Negative glucorticoid regulation of cyclic adenosine 3', 5'-monophosphate-stimulated corticotropin-releasing hormone-reporter expression in AtT-20 cells.
Negative glucorticoid regulation of cyclic adenosine 3', 5'-monophosphate-stimulated corticotropin-releasing hormone-reporter expression in AtT-20 cells.
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DOI：
10.1210/mend.10.3.8833660
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发表时间：
1996-03
影响因子：
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通讯作者：
H. Guardiola-Diaz;J. S. Kolinske;L. H. Gates;A. Seasholtz
中科院分区：
文献类型：
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作者：
H. Guardiola-Diaz;J. S. Kolinske;L. H. Gates;A. Seasholtz
糖皮质激素对CRH基因表达的负调节是下丘脑-垂体-肾上腺轴的关键控制因素。在这项研究中，在AtT-20细胞中，cAMP诱导的CRH-报告基因表达的糖皮质激素抑制介导的分子机制进行了研究。在这些细胞中，地塞米松以剂量依赖性方式降低毛喉素诱导的CRH报告活性表达。这种抑制是由糖皮质激素受体（GR）介导的，不需要持续的蛋白质合成。利用体外DNA酶I保护试验，在CRH 5 '侧翼和5'非翻译区内鉴定了GR DNA结合结构域的几个结合位点。这些位点独立地突变和/或缺失。在转染细胞中的功能研究表明，没有受保护的DNA序列介导糖皮质激素调节，并且介导负糖皮质激素调节的调节元件包含在相对于主要转录起始位点的-248至+4 bp的CRH DNA序列内。为了进一步定位响应糖皮质激素的DNA序列，将含有不同量的人CRH 5 ′侧翼序列的DNA片段插入到SV 40启动子的5 ′端。含有CRH cAMP反应元件的18-bp DNA片段足以赋予正cAMP调节和cAMP刺激表达的糖皮质激素抑制SV 40启动子。这些结果表明，糖皮质激素抑制毛喉素激活CRH报告基因表达AtT-20细胞发生通过干扰cAMP介导的基因表达的激活，可能通过直接或间接的GR和cAMP反应元件结合蛋白之间的相互作用。
The negative glucocorticoid regulation of CRH gene expression is a critical control element in the hypothalamic-pituitary-adrenal axis. In this study, the molecular mechanisms mediating the glucocorticoid repression of cAMP-induced CRH-reporter expression in AtT-20 cells have been examined. In these cells, dexamethasone decreases forskolin-induced expression of CRH-reporter activity in a dose-dependent manner. This repression is mediated by the glucocorticoid receptor (GR) and does not require ongoing protein synthesis. Several binding sites for the GR DNA-binding domain were identified within the CRH 5'-flanking and 5'-untranslated regions utilizing in vitro DNase I protection assays. These sites were independently mutated and/or deleted. Functional studies in transfected cells suggest that none of the protected DNA sequences mediate the glucocorticoid regulation and that the regulatory element(s) mediating negative glucocorticoid regulation is contained within the CRH DNA sequences from -248 to +4 bp relative to the major transcription initiation site. To further localize the DNA sequence(s) responsive to glucocorticoids, DNA fragments containing various amounts of human CRH 5'flanking sequences were inserted 5' to the SV40 promoter. An 18-bp DNA fragment containing the CRH cAMP-responsive element is sufficient to confer both positive cAMP regulation and glucocorticoid repression of cAMP-stimulated expression to the SV40 promoter. These results suggest that glucocorticoid repression of forskolin-activated CRH-reporter expression in AtT-20 cells occurs via interference with the cAMP-mediated activation of gene expression, possibly via direct or indirect interactions between the GR and the cAMP-responsive element-binding proteins.