Alteration of cardiac and renal functions in transgenic mice overexpressing human mineralocorticoid receptor.

The mineralocorticoid receptor (MR), a ligand-dependent transcription factor, mediates aldosterone actions in a large variety of tissues. To explore the functional implication of MR in pathophysiology, transgenic mouse models were generated using the proximal human MR (hMR) promoter to drive expression of hMR in aldosterone target tissues. Tissue-specific analysis of transgene expression in two independent transgenic animal (TG) lines by ribonuclease protection assays revealed that hMR is expressed in all mineralocorticoid-sensitive tissues, most notably in the kidney and the heart. TG exhibit both renal and cardiac abnormalities. Enlarged kidneys were histologically associated with renal tubular dilation and cellular vacuolization whose prevalence increased with aging. Renal clearance studies also disclosed a significant decrease in urinary potassium excretion rate in TG. hMR-expressing animals had normal blood pressure but developed mild dilated cardiomyopathy (increased left ventricle diameters and decreased shortening fraction), which was accompanied by a significant increase in heart rate. Differential gene expression analysis revealed a 2- to 5-fold increase in cardiac expression of atrial natriuretic peptide, serum- and glucocorticoid-induced kinase, and early growth response gene 1 as detected by microarrays; renal serum- and glucocorticoid-induced kinase was also induced significantly. Altogether, TG exhibited specific alteration of renal and cardiac functions, thus providing useful pathophysiological models to gain new insights into the tissue-specific mineralocorticoid signaling pathways.

The mineralocorticoid receptor mediates aldosterone-induced differentiation of T37i cells into brown adipocytes.

By use of targeted oncogenesis, a brown adipocyte cell line was derived from a hibernoma of a transgenic mouse carrying the proximal promoter of the human mineralocorticoid receptor (MR) linked to the SV40 large T antigen. T37i cells remain capable of differentiating into brown adipocytes upon insulin and triiodothyronine treatment as judged by their ability to express uncoupling protein 1 and maintain MR expression. Aldosterone treatment of undifferentiated cells induced accumulation of intracytoplasmic lipid droplets and mitochondria. This effect was accompanied by a significant and dose-dependent increase in intracellular triglyceride content (half-maximally effective dose 10(-9) M) and involved MR, because it was unaffected by RU-38486 treatment but was totally abolished in the presence of aldosterone antagonists (spironolactone, RU-26752). The expression of early adipogenic gene markers, such as lipoprotein lipase, peroxisome proliferator-activated receptor-gamma, and adipocyte-specific fatty acid binding protein 2, was enhanced by aldosterone, confirming activation of the differentiation process. We demonstrate that, in the T37i cell line, aldosterone participates in the very early induction of brown adipocyte differentiation. Our findings may have a broader biological significance and suggest that MR is not only implicated in maintaining electrolyte homeostasis but could also play a role in metabolism and energy balance.

Transgenic mouse models to study human mineralocorticoid receptor function in vivo.

The mineralocorticoid receptor (MR) is a transcription factor that mediates aldosterone action. MR is expressed in a wide variety of tissues, most notably in sodium-transporting epithelia, but also in nonepithelial cells of the cardiovascular and central nervous systems. However, molecular mechanisms underlying mineralocorticoid signaling and the primary mineralocorticoid-regulated genes are not fully identified. We recently showed that the human MR (hMR) gene possesses two first 5'-untranslated exons 1alpha and 1beta, and demonstrated that the 5'-flanking regions of these exons, named P1 and P2, respectively, are functional promoters that differ by their basal and corticosteroid-regulated transcriptional activities. To gain insight into the tissue-specific expression and function of MR, we have established transgenic mouse models using both targeted oncogenesis and receptor overexpression strategies. P1 and P2 promoters were used to direct expression of the large T antigen (TAg) of SV40 in constitutively MR-expressing cells. P1.TAg mice developed lethal hibernomas, while P2.TAg animals died from cerebral neuroectodermal tumors and leiomyosarcomas. Quantification of TAg messenger RNA levels revealed that P1 and P2 were differentially utilized. P1 promoter was transcriptionally active in all MR-expressing tissues and importantly directed an appropriate transgene expression in the distal nephron. Conversely, P2 activity was weak and spatially restricted. Several immortalized cell lines were established, thus constituting valuable models to investigate on aldosterone-regulated proteins. We also used P1 and P2 to target overexpression of hMR cDNA in mice. Phenotypic characterization of these mice is currently under investigation. Some transgenic lines should represent useful systems to further explore multiple functions of MR in vivo.

Targeted oncogenesis reveals a distinct tissue-specific utilization of alternative promoters of the human mineralocorticoid receptor gene in transgenic mice.

The human mineralocorticoid receptor (hMR) is a nuclear receptor mediating aldosterone action, whose expression is driven by two alternative promoters, P1 and P2, flanking the two first 5'-untranslated exons. In vivo characterization of hMR regulatory regions was performed by targeted oncogenesis in mice using P1 or P2 directing expression of the large T antigen of SV40 (TAg). While transgenic P1.TAg founders rapidly developed lethal hibernomas from brown fat, cerebral primitive neuroectodermal tumors and facial leiomyosarcomas occurred in P2.TAg mice. Quantitative analyses of mouse MR (mMR) and transgene expression indicate that P1 promoter was transcriptionally active in all MR-expressing tissues, directing strong TAg expression in testis and salivary glands, moderate in lung, brain, uterus, liver, and heart but, unlike mMR, rather low in colon and kidney. Importantly, the renal transgene expression colocalized with mMR in the distal nephron. In contrast, P2 promoter was approximately 10 times less potent than P1, with no activity in the brain and colon. Several immortalized cell lines were established from both neoplastic and normal tissues of transgenic mice. These cells exhibited differentiated characteristics and maintained MR expression, thus providing useful models for further studies exploring the widespread expression and functions of MR. Our results demonstrate that hMR gene expression in vivo is controlled by complex regulatory mechanisms involving distinct tissue-specific utilization of alternative promoters.

Hibernoma development in transgenic mice identifies brown adipose tissue as a novel target of aldosterone action.

Aldosterone is a major regulator of salt balance and blood pressure, exerting its effects via the mineralocorticoid receptor (MR). To analyze the regulatory mechanisms controlling tissue-specific expression of the human MR (hMR) in vivo, we have developed transgenic mouse models expressing the SV40 large T antigen (TAg) under the control of each of the two promoters of the hMR gene (P1 or P2). Unexpectedly, all five P1-TAg founder animals died prematurely from voluminous malignant liposarcomas originating from brown adipose tissue, as evidenced by the expression of the mitochondrial uncoupling protein ucp1, indicating that the proximal P1 promoter was transcriptionally active in brown adipocytes. No such hibernoma occurred in P2-TAg transgenic mice. Appropriate tissue-specific usage of P1 promoter sequences was confirmed by demonstrating the presence of endogenous MR in both neoplastic and normal brown adipose tissue. Several cell lines were derived from hibernomas; among them, the T37i cells can undergo terminal differentiation into brown adipocytes, which remain capable of expressing ucp1 upon adrenergic or retinoic acid stimulation. These cells possess endogenous functional MR, thus providing a new model to explore molecular mechanisms of mineralocorticoid action. Our data broaden the known functions of aldosterone and suggest a potential role for MR in adipocyte differentiation and regulation of thermogenesis.

Characterization of the human mineralocorticoid receptor gene 5'-regulatory region: evidence for differential hormonal regulation of two alternative promoters via nonclassical mechanisms.

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.