Mineralocorticoid Receptor Protein Research Articles

Mineralocorticoid receptor overactivation in diabetes mellitus: role of O-GlcNAc modification.

Hypertension is a significant risk factor for microangiopathy and cardiovascular complications in diabetic patients. The efficacy of mineralocorticoid receptor (MR) antagonists in impeding the advancement of diabetic nephropathy, along with the reduction in active renin concentration observed in diabetic retinopathy, strongly implies the involvement of MR overactivation in diabetic complications. This review provides a comprehensive review of various mechanisms proposed for MR overactivation in diabetes mellitus. In particular, it focuses on post-translational MR modifications, including O-linkedN-acetylglucosamine modification and phosphorylation, which have been implicated in MR protein stabilization and overactivation under conditions of high glucose. Given the role of MR overactivation in hyperglycemia, it emerges as a promising therapeutic target for preventing diabetic complications. Post-translational modifications (PTMs), such as O-GlcNAcylation and phosphorylation, are related to MR overactivation in diabetes and metabolic syndrome. Aldosterone binding promotes the proteasomal degradation of MR. Under conditions of high glucose, O-GlcNAcylation, and PKCβ-mediated MR phosphorylation are increased. Salt loading and oxidative stress also increase MR phosphorylation through the EGER/ERK pathway. PTMs inhibit ubiquitin attachment to the MR and interfere with the receptor's aldosterone-induced proteasomal degradation. Consequently, they increase the sensitivity of the MR to aldosterone and exacerbate aldosterone-associated complications.

First Evidence of Mineralocorticoid Receptor Gene and Protein Expression in Rat and Human Thyroid Tissues and Cell Cultures.

Aldosterone (Aldo) exerts its action through binding with the mineralocorticoid receptor (MR). Clinically, a link between primary aldosteronism (PA) and thyroid diseases has been hypothesised. However, the presence and activity of MR on the thyroid have not yet been demonstrated. We investigated the gene/protein expression and activation of MR in primary thyroid cell cultures (normal rat thyroid [FRTL-5] and human papillary thyroid cancer [PTC] cell lines, BCPAP and K1) through qRT-PCR analysis, immunofluorescence, and confocal microscopy. We also studied the effects of Aldo on thyroid-specific and inflammation genes in vitro. Paired human normal and neoplastic thyroid tissues were also studied. We demonstrated both gene and protein expression and activation of MR in normal rat thyroid and human PTC lines. Incubation with Aldo induced an acute increase in IL-6 expression in both the FRTL-5 and BCPAP lines, which was antagonised by spironolactone, and an acute and late upregulation of thyroid-specific genes in FRTL-5. MR was also expressed at both gene and protein levels in normal human thyroid tissues and in PTC, with a progressive decline during neoplastic tumourigenesis, particularly in more aggressive histotypes. We present the first evidence of MR gene and protein expression in both normal and pathological thyroid cells and tissues. We have shown that MR is present and functionally activated in thyroid tissue. Binding of Aldo to MR induces the expression of inflammatory and thyroid-specific genes, and the thyroid may thus be considered a novel mineralocorticoid target tissue.

Mineralocorticoid receptor knockout in Schwann cells alters myelin sheath thickness.

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.

Neutral Effect of Skeletal Muscle Mineralocorticoid Receptor on Glucose Metabolism in Mice.

The mineralocorticoid receptor (MR) is able to regulate the transcription of a number of genes in the myotube, although its roles in skeletal muscle (SM) metabolism still await demonstration. SM represents a major site for glucose uptake, and its metabolic derangements play a pivotal role in the development of insulin resistance (IR). The aim of this study was to investigate the contribution of SM MR in mediating derangements of glucose metabolism in a mouse model of diet-induced obesity. We observed that mice fed a high-fat diet (HFD mice) showed impaired glucose tolerance compared to mice fed a normal diet (ND mice). Mice fed a 60% HFD treated with the MR antagonist Spironolactone (HFD + Spiro) for 12 weeks revealed an improvement in glucose tolerance, as measured with an intraperitoneal glucose tolerance test, compared with HFD mice. To investigate if blockade of SM MR could contribute to the favorable metabolic effects observed with pharmacological MR antagonism, we analyzed MR expression in the gastrocnemius, showing that SM MR protein abundance is downregulated by HFD compared to ND mice and that pharmacological treatment with Spiro was able to partially revert this effect in HFD + Spiro mice. Differently from what we have observed in adipose tissue, where HDF increased adipocyte MR expression, SM MR protein was down-regulated in our experimental model, suggesting a completely different role of SM MR in the regulation of glucose metabolism. To confirm this hypothesis, we investigated the effects of MR blockade on insulin signaling in a cellular model of IRin C2C12 myocytes, which were treated with or without Spiro. We confirmed MR protein downregulation in insulin-resistant myotubes. We also analyzed Akt phosphorylation upon insulin stimulation, and we did not observe any difference between palmitate- and palmitate + Spiro-treated cells. These results were confirmed by in vitro glucose uptake analysis. Taken together, our data indicate that reduced activity of SM MR does not improve insulin signaling in mouse skeletal myocytes and does not contribute to the favorable metabolic effects on glucose tolerance and IR induced by systemic pharmacological MR blockade.

PS-BPB08-7: MECHANISMS OF MINERALOCORTICOID RECEPTOR-ASSOCIATED HYPERTENSION IN DIABETES MELLITUS: ROLE OF O-GLCNAC MODIFICATION

Objective: Recently administration of non-steroidal mineralocorticoid receptor (MR) antagonist with maximal tolerable doses of the renin-angiotensin system inhibitors significantly reduced renal as well as cardiovascular composite outcomes in patients with type 2 diabetes and chronic kidney diseases. However, molecular mechanisms of how MR is activated are not understood. Post-translational modification of O-linked-N-acetylglucosamine (O-GlcNAc) has emerged as an essential glucose-sensing mechanism. The aim of this study was to clarify whether MR is modified by O-GlcNAc and figure out the significance of this modification on MR in the context of diabetes mellitus. Design and method: 1) The MR transcriptional activities were investigated in reporter assays and real time RT-PCR. Levels of expression of MR were examined with western blot analysis. Cells were cultured in 5 mM or 30 mM glucose conditions and additionally treated with PUGNAc, an O-GlcNAcase inhibitor, or 6-diazo-5-oxonorleucine (DON), a glucosamine-fructose-6-phosphate amidotransferase inhibitor. 2) O-GlcNAc modification of MR was investigated with coimmunoprecipitation assays. We mapped actual O-GlcNAc sites on MR by liquid chromatography-tandem mass spectrometry and generated several MR mutants. 3) We evaluated the expression levels of MR and the total O-GlcNAc levels in kidneys of 9-week-old db/db mice. We also intraperitoneally administered 3 g/kg DON to them for five days and evaluated the same subjects. Results: 1) Treatments with either 30 mM glucose or PUGNAc enhanced 10–10 M aldosterone-mediated MR transactivation by 2-fold and increased endogenous SGK1 mRNA levels. These changes were accompanied by increased expression levels of MR protein. In contrast, treatment with DON lowered a reporter activity and the expression levels of MR. 2) Coimmunoprecipitation assays showed that MR was modified by O-GlcNAc and this modification of MR was increased by 30 mM glucose and PUGNAc treatment. The MR mutant of multiple O-GlcNAc sites (S295A/S296A/S298A/S299A) showed the decrease of its O-GlcNAc level, which was accompanied by lowering their expression levels and transcriptional activities by 40%. 3) The levels of MR protein increased by 2.5-fold in parallel with O-GlcNAc levels in kidneys of db/db mice. In addition, mRNA levels of SGK1 increased by 2-fold in db/db mice. Conversely, administration of DON decreased the expression levels of MR parallel to the total O-GlcNAc levels in kidneys. Conclusions: We showed for the first time that MR is a target for O-GlcNAc modification. High glucose conditions enhance O-GlcNAc modification of MR, which are associated with increased MR levels and its transcriptional activities in vitro and in vivo. The epigenetic modification of MR may account for the pathological MR activation in diabetes mellitus.

Mechanisms of mineralocorticoid receptor-associated hypertension in diabetes mellitus: the role of O-GlcNAc modification.

This study investigated the mechanism underlying the beneficial effects of mineralocorticoid receptor (MR) antagonists in patients with resistant hypertension and diabetic nephropathy by examining post-translational modification of the MR by O-linked-N-acetylglucosamine (O-GlcNAc), which is strongly associated with type 2 diabetes. Coimmunoprecipitation assays in HEK293T cells showed that MR is a target of O-GlcNAc modification (O-GlcNAcylation). The expression levels and transcriptional activities of the receptor increased in parallel with its O-GlcNAcylation under high-glucose conditions. Liquid chromatography-tandem mass spectrometry revealed O-GlcNAcylation of the MR at amino acids 295-307. Point mutations in those residues decreased O-GlcNAcylation, and both the protein levels and transcriptional activities of MR. In db/db mouse kidneys, MR protein levels increased in parallel with overall O-GlcNAc levels of the tissue, accompanied by increased SGK1 mRNA levels. The administration of 6-diazo-5-oxo-L-norleucin, an inhibitor of O-GlcNAcylation, reduced tissue O-GlcNAc levels and MR protein levels in db/db mice. Thus, our study showed that O-GlcNAcylation of the MR directly increases protein levels and transcriptional activities of the receptor under high-glucose conditions in vitro and in vivo. These findings provide a novel mechanism of MR as a target for prevention of complications associated with diabetes mellitus.

The mineralocorticoid receptor signal could be a new molecular target for the treatment of diabetic retinal complication

ABSTRACT Background Activation of the mineralocorticoid receptor (MR) is involved in the pathophysiology of diabetic vascular complications. In recent years, it has been indicated that the MR expressed in retinal Müller cells plays an important role in regulating the potassium and water balance in the retina. Therefore, it has also been speculated that abnormal MR signaling contributes to edematous diseases of the retina. Research design and methods We examined the effect of high-glucose conditions on MR protein and mRNA levels in human retinal Müller cells and changes in cell size in vitro. We also investigated MR transcriptional activity and signaling in high-glucose conditions. Results The MR protein increased by 2.2-fold with high-glucose treatment. Additionally, high-glucose treatment induced Müller cell swelling. Aldosterone-induced MR transcriptional activity was enhanced in high-glucose conditions, resulting in the upregulation of αENaC, AQP4, and Kir4.1 mRNA. Treatment with an MR antagonist led to the suppression of aldosterone-induced cell swelling, MR transcriptional activity, and upregulation of the target genes in high-glucose conditions. Conclusions High glucose induces Müller cell swelling through activation of MR signaling, which could be associated with aggravation of macular edema. Thus, Müller cell swelling and diabetic macular edema may represent a target for treatment with MR antagonists.

Global PER1 knockout Dahl Salt Sensitive rats show increased expression of renal Edn1 mRNA and Endothelin‐1 peptide

BackgroundPreviously, we showed that global knockout of the circadian clock protein PERIOD1 (PER1) in Dahl Salt Sensitive (SS) rats resulted in worsening of the SS hypertensive phenotype (1). PER1 KO rats also exhibited evidence of renal injury and increased plasma aldosterone levels after three weeks on a high salt diet (4% NaCl). Because we previously showed that PER1 negatively regulates the peptide hormone Endothelin‐1 (ET‐1) via transcriptional control of the ET‐1‐encoding gene Edn1, we wanted to investigate ET‐1 regulation in this novel rat model. Interestingly, Edn1 is also positively regulated by aldosterone through the mineralocorticoid receptor (MR).HypothesisGiven that ET‐1 signaling can be pro‐hypertensive and profibrotic in the kidney, we hypothesized that ET‐1 peptide levels in the kidney may be upregulated as part of the phenotype of the PER1 KO SS rats.MethodsProtein and total RNA were isolated from the kidney samples of control and PER1 KO SS rats on normal diet (0.4% NaCl) or after three weeks on a high salt (4% NaCl) diet. Renal ET‐1 peptide and plasma aldosterone levels were measured by ELISA and quantitative PCR was performed to quantify the renal mRNA levels of ET‐1 and its receptors ETA and ETB, MR, and GR. Renal MR protein levels were measured by Western Blot and quantified by relative densitometry. Data were evaluated for genotype or diet differences using ANOVA.ResultsRenal ET‐1 peptide levels were significantly higher in PER1 KO rats on high salt diet when compared to control SS rats (p=0.0003), as were plasma aldosterone levels (p=0.0156). There was also a significant genotype difference in Edn1 mRNA levels, with increased levels in PER1 KO rats on a high salt diet (p=0.0389). In contrast, significant differences were not observed in the mRNA levels of ET‐1 receptors Ednra and Ednrb between genotypes or diets. Importantly, quantification of renal MR protein levels showed marked increase with genotype and treatment effects (genotype: p=0.0002, treatment: p=0.0133).ConclusionsThese data suggest that PER1 KO results in increased ET‐1 in the kidney, due to increased transcription of the Edn1 gene, with increased plasma aldosterone and renal MR protein levels. This apparent negative regulation of ET‐1 by PER1, coupled with increased MR and aldosterone levels, is likely to contribute to the hypertensive and renal injury phenotype of PER1 KO SS rats on a high salt diet. Together these data show an important role for PER1 and its target ET‐1 in salt‐sensitive hypertension and the accompanying renal injury.Reference1 Spires DR, Zietara A, Levchenko V, Gumz ML, Staruschenko A. Knockout of Per1 Exacerbates the Hypertensive Phenotype of the Dahl Salt Sensitive Rat. FASEB J. 2020;34(S1):1.

Identification of Mineralocorticoid Receptors, Aldosterone, and Its Processing Enzyme CYP11B2 on Parasympathetic and Sympathetic Neurons in Rat Intracardiac Ganglia.

Recent interest has focused on the mineralocorticoid receptor (MR) and its impact on the myocardium and the performance of the heart. However, there is a lack of evidence about MR expression and its endogenous ligand aldosterone synthesis with specific regard to the intrinsic cardiac nervous system. Therefore, we looked for evidence of MR and aldosterone in sympathetic and parasympathetic neurons of intracardiac ganglia. Tissue samples from rat heart atria were subjected to conventional reverse-transcriptase polymerase chain reaction (PCR), Western blot, and double immunofluorescence confocal analysis of MR, corticosterone-inactivating enzyme 11β-hydroxysteroid-dehydrogenase-2 (11β-HSD2), aldosterone, and its processing enzyme CYP11B2 together with the neuronal markers vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH). Our results demonstrated MR, 11β-HSD2, and CYP11B2 specific mRNA and protein bands in rat heart atria. Double immunofluorescence labeling revealed coexpression of MR immunoreactivity with VAChT in large diameter parasympathetic principal neurons. In addition, MR immunoreactivity was identified in TH-immunoreactive small intensely fluorescent (SIF) cells and in nearby VAChT- and TH-immunoreactive nerve terminals. Interestingly, the aldosterone and its synthesizing enzyme CYP11B2 and 11β-HSD2 colocalized in MR– immunoreactive neurons of intracardiac ganglia. Overall, this study provides first evidence for the existence of not only local expression of MR, but also of 11β-HSD2 and aldosterone with its processing enzyme CYP11B2 in the neurons of the cardiac autonomic nervous system, suggesting a possible modulatory role of the mineralocorticoid system on the endogenous neuronal activity on heart performance.

