Hou Tieying, Ray Sutapa, Brasier Allan R
Department of Biochemistry, and Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1060, USA.
J Biol Chem. 2007 Dec 21;282(51):37091-102. doi: 10.1074/jbc.M706458200. Epub 2007 Oct 23.
The signal transducer and activator of transcription 3 (STAT3) is an IL-6-inducible transcription factor that mediates the hepatic acute phase response (APR). Using gamma-fibrinogen (FBG) as a model of the APR, we investigated the requirement of an IL-6-inducible complex of STAT3 with cyclin-dependent kinase 9 (CDK9) on gamma-FBG expression in HepG2 hepatocarcinoma cells. IL-6 induces rapid nuclear translocation of Tyr-phosphorylated STAT3 that forms a nuclear complex with CDK9 in nondenaturing co-immunoprecipitation and confocal colocalization assays. To further understand this interaction, we found that CDK9-STAT3 binding is mediated via both STAT NH2-terminal modulatory and COOH-terminal transactivation domains. Both IL-6-inducible gamma-FBG reporter gene and endogenous mRNA expression are significantly decreased after CDK9 inhibition using the potent CDK inhibitor, flavopiridol (FP), or specific CDK9 siRNA. Moreover, chromatin immunoprecipitation (ChIP) experiments revealed an IL-6-inducible STAT3 and CDK9 binding to the proximal gamma-FBG promoter as well as increased loading of RNA Pol II and phospho-Ser2 CTD Pol II on the TATA box and coding regions. Finally, FP specifically and efficiently inhibits association of phospho-Ser2 CTD RNA Pol II on the gamma-FBG promoter, indicating that CDK9 kinase activity mediates IL-6-inducible CTD phosphorylation. Our data indicate that IL-6 induces a STAT3.CDK9 complex mediated by bivalent STAT3 domains and CDK9 kinase activity is necessary for licensing Pol II to enter a transcriptional elongation mode. Therefore, disruption of IL-6 signaling by CDK9 inhibitors could be a potential therapeutic strategy for inflammatory disease.
信号转导及转录激活因子3（STAT3）是一种白细胞介素6诱导的转录因子，介导肝脏急性期反应（APR）。我们以γ-纤维蛋白原（FBG）作为急性期反应的模型，研究了白细胞介素6诱导的STAT3与细胞周期蛋白依赖性激酶9（CDK9）形成的复合物对HepG2肝癌细胞中γ-FBG表达的需求。在非变性共免疫沉淀和共聚焦共定位分析中，白细胞介素6诱导酪氨酸磷酸化的STAT3快速核转位，其与CDK9形成核复合物。为了进一步了解这种相互作用，我们发现CDK9-STAT3结合是通过STAT的氨基末端调节域和羧基末端反式激活域介导的。使用强效CDK抑制剂黄酮哌啶醇（FP）或特异性CDK9小干扰RNA（siRNA）抑制CDK9后，白细胞介素6诱导的γ-FBG报告基因和内源性mRNA表达均显著降低。此外，染色质免疫沉淀（ChIP）实验显示，白细胞介素6诱导STAT3和CDK9结合至γ-FBG近端启动子，同时RNA聚合酶II（RNA Pol II）和磷酸化丝氨酸2羧基末端结构域（CTD）的RNA Pol II在TATA盒和编码区的负载增加。最后，FP特异性且有效地抑制了γ-FBG启动子上磷酸化丝氨酸2 CTD RNA Pol II的结合，表明CDK9激酶活性介导白细胞介素6诱导的CTD磷酸化。我们的数据表明，白细胞介素6诱导由双价STAT3结构域介导的STAT3.CDK9复合物，且CDK9激酶活性是使Pol II进入转录延伸模式所必需的。因此，CDK9抑制剂破坏白细胞介素6信号传导可能是炎症性疾病的一种潜在治疗策略。
