Involvement of Transducer of Regulated cAMP Response Element-Binding Protein Activity on Corticotropin Releasing Hormone Transcription
Involvement of Transducer of Regulated cAMP Response Element-Binding Protein Activity on Corticotropin Releasing Hormone Transcription
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DOI：
10.1210/en.2009-0963
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发表时间：
2010-03-01
期刊：
影响因子：
4.8
通讯作者：
Aguilera, Greti
中科院分区：
文献类型：
--
作者：
Liu, Ying;Coello, Ana G.;Aguilera, Greti
我们最近表明，磷酸化cAMP反应元件结合蛋白（CREB）是必不可少的，但不足以激活CRH转录，这表明需要一个辅激活因子。在这里，我们测试的假设，CREB辅激活因子，调节CREB活性（TORC）的换能器，需要激活CRH转录，使用细胞系4 B和下丘脑神经元的原代培养物。4 B细胞的免疫组织化学和蛋白质印迹实验显示，TORC 1，TORC 2，和TORC 3的毛喉素的时间依赖性核转位[而不是由佛波酯，佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）]在浓度依赖性的方式。在报告基因检测中，TORC 1或TORC 2的共转染增强了毛喉素对CRH启动子活性的刺激作用，但在PMA处理的细胞中没有效果。使用沉默RNA敲除内源性TORC显著抑制4 B细胞中毛喉素激活的CRH启动子活性，以及毛喉素在原代神经元培养物中诱导内源性CRH初级转录物。免疫共沉淀和染色质免疫沉淀实验显示，在4 B细胞的CREB和TORC在细胞核中的协会，和招聘的TORC 2的CRH启动子，20分钟孵育后与毛喉素。这些研究证明了TORC核转位与CRH启动子的关联和CRH转录的激活之间的相关性。这些数据表明，TORC是CRH启动子的转录激活所需的CREB共激活剂。此外，在PMA处理过程中TORC的细胞质保留可能解释了尽管有效磷酸化CREB，但佛波酯未能激活CRH转录的原因。（内分泌学151：1109-1118，2010）
We have recently shown that phospho-cAMP response element-binding protein ( CREB) is essential but not sufficient for activation of CRH transcription, suggesting the requirement of a coactivator. Here, we test the hypothesis that the CREB coactivator, transducer of regulated CREB activity (TORC), is required for activation of CRH transcription, using the cell line 4B and primary cultures of hypothalamic neurons. Immunohistochemistry and Western blot experiments in 4B cells revealed time-dependent nuclear translocation of TORC1, TORC 2, and TORC3 by forskolin [ but not by the phorbol ester, phorbol 12-myristate 13-acetate (PMA)] in a concentration-dependent manner. In reporter gene assays, cotransfection of TORC1 or TORC2 potentiated the stimulatory effect of forskolin on CRH promoter activity but had no effect in cells treated with PMA. Knockout of endogenous TORC using silencing RNA markedly inhibited forskolin-activated CRH promoter activity in 4B cells, as well as the induction of endogenous CRH primary transcript by forskolin in primary neuronal cultures. Coimmunoprecipitation and chromatin immunoprecipitation experiments in 4B cells revealed association of CREB and TORC in the nucleus, and recruitment of TORC2 by the CRH promoter, after 20-min incubation with forskolin. These studies demonstrate a correlation between nuclear translocation of TORC with association to the CRH promoter and activation of CRH transcription. The data suggest that TORC is required for transcriptional activation of the CRH promoter by acting as a CREB coactivator. In addition, cytoplasmic retention of TORC during PMA treatment is likely to explain the failure of phorbolesters to activate CRH transcription in spite of efficiently phosphorylating CREB. ( Endocrinology 151: 1109-1118, 2010)