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  Gastroenterology  Volume 129, Issue 5, Pages 1616-1632 (November 2005) DOI: 10.1053/j.gastro.2005.07.055 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Acetylation of STAT3 in cellulo. (A) Acetylation of endogenous STAT3 in HepG2 NE. NEs of IL-6–stimulated (20 min) HepG2 cells were immunoprecipitated (IP) either with rabbit preimmune serum (PI, lane 1) or with anti-STAT3 Ab (lane 2). Western immunoblot analysis was performed on the immune complexes with anti-AcLys–specific Ab (upper panel). The blots then were stripped and stained with anti-STAT3 Ab (lower panel) to identify the band and show the specificity of immunoprecipitation. Acetylated bovine serum albumin (AcBSA) and non-AcBSA also were used as a positive and negative control in this experiment (data not shown). (B) Acetylation of transfected STAT3. HepG2 cells were cotransfected with pEF6-STAT3(1–770) and pCMVβ-p300 eukaryotic expression vectors. After 20 minutes of IL-6 stimulation, extracts were immunoprecipitated either with anti-V5 Ab (V5, lane 2) or an unrelated monoclonal Ab (Un, lane 1). Western blot was performed with anti-AcLys Ab. (C) Metabolic labeling of STAT3 with [3H] acetic acid and p300. HCT116 cells were transfected with pCMVβp300 and either pEF6-STAT3(1–770) (lane 2) or empty vector (pEF6-V5, lane 1). After labeling with [3H] acetic acid, whole-cell extracts were immunoprecipitated with anti-V5 Ab, fractionated by SDS-PAGE, and detected by autoradiography after fluorographic enhancement. (D) Specificity of p300-mediated STAT3 acetylation. HepG2 cells were transfected with pEF6-STAT3(1–770) alone (lane 1); pEF6-STAT3(1-770), CMVβp300, and pCMVE1A (lane 2); or pEF6-STAT3(1–770) and pCMVβp300 (lane 3). After immunoprecipitation with anti-V5 Ab, Western blot was performed with anti-AcLys Ab (upper panel). Lower panel shows input for the immunoprecipitation indicating equivalent expression of STAT3(1–770). All experiments were repeated 3 times with similar results. Gastroenterology 2005 129, 1616-1632DOI: (10.1053/j.gastro.2005.07.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 p300-dependent acetylation of STAT3 in vitro. (A) pEF6-STAT3 (1–770) was used to prime a T7 polymerase–based in vitro transcription–translation reaction. The product was subjected to in vitro acetylation with p300 HAT and [3H] acetyl CoA. [3H] incorporation was quantitated by spotting onto P81 phosphocellulose paper. Shown are counts/min over background determined by scintillographic counting from a representative experiment. , STAT3; ■, histone. (B) [3H] incorporation into STAT3. Sample was prepared and treated as in A. Shown is an autoradiographic exposure after SDS-PAGE with fluorographic enhancement. Note incorporation into the 91-kilodalton STAT3 band. The data shown are representative of 1 of 3 experiments. Gastroenterology 2005 129, 1616-1632DOI: (10.1053/j.gastro.2005.07.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Identification of Ac-acceptor Lys residues. (A) Acetylation assay with STAT3 deletion constructs. HCT116 cells were transfected with CMVβp300 and either pEF6-STAT3(1–770), lane 1; pEF6-STAT3(130–770), lane 2; pEF6-STAT3(1–688), lane 3; or pEF6-STAT3(1–585), lane 4. Immunoprecipitation was performed with anti-V5 Ab. SDS-PAGE fractionated immune complexes then were probed with either anti-AcLys Ab (upper panel) or anti-V5 conjugated horseradish-peroxidase Ab (lower panel). (B) Peptide HAT assay. Equal amounts of 2 peptides spanning Lys residues in the STAT3 NH2 terminus, corresponding to residues 43-53 (K 49) and 83-101 (K 87/97) were subjected to HAT assay in the presence of recombinant p300 HAT domain and [3H] acetyl CoA. [3H] incorporation was detected by spotting onto p81 phosphocellulose paper and scintillation counting. ■, STAT3(43–53); , STAT3(83–101). (C) MALDI-TOF MS analysis of STAT3 (83–101). Synthetic STAT3 (83–101) acetylated with recombinant p300 HAT was subjected to MALDI-TOF MS. Shown is a mass spectrum. Y-axis, relative signal intensity; X-axis, mass to charge (m/z). The peak corresponding to the unmodified peptide (m/z = 2447.1) and the peak corresponding to the monoacetylated form (m/z = 2490) are indicated. (D) Acetylation assay with STAT3 Lys site mutants. Eukaryotic expression vectors pEF6-STAT3 WT (lane 1), pEF6-STAT3 K49R (lane 2), pEF6-STAT3 K87R (lane 3), or pEF6-STAT3 K49R/K87R (lane 4) were cotransfected with CMVβp300 and assayed for AcSTAT3 by anti-V5 immunoprecipitation/anti-AcLys immunoblot assay. Upper panel shows Western immunoblot with anti-AcLys Ab; lower panel is blotted with anti-V5–conjugated horseradish-peroxidase Ab as a protein expression control. Gastroenterology 2005 129, 1616-1632DOI: (10.1053/j.gastro.2005.07.