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.

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.

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.

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.

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.

Hypermethylator Phenotype and Ectopic GIP Receptor in GNAS Mutation-Negative Somatotropinomas.

CONTEXT: Besides GNAS gene mutations, the molecular pathogenesis of somatotroph adenomas responsible for gigantism and acromegaly remains elusive. OBJECTIVE: To investigate alternative driver events in somatotroph tumorigenesis, focusing on a subgroup of acromegalic patients with a paradoxical increase in growth hormone (GH) secretion after oral glucose, resulting from ectopic glucose-dependent insulinotropic polypeptide receptor (GIPR) expression in their somatotropinomas. DESIGN, SETTING, AND PATIENTS: We performed combined molecular analyses, including array-comparative genomic hybridization, RNA/DNA fluorescence in situ hybridization, and RRBS DNA methylation analysis on 41 somatotropinoma samples from 38 patients with acromegaly and three sporadic giants. Ten patients displayed paradoxical GH responses to oral glucose. RESULTS: GIPR expression was detected in 13 samples (32%), including all 10 samples from patients with paradoxical GH responses. All GIPR-expressing somatotropinomas were negative for GNAS mutations. GIPR expression occurred through transcriptional activation of a single allele of the GIPR gene in all GIPR-expressing samples, except in two tetraploid samples, where expression occurred from two alleles per nucleus. In addition to extensive 19q duplications, we detected in four samples GIPR locus microamplifications in a certain proportion of nuclei. We identified an overall hypermethylator phenotype in GIPR-expressing samples compared with GNAS-mutated adenomas. In particular, we observed hypermethylation in the GIPR gene body, likely driving its ectopic expression. CONCLUSIONS: We describe a distinct molecular subclass of somatotropinomas, clinically revealed by a paradoxical increase of GH to oral glucose related to pituitary GIPR expression. This ectopic GIPR expression occurred through hypomorphic transcriptional activation and is likely driven by GIPR gene microamplifications and DNA methylation abnormalities.

Glucocorticoids stimulate hypothalamic dynorphin expression accounting for stress-induced impairment of GnRH secretion during preovulatory period.

Stress-induced reproductive dysfunction is frequently associated with increased glucocorticoid (GC) levels responsible for suppressed GnRH/LH secretion and impaired ovulation. Besides the major role of the hypothalamic kisspeptin system, other key regulators may be involved in such regulatory mechanisms. Herein, we identify dynorphin as a novel transcriptional target of GC. We demonstrate that only priming with high estrogen (E2) concentrations prevailing during the late prooestrus phase enables stress-like GC concentrations to specifically stimulate Pdyn (prodynorphin) expression both in vitro (GT1-7 mouse hypothalamic cell line) and ex vivo (ovariectomized E2-supplemented mouse brains). Our results indicate that stress-induced GC levels up-regulate dynorphin expression within a specific kisspeptin neuron-containing hypothalamic region (antero-ventral periventricular nucleus), thus lowering kisspeptin secretion and preventing preovulatory GnRH/LH surge at the end of the prooestrus phase. To further characterize the molecular mechanisms of E2 and GC crosstalk, chromatin immunoprecipitation experiments and luciferase reporter gene assays driven by the proximal promoter of Pdyn show that glucocorticoid receptors bind specific response elements located within the Pdyn promoter, exclusively in presence of E2. Altogether, our work provides novel understanding on how stress affects hypothalamic-pituitary-gonadal axis and underscores the role of dynorphin in mediating GC inhibitory actions on the preovulatory GnRH/LH surge to block ovulation.

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.

[Differences of Sex Development (DSD): Controversies and Challenges]. / Controverses et challenges sur le développement sexuel différent (DSD).

DSD for "Differences of Sex Development" or "Sexual Differences Development" refers to situations where chromosomal, gonadal or anatomical sex is atypical. DSD 46,XX are mainly represented by congenital adrenal hyperplasia (HCS) and are not a diagnostic issue. DSD 46,XY involve genes for the determination and differenciation of the bipotential gonad, making sometimes difficult the choice of sex at birth. They remain without diagnosis in about half of the cases, despite the new genetic techniques (exome, NGS). The management of DSD is complex as well as are the long-term consequences, particularly in terms of options for medical or surgical treatments, fertility and quality of life of patients that should be discussed. This review describes the main causes of DSD and the recent issues of their clinical management. It addresses the difficult question of identity of these patients, in a society that leaves no place for difference.

Genomic Alterations and Complex Subclonal Architecture in Sporadic GH-Secreting Pituitary Adenomas.

