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Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-gamma-treated RAW264.7 murine macrophages.

ABSTRACT: We hypothesized that acetylation of the Stat1 regulates interferon-gamma (IFN-gamma) mediated macrophage expression of inducible nitric oxide synthase (iNOS).RAW 264.7 iNOS expression was induced with IFN-gamma. Deacetylase inhibitors trichostatin A (TSA) or valproic acid (VPA) were added. Stat1 and iNOS mRNA and protein were measured. Acetylated Stat1 was determined by immunoprecipitation. Chromatin immunoprecipitation assessed in vivo binding of Stat1 to the iNOS promoter.IFN-gamma significantly increased nitrite, iNOS protein and iNOS mRNA, and iNOS promoter activation. (P < .01 vs control for nitrite, protein, and mRNA). TSA-mediated acetylation decreased these to levels that were not different from controls. IFN-gamma increased acetylated Stat1 by 5-fold (P < .02 vs control); TSA + IFN-gamma caused an additional 4-fold increase in acetylated Stat1 (P < .05 vs IFN alone). Stat1 binding to the iNOS promoter increased 8-fold with IFN-gamma (P < .01 vs control). In TSA + IFN-gamma, Stat1 binding was not different from controls. Although less potent than TSA, VPA also significantly decreased nitrite, iNOS protein, iNOS mRNA, Stat1 acetylation, and Stat1 binding.Acetylation of Stat1 protein correlates with decreased Stat1 binding to the iNOS promoter with resultant inhibition of IFN-gamma-mediated iNOS expression. Acetylation of the Stat1 protein may downregulate iNOS expression in proinflammatory states.

SUBMITTER: Guo L

PROVIDER: S-EPMC2034510 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-gamma-treated RAW264.7 murine macrophages.

Guo Lucie L Guo Hongtao H Gao Chengjiang C Mi Zhiyong Z Russell William B WB Kuo Paul C PC

Surgery 20070801 2

<h4>Background</h4>We hypothesized that acetylation of the Stat1 regulates interferon-gamma (IFN-gamma) mediated macrophage expression of inducible nitric oxide synthase (iNOS).<h4>Methods</h4>RAW 264.7 iNOS expression was induced with IFN-gamma. Deacetylase inhibitors trichostatin A (TSA) or valproic acid (VPA) were added. Stat1 and iNOS mRNA and protein were measured. Acetylated Stat1 was determined by immunoprecipitation. Chromatin immunoprecipitation assessed in vivo binding of Stat1 to the ...[more]

PMID: 17689680

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Project description:Interferon gamma (IFN-?) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-? confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-?. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-?-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-?-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN-?. Instead, we found that 2DG rescues bacterial replication by inhibiting the expression of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune-responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirmed that loss of both iNOS and IRG1, but not individual deficiency in either gene, partially reduced IFN-?-mediated restriction of L. pneumophila Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we found that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3, and NOX2) resulted in a total loss of L. pneumophila restriction by IFN-? in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-?-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages.IMPORTANCE Legionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN-?) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has identified the exact molecular factors induced by IFN-? that are required for its activity. We generated macrophages lacking different combinations of IFN-?-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN-?-mediated restriction of L. pneumophila We identified six genes that comprise the totality of the IFN-?-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN-? and highlight how redundancy downstream of IFN-? is key to prevent exploitation of macrophages by pathogens.

Dataset Information

Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-gamma-treated RAW264.7 murine macrophages.

Publications

Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-gamma-treated RAW264.7 murine macrophages.

Similar Datasets

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