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.

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.

Mineralocorticoid Receptor Antagonism by Finerenone Attenuates Established Pulmonary Hypertension in Rats.

BACKGROUND: We studied the ability of the nonsteroidal MR (mineralocorticoid receptor) antagonist finerenone to attenuate vascular remodeling and pulmonary hypertension using two complementary preclinical models (the monocrotaline and sugen/hypoxia rat models) of severe pulmonary hypertension. METHODS: We first examined the distribution pattern of MR in the lungs of patients with pulmonary arterial hypertension (PAH) and in monocrotaline and sugen/hypoxia rat lungs. Subsequent studies were performed to explore the effect of MR inhibition on proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. To validate the functional importance of MR activation in the pulmonary vascular remodeling characteristic of pulmonary hypertension, mice overexpressing human MR (hMR+) were studied, and curative treatments with finerenone (1 mg/kg per day by gavage), started 2 weeks after monocrotaline injection or 5 weeks after Sugen injection were realized. RESULTS: We demonstrated that MR is overexpressed in experimental and human PAH and that its inhibition following small interfering RNA-mediated MR silencing or finerenone treatment attenuates proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. In addition, we obtained evidence that hMR+ mice display increased right ventricular systolic pressure, right ventricular hypertrophy, and remodeling of pulmonary arterioles. Consistent with these observations, curative treatments with finerenone partially reversed established pulmonary hypertension, reducing total pulmonary vascular resistance and vascular remodeling. Finally, we found that continued finerenone treatment decreases inflammatory cell infiltration and vascular cell proliferation in monocrotaline and sugen/hypoxia rat lungs. CONCLUSIONS: Finerenone treatment appears to be a potential therapy for PAH worthy of investigation and evaluation for clinical use in conjunction with current PAH treatments.

miR-324-5p and miR-30c-2-3p Alter Renal Mineralocorticoid Receptor Signaling under Hypertonicity.

The Mineralocorticoid Receptor (MR) mediates the sodium-retaining action of aldosterone in the distal nephron, but mechanisms regulating MR expression are still poorly understood. We previously showed that RNA Binding Proteins (RBPs) regulate MR expression at the post-transcriptional level in response to variations of extracellular tonicity. Herein, we highlight a novel regulatory mechanism involving the recruitment of microRNAs (miRNAs) under hypertonicity. RT-qPCR validated miRNAs candidates identified by high throughput screening approaches and transfection of a luciferase reporter construct together with miRNAs Mimics or Inhibitors demonstrated their functional interaction with target transcripts. Overexpression strategies using Mimics or lentivirus revealed the impact on MR expression and signaling in renal KC3AC1 cells. miR-324-5p and miR-30c-2-3p expression are increased under hypertonicity in KC3AC1 cells. These miRNAs directly affect Nr3c2 (MR) transcript stability, act with Tis11b to destabilize MR transcript but also repress Elavl1 (HuR) transcript, which enhances MR expression and signaling. Overexpression of miR-324-5p and miR-30c-2-3p alter MR expression and signaling in KC3AC1 cells with blunted responses in terms of aldosterone-regulated genes expression. We also confirm that their expression is increased by hypertonicity in vivo in the kidneys of mice treated with furosemide. These findings may have major implications for the pathogenesis of renal dysfunctions, sodium retention, and mineralocorticoid resistance.

Progesterone receptor isoform ratio dictates antiprogestin/progestin effects on breast cancer growth and metastases: A role for NDRG1.

Progesterone receptors (PRs) ligands are being tested in luminal breast cancer. There are mainly two PR isoforms, PRA and PRB, and their ratio (PRA/PRB) may be predictive of antiprogestin response. Our aim was to investigate: the impact of the PR isoform ratio on metastatic behaviour, the PR isoform ratio in paired primary tumours and lymph node metastases (LNM) and, the effect of antiprogestin/progestins on metastatic growth. Using murine and human metastatic models, we demonstrated that tumours with PRB > PRA (PRB-H) have a higher proliferation index but less metastatic ability than those with PRA > PRB (PRA-H). Antiprogestins and progestins inhibited metastatic burden in PRA-H and PRB-H models, respectively. In breast cancer samples, LNM retained the same PRA/PRB ratio as their matched primary tumours. Moreover, PRA-H LNM expressed higher total PR levels than the primary tumours. The expression of NDRG1, a metastasis suppressor protein, was higher in PRB-H compared to PRA-H tumours and was inversely regulated by antiprogestins/progestins. The binding of the corepressor SMRT at the progesterone responsive elements of the NDRG1 regulatory sequences, together with PRA, impeded its expression in PRA-H cells. Antiprogestins modulate the interplay between SMRT and AIB1 recruitment in PRA-H or PRB-H contexts regulating NDRG1 expression and thus, metastasis. In conclusion, we provide a mechanistic interpretation to explain the differential role of PR isoforms in metastatic growth and highlight the therapeutic benefit of using antiprogestins in PRA-H tumours. The therapeutic effect of progestins in PRB-H tumours is suggested.

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.

Adrenomedullin: new inhibitory regulator for cortisol synthesis and secretion.

Preterm birth is associated with immaturity of several crucial physiological functions notably those prevailing in the lung and kidney. Recently, a steroid secretion deficiency was identified in very preterm neonates, associated with a partial yet transient deficiency in 11ß-hydroxylase activity, sustaining cortisol synthesis. However, the P450c11ß enzyme is expressed in preterm adrenal glands, we hypothesized an inhibition of cortisol production by adrenomedullin (ADM), a peptide highly produced in neonates and whose effect on steroidogenesis remains poorly known. We studied the effects of ADM on three models: 104 cord-blood samples of the PREMALDO neonate cohort, genetically targeted mice overexpressing ADM, and two human adrenocortical cell lines (H295R and HAC15 cells). Mid-regional-proADM (MR-proADM) quantification in cord-blood samples showed strong negative correlation with gestational age (P = 0.0004), cortisol production (P < 0.0001), and 11ß-hydroxylase activity index (P < 0.0001). Mean MR-proADM was higher in very preterm than in term neonates (1.12 vs 0.60 nmol/L, P < 0.0001). ADM-overexpression mice revealed a lower 11ß-hydroxylase activity index (P < 0.05). Otherwise, aldosterone levels measured by LC-MS/MS were higher in ADM-overexpression mice (0.83 vs 0.46 ng/mL, P < 0.05). More importantly, the negative relationship between adrenal ADM expression and aldosterone production found in control was lacking in the ADM-overexpression mice. Finally, LC-MS/MS and gene expression studies on H295R and HAC15 cells revealed an ADM-induced inhibition of both cortisol secretion in cell supernatants and CYP11B1 expression. Collectively, our results converge toward an inhibitory effect of ADM on glucocorticoid synthesis in humans and should be considered to explain the steroid secretion deficiency observed at birth in premature newborns.

The invention of aldosterone, how the past resurfaces in pediatric endocrinology.

Sodium and water homeostasis are drastically modified at birth, in mammals, by the transition from aquatic life to terrestrial life. Accumulating evidence during the past ten years underscores the central role for the mineralocorticoid signaling pathway, in the fine regulation of this equilibrium, at this critical period of development. Interestingly, regarding evolution, while the mineralocorticoid receptor is expressed in fish, the appearance of its related ligand, aldosterone, coincides with terrestrial life, as it is first detected in lungfish and amphibian. Thus, aldosterone is likely one of the main hormones regulating the transition from an aquatic environment to an air environment. This review will focus on the different actors of the mineralocorticoid signaling pathway from aldosterone secretion in the adrenal gland, to mineralocorticoid receptor expression in the kidney, summarizing their regulation and roles throughout fetal and neonatal development, in the light of evolution.

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.

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.

Changes in circulating miRNA19a-3p precede insulin resistance programmed by intra-uterine growth retardation in mice.

OBJECTIVE: Individuals born with intrauterine growth retardation (IUGR) are more prone to cardio-metabolic diseases as adults, and environmental changes during the perinatal period have been identified as potentially crucial factors. We have studied in a preclinical model early-onset molecular alterations present before the development of a clinical phenotype. METHODS: We used a preclinical mouse model of induced IUGR, in which we modulated the nutrition of the pups during the suckling period, to modify their susceptibility to cardio-metabolic diseases in adulthood. RESULTS: Mice born with IUGR that were overfed (IUGR-O) during lactation rapidly developed obesity, hepatic steatosis and insulin resistance, by three months of age, whereas those subjected to nutrition restriction during lactation (IUGR-R) remained permanently thin and highly sensitive to insulin. Mice born with IUGR and fed normally during lactation (IUGR-N) presented an intermediate phenotype and developed insulin resistance by 12 months of age. Molecular alterations to the insulin signaling pathway with an early onset were observed in the livers of adult IUGR-N mice, nine months before the appearance of insulin resistance. The implication of epigenetic changes was revealed by ChIP sequencing, with both posttranslational H3K4me3 histone modifications and microRNAs involved. CONCLUSIONS: These two changes lead to the coherent regulation of insulin signaling, with a decrease in Akt gene transcription associated with an increase in the translation of its inhibitor, Pten. Moreover, we found that the levels of the implicated miRNA19a-3p also decreased in the blood of young adult IUGR mice nine months before the appearance of insulin resistance, suggesting a possible role for this miRNA as an early circulating biomarker of metabolic fate of potential use for precision medicine.

The novel non-steroidal MR antagonist finerenone improves metabolic parameters in high-fat diet-fed mice and activates brown adipose tissue via AMPK-ATGL pathway.

Mineralocorticoid receptor antagonists (MRAs) are recommended for the treatment of heart failure and hypertension, mainly due to their natriuretic and anti-fibrotic mode of action. Rodent studies have shown that MRAs can prevent adverse metabolic consequences of obesity but an elucidation of underlying molecular mechanisms is missing. Here, we investigated metabolic effects of the novel non-steroidal MRA finerenone (FIN) in a mouse model of high-fat diet (HFD)-induced obesity and the signaling pathways activated by MR antagonism at level of interscapular brown adipose tissue (iBAT). C57BL/6J male mice were fed a normal diet or a HFD (with60% kcal from fat) containing or not FIN for 3 months. Metabolic parameters, adipose tissue morphology, gene and protein expression analysis were assessed. We also used brown adipocyte cultures (T37i cells) to investigate the effects of FIN-mediated MR antagonism upon lipid and mitochondrial metabolism. HFD + FIN-treated mice showed improved glucose tolerance together with increased multilocularity and higher expression of thermogenic markers at the level of iBAT, without differences in white adipose depots, suggesting an iBAT-specific effect of FIN. Mechanistically, FIN increased activation of AMP-activated protein kinase which, in turn, stimulated adipose triglyceride lipase activation, with subsequent increased expression of uncoupling protein-1 in brown adipocytes.

Preterm birth is associated with epigenetic programming of transgenerational hypertension in mice.

Renal and cardiovascular complications of prematurity are well established, notably the development of hypertension in adulthood. However, the underlying molecular mechanisms remain poorly understood. Our objective was to investigate the impact of prematurity on the ontogenesis of renal corticosteroid pathways, to evaluate its implication in perinatal renal complications and in the emergence of hypertension in adulthood. Swiss CD1 pregnant mice were injected with lipopolysaccharides at 18 days of gestation (E18) to induce prematurity at E18.5. Pups were sacrificed at birth, 7 days and 6 months of life. Second (F2) and third (F3) generations, established by mating prematurely born adult females with wild-type males, were also analyzed. Former preterm males developed hypertension at M6 (P < 0.0001). We found robust activation of renal corticosteroid target gene transcription at birth in preterm mice (αENaC (+45%), Gilz (+85%)), independent of any change in mineralocorticoid or glucocorticoid receptor expression. The offspring of the preterm group displayed increased blood pressure in F2 and F3, associated with increased renal Gilz mRNA expression, despite similar MR or GR expression and plasma corticosteroid levels measured by LC-MS/MS. Gilz promoter methylation measured by methylated DNA immunoprecipitation-qPCR was reduced with a negative correlation between methylation and expression (P = 0.0106). Our study demonstrates prematurity-related alterations in renal corticosteroid signaling pathways, with transgenerational inheritance of blood pressure dysregulation and epigenetic Gilz regulation up to the third generation. This study provides a better understanding of the molecular mechanisms involved in essential hypertension, which could partly be due to perinatal epigenetic programming from previous generations.

GENETICS IN ENDOCRINOLOGY: Glucocorticoid resistance syndrome

Glucocorticoids (GC) such as cortisol regulate multiple physiological functions, notably those involved in development, metabolism, inflammatory processes and stress, and exert their effects upon binding to the glucocorticoid receptor (GR, encoded by NR3C1 gene in humans). GC signaling follows several consecutive steps leading to target gene transactivation, including ligand binding, nuclear translocation of ligand-activated GR complexes, DNA binding, and recruitment of functional transcriptional machinery. Generalized glucocorticoid resistance syndrome, due to GR loss-of-function mutations, may be related to the impairment of one of the GC signaling steps. To date, 31 NR3C1 loss-of-function mutations have been reported in patients presenting with various clinical signs such as hypertension, adrenal hyperplasia, hirsutism or metabolic disorders associated with biological hypercortisolism but without Cushing syndrome signs and no negative regulatory feedback loop on the hypothalamic-pituitary-adrenal axis. Functional characterization of GR loss-of-function mutations often demonstrates GR haploinsufficiency and a decrease of GR target gene induction in relevant cell types. The main signs at presentation are very variable from resistant hypertension, bilateral adrenal hyperplasia likely related to increased ACTH levels but not exclusively, hirsutism to isolated renin-angiotensin-aldosterone system abnormalities in a context of 11ßHSD2 deficiency. Some mutated GR patients are obese or overweight together with a healthier metabolic profile that remains to be further explored in future studies. Deciphering the molecular mechanisms altered by GR mutations should enhance our knowledge on GR signaling and ultimately facilitate management of GC-resistant patients. This review also focuses on the criteria facilitating identification of novel NR3C1 mutations in selected patients.

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.

Urinary steroidomic profiles by LC-MS/MS to monitor classic 21-Hydroxylase deficiency.

21-hydroxylase deficiency, the most common enzyme defect associated with congenital adrenal hyperplasia (CAH) is characterized by an impairment of both aldosterone and cortisol biosynthesis. Close clinical and biological monitoring of Hydrocortisone (HC) and 9α-Fludrocortisone (FDR) replacement therapies is required to achieve an optimal treatment. As frequent and repeated reassessments of plasma steroids, 17-hydroxyprogesterone (17-OHP), androstenedione (Δ4-A) and testosterone (TESTO) is needed in childhood, urine steroid profiling could represent an interesting non-invasive alternative. We developed and validated a LC-MS/MS method for the measurement of 23-urinary mineralocorticoids, glucocorticoids and adrenal androgens. The usefulness of steroid profiling was investigated on single 08h00 am-collected spot urine for discriminating between 61 CAH patients and their age- and sex-matched controls. CAH patients were split into two groups according to their 08h00 am-plasma concentrations of 17-OHP: below (controlled patients, n = 26) and above 20 ng/mL (uncontrolled patients, n = 35). The lower limit of quantification and the wide analytical range allows to assay both free and total concentrations of the main urinary adreno-corticoids and their tetra-hydrometabolites. Extraction recoveries higher than 75% and intra-assay precision below 20% were found for most steroids. Urinary steroids upstream of the 21-hydroxylase defect were higher in uncontrolled CAH patients. Among CAH patients, plasma and urinary 17-OHP were closely correlated. As compared to controls, steroids downstream of the enzyme defect collapsed in CAH patients. This fall was more pronounced in controlled than in uncontrolled patients. Androgens (Δ4-A, TESTO and the sum etiocholanolone + androsterone) accumulated in uncontrolled CAH patients. A strong relationship was observed between plasma and urinary levels of androstenedione. Daily doses and urinary excretion of both FDR and HC were similar in both CAH groups. Urinary FDR was inversely related to the sodium-to-potassium ratio in urine. A partial least squares discriminant analysis model allowed to classify the patient's classes unaffected, controlled and un-controlled CAH patients based on urinary steroidomic profiles. Our LC-MS/MS method successfully established steroid profiling in urine and represents a useful and non-invasive tool for discriminating CAH patients according to treatment efficiency.

Insuffisance surrénalienne secondaire : actualités diagnostiques et thérapeutiques: News in diagnosis and therapeutics of secondary adrenal insufficiency.

Immunotherapy and opioids treatment are new causes of secondary adrenal insufficiency (SAI). Prevalence of SAI with immunotherapy is more frequent with combined therapy (8% vs 4 to 10% with CTLA4 blocking antibody and 1% with PD1 blocking antibody). Although hypophysitis are more frequently observed with CTLA4 blocking antibody, some cases of Isolated SAI have been reported in patients treated by PD1 blocking antibody. SAI could be transient, requiring long-term monitoring. The use of opioid analgesics is increasing in many countries, thus becoming a public health problem. Prevalence of opioid-related SAI is unclear but recent prospective studies reveal a prevalence between 5 and 20%. The main risk factor to develop this pathology is morphine-equivalent daily dose. Diagnosis relies on 8.00 am plasma cortisol measurement and cortisol increase after Synacthen® administration. Recent cortisol immuno-assays, in agreement with mass spectrometry, give lower reference values, encouraging reevaluation of the current cut-off of 500 nmol/L. New modified-release hydrocortisone preparations have been recently developed to better mimic the physiological cortisol rhythm and to improve compliance in adrenocortical deficient patients. Nowadays, continuous subcutaneous hydrocortisone infusion seems to be a unique replacement therapy allowing adequate circadian biorhythm but should be restricted to specific patients due to the complexity of this substituting strategy. © 2019 Published by Elsevier Masson SAS. All rights reserved. Cet article fait partie du numéro supplément Les Must de l'Endocrinologie 2019 réalisé avec le soutien institutionnel de Ipsen-Pharma.

Impaired 11ß-Hydroxysteroid Dehydrogenase Type 2 in Glucocorticoid-Resistant Patients.

CONTEXT: Six patients carrying heterozygous loss-of-function mutations of glucocorticoid (GC) receptor (GR) presented with hypercortisolism, associated with low kalemia, low plasma renin, and aldosterone levels, with or without hypertension, suggesting a pseudohypermineralocorticism whose mechanisms remain unclear. We hypothesize that an impaired activity of the 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2; encoded by the HSD11B2 gene), catalyzing cortisol (F) inactivation, may account for an inappropriate activation of a renal mineralocorticoid signaling pathway in these GC-resistant patients. OBJECTIVE: We aim at studying the GR-mediated regulation of HSD11B2. DESIGN: The HSD11B2 promoter was subcloned and luciferase reporter assays evaluated GR-dependent HSD11B2 regulation, and 11ß-HSD2 expression/activity was studied in human breast cancer MCF7 cells, endogenously expressing this enzyme. RESULTS: Transfection assays revealed that GR transactivated the long (2.1-kbp) HSD11B2 promoter construct, whereas a defective 501H GR mutant was unable to stimulate luciferase activity. GR-mediated transactivation of the HSD11B2 gene was inhibited by the GR antagonist RU486. A threefold increase in HSD11B2 mRNA levels was observed after dexamethasone (DXM) treatment of MCF7 cells, inhibited by RU486 or by actinomycin, supporting a GR-dependent transcription. Chromatin immunoprecipitation further demonstrated a DXM-dependent GR recruitment onto the HSD11B2 promoter. 11ß-HSD2 activity, evaluated by the cortisone/F ratio, quantified by liquid chromatography/tandem mass spectrometry, was 10-fold higher in the supernatant of DXM-treated cells than controls, consistent with a GR-dependent stimulation of 11ß-HSD2 catalytic activity. CONCLUSION: Collectively, we demonstrate that 11ß-HSD2 expression and activity are transcriptionally regulated by GR. In the context of GR haploinsufficiency, these findings provide evidence that defective GR signaling may account for apparent mineralocorticoid excess in GC-resistant patients.

Functional Characterization of Glucocorticoid Receptor Variants Is Required to Avoid Misinterpretation of NGS Data.

Recent advances in genetic analysis technologies such as next-generation sequencing (NGS) have considerably increased the incidental discovery of genetic abnormalities. Six heterozygous missense mutations of the human glucocorticoid receptor (GR; encoded by the NR3C1 gene) have been identified in the context of genetic screening of endocrine pathologies. GR, a nuclear receptor, hormone-induced transcription factor, is involved in many physiological processes. Nevertheless, the pathogenic significance of incidentally discovered mutations remains obscure. The aim of this work was to characterize these variants by evaluating their functional impact on GR signaling. Six original GR variants, located in exon 2, led to amino acid substitutions of the N-terminal domain of GR (F65V, M86V, A229T, A304E, N374S, and R386Q), excluding mainly the activation function tau core 1 domain, the potential site of functional interaction with transcriptional coregulators. Transient cotransfection in HEK293T cells of mutated GR-expressing vectors and a luciferase reporter established dose-response curves for dexamethasone. This excluded any major transactivation abnormality of the mutated GRs (ligand concentration leading to 50% maximal transactivation capacity ≈ 0.2 nM), with maximal transactivation capacity identical to that of the wild-type (WT) GR and without modification of the potentiation of transcriptional coactivator steroid receptor coactivator 2 except in N374S. Moreover, protein expression of mutated GRs and their cytonuclear translocation studied by immunocytochemistry were almost unchanged compared with WT GR. These results underline the silent nature of these missense GR variants and call for cautious interpretation of the discovery of genetic incidentalomas by NGS in the absence of detailed characterization in order to appropriately assess their functional impact on a particular signaling pathway.

Identification of two independent SUMO-interacting motifs in Fas-associated factor 1 (FAF1): Implications for mineralocorticoid receptor (MR)-mediated transcriptional regulation.

Fas-associated factor 1 (FAF1) was originally isolated as a Fas-associated factor and was subsequently found to interact with numerous other proteins that are involved in various cellular events including Fas-mediated apoptosis, nuclear factor (NF)-κB, Wnt/ß-catenin, and transforming growth factor (TGF)-ß signaling pathways, mineralocorticoid receptor (MR)-mediated transactivation, and ubiquitin-dependent processes. Herein, we defined two small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) within FAF1 and demonstrated to be crucial for transcriptional modulation of the MR. Our study demonstrated that the SIMs of FAF1 do not play a significant role in regulating its subcellular localization, Fas-mediated apoptosis, or NF-κB or Wnt/ß-catenin pathways. Remarkably, FAF1 interacts with the sumoylated MR and represses aldosterone-activated MR transactivation in a SIM-dependent manner. Moreover, silencing of endogenous FAF1 in cells resulted in an increase in the induction of MR target genes by aldosterone, indicating that FAF1 functions as an MR co-repressor. We further provide evidence to suggest that the mechanisms of FAF1/SIM-mediated MR transrepression involve inhibition of MR N/C interactions and promotion of MR polyubiquitination and degradation. Sumoylation has been linked to impacting of repressive properties on several transcription factors and cofactors. Our findings therefore provide mechanistic insights underlying SUMO-dependent transcriptional repression of the MR.