Mineralocorticoid Receptor Binding Research Articles

Glucocorticoid receptor modulator CORT125385 alleviates diet-induced hepatosteatosis in male and female mice

Non-alcoholic fatty liver disease (NAFLD) is a common condition that can progress to the more severe conditions like non-alcoholic steatohepatitis (NASH) for which limited effective therapeutic options are available. In this study, we set out to evaluate the novel glucocorticoid receptor modulator CORT125385, an analogue of the previously studied miricorilant but without mineralocorticoid receptor binding activity. Male and female mice that received high-fat diet and fructose water were treated with either vehicle, CORT125385 or mifepristone. We found that CORT125385 significantly lowered hepatic triglyceride levels in male mice, and hepatic triglyceride and cholesterol levels in female mice. Mifepristone treatment had no effect in male mice, but significantly lowered hepatic triglyceride and cholesterol levels in female mice. In reporter assays in vitro, CORT125385 showed weak partial agonism on the progesterone receptor (PR) at high doses, as well as PR antagonism at a potency 1000-fold lower than mifepristone. In vivo, CORT125385 treatment did not influence PR-responsive gene expression in the oviduct, while mifepristone treatment strongly influenced these genes in the oviduct, thus excluding in vivo PR cross-reactivity of CORT125385 at a therapeutically active dose. We conclude that CORT125385 is a promising glucocorticoid receptor modulator that effectively reduces liver steatosis in male and female mice without affecting other steroid receptors at doses that lower hepatic lipid content.

An allostatic epigenetic memory on chromatin footprints after double-hit acute stress

Stress induces allostatic responses, whose limits depend on genetic background and the nature of the challenges. Allostatic load reflects the cumulation of these reponses over the course of life. Acute stress is usually associated with adaptive responses, although, depending on the intensity of the stress and individual differences , some may experience maladaptive coping that persists through life and may influence subsequent responses to stressful events, as is the case of post-traumatic stress disorder. We investigated the behavioral traits and epigenetic signatures in a double-hit mouse model of acute stress in which heterotypic stressors (acute swim stress and acute restraint stress) were applied within a 7-day interval period. The ventral hippocampus was isolated to study the footprints of chromatin accessibility driven by exposure to double-hit stress. Using ATAC sequencing to determine regions of open chromatin, we showed that depending on the number of acute stressors, several gene sets related to development, immune function, cell starvation, translation, the cytoskeleton, and DNA modification were reprogrammed in both males and females. Chromatin accessibility for transcription factor binding sites showed that stress altered the accessibility for androgen, glucocorticoid, and mineralocorticoid receptor binding sites (AREs/GREs) at the genome-wide level, with double-hit stressed mice displaying a profile unique from either single hit of acute stress. The investigation of AREs/GREs adjacent to gene coding regions revealed several stress-related genes, including Fkbp5, Zbtb16, and Ddc, whose chromatin accessibility was affected by prior exposure to stress. These data demonstrate that acute stress is not truly acute because it induces allostatic signatures that persist in the epigenome and may manifest when a second challenge hits later in life.

Administration of finerenone in chronic kidney disease

Spironolactone is a first-generation and non-selective mineralocorticoid receptor antagonist (MRA). It is extensively well-studied and recommended due to increased accessibility for patients. Unfortunately, it is often discontinued in several cases due to its association with hyperkalemia. The apparent benefit of eplerenone over spironolactone is its mineralocorticoid receptor (MR) selectivity. However, it is also characterized by low-potency and higher cost compared to spironolactone. The high adverse-effect profile of spironolactone and eplerenone has led to the innovation of novel medications such as non-steroidal MRAs. Among these medications, finerenone is the most advanced agent. Finerenone is associated with decreased proteinuria, reduced risk of hyperkalemia and increased preservation of renal function with comparable benefit in heart failure compared to selective and nonselective MRAs. The nonsteroidal structure of finerenone affects mineralocorticoid receptor binding, lipophilicity and polarity which have potent effects on distribution, the degree of attachment to blood proteins, transportation, and tissue diffusion.

