Biochemical characterization and modulation of LH/CG-receptor during human trophoblast differentiation.: LH/CG-R in human trophoblast differentiation.

Due to the key role of the human chorionic gonadotropin hormone (hCG) in placental development, the aim of this study was to characterize the human trophoblastic luteinizing hormone/chorionic gonadotropin receptor (LH/CG-R) and to investigate its expression using the in vitro model of human cytotrophoblast differentiation into syncytiotrophoblast. We confirmed by in situ immunochemistry and in cultured cells, that LH/CG-R is expressed in both villous cytotrophoblasts and syncytiotrophoblasts. However, LH/CG-R expression decreased during trophoblast fusion and differentiation, while the expression of hCG and hPL (specific markers of syncytiotrophoblast formation) increased. A decrease in LH/CG-R mRNA during trophoblast differentiation was observed by means of semi-quantitative RT-PCR with two sets of primers. A corresponding decrease ( approximately 60%) in LH/CG-R protein content was shown by Western-blot and immunoprecipitation experiments. The amount of the mature form of LH/CG-R, detected as a 90-kDa band specifically binding (125)I-hCG, was lower in syncytiotrophoblasts than in cytotrophoblasts. This was confirmed by Scatchard analysis of binding data on cultured cells. Maximum binding at the cell surface decreased from 3,511 to about 929 molecules/seeded cells with a kDa of 0.4-0.5 nM. Moreover, on stimulation by recombinant hCG, the syncytiotrophoblast produced less cyclic AMP than cytotrophoblasts, indicating that LH/CG-R expression is regulated during human villous trophoblast differentiation. J. Cell. Physiol. 212: 26-35, 2007. (c) 2007 Wiley-Liss, Inc

IMPLICATION D’UNE FORME D’hCG ANORMALE ET DE SON RÉCEPTEUR (R-LH/CG) DANS LE DÉVELOPPEMENT PLACENTAIRE LORS D’UNE GROSSESSE ASSOCIÉE À UNE TRISOMIE 21

