Department of Physiology, Medical College of Georgia, 1120 15th Street, Augusta, Georgia 30912, USA.
Endocrinology. 2010 Mar;151(3):1060-70. doi: 10.1210/en.2009-0977. Epub 2010 Jan 22.
Aldosterone synthesis is regulated by angiotensin II (Ang II) and K(+) acting in the adrenal zona glomerulosa, in part through the regulation of aldosterone synthase (CYP11B2). Here, we analyzed the role of cAMP response element (CRE)-binding proteins (CREBs) in the regulation of CYP11B2. Expression analysis of activator transcription factor (ATF)/CREB family members, namely the ATF1 and ATF2, the CREB, and the CRE modulator, in H295R cells and normal human adrenal tissue was performed using quantitative real-time PCR. Ang II-induced phosphorylation of ATF/CREB members was analyzed by Western blot analysis, and their subsequent binding to the CYP11B2 promoter using chromatin immunoprecipitation assay. Aldosterone production and CYP11B2 expression were measured in small interfering RNA-transfected cells to knockdown the expression of ATF/CREB members. CYP11B2 promoter activity was measured in H295R cells cotransfected with NURR1 (NR4A2) alone or with constitutively active vectors for ATF/CREB members. Ang II induced phosphorylation of ATF1, ATF2, and CRE modulator in a time-dependent manner. Based on chromatin immunoprecipitation analysis, there was an increased association of these proteins with the CYP11B2 promoter after Ang II and K(+) treatment. Phosphorylated ATF/CREB members also bound the CYP11B2 promoter. Knockdown of ATF/CREB members reduced Ang II and K(+) induction of adrenal cell CYP11B2 mRNA expression and aldosterone production. The constitutively active ATF/CREB vectors increased the promoter activity of CYP11B2 and had a synergistic effect with NURR1. In summary, these results suggest that ATF/CREB and NGFI-B family members play a crucial role in the transcriptional regulation of CYP11B2 and adrenal cell capacity to produce aldosterone.
醛固酮的合成受血管紧张素 II（Ang II）和 K+在肾上腺球状带中的作用调节，部分通过调节醛固酮合酶（CYP11B2）。在这里，我们分析了 cAMP 反应元件（CRE）结合蛋白（CREB）在 CYP11B2 调节中的作用。使用定量实时 PCR 分析 H295R 细胞和正常人类肾上腺组织中激活转录因子（ATF）/CREB 家族成员，即 ATF1 和 ATF2、CREB 和 CRE 调节剂的表达分析。通过 Western blot 分析分析 Ang II 诱导的 ATF/CREB 成员的磷酸化，并用染色质免疫沉淀测定分析它们随后与 CYP11B2 启动子的结合。在转染小干扰 RNA 的细胞中测量醛固酮产生和 CYP11B2 表达，以敲低 ATF/CREB 成员的表达。在单独转染 NURR1（NR4A2）或转染 ATF/CREB 成员组成型激活载体的 H295R 细胞中测量 CYP11B2 启动子活性。Ang II 以时间依赖性方式诱导 ATF1、ATF2 和 CRE 调节剂的磷酸化。基于染色质免疫沉淀分析，在 Ang II 和 K+处理后，这些蛋白质与 CYP11B2 启动子的结合增加。磷酸化的 ATF/CREB 成员也与 CYP11B2 启动子结合。ATF/CREB 成员的敲低降低了 Ang II 和 K+诱导的肾上腺细胞 CYP11B2 mRNA 表达和醛固酮产生。组成型激活的 ATF/CREB 载体增加了 CYP11B2 的启动子活性，并与 NURR1 具有协同作用。总之，这些结果表明，ATF/CREB 和 NGFI-B 家族成员在 CYP11B2 和肾上腺细胞产生醛固酮的转录调节中发挥关键作用。
