IFN-gamma and IL-4 differently regulate inducible NO synthase gene expression through IRF-1 modulation.
NO is a labile radical involved in several immunological, antimicrobial and inflammatory processes. In macrophages, NO formation is catalyzed by the cytokine-inducible enzyme inducible NO synthase (iNOS). The importance of IFN regulatory factor (IRF)-1 and of the signal transducers and activators of transcription (STAT)-1 for the induction of iNOS gene expression in response to IFN-gamma has been well defined. Here, we investigated the molecular events responsible for the inhibition of iNOS gene expression by IL-4 in the murine macrophage cell line RAW264.7. Unidirectional deletion analysis on iNOS promoter demonstrated that an IFN-stimulated responsive element (ISRE), contained in the -980 to -765 bp region of the iNOS promoter, may be involved in the IL-4-mediated inhibition of IFN-gamma-inducible iNOS transcription. Accordingly, the IFN-gamma-induced binding activity of IRF-1 to the ISRE sequence was reduced in cells pre-treated with IL-4, while the binding activity of STAT-1 to the STAT-binding element (SBE) within the same region of the iNOS promoter remained unaffected. Moreover, IL-4 even down-regulated IFN-gamma-inducible expression of IRF-1 mRNA. This could be related to a transcriptional mechanism by which IL-4 and IFN-gamma differentially influence the trans-acting activity of the STAT factors binding to SBE within the IRF-1 promoter. SBE is targeted by IFN-gamma-inducible STAT-1 and by IL-4-inducible STAT-6. Although STAT-6 has no trans-acting function on iNOS gene expression, it is able to inhibit the IFN-gamma-induced expression of IRF-1. Thus, IL-4 may down-regulate IFN-gamma-inducible iNOS transcription by activation of STAT-6 which in turn inhibits IRF-1 expression.
Coccia
E M
EM
Laboratory of Virology and Immunology, Istituto Superiore di Sanità, Rome 00161, Italy. e.coccia.iss.it
Stellacci
E
E
Marziali
G
G
Weiss
G
G
Battistini
A
A
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000494:pharmacology*; D015847:Interleukin-4 / Q000494:pharmacology*; D051379:Mice; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D010750:Phosphoproteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D010750:Phosphoproteins; D014157:Transcription Factors; D015847:Interleukin-4; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse