Specific hydroxylations determine selective corticosteroid recognition by human glucocorticoid and mineralocorticoid receptors

AbstractThe ligand binding domains of the human mineralocorticoid receptor (hMR) and glucocorticoid receptor (hGR) display a high sequence homology. Aldosterone and cortisol, the major mineralocorticoid and glucocorticoid hormones, are very closely related, leading to the cross-binding of these hormones to both receptors. The present study reports on the mechanism by which hMR and hGR are activated preferentially by their cognate hormones. We found that the ability of corticosteroids to stimulate the receptor’s transactivation function is depending on the stability of the steroid-receptor complexes. In the light of a hMR structural model we propose that contacts through the corticosteroid C21 hydroxyl group are sufficient to stabilize hMR but not hGR and that additional contacts through the C11- and C17-hydroxyl groups are required for hGR

Normosmic Congenital Hypogonadotropic Hypogonadism Due to TAC3/TACR3 Mutations: Characterization of Neuroendocrine Phenotypes and Novel Mutations

CONTEXT: TAC3/TACR3 mutations have been reported in normosmic congenital hypogonadotropic hypogonadism (nCHH) (OMIM #146110). In the absence of animal models, studies of human neuroendocrine phenotypes associated with neurokinin B and NK3R receptor dysfunction can help to decipher the pathophysiology of this signaling pathway. OBJECTIVE: To evaluate the prevalence of TAC3/TACR3 mutations, characterize novel TACR3 mutations and to analyze neuroendocrine profiles in nCHH caused by deleterious TAC3/TACR3 biallelic mutations. RESULTS: From a cohort of 352 CHH, we selected 173 nCHH patients and identified nine patients carrying TAC3 or TACR3 variants (5.2%). We describe here 7 of these TACR3 variants (1 frameshift and 2 nonsense deleterious mutations and 4 missense variants) found in 5 subjects. Modeling and functional studies of the latter demonstrated the deleterious consequence of one missense mutation (Tyr267Asn) probably caused by the misfolding of the mutated NK3R protein. We found a statistically significant (p<0.0001) higher mean FSH/LH ratio in 11 nCHH patients with TAC3/TACR3 biallelic mutations than in 47 nCHH patients with either biallelic mutations in KISS1R, GNRHR, or with no identified mutations and than in 50 Kallmann patients with mutations in KAL1, FGFR1 or PROK2/PROKR2. Three patients with TAC3/TACR3 biallelic mutations had an apulsatile LH profile but low-frequency alpha-subunit pulses. Pulsatile GnRH administration increased alpha-subunit pulsatile frequency and reduced the FSH/LH ratio. CONCLUSION: The gonadotropin axis dysfunction associated with nCHH due to TAC3/TACR3 mutations is related to a low GnRH pulsatile frequency leading to a low frequency of alpha-subunit pulses and to an elevated FSH/LH ratio. This ratio might be useful for pre-screening nCHH patients for TAC3/TACR3 mutations

Les hormones corticostéroïdes : mécanismes impliqués dans la reconnaissance de l’aldostérone par le récepteur aux minéralocorticoïdes

L’aldostérone et le cortisol, principales hormones minéralocorticoïdes et glucocorticoïdes chez l’Homme, ont des structures très proches. Les deux hormones se lient au récepteur aux minéralocorticoïdes (RM) avec la même affinité, néanmoins le RM est activé préférentiellement par l’aldostérone, ce qui suggère que l’interaction de ces deux hormones avec le RM met en jeu des liaisons en partie distinctes. La construction d’un modèle tridimensionnel du domaine de liaison à l’hormone du RM humain, à partir de données cristallographiques du récepteur à l’acide rétinoïque associé à son ligand, a permis l’identification de plusieurs résidus du RM impliqués dans l’interaction avec l’aldostérone et le cortisol. Les résidus Gin 776 et Arg 817 font des liaisons hydrogène avec la fonction cétone en C3 du stéroïde et le résidu Asn 770 avec l’hydroxyle en C21. L’analyse de l’activité du récepteur sauvage ou muté en réponse à des stéroïdes hydroxylés en différentes positions du squelette stéroïdien ont permis d’arriver aux conclusions suivantes : I) l’interaction entre le résidu Asn 770 du RM et la fonction hydroxyle en C21 des corticostéroïdes est déterminante pour stabiliser le RM dans sa conformation active; 2) la présence d’un l’hémiacétal en 11-18 de l’aldostérone renforce la stabilité de la conformation active du RM, alors que les hydroxylés en 11β et en 17α du cortisol diminue sa stabilité. Ces résultats sont discutés à la lumière d’un modèle d’activation du RM

Cistrome of the Aldosterone-activated Mineralocorticoid Receptor in Human Renal Cells: Cistrome of Human Renal Mineralocorticoid Receptor

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

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

International audienceAldosterone, 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