with IFN-γ and TNF-α. However, specific antagonism of the p42mapk/erk2 and p38mapk with PD98059 and SKF86002, respectively, had no effect on the induction of iNOS expression. In contrast, blockade of all three kinases with N-acetylcysteine completely blocked the induction of iNOS expression. In addition, specific antagonism of the JNK/SAPK upstream kinases MEKK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase) and MKK4 (mitogen-activated protein kinase kinase 4) with dominant inhibitory mutants blocked transcriptional activation of the iNOS promoter in response to costimulation with IFN-γ and TNF-α. Collectively, these findings support the involvement of p46 JNK/SAPK and its upstream kinases in regulating the induction of iNOS following ligation of the TNF-α receptor CD120a (p55) in the presence of IFN-γ.