STAT3 NH2-terminal acetylation is activated by the hepatic acute-phase response and required for IL-6 induction of angiotensinogen

Sutapa Ray, Istvan Boldogh, Allan R. Brasier

Research output: Contribution to journalArticle

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Abstract

Background & Aims: The signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors mediating acute-phase response (APR) of the human angiotensinogen (hAGT) gene in hepatocytes. The mechanisms of how STAT3 activates target genes are unknown. Here we analyzed the biochemistry of STAT3 activation by interleukin (IL)-6 in hepatocellular carcinoma HepG2 and Balb/C mice. Methods: Immunoprecipitation- Western assays and Matrix Assisted Laser Desorption-Time of Flight mass spectrometry determined sites of STAT3 acetylation by the 300-kilodalton target of E1A (p300) co-activator. The subcellular localization of acetylation- deficient STAT3 molecules were studied by microscopic imaging, effects on DNA binding measured by gel shift and chromatin immunoprecipitation (ChIP) assays, and gene transactivation by Northern blot and reporter assays. Results: Two Lys residues at amino acids 49 and 87 in the STAT3 NH2 terminus are acetylated by p300. Lys-to-Arg point mutations (STAT3 K49R/K87R) had no effect on inducible DNA binding, but blocked p300-mediated acetyl(Ac)-STAT3 formation and abrogated IL-6-induced hAGT activation. Although STAT3 K49R/K87R rapidly translocated into the nucleus, it did not bind p300 and had delayed cytoplasmic redistribution. ChIP assays show IL-6-inducible acetylated STAT3 and p300 binding to the native hAGT promoter. Activation of the APR in mice induces nuclear Tyr phosphorylated and acetylated STAT3 in hepatic nuclei. We also observed that STAT3 interacts with histone deacetylases (HDACs), specifically HDAC 1, that down-regulate IL-6-induced hAGT transactivation. Conclusions: IL-6-induced target gene activation requires p300-mediated STAT3 acetylation, and HDACs are involved in the termination of STAT3 action. These studies indicate the acetylation-deacetylation reaction as a novel signaling mechanism controlling the IL-6-STAT3 pathway in the hepatic APR.

Original languageEnglish (US)
Pages (from-to)1616-1632
Number of pages17
JournalGastroenterology
Volume129
Issue number5
DOIs
StatePublished - Nov 2005

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Angiotensinogen
Acute-Phase Reaction
Acetylation
Interleukin-6
Histone Deacetylases
Liver
Transcriptional Activation
Chromatin Immunoprecipitation
Genes
DNA
Transducers
Immunoprecipitation
Point Mutation
Northern Blotting
Biochemistry
Hepatocytes
Hepatocellular Carcinoma
Mass Spectrometry
Lasers
Transcription Factors

ASJC Scopus subject areas

  • Gastroenterology

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STAT3 NH2-terminal acetylation is activated by the hepatic acute-phase response and required for IL-6 induction of angiotensinogen. / Ray, Sutapa; Boldogh, Istvan; Brasier, Allan R.

In: Gastroenterology, Vol. 129, No. 5, 11.2005, p. 1616-1632.

Research output: Contribution to journalArticle

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abstract = "Background & Aims: The signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors mediating acute-phase response (APR) of the human angiotensinogen (hAGT) gene in hepatocytes. The mechanisms of how STAT3 activates target genes are unknown. Here we analyzed the biochemistry of STAT3 activation by interleukin (IL)-6 in hepatocellular carcinoma HepG2 and Balb/C mice. Methods: Immunoprecipitation- Western assays and Matrix Assisted Laser Desorption-Time of Flight mass spectrometry determined sites of STAT3 acetylation by the 300-kilodalton target of E1A (p300) co-activator. The subcellular localization of acetylation- deficient STAT3 molecules were studied by microscopic imaging, effects on DNA binding measured by gel shift and chromatin immunoprecipitation (ChIP) assays, and gene transactivation by Northern blot and reporter assays. Results: Two Lys residues at amino acids 49 and 87 in the STAT3 NH2 terminus are acetylated by p300. Lys-to-Arg point mutations (STAT3 K49R/K87R) had no effect on inducible DNA binding, but blocked p300-mediated acetyl(Ac)-STAT3 formation and abrogated IL-6-induced hAGT activation. Although STAT3 K49R/K87R rapidly translocated into the nucleus, it did not bind p300 and had delayed cytoplasmic redistribution. ChIP assays show IL-6-inducible acetylated STAT3 and p300 binding to the native hAGT promoter. Activation of the APR in mice induces nuclear Tyr phosphorylated and acetylated STAT3 in hepatic nuclei. We also observed that STAT3 interacts with histone deacetylases (HDACs), specifically HDAC 1, that down-regulate IL-6-induced hAGT transactivation. Conclusions: IL-6-induced target gene activation requires p300-mediated STAT3 acetylation, and HDACs are involved in the termination of STAT3 action. These studies indicate the acetylation-deacetylation reaction as a novel signaling mechanism controlling the IL-6-STAT3 pathway in the hepatic APR.",
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AB - Background & Aims: The signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors mediating acute-phase response (APR) of the human angiotensinogen (hAGT) gene in hepatocytes. The mechanisms of how STAT3 activates target genes are unknown. Here we analyzed the biochemistry of STAT3 activation by interleukin (IL)-6 in hepatocellular carcinoma HepG2 and Balb/C mice. Methods: Immunoprecipitation- Western assays and Matrix Assisted Laser Desorption-Time of Flight mass spectrometry determined sites of STAT3 acetylation by the 300-kilodalton target of E1A (p300) co-activator. The subcellular localization of acetylation- deficient STAT3 molecules were studied by microscopic imaging, effects on DNA binding measured by gel shift and chromatin immunoprecipitation (ChIP) assays, and gene transactivation by Northern blot and reporter assays. Results: Two Lys residues at amino acids 49 and 87 in the STAT3 NH2 terminus are acetylated by p300. Lys-to-Arg point mutations (STAT3 K49R/K87R) had no effect on inducible DNA binding, but blocked p300-mediated acetyl(Ac)-STAT3 formation and abrogated IL-6-induced hAGT activation. Although STAT3 K49R/K87R rapidly translocated into the nucleus, it did not bind p300 and had delayed cytoplasmic redistribution. ChIP assays show IL-6-inducible acetylated STAT3 and p300 binding to the native hAGT promoter. Activation of the APR in mice induces nuclear Tyr phosphorylated and acetylated STAT3 in hepatic nuclei. We also observed that STAT3 interacts with histone deacetylases (HDACs), specifically HDAC 1, that down-regulate IL-6-induced hAGT transactivation. Conclusions: IL-6-induced target gene activation requires p300-mediated STAT3 acetylation, and HDACs are involved in the termination of STAT3 action. These studies indicate the acetylation-deacetylation reaction as a novel signaling mechanism controlling the IL-6-STAT3 pathway in the hepatic APR.

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