Damien Le Menuet

The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology

The last decade has witnessed tremendous progress in the understanding of the mineralocorticoid receptor (MR), its molecular mechanism of action, and its implications for physiology and pathophysiology. After the initial cloning of MR, and identification of its gene structure and promoters, it now appears as a major actor in protein-protein interaction networks. The role of transcriptional coregulators and the determinants of mineralocorticoid selectivity have been elucidated. Targeted oncogenesis and transgenic mouse models have identified unexpected sites of MR expression and novel roles for MR in non-epithelial tissues. These experimental approaches have contributed to the generation of new cell lines for the characterization of aldosterone signaling pathways, and have also facilitated a better understanding of MR physiology in the heart, vasculature, brain and adipose tissues. This review describes the structure, molecular mechanism of action and transcriptional regulation mediated by MR, emphasizing the most recent developments at the cellular and molecular level. Finally, through insights obtained from mouse models and human disease, its role in physiology and pathophysiology will be reviewed. Future investigations of MR biology should lead to new therapeutic strategies, modulating cell-specific actions in the management of cardiovascular disease, neuroprotection, mineralocorticoid resistance, and metabolic disorders.

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 signaling pathway throughout development: Expression, regulation and pathophysiological implications

The mineralocorticoid signaling pathway has gained interest over the past few years, considering not only its implication in numerous pathologies but also its emerging role in physiological processes during kidney, brain, heart and lung development. This review aims at describing the setting and regulation of aldosterone biosynthesis and the expression of the mineralocorticoid receptor (MR), a nuclear receptor mediating aldosterone action in target tissues, during the perinatal period. Specificities concerning MR expression and regulation during the development of several major organs are highlighted. We provide evidence that MR expression is tightly controlled in a tissue-specific manner during development, which could have major pathophysiological implications in the neonatal period.

Expression and function of the human mineralocorticoid receptor : Lessons from transgenic mouse models

The human mineralocorticoid receptor (hMR), a ligand-dependent transcription factor (NR3C2) which belongs to the nuclear receptor superfamily, mediates most of the known effects of aldosterone. Beside its involvement in the regulation of sodium balance and the control of blood pressure, aldosterone-hMR tandem also exerts important regulatory functions on the cardiovascular and central nervous systems. To study the molecular mechanisms involved in the tissue-specific expression of hMR and explore its functional implication in pathophysiology, transgenic mouse models have been generated using both targeted oncogenesis and MR overexpression. We have previously demonstrated that the transcription of hMR is directed by two alternative promoters, P1 and P2, which correspond to the 5-flanking regions of the untranslated exons 1alpha and 1beta of the hMR gene, respectively. Utilization of P1 and P2 to drive expression of the SV40 large T antigen (TAg) in transgenic mice led us (i) to determine distinct tissue-specific patterns of promoter usage; (ii) to identify novel sites of MR expression including brown adipose tissue, thus providing a new functional link between aldosterone and energy homeostasis; (iii) to generate original immortalized cell lines derived from numerous aldosterone-sensitive tissues most notably distal nephron, brown fat, skin, liver, lung, brain, heart, blood vessels and inner ear. These differentiated cell lines represent suitable models to further explore cell-specific mineralocorticoid responses and cross-talk with other signaling pathways. Generation of transgenic mice in which hMR expression was directed by P1 promoter demonstrated the importance of MR in the cardiac and renal function. Morphological and functional alterations of the renal tubule were observed with basal decreased sodium/potassium ratio exacerbated under sodium depletion. Hypokinetic dilated cardiomyopathies were associated with tachycardia, arrhythmia but normal arterial blood pressure emphasizing the direct role of MR on cardiomyocyte function. Taken together, transgenic animal models constitute valuable experimental systems to gain new insights into the widespread and pleiotropic in vivo functions of MR.

The neuronal mineralocorticoid receptor: from cell survival to neurogenesis.

