Zennaro M C, Le Menuet D, Lombès M
INSERM U 246, Institut Féderatif de Recherche Cellules Epithéliales Faculté de Médecine Xavier Bichat, Paris, France.
Mol Endocrinol. 1996 Dec;10(12):1549-60. doi: 10.1210/mend.10.12.8961265.
The mineralocorticoid receptor (MR) is a ligand-dependent transcription factor involved in the regulation of sodium homeostasis. Two distinct mRNA isoforms of the human MR (hMR) differing in their untranslated 5'-ends have recently been identified, suggesting the existence of alternative promoters. To eludicate the regulatory mechanisms controlling hMR gene expression, we have isolated and characterized approximately 15 kb of hMR 5'-flanking region. Various deletion mutants of regions located immediately upstream of the untranslated exons 1 alpha and 1 beta (P1: 1 kb and P2: 1.7 kb, respectively) were inserted into a luciferase reporter gene and used in transient transfection experiments in CV-1 and human differentiated renal H5 cells. Both regions were shown to possess significant functional promoter activity, more pronounced in renal cells, although P1 directed higher levels of basal transcription. Cotransfection experiments with hMR or human glucocorticoid receptor (hGR) revealed that, while both promoters were glucocorticoid inducible, only the distal P2 promoter was stimulated by aldosterone in a dose- and hMR-dependent manner. Furthermore, we demonstrate that hMR and hGR are able to synergistically activate the P2 promoter, consistent with cooperativity between the two transduction pathways. Mineralocorticoid induction was localized to a region between -318 and +123 bp of P2. This region does not contain any consensus hormone responsive element, and direct binding of hMR to this DNA sequence was not observed, indicating that mineralocorticoid-induced transcriptional enhancement is mediated by nonclassical mechanisms. On the other hand, Sp1 and AP-2 bind to definite sequences on both promoters, suggesting that they represent important regulators of hMR promoter activity. Our results indicate that hMR gene expression is under the control of complex regulatory mechanisms involving alternative promoters and differential hormonal control, which might allow tissue-specific modulation of aldosterone action.
盐皮质激素受体（MR）是一种参与钠稳态调节的配体依赖性转录因子。最近已鉴定出人类MR（hMR）的两种不同mRNA亚型，它们的5'-非翻译区不同，这表明存在替代启动子。为了阐明控制hMR基因表达的调控机制，我们分离并鉴定了约15 kb的hMR 5'-侧翼区域。将位于非翻译外显子1α和1β上游紧邻区域的各种缺失突变体（分别为P1：1 kb和P2：1.7 kb）插入荧光素酶报告基因，并用于CV-1和人分化肾H5细胞的瞬时转染实验。结果显示，这两个区域均具有显著的功能性启动子活性，在肾细胞中更明显，尽管P1指导更高水平的基础转录。与hMR或人类糖皮质激素受体（hGR）的共转染实验表明，虽然两个启动子均受糖皮质激素诱导，但只有远端的P2启动子以剂量和hMR依赖性方式被醛固酮刺激。此外，我们证明hMR和hGR能够协同激活P2启动子，这与两条转导途径之间的协同作用一致。盐皮质激素诱导定位于P2的-318至+123 bp之间的区域。该区域不包含任何共有激素反应元件，并且未观察到hMR与该DNA序列的直接结合，表明盐皮质激素诱导的转录增强是由非经典机制介导的。另一方面，Sp1和AP-2与两个启动子上的特定序列结合，表明它们代表hMR启动子活性的重要调节因子。我们的结果表明，hMR基因表达受涉及替代启动子和差异激素控制的复杂调控机制的控制，这可能允许醛固酮作用的组织特异性调节。
