Immunomodulation by maternal autoantibodies of the fetal serotoninergic 5-HT4 receptor and its consequences in early BALB/c mouse embryonic development

<p>Abstract</p> <p>Background</p> <p>The presence of functional 5-HT₄receptors in human and its involvement in neonatal lupus erythematosus (NLE) have prompted us to study the receptor expression and role during embryogenesis. Earlier we managed to demonstrate that female BALB/c mice immunized against the second extracellular loop (SEL) of the 5-HT₄receptor gave birth to pups with heart block. To explain this phenomenon we investigated the expression of 5-HT₄receptors during mouse embryogenesis. At the same time we looked whether the consequence of 5-HT₄receptor immunomodulation observed earlier is in relation to receptor expression.</p> <p>We studied the expression of 5-HT₄receptor at the mRNA level and its two isoforms 5-HT_4(a)and 5-HT_4(d)at the protein level in embryos from BALB/c mice, at 8^th, 12^th, 18^thgestation days (GD) and 1 day post natal (DPN). Simultaneously the receptor activity was inhibited by rising antibodies, in female mice against SEL of the receptor. The mice were mated and embryos were collected at 8^th, 12^th, 18^thGD and 1 DPN.</p> <p>Results</p> <p>5-HT₄receptor mRNA increased in brain from 12^thGD to 1 DPN. Its expression gradually decreased in heart and disappeared at birth. This was consistent with expression of the receptor isoforms 5-HT_{4(a) and (d)}. Abnormalities like decreased number of embryos, growth delay, spina bifida and sinus arrhythmia from 12^thGD were documented in pups of mice showing anti-5-HT₄receptor antibodies.</p> <p>Conclusion</p> <p>serotoninergic 5-HT₄receptor plays an important role in mouse foetal development. In BALB/c mice there is a direct relation between the expression of receptor and the deleterious effect of maternal anti-5-HT₄receptor autoantibodies in early embryogenesis.</p

Modeling Epac1 interactions with the allosteric inhibitor AM-001 by co-solvent molecular dynamics

The exchange proteins activated by cAMP (EPAC) are implicated in a large variety of physiological processes and they are considered as promising targets for a wide range of therapeutic applications. Several recent reports provided evidence for the therapeutic effectiveness of the inhibiting EPAC1 activity cardiac diseases. In that context, we recently characterized a selective EPAC1 antagonist named AM-001. This compound was featured by a non-competitive mechanism of action but the localization of its allosteric site to EPAC1 structure has yet to be investigated. Therefore, we performed cosolvent molecular dynamics with the aim to identify a suitable allosteric binding site. Then, the docking and molecular dynamics were used to determine the binding of the AM-001 to the regions highlighted by cosolvent molecular dynamics for EPAC1. These analyses led us to the identification of a suitable allosteric AM-001 binding pocket at EPAC1. As a model validation, we also evaluated the binding poses of the available AM-001 analogues, with a different biological potency. Finally, the complex EPAC1 with AM-001 bound at the putative allosteric site was further refined by molecular dynamics. The principal component analysis led us to identify the protein motion that resulted in an inactive like conformation upon the allosteric inhibitor binding

Elevated cAMP Protects against Diclofenac-Induced Toxicity in Primary Rat Hepatocytes:A Protective Effect Mediated by the Exchange Protein Directly Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Factors

Background: Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism including the classical protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). Aim: Investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Methods: Rat hepatocytes were exposed to 400 µmol/L diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by qPCR and Western blot, respectively. The cAMP-elevating agent forskolin, the pan-phosphodiesterase inhibitor IBMX and EPAC inhibitors CE3F4 and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Results: Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition, but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP while both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. Conclusion: cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI. Significance Statement Our study shows two main highlights. First, elevated cAMP levels protect against diclofenac-induced apoptosis in primary hepatocytes via maintenance of mitochondrial integrity. In addition, we propose the existence of mitochondrial cAMP-EPAC microdomains in rat hepatocytes, opening new avenues for targeted therapy in DILI. Both EPAC1 and EPAC2, but not PKA, are responsible for this protective effect. Our findings present cAMP-EPAC as a potential target for the treatment of drug-induced liver injury (DILI) and liver injury involving mitochondrial dysfunction

Catecholamines facilitate VEGF-dependent angiogenesis via β2-adrenoceptor-induced Epac1 and PKA activation