Purpose: The molecular pathogenesis of growth hormone-secreting pituitary adenomas is not fully understood. Cytogenetic alterations might serve as alternative driver events in GNAS mutation-negative somatotroph tumors. Experimental Design: We performed cytogenetic profiling of pituitary adenomas obtained from 39 patients with acromegaly and four patients with sporadic gigantism by using array comparative genomic hybridization analysis. We explored intratumor DNA copy-number heterogeneity in two tumor samples by using DNA fluorescence in situ hybridization (FISH). Results: Based on copy-number profiles, we found two groups of adenomas: a low-copy-number alteration (CNA) group (<12% of genomic disruption, 63% of tumors) and a high-CNA group (24% to 45% of genomic disruption, 37% of tumors). Arm-level CNAs were the most common abnormalities. GNAS mutation-positive adenomas belonged exclusively to the low-CNA group, whereas a subgroup of GNAS mutation-negative adenomas had a high degree of genomic disruption. We detected chromothripsis-related CNA profiles in two adenoma samples from an AIP mutation-positive patient with acromegaly and a patient with sporadic gigantism. RNA sequencing of these two samples identified 17 fusion transcripts, most of which resulted from chromothripsis-related chromosomal rearrangements. DNA FISH analysis of these samples demonstrated a subclonal architecture with up to six distinct cell populations in each tumor. Conclusion: Somatotroph pituitary adenomas display substantial intertumor and intratumor DNA copy-number heterogeneity, as revealed by variable CNA profiles and complex subclonal architecture. The extensive cytogenetic burden in a subgroup of GNAS mutation-negative somatotroph adenomas points to an alternative tumorigenic pathway linked to genomic instability.

Significant prevalence of NR3C1 mutations in incidentally discovered bilateral adrenal hyperplasia: results of the French MUTA-GR Study.

BACKGROUND: Recently discovered mutations of NR3C1 gene, encoding for the GR, in patients with glucocorticoid resistance and bilateral adrenal incidentalomas prompted us to investigate whether GR mutations might be associated with adrenal hyperplasia. OBJECTIVE: The multicenter French Clinical Research Program (Muta-GR) was set up to determine the prevalence of GR mutations and polymorphisms in patients harboring bilateral adrenal incidentalomas associated with hypertension and/or biological hypercortisolism without clinical Cushing's signs. RESULTS: One hundred patients were included in whom NR3C1 sequencing revealed five original heterozygous GR mutations that impaired GR signaling in vitro. Mutated patients presented with mild glucocorticoid resistance defined as elevated urinary free cortisol (1.7 ± 0.7 vs 0.9 ± 0.8 upper limit of normal range, P = 0.006), incomplete 1 mg dexamethasone suppression test without suppressed 8-AM adrenocorticotrophin levels (30.9 ± 31.2 vs 16.2 ± 17.5 pg/mL) compared to the non-mutated patients. Potassium and aldosterone levels were lower in mutated patients (3.6 ± 0.2 vs 4.1 ± 0.5 mmol/L, P = 0.01, and 17.3 ± 9.9 vs 98.6 ± 115.4 pg/mL, P = 0.0011, respectively) without elevated renin levels, consistent with pseudohypermineralocorticism. Ex vivo characterization of mutated patients' fibroblasts demonstrated GR haploinsufficiency as revealed by below-normal glucocorticoid induction of FKBP5 gene expression. There was no association between GR polymorphisms and adrenal hyperplasia in this cohort, except an over-representation of BclI polymorphism. CONCLUSION: The 5% prevalence of heterozygous NR3C1 mutations discovered in our series is higher than initially thought and encourages GR mutation screening in patients with adrenal incidentalomas to unambiguously differentiate from Cushing's states and to optimize personalized follow-up.

Isoform specificity of progesterone receptor antibodies.

Progesterone receptors (PR) are prognostic and predictive biomarkers in hormone-dependent cancers. Two main PR isoforms have been described, PRB and PRA, that differ only in that PRB has 164 extra N-terminal amino acids. It has been reported that several antibodies empirically exclusively recognize PRA in formalin-fixed paraffin-embedded (FFPE) tissues. To confirm these findings, we used human breast cancer xenograft models, T47D-YA and -YB cells expressing PRA or PRB, respectively, MDA-MB-231 cells modified to synthesize PRB, and MDA-MB-231/iPRAB cells which can bi-inducibly express either PRA or PRB. Cells were injected into immunocompromised mice to generate tumours exclusively expressing PRA or PRB. PR isoform expression was verified using immunoblots. FFPE samples from the same tumours were studied by immunohistochemistry using H-190, clone 636, clone 16, and Ab-6 anti-PR antibodies, the latter exclusively recognizing PRB. Except for Ab-6, all antibodies displayed a similar staining pattern. Our results indicate that clones 16, 636, and the H-190 antibody recognize both PR isoforms. They point to the need for more stringency in evaluating the true specificity of purported PRA-specific antibodies as the PRA/PRB ratio may have prognostic and predictive value in breast cancer.

Adrenal GIPR expression and chromosome 19q13 microduplications in GIP-dependent Cushing's syndrome.

GIP-dependent Cushing's syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Here we performed molecular analyses on adrenocortical adenomas and bilateral macronodular adrenal hyperplasias obtained from 14 patients with GIP-dependent adrenal Cushing's syndrome and one patient with GIP-dependent aldosteronism. GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had a 19q duplication only. We demonstrated that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in adenoma cells. Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations.

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.

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.

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.

A novel non genomic glucocorticoid signaling mediated by a membrane palmitoylated glucocorticoid receptor cross talks with GnRH in gonadotrope cells.

Glucocorticoid hormones (GC) are the main stress mediators associated with reproductive disorders. GC exert their effects through activation of the glucocorticoid receptor (GR) principally acting as a transcription factor. Beside well-established GR-mediated genomic actions, several lines of evidence suggest a role for rapid membrane-initiated GC signaling in gonadotrope cells triggered by a membrane-associated GR. Herein, we demonstrate the existence of a specific membrane-initiated GC signaling in LßT2 gonadotrope cells involving two related phosphoproteins: Ca2+/Calmodulin-dependent protein kinase II (CaMKII) and synapsin-I. Within 5 min, LßT2 cells treated with stress range of 10-7 M Corticosterone or a membrane impermeable-GC, BSA-conjugated corticosterone, exhibited a 2-fold increase in levels of phospho-CaMKII and phospho-synapsin-I. Biochemical approaches revealed that this rapid signaling is promoted by a palmitoylated GR. Importantly, GC significantly alter GnRH-induced CaMKII phosphorylation, consistent with a novel cross-talk between the GnRH receptor and GC. This negative effect of GC on GnRH signaling was further observed on LH release by mouse pituitary explants. Altogether, our work provides new findings in GC field by bringing novel understanding on how GR integrates plasma membrane, allowing GC membrane-initiated signaling that differs in presence of GnRH to disrupt GnRH-dependent signaling and LH secretion.

Glucocorticoid receptor represses brain-derived neurotrophic factor expression in neuron-like cells.

Brain-derived neurotrophic factor (BDNF) is involved in many functions such as neuronal growth, survival, synaptic plasticity and memorization. Altered expression levels are associated with many pathological situations such as depression, epilepsy, Alzheimer's, Huntington's and Parkinson's diseases. Glucocorticoid receptor (GR) is also crucial for neuron functions, via binding of glucocorticoid hormones (GCs). GR actions largely overlap those of BDNF. It has been proposed that GR could be a regulator of BDNF expression, however the molecular mechanisms involved have not been clearly defined yet. Herein, we analyzed the effect of a GC agonist dexamethasone (DEX) on BDNF expression in mouse neuronal primary cultures and in the newly characterized, mouse hippocampal BZ cell line established by targeted oncogenesis. Mouse Bdnf gene exhibits a complex genomic structure with 8 untranslated exons (I to VIII) splicing onto one common and unique coding exon IX. We found that DEX significantly downregulated total BDNF mRNA expression by around 30%. Expression of the highly expressed exon IV and VI containing transcripts was also reduced by DEX. The GR antagonist RU486 abolished this effect, which is consistent with specific GR-mediated action. Transient transfection assays allowed us to define a short 275 bp region within exon IV promoter responsible for GR-mediated Bdnf repression. Chromatin immunoprecipitation experiments demonstrated GR recruitment onto this fragment, through unidentified transcription factor tethering. Altogether, GR downregulates Bdnf expression through direct binding to Bdnf regulatory sequences. These findings bring new insights into the crosstalk between GR and BDNF signaling pathways both playing a major role in physiology and pathology of the central nervous system.

Familial Multiplicity of Estrogen Insensitivity Associated With a Loss-of-Function ESR1 Mutation.

Context: Estrogens influence many physiological processes in mammals, including reproduction. Estrogen peripheral actions are mainly mediated through estrogen receptors (ERs) α and ß, encoded by ESR1 and ESR2 genes, respectively. Objective: The study's aim was to describe a family in which 3 members presented with estrogen insensitivity. Design and Setting: Clinical evaluation and genetic and mutational analysis were performed in an academic medical center. Patients and Interventions: An ESR1 mutation was identified in 2 sisters and 1 brother, originating from a consanguineous Algerian family, who did not enter puberty and presented with delayed bone maturation consistent with estrogen insensitivity. The 2 sisters had enlarged multicystic ovaries. Hormonal evaluation as well as genetic and mutational analysis were performed. Results: Hormonal evaluation revealed extremely high plasma 17ß-estradiol (>50-fold normal range) associated with elevated gonadotropin levels (greater than threefold normal range), highly suggestive of estrogen resistance. The 3 affected patients carried a homozygous mutation of a highly conserved arginine 394 for which histidine was substituted through an autosomal recessive mode of transmission. Structural and functional analysis of the mutant ERα revealed strongly reduced transcriptional activity and the inability to securely anchor the activating hormone, estradiol, compared with wild-type ERα. A group of other potential ER activating ligands were tested, but none overcame the estrogen insensitivity in these patients. Conclusion: Description and analysis of this family of patients with mutant ERα provide additional clinical findings toward identification and characterization of what was previously thought to be a highly rare clinical condition.