Stat1 acetylation inhibits inducible nitric oxide synthase expression in interferon-gamma-treated RAW264.7 murine macrophages.
BACKGROUND
We hypothesized that acetylation of the Stat1 regulates interferon-gamma (IFN-gamma) mediated macrophage expression of inducible nitric oxide synthase (iNOS).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Guo
Lucie
L
Department of Surgery, Duke University Medical Center, Durham, NC, USA.
Guo
Hongtao
H
Gao
Chengjiang
C
Mi
Zhiyong
Z
Russell
William B
WB
Kuo
Paul C
PC
D000107:Acetylation; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects / Q000276:immunology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000276:immunology; D007371:Interferon-gamma / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic / Q000502:physiology; D050794:STAT1 Transcription Factor / Q000378:metabolism*
D000998:Antiviral Agents; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse
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