Chronic stress has been associated with the progression of cancer and antagonists for β-adrenoceptors (βAR) are regarded as therapeutic option. As they are also used to treat hemangiomas as well as retinopathy of prematurity, a role of endothelial β2AR in angiogenesis can be envisioned. We therefore investigated the role of β2AR-induced cAMP formation by analyzing the role of the cAMP effector molecules exchange factor directly activated by cAMP 1 (Epac1) and protein kinase A (PKA) in endothelial cells (EC). Epac1-deficient mice showed a reduced amount of pre-retinal neovascularizations in the model of oxygen-induced retinopathy, which is predominantly driven by vascular endothelial growth factor (VEGF). siRNA-mediated knockdown of Epac1 in human umbilical vein EC (HUVEC) decreased angiogenic sprouting by lowering the expression of the endothelial VEGF-receptor-2 (VEGFR-2). Conversely, Epac1 activation by β2AR stimulation or the Epac-selective activator cAMP analog 8-p-CPT-2'-O-Me-cAMP (8-pCPT) increased VEGFR-2 levels and VEGF-dependent sprouting. Similar to Epac1 knockdown, depletion of the monomeric GTPase Rac1 decreased VEGFR-2 expression. As Epac1 stimulation induces Rac1 activation, Epac1 might regulate VEGFR-2 expression through Rac1. In addition, we found that PKA was also involved in the regulation of angiogenesis in EC since the adenylyl cyclase (AC) activator forskolin (Fsk), but not 8-pCPT, increased sprouting in Epac1-depleted HUVEC and this increase was sensitive to a selective synthetic peptide PKA inhibitor. In accordance, β2AR- and AC-activation, but not Epac1 stimulation increased VEGF secretion in HUVEC.Our data indicate that high levels of catecholamines, which occur during chronic stress, prime the endothelium for angiogenesis through a β2AR-mediated increase in endothelial VEGFR-2 expression and VEGF secretion

Pharmacological Inhibition of Epac1 Averts Ferroptosis Cell Death by Preserving Mitochondrial Integrity

Exchange proteins directly activated by cAMP (Epac) proteins are implicated in a wide range of cellular functions including oxidative stress and cell survival. Mitochondrial-dependent oxidative stress has been associated with progressive neuronal death underlying the pathology of many neurodegenerative diseases. The role of Epac modulation in neuronal cells in relation to cell survival and death, as well as its potential effect on mitochondrial function, is not well established. In immortalized hippocampal (HT-22) neuronal cells, we examined mitochondria function in the presence of various Epac pharmacological modulators in response to oxidative stress due to ferroptosis. Our study revealed that selective pharmacological modulation of Epac1 or Epac2 isoforms, exerted differential effects in erastin-induced ferroptosis conditions in HT-22 cells. Epac1 inhibition prevented cell death and loss of mitochondrial integrity induced by ferroptosis, while Epac2 inhibition had limited effects. Our data suggest Epac1 as a plausible therapeutic target for preventing ferroptosis cell death associated with neurodegenerative diseases

Epac1 links prostaglandin E2 to β-catenin-dependent transcription during epithelial-to-mesenchymal transition

In epithelial cells, β-catenin is localized at cell-cell junctions where it stabilizes adherens junctions. When these junctions are disrupted, β-catenin can translocate to the nucleus where it functions as a transcriptional cofactor. Recent research has indicated that PGE2 enhances the nuclear function of β-catenin through cyclic AMP. Here, we aim to study the role of the cyclic AMP effector Epac in β-catenin activation by PGE2 in non-small cell lung carcinoma cells.We show that PGE2 induces a down-regulation of E-cadherin, promotes cell migration and enhances β-catenin translocation to the nucleus. This results in β-catenin-dependent gene transcription. We also observed increased expression of Epac1. Inhibition of Epac1 activity using the CE3F4 compound or Epac1 siRNA abolished the effects of PGE2 on β-catenin. Further, we observed that Epac1 and β-catenin associate together. Expression of an Epac1 mutant with a deletion in the nuclear pore localization sequence prevents this association. Furthermore, the scaffold protein Ezrin was shown to be required to link Epac1 to β-catenin.This study indicates a novel role for Epac1 in PGE2-induced EMT and subsequent activation of β-catenin

Caractérisation fonctionnelle du facteur d'échange Epac, une nouvelle cible cardiaque de l'AMPc

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

Étude structurale et fonctionnelle de la dimérisation du récepteur 5-HT 4 de la sérotonine

Serotonin 5-HT4 receptors (5-HT4R) are members of G protein-coupled receptors (GPCRs). Given the recent recognition that many GPCRs can dimerize, the present study was undertaken to determine whether 5-HT4R can form dimers and what are the molecular determinants involved in the dimerization process. Using co-immunoprecipitation and bioluminescence resonance energy transfer, we showed that the human 5-HT4R isoforms can form constitutive homodimers and heterodimers. In addition, we showed heterodimer formation between the 5-HT4(d)R and the beta2-adrenergic receptor. The 5-HT4(d)R dimer is sensible to dithiothreitol (reducing agent). Mutation of two cysteines in the 3 and 4 transmembrane domains, inhibits 5-HT4 receptor dimerization and brings about reticulum endoplasmic retention. Specific 5-HT4 receptor bivalent ligand do not have special affinity or efficacy for the 5-HT4 receptor but are able to stabilize receptor dimerization.Les récepteurs 5-HT4 de la sérotonine sont des récepteurs couplés aux protéines G (RCPG). Les RCPGs peuvent se dimériser, ce qui influence leurs propriétés pharmacologiques. Les récepteurs 5-HT4 étant impliqués dans d'importants processus physiologiques, nous avons étudié leur processus de dimérisation. Nous avons montré par co-immunoprécipitation et Bioluminescence Resonnance Energy Transfert, que les isoformes 5-HT4 s'homodimérisent et s'hétérodimérisent constitutivement, entre elles et avec le récepteur b2-adrénergique. Le dimère de récepteur 5-HT4 est sensible au dithiothréitol (agent réducteur de ponts disulfures). La mutation de deux cystéines localisées dans les domaines transmembranaires 3 et 4, inhibe la dimérisation du récepteur 5-HT4 et entraîne sa rétention dans le réticulum endoplasmique. Des ligands bivalents spécifiques du récepteur 5-HT4 ne présentent pas d'affinité ou d'efficacité particulière pour le récepteur 5-HT4 mais peuvent stabiliser les dimères de récepteurs.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Influence des récepteurs 5-HT4 de la sérotonine sur la maturation du précurseur de la protéine ß-amyloïde associée à la maladie d'Alzheimer

Les récepteurs 5-HT4 de la sérotonine sont des récepteurs à 7 domaines transmembranaires positivement couplés à l'adénylate cyclase. Dans une première partie de ce travail, nous avons montré que dans plusieurs lignées cellulaires et dans des cultures primaires de neurones corticaux de souris, l'activation du récepteur 5-HT4 augmente la libération d'une protéine clé de la maladie d'Alzheimer, la protéine non amyloïdogénique sAPPa. Cet effet est dépendant de l'AMPc mais indépendant de la PKA. Dans une seconde partie nous montrons que cette sécrétion de protéine sAPPa induite par le récepteur 5-HT4 met en jeu une nouvelle voie de signalisation du récepteur 5-HT4 impliquant le facteur d'échange sensible à l'AMPc, Epac, et les petites protéines G, Rap1 et Rac. Ces résultats ouvrent un nouvel axe de réflexion sur l'implication potentielle des récepteurs 5-HT4 dans les perturbations de la mémoire chez les patients atteints de la maladie d'AlzheimerThe serotonin 5-HT4 receptors are heptahelical transmembrane receptors that are positively coupled to adenylyl cyclase. In the first part of this work, we showed that the human 5-HT4 receptor stimulated the secretion of the non-amyloïdogenic soluble form of the amyloid precursor protein (sAPPa), a key protein involved in Alzheimer's disease which has neuroprotective and memory enhancing effects. In the second part of this work we showed that this process which was cAMP dependent but PKA independent, involved a new signaling pathway of the 5-HT4 receptors that involved the small G proteins, Rac and Rap 1. Rac activation results from activation of Rap1 through the cAMP-activated guanine nucleotide exchange factor Epac. Futhermore, we showed that secretion of sAPPa induced by the human 5-HT4 receptor is due to its specific effect on a a-secretase activity. These findings may lead to the identification of new targets for the regulation of APP processingCHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF