Small-molecule allosteric activators of PDE4 long form cyclic AMP phosphodiesterases

Cyclic AMP (cAMP) phosphodiesterase-4 (PDE4) enzymes degrade cAMP and underpin the compartmentalization of cAMP signaling through their targeting to particular protein complexes and intracellular locales. We describe the discovery and characterization of a small-molecule compound that allosterically activates PDE4 long isoforms. This PDE4-specific activator displays reversible, noncompetitive kinetics of activation (increased Vmax with unchanged Km), phenocopies the ability of protein kinase A (PKA) to activate PDE4 long isoforms endogenously, and requires a dimeric enzyme assembly, as adopted by long, but not by short (monomeric), PDE4 isoforms. Abnormally elevated levels of cAMP provide a critical driver of the underpinning molecular pathology of autosomal dominant polycystic kidney disease (ADPKD) by promoting cyst formation that, ultimately, culminates in renal failure. Using both animal and human cell models of ADPKD, including ADPKD patient-derived primary cell cultures, we demonstrate that treatment with the prototypical PDE4 activator compound lowers intracellular cAMP levels, restrains cAMP-mediated signaling events, and profoundly inhibits cyst formation. PDE4 activator compounds thus have potential as therapeutics for treating disease driven by elevated cAMP signaling as well as providing a tool for evaluating the action of long PDE4 isoforms in regulating cAMP-mediated cellular processes

STAT5 drives abnormal proliferation in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) leads to renal failure. The hallmark of ADPKD is increased epithelial proliferation, which has been proposed to be due to atypical signaling including abnormal JAK-STAT activity. However, the relative contribution of JAK-STAT family members in promoting proliferation in ADPKD is unknown. Here, we present siRNA JAK-STAT-focused screens discovering a previously unknown proliferative role for multiple JAK-STAT components (including STAT1, STAT2, STAT4, STAT5a, and STAT5b). Amongst these, we selected to study the growth hormone/growth hormone receptor/STAT5-axis because of its known role as a regulator of growth in nonrenal tissues. Loss of STAT5 function, facilitated by pharmacological inhibition or siRNAs, significantly reduced proliferation with an associated reduction in cyst growth in vitro. To study whether STAT5 is abnormally activated in vivo, we analyzed its expression using two independent mouse models of ADPKD. STAT5 was nuclear, thus activated, in renal epithelial cyst lining cells in both models. To test whether forced activation of STAT5 can modulate proliferation of renal cells in vivo, irrespective of the Pkd1 status, we overexpressed growth hormone. These mice showed increased STAT5 activity in renal epithelial cells, which correlated with de novo expression of cyclin D1, a STAT5 target gene. Chromatin immunoprecipitation experiments revealed that STAT5 transcriptionally activated cyclin D1 in a growth hormone-dependent fashion, thus providing a mechanism into how STAT5 enhances proliferation. Finally, we provide evidence of elevated serum growth hormone in Pkd1 mutant mice. Thus, the growth hormone/STAT5 signaling axis is a novel therapeutic target in ADPKD

