RESUMEN
Myelination allows fast and synchronized nerve influxes and is provided by Schwann cells (SCs) in the peripheral nervous system. Glucocorticoid hormones are major regulators of stress, metabolism and immunity affecting all tissues. They act by binding to two receptors, the low-affinity glucocorticoid receptor (GR) and the high-affinity mineralocorticoid receptor (MR). Little is known about the effect of glucocorticoid hormones on the PNS, and this study focuses on deciphering the role of MR in peripheral myelination. In this work, the presence of a functional MR in SCs is demonstrated and the expression of MR protein in mouse sciatic nerve SC is evidenced. Besides, knockout of MR in SC (SCMRKO using Cre-lox system with DesertHedgeHog (Dhh) Cre promoter) was undertaken in mice. SCMRKO was not associated with alterations of performance in motor behavioral tests on 2- to 6-month-old male mice compared to their controls. No obvious modifications of myelin gene expression or MR signaling gene expression were observed in the SCMRKO sciatic nerves. Nevertheless, Gr transcript and GR protein amounts were significantly increased in SCMRKO nerves compared to controls, suggesting a possible compensatory effect. Besides, an increase in myelin sheath thickness was noted for axons with perimeters larger than 15 µm in SCMRKO illustrated by a significant 4.5% reduction in g-ratio (axon perimeter/myelin sheath perimeter). Thus, we defined MR as a new player in peripheral system myelination and in SC homeostasis.
Asunto(s)
Vaina de Mielina , Receptores de Mineralocorticoides , Masculino , Ratones , Animales , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Glucocorticoides/metabolismo , Ratones Noqueados , Células de Schwann/metabolismo , Nervio CiáticoRESUMEN
Glucocorticoids are amongst the most used drugs to treat retinal diseases of various origins. Yet, the transcriptional regulations induced by glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation in retinal pigment epithelium cells (RPE) that form the outer blood-retina barrier are unknown. Levels of endogenous corticoids, ligands for MR and GR, were measured in human ocular media. Human RPE cells derived from induced pluripotent stem cells (iRPE) were used to analyze the pan-transcriptional regulations induced by aldosterone-an MR-specific agonist, or cortisol or cortisol + RU486-a GR antagonist. The retinal phenotype of transgenic mice that overexpress the human MR (P1.hMR) was analyzed. In the human eye, the main ligand for GR and MR is cortisol. The iRPE cells express functional GR and MR. The subset of genes regulated by aldosterone and by cortisol + RU-486, and not by cortisol alone, mimics an imbalance toward MR activation. They are involved in extracellular matrix remodeling (CNN1, MGP, AMTN), epithelial-mesenchymal transition, RPE cell proliferation and migration (ITGB3, PLAUR and FOSL1) and immune balance (TNFSF18 and PTX3). The P1.hMR mice showed choroidal vasodilation, focal alteration of the RPE/choroid interface and migration of RPE cells together with RPE barrier function alteration, similar to human retinal diseases within the pachychoroid spectrum. RPE is a corticosteroid-sensitive epithelium. MR pathway activation in the RPE regulates genes involved in barrier function, extracellular matrix, neural regulation and epithelial differentiation, which could contribute to retinal pathology.
Asunto(s)
Aldosterona/metabolismo , Hidrocortisona/metabolismo , Células Madre Pluripotentes/metabolismo , Receptores de Mineralocorticoides/metabolismo , Enfermedades de la Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Animales , Transición Epitelial-Mesenquimal , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Humanos , Ratones , Ratones Transgénicos , Células Madre Pluripotentes/patología , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Enfermedades de la Retina/genética , Enfermedades de la Retina/patología , Epitelio Pigmentado de la Retina/patologíaRESUMEN
OBJECTIVES: The mineralocorticoid receptor (MR), a hormone-activated transcription factor, besides its role in controlling hydroelectrolytic homeostasis, exerts pro-adipogenic and anti-thermogenic effects, inhibiting mitochondrial-uncoupling protein UCP1 expression in brown adipocytes. The aim of this study was to gain insight into the molecular mechanisms by which MR participates in such metabolic regulation. METHODS: We evaluated in vivo MR effects on cold-induced UCP1 expression in MR-overexpressing mice. Expression profiles of several transcriptional coregulators were analyzed during differentiation of the brown adipocyte T37i cell line. Given that UCP1 expression is inversely controlled by catecholamines/retinoic acid and corticosteroids, we investigated the mechanisms of MR's inhibitory effect on UCP1 transcription in T37i cells. Chromatin immunoprecipitation (ChIP) experiments enabled us to explore MR interaction with UCP1 promoter regions. RESULTS: Cold-induced UCP1 expression was blunted in the brown fat of MR-overexpressing mice. Along with induction of increasing mRNA levels for specific adipocyte markers during T37i differentiation, MR coactivator transcript levels significantly increased in intermediate states of differentiation, whereas expression of MR corepressors transiently increased approximately 2-fold. Such a simultaneous transient peak in coregulator expression is consistent with physiologically relevant cooperation occurring during brown adipogenesis. ChIP demonstrated that, after retinoic acid stimulation and aldosterone exposure, MR and PPARγ concomitantly bind to specific UCP1 promoter motifs. CONCLUSION: Our studies demonstrate that MR exerts a pivotal metabolic role by controlling energy expenditure, and provide novel information on how MR participates in the regulation of brown adipocyte function.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Receptores de Mineralocorticoides/fisiología , Termogénesis/fisiología , Transcripción Genética/fisiología , Proteína Desacopladora 1/genética , Adipocitos/química , Adipocitos/fisiología , Adipogénesis/fisiología , Aldosterona/farmacología , Animales , Diferenciación Celular/fisiología , Línea Celular , Frío , Metabolismo Energético/fisiología , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Humanos , Masculino , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/análisis , Receptores de Mineralocorticoides/genética , Termogénesis/genética , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacologíaRESUMEN
The brain-derived neurotrophic factor (BDNF) is a key player in brain functions such as synaptic plasticity, stress, and behavior. Its gene structure in rodents contains 8 untranslated exons (I to VIII) whose expression is finely regulated and which spliced onto a common and unique translated exon IX. Altered Bdnf expression is associated with many pathologies such as depression, Alzheimer's disease and addiction. Through binding to glucocorticoid receptor (GR), glucocorticoids play a pivotal role for stress responses, mood and neuronal plasticity. We recently showed in neuronal primary culture and in the immortalized neuronal-like BZ cells that GR repressed Bdnf expression, notably the bdnf exon IV containing mRNA isoform (Bdnf4) via GR binding to a short 275-bp sequence of Bdnf promoter. Herein, we demonstrate by transient transfection experiments and mutagenesis in BZ cells that GR interacts with an early growth response protein 1 (EGR1) response element (EGR-RE) located in the transcription start site of Bdnf exon IV promoter. Using Chromatin Immunoprecipitation, we find that both GR and EGR1 bind to this promoter sequence in a glucocorticoid-dependent manner and demonstrate by co-immunoprecipitation that GR and EGR1 are interacting physically. Interestingly, EGR1 has been widely characterized as a regulator of brain plasticity. In conclusion, we deciphered a mechanism by which GR downregulates Bdnf expression, identifying a novel functional crosstalk between glucocorticoid pathways, immediate early growth response proteins and Bdnf. As all these factors are well-recognized germane for brain pathophysiology, these findings may have significant implications in neurosciences as well as in therapeutics.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación de la Expresión Génica , Receptores de Glucocorticoides/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Exones , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Neuronas/metabolismo , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Brain-derived neurotrophic factor (BDNF) is involved in many functions such as neuronal growth, survival, synaptic plasticity and memorization. Altered expression levels are associated with many pathological situations such as depression, epilepsy, Alzheimer's, Huntington's and Parkinson's diseases. Glucocorticoid receptor (GR) is also crucial for neuron functions, via binding of glucocorticoid hormones (GCs). GR actions largely overlap those of BDNF. It has been proposed that GR could be a regulator of BDNF expression, however the molecular mechanisms involved have not been clearly defined yet. Herein, we analyzed the effect of a GC agonist dexamethasone (DEX) on BDNF expression in mouse neuronal primary cultures and in the newly characterized, mouse hippocampal BZ cell line established by targeted oncogenesis. Mouse Bdnf gene exhibits a complex genomic structure with 8 untranslated exons (I to VIII) splicing onto one common and unique coding exon IX. We found that DEX significantly downregulated total BDNF mRNA expression by around 30%. Expression of the highly expressed exon IV and VI containing transcripts was also reduced by DEX. The GR antagonist RU486 abolished this effect, which is consistent with specific GR-mediated action. Transient transfection assays allowed us to define a short 275 bp region within exon IV promoter responsible for GR-mediated Bdnf repression. Chromatin immunoprecipitation experiments demonstrated GR recruitment onto this fragment, through unidentified transcription factor tethering. Altogether, GR downregulates Bdnf expression through direct binding to Bdnf regulatory sequences. These findings bring new insights into the crosstalk between GR and BDNF signaling pathways both playing a major role in physiology and pathology of the central nervous system.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Regulación hacia Abajo , Neuronas/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Secuencia de Bases , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Línea Celular , Dexametasona/farmacología , Regulación hacia Abajo/efectos de los fármacos , Exones/genética , Glucocorticoides/farmacología , Hipocampo/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Elementos de Respuesta/genéticaRESUMEN
Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads.
Asunto(s)
Gametogénesis/fisiología , Gónadas/crecimiento & desarrollo , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares/metabolismo , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Gónadas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismoRESUMEN
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 11ß 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.
Asunto(s)
Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Receptores de Mineralocorticoides/metabolismo , Animales , Supervivencia Celular , Cognición , Humanos , Degeneración Nerviosa/patologíaRESUMEN
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 11ß-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.
Asunto(s)
Dieta Alta en Grasa , Expresión Génica , Macrófagos/fisiología , Obesidad/etiología , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/fisiología , Adipocitos/patología , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Glucemia/análisis , Diferenciación Celular/fisiología , Células Cultivadas , Medios de Cultivo Condicionados , Metabolismo Energético/fisiología , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/sangre , Macrófagos/patología , Masculino , Ratones , Ratones Transgénicos , Obesidad/prevención & control , PPAR gamma/genética , PPAR gamma/fisiología , Transducción de Señal , Aumento de Peso/fisiologíaRESUMEN
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.
Asunto(s)
Aldosterona/genética , Regulación del Desarrollo de la Expresión Génica , Riñón/metabolismo , Receptores de Mineralocorticoides/genética , Transducción de Señal/fisiología , Aldosterona/biosíntesis , Aldosterona/sangre , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Femenino , Feto , Corazón/embriología , Humanos , Recién Nacido , Riñón/embriología , Riñón/fisiopatología , Pulmón/embriología , Pulmón/metabolismo , Embarazo , Receptores de Mineralocorticoides/metabolismo , Sistema Renina-Angiotensina/fisiologíaRESUMEN
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 ß-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 ß-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.
Asunto(s)
Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Neuronas/citología , Receptores de Mineralocorticoides/biosíntesis , Animales , Apoptosis , Diferenciación Celular , Supervivencia Celular , Humanos , Ratones , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Neuronas/metabolismo , Estrés Oxidativo , ARN Interferente Pequeño/metabolismoRESUMEN
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.
Asunto(s)
Enfermedad/etiología , Células Madre Embrionarias/citología , Modelos Teóricos , Receptores de Mineralocorticoides/fisiología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Enfermedad/genética , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Humanos , Ratones , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismoRESUMEN
The pluripotency gene Oct4 encodes a key transcription factor that maintains self-renewal of embryonic stem cell (ESC) and is downregulated upon differentiation of ESCs and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, mediates Oct4 gene expression. Here, we show that the orphan nuclear receptor germ cell nuclear factor (GCNF) initiates Oct4 repression and DNA methylation by the differential recruitment of methyl-CpG binding domain (MBD) and DNA methyltransferases (Dnmts) to the Oct4 promoter. When compared with wild-type ESCs and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in retinoic acid (RA)-differentiated GCNF(-/-) ESCs and GCNF(-/-) embryos. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified MBD3, MBD2, and de novo Dnmts as GCNF interacting factors. Upon differentiation, endogenous GCNF binds to the Oct4 proximal promoter and differentially recruits MBD3 and MBD2 as well as Dnmt3A. In differentiated GCNF(-/-) ESCs, recruitment of MBD3 and MBD2 as well as Dnmt3A to Oct4 promoter is lost and subsequently Oct4 repression and DNA methylation failed to occur. Hypomethylation of the Oct4 promoter is also observed in RA-differentiated MBD3(-/-) and Dnmt3A(-/-) ESCs, but not in MBD2(-/-) and Dnmt3B(-/-) ESCs. Thus, recruitment of MBD3, MBD2, and Dnmt3A by GCNF links two events: gene-specific repression and DNA methylation, which occur differentially at the Oct4 promoter. GCNF initiates the repression and epigenetic modification of Oct4 gene during ESC differentiation.
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ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/fisiología , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular/fisiología , Línea Celular Tumoral , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Proteínas de Unión al ADN/genética , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Datos de Secuencia Molecular , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
AIMS: Cardiac 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. METHODS AND RESULTS: During 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. CONCLUSION: We 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.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/metabolismo , Frecuencia Cardíaca , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Receptores de Mineralocorticoides/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Línea Celular , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/genética , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Potenciales de la Membrana , Ratones , Ratones Transgénicos , Antagonistas de Receptores de Mineralocorticoides/farmacología , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/genética , Miocitos Cardíacos/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , ARN Mensajero/metabolismo , Receptores de Mineralocorticoides/genética , Espironolactona/farmacología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/metabolismo , Neuronas/metabolismo , Receptores de Mineralocorticoides/genética , Aldosterona/farmacología , Animales , Línea Celular , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica , Ratones , Microscopía Confocal , Microscopía Fluorescente , Neuronas/citología , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
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.
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Encefalopatías/metabolismo , Enfermedades Cardiovasculares/metabolismo , Núcleo Celular/metabolismo , Enfermedades Metabólicas/metabolismo , Receptores de Mineralocorticoides/metabolismo , Activación Transcripcional/fisiología , Animales , HumanosRESUMEN
The pluripotent factor Oct4 is a key transcription factor that maintains embryonic stem (ES) cell self-renewal and is down-regulated upon the differentiation of ES cells and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, are involved in the regulation of Oct4 gene expression. Here we show that the orphan nuclear receptor GCNF initiates Oct4 repression and DNA methylation by the differential recruitment of MBD (methylated CpG binding domain) factors to the promoter. Compared with wild-type ES cells and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in RA-differentiated GCNF(-/-) ES cells. The Oct4 gene is reexpressed in some somatic cells of GCNF(-/-) embryos, showing that it has not been properly silenced coincident with reduced DNA methylation of its promoter. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified methyl-DNA binding proteins, MBD3 and MBD2, as GCNF-interacting factors. In P19 and ES cells, upon differentiation, endogenous GCNF binds to the Oct4 proximal promoter and differentially recruits MBD3 and MBD2. In differentiated GCNF(-/-) ES cells, recruitment of MBD3 and MBD2 to the Oct4 promoter is lost, and repression of Oct4 expression and DNA methylation fails to occur. RNA interference-mediated knockdown of MBD3 and/or MBD2 expression results in reduced Oct4 repression in differentiated P19 and ES cells. Repression of Oct4 expression and recruitment of MBD3 are maintained in de novo DNA methylation-deficient ES cells (Dnmt3A/3B-null cells), while MBD2 recruitment is lost. Thus, recruitment of MBD3 and MBD2 by GCNF links two events, gene-specific repression and DNA methylation, which occur differentially at the Oct4 promoter. GCNF initiates the repression and epigenetic modification of Oct4 gene during ES cell differentiation.
Asunto(s)
Islas de CpG , Metilación de ADN , Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Silenciador del Gen , Factor 3 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Factor 3 de Transcripción de Unión a Octámeros/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Islas de CpG/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Transporte de Proteínas/genéticaRESUMEN
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.