Mineralocorticoid Receptor Ligand Research Articles

9304 Integration of Glucocorticoid and Mineralocorticoid Receptor Signaling in Triple-negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

7873 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

9304 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

Differential Effect of Aldosterone or Mineralocorticoid Receptor Overexpression on Retinal Inflammation.

Overactivation of the mineralocorticoid receptor (MR) pathway is proinflammatory and contributes to the pathogenesis of diabetic retinopathy and of age-related macular degeneration. Excess of aldosterone, the specific MR ligand, is known to stimulate the production of proinflammatory cytokines and chemokines in extrarenal tissues and cells. In the RPE/choroid complex, aldosterone upregulated genes encoding proteins of the inflammatory response and downregulated genes encoding proteins involved in synaptic activity and neurotransmitters. Yet, cortisol, which is the main MR ligand in the eye, is a potent anti-inflammatory endogenous glucocorticoid. The aim of the present work was to better understand the role of MR activation in retinal inflammation either by acute injection of aldosterone or overexpression of the receptor. We first analyzed the retinal transcriptomic regulation induced by acute intraocular injection of aldosterone in the rat. Then, we used a transgenic rat overexpressing human MR (hMR) to also conduct retinal transcriptomic analysis as well as histological evaluation of the retina, retinal pigment epithelium and choroid. Our results show that acute intravitreal injection of aldosterone is highly proinflammatory, upregulating pathways related to microglial activation, oxidative stress, cell death, and downregulating pathways related to glial/neuronal cells activity and proper neurotransmission. On the other hand, hMR overexpression mediates a low-grade inflammation in the retina, associated with notable choroidal inflammation and choroidal neuropathy. Consequences of hMR overexpression or aldosterone-injection on retinal transcriptome reveal very distinct pathological mechanisms, with only a few common genes regulated, most of them not being regulated in the same way. Although aldosterone is highly proinflammatory in the retina, MR overactivation in its physiologic milieu mediates a low-grade inflammation in the neural retina.

Cardiovascular Disease in Obstructive Sleep Apnea: Putative Contributions of Mineralocorticoid Receptors.

Obstructive sleep apnea (OSA) is a chronic and highly prevalent condition that is associated with oxidative stress, inflammation, and fibrosis, leading to endothelial dysfunction, arterial stiffness, and vascular insulin resistance, resulting in increased cardiovascular disease and overall mortality rates. To date, OSA remains vastly underdiagnosed and undertreated, with conventional treatments yielding relatively discouraging results for improving cardiovascular outcomes in OSA patients. As such, a better mechanistic understanding of OSA-associated cardiovascular disease (CVD) and the development of novel adjuvant therapeutic targets are critically needed. It is well-established that inappropriate mineralocorticoid receptor (MR) activation in cardiovascular tissues plays a causal role in a multitude of CVD states. Clinical studies and experimental models of OSA lead to increased secretion of the MR ligand aldosterone and excessive MR activation. Furthermore, MR activation has been associated with worsened OSA prognosis. Despite these documented relationships, there have been no studies exploring the causal involvement of MR signaling in OSA-associated CVD. Further, scarce clinical studies have exclusively assessed the beneficial role of MR antagonists for the treatment of systemic hypertension commonly associated with OSA. Here, we provide a comprehensive overview of overlapping mechanistic pathways recruited in the context of MR activation- and OSA-induced CVD and propose MR-targeted therapy as a potential avenue to abrogate the deleterious cardiovascular consequences of OSA.

Importance of Micromilieu for Pathophysiologic Mineralocorticoid Receptor Activity-When the Mineralocorticoid Receptor Resides in the Wrong Neighborhood.

The mineralocorticoid receptor (MR) is a member of the steroid receptor family and acts as a ligand-dependent transcription factor. In addition to its classical effects on water and electrolyte balance, its involvement in the pathogenesis of cardiovascular and renal diseases has been the subject of research for several years. The molecular basis of the latter has not been fully elucidated, but an isolated increase in the concentration of the MR ligand aldosterone or MR expression does not suffice to explain long-term pathologic actions of the receptor. Several studies suggest that MR activity and signal transduction are modulated by the surrounding microenvironment, which therefore plays an important role in MR pathophysiological effects. Local changes in micromilieu, including hypoxia, ischemia/reperfusion, inflammation, radical stress, and aberrant salt or glucose concentrations affect MR activation and therefore may influence the probability of unphysiological MR actions. The surrounding micromilieu may modulate genomic MR activity either by causing changes in MR expression or MR activity; for example, by inducing posttranslational modifications of the MR or novel interaction with coregulators, DNA-binding sites, or non-classical pathways. This should be considered when developing treatment options and strategies for prevention of MR-associated diseases.

Glucocorticoid Hormones Regulate Pendrin Abundance and Subcellular Distribution

IntroductionFollowing a low salt diet or aldosterone administration, total and apical plasma membrane pendrin abundance increase through a mechanism thought to involve aldosterone binding to the mineralocorticoid receptor (MR). However, in other cells, aldosterone binding to the MR depends on 11 b Hydroxysteroid Dehydrogenase 2 (11 b HSD2) to oxidize and inactivate glucocorticoids, which are also MR ligands. Although 11 b HSD2 abundance is low or possibly absent in intercalated cells (ICs), it’s abundance might be sufficient to inactivate glucocorticoids, making aldosterone the preferred ligand. If so, 11 b HSD2 gene ablation should reduce pendrin expression. Alternatively, the IC MR might preferentially bind another ligand such as glucocorticoids. To test these possibilities, we examined the effect of 11 b HSD2 gene ablation as well as glucocorticoid administration on pendrin abundance and subcellular distribution.Methods11 b HSD2 KO rats and age‐, and sex matched wild type rats on the same background (controls) were given either a diet with a low or high NaCl content for 10 days. Wild type mice were adrenalectomized and given a diet with either zero, normal, or high (i.e. stress level) doses of corticosterone or dexamethasone for 10 days. Pendrin total protein abundance and subcellular distribution were determined in rats and mice from each group by immunoblot and immunohistochemistry.ResultsBoth pendrin total protein abundance and pendrin’s relative abundance in the most apical region of the cell were greater in the 11 b HSD2 null than in control rats following either a NaCl‐replete or ‐restricted diet. Moreover, we observed a dose‐dependent increase in both total pendrin abundance and pendrin abundance in the most apical region of the cell following either dexamethasone or corticosterone administration, although the increase appeared greater in mice given corticosterone than dexamethasone.ConclusionsGlucocorticoids increase pendrin total protein abundance and its relative abundance in the most apical region of the cell. These results might explain, in part, why pendrin is upregulated in the known absence of 11 b HSD2.

Pathogenic Effects of Mineralocorticoid Pathway Activation in Retinal Pigment Epithelium.

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.

Mineralocorticoid Receptor Pathway and Its Antagonism in a Model of Diabetic Retinopathy.

Diabetic retinopathy remains a major cause of vision loss worldwide. Mineralocorticoid receptor (MR) pathway activation contributes to diabetic nephropathy, but its role in retinopathy is unknown. In this study, we show that MR is overexpressed in the retina of type 2 diabetic Goto-Kakizaki (GK) rats and humans and that cortisol is the MR ligand in human eyes. Lipocalin 2 and galectin 3, two biomarkers of diabetes complications regulated by MR, are increased in GK and human retina. The sustained intraocular delivery of spironolactone, a steroidal mineralocorticoid antagonist, decreased the early and late pathogenic features of retinopathy in GK rats, such as retinal inflammation, vascular leakage, and retinal edema, through the upregulation of genes encoding proteins known to intervene in vascular permeability such as Hey1, Vldlr, Pten, Slc7a1, Tjp1, Dlg1, and Sesn2 but did not decrease VEGF. Spironolactone also normalized the distribution of ion and water channels in macroglial cells. These results indicate that MR is activated in GK and human diabetic retina and that local MR antagonism could be a novel therapeutic option for diabetic retinopathy.

The mineralocorticoid receptor-an emerging player in metabolic syndrome?

Metabolic syndrome is a cluster of conditions that increase the risk of cardiovascular diseases, and comprises obesity, hypertension, impaired glucose metabolism and dyslipidaemia. It is well recognised that the mineralocorticoid receptor (MR) plays an important role in blood pressure regulation via its effect on salt and water retention in renal tubules, with hypertension being a key feature in primary aldosteronism patients with excess adrenal production of aldosterone, the primary ligand for MRs in the epithelial tissues. MRs are also expressed in a number of non-epithelial tissues including adipose tissue; in these tissues, glucocorticoids or cortisol can also activate MRs due to low levels of 11-beta-hydroxysteroid-dehydrogenase type 2 (11-βHSD2), the enzyme which inactivates cortisol. There is increasing evidence suggesting that over-activation of MRs plays a role in the pathophysiology of the other components of metabolic syndrome, promoting adiposity, inflammation and glucose intolerance, and that MR antagonists may confer beneficial effects on energy and substrate homeostasis and cardiometabolic diseases. This review discusses the advances in the literature shedding light on the MR as an emerging player in metabolic syndrome.

CASZ1b is a novel transcriptional corepressor of mineralocorticoid receptor.

Aldosterone is a biological ligand for mineralocorticoid receptor (MR) that elevates blood pressure by promoting sodium reabsorption in the kidneys. However, the molecular mechanisms of aldosterone-MR-mediated transcription and the role of this transcription in hypertension remain largely unknown. In this study, we aimed to identify novel MR coregulators and elucidate one of the molecular mechanisms of hypertension. We purified MR-interacting factors from HEK293F cells stably expressing FLAG-MR through a biochemical approach and identified the zinc finger protein castor homolog 1 isoform b (CASZ1b) as a candidate novel MR coregulator via liquid chromatography-tandem mass spectrometry analysis. The CASZ1 gene has been implicated in hypertension in genome-wide single-nucleotide polymorphism studies, but its role in the development of hypertension remains unclear. We found that CASZ1b colocalized with MR in the kidneys and interacted with MR in an aldosterone-dependent manner. In luciferase assays using HEK293F cells, overexpression of CASZ1b reduced aldosterone-dependent MR transcriptional activity by ~50%. In contrast, knockdown of CASZ1b via RNA interference increased the expression levels of the aldosterone-induced MR target genes epithelial Na+ channel-α (ENaCα) and serum/glucocorticoid regulated kinase 1 (SGK1) by approximately twofold and 2.3-fold, respectively. Upon aldosterone-MR binding, CASZ1b interacted with MR and formed a protein complex with nucleosome remodeling deacetylase (Mi-2/NuRD), a corepressor complex with chromatin remodeling and histone deacetylation activity, which suppressed ENaCα and SGK1. These findings reveal a critical role of CASZ1b in regulating MR-mediated transcriptional activity and provide new insights into the pathophysiology of hypertension.

Interleukin 6‐Mediated Mineralocorticoid Receptor Translocation and Total Sodium Uptake Occurs via JAK2/STAT3

Hypertension is an inflammatory disease that is a leading cause of death and disability worldwide and is characterized by increased sodium (Na+) reabsorption. The beneficial effect of mineralocorticoid receptor (MR) antagonists in reducing blood pressure suggests a role for the MR in hypertension. Interestingly, the classical MR ligand, aldosterone (Aldo) is not always increased during hypertension. Elevated levels of the pro‐inflammatory cytokine, interleukin 6 (IL‐6); however, have been observed in the serum of hypertensive individuals. Cytokines elicit intracellular effects through various signaling mechanisms, including the Janus Kinase (JAK)/Signal Transducer and Activation of Transcription (STAT) pathway. There are multiple isoforms of each of these kinases that have been implicated in other IL‐6‐mediated inflammatory responses in the kidney; however, no empirical studies thus far have investigated how IL‐6 increases Na+ chloride cotransporter (NCC)‐mediated Na+ reabsorption. We hypothesize that the JAK2/STAT3 signaling pathway is involved in MR‐mediated activation of distal Na+ transporters. Using an in vitro model of a late distal convoluted tubule cell line, we have previously we have shown that IL‐6 activates JAK2 and STAT3 kinases using immunofluorescence probing and western blot techniques. We have also previously demonstrated that IL‐6 transactivates the MR and increases its activity via a JAK/STAT3 mechanism in transiently transfected mDCT15 cells using luciferase reporter assay studies.To visualize the effect of IL‐6‐mediated JAK2/STAT3 induction of MR nuclear translocation, we transfected mDCT15 cells with eGFP‐tagged MR constructs. Cells were incubated and treated with either: vehicle, IL‐6 [100ng/mL], IL‐6 + NSC33994 (JAK2 inhibitor, [10μM]), or IL‐6 + NSC74859 (STAT3 inhibitor, [50μM]) for 30 min. Images were collected using confocal microscopy. To demonstrate the role of IL‐6‐stimulated JAK2/STAT3 signaling on total Na+ reabsorption, we performed Na+ uptake studies using CoroNa Green (fluorescent Na+ indicator, 6μM, 30 min). mDCT15 cells were treated with: vehicle, IL‐6 (100ng/mL), IL‐6 receptor subunit gp130 inhibitor (SC144, 2 μM), JAK2 inhibitor (NSC33994, 10 μM), STAT3 inhibitor (NSC, 50μM) or an MR antagonist (spironolactone, 10 nM).In the presence of both JAK2 and STAT3 inhibitors, IL‐6 MR translocation was reduced (JAK2) or completely inhibited (STAT3). When IL‐6 receptor, STAT3, and MR activity was inhibited, we observed reduced Na+ uptake levels. This suggests that IL‐6 activation of the MR increases distal Na+ uptake via an IL‐6 induced JAK/STAT3 mechanism. Overall, we are working to identify a novel mechanism of IL‐6 mediated distal Na+ transporter activation via MR induction, in order to demonstrate an alternative mechanism for increased Na+ reabsorption in hypertension when serum aldosterone levels are not increased, but elevated cytokine levels are observed. Together, our data suggests a mechanism for IL‐6‐mediated activation of the MR via a JAK2/STAT3 signaling pathway.Support or Funding InformationNIH DK115660, American Society of Nephrology Gottschalk to BMWThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sleep-EEG in patients with primary aldosteronism in comparison to healthy controls and patients with depression

The mineralocorticoid receptor (MR)/glucocorticoid receptor balance plays an important role in the pathophysiology of anxiety and depression. Aldosterone, a primary MR ligand, seems to be related to the pathophysiology of anxiety and depressive symptoms. The objective of this study was to investigate effects of aldosterone excess on sleep EEG, as sleep EEG is a tool to gain insight into psychoneuroendocrine function. Here, 19 untreated patients (9 males, 10 females) suffering from primary aldosteronism were investigated using sleep EEG and several rating scales for anxiety, depression, quality of life and sleep before starting specific treatment. Parameters were compared to age and sex matched healthy controls and patients with depression and correlated with laboratory findings and blood pressure. Patients had higher values for anxiety and depression compared to the general population, although a psychiatric disorder in their history was ruled out. Although sleep disturbances were reported in the Pittsburgh sleep quality index, sleep EEG did not show significant changes between patients and healthy controls. No depression specific pattern in sleep EEG was found. But in contrast to females, several sleep-EEG parameters of male PA patients differed significantly from patients with depression. There was a significant correlation between blood pressure and the severity of depression and anxiety in females. Correlation analysis between blood pressure and rating scales indicate a relationship between blood pressure and anxiety in women. In conclusion, these data suggest gender related effects of aldosterone excess in males and females.

Aldosterone Research: 65 Years, and Counting.

Aldosterone was characterized as the major mineralocorticoid hormone 65 years ago, and since then its physiologic role in epidural electrolyte homeostasis the province of nephrologists. In epithelia it acts via the mineralocorticoid receptor (MR) to retain Na+ and excrete K+; MRs, however, are widely expressed in organs not known to be aldosterone target tissues. MRs are not merely "aldosterone receptors," as they have equivalently high affinity for the physiologic glucocorticoids, and for progesterone. In epithelia (plus in the blood vessel wall and in the nucleus tractus solitarius of the brain) MRs are "protected" by coexpression of the enzyme 11β-hydroxysteroid dehydrogenase. This enzyme converts cortisol-which circulates at much higher concentrations than aldosterone-to receptor-inactive cortisone, thus allowing aldosterone selectively to activate "protected" MR. In tissues which do not express 11β-hydroxysteroid dehydrogenase, the default MR ligand is cortisol, which circulates at ≥100-fold higher plasma free concentrations than aldosterone. In such tissues there is as yet scant evidence for the physiologic role of cortisol-occupied MR: over the past decade, however, it has become clear that in damaged tissues cortisol can act as an MR-agonist, mimicking the effects seen with aldosterone under experimental conditions, in vitro and in vivo. Many pathophysiologic roles have been attributed to aldosterone: on the current evidence there are none outside its long established epithelial actions, those on the blood vessel wall and on the nucleus tractus solitarius.

The mineralocorticoid receptor as a modulator of innate immunity and atherosclerosis.

The mineralocorticoid receptor (MR) is a member of the nuclear receptor steroid-binding family. The classical MR ligand aldosterone controls electrolyte and fluid homeostasis after binding in renal epithelial cells. However, more recent evidence suggests that activation of extrarenal MRs by aldosterone negatively impacts cardiovascular health independent of its effects on blood pressure: high levels of aldosterone associate with an increased cardiovascular event rate, where MR antagonists exert beneficial effects on cardiovascular mortality. The most important cause for cardiovascular events is atherosclerosis that is currently considered a low-grade inflammatory disorder of the arterial wall. In this inflammatory process, the innate immune system plays a deciding role, with the monocyte-derived macrophage being the most abundant cell in the atherosclerotic plaque. Intriguingly, both monocytes and macrophages express the MR, and a growing body of evidence shows that these cells are skewed into a pro-inflammatory and pro-atherosclerotic phenotype via MR stimulation. In this review, we detail the current perspective on the role of the monocyte and macrophage MR in atherosclerosis development and provide a comprehensive framework of the effects of MR activation of the innate immune system that might drive the pro-atherosclerotic outcome.

IL‐6 Activates the Mineralocorticoid Receptor via the JAK2/STAT3 Pathway

Hypertension is an inflammatory disease that is a leading cause of death and disability worldwide and is characterized by increased sodium (Na+) reabsorption. The beneficial effect of mineralocorticoid receptor (MR) antagonists in reducing blood pressure suggests a role for the MR in hypertension. Interestingly, the classical MR ligand, aldosterone (Aldo) is not always increased during hypertension. However, the pro‐inflammatory cytokine interleukin 6 (IL‐6) is elevated in the serum of hypertensive individuals. Previously we have shown that IL‐6 increases MR‐dependent Na+ uptake in vitro, and increases expression of the (Na+) chloride cotransporter (NCC) in vivo. IL‐6 signals through the IL‐6 receptor a (IL‐6Ra) and the small signaling molecule, gp130, activating multiple intracellular signaling pathways including the Janus Kinase (JAK)/Signal Transducer and Activation of Transcription (STAT) signaling pathway. There are multiple isoforms of each of these kinases which have been implicated in other IL‐6‐mediated inflammatory responses in the kidney. However, no studies have investigated how IL‐6 increases NCC‐mediated Na+ reabsorption, or the specific JAK/STAT pathway combination that may mediate this process. We hypothesize that the JAK2/STAT3 signaling pathway is involved in MR‐mediated activation of NCC.We used an in vitro model of the late distal convoluted tubule (DCT2), mDCT15 cells. Cells were grown to confluence (60%), and then treated with IL‐6 (100 ng/mL, 24 hrs). Cells were stained for phosphor‐JAK2 and ‐STAT3 levels. We observed a significant increase in activation levels of this pathway. Phospho‐JAK2 levels were more than doubled (211%) following IL‐6 treatment, suggesting strong activation of the JAK2/STAT3 pathway following IL‐6 in distal nephron epithelial cells.To demonstrate IL‐6‐mediated MR activation via the JAK2/STAT3 pathway, mDCT15 cells were transfected with mineralocorticoid response element (MRE)‐luciferase reporter constructs (5 mg), and treated with: vehicle, Aldo (100nM), IL‐6 (100 ng/mL) and/or the MR antagonist (spironolactone, 10 nM), the gp130 inhibitor (SC144, 2μM) and the STAT3 inhibitor (NSC74859, 100 μM) or the JAK1/2 inhibitor (Ruxolitinib, 250 nM). Luciferase assays were performed on whole cell lysates (48 hrs). We found that IL‐6 increased MRE activity (36%) over vehicle. When cells were co‐incubated with inhibitors for gp130 or STAT3, we saw a decrease in MRE activity to levels lower than vehicle. We did not observe a decrease in MRE activity when incubated with the JAK1/2 inhibitor. Together, our data suggests a mechanism for IL‐6‐mediated activation of the MR via a gp130‐JAK2/STAT3 signaling pathway. Understanding this complicated intracellular signaling pathway will allow for targeted therapies to inhibit IL‐6 induced activation of the MR and possible increases in Na+ reabsorption and hypertension.Support or Funding InformationEmory University SUPERR 5R25DK101390‐05 to HCM; NIDDK085097 and VA MERIT I01BX002322 to RSH and 2T32DK7656‐2 to BMWThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel Pathways of Endocrine Disruption Through Pesticides Interference With Human Mineralocorticoid Receptors.

Mineralocorticoid receptor (MR) is one member of the steroid receptor family. In addition to its important role in Na+/K+ homeostasis, MR is reported as a tumor-suppressor in carcinogenesis. So far, little was known about the ability of pesticides to interfere with MR. In this study, a total of 43 pesticides and/or metabolites were investigated for their potential effects on human MR. None of the tested pesticides exhibited MR agonistic potency, whereas 16 compounds showed antagonistic activities. Further investigations indicated that these 16 chemicals individually antagonized aldosterone-induced alkaline phosphatase expression in vascular smooth muscle cells and aldosterone-inhibited hepatocellular carcinoma cell proliferation at higher concentrations, and the mixture of these 16 pesticides at environmentally relevant concentrations significantly disrupted MR activity. The additional quantitative mixture experiments indicated a good agreement between the combined anti-mineralocorticoidic activities of 16 pesticides and the responses predicted by concentration addition model instead of independent action model. The interruption of nuclear translocation of MR was clarified as a main mechanism for the anti-mineralocorticoidic activities by these pesticides. These data suggest that the health risk may increase when multiple MR antagonists cooperate following concentration addition model and exhibit a combined effect. Our findings emphasize that comprehensive risk assessment of adverse effects of environmental MR ligands on human health should be considered.

Spironolactone Prevents Endothelial Nitric Oxide Synthase Uncoupling and Vascular Dysfunction Induced by β-Adrenergic Overstimulation

Sustained stimulation of β-adrenoceptors (β-ARs) and activation of renin–angiotensin–aldosterone system are common features of cardiovascular diseases with rising sympathetic activation, including essential hypertension, myocardial infarction, and heart failure. In this study, we investigated the role of AT1 receptor and mineralocorticoid receptor (MR) in the vascular alterations caused by β-AR overstimulation. β-AR overstimulation with associated cardiac hypertrophy and increased vasoconstrictor response to phenylephrine in aorta were modeled in rats by 7-day isoproterenol treatment. The increased vasoconstrictor response to phenylephrine in this model was blunted by the MR antagonist spironolactone, but not by the AT1 receptor antagonist losartan, despite the blunting of cardiac hypertrophy with both drugs. Spironolactone, but not losartan, restored NO bioavailability in association with lower endothelial nitric oxide synthase–derived superoxide production, increased endothelial nitric oxide synthase dimerization, and aortic HSP90 upregulation. MR genomic and nongenomic functions were activated in aortas from isoproterenol-treated rats. Isoproterenol did not modify plasma levels of MR ligands aldosterone and corticosterone but rather increased perivascular adipose tissue–derived corticosterone in association with increased expression of 11β-hydroxysteroid dehydrogenase type 1. The anticontractile effect of aortic perivascular adipose tissue was impaired by β-AR overstimulation and restored by MR blockade. These results suggest that activation of vascular MR signaling contributes to the vascular dysfunction induced by β-AR overstimulation associated with endothelial nitric oxide synthase uncoupling. These findings reveal an additional explanation for the protective effects of MR antagonists in cardiovascular disorders with sympathetic activation.

Identification of spirooxindole and dibenzoxazepine motifs as potent mineralocorticoid receptor antagonists

Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR.

Phosphorylation of Mineralocorticoid Receptor at Residue S839 Impairs Aldosterone‐dependent Gene Transactivation Coupling in a Dominant Negative Manner

The mineralocorticoid receptor (MR), a member of the nuclear receptor family, transduces the biological effects of the corticosteroid hormone aldosterone. Aldosterone interaction with MR ligand binding domain (LBD) induces nuclear translocation, dimerization and gene transactivation. It has recently been demonstrated that phosphorylation of S843, a residue near LBD, inactivates human MR. In this work we examined the mechanisms involved in MR modulation by S843p using mouse MR phosphomimetic mutant S839D (equivalent to S843 in human). MR‐S839D is inactive and significantly decreases wild type MR activity when both forms are coexpressed, consistent with a dominant negative role of MR‐S839D in the dimer. Surprisingly, aldosterone is able to induce nuclear translocation of MR‐S839D, although at a slower rate than wild type receptors. Therefore aldosterone is still able to bind to MR‐S839D but it is inefficient for gene transactivation. In silico modeling of MR LBD with S839D mutation or S839p show the same effect, namely a small decrease in steady‐state agonist docking energy. Our results suggest that the effect of S839p cannot be fully explained by decreased aldosterone binding affinity and may imply a defect in transactivation coupling.