Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes

Abstract

Treatment of mouse astrocyte cultures with combined interleukin (IL)- 1α and tumor necrosis factor (TNF)-α induced expression of inducible nitric-oxide synthase (iNOS), resulting in sustained release of large amounts of nitric oxide, whereas TNF-α and IL-1α individually were unable to induce iNOS expression in astrocytes. The role of MAPK cascades and of NF-κB activation in the early intracellular signal transduction involved in iNOS transcription in TNF-α/IL-1α-stimulated astrocytes was investigated. TNF- α and IL-1α activated all p42/44(MAPK), p38(MAPK), and p54(JNK) pathways as determined by immunoprecipitation kinase assays using specific antibodies and substrates. The p38(MAPK) pathway is specifically involved in TNF-αd/IL- 1α-induced iNOS expression, since iNOS protein and nitric oxide release in the presence of a specific inhibitor of p38(MAPK), 4-(4-fluorophenyl) -2-2- (4-hydroxyphenyl)-5-(4-pyridyl) -imidazole (FHPI), were dramatically diminished. In contrast, PD98059, a specific inhibitor of MEK1 had no effect on iNOS expression. p38(MAPK) did not couple NF-κB to iNOS transcription, but NF-κB had a clear role in iNOS transcription regulation. Northern blot analysis showed that the p38(MAPK) pathway controlled iNOS expression at the transcriptional level, since iNOS mRNA was reduced in the presence of FHPI in TNF-α/IL-1α-stimulated astrocytes. iNOS expression was investigated with TNF receptor (TNFR)-1- and TNFR-2-deficient mice. The TNF-α activity in TNF- α/IL-1α-stimulated astrocytes was exclusively mediated through TNFR-1, most likely because TNFR-2-mediated signals in astrocytes did not connect to the p38(MAPK) pathway. These data suggest that TNF-α/IL-α1-induced iNOS expression depends on a yet undetermined second pathway in addition to p38(MAPK).