Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, 10 Center Drive, Bethesda, Maryland 20892, USA.
Endocrinology. 2010 Mar;151(3):1109-18. doi: 10.1210/en.2009-0963. Epub 2010 Jan 15.
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.
我们最近表明，磷酸化 cAMP 反应元件结合蛋白（CREB）对于 CRH 转录的激活是必需的，但不是充分的，这表明需要一个共激活剂。在这里，我们使用细胞系 4B 和下丘脑神经元的原代培养物来测试假设，即 CREB 共激活剂，调节的 CREB 活性转导子（TORC），对于 CRH 转录的激活是必需的。4B 细胞中的免疫组织化学和 Western blot 实验表明， forskolin [而不是佛波酯，十四烷酰佛波醇 12-乙酸酯（PMA）]以浓度依赖性方式引起 TORC1、TORC2 和 TORC3 的时间依赖性核转位。在报告基因测定中，共转染 TORC1 或 TORC2 增强了 forskolin对 CRH 启动子活性的刺激作用，但在用 PMA 处理的细胞中没有作用。使用沉默 RNA 敲除内源性 TORC 可显著抑制 4B 细胞中 forskolin 激活的 CRH 启动子活性，以及原代神经元培养物中 forskolin 诱导的内源性 CRH 初级转录物。4B 细胞中的共免疫沉淀和染色质免疫沉淀实验表明，在与 forskolin 孵育 20 分钟后，细胞核中 CREB 和 TORC 之间存在关联，并且 TORC2 被 CRH 启动子募集。这些研究表明，TORC 与核内 TORC 与 CRH 启动子的关联以及 CRH 转录的激活之间存在相关性。数据表明，TORC 通过作为 CREB 共激活剂起作用，是 CRH 启动子转录激活所必需的。此外，在 PMA 处理期间 TORC 的细胞质保留可能解释了尽管有效地磷酸化 CREB，但佛波酯未能激活 CRH 转录的原因。
