CHARACTERIZATION OF THE HUMAN MINERALOCORTICOID RECEPTOR GENE 5'-REGULATORY REGION - EVIDENCE FOR DIFFERENTIAL HORMONAL-REGULATION OF 2 ALTERNATIVE PROMOTERS VIA NONCLASSICAL MECHANISMS

The mineralocorticoid receptor (MR) is a ligand-dependent transcriptio n factor involved in the regulation of sodium homeostasis. Two distinc t mRNA isoforms of the human MR (hMR) differing in their untranslated 5'-ends have recently been identified, suggesting the existence of alt ernative promoters. To eludicate the regulatory mechanisms controlling hMR gene expression, we have isolated and characterized similar to 15 kb of hMR 5'-flanking region. Various deletion mutants of regions loc ated immediately upstream of the untranslated exons lar and 1 beta (P1 : 1 kb and P2: 1.7 kb, respectively) were inserted into a luciferase r eporter gene and used in transient transfection experiments in CV-1 an d human differentiated renal H5 cells. Both regions were shown to poss ess significant functional promoter activity, more pronounced in renal cells, although P1 directed higher levels of basal transcription. Cot ransfection experiments with hMR or human glucocorticoid receptor (hGR ) revealed that, while both promoters were glucocorticoid inducible, o nly the distal P2 promoter was stimulated by;aldosterone in a dose- an d hMR-dependent manner. Furthermore, we demonstrate that hMR and hGR a re able to synergistically activate the P2 promoter, consistent with c ooperativity between the two transduction pathways. Mineralocorticoid induction was localized to a region between -318 and +123 bp of P2. Th is region does not contain any consensus hormone responsive element, a nd direct binding of hMR to this DNA sequence was not observed, indica ting that mineralocorticoid-induced transcriptional enhancement is med iated by nonclassical mechanisms. On the other hand, Spl and AP-2 bind to definite sequences on both promoters, suggesting that they represe nt important regulators of hMR promoter activity. Our results indicate that hMR gene expression is under the control of complex regulatory m echanisms involving alternative promoters and differential hormonal co ntrol, which might allow tissue-specific modulation of aldosterone act ion.