Mécanismes de régulation de la synthèse des fibres élastiques artérielles

L'élastine, principal constituant des fibres élastiques, apporte l'élasticité nécessaire aux tissus pour résister aux contraintes mécaniques. Un défaut quantitatif en élastine peut entraîner le développement de certaines pathologies comme les sténoses aortiques supravalvulaires. Comprendre ses mécanismes de régulation permettrait de trouver de nouvelles molécules capables d'augmenter sa synthèse pour traiter ces conditions pathologiques. Précédemment, des études de liaisons génétiques et d'expressions différentielles ont montré que certains gènes impliqués dans la régulation de la concentration intracellulaire en potassium ([K+];) et en calcium ([Ca2+];) pouvaient également l'être dans la régulation de la synthèse de l'élastine. Au cours de cette thèse, il a été montré que les ouvreurs de canaux potassiques ATP-dépendants augmentent in vitro la transcription du gène de l'élastine ainsi que la stabilité de ses ARNm dans les CML et in vivo la quantité d'élastine incorporée dans les lames élastiques de l'aorte de jeunes rats. La description du mécanisme impliqué cette régulation transcriptionnelle de l'élastine par l'utilisation d'un ionophore de calcium a montré qu'elle implique l'activation de la voie ERK1/2 / AP1. In vitro, la répression des facteurs de transcription AP1 augmente l'expression de l'élastine tout comme le fait l'inhibition de ERK1/2 qui, par ailleurs, augmente in vivo la synthèse et la quantité d'élastine dans l'aorte de rat. En conclusion, ces travaux ont permis de mettre en évidence, in vitro et in vivo, l'intérêt d'utiliser des ouvreurs de canaux potassiques et des inhibiteurs de la voie ERK1/2 en vue d'augmenter la synthèse d'élastine dans l'aorte.Elastin is the main component of elastic fibers, conferring elastic properties on tissues submitted to mechanical distortion. A quantitative defect of elastin could lead to arterial pathologies such as supravalvular aortic stenosis. Understanding the mechanisms regulating elastin synthesis would be of interest in the search for new molecules capable of stimulating elastogenesis to treat these pathological conditions. Previously, quantitative trait loci analysis and micro-array studies revealed that many genes implicated in the regulation of intracellular concentrations of potassium ([K+]) and calcium ([Ca2+]) could be implicated in the regulation of elastin synthesis. In this work, we have demonstrated that ATP dependant potassium channel openers increase elastin gene transcription and stability of its mRNA in vitro, and increase aortic elastin content of young rats in vivo. The mechanisms implicated in this transcriptional regulation were investigated using a calcium ionophore, studies which showed that it requires activation of the ERK1/2 / AP1 pathway. In vitro, inhibition of AP1 transcription factors or ERK1/2 phosphorylation increased elastin synthesis. In vivo, inhibition of ERK1/2 phosphorylation increased the elastin content of the rat aorta. In conclusion, this work demonstrates, both in vitro and in vivo, the interest of using potassium channel openers and ERK1/2 inhibitors to increase aortic elastin synthesis in conditions where this protein is deficient.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Potassium Channel Openers Increase Aortic Elastic Fiber Formation and Reverse the Genetically Determined Elastin Deficit in the BN Rat

International audienceHypertension is a cardiovascular disorder that appears in more than half of the patients with Williams-Beuren syndrome, hemizygous for the elastin gene among 26 to 28 other genes. It was shown that the antihypertensive drug minoxidil, an ATP-dependent potassium channel opener, enhances elastic fiber formation; however, no wide clinical application was developed because of its adverse side effects. The Brown Norway rat was used here as an arterial elastin-deficient model. We tested 3 different potassium channel openers, minoxidil, diazoxide, and pinacidil, and 1 potassium channel blocker, glibenclamide, on cultured smooth muscle cells from Brown Norway rat aorta. All tested potassium channel openers increased mRNAs encoding proteins and enzymes involved in elastic fiber formation, whereas glibenclamide had the opposite effect. The higher steady-state level of tropoelastin mRNA in minoxidil-treated cells was attributable to an increase in both transcription and mRNA stability. Treatment of Brown Norway rats for 10 weeks with minoxidil or diazoxide increased elastic fiber content and decreased cell number in the aortic media, without changing collagen content. The minoxidil-induced cardiac hypertrophy was reduced when animals simultaneously received irbesartan, an angiotensin II-receptor antagonist. This side effect of minoxidil was not observed in diazoxide-treated animals. In conclusion, diazoxide, causing less undesirable side effects than minoxidil, or coadministration of minoxidil and irbesartan, increases elastic fiber content, decreases cell number in the aorta and, thus, could be suitable for treating vascular pathologies characterized by diminished arterial elastin content and simultaneous hypertension

Genetic inactivation of Semaphorin 3C protects mice from acute kidney injury

International audienceTo guide the development of therapeutic interventions for acute kidney injury, elucidating the deleterious pathways of this global health problem is highly warranted. Emerging evidence has indicated a pivotal role of endothelial dysfunction in the etiology of this disease. We found that the class III semaphorin SEMA3C was ectopically upregulated with full length protein excreted into the blood and truncated protein secreted into the urine upon kidney injury and hypothesized a role for SEAM3C in acute kidney injury. Sema3c was genetically abrogated during acute kidney injury and subsequent kidney morphological and functional defects in two well-characterized models of acute kidney injury; warm ischemia/reperfusion and folic acid injection were analyzed. Employing a beta actin-dependent, inducible knockout of Sema3c, we demonstrate that in acute kidney injury SEMA3C promotes interstitial edema, leucocyte infiltration and tubular injury. Additionally, intravital microscopy combined with Evans Blue dye extravasation and primary culture of magnetically sorted peritubular endothelial cells identified a novel role for SEMA3C in promoting vascular permeability. Thus, our study points to microvascular permeability as an important driver of injury in acute kidney injury, and to SEMA3C as a novel permeability factor and potential target for therapeutic intervention