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Functional activity of acetylation-defective STAT3 K49R/K87R mutation. (A) STAT3 K49R/K87R rapidly translocates into the nucleus. HepG2 cells transfected with pEF6-STAT3 WT (top row) or pEF6-STAT3 K49R/K87R (bottom row) were stimulated with IL-6 for indicated times. Cells were stained with fluorescein isothiocyanate–anti-V5 Ab. Shown is confocal immunofluorescence of representative cells. (B) STAT3 K49R/K87R affects nuclear abundance of phosphoTyr STAT3. HepG2 cells were transfected with either pEF6-STAT3(1–770) (left panel) or pEF6-STAT3 K49R/K87R (right panel) and stimulated with IL-6 for the indicated times. NE were immunoprecipitated with anti-V5 Ab. Immunoprecipitates were assayed by Western blot with either anti-pTyrSTAT3 (Santa Cruz, B7) or anti-AcLys Abs. Bottom lanes, 20% input as a loading control. (C) STAT3 K49R/K87R inhibits IL-6–mediated reporter transcription. HepG2 cells were transfected transiently with 4 μg of (hAPRE1)5 LUC reporter vector and increasing concentrations of pEF6-STAT3 K49R/K87R (lanes 2–6). WT pEF6-STAT3(1–770) served as a positive control (lane 1). Cells were stimulated with IL-6 (8 ng/mL) and luciferase activity was measured 24 hours later. Data are means ± SD (n = 3 experiments). *Significant difference from WT STAT3 expression vector (P < .05, Student t test). Inset, Western blot using anti–V5-conjugated horseradish-peroxidase Ab shows the relative expression of WT and mutant STAT3 isoforms. (D) Sequence specificity for IL-6 induction of hAPRE1-LUC reporter activity. HepG2 cells were transfected with either WT (hAPRE1)5-LUC (lane 1) or STAT binding site mutation (hAPRE1)5-Mut LUC (lanes 2–4) in the presence of either empty vector (lane 2), WT STAT3 (lane 3), or STAT3 K49R/K87R (lane 4). Cells were stimulated with IL-6 and harvested after 24 hours of stimulation for luciferase assay. Shown is mean ± SD of triplicate assay repeated twice. *P < .05 compared with unstimulated values, Student t test. □, None; ■, IL-6. (E) Effect of STAT3 K49R/K87R on endogenous IL-6–inducible gene. HepG2 cells were transfected with empty vector (lanes 1 and 2) or pEF6-STAT3 K49R/K87R (lanes 3 and 4), and IL-6 stimulated for 72 hours, a time when peak IL-6–inducible hAGT gene expression is seen.11 Shown is a Northern blot analysis. Top panel is the autoradiogram after hybridization with 32P-labeled hAGT cDNA probe. Bottom panel is the same blot hybridized with 32P-labeled 18S probe as an internal control. Gastroenterology 2005 129, 1616-1632DOI: (10.1053/j.gastro.2005.07.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 (A) Effect of acetylation-deficient STAT3 on DNA binding. HepG2 cells were transfected either with pEF6-STAT3(1–770) (lanes 1–5) or pEF6-STAT3 K49R/K87R (lanes 7–10) and stimulated with IL-6 (8 ng/mL, 15 min; lanes 2–5 and 6–10). Lanes 1 and 6 are unstimulated controls. NEs from homogeneous population of HepG2 cell transfectants were used to bind 32P-labeled WT SIE in EMSA. Lanes 3 and 4 and lanes 9 and 10 are competition with unlabeled WT SIE, whereas lanes 5 and 11 are competition with mutant SIE. Both specific complexes compete with similar affinities. (B) Ectopically expressed WT and mutant STAT3 bind to native hAGT promoter. HepG2 cells were transfected with either pEF-WT STAT3 or pEF-STAT3 STAT3 K49R/K87R and protein-DNA cross-linking was performed on control (−) or IL-6–stimulated (+) (for 20 min) cells. Chromatin was immunoprecipitated with IgG, anti-STAT3 Ab, or anti-V5 Ab as indicated. Shown is ethidium bromide–stained gel of PCR reaction using hAGT-specific primer pairs. Both STAT3 WT and −K49R/K87R inducibly bind endogenous gene targets. (C) STAT3 K49R/K87R is deficient in co-activator recruitment. AcLys87 STAT3 associates with p300. IL-6–stimulated HepG2 cells were fixed and stained with anti-AcLys87 STAT3 or anti-p300 antibodies. Shown are the immunofluorescent images from a confocal plane through the nucleus. Right panel is the superimposed image (Merge). (D) STAT3 K49R/K87R does not strongly bind p300. HepG2 cells were transfected with pCMVβp300 and either pEF6-STAT3(1–770) (lane 1) or pEF6-STAT3 K49R/K87R (lane 2). IL-6–stimulated (20 min) NEs were prepared and immunoprecipitated with anti-p300 Ab. Immune complexes were analyzed by Western blot for STAT3 association using anti–V5-conjugated horseradish-peroxidase Ab. Lower panel: Western blot of transfected lysates indicates similar expression levels of V5-tagged WT and mutant STAT3. Gastroenterology 2005 129, 1616-1632DOI: (10.1053/j.gastro.2005.07.