Molecular cloning, characterization, and gene expression behavior of glucocorticoid and mineralocorticoid receptors from the Chinese alligator (Alligator sinensis).

The Chinese alligator is an endemic crocodilian species in China. We isolated and obtained the glucocorticoid and mineralocorticoid receptorgenes coding from the kidney ofAlligator sinensis by nested polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). The glucocorticoid receptor (GR) gene has 2343 base pairs encoding 780 amino acids, while the mineralocorticoid receptor (MR) gene is2958 bp in length encoding 985 amino acids. Quantitative real-time PCR was used to detect the distribution of messenger RNA (mRNA) levels. The maximum mRNA expressions were observed in the ovary and kidney, suggesting that these receptors may be involved in basic cellular functions or stress response of alligators. Besides this, RT-qPCR was performed to analyze the abundance of GR and MR mRNA transcripts in early embryonic development of the Chinese alligator in the kidney, liver, and heart. The mRNA levels of GR and MR at earlier stages in kidney, liver, and heart indicates that they might involve in the transcriptional regulation of early embryos and activate many precise developmental effects in fetal tissues. We also measured the protein expression in the liver embryonic developmental stages and found that the GR and MR proteins were restricted to both the nuclei and cytoplasm. The protein expression levels in the liver at different embryonic developmental stages have extremely prominent differences. Taken together, our results showed the full coding regions of GR and MR, their characteristics, and embryonic developmental mRNA and protein expressions of both genes in A. sinensis. This study could provide the necessary information for further investigating the diverse functions of GR and MR in A. sinensis.

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.

Aldosterone-induced microRNAs act as feedback regulators of mineralocorticoid receptor signaling in kidney epithelia.

The final steps in the Renin-Angiotensin-Aldosterone signaling System (RAAS) involve binding of the corticosteroid hormone, aldosterone to its mineralocorticoid receptor (MR). The bound MR interacts with response elements to induce or repress the transcription of aldosterone-regulated genes. A well characterized aldosterone-induced gene is the serum and glucocorticoid-induced kinase (SGK1), which acts downstream to increase sodium transport in distal kidney nephron epithelial cells. The role of microRNAs (miRs) induced by extended aldosterone stimulation in regulating MR and SGK1 has not been reported. In these studies, miRs predicted to bind to the 3'-UTR of mouse MR were profiled by qRT-PCR after aldosterone stimulation. The miR-466a/b/c/e family was upregulated in mouse kidney cortical collecting duct epithelial cells. A luciferase reporter assay confirmed miR-466 binding to both MR and SGK1 3'-UTRs. Inhibition of miR-466 increased MR and SGK1 mRNA and protein levels. Inhibiting miR-466b and preventing its upregulation after aldosterone stimulation increased amiloride-sensitive sodium transport and sensitivity to aldosterone stimulation. In vivo upregulation of miR-466 was confirmed in distal nephrons of mice on low Na+ diets. Repression of MR and SGK1 by aldosterone-induced miRs may represent a negative feedback loop that contributes to a form of aldosterone escape in vivo.

Three Brothers With Autism Carry a Stop‐Gain Mutation in the HPA‐Axis Gene NR3C2

Whole exome sequencing and copy-number variant analysis was performed on a family with three brothers diagnosed with autism. Each of the siblings shares an alteration in the nuclear receptor subfamily 3 group C member 2 (NR3C2) gene that is predicted to result in a stop-gain mutation (p.Q919X) in the mineralocorticoid receptor (MR) protein. This variant was maternally inherited and provides further evidence for a connection between the NR3C2 and autism. Interestingly, the NR3C2 gene encodes the MR protein, a steroid hormone-regulated transcription factor that acts in the hypothalamic-pituitary-adrenal axis and has been connected to stress and anxiety, both of which are features often seen in individuals with autism. Autism Res 2020, 13: 523-531. © 2020 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Given the complexity of the genetics underlying autism, each gene contributes to risk in a relatively small number of individuals, typically less than 1% of all autism cases. Whole exome sequencing of three brothers with autism identified a rare variant in the nuclear receptor subfamily 3 group C member 2 gene that is predicted to strongly interfere with its normal function. This gene encodes the mineralocorticoid receptor protein, which plays a role in how the body responds to stress and anxiety, features that are often elevated in people diagnosed with autism. This study adds further support to the relevance of this gene as a risk factor for autism.

Androgen and mineralocorticoid receptors are present on the germinal disc region in laying hens: Potential mediators of sex ratio adjustment in birds?

Female birds skew offspring sex ratios based on environmental and social stimuli; however, the mechanism mediating this phenomenon remains unknown. Growing evidence suggests that testosterone and corticosterone may influence meiosis, as they skew sex ratios when given immediately before chromosomal segregation. It is unclear if these hormones act on the germinal disc (GD) or through a downstream mediator. It is also unknown whether the GD contains receptors for these hormones. If testosterone and/or corticosterone act on the GD to skew sex ratios, then the GD should have receptors for them and that receptor levels should be higher in the GD regions compared to other follicular regions. Furthermore, fluctuations of receptor levels should occur near meiotic segregation. We collected ovarian follicles at 5 h pre-ovulation (just before meiotic segregation) and 20 h pre-ovulation (when sex chromosomes are arrested), and measured androgen receptor (AR) and mineralocorticoid receptor (MR) protein levels via Western blot. ARs and MRs were on the follicle in the GD and non-GD regions, and at 5 h and 20 h pre-ovulation. Both AR and MR protein levels were higher in the GD region than the non-GD region at both time points, but did not differ between time points. These results suggest that hen ovarian follicles have receptors for testosterone and corticosterone, and that the ability for testosterone to respond may be specifically higher in the GD-region, providing further support for the role of testosterone in the alteration of meiotic segregation.

Glucocorticoid and Mineralocorticoid Receptors in the Brain: A Transcriptional Perspective.

Adrenal glucocorticoid hormones are crucial for maintenance of homeostasis and adaptation to stress. They act via the mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs)—members of the family of nuclear receptors. MRs and GRs can mediate distinct, sometimes opposite, effects of glucocorticoids. Both receptor types can mediate nongenomic steroid effects, but they are best understood as ligand-activated transcription factors. MR and GR protein structure is similar; the receptors can form heterodimers on the DNA at glucocorticoid response elements (GREs), and they share a number of target genes. The transcriptional basis for opposite effects on cellular physiology remains largely unknown, in particular with respect to MR-selective gene transcription. In this review, we discuss proven and potential mechanisms of transcriptional specificity for MRs and GRs. These include unique GR binding to “negative GREs,” direct binding to other transcription factors, and binding to specific DNA sequences in conjunction with other transcription factors, as is the case for MRs and NeuroD proteins in the brain. MR- and GR-specific effects may also depend on specific interactions with transcriptional coregulators, downstream mediators of transcriptional receptor activity. Current data suggest that the relative importance of these mechanisms depends on the tissue and physiological context. Insight into these processes may not only allow a better understanding of homeostatic regulation but also the development of drugs that target specific aspects of disease.

Aldosterone‐Induced MicroRNAs Act as Negative Feedback Regulators for the Mineralocorticoid Receptor in Kidney Distal Nephron Epithelia

Regulated sodium (Na+) homeostasis in the kidney is achieved by the action of the renin‐angiotensin‐aldosterone signaling cascade. The terminal signaling hormone, aldosterone binds to the mineralocorticoid receptor (MR) to increase Na+ uptake across tubule epithelial cells by altering the expression and function of channels, transporters and regulatory proteins acting in concert. Along with a change in gene and protein expression, aldosterone regulates the expression of non‐coding RNAs including microRNAs (miRs). The primary function of miRs is to repress target mRNA expression and decrease protein abundance. Extended aldosterone stimulation (1–7 days) is known to reduce MR expression (protein and mRNA) in cell and animal models. To investigate if miRs were involved in this negative feedback regulation of the MR, we profiled the expression of miRs predicted to bind to the MR‐3’untranslated region (UTR) in mouse cortical collecting duct (mCCD) epithelial cells stimulated with aldosterone (50nM). Of the significantly altered miRs, the miR‐466a‐e family members were most highly upregulated (>5 fold). This upregulation was verified in vivo in CCD cells isolated from mouse kidneys of animals placed on low Na+ diets for 3 days. Luciferase assays confirmed miR‐466 binding to the MR‐UTR. Preventing the upregulation of miR‐466 after aldosterone stimulation, using miR inhibitors increased MR protein expression. Aldosterone‐stimulated sodium transport via the epithelial sodium channel in miR‐466 inhibited CCD cells was significantly higher than control transfected cells. The Na+‐stimulation was sustained over extended periods (24–72hrs) and miR‐466 inhibited cells had increased aldosterone sensitivity. In conclusion, we have identified a role for the miR‐466's in the negative feedback regulation of MR after extended aldosterone stimulation. Inhibition of miR‐466 released this feedback regulation to allow for a larger, more sensitive and more sustained response to aldosterone stimulation.Support or Funding InformationNIDDK R01DK103776 (JH) NIDDK DK102843 (MB)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The mineralocorticoid receptor contributes to barrier function of a model fish gill epithelium.

Cortisol-induced epithelial tightening of a primary cultured rainbow trout gill epithelium model occurs in association with reduced paracellular permeability and increased abundance of select barrier-forming tight junction (TJ) proteins. Corticosteroid receptor (CR) pharmacological blocker studies have suggested that to produce this tightening effect, cortisol acts on the mineralocorticoid receptor (MR) as well as glucocorticoid receptors (GRs). This study considered how cortisol influences model gill epithelium permeability and TJ properties by transcriptional knockdown of the gene encoding the MR (mr-KD) using double-stranded RNA. Following mr-KD, a significant reduction in MR protein abundance was observed in the epithelium. The mr-KD epithelium demonstrated reduced transepithelial resistance (TER) and an increase in the paracellular flux of [3H]polyethylene glycol (MW 400 kDa, PEG-400). Concurrently, mRNA abundance of gr2 and 11βhsd increased, indicating a possible compensatory response to mr-KD. Transcript abundance of claudin (cldn)-6, -8d, -23a and -28b decreased while that of cldn-20a increased in mr-KD preparations. Cortisol-induced epithelial tightening was enhanced in mr-KD preparations, suggesting that alterations in CRs and TJ composition augmented model epithelium barrier function in response to lowered MR abundance. Cortisol treatment significantly increased the transcript and protein abundance of TJ proteins such as Cldn-8d and -28b. However, in mr-KD preparations, Cldn-28b protein abundance did not significantly alter in response to cortisol treatment, while Cldn-8d abundance was significantly elevated. Data suggest that mr-KD compromises normal barrier function of a primary cultured rainbow trout gill epithelium in both the presence and absence of cortisol and that Cldn-28b protein abundance may be modulated by cortisol via the MR only.

Minor role of mature adipocyte mineralocorticoid receptor in high-fat diet-induced obesity.

Obesity is a major risk factor that contributes to the development of cardiovascular disease and type 2 diabetes. Mineralocorticoid receptor (MR) expression is increased in the adipose tissue of obese patients and several studies provide evidence that MR pharmacological antagonism improves glucose metabolism in genetic and diet-induced mouse models of obesity. In order to investigate whether the lack of adipocyte MR is sufficient to explain these beneficial metabolic effects, we generated a mouse model with inducible adipocyte-specific deletion of Nr3c2 gene encoding MR (adipo-MRKO). We observed a significant, yet not complete, reduction of Nr3c2 transcript and MR protein expression in subcutaneous and visceral adipose depots of adipo-MRKO mice. Notably, only mature adipocyte fraction lacks MR, whereas the stromal vascular fraction maintains normal MR expression in our mouse model. Adipo-MRKO mice fed a 45% high-fat diet for 14 weeks did not show any significant difference in body weight and fat mass compared to control littermates. Glucose and insulin tolerance tests revealed that mature adipocyte MR deficiency did not improve insulin sensitivity in response to a metabolic homeostatic challenge. Accordingly, no significant changes were observed in gene expression profile of adipogenic and inflammatory markers in adipose tissue of adipo-MRKO mice. Moreover, pharmacological MR antagonism in mature primary murine adipocytes, which differentiated ex vivo from WT mice, did not display any effect on adipokine expression. Taken together, these data demonstrate that the depletion of MR in mature adipocytes displays a minor role in diet-induced obesity and metabolic dysfunctions.

RNA sequencing of kidney distal tubule cells reveals multiple mediators of chronic aldosterone action.

The renal aldosterone-sensitive distal tubule (ASDT) is crucial for sodium reabsorption and blood pressure regulation. The ASDT consists of the late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct. Due to difficulties in isolating epithelial cells from the ASDT in large quantities, few transcriptome studies have been performed on this segment. Moreover, no studies exist on isolated DCT2 and CNT cells (excluding intercalated cells), and the role of aldosterone for regulating the transcriptome of these specific cell types is largely unknown. A mouse model expressing eGFP in DCT2/CNT/initial cortical collecting duct (iCCD) principal cells was exploited to facilitate the isolation of these cells in high number and purity. Combined with deep RNA sequencing technology, a comprehensive catalog of chronic aldosterone-regulated transcripts from enriched DCT2/CNT/iCCD principal cells was generated. There were 257 significantly downregulated and 290 upregulated transcripts in response to aldosterone ( P < 0.05). The RNA sequencing confirmed aldosterone regulation of well-described aldosterone targets including Sgk1 and Tsc22d3. Changes in selected transcripts such as S100a1 and Cldn4 were confirmed by RT-qPCR. The RNA sequencing showed downregulation of Nr3c2 encoding the mineralocorticoid receptor (MR), and cell line experiments showed a parallel decrease in MR protein. Furthermore, a large number of transcripts encoding transcription factors were downregulated. An extensive mRNA transcriptome reconstruction of an enriched CNT/iCCD principal cell population was also generated. The results provided a comprehensive database of aldosterone-regulated transcripts in the ASDT, allowing development of novel hypotheses for the action of aldosterone.

Epidermal growth factor receptor/extracellular signal-regulated kinase pathway enhances mineralocorticoid receptor transcriptional activity through protein stabilization.

Activation of mineralocorticoid receptor (MR) is evoked by aldosterone, and it induces hypertension and cardiovascular disease when it's concomitant with excessive salt loading. We have proposed the notion of "MR-associated hypertension", in which add-on therapy of MR blockers is effective even though serum aldosterone level is within normal range. To elucidate its underlying molecular mechanism, we focused on the effect of epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK) activation on MR activity. Epidermal growth factor (EGF) administration increased MR transcriptional activity through EGFR/ERK pathway and increased protein level by counteracting MR ubiquitylation invitro. EGF administration invivo also increased MR protein level and target gene expression in kidney, which were decreased by EGFR inhibitor. In addition, the administration of EGFR inhibitor lowered systolic blood pressure and MR activity in DOCA/salt-treated mice. In conclusion, EGFR/ERK pathway activation is considered as one of the underlying mechanisms of aberrant MR activation and EGFR/ERK pathway blockade could be an alternative approach for the prevention of MR-related cardiovascular events.