Ray Sutapa, Boldogh Istvan, Brasier Allan R
Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-8709, USA.
Gastroenterology. 2005 Nov;129(5):1616-32. doi: 10.1053/j.gastro.2005.07.055.
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.
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.
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.
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.
信号转导及转录激活因子（STATs）是细胞质转录因子，介导肝细胞中人血管紧张素原（hAGT）基因的急性期反应（APR）。STAT3激活靶基因的机制尚不清楚。在此，我们分析了白细胞介素（IL）-6在肝癌HepG2细胞和Balb/C小鼠中激活STAT3的生物化学过程。
免疫沉淀-蛋白质印迹分析和基质辅助激光解吸-飞行时间质谱法确定E1A的300千道尔顿靶标（p300）共激活因子对STAT3乙酰化的位点。通过显微镜成像研究乙酰化缺陷型STAT3分子的亚细胞定位，通过凝胶迁移和染色质免疫沉淀（ChIP）分析测量对DNA结合的影响，并通过Northern印迹和报告基因分析检测基因反式激活。
STAT3氨基末端第49和87位氨基酸处的两个赖氨酸残基被p300乙酰化。赖氨酸到精氨酸的点突变（STAT3 K49R/K87R）对诱导型DNA结合没有影响，但阻断了p300介导的乙酰化（Ac）-STAT3形成，并消除了IL-6诱导的hAGT激活。尽管STAT3 K49R/K87R迅速转运到细胞核中，但它不与p300结合，并且细胞质再分布延迟。ChIP分析显示IL-6诱导的乙酰化STAT3和p300与天然hAGT启动子结合。小鼠中APR的激活诱导肝细胞核中酪氨酸磷酸化和乙酰化的STAT3。我们还观察到STAT3与组蛋白去乙酰化酶（HDACs）相互作用，特别是HDAC 1，其下调IL-6诱导的hAGT反式激活。
IL-6诱导的靶基因激活需要p300介导的STAT3乙酰化，并且HDACs参与STAT3作用的终止。这些研究表明乙酰化-去乙酰化反应是控制肝脏APR中IL-6-STAT3途径的一种新型信号传导机制。
