Requirement of tumor necrosis factor alpha and nuclear factor-kappaB in the induction by IFN-gamma of inducible nitric oxide synthase in macrophages.
IFN-gamma induces NO production, inducible NO synthase (iNOS) protein, and promoter expression in mouse macrophage cells. Mutation of IFN regulatory factor 1 responsive element, gamma-activated site, as well as NF-kappaB elements in the murine iNOS promoter strongly reduced IFN-gamma-induced iNOS transcriptional activity. The role of NF-kappaB activation in iNOS induction by IFN-gamma was corroborated by overexpression of the NF-kappaB inhibitory protein IkappaBalpha, which inhibited iNOS promoter activity induced by IFN-gamma. In addition, IFN-gamma treatment induced p65 binding to the iNOS promoter by chromatin immunoprecipitation assay and NF-kappaB binding to DNA by EMSA, although with a delayed kinetics, suggesting an indirect autocrine role for another cytokine produced in response to IFN-gamma. It is interesting that we found that IFN-gamma induced TNF-alpha secretion, and the induction of iNOS expression by IFN-gamma was abolished in primary peritoneal macrophages from TNF-alpha-deficient (TNF-alpha-/-) mice or in RAW 264.7 cells treated with anti-TNF-alpha neutralizing antibodies. Moreover, exogenous addition of recombinant mouse TNF-alpha restored iNOS expression induced by IFN-gamma in TNF-alpha-/- mice. It is intriguing that NF-kappaB binding to DNA in response to IFN-gamma treatment was absent in TNF-alpha-/- mice. Taken together, our data suggest that the TNF-alpha produced in response to IFN-gamma is required for iNOS induction by activating NF-kappaB transcription factor.
Vila-del Sol
Virginia
V
Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain.
Díaz-Muñoz
Manuel D
MD
Fresno
Manuel
M
D000818:Animals; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic; D007371:Interferon-gamma / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II