L’effet anti-thermogénique du récepteur minéralocorticoïde in vivo et in vitro par inhibition de l’expression d’UCP1

International audienceObjectifs : Le but de cette étude était de mieux comprendre les mécanismes moléculaires sous-tendant les effets métaboliques du récepteur minéralocorticoïde (MR), un facteur de transcription qui exerce un rôle pro-adipogénique et anti-thermogénique en inhibant l’expression de la protéine découplante mitochondriale de type 1 UCP1.Méthodes : L’expression d’UCP1, induite par le froid, a été étudiée dans un modèle de souris transgéniques surexprimant le MR. Nous avons également déterminé le profil d’expression de certains corégulateurs au cours de l’adipogenèse en utilisant les cellules T37i, une lignée murine préadipocytaire brune. La précipitation de la chromatine (ChiP) a permis d’évaluer la capacité du MR à interagir avec certaines séquences régulatrices du promoteur d’UCP1.Résultats : Nous montrons que la surexpression du MR in vivo inhibe l’expression d’UCP1 induite par le froid dans le tissu adipeux brun. Le profil d’expression de certains coactivateurs/corégulateurs du MR diffère clairement en fonction du caractère transitoire ou soutenu de leur niveau d’expression au cours de l’adipogenèse brune, témoignant d’une coopération fonctionnelle en rapport avec la maturation adipocytaire. L’activation du MR inhibe l’expression d’UCP1 induite par les catécholamines et l’acide rétinoïque. La ChiP a démontré l’interaction du MR et de PPARγ sur le promoteur d’UCP1, identifiant le mécanisme d’inhibition de l’expression d’UCP1 par le MR activé par l’aldostérone.Conclusion : L’ensemble de ces résultats montre que le MR joue un rôle métabolique central en contrôlant la fonction adipocytaire brune et la dépense énergétique.Objectives : The mineralocorticoid receptor (MR), a hormone-activated transcription factor, besides its role in controlling hydroelectrolytic homeostasis, exerts pro-adipogenic and anti-thermogenic effects, inhibiting mitochondrial-uncoupling protein UCP1 expression in brown adipocytes. The aim of this study was to gain insight into the molecular mechanisms by which MR participates in such metabolic regulation.Methods : We evaluated in vivo MR effects on cold-induced UCP1 expression in MR-overexpressing mice. Expression profiles of several transcriptional coregulators were analyzed during differentiation of the brown adipocyte T37i cell line. Given that UCP1 expression is inversely controlled by catecholamines/retinoic acid and corticosteroids, we investigated the mechanisms of MR's inhibitory effect on UCP1 transcription in T37i cells. Chromatin immunoprecipitation (ChIP) experiments enabled us to explore MR interaction with UCP1 promoter regions.Results : Cold-induced UCP1 expression was blunted in the brown fat of MR-overexpressing mice. Along with induction of increasing mRNA levels for specific adipocyte markers during T37i differentiation, MR coactivator transcript levels significantly increased in intermediate states of differentiation, whereas expression of MR corepressors transiently increased approximately 2-fold. Such a simultaneous transient peak in coregulator expression is consistent with physiologically relevant cooperation occurring during brown adipogenesis. ChIP demonstrated that, after retinoic acid stimulation and aldosterone exposure, MR and PPARγ concomitantly bind to specific UCP1 promoter motifs.Conclusion : Our studies demonstrate that MR exerts a pivotal metabolic role by controlling energy expenditure, and provide novel information on how MR participates in the regulation of brown adipocyte function

Hypertonicity Compromises Renal Mineralocorticoid Receptor Signaling through Tis11b-Mediated Post-Transcriptional Control

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

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

International audienceMineralocorticoid 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

International audienceAldosterone 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

Chromosomal Translocation Formation Is Sufficient to Produce Fusion Circular RNAs Specific to Patient Tumor Cells

Summary: Circular RNAs constitute a unique class of RNAs whose precise functions remain to be elucidated. In particular, cancer-associated chromosomal translocations can give rise to fusion circular RNAs that play a role in leukemia progression. However, how and when fusion circular RNAs are formed and whether they are being selected in cancer cells remains unknown. Here, we used CRISPR/Cas9 to generate physiological translocation models of NPM1-ALK fusion gene. We showed that, in addition to generating fusion proteins and activating specific oncogenic pathways, chromosomal translocation induced by CRISPR/Cas9 led to the formation of de novo fusion circular RNAs. Specifically, we could recover different classes of circular RNAs composed of different circularization junctions, mainly back-spliced species. In addition, we identified fusion circular RNAs identical to those found in related patient tumor cells providing evidence that fusion circular RNAs arise early after chromosomal formation and are not just a consequence of the oncogenesis process. : Molecular Biology; Molecular Genetics; Cancer Subject Areas: Molecular Biology, Molecular Genetics, Cance

The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

International audienceTPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3'-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein-protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function