The mineralocorticoid receptor modulates timing and location of genomic binding by glucocorticoid receptor in response to synthetic glucocorticoids in keratinocytes.

Glucocorticoids (GCs) exert potent antiproliferative and anti-inflammatory properties, explaining their therapeutic efficacy for skin diseases. GCs act by binding to the GC receptor (GR) and the mineralocorticoid receptor (MR), co-expressed in classical and non-classical targets including keratinocytes. Using knockout mice, we previously demonstrated that GR and MR exert essential nonoverlapping functions in skin homeostasis. These closely related receptors may homo- or heterodimerize to regulate transcription, and theoretically bind identical GC-response elements (GRE). We assessed the contribution of MR to GR genomic binding and the transcriptional response to the synthetic GC dexamethasone (Dex) using control (CO) and MR knockout (MREKO ) keratinocytes. GR chromatin immunoprecipitation (ChIP)-seq identified peaks common and unique to both genotypes upon Dex treatment (1 h). GREs, AP-1, TEAD, and p53 motifs were enriched in CO and MREKO peaks. However, GR genomic binding was 35% reduced in MREKO , with significantly decreased GRE enrichment, and reduced nuclear GR. Surface plasmon resonance determined steady state affinity constants, suggesting preferred dimer formation as MR-MR > GR-MR ~ GR-GR; however, kinetic studies demonstrated that GR-containing dimers had the longest lifetimes. Despite GR-binding differences, RNA-seq identified largely similar subsets of differentially expressed genes in both genotypes upon Dex treatment (3 h). However, time-course experiments showed gene-dependent differences in the magnitude of expression, which correlated with earlier and more pronounced GR binding to GRE sites unique to CO including near Nr3c1. Our data show that endogenous MR has an impact on the kinetics and differential genomic binding of GR, affecting the time-course, specificity, and magnitude of GC transcriptional responses in keratinocytes.

The choroidal nervous system: a link between mineralocorticoid receptor and pachychoroid.

Central serous chorioretinopathy (CSCR) belongs to the pachychoroid spectrum, a pathological phenotype of the choroidal vasculature, in which blood flow is under the choroidal nervous system (ChNS) regulation. The pathogenesis of CSCR is multifactorial, with the most recognised risk factor being intake of glucocorticoids, which activate both the gluco- and the mineralocorticoid (MR) receptors. As MR over-activation is pathogenic in the retina and choroid, it could mediate the pathogenic effects of glucocorticoids in CSCR. But the role of MR signalling in pachychoroid is unknown and whether it affects the ChNS has not been explored. Using anatomo-neurochemical characterisation of the ChNS in rodents and humans, we discovered that beside innervation of arteries, choroidal veins and choriocapillaris are also innervated, suggesting that the entire choroidal vasculature is under neural control. The numerous synapses together with calcitonin gene-related peptide (CGRP) vesicles juxtaposed to choroidal macrophages indicate a neuro-immune crosstalk. Using ultrastructural approaches, we show that transgenic mice overexpressing human MR, display a pachychoroid-like phenotype, with signs of choroidal neuropathy including myelin abnormalities, accumulation and enlargement of mitochondria and nerves vacuolization. Transcriptomic analysis of the RPE/choroid complex in the transgenic mice reveals regulation of corticoids target genes, known to intervene in nerve pathophysiology, such as Lcn2, rdas1/dexras1, S100a8 and S100a9, rabphilin 3a (Rph3a), secretogranin (Scg2) and Kinesin Family Member 5A (Kif5a). Genes belonging to pathways related to vasculature development, hypoxia, epithelial cell apoptosis, epithelial mesenchymal transition, and inflammation, support the pachychoroid phenotype and highlight downstream molecular targets. Hypotheses on the imaging phenotype of pachychoroid in humans are put forward in the light of these new data. Our results provide evidence that MR overactivation causes a choroidal neuropathy that could explain the pachychoroid phenotype found in transgenic mice overexpressing human MR. In patients with pachychoroid and CSCR in which systemic dysautonomia has been demonstrated, MR-induced choroidal neuropathy could be the missing link between corticoids and pachychoroid.

Antagonistic effects of finerenone and spironolactone on the aldosterone-regulated transcriptome of human kidney cells.

Aldosterone, the main mineralocorticoid hormone in humans, plays a pivotal role in the control of water and salt reabsorption via activation of the mineralocorticoid receptor (MR). Alterations in MR signaling pathway lead to renal dysfunction, including chronic kidney disease and renal fibrosis, that can be prevented or treated with mineralocorticoid receptor antagonists (MRAs). Here, we used RNA-Sequencing to analyze effects of two MRAs, spironolactone and finerenone, on the aldosterone-induced transcriptome of a human renal cell line stably expressing the MR. Bioinformatics analysis of the data set reveals the identity of hundreds of genes induced or repressed by aldosterone. Their regulation is modulated in a time-dependent manner and, for the induced genes, depends on the aldosterone-driven direct binding of the MR onto its genomic targets that we have previously characterized. Although both MRAs block aldosterone-induced as well as aldosterone-repressed genes qualitatively similarly, finerenone has a quantitatively more efficient antagonism on some aldosterone-induced genes. Our data provide the first complete transcriptome for aldosterone on a human renal cell line and identifies pro-inflammatory markers (IL6, IL11, CCL7, and CXCL8) as aldosterone-repressed genes.

Sexual Dimorphism of Corticosteroid Signaling during Kidney Development.

Sexual dimorphism involves differences between biological sexes that go beyond sexual characteristics. In mammals, differences between sexes have been demonstrated regarding various biological processes, including blood pressure and predisposition to develop hypertension early in adulthood, which may rely on early events during development and in the neonatal period. Recent studies suggest that corticosteroid signaling pathways (comprising glucocorticoid and mineralocorticoid signaling pathways) have distinct tissue-specific expression and regulation during this specific temporal window in a sex-dependent manner, most notably in the kidney. This review outlines the evidence for a gender differential expression and activation of renal corticosteroid signaling pathways in the mammalian fetus and neonate, from mouse to human, that may favor mineralocorticoid signaling in females and glucocorticoid signaling in males. Determining the effects of such differences may shed light on short term and long term pathophysiological consequences, markedly for males.

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.

Interaction between accumulated 21-deoxysteroids and mineralocorticoid signaling in 21-hydroxylase deficiency.

21-Hydroxylase deficiency (21OHD) is a rare genetic disorder in which salt-wasting syndrome occurs in 75% of cases, due to inability to synthesize cortisol and aldosterone. Recent mass spectrometry progress allowed identification of 21-deoxysteroids, i.e., 17-hydroxyprogesterone (17OHP), 21-deoxycortisol (21DF), and 21-deoxycorticosterone (21DB). We hypothesized that they may interfere with mineralocorticoid signaling and fludrocortisone therapy in patients with congenital adrenal hyperplasia (CAH) without effective glucocorticoid replacement and ACTH suppression. Our goal was to quantify circulating 21-deoxysteroids in a pediatric cohort with CAH related to 21OHD and to examine their impact on mineralocorticoid receptor (MR) activation. Twenty-nine patients with salt-wasting phenotype were classified in two groups according to their therapeutic control. During routine follow-up, 17OHP, 21DF, 21DB, and cortisol levels were quantified by liquid chromatography with tandem mass spectrometry before hydrocortisone intake and 1 and 2.5 h following treatment administration. Luciferase reporter gene assays were performed on transfected HEK293T cells while in silico modeling examined structural interactions between these steroids within ligand-binding domain of MR. Plasma 17OHP, 21DF, and 21DB accumulate in uncontrolled patients reaching micromolar concentrations even after hydrocortisone intake. 21DF and 21DB act as partial MR agonists with antagonist features similar to 17OHP, consistent with altered anchoring to Asn770 and unfavorable contact with Ala773 in ligand-binding pocket of MR. Our results demonstrate a complex interaction between all accumulating 21-deoxysteroids in uncontrolled 21OHD patients and mineralocorticoid signaling and suggest that appropriate steroid profiling should optimize management and follow-up of such patients, as keeping those steroids to low plasma levels should attest therapeutic efficacy and prevent interference with MR signaling.

Specific Activation of the Alternative Cardiac Promoter of Cacna1c by the Mineralocorticoid Receptor.

RATIONALE: The MR (mineralocorticoid receptor) antagonists belong to the current therapeutic armamentarium for the management of cardiovascular diseases, but the mechanisms conferring their beneficial effects are poorly understood. Part of the cardiovascular effects of MR is because of the regulation of L-type Cav1.2 Ca2+ channel expression, which is generated by tissue-specific alternative promoters as a long cardiac or short vascular N-terminal transcripts. OBJECTIVE: To analyze the molecular mechanisms by which aldosterone, through MR, modulates Cav1.2 expression and function in a tissue-specific manner. METHODS AND RESULTS: In primary cultures of neonatal rat ventricular myocytes, aldosterone exposure for 24 hours increased in a concentration-dependent manner long cardiac Cav1.2 N-terminal transcripts expression at both mRNA and protein levels, correlating with enhanced concentration-, time-, and MR-dependent P1-promoter activity. In silico analysis and mutagenesis identified MR interaction with both specific activating and repressing DNA-binding elements on the P1-promoter. The relevance of this regulation is confirmed both ex and in vivo in transgenic mice harboring the luciferase reporter gene under the control of the cardiac P1-promoter. Moreover, we show that this cis-regulatory mechanism is not limited to the heart. Indeed, in smooth muscle cells from different vascular beds, in which the short vascular Cav1.2 N-terminal transcripts is normally the major isoform, we found that MR signaling activates long cardiac Cav1.2 N-terminal transcripts expression through P1-promoter activation, leading to vascular contractile dysfunction. These results were further corroborated in hypertensive aldosterone/salt rodent models, showing notably a positive correlation between blood pressure and cardiac P1-promoter activity in aorta. This new vascular long cardiac Cav1.2 N-terminal transcripts molecular signature reduced sensitivity to the Ca2+ channel blocker, nifedipine, in aldosterone-treated vessels. CONCLUSIONS: Our results reveal that MR acts as a transcription factor to translate aldosterone signal into specific cardiac P1-promoter activation that might influence the therapeutic outcome of cardiovascular diseases.

Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease.

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.

Corticosteroid receptors adopt distinct cyclical transcriptional signatures.

Mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) are two closely related hormone-activated transcription factors that regulate major pathophysiologic functions. High homology between these receptors accounts for the crossbinding of their corresponding ligands, MR being activated by both aldosterone and cortisol and GR essentially activated by cortisol. Their coexpression and ability to bind similar DNA motifs highlight the need to investigate their respective contributions to overall corticosteroid signaling. Here, we decipher the transcriptional regulatory mechanisms that underlie selective effects of MRs and GRs on shared genomic targets in a human renal cellular model. Kinetic, serial, and sequential chromatin immunoprecipitation approaches were performed on the period circadian protein 1 ( PER1) target gene, providing evidence that both receptors dynamically and cyclically interact at the same target promoter in a specific and distinct transcriptional signature. During this process, both receptors regulate PER1 gene by binding as homo- or heterodimers to the same promoter region. Our results suggest a novel level of MR-GR target gene regulation, which should be considered for a better and integrated understanding of corticosteroid-related pathophysiology.-Le Billan, F., Amazit, L., Bleakley, K., Xue, Q.-Y., Pussard, E., Lhadj, C., Kolkhof, P., Viengchareun, S., Fagart, J., Lombès, M. Corticosteroid receptors adopt distinct cyclical transcriptional signatures.

RNA-binding protein HuR enhances mineralocorticoid signaling in renal KC3AC1 cells under hypotonicity.

Mineralocorticoid receptor (MR) mediates the sodium-retaining action of aldosterone in the distal nephron. Herein, we decipher mechanisms by which hypotonicity increases MR expression in renal principal cells. We identify HuR (human antigen R), an mRNA-stabilizing protein, as an important posttranscriptional regulator of MR expression. Hypotonicity triggers a rapid and reversible nuclear export of HuR in renal KC3AC1 cells, as quantified by high-throughput microscopy. We also identify a key hairpin motif in the 3'-untranslated region of MR transcript, pivotal for the interaction with HuR and its stabilizing function. Next, we show that hypotonicity increases MR recruitment onto Sgk1 promoter, a well-known MR target gene, thereby enhancing aldosterone responsiveness. Our data shed new light on the crucial role of HuR as a stabilizing factor for the MR transcript and provide evidence for a short autoregulatory loop in which expression of a nuclear receptor transcriptionally regulating water and sodium balance is controlled by osmotic tone.

Sex-Specificity of Mineralocorticoid Target Gene Expression during Renal Development, and Long-Term Consequences.

Sex differences have been identified in various biological processes, including hypertension. The mineralocorticoid signaling pathway is an important contributor to early arterial hypertension, however its sex-specific expression has been scarcely studied, particularly with respect to the kidney. Basal systolic blood pressure (SBP) and heart rate (HR) were measured in adult male and female mice. Renal gene expression studies of major players of mineralocorticoid signaling were performed at different developmental stages in male and female mice using reverse transcription quantitative PCR (RT-qPCR), and were compared to those of the same genes in the lung, another mineralocorticoid epithelial target tissue that regulates ion exchange and electrolyte balance. The role of sex hormones in the regulation of these genes was also investigated in differentiated KC3AC1 renal cells. Additionally, renal expression of the 11 ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) protein, a regulator of mineralocorticoid specificity, was measured by immunoblotting and its activity was indirectly assessed in the plasma using liquid-chromatography coupled to mass spectrometry in tandem (LC-MSMS) method. SBP and HR were found to be significantly lower in females compared to males. This was accompanied by a sex- and tissue-specific expression profile throughout renal development of the mineralocorticoid target genes serum and glucocorticoid-regulated kinase 1 (Sgk1) and glucocorticoid-induced leucine zipper protein (Gilz), together with Hsd11b2, Finally, the implication of sex hormones in this sex-specific expression profile was demonstrated in vitro, most notably for Gilz mRNA expression. We demonstrate a tissue-specific, sex-dependent and developmentally-regulated pattern of expression of the mineralocorticoid pathway that could have important implications in physiology and pathology.

Finerenone Impedes Aldosterone-dependent Nuclear Import of the Mineralocorticoid Receptor and Prevents Genomic Recruitment of Steroid Receptor Coactivator-1.

Aldosterone regulates sodium homeostasis by activating the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Hyperaldosteronism leads todeleterious effects on the kidney, blood vessels, and heart. Although steroidal antagonists such as spironolactone and eplerenone are clinically useful for the treatment of cardiovascular diseases, they are associated with several side effects. Finerenone, a novel nonsteroidal MR antagonist, is presently being evaluated in two clinical phase IIb trials. Here, we characterized the molecular mechanisms of action of finerenone and spironolactone at several key steps of the MR signaling pathway. Molecular modeling and mutagenesis approaches allowed identification of Ser-810 and Ala-773 as key residues for the high MR selectivity of finerenone. Moreover, we showed that, in contrast to spironolactone, which activates the S810L mutant MR responsible for a severe form of early onset hypertension, finerenone displays strict antagonistic properties. Aldosterone-dependent phosphorylation and degradation of MR are inhibited by both finerenone and spironolactone. However, automated quantification of MR subcellular distribution demonstrated that finerenone delays aldosterone-induced nuclear accumulation of MR more efficiently than spironolactone. Finally, chromatin immunoprecipitation assays revealed that, as opposed to spironolactone, finerenone inhibits MR, steroid receptor coactivator-1, and RNA polymerase II binding at the regulatory sequence of the SCNN1A gene and also remarkably reduces basal MR and steroid receptor coactivator-1 recruitment, unraveling a specific and unrecognized inactivating mechanism on MR signaling. Overall, our data demonstrate that the highly potent and selective MR antagonist finerenone specifically impairs several critical steps of the MR signaling pathway and therefore represents a promising new generation MR antagonist.

Cistrome of the aldosterone-activated mineralocorticoid receptor in human renal cells.

Aldosterone exerts its effects mainly by activating the mineralocorticoid receptor (MR), a transcription factor that regulates gene expression through complex and dynamic interactions with coregulators and transcriptional machinery, leading to fine-tuned control of vectorial ionic transport in the distal nephron. To identify genome-wide aldosterone-regulated MR targets in human renal cells, we set up a chromatin immunoprecipitation (ChIP) assay by using a specific anti-MR antibody in a differentiated human renal cell line expressing green fluorescent protein (GFP)-MR. This approach, coupled with high-throughput sequencing, allowed identification of 974 genomic MR targets. Computational analysis identified an MR response element (MRE) including single or multiple half-sites and palindromic motifs in which the AGtACAgxatGTtCt sequence was the most prevalent motif. Most genomic MR-binding sites (MBSs) are located >10 kb from the transcriptional start sites of target genes (84%). Specific aldosterone-induced recruitment of MR on the first most relevant genomic sequences was further validated by ChIP-quantitative (q)PCR and correlated with concomitant and positive aldosterone-activated transcriptional regulation of the corresponding gene, as assayed by RT-qPCR. It was notable that most MBSs lacked MREs but harbored DNA recognition motifs for other transcription factors (FOX, EGR1, AP1, PAX5) suggesting functional interaction. This work provides new insights into aldosterone MR-mediated renal signaling and opens relevant perspectives for mineralocorticoid-related pathophysiology.

Glucocorticoids stimulate endolymphatic water reabsorption in inner ear through aquaporin 3 regulation.

Menière's disease, clinically characterized by fluctuating, recurrent, and invalidating vertigo, hearing loss, and tinnitus, is linked to an increase in endolymph volume, the so-called endolymphatic hydrops. Since dysregulation of water transport could account for the generation of this hydrops, we investigated the role of aquaporin 3 (AQP3) in water transport into endolymph, the K-rich, hyperosmotic fluid that bathes the apical ciliated membrane of sensory cells, and we studied the regulatory effect of dexamethasone upon AQP3 expression and water fluxes. The different AQP subtypes were identified in inner ear by RT-PCR. AQP3 was localized in human utricle and mouse inner ear by immunohistochemistry and confocal microscopy. Unidirectional transepithelial water fluxes were studied by means of (3)H2O transport in murine EC5v vestibular cells cultured on filters, treated or not with dexamethasone (10(-7) M). The stimulatory effect of dexamethasone upon AQP3 expression was assessed in EC5v cells and in vivo in mice. AQP3 was unambiguously detected in human utricle and was highly expressed in both endolymph secretory structures of the mouse inner ear, and EC5v cells. We demonstrated that water reabsorption, from the apical (endolymphatic) to the basolateral (perilymphatic) compartments, was stimulated by dexamethasone in EC5v cells. This was accompanied by a glucocorticoid-dependent increase in AQP3 expression at both messenger RNA (mRNA) and protein level, presumably through glucocorticoid receptor-mediated AQP3 transcriptional activation. We show that glucocorticoids enhance AQP3 expression in human inner ear and stimulate endolymphatic water reabsorption. These findings should encourage further clinical trials evaluating glucocorticoids efficacy in Menière's disease.

Germline and somatic genetic variations of TNFAIP3 in lymphoma complicating primary Sjogren's syndrome.

Several autoimmune diseases, including primary Sjögren's syndrome (pSS), are associated with an increased risk for lymphoma. Polymorphisms of TNFAIP3, which encodes the A20 protein that plays a key role in controlling nuclear factor κB activation, have been associated with several autoimmune diseases. Somatic mutations of TNFAIP3 have been observed in the mucosa-associated lymphoid tissue lymphoma subtype frequently associated with pSS. We studied germline and somatic abnormalities of TNFAIP3 in 574 patients with pSS, including 25 with lymphoma. Nineteen additional patients with pSS and lymphoma were available for exome sequence analysis. Functional abnormalities of A20 were assessed by gene reporter assays. The rs2230926 exonic variant was associated with an increased risk for pSS complicated by lymphoma (odds ratio, 3.36 [95% confidence interval, 1.34-8.42], and odds ratio, 3.26 [95% confidence interval, 1.31-8.12], vs controls and pSS patients without lymphoma, respectively; P = .011). Twelve (60%) of the 20 patients with paired germline and lymphoma TNFAIP3 sequence data had functional abnormalities of A20: 6 in germline DNA, 5 in lymphoma DNA, and 1 in both. The frequency was even higher (77%) among pSS patients with mucosa-associated lymphoid tissue lymphoma. Some of these variants showed impaired control of nuclear factor κB activation. These results support a key role for germline and somatic variations of A20 in the transformation between autoimmunity and lymphoma.

Hypertonicity compromises renal mineralocorticoid receptor signaling through Tis11b-mediated post-transcriptional control.

The mineralocorticoid receptor (MR) mediates the Na(+)-retaining action of aldosterone. MR is highly expressed in the distal nephron, which is submitted to intense variations in extracellular fluid tonicity generated by the corticopapillary gradient. We previously showed that post-transcriptional events control renal MR abundance. Here, we report that hypertonicity increases expression of the mRNA-destabilizing protein Tis11b, a member of the tristetraprolin/ZFP36 family, and thereby, decreases MR expression in renal KC3AC1 cells. The 3'-untranslated regions (3'-UTRs) of human and mouse MR mRNA, containing several highly conserved adenylate/uridylate-rich elements (AREs), were cloned downstream of a reporter gene. Luciferase activities of full-length or truncated MR Luc-3'-UTR mutants decreased drastically when cotransfected with Tis11b plasmid, correlating with an approximately 50% shorter half-life of ARE-containing transcripts. Using site-directed mutagenesis and RNA immunoprecipitation, we identified a crucial ARE motif within the MR 3'-UTR, to which Tis11b must bind for destabilizing activity. Coimmunoprecipitation experiments suggested that endogenous Tis11b physically interacts with MR mRNA in KC3AC1 cells, and Tis11b knockdown prevented hypertonicity-elicited repression of MR. Moreover, hypertonicity blunted aldosterone-stimulated expression of glucocorticoid-induced leucine-zipper protein and the α-subunit of the epithelial Na(+) channel, supporting impaired MR signaling. Challenging the renal osmotic gradient by submitting mice to water deprivation, diuretic administration, or high-Na(+) diet increased renal Tis11b and decreased MR expression, particularly in the cortex, thus establishing a mechanistic pathway for osmotic regulation of MR expression in vivo. Altogether, we uncovered a mechanism by which renal MR expression is regulated through mRNA turnover, a post-transcriptional control that seems physiologically relevant.

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

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

A novel mutation in the NR3C1 gene associated with reversible glucocorticoid resistance.

OBJECTIVE: Glucocorticoid resistance is a rare endocrine disease caused by variants of the NR3C1 gene encoding the glucocorticoid receptor (GR). We identified a novel heterozygous variant (GRR569Q) in a patient with uncommon reversible glucocorticoid resistance syndrome. METHODS: We performed ex vivo functional characterization of the variant in patient fibroblasts and in vitro through transient transfection in undifferentiated HEK 293T cells to assess transcriptional activity, affinity, and nuclear translocation. We studied the impact of the variant on the tertiary structure of the ligand-binding domain through 3D modeling. RESULTS: The patient presented initially with an adrenal adenoma with mild autonomous cortisol secretion and undetectable adrenocorticotropin hormone (ACTH) levels. Six months after surgery, biological investigations showed elevated cortisol and ACTH (urinary free cortisol 114 µg/24 h, ACTH 10.9 pmol/L) without clinical symptoms, evoking glucocorticoid resistance syndrome. Functional characterization of the GRR569Q showed decreased expression of target genes (in response to 100 nM cortisol: SGK1 control +97% vs patient +20%, P < .0001) and impaired nuclear translocation in patient fibroblasts compared to control. Similar observations were made in transiently transfected cells, but higher cortisol concentrations overcame glucocorticoid resistance. GRR569Q showed lower ligand affinity (Kd GRWT: 1.73 nM vs GRR569Q: 4.61 nM). Tertiary structure modeling suggested a loss of hydrogen bonds between H3 and the H1-H3 loop. CONCLUSION: This is the first description of a reversible glucocorticoid resistance syndrome with effective negative feedback on corticotroph cells regarding increased plasma cortisol concentrations due to the development of mild autonomous cortisol secretion.

Double Myod and Igf2 inactivation promotes brown adipose tissue development by increasing Prdm16 expression.

Brown fat or brown adipose tissue (BAT), found in newborn mammals as small depots localized in the interscapular region, plays a prominent role in regulating thermogenesis perinatally. The physiological importance of functional BAT has been recently reasserted in human adults. Because myoblasts and adipoblasts emerge from a common mesodermal precursor, we investigated developmental determination and the reciprocal relationship between muscle and adipocyte commitment. Here we show that a mutant mouse defective for both Igf2 and Myod genes exhibits massive BAT hypertrophy compared with wild-type and single-mutant newborns. The increased adipocyte proliferation in BAT of double-mutant newborns was associated with overexpression of the brown fat-specific marker Ucp1. More strikingly, expression of the master key gene Prdm16 involved in the switch between myogenic and brown adipogenic lineages was drastically enhanced. We further demonstrate that concomitant Myod and Igf2 inactivation accelerates differentiation of a brown preadipocyte cell line and induces lipid accumulation and increased Ucp1 and Prdm16 expression. This in vitro approach brings additional support for the implication of both Myod and Igf2 in BAT development. These results provide the first in vivo evidence that a myogenic regulator together with a growth factor act simultaneously but through independent pathways to repress Prdm16, which opens potential therapeutic perspectives for human metabolic disorders.

Beige differentiation of adipose depots in mice lacking prolactin receptor protects against high-fat-diet-induced obesity.

Stimulating conversion of white fat to metabolically active adipocytes (beige fat) constitutes a promising strategy against weight gain and its deleterious associated-disorders. We provide direct evidence that prolactin (PRL), best known for its actions on the mammary gland, plays a pivotal role in energy balance through the control of adipocyte differentiation and fate. Here we show that lack of prolactin receptor (PRLR) causes resistance to high-fat-diet-induced obesity due to enhanced energy expenditure and increased metabolic rate. Mutant mice displayed reduced fat mass associated with appearance of massive brown-like adipocyte foci in perirenal and subcutaneous but not in gonadal fat depots under a high-fat diet. Positron emission tomography imaging further demonstrated the occurrence of these thermogenic brown fat depots in adult mice, providing additional support for recruitable brown-like adipocytes (beigeing) in white fat depots. Consistent with the activation of brown adipose tissue, PRLR inactivation increases expression of master genes controlling brown adipocyte fate (PRDM16) and mitochondrial function (PGC1α, UCP1). Altered pRb/Foxc2 expression suggests that this PRL-regulated pathway may contribute to beige cell commitment. Together, these results provide direct genetic evidence that PRLR affects energy balance and metabolic adaptation in rodents via effects on brown adipose tissue differentiation and function.