Mineralocorticoid receptor (MR), a hormone-activated transcription factor belonging to the nuclear receptor superfamily, exerts widespread actions in many tissues such as tight epithelia, the cardiovascular system, adipose tissues and macrophages. In the mammalian brain, MR is present in the limbic areas where it is highly expressed in neurons of the hippocampus and mostly absent in other regions while the glucocorticoid receptor (GR) expression is ubiquitous. MR binds both aldosterone and glucocorticoids, the latter having a ten-fold higher affinity for MR than for the closely related GR. However, owing to the minimal aldosterone transfer across the blood brain barrier and the absence of neuronal 11β hydroxysteroid dehydrogenase type 2 as an intracellular gate-keeper, neuronal MR appears to be fully occupied even at low physiological glucocorticoid levels while GR activation only occurs at high glucocorticoid concentrations, i.e. at the peak of the circadian rhythm or under stress. This defined a one hormone/two receptors system that works in balance, modulating a large spectrum of actions in the central nervous system. MR and GR are involved in the stress responses, the regulation of neuron excitability, long term potentiation, neuroprotection and neurogenesis in the dentate gyrus. MR thus constitutes a key factor in the arising of higher cognitive functions such as memorization, learning and mood. This review presents an overview of various roles of MR in the central nervous system which are somewhat less studied than that of GR, in the light of recent data obtained using cellular models, animal models and clinical investigations.

Regulation of mineralocorticoid receptor expression during neuronal differentiation of murine embryonic stem cells

Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem cell (ES) lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene green fluorescent protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted us to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites), and the increase in the expression of neuronal markers (nestin, beta-tubulin III, and microtubule-associated protein-2) as demonstrated by immunocytochemistry and quantitative PCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively, and correlated with MR expression. Finally, although progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by small interfering RNA. In conclusion, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases.

Mineralocorticoid receptor overexpression in embryonic stem cell-derived cardiomyocytes increases their beating frequency

AIMSnCardiac mineralocorticoid receptor (MR) activation triggers adverse cardiovascular events that could be efficiently prevented by mineralocorticoid antagonists. To gain insights into the pathophysiological role of MR function, we established embryonic stem (ES) cell lines from blastocysts of transgenic mice overexpressing the human MR driven by its proximal P1 or distal P2 promoter and presenting with cardiomyopathy, tachycardia, and arrhythmia. Cardiomyocyte differentiation allowed us to investigate the molecular mechanisms contributing to MR-mediated cardiac dysfunction.nnnMETHODS AND RESULTSnDuring cardiac differentiation, wild-type (WT) and recombinant ES cell cultures and excised beating patches expressed endogenous MR along with cardiac gene markers. The two-fold increase in MR protein detected in P1.hMR and P2.hMR cardiomyocytes led to a parallel increase in the spontaneous beating frequency of hMR-overexpressing cardiomyocytes compared with WT. The MR-mediated chronotropic effect was ligand-independent, could be partially repressed by spironolactone, and was accompanied by a significant two- to four-fold increase in mRNA and protein levels of the pacemaker channel HCN1, generating depolarizing If currents, thus revealing a potential new MR target. This was associated with modification in the expression of HCN4, the inward-rectifier potassium channel Kir2.1, and the L-type voltage-dependent calcium channel Cav1.2.nnnCONCLUSIONnWe demonstrate that the amplification of MR signalling in ES-derived cardiomyocytes has a major impact on cardiomyocyte contractile properties through an important remodelling of ion channel expression, contributing to arrhythmias. Our results highlight the prominent role of MR function in cardiac physiology and support the benefit of MR antagonists in the management of cardiac dysfunctions.

Mineralocorticoid Receptor Overexpression Facilitates Differentiation and Promotes Survival of Embryonic Stem Cell-Derived Neurons

Mineralocorticoid receptor (MR), highly expressed in the hippocampus, binds corticosteroid hormones and coordinately participates, with the glucocorticoid receptor, to the control of stress responses, memorization, and behavior. To investigate the impact of MR in neuronal survival, we generated murine embryonic stem (ES) cells that overexpress human MR (hMR) (P1-hMR) and are induced to differentiate into mature neurons. We showed that recombinant MR expression increased throughout differentiation and is 2-fold higher in P1-hMR ES-derived neurons compared with wild-type controls, whereas glucocorticoid receptor expression was unaffected. Although proliferation and early neuronal differentiation were comparable in P1-hMR and wild-type ES cells, MR overexpression was associated with higher late neuronal marker expression (microtubule-associated protein 2 and β-tubulin III). This was accompanied by a shift towards neuron survival with an increased ratio of anti- vs. proapoptotic molecules and 50% decreased caspase 3 activity. Knocking down MR overexpression by small interfering RNA drastically reversed neuroprotective effects with reduced Bcl(2)/Bax ratio and decreased microtubule-associated protein 2 expression. P1-hMR neurons were protected against oxidative stress-induced apoptosis through reduced caspase 3 activation and drastically increased Bcl(2)/Bax ratio and β-tubulin III expression. We demonstrated the involvement of MR in neuronal differentiation and survival and identify MR as an important neuroprotective mediator opening potential pharmacological strategies.

Paradoxical resistance to high-fat diet-induced obesity and altered macrophage polarization in mineralocorticoid receptor-overexpressing mice

The mineralocorticoid receptor (MR) exerts proadipogenic and antithermogenic effects in vitro, yet its in vivo metabolic impact remains elusive. Wild type (WT) and transgenic (Tg) mice overexpressing human MR were subjected to standard chow (SC) or high-fat diet (HFD) for 16 wk. Tg mice had a lower body weight gain than WT animals and exhibited a relative resistance to HFD-induced obesity. This was associated with a decrease in fat mass, an increased population of smaller adipocytes, and an improved glucose tolerance compared with WT animals. Quantitative RT-PCR studies revealed decreased expression of PPARγ2, a master adipogenic gene, and of glucocorticoid receptor and 11β-hydroxysteroid dehydrogenase type 1, consistent with an impaired local glucocorticoid signaling in adipose tissues (AT). This paradoxical resistance to HFD-induced obesity was not related to an adipogenesis defect since differentiation capacity of Tg preadipocytes isolated from stroma-vascular fractions was unaltered, suggesting that other nonadipocyte factors might compromise AT development. Although AT macrophage infiltration was not different between genotypes, Tg mice exhibited a distinct macrophage polarization, as revealed by FACS analysis and CD11c/CD206 expression studies. We further demonstrated that Tg macrophage-conditioned medium partially impaired preadipocyte differentiation. Therefore, we propose that modification of M1/M2 polarization of hMR-overexpressing macrophages could account in part for the metabolic phenotype of Tg mice. Collectively, our results provide evidence that MR exerts a pivotal immunometabolic role by controlling adipocyte differentiation processes directly but also indirectly through macrophage polarization regulation. Our findings should be taken into account for the pharmacological treatment of metabolic disorders.

Mineralocorticoid receptor and embryonic stem cell models: molecular insights and pathophysiological relevance.

Mineralocorticoid receptor (MR) signaling is pivotal for numerous physiological processes and implicated in various pathological conditions concerning among others, tight epithelia, central nervous and cardiovascular systems. For decades, the pleiotropic actions of MR have been investigated using animal and cellular models as well as by clinical studies. Here is reviewed and contextualized the utilization of a strategy that recently emerged to analyze the complexity of MR signaling: the derivation and differentiation of mouse embryonic stem (ES) cell models. ES cells were derived from wild-type or transgenic MR overexpressing animals. Undifferentiated ES cells were differentiated into cardiomyocytes, neurons and adipocytes, these cell types being important pathophysiological targets of MR. These approaches have already brought new insights concerning MR effect on cardiomyocyte contractility and ionic channel remodeling, in the regulation of neuronal MR expression and its positive role on neuron survival. Differentiated ES cell models thus constitute powerful and promising tools to further decipher the molecular mechanisms of cell-specific MR actions.