In women bearing a trisomy 21 -affected fetus, there are severe abnormalities during the placental development, leading to a defect in the formation and functionality of the syncytiotrophoblast (ST). ST, which arises from fusion and differentiation of cytotrophoblasts (CT), plays an essential role in the maintenance of the pregnancy by transporting the nutrients necessary to fetal development and secreting in maternal blood the placental hormone hCG. In T21-affected pregnancies, CT don't fused properly and the resulting ST secretes an abnormal and weakly bioactive hCG. To better understand the defect of ST formation occurring in placental development of T21 -affected pregnancies, we have studied the functionality and the interaction of the abnormal hCG with its receptor (LH/CG-R). In this study, we describe for the first time that in T21, it exists a severe decrease in the number of mature LH/CG-R expressed at the cell surface and that abnormal secreted hCG can bind to its receptor. Indeed, in T21, decrease of LH/CG-R expression associated with a weakly bioactive hCG may be involved in the ST formation defect as illustrated by the fact that specific inhibition of LH/CG-R expression by siRNA in normal CT mimics the T21 phenotype (defect of ST formation). More interestingly, treatment of T21-affected CT by a normal exogenous hCG reverses the T21 phenotype, allowing CT to fuse and form a functional ST.Le développement du placenta humain lors d’une grossesse associée à une trisomie 21 est caractérisé par différentes anomalies, conduisant notamment à un défaut de formation et de fonctionnalité du syncytiotrophoblaste. Le syncytiotrophoblaste, qui provient de la fusion et de la différenciation des cytotrophoblastes, joue un rôle essentiel dans le maintien de la grossesse en assurant le transport des nutriments indispensables au développement fœtal et en sécrétant dans le sang maternel les hormones de la grossesse comme l’hCG. Les cytotrophoblastes issus de placentas trisomiques fusionnent peu ou avec un certain retard et le syncytiotrophoblaste résultant est à l’origine d’une synthèse et d’une sécrétion d’hCG diminuée, anormalement glycosylée et biologiquement moins active par rapport aux cellules issues de placenta normal. Cependant, lors d’une grossesse associée à une trisomie 21, l’hCG est retrouvée augmentée dans le sang maternel. Nous sommes donc confronté à un paradoxe: d’un côté une synthèse d’hCG diminuée et de l’autre, un taux sérique maternel augmenté. Nous avons montré que ce paradoxe ne s’explique pas par une anomalie de clairance de l’hCG anormalement glycosylée, ni par une augmentation de sa demi-vie plasmatique après expulsion du placenta. Il semblerait donc qu’il existe une élimination anormale de l’hCG par le placenta dans les grossesses affectées par une trisomie 21. Pour mieux comprendre le défaut de formation du syncytiotrophoblaste observé dans les cellules trophoblastiques trisomiques et le paradoxe observé entre la faible production d’hCG et son taux sérique maternel anormalement élevé, nous avons étudié la fonctionnalité et l’intéraction de l’hCG anormale avec son récepteur (R-LH/CG). Nous avons montré pour la première fois que l’expression du R-LH/CG est régulée au cours de la différenciation trophoblastique. De plus, nous avons caractérisé d’un point de vue biochimique et moléculaire le R-LH/CG dans les cytotrophoblastes trisomiques, montrant qu’il existe une très nette diminution de l’expression de la forme mature du R-LH/CG dans les cellules trophoblastiques trisomiques. L’utilisation de RNAi inhibant spécifiquement l’expression du R-LH/CG dans les cytotrophoblastes normaux, nous permet de mimer le phénotype observé dans les cellules trophoblastiques trisomiques (le défaut de formation syncytiale). Mais plus intéressant, nous avons montré que le défaut de différenciation observé dans les cellules trisomiques est réversible par l’action d’une hCG recombinante fonctionnelle, entraînant la fusion et la différenciation des cytotrophoblastes issus de grossesses associées à une trisomie 21. L’ensemble de nos travaux montre que la diminution de l’expression du R-LH/CG associée à une hCG anormalement glycosylée et biologiquement moins active semble être impliquée dans le défaut de formation syncytiale et dans le recaptage de l’hormone par le placenta, permettant alors d’expliquer le paradoxe de l’hCG dans la trisomie 21

REVIEW Specificity and spatial dynamics of protein kinase A signaling organized by A-kinase-anchoring proteins

International audienceProtein phosphorylation is the most common post-translational modification observed in cell signaling and is controlled by the balance between protein kinase and phosphatase activities. The cAMP-protein kinase A (PKA) pathway is one of the most studied and well-known signal pathways. To maintain a high level of specificity, the cAMP-PKA pathway is tightly regulated in space and time. A-kinase-anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity in the mediation of biological effects controlled by the cAMP-PKA pathway. AKAPs also serve as scaffolding proteins that assemble PKA together with signal terminators such as phosphoprotein phosphatases and cAMP-specific phosphodiesterases as well as components of other signaling pathways into multiprotein-signaling complexes

An overview of molecular events occurring in human trophoblast fusion

International audienceDuring human placentation, mononuclear cytotrophoblasts fuse to form a multinucleated syncytia ensuring hormonal production and nutrient exchanges between the maternal and fetal circulation. Syncytia formation is essential for the maintenance of pregnancy and for fetal growth. The trophoblast cell fusion process first requires the acquisition of cell fusion properties, then cells set up plasma membrane protein macrocomplexes and fusogen machinery that trigger cellecell fusion. Numerous proteins have been shown to be directly involved in the initiation of trophoblast cell fusion. These proteins must expressed at the right time and in the right place to trigger cellecell fusion. In this review, we describe the role of certain fusogenic protein macrocomplexes that form the scaffold for the fusogen machinery underlying human trophoblastic-lipid mixing and merging of cell contents that lead to cell fusion in physiological conditions

Anchored PKA as a gatekeeper for gap junctions

International audienceAnchored protein kinase A (PKA) bound to A Kinase Anchoring Protein (AKAP) mediates effects of localized increases in cAMP in defined subcellular microdomains and retains the specificity in cAMP-PKA signaling to distinct extracellular stimuli. Gap junctions are pores between adjacent cells constituted by connexin proteins that provide means of communication and transfer of small molecules. While the PKA signaling is known to promote human trophoblast cell fusion, the gap junction communication through connexin 43 (Cx43) is a prerequisite for this process. We recently demonstrated that trophoblast fusion is regulated by ezrin, a known AKAP, which binds to Cx43 and delivers PKA in the vicinity gap junctions. We found that disruption of the ezrin-Cx43 interaction abolished PKA-dependent phosphorylation of Cx43 as well as gap junction communication and subsequently cell fusion. We propose that the PKA-ezrin-Cx43 macromolecular complex regulating gap junction communication constitutes a general mechanism to control opening of Cx43 gap junctions by phosphorylation in response to cAMP signaling in various cell types

Implication d'une forme d'hCG anormale et de son récepteur (R-LH/CG) dans le développement placentaire lors d'une grossesse associée à une trisomie 21

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Cardiac cAMP-PKA Signaling Compartmentalization in Myocardial Infarction

Under physiological conditions, cAMP signaling plays a key role in the regulation of cardiac function. Activation of this intracellular signaling pathway mirrors cardiomyocyte adaptation to various extracellular stimuli. Extracellular ligand binding to seven-transmembrane receptors (also known as GPCRs) with G proteins and adenylyl cyclases (ACs) modulate the intracellular cAMP content. Subsequently, this second messenger triggers activation of specific intracellular downstream effectors that ensure a proper cellular response. Therefore, it is essential for the cell to keep the cAMP signaling highly regulated in space and time. The temporal regulation depends on the activity of ACs and phosphodiesterases. By scaffolding key components of the cAMP signaling machinery, A-kinase anchoring proteins (AKAPs) coordinate both the spatial and temporal regulation. Myocardial infarction is one of the major causes of death in industrialized countries and is characterized by a prolonged cardiac ischemia. This leads to irreversible cardiomyocyte death and impairs cardiac function. Regardless of its causes, a chronic activation of cardiac cAMP signaling is established to compensate this loss. While this adaptation is primarily beneficial for contractile function, it turns out, in the long run, to be deleterious. This review compiles current knowledge about cardiac cAMP compartmentalization under physiological conditions and post-myocardial infarction when it appears to be profoundly impaired

Compartimentalisation de la signalisation AMPc dans l'infarctus du myocarde

Under physiological conditions cAMP signaling plays a key role in the regulation of cardiac function. Activation of this intracellular signaling pathway mirrors cardiomyocyte adaptation to various extracellular stimuli. Extracellular ligand binding to seven-transmembrane receptors (a.k.a., GPCR) with G-proteins and adenylyl cyclases (ACs) modulate intracellular cAMP content. Subsequently, this second messenger triggers activation of specific intracellular downstream effectors that ensure a proper cellular response. Therefore, it is essential for the cell to keep the cAMP signaling highly regulated in space and time. The temporal regulation depends on activity of ACs and phosphodiesterases. By scaffolding key components of the cAMP signaling machinery, A-kinase anchoring proteins (AKAPs) coordinate both the spatial and temporal regulation. Myocardial infarction is one of the major causes of death in industrialized countries and is characterized by a prolonged cardiac ischemia. This leads to irreversible cardiomyocyte death and impairs cardiac function. Regardless of its causes, a chronic activation of cardiac cAMP signaling is established to compensate this loss. While this adaptation is primary beneficial for contractile function, it turns in the long run to be deleterious. This review compiles current knowledge about cardiac cAMP compartmentalization under physiological conditions and post-myocardial infarction when it appears to be profoundly impaired