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

doi:10.1016/j.surg.2007.02.016

Surgery

Volume 142, Issue 2, August 2007, Pages 156-162

https://doi.org/10.1016/j.surg.2007.02.016 Get rights and content

Background

We hypothesized that acetylation of the Stat1 regulates interferon-γ (IFN-γ) mediated macrophage expression of inducible nitric oxide synthase (iNOS).

Methods

RAW 264.7 iNOS expression was induced with IFN-γ. 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.

Results

IFN-γ 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-γ increased acetylated Stat1 by 5-fold (P < .02 vs control); TSA + IFN-γ 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-γ (P < .01 vs control). In TSA + IFN-γ, 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.

Conclusions

Acetylation of Stat1 protein correlates with decreased Stat1 binding to the iNOS promoter with resultant inhibition of IFN-γ–mediated iNOS expression. Acetylation of the Stat1 protein may downregulate iNOS expression in proinflammatory states.

Section snippets

Cell culture

RAW 264.7 macrophages were maintained in Dulbecco modified Eagle medium (DMEM) with 10% heat-inactivated fetal calf serum, 100-U/mL penicillin, and 100-μg/mL streptomycin, and incubated in 5% CO₂–95% air at 37°C. IFN-γ (500 u) was used to induce NO synthesis. In selected instances, the deacetylase inhibitors trichostatin A (TSA; 200 nmol/L) and valproic acid (VPA; 1.5 mmol/L) were used. After incubation for 3, 12, and/or 24 hours, the supernatants and cells were harvested.

Assay of NO production

After stimulation, 50

Results

RAW264.7 murine macrophages were exposed to IFN-γ (500 U/mL) for 0, 3, 12, and 24 hours. Unstimulated cells served as controls. TSA and VPA were added in the presence and absence of IFN-γ to inhibit deacetylase activity. NO was measured as nitrite, the NO metabolite (Fig 1, A). In the presence of IFN-γ, NO production was readily detected after 12 and 24 hours with concentrations of 62 ± 2.3 μmol/L and 88 ± 4.8 μmol/L, respectively (P < .01 vs control at 12 and 24 hours). In IFN + TSA cells, NO

Discussion

In the current study, we demonstrate that acetylation of Stat1 in IFN-γ–stimulated RAW 264.7 murine macrophages accompanies decreased binding to the iNOS promoter GAS binding site, decreased iNOS mRNA and protein expression, and decreased NO production. This result occurs despite increased levels of activated Stat1 that is phosphorylated at Tyr 701. Stat1 Tyr701 phosphorylation induces Stat1 dimerization, nuclear translocation, and DNA binding, which suggests that a hierarchy of acetylation

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: Supported in part by grants GM65113 (P.C.K.), AI44629 (P.C.K.), and DK070642 (P.C.K.) from the National Institutes of Health.

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Society of University SurgeonStat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-γ–treated RAW264.7 murine macrophages

Background

Methods

Results

Conclusions

Section snippets

Cell culture

Assay of NO production

Results

Discussion

J Crit Care

Biochem Biophys Res Commun

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

Cell

J Biol Chem

The high output nitric oxide pathway: role and regulation

J Leukocyte Biol

Nitric oxide and shock

Dis Mon

Transcriptional responses to polypeptide ligands: the JAK-STAT pathway

Annu Rev Biochem

Acetylation: a regulatory modification to rival phosphorylation?

EMBO J

Society of University Surgeon
Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-γ–treated RAW264.7 murine macrophages