RESUMEN
The expression of inducible nitric oxide synthase (iNOS) by macrophages is stimulated by coexposure to IFN-gamma and a number of stimuli, including TNF-alpha. Recent work has shown that TNF-alpha 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-gamma and TNF-alpha. All three kinases were activated during costimulation with IFN-gamma and TNF-alpha. 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-gamma and TNF-alpha. 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-alpha receptor CD120a (p55) in the presence of IFN-gamma.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Interferón gamma/fisiología , MAP Quinasa Quinasa 4 , Quinasa 1 de Quinasa de Quinasa MAP , Macrófagos/enzimología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Óxido Nítrico Sintasa/biosíntesis , Factor de Necrosis Tumoral alfa/fisiología , Células 3T3 , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Activación Enzimática/inmunología , Inducción Enzimática , Regulación de la Expresión Génica/inmunología , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Ratones Endogámicos C3H , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Mutación , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Regiones Promotoras Genéticas/inmunología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
A pleiotropic cytokine, tumor necrosis factor-alpha (TNF alpha), regulates the expression of multiple macrophage gene products and thus contributes a key role in host defense. In this study, we have investigated the specificity and mechanism of activation of members of the c-Jun-NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of mitogen-activated protein kinases (MAPKs) in mouse macrophages in response to stimulation with TNF alpha. Exposure of macrophages to TNF alpha stimulated a preferential increase in catalytic activity of the p46 JNK/SAPK isoform compared with the p54 JNK/SAPK isoform as determined by: (i) separation of p46 and p54 JNK/SAPKs by anion exchange liquid chromatography and (ii) selective immunodepletion of the p46 JNK/SAPK from macrophage lysates. To investigate the level of regulation of p46 JNK/SAPK activation, we determined the ability of MKK4/SEK1/JNKK, an upstream regulator of JNK/SAPKs, to phosphorylate recombinant kinase-inactive p46 and p54 JNK/SAPKs. Endogenous MKK4 was able to transphosphorylate both isoforms. In addition, both the p46 and p54 JNK/SAPK isoforms were phosphorylated on their TPY motif in response to TNF alpha stimulation as reflected by immunoblotting with a phospho-specific antibody that recognizes both kinases. Collectively, these results suggest that the level of control of p46 JNK/SAPK activation is distal not only to MKK4 but also to the p54 JNK/SAPK. Preferential isoform activation within the JNK/SAPK subfamily of MAPKs may be an important mechanism through which TNF alpha regulates macrophage phenotypic heterogeneity and differentiation.