Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes.
Treatment of mouse astrocyte cultures with combined interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha induced expression of inducible nitric-oxide synthase (iNOS), resulting in sustained release of large amounts of nitric oxide, whereas TNF-alpha and IL-1alpha individually were unable to induce iNOS expression in astrocytes. The role of MAPK cascades and of NF-kappaB activation in the early intracellular signal transduction involved in iNOS transcription in TNF-alpha/IL-1alpha-stimulated astrocytes was investigated. TNF-alpha and IL-1alpha 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-alpha/IL-1alpha-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-kappaB to iNOS transcription, but NF-kappaB 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-alpha/IL-1alpha-stimulated astrocytes. iNOS expression was investigated with TNF receptor (TNFR)-1- and TNFR-2-deficient mice. The TNF-alpha activity in TNF-alpha/IL-1alpha-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-alpha/IL-1alpha-induced iNOS expression depends on a yet undetermined second pathway in addition to p38(MAPK).
Da Silva
J
J
Department of Central Nervous System Diseases, PRPN, F. Hoffmann-La Roche, Ltd., 4070 Basel, Switzerland. jean.da_silva@roche.com
Pierrat
B
B
Mary
J L
JL
Lesslauer
W
W
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D020928:Mitogen-Activated Protein Kinases; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D004791:Enzyme Inhibitors; D007093:Imidazoles; D007375:Interleukin-1; D016328:NF-kappa B; D011500:Protein Synthesis Inhibitors; D011725:Pyridines; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019001:Nitric Oxide Synthase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole