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.

Loss of cAbl Tyrosine Kinase in Pulmonary Arterial Hypertension Causes Dysfunction of Vascular Endothelial Cells.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by the dysfunction of pulmonary endothelial cells (ECs) and obstructive vascular remodeling. cAbl (non-receptor tyrosine kinase c-Abelson) plays central roles in regulating cell-cycle arrest, apoptosis, and senescence after cellular stress. We hypothesized that cAbl is downactivated in experimental and human PAH, thus leading to reduced DNA integrity and angiogenic capacity of pulmonary ECs from patients with PAH (PAH-ECs). We found cAbl and phosphorylated cAbl concentrations to be lower in the endothelium of remodeled pulmonary vessels in the lungs of patients with PAH than in control subjects. Similar observations were obtained for the lungs of Sugen + hypoxia and monocrotaline rats with established pulmonary hypertension. These in situ abnormalities were also replicated in vitro, with cultured PAH-ECs displaying lower cAbl expression and activity and an altered DNA damage response and capacity of tube formation. Downregulation of cAbl by RNA interference in control ECs or its inhibition with dasatinib resulted in genomic instability and the failure to form tubes, whereas upregulation of cAbl with 5-(1,3-diaryl-1H-pyrazol-4-yl) hydantoin reduced DNA damage and apoptosis in PAH-ECs. Finally, we establish the existence of cross-talk between cAbl and bone morphogenetic protein receptor type II. This work identifies the loss of cAbl signaling as a novel contributor to pulmonary EC dysfunction associated with PAH.

ATG7 and ATG9A loss-of-function variants trigger autophagy impairment and ovarian failure.

PURPOSE: Primary ovarian insufficiency (POI) is a frequent disorder that affects ~1% of women under 40 years of age. POI, which is characterized by the premature depletion of ovarian follicles and elevated plasma levels of follicle-stimulating hormone (FSH), leads to infertility. Although various etiological factors have been described, including chromosomal abnormalities and gene variants, most cases remain idiopathic. The aim of the present study was to identify and validate functionally new sequence variants in ATG (autophagy-related genes) leading to POI. METHODS: We have reanalyzed, in silico, the exome sequencing data from a previously reported work performed in 69 unrelated POI women. Functional experiments using a classical hallmark of autophagy, the microtubule-associated protein 1 light chain 3ß (LC3), were then used to link these genes to this lysosomal degradation pathway. RESULTS: We venture a functional link between ATG7 and ATG9A variants and POI. We demonstrated that variant ATG7 and ATG9A led to a decrease in autophagosome biosynthesis and consequently to an impairment of autophagy, a key biological process implicated in the preservation of the primordial follicles forming the ovarian reserve. CONCLUSION: Our results unveil that impaired autophagy is a novel pathophysiological mechanism involved in human POI.

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.

SRC-3Delta4 mediates the interaction of EGFR with FAK to promote cell migration.

EGF induces signal transduction between EGFR and FAK, and FAK is required for EGF-induced cell migration. It is unknown, however, what factor mediates the interaction between EGFR and FAK and leads to EGF-induced FAK phosphorylation. Here, we identify SRC-3Delta4, a splicing isoform of the SRC-3 oncogene, as a signaling adaptor that links EGFR and FAK and promotes EGF-induced phosphorylations of FAK and c-Src. We identify three PAK1-mediated phosphorylations in SRC-3Delta4 that promote the localization of SRC-3Delta4 to the plasma membrane and mediate the interactions with EGFR and FAK. Importantly, overexpression of SRC-3Delta4 promotes MDA-MB231-induced breast tumor metastasis. Our findings identify phosphorylated SRC-3Delta4 as a missing adaptor between EGFR and its downstream signaling molecule FAK to coordinately regulate EGF-induced cell migration. Our study also reveals that a nuclear receptor coactivator can act in the periphery of a cell to directly mediate activation of an enzyme.

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.

Three Novel Heterozygous Point Mutations of NR3C1 Causing Glucocorticoid Resistance.

Generalized glucocorticoid resistance is associated with glucocorticoid receptor (GR; NR3C1) mutations. Three novel heterozygous missense NR3C1 mutations (R477S, Y478C, and L672P) were identified in patients presenting with adrenal incidentalomas, glucocorticoid excess without Cushing syndrome. Dexamethasone (DXM) binding studies demonstrated that the affinity of GRR477S and GRY478C mutants, located in the DNA-binding domain (DBD) of GR, was similar to wild-type GR (Kd  = 2-3 nM). In contrast, GRL672P mutant, located in the ligand-binding domain (LBD) of GR, was unable to bind glucocorticoids and was more sensitive to protein degradation. GR subcellular distribution revealed a marked decrease in DXM-induced nuclear translocation of GRR477S and GRY478C mutants, whereas GRL672P remained exclusively cytoplasmic. Chromatin immunoprecipitation demonstrated impaired recruitment of DBD mutants onto the regulatory sequence of FKBP5. Transactivation assays disclosed the lack of transcriptional activity of GRR477S and GRL672P , whereas GRY478C had a reduced transactivation capacity. Three-dimensional modeling indicated that R477S lost two essential hydrogen bonds with DNA, Y478C resulted in altered interaction with surrounding amino-acids, destabilizing DBD, whereas L672P altered the H8 helix folding, leading to unstructured LBD. This study identifies novel NR3C1 mutations with their molecular consequences on altered GR signaling and suggests that genetic screening of NR3C1 should be conducted in patients with subclinical hypercorticism.

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.

Prevalence of KISS1 Receptor mutations in a series of 603 patients with normosmic congenital hypogonadotrophic hypogonadism and characterization of novel mutations: a single-centre study.

STUDY QUESTION: What is the exact prevalence of Kisspeptin Receptor (KISS1R) mutations in the population of patients with normosmic congenital hypogonadotrophic hypogonadism (nCHH) by comparison with other genes, involved in gonadotrophin-releasing hormone (GnRH) release or action? SUMMARY ANSWER: KISS1R mutants are responsible for the nCHH phenotype in only a small minority of cases and were less prevalent than GnRH Receptor (GNRHR) mutations. WHAT IS KNOWN ALREADY: The respective prevalence of each of the genetic causes of nCHH is unclear. Large series of patients are very rare and suffer from heterogeneity of the population of CHH studied. STUDY DESIGN, SIZE, DURATION: Patients with nCHH were consecutively enrolled in a single French referral centre and were gradually tested for KISS1R between January 2006 and April 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 603 patients with nCHH (399 men and 204 women) were diagnosed at the Bicêtre Hospital and underwent KISS1R analysis. The GNRHR, tachykinin receptor 3 (TACR3), gonadotrophin-releasing hormone 1 (GNRH1), tachykinin 3 (TAC3) and KISS1 genes were also sequenced. Functional characterization of KISS1R mutations included a study of signal transduction using a reporter gene (serum response element-luciferase (SRE-Luc) involved in the mitogen-activated protein (MAP) kinase pathway. MAIN RESULTS AND THE ROLE OF CHANCE: We detected 15 KISS1R variants (10 novel), in 12 of the 603 patients (2.0%, 95% CI [0.9-3.1]. KISS1R mutations were less prevalent than GNRHR (4.7%) and TACR3 (2.6%) mutations but more prevalent than GNRH1 (1.5%), TAC3 (1.0%) and KISS1 (0%) mutations. KISS1R mutants were present in the biallelic state in 8 of the 12 patients concerned. Among 5 men with biallelic KISS1R mutations, 4 had either micropenis or cryptorchidism. In vitro analysis of the 5 new variants present in the biallelic state (C95W, Y103*, C115W, P176R and A287E) showed a loss of function. LIMITATIONS, REASONS FOR CAUTION: The prevalence of TACR3, GNRH1, TAC3 and KISS1 mutations was calculated from a smaller number of nCHH patients than KISS1R and GNRHR. This should prompt caution concerning the reported prevalence of mutations in these four genes. WIDER IMPLICATIONS OF THE FINDINGS: We show that KISS1R mutants are responsible for the nCHH phenotype in only a small minority of cases. Together, the genes analysed here were mutated in fewer than 15% of patients, suggesting a role of other genes in nCHH. The presence of cryptorchidism and/or micropenis in the majority of men with biallelic KISS1R mutations strongly suggests that this gene is essential for prenatal GnRH secretion. STUDY FUNDING, COMPETING INTERESTS: This work was supported in part by grants from Paris-Sud University (Bonus Qualité Recherche, and Attractivité grants) to J.B., French Ministry of Health, Hospital Clinical Research Program on Rare Diseases. Assistance Publique Hôpitaux de Paris, Programme Hospitalier de Recherche Clinique (PHRC # P081212 HYPOPROTEO) to J.Y. C.P. was supported by student fellowships 'Année Recherche' from Agence Régionale de Santé Provence Alpes Côtes d'Azur. The authors have nothing to disclose.

Atypical protein kinase C regulates dual pathways for degradation of the oncogenic coactivator SRC-3/AIB1.

SRC-3/AIB1 is a steroid receptor coactivator with potent growth-promoting activity, and its overexpression is sufficient to induce tumorigenesis. Previous studies indicate that the cellular level of SRC-3 is tightly regulated by both ubiquitin-dependent and ubiquitin-independent proteasomal degradation pathways. Atypical protein kinase C (aPKC) is frequently overexpressed in cancers. In the present study, we show that aPKC phosphorylates and specifically stabilizes SRC-3 in a selective ER-dependent manner. We further demonstrate that an acidic residue-rich region in SRC-3 is an important determinant for aPKC-mediated phosphorylation and stabilization. The mechanism of the aPKC-mediated stabilization appears due to a decreased interaction between SRC-3 and the C8 subunit of the 20S core proteasome, thus preventing SRC-3 degradation. Our results demonstrate a potent signaling mechanism for regulating SRC-3 levels in cells by coordinate enzymatic inhibition of both ubiquitin-dependent and ubiquitin-independent proteolytic pathways.

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.

Loss of KDM1A in GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome: a multicentre, retrospective, cohort study.

BACKGROUND: GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome is caused by aberrant expression of the GIP receptor in adrenal lesions. The bilateral nature of this disease suggests germline genetic predisposition. We aimed to identify the genetic driver event responsible for GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. METHODS: We conducted a multicentre, retrospective, cohort study at endocrine hospitals and university hospitals in France, Canada, Italy, Greece, Belgium, and the Netherlands. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. Adrenal samples from patients with primary bilateral macronodular adrenal hyperplasia who had undergone an adrenalectomy for overt or mild Cushing's syndrome without evidence of food-dependent cortisol production and those with GIP-dependent unilateral adrenocortical adenomas were used as control groups. We performed whole genome, whole exome, and targeted next generation sequencing, and copy number analyses of blood and adrenal DNA from patients with familial or sporadic disease. We performed RNA sequencing on adrenal samples and functional analyses of the identified genetic defect in the human adrenocortical cell line H295R. FINDINGS: 17 patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome were studied. The median age of patients was 43·3 (95% CI 38·8-47·8) years and most patients (15 [88%]) were women. We identified germline heterozygous pathogenic or most likely pathogenic variants in the KDM1A gene in all 17 patients. We also identified a recurrent deletion in the short p arm of chromosome 1 harboring the KDM1A locus in adrenal lesions of these patients. None of the 29 patients in the control groups had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in adrenal lesions. Global gene expression analysis showed GIP receptor upregulation with a log2 fold change of 7·99 (95% CI 7·34-8·66; p=4·4 × 10-125), and differential regulation of several other G protein-coupled receptors in GIP-dependent primary bilateral macronodular hyperplasia samples compared with control samples. In vitro pharmacological inhibition and inactivation of KDM1A by CRISPR-Cas9 genome editing resulted in an increase of GIP receptor transcripts and protein in human adrenocortical H295R cells. INTERPRETATION: We propose that GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome results from a two-hit inactivation of KDM1A, consistent with the tumour suppressor gene model of tumorigenesis. Genetic testing and counselling should be offered to these patients and their relatives. FUNDING: Agence Nationale de la Recherche, Fondation du Grand défi Pierre Lavoie, and the French National Cancer Institute.

Specific amino acid residues in the basic helix-loop-helix domain of SRC-3 are essential for its nuclear localization and proteasome-dependent turnover.

SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM-1 is a primary transcriptional coactivator for the estrogen receptor. Here we report that deletion of the SRC-3 basic helix-loop-helix (bHLH) domain blocks its proteasome-dependent turnover. We further identified two residues (K17 and R18) in the SRC-3 bHLH domain that are essential for its stability. Moreover, we found that the bHLH domain contains a bipartite nuclear localization signal (NLS). SRC-3 NLS mutants block its translocation into the nucleus, and this correlates with its insensitivity to proteasome-dependent turnover. SRC-3 shows a time-dependent decay in the presence of cycloheximide which is not apparent for the cytoplasmic mutant. Fusion of a simian virus 40 T antigen NLS to the cytoplasmic localized SRC-3 mutant drives it back into the nucleus and restores its proteasomal sensitivity. In addition, the cytoplasmic mutants are inactive for transcriptional coactivation and cancer cell growth. Taken together, our data indicate that proteasome-dependent turnover of SRC-3 occurs in the nucleus and that two amino acid residues in the bHLH domain provide a signal for its nuclear localization and proteasome-dependent degradation as well as for regulation of SRC-3 transcriptional coactivator capacity.

Regulation of SRC-3 intercompartmental dynamics by estrogen receptor and phosphorylation.

The steroid receptor coactivator 3 gene (SRC-3) (AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family transcription coactivator and a known oncogene. Despite its importance, the functional regulation of SRC-3 remains poorly understood within a cellular context. Using a novel combination of live-cell, high-throughput, and fluorescent microscopy, we report SRC-3 to be a nucleocytoplasmic shuttling protein whose intracellular mobility, solubility, and cellular localization are regulated by phosphorylation and estrogen receptor alpha (ERalpha) interactions. We show that both chemical inhibition and small interfering RNA reduction of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by epidermal growth factor signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known participants in the phosphocode that regulates SRC-3 activity. Accordingly, the cytoplasmic localization of a nonphosphorylatable SRC-3 mutant further supported these results. In the presence of ERalpha, U0126 also dramatically reduces (i) ligand-dependent colocalization of SRC-3 and ERalpha, (ii) the formation of ER-SRC-3 complexes in cell lysates, and (iii) SRC-3 targeting to a visible, ERalpha-occupied and -regulated prolactin promoter array. Taken together, these results indicate that phosphorylation coordinates SRC-3 coactivator function by linking the probabilistic formation of transient nuclear receptor-coactivator complexes with its molecular dynamics and cellular compartmentalization. Technically and conceptually, these findings have a new and broad impact upon evaluating mechanisms of action of gene regulators at a cellular system level.

Crosstalk Between Glucocorticoid Receptor and Early-growth Response Protein 1 Accounts for Repression of Brain-derived Neurotrophic Factor Transcript 4 Expression.

The brain-derived neurotrophic factor (BDNF) is a key player in brain functions such as synaptic plasticity, stress, and behavior. Its gene structure in rodents contains 8 untranslated exons (I to VIII) whose expression is finely regulated and which spliced onto a common and unique translated exon IX. Altered Bdnf expression is associated with many pathologies such as depression, Alzheimer's disease and addiction. Through binding to glucocorticoid receptor (GR), glucocorticoids play a pivotal role for stress responses, mood and neuronal plasticity. We recently showed in neuronal primary culture and in the immortalized neuronal-like BZ cells that GR repressed Bdnf expression, notably the bdnf exon IV containing mRNA isoform (Bdnf4) via GR binding to a short 275-bp sequence of Bdnf promoter. Herein, we demonstrate by transient transfection experiments and mutagenesis in BZ cells that GR interacts with an early growth response protein 1 (EGR1) response element (EGR-RE) located in the transcription start site of Bdnf exon IV promoter. Using Chromatin Immunoprecipitation, we find that both GR and EGR1 bind to this promoter sequence in a glucocorticoid-dependent manner and demonstrate by co-immunoprecipitation that GR and EGR1 are interacting physically. Interestingly, EGR1 has been widely characterized as a regulator of brain plasticity. In conclusion, we deciphered a mechanism by which GR downregulates Bdnf expression, identifying a novel functional crosstalk between glucocorticoid pathways, immediate early growth response proteins and Bdnf. As all these factors are well-recognized germane for brain pathophysiology, these findings may have significant implications in neurosciences as well as in therapeutics.