The DEXA-CORT trial: study protocol of a randomised placebo-controlled trial of hydrocortisone in patients with brain tumour on the prevention of neuropsychiatric adverse effects caused by perioperative dexamethasone

IntroductionThe synthetic glucocorticoid dexamethasone can induce serious neuropsychiatric adverse effects. Dexamethasone activates the glucocorticoid receptor (GR) but, unlike endogenous cortisol, not the mineralocorticoid receptor (MR). Moreover, dexamethasone suppresses cortisol production,...

Myeloid Mineralocorticoid Receptor Transcriptionally Regulates P-Selectin Glycoprotein Ligand-1 and Promotes Monocyte Trafficking and Atherosclerosis.

MR (mineralocorticoid receptor) activation associates with increased risk of cardiovascular ischemia while MR inhibition reduces cardiovascular-related mortality and plaque inflammation in mouse atherosclerosis. MR in myeloid cells (My-MR) promotes inflammatory cell infiltration into injured tissues and atherosclerotic plaque inflammation by unclear mechanisms. Here, we examined the role of My-MR in leukocyte trafficking and the impact of sex. We confirm in vivo that My-MR deletion (My-MR-KO) in ApoE-KO mice decreased plaque size. Flow cytometry revealed fewer plaque macrophages with My-MR-KO. By intravital microscopy, My-MR-KO significantly attenuated monocyte slow-rolling and adhesion to mesenteric vessels and decreased peritoneal infiltration of myeloid cells in response to inflammatory stimuli in male but not female mice. My-MR-KO mice had significantly less PSGL1 (P-selectin glycoprotein ligand 1) mRNA in peritoneal macrophages and surface PSGL1 protein on circulating monocytes in males. In vitro, MR activation with aldosterone significantly increased PSGL1 mRNA only in monocytes from MR-intact males. Similarly, aldosterone induced, and MR antagonist spironolactone inhibited, PSGL1 expression in human U937 monocytes. Mechanistically, aldosterone stimulated MR binding to a predicted MR response element in intron-1 of the PSGL1 gene by ChIP-qPCR. Reporter assays demonstrated that this PSGL1 MR response element is necessary and sufficient for aldosterone-activated, MR-dependent transcriptional activity. These data identify PSGL1 as a My-MR target gene that drives leukocyte trafficking to enhance atherosclerotic plaque inflammation. These novel and sexually dimorphic findings provide insight into increased ischemia risk with MR activation, cardiovascular protection in women, and the role of MR in atherosclerosis and tissue inflammation.

Distinct regulation of hippocampal neuroplasticity and ciliary genes by corticosteroid receptors

Glucocorticoid hormones (GCs) — acting through hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) — are critical to physiological regulation and behavioural adaptation. We conducted genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus to elucidate MR- and GR-regulated genes under circadian variation or acute stress. In a subset of genes, these physiological conditions resulted in enhanced MR and/or GR binding to DNA sequences and associated transcriptional changes. Binding of MR at a substantial number of sites however remained unchanged. MR and GR binding occur at overlapping as well as distinct loci. Moreover, although the GC response element (GRE) was the predominant motif, the transcription factor recognition site composition within MR and GR binding peaks show marked differences. Pathway analysis uncovered that MR and GR regulate a substantial number of genes involved in synaptic/neuro-plasticity, cell morphology and development, behavior, and neuropsychiatric disorders. We find that MR, not GR, is the predominant receptor binding to >50 ciliary genes; and that MR function is linked to neuronal differentiation and ciliogenesis in human fetal neuronal progenitor cells. These results show that hippocampal MRs and GRs constitutively and dynamically regulate genomic activities underpinning neuronal plasticity and behavioral adaptation to changing environments.

Mineralocorticoid receptors dampen glucocorticoid receptor sensitivity to stress via regulation of FKBP5.