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Acetylated STAT3 and co-activator p300 bind to the native angiotensinogen promoter. (A) ChIP assay of STAT3. Protein-DNA cross-linked extracts of control and IL-6–stimulated HepG2 cells (20 min) were immunoprecipitated with IgG, anti-STAT3 (S3) Ab, or anti-AcSTAT3 (AcS3) Ab. Shown is ethidium bromide–stained gel of PCR reaction using hAGT-specific primer pairs. (B) Inducible p300 and Ac histone 3 (H3) formation on hAGT. Protein-DNA cross-links after 0, 30, 60, and 120 minutes of IL-6 stimulation were immunoprecipitated with IgG, anti-P300 Ab, or anti-AcH3 Ab. PCRs were performed as in A. PC indicates positive control for genomic DNA PCR. Gastroenterology 2005 129, 1616-1632DOI: (10.1053/j.gastro.2005.07.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Formation of AcSTAT3 in LPS-induced APR. (A) Immunoprecipitation–Western blot for STAT3 modifications. NEs were prepared from LPS-injected Balb/C mice 0, 2, and 4 hours after injection. One milligram of nuclear extract was immunoprecipitated with anti-STAT3 Ab (C20; Santa Cruz), and the presence of Tyr phosphorylated STAT3 or AcSTAT3 was determined by Western blot. For AcSTAT3, both anti-AcSTAT3 and anti-AcLys antibodies were used. (B) LPS untreated and (C) treated mouse liver sections were stained with fluorescein isothiocyanate–anti-STAT3 or anti-phosphor-Tyrosine STAT3 or acetylated STAT3 Ab. Shown is confocal immunofluorescence of representative cells. Gastroenterology 2005 129, 1616-1632DOI: (10.1053/j.gastro.2005.07.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 In cellulo interaction of STAT3 with HDACs. (A) HCT116 cells were cotransfected with expression vector of WT STAT3 and FLAG-tagged HDACs 1–6. The immunoprecipitates of cell extracts with FLAG antibody were analyzed for V5-tagged STAT3. Lane 1 is the cell extract with pcDNA3 only and lanes 2–7 are the cell extracts of STAT3 along with HDACs 1–6 (upper panel). Lower panel indicates expression levels of FLAG-tagged HDACs. (B) Effect of HDAC expression on IL-6–induced hAGT activation. HepG2 cells were transfected with (−991/+22) hAGT/LUC promoter reporter construct in the absence or presence of HDACs 1–6 and stimulated with IL-6. Twenty-four hours after stimulation cells were harvested and luciferase activity was measured. Data are means ± SD of 1 of 3 experiments (n = 3). *Significant difference from WT STAT3 expression vector (P < .05, Student t test). □, (−) IL-6; ■, (+) IL-6. (C) HepG2 cells were transfected with WT STAT3 and stimulated with IL-6 for indicated times. After immunoprecipitation of whole-cell extract with anti-V5 Ab, the immune complexes were assayed for HDAC-1 by Western immunoblot. Shown is the blot probed with anti–HDAC-1 Ab (Upstate Biotechnology). Gastroenterology 2005 129, 1616-1632DOI: (10.1053/j.gastro.2005.07.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Model for the role of acetylation/deacetylation in the Jak/STAT pathway. In response to ligand (IL-6) stimulation, STAT3 undergoes tyrosine phosphorylation, dimerization, and nuclear translocation. Phosphorylated STAT3 dimers recruit the co-activator p300/CREB-binding protein, which, in turn, acetylates the NH2 terminus of STAT3. HDAC 1– and 4–mediated deacetylation of STAT3 molecule serves to dissociate p300 and target the protein to the rapid Crm1-dependent nuclear export pathway. In this way, acetylation/deacetylation serves as an intranuclear molecular switch controlling transcriptional competence of tyrosine phosphorylated STAT3. P, tyrosine phosphorylation; p300, 300-kilodalton target of E1A. Gastroenterology 2005 129, 1616-1632DOI: (10.1053/j.gastro.2005.07.055) Copyright © 2005 American Gastroenterological Association Terms and Conditions