Potentiation of mitotane action by rosuvastatin: New insights for adrenocortical carcinoma management.

Mitotane (also termed o,p'­DDD) is the most effective therapy for advanced adrenocortical carcinoma (ACC). Mitotane­induced dyslipidemia is treated with statins. Mitotane and statins are known to exert anti­proliferative effects in vitro; however, the effects of statins have never been directly evaluated in patients with ACC and ACC cells, at least to the best of our knowledge. Thus, in this study, we aimed to examine the effects of the rosuvastatin on ACC cells. It has been shown that the combined use of mitotane and statins significantly increases the tumor control rate in patients with ACC; however, it would be of interest to elucidate the molecular mechanisms involved in this potentiation. In this study, we examined the effects of mitotane, rosuvastatin and their combination in NCI­H295R human ACC cells using proliferation assays, gene expression analyses and free intracellular cholesterol measurements. The results revealed that mitotane dose­dependently reduced cell viability, induced apoptosis and increased intracellular free cholesterol levels, considered as one of the key features of mitotane action, while rosuvastatin alone reduced cell viability and increased apoptosis at high concentrations. We also demonstrated that rosuvastatin potentiated the effects of mitotane by reducing cell viability, inducing apoptosis, increasing intracellular free cholesterol levels, and by decreasing the expression of 3­hydroxy­3­methylglutaryl­CoA reductase (HMGCR) and ATP binding cassette subfamily a member 1 (ABCA1), genes involved in cholesterol metabolism, and inhibiting steroidogenesis. Collectively, potentiating the effects of mitotane with the use of rosuvastatin may provide novel therapeutic strategies for ACC, given that the combination of these drugs, pending clinical validation, may lead to the better management of ACC.

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.

Ligand-controlled interaction of histone acetyltransferase binding to ORC-1 (HBO1) with the N-terminal transactivating domain of progesterone receptor induces steroid receptor coactivator 1-dependent coactivation of transcription.

Modulators of cofactor recruitment by nuclear receptors are expected to play an important role in the coordination of hormone-induced transactivation processes. To identify such factors interacting with the N-terminal domain (NTD) of the progesterone receptor (PR), we used this domain as bait in the yeast Sos-Ras two-hybrid system. cDNAs encoding the C-terminal MYST (MOZ-Ybf2/Sas3-Sas2-Tip60 acetyltransferases) domain of HBO1 [histone acetyltransferase binding to the origin recognition complex (ORC) 1 subunit], a member of the MYST acetylase family, were thus selected from a human testis cDNA library. In transiently transfected CV1 cells, the wild-type HBO1 [611 amino acids (aa)] enhanced transcription mediated by steroid receptors, notably PR, mineralocorticoid receptor, and glucocorticoid receptor, and strongly induced PR and estrogen receptor coactivation by steroid receptor coactivator 1a (SRC-1a). As assessed by two-hybrid and glutathione-S-transferase pull-down assays, the HBO1 MYST acetylase domain (aa 340-611) interacts mainly with the NTD, and also contacts the DNA-binding domain and the hinge domains of hormone-bound PR. The HBO1 N-terminal region (aa 1-340) associates additionally with PR ligand-binding domain (LBD). HBO1 was found also to interact through its NTD with SRC-1a in the absence of steroid receptor. The latter coassociation enhanced specifically activation function 2 activation function encompassed in the LBD. Conversely, the MYST acetylase domain specifically enhanced SRC-1 coupling with PR NTD, through a hormone-dependent mechanism. In human embryonic kidney 293 cells expressing human PRA or PRB, HBO1 raised selectively an SRC-1-dependent response of PRB but failed to regulate PRA activity. We show that HBO1 acts through modification of an LBD-controlled structure present in the N terminus of PRB leading to the modulation of SRC-1 functional coupling with activation function 3-mediated transcription. Importantly, real-time RT-PCR analysis also revealed that HBO1 enhanced SRC-1 coactivation of PR-dependent transcription of human endogenous genes such as alpha-6 integrin and 11beta-hydroxydehydrogenase 2 but not that of amphiregulin. Immunofluorescence and confocal microscopy of human embryonic kidney-PRB cells demonstrated that the hormone induces the colocalization of HBO1 with PR-SRC-1 complex into nuclear speckles characteristic of PR-mediated chromatin remodeling. Our results suggest that HBO1 might play an important physiological role in human PR signaling.

HuR-Dependent Editing of a New Mineralocorticoid Receptor Splice Variant Reveals an Osmoregulatory Loop for Sodium Homeostasis.

Aldosterone and the Mineralocorticoid Receptor (MR) control hydroelectrolytic homeostasis and alterations of mineralocorticoid signaling pathway are involved in the pathogenesis of numerous human diseases, justifying the need to decipher molecular events controlling MR expression level. Here, we show in renal cells that the RNA-Binding Protein, Human antigen R (HuR), plays a central role in the editing of MR transcript as revealed by a RNA interference strategy. We identify a novel Δ6 MR splice variant, which lacks the entire exon 6, following a HuR-dependent exon skipping event. Using isoform-specific TaqMan probes, we show that Δ6 MR variant is expressed in all MR-expressing tissues and cells and demonstrate that extracelullar tonicity regulates its renal expression. More importantly, this splice variant exerts dominant-negative effects on transcriptional activity of the full-length MR protein. Collectively, our data highlight a crucial role of HuR as a master posttranscriptional regulator of MR expression in response to osmotic stress. We demonstrate that hypotonicity, not only enhances MR mRNA stability, but also decreases expression of the Δ6 MR variant, thus potentiating renal MR signaling. These findings provide compelling evidence for an autoregulatory feedback loop for the control of sodium homeostasis through posttranscriptional events, likely relevant in renal pathophysiological situations.

Identifying mitotane-induced mitochondria-associated membranes dysfunctions: metabolomic and lipidomic approaches.

Mitotane (o,p'DDD), the most effective drug in adrenocortical carcinoma, concentrates into the mitochondria and impacts mitochondrial functions. To address the molecular mechanisms of mitotane action and to identify its potential target, metabolomic and lipidomic approaches as well as imaging analyses were employed in human adrenocortical H295R cells allowing identification of Mitochondria-Associated Membranes dysfunction as a critical impact of mitotane. Study of intracellular energetic metabolites by NMR spectroscopy showed that mitotane significantly decreased aspartate while concomitantly increased glutamate content in a time- and concentration-dependent manner. Such alterations were very likely linked to the previously described, mitotane-induced respiratory chain defect. Lipidomic studies of intracellular and intramitochondrial phospholipids revealed that mitotane exposure markedly reduced the phosphatidylserine/phosphatidylethanolamine ratio, indicative of a dysfunction of phosphatidylserine decarboxylase located in Mitochondria-Associated Membranes. Expression levels of Mitochondria-Associated Membranes proteins phosphatidylserine decarboxylase, DRP1, ATAD3A or TSPO were greatly reduced by mitotane as assessed by western blot analyses. Mitotane exposure markedly altered endogenous Mitochondria-Associated Membranes integrity and reduced the magnitude of mitochondria and the endoplasmic reticulum interactions as demonstrated by high resolution deconvolution microscopy and quantification. Finally, we showed that PK11195, a pharmacological inhibitor of the cholesterol translocator TSPO, embedded in Mitochondria-Associated Membranes, exerts a synergetic effect with mitotane in inducing Mitochondria-Associated Membranes disruption, apoptosis and in inhibiting steroid secretion. Altogether, our results demonstrate Mitochondria-Associated Membranes dysfunction in H295R cells treated with mitotane and that TSPO inhibition significantly potentiates mitotane antitumoral and antisecretory actions in vitro. This constitutes a potential and promising pharmacological strategy for patients with adrenocortical carcinoma.