SUMMARYResponding to different dynamic levels of stress is critical for mammalian survival. Disruption of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) signaling is proposed to underlie hypothalamic-pituitary-adrenal (HPA) axis dysregulation observed in stress-related psychiatric disorders. In this study, we show that FK506-binding protein 51 (FKBP5) plays a critical role in fine-tuning MR:GR balance in the hippocampus. Biotinylated-oligonucleotide immunoprecipitation in primary hippocampal neurons reveals that MR binding, rather than GR binding, to the Fkbp5 gene regulates FKBP5 expression during baseline activity of glucocorticoids. Notably, FKBP5 and MR exhibit similar hippocampal expression patterns in mice and humans, which are distinct from that of the GR. Pharmacological inhibition and region- and cell type-specific receptor deletion in mice further demonstrate that lack of MR decreases hippocampal Fkbp5 levels and dampens the stress-induced increase in glucocorticoid levels. Overall, our findings demonstrate that MR-dependent changes in baseline Fkbp5 expression modify GR sensitivity to glucocorticoids, providing insight into mechanisms of stress homeostasis.

Zi Shen Huo Luo Formula Prevents Aldosterone-Induced Cardiomyocyte Hypertrophy and Cardiac Fibroblast Proliferation by Regulating the Striatin-Mediated MR/EGFR/ERK Signaling Pathway.

Inappropriate activation of the renin-angiotensin-aldosterone system (RAAS) is an important factor in the development of hypertension. Excessive aldosterone can lead to myocardial extracellular matrix collagen proliferation, fibrosis, and cardiomyocyte hypertrophy and aggravate maladaptive remodeling. The results of our previous clinical and animal experiments suggested that Zi Shen Huo Luo Formula (ZSHLF) combined with perindopril can effectively control the process of left ventricular hypertrophy (LVH). The purpose of this study was to investigate whether ZSHLF-treated serum inhibits the membrane localization of the striatin-mediated mineralocorticoid receptor (MR) and affects MR-mediated nongenomic effects and the downstream epidermal growth factor receptor (EGFR)/extracellular regulated kinase (ERK) signaling pathways, thereby improving aldosterone-induced myocardial remodeling. Serum containing ZSHLF was prepared and used to treat rat cardiomyocytes and cardiac fibroblasts in vitro after aldosterone induction and striatin knockdown by small interfering RNA (siRNA). Cell-based assays were carried out to determine the cardiomyocyte surface area and assess the proliferation rate and hydroxyproline secretion of cardiac fibroblasts. Quantitative real-time PCR (qRT-PCR), immunoprecipitation (IP), and Western blotting were performed to evaluate the striatin-mediated MR/EGFR/ERK signaling pathway. In the present study, ZSHLF attenuated the aldosterone-induced hypertrophy of cardiomyocytes and inhibited the proliferation and collagen synthesis of cardiac fibroblasts. ZSHLF also reduced striatin mRNA expression and inhibited striatin and MR binding, membrane MR protein expression, and EGFR and ERK1/2 phosphorylation. Furthermore, after striatin silencing with siRNA, some of the effects of ZSHLF were not changed significantly. In conclusion, ZSHLF inhibits the downstream EGFR/ERK signaling pathway by blocking the striatin-mediated membrane localization of MR, which may be an important molecular mechanism by which ZSHLF improves aldosterone-induced myocardial remodeling.

Unexpected effects of metyrapone on corticosteroid receptor interaction with the genome and subsequent gene transcription in the hippocampus of male rats.

Glucocorticoid hormones (GCs) play a pivotal role in many stress-related biological processes. In the hippocampus, GCs act through mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) to modify gene transcription. The involvement of GCs in biological processes has been investigated using the corticosterone (CORT)-synthesis blocker metyrapone. How metyrapone affects the action of GC at the genomic level still remains unclear. Therefore, we investigated the effects of this enzyme blocker on plasma CORT levels and hippocampal MR and GR binding to GC responsive elements (GREs) within the GC target genes Fkbp5 (FK506-binding protein 5), Per1 (Period 1) and Sgk1 (Serum- and glucocorticoid-activated kinase 1), as well as the transcriptional responses of these genes under control and acute stress conditions in rats. For comparison, we also investigated these endpoints in rats that had undergone adrenalectomy (ADX). Although metyrapone had no effect on baseline levels of CORT, the drug increased MR and GR to GRE binding within the GC target genes and the transcriptional activity of these genes. As expected, acute forced swim (FS) stress strongly increased plasma CORT levels, hippocampal MR and GR to GRE binding within Fkbp5, Per1 and Sgk1, and the transcriptional activity (mainly hnRNA levels) of these genes. Metyrapone attenuated, but did not abolish, these effects of stress on plasma CORT and MR and GR to GRE binding. The drug effects on FS-induced transcriptional activity were gene-dependent with a reduction seen in Fkbp5 hnRNA (but not Fkbp5 mRNA), an enhancement in Per1 hnRNA (but not Per1 mRNA), and no effect on both Sgk1 hnRNA and mRNA levels. ADX however completely abrogated the effects of FS on plasma CORT, as well as hippocampal MR and GR to GRE binding and transcriptional responses. Thus, in contrast to ADX, metyrapone produced inconsistent effects on GC-sensitive genomic endpoints that question its suitability as a tool in neuroendocrine and other research.

Identification of mineralocorticoid receptor target genes in the mouse hippocampus

Brain mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) respond to the same glucocorticoid hormones but can have differential effects on cellular function. Several lines of evidence suggest that MR‐specific target genes must exist and might underlie the distinct effects of the receptors. The present study aimed to identify MR‐specific target genes in the hippocampus, a brain region where MR and GR are co‐localised and play a role in the stress response. Using genome‐wide binding of both receptor types, we previously identified MR‐specific, MR‐GR overlapping and GR‐specific putative target genes. We now report altered gene expression levels of such genes in the hippocampus of forebrain MR knockout (fbMRKO) mice, killed at the time of their endogenous corticosterone peak. Of those genes associated with MR‐specific binding, the most robust effect was a 50% reduction in Jun dimerization protein 2 (Jdp2) mRNA levels in fbMRKO mice. Down‐regulation was also observed for the MR‐specific Nitric oxide synthase 1 adaptor protein (Nos1ap) and Suv3 like RNA helicase (Supv3 l1). Interestingly, the classical glucocorticoid target gene FK506 binding protein 5 (Fkbp5), which is associated with MR and GR chromatin binding, was expressed at substantially lower levels in fbMRKO mice. Subsequently, hippocampal Jdp2 was confirmed to be up‐regulated in a restraint stress model, posing Jdp2 as a bona fide MR target that is also responsive in an acute stress condition. Thus, we show that MR‐selective DNA binding can reveal functional regulation of genes and further identify distinct MR‐specific effector pathways.

Mineralocorticoid Receptor May Regulate Glucose Homeostasis through the Induction of Interleukin-6 and Glucagon-Like peptide-1 in Pancreatic Islets.

Because the renin-angiotensin-aldosterone system influences glucose homeostasis, the mineralocorticoid receptor (MR) signal in pancreatic islets may regulate insulin response upon glucose load. Glucagon-like peptide-1 (GLP-1) production is stimulated by interleukin-6 (IL-6) in pancreatic α-cells. To determine how glucose homeostasis is regulated by interactions of MR, IL-6 and GLP-1 in islets, we performed glucose tolerance and histological analysis of islets in primary aldosteronism (PA) model rodents and conducted in vitro experiments using α-cell lines. We measured active GLP-1 concentration in primary aldosteronism (PA) patients before and after the administration of MR antagonist eplerenone. In PA model rodents, aldosterone decreased insulin-secretion and the islet/pancreas area ratio and eplerenone added on aldosterone (E+A) restored those with induction of IL-6 in α-cells. In α-cells treated with E+A, IL-6 and GLP-1 concentrations were increased, and anti-apoptotic signals were enhanced. The E+A-treatment also significantly increased MR and IL-6 mRNA and these upregulations were blunted by MR silencing using small interfering RNA (siRNA). Transcriptional activation of the IL-6 gene promoter by E+A-treatment required an intact MR binding element in the promoter. Active GLP-1 concentration was significantly increased in PA patients after eplerenone treatment. MR signal in α-cells may stimulate IL-6 production and increase GLP-1 secretion, thus protecting pancreatic β-cells and improving glucose homeostasis.

Mechanistic Insights in NeuroD Potentiation of Mineralocorticoid Receptor Signaling.

Mineralocorticoid receptor (MR)-mediated signaling in the brain has been suggested as a protective factor in the development of psychopathology, in particular mood disorders. We recently identified genomic loci at which either MR or the closely related glucocorticoid receptor (GR) binds selectively, and found members of the NeuroD transcription factor family to be specifically associated with MR-bound DNA in the rat hippocampus. We show here using forebrain-specific MR knockout mice that GR binding to MR/GR joint target loci is not affected in any major way in the absence of MR. Neurod2 binding was also independent of MR binding. Moreover, functional comparison with MyoD family members indicates that it is the chromatin remodeling aspect of NeuroD, rather than its direct stimulation of transcription, that is responsible for potentiation of MR-mediated transcription. These findings suggest that NeuroD acts in a permissive way to enhance MR-mediated transcription, and they argue against competition for DNA binding as a mechanism of MR- over GR-specific binding.

Liquiritinapioside – A mineralocorticoid-like substance from liquorice

Surface plasmon resonance (SPR) analysis of the main components of liquorice was performed and a novel strong mineralocorticoid receptor (MR) agonist, namely liquiritinapioside (LA), whose the binding constant was 1.093 × 10−5 M, was reported. As a supplement, LA has been further demonstrated to have a strong MR binding capacity through competitive binding experiments (the enrichment factor of LA was 10.22%). This study also validated the activity of LA on H9c2 cells. The expression of collagen I and the results of Masson staining were used to determine the ability of this substance to cause H9c2 cell fibrosis. Moreover, western blotting was used to verify the mechanism of compound-induced myocardial fibrosis. Overall, the attained results showed that LA could induce the activation of the TGF-β1/p38 MAPK signalling pathway through the MR to induce H9c2 cell fibrosis.

Up-regulation of claudin-2 expression by aldosterone in colonic epithelial cells of mice fed with NaCl-depleted diets

Dietary NaCl depletion increases Na+ absorption and K+ secretion in the colon, but the mechanisms are not fully understood. In mice fed with NaCl-depleted diets, the expression of claudin-2 and -7 increased compared to those in control mice. Aldosterone (ALD) concentration was also increased. We examined the regulatory mechanism of claudin expression by ALD using the murine colonic epithelial MCE301 cells. ALD dose-dependently increased claudin-2 expression without affecting the expression of claudin-4, -7, -8, and -15. ALD increased nuclear distribution of mineralocorticoid receptor (MR), which was inhibited by spironolactone, an MR antagonist. The ALD-induced elevation of claudin-2 mRNA and protein expression was inhibited by spironolactone, but not by RU-486, a glucocorticoid receptor antagonist. Luciferase reporter assay showed that ALD interacts with the promoter region between -2,021 and -2,008 of human claudin-2. The binding of MR on the promoter region of claudin-2 was increased by ALD, which was inhibited by spironolactone in chromatin immunoprecipitation assay. Our data suggest that ALD acts on MR and increases paracellular permeability to ions mediated by the elevation of claudin-2 expression in the colon. NaCl depletion may increase ALD secretion from adrenal cortex, resulting in the elevation of paracellular permeability to cations in the colon.

NeuroD Factors Discriminate Mineralocorticoid From Glucocorticoid Receptor DNA Binding in the Male Rat Brain.

In the limbic brain, mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) both function as receptors for the naturally occurring glucocorticoids (corticosterone/cortisol) but mediate distinct effects on cellular physiology via transcriptional mechanisms. The transcriptional basis for specificity of these MR- vs GR-mediated effects is unknown. To address this conundrum, we have identified the extent of MR/GR DNA-binding selectivity in the rat hippocampus using chromatin immunoprecipitation followed by sequencing. We found 918 and 1450 nonoverlapping binding sites for MR and GR, respectively. Furthermore, 475 loci were co-occupied by MR and GR. De novo motif analysis resulted in a similar binding motif for both receptors at 100% of the target loci, which matched the known glucocorticoid response element (GRE). In addition, the Atoh/NeuroD consensus sequence was found in co-occurrence with all MR-specific binding sites but was absent for GR-specific or MR-GR overlapping sites. Basic helix-loop-helix family members Neurod1, Neurod2, and Neurod6 showed hippocampal expression and were hypothesized to bind the Atoh motif. Neurod2 was detected at rat hippocampal MR binding sites but not at GR-exclusive sites. All three NeuroD transcription factors acted as DNA-binding-dependent coactivators for both MR and GR in reporter assays in heterologous HEK293 cells, likely via indirect interactions with the receptors. In conclusion, a NeuroD family member binding to an additional motif near the GRE seems to drive specificity for MR over GR binding at hippocampal binding sites.

Acute stress enhances heterodimerization and binding of corticosteroid receptors at glucocorticoid target genes in the hippocampus

A stressful event results in secretion of glucocorticoid hormones, which bind to mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) in the hippocampus to regulate cognitive and affective responses to the challenge. MRs are already highly occupied by low glucocorticoid levels under baseline conditions, whereas GRs only become substantially occupied by stress- or circadian-driven glucocorticoid levels. Currently, however, the binding of MRs and GRs to glucocorticoid-responsive elements (GREs) within hippocampal glucocorticoid target genes under such physiological conditions in vivo is unknown. We found that forced swim (FS) stress evoked increased hippocampal RNA expression levels of the glucocorticoid-responsive genes FK506-binding protein 5 (Fkbp5), Period 1 (Per1), and serum- and glucocorticoid-inducible kinase 1 (Sgk1). Chromatin immunoprecipitation (ChIP) analysis showed that this stressor caused substantial gene-dependent increases in GR binding and surprisingly, also MR binding to GREs within these genes. Different acute challenges, including novelty, restraint, and FS stress, produced distinct glucocorticoid responses but resulted in largely similar MR and GR binding to GREs. Sequential and tandem ChIP analyses showed that, after FS stress, MRs and GRs bind concomitantly to the same GRE sites within Fkbp5 and Per1 but not Sgk1 Thus, after stress, MRs and GRs seem to bind to GREs as homo- and/or heterodimers in a gene-dependent manner. MR binding to GREs at baseline seems to be restricted, whereas after stress, GR binding may facilitate cobinding of MR. This study reveals that the interaction of MRs and GRs with GREs within the genome constitutes an additional level of complexity in hippocampal glucocorticoid action beyond expectancies based on ligand-receptor interactions.

Chronic Psychosocial Stress and Negative Feedback Inhibition: Enhanced Hippocampal Glucocorticoid Signaling despite Lower Cytoplasmic GR Expression.

Chronic subordinate colony housing (CSC), a pre-clinically validated mouse model for chronic psychosocial stress, results in increased basal and acute stress-induced plasma adrenocorticotropic hormone (ACTH) levels. We assessed CSC effects on hippocampal glucocorticoid (GC) receptor (GR), mineralocorticoid receptor (MR), and FK506 binding protein (FKBP51) expression, acute heterotypic stressor-induced GR translocation, as well as GC effects on gene expression and cell viability in isolated hippocampal cells. CSC mice showed decreased GR mRNA and cytoplasmic protein levels compared with single-housed control (SHC) mice. Basal and acute stress-induced nuclear GR protein expression were comparable between CSC and SHC mice, as were MR and FKBP51 mRNA and/or cytoplasmic protein levels. In vitro the effect of corticosterone (CORT) on hippocampal cell viability and gene transcription was more pronounced in CSC versus SHC mice. In summary, CSC mice show an, if at all, increased hippocampal GC signaling capacity despite lower cytoplasmic GR protein expression, making negative feedback deficits in the hippocampus unlikely to contribute to the increased ACTH drive following CSC.

Are receptor concentrations correlated across tissues within individuals? A case study examining glucocorticoid and mineralocorticoid receptor binding.

Hormone receptors are a necessary (although not sufficient) part of the process through which hormones like corticosterone create physiological responses. However, it is currently unknown to what extent receptor concentrations across different target tissues may be correlated within individual animals. In this study, we examined this question using a large dataset of radioligand binding data for glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs) in 13 different tissues in the house sparrow (Passer domesticus) (n=72). Our data revealed that individual house sparrows tended to exhibit higher or lower receptor binding across all tissues, which could be part of what creates the physiological and behavioral syndromes associated with different hormonal profiles. However, although statistically significant, the correlations between tissues were very weak. Thus, when each tissue was independently regressed on receptor concentrations in the other tissues, multivariate analysis revealed significant relationships only for sc fat (for GR) and whole brain, hippocampus, kidney, omental fat, and sc fat (for MR). We also found significant pairwise correlations only between receptor concentrations in brain and hippocampus, and brain and kidney (both for MR). This research reveals that although there are generalized individual consistencies in GR and MR concentrations, possibly due to such factors as hormonal regulation and genetic effects, the ability of 2 different tissues to respond to the same hormonal signal appears to be affected by additional factors that remain to be identified.

Epigenetics of epithelial Na+channel-dependent sodium uptake and blood pressure regulation

The epithelial Na(+) channel (ENaC) consists of α, β, γ subunits. Its expression and function are regulated by aldosterone at multiple levels including transcription. ENaC plays a key role in Na(+) homeostasis and blood pressure. Mutations in ENaC subunit genes result in hypertension or hypotension, depending on the nature of the mutations. Transcription of αENaC is considered as the rate-limiting step in the formation of functional ENaC. As an aldosterone target gene, αENaC is activated upon aldosterone- mineralocorticoid receptor binding to the cis-elements in the αENaC promoter, which is packed into chromatin. However, how aldosterone alters chromatin structure to induce changes in transcription is poorly understood. Studies by others and us suggest that Dot1a-Af9 complex represses αENaC by directly binding and regulating targeted histone H3 K79 hypermethylation at the specific subregions of αENaC promoter. Aldosterone decreases Dot1a-Af9 formation by impairing expression of Dot1a and Af9 and by inducing Sgk1, which, in turn, phosphorylates Af9 at S435 to weaken Dot1a-Af9 interaction. MR attenuates Dot1a-Af9 effect by competing with Dot1a for binding Af9. Af17 relieves repression by interfering with Dot1a-Af9 interaction and promoting Dot1a nuclear export. Af17(-/-) mice exhibit defects in ENaC expression, renal Na(+) retention, and blood pressure control. This review gives a brief summary of these novel findings.

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one derivatives. Screening of alternative azole rings identified 1,3-dimethyl pyrazole 6a as a lead compound with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR binding activity while maintaining the agonistic response at the lower level. Among these compounds, 6-[1-(2,2-difluoro-3-hydroxypropyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone receptors, and good pharmacokinetic profiles. Oral administration of 37a in deoxycorticosterone acetate-salt hypertensive rats showed a significant blood pressure-lowering effect with no signs of antiandrogenic effects.