Evans Andrew N, Liu Ying, Macgregor Robert, Huang Victoria, Aguilera Greti
Section on Endocrine Physiology, Eunice Kennedy Shriver Institute of Child Health and Human Development, National Institutes of Health, Building 10/CRC, Room 1E-3330, Bethesda, Maryland 20892.
Mol Endocrinol. 2013 Nov;27(11):1796-807. doi: 10.1210/me.2013-1095. Epub 2013 Sep 24.
Negative glucocorticoid feedback is essential for preventing the deleterious effects of excessive hypothalamic pituitary adrenal axis axis activation, with an important target being CRH transcription in the hypothalamic paraventricular nucleus. The aim of these studies was to determine whether glucocorticoids repress CRH transcription directly in CRH neurons, by examining glucocorticoid effects on glucocorticoid receptor (GR)-CRH promoter interaction and the activation of proteins required for CRH transcription. Immunoprecipitation of hypothalamic chromatin from intact or adrenalectomized rats subjected to either stress or corticosterone injections showed minor association of the proximal CRH promoter with the GR compared with that with phospho-CREB (pCREB). In contrast, the Period-1 (Per1, a glucocorticoid-responsive gene) promoter markedly recruited GR. Stress increased pCREB recruitment by the CRH but not the Per1 promoter, irrespective of circulating glucocorticoids. In vitro, corticosterone pretreatment (30 minutes or 18 hours) only slightly inhibited basal and forskolin-stimulated CRH heteronuclear RNA in primary hypothalamic neuronal cultures and CRH promoter activity in hypothalamic 4B cells. In 4B cells, 30 minutes or 18 hours of corticosterone exposure had no effect on forskolin-induced nuclear accumulation of the recognized CRH transcriptional regulators, pCREB and transducer of regulated CREB activity 2. The data show that inhibition of CRH transcription by physiological glucocorticoids in vitro is minor and that direct interaction of GR with DNA in the proximal CRH promoter may not be a major mechanism of CRH gene repression. Although GR interaction with distal promoter elements may have a role, the data suggest that transcriptional repression of CRH by glucocorticoids involves protein-protein interactions and/or modulation of afferent inputs to the hypothalamic paraventricular nucleus.
负性糖皮质激素反馈对于预防下丘脑-垂体-肾上腺轴过度激活的有害影响至关重要，其中一个重要靶点是下丘脑室旁核中的促肾上腺皮质激素释放激素（CRH）转录。这些研究的目的是通过检查糖皮质激素对糖皮质激素受体（GR）-CRH启动子相互作用以及CRH转录所需蛋白质激活的影响，来确定糖皮质激素是否直接在CRH神经元中抑制CRH转录。对完整或肾上腺切除的大鼠进行应激或注射皮质酮后，对下丘脑染色质进行免疫沉淀，结果显示，与磷酸化CREB（pCREB）相比，近端CRH启动子与GR的结合较弱。相反，周期蛋白1（Per1，一种糖皮质激素反应基因）启动子能显著募集GR。无论循环糖皮质激素水平如何，应激都会增加CRH启动子而非Per1启动子对pCREB的募集。在体外，皮质酮预处理（30分钟或18小时）仅轻微抑制原代下丘脑神经元培养物中基础和福斯可林刺激的CRH异核RNA以及下丘脑4B细胞中CRH启动子活性。在4B细胞中，暴露于皮质酮30分钟或18小时对福斯可林诱导的公认CRH转录调节因子pCREB和调节性CREB活性转导蛋白2的核积累没有影响。数据表明，生理浓度的糖皮质激素在体外对CRH转录的抑制作用较小，并且GR与近端CRH启动子中DNA的直接相互作用可能不是CRH基因抑制的主要机制。尽管GR与远端启动子元件的相互作用可能起作用，但数据表明糖皮质激素对CRH的转录抑制涉及蛋白质-蛋白质相互作用和/或对下丘脑室旁核传入输入的调节。
