Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level.

FKBP12.6, a binding protein to the immunosuppressant FK506, which also binds the ryanodine receptor (RyR2) in the heart, has been proposed to regulate RyR2 function and to have antiarrhythmic properties. However, the level of FKBP12.6 expression in normal hearts remains elusive and some controversies still persist regarding its effects, both in basal conditions and during ß-adrenergic stimulation. We quantified FKBP12.6 in the left ventricles (LV) of WT (wild-type) mice and in two novel transgenic models expressing distinct levels of FKBP12.6, using a custom-made specific anti-FKBP12.6 antibody and a recombinant protein. FKBP12.6 level in WT LV was very low (0.16 ± 0.02 nmol/g of LV), indicating that <15% RyR2 monomers are bound to the protein. Mice with 14.1 ± 0.2 nmol of FKBP12.6 per g of LV (TG1) had mild cardiac hypertrophy and normal function and were protected against epinephrine/caffeine-evoked arrhythmias. The ventricular myocytes showed higher [Ca2+]i transient amplitudes than WT myocytes and normal SR-Ca2+ load, while fewer myocytes showed Ca2+ sparks. TG1 cardiomyocytes responded to 50 nM Isoproterenol increasing these [Ca2+]i parameters and producing RyR2-Ser2808 phosphorylation. Mice with more than twice the TG1 FKBP12.6 value (TG2) showed marked cardiac hypertrophy with calcineurin activation and more arrhythmias than WT mice during ß-adrenergic stimulation, challenging the protective potential of high FKBP12.6. RyR2R420Q CPVT mice overexpressing FKBP12.6 showed fewer proarrhythmic events and decreased incidence and duration of stress-induced bidirectional ventricular tachycardia. Our study, therefore, quantifies for the first time endogenous FKBP12.6 in the mouse heart, questioning its physiological relevance, at least at rest due its low level. By contrast, our work demonstrates that with caution FKBP12.6 remains an interesting target for the development of new antiarrhythmic therapies.

Two-pore channels (TPCs) acts as a hub for excitation-contraction coupling, metabolism and cardiac hypertrophy signalling.

Ca2+ signaling is essential for cardiac contractility and excitability in heart function and remodeling. Intriguingly, little is known about the role of a new family of ion channels, the endo-lysosomal non-selective cation "two-pore channel" (TPCs) in heart function. Here we have used double TPC knock-out mice for the 1 and 2 isoforms of TPCs (Tpcn1/2-/-) and evaluated their cardiac function. Doppler-echocardiography unveils altered left ventricular (LV) systolic function associated with a LV relaxation impairment. In cardiomyocytes isolated from Tpcn1/2-/- mice, we observed a reduction in the contractile function with a decrease in the sarcoplasmic reticulum Ca2+ content and a reduced expression of various key proteins regulating Ca2+ stores, such as calsequestrin. We also found that two main regulators of the energy metabolism, AMP-activated protein kinase and mTOR, were down regulated. We found an increase in the expression of TPC1 and TPC2 in a model of transverse aortic constriction (TAC) mice and in chronically isoproterenol infused WT mice. In this last model, adaptive cardiac hypertrophy was reduced by Tpcn1/2 deletion. Here, we propose a central role for TPCs and lysosomes that could act as a hub integrating information from the excitation-contraction coupling mechanisms, cellular energy metabolism and hypertrophy signaling.

Housekeeping Proteins Exhibit a High Level of Expression Variability Within Control Group and Between Ischemic Human Heart Biopsies.

Background Human cardiac biopsies are widely used in clinical and fundamental research to decipher molecular events that characterize cardiac physiological and pathophysiological states. One of the main approaches relies on the analysis of semiquantitative immunoblots that reveals alterations in protein expression levels occurring in diseased hearts. To maintain semiquantitative results, expression level of target proteins must be standardized. The expression of HKP (housekeeping proteins) is commonly used to this purpose. Methods and Results We evaluated the stability of HKP expression (actin, ß-tubulin, GAPDH, vinculin, and calsequestrin) and total protein staining within control (coefficient of variation) and comparatively with ischemic human heart biopsies (P value). All HKP exhibited a high level of intragroup (ie, actin, ß-tubulin, and GAPDH) and/or intergroup variability (ie, GAPDH, vinculin, and calsequestrin). Among all, we found total protein staining to exhibit the highest degree of stability within and between groups, which makes this reference the best to study protein expression level in human biopsies from ischemic hearts and age-matched controls. In addition, we illustrated that using an inappropriate reference protein marker misleads interpretation on SERCA2 (sarco/endoplasmic reticulum Ca2+ ATPase) and cMyBPC (cardiac myosin binding protein-C) expression level after myocardial infarction. Conclusions These reemphasize the need to standardize the level of protein expression with total protein staining in comparative immunoblot studies on human samples from control and diseased hearts.

Uptake-leak balance of SR Ca2+ determines arrhythmogenic potential of RyR2R420Q+/- cardiomyocytes.

Mutations of the RyR2 are channelopathies that can predispose to life threatening catecholaminergic polymorphic ventricular tachycardias (CPVTs) during exercise or stress. However, the cellular and molecular mechanisms that are causal for the arrhythmias downstream of the ß-adrenergic receptor (ß-AR) activation are not defined. They may be specific and different for each particular RyR2 mutation. Obvious possibilities are the phosphorylation of the mutated RyR2s or the stimulation of the SR Ca2+ pump (SERCA), which could increase SR Ca2+ loading. Potentially arrhythmogenic Ca2+ signals, such as Ca2+ waves, were recorded and analyzed from WT and RyR2R420Q+/- mouse cardiomyocytes with confocal microscopy after field stimulation at 1 Hz. In RyR2R420Q+/- cardiomyocytes we found a higher occurrence and frequency of Ca2+ waves, particularly upon ß-AR stimulation with isoproterenol. This was accompanied by a shorter latency to the first spontaneous wave. Wave velocity from raw traces, as well as amplitude and decay time constant (τ) analyzed in de-skewed traces were comparable in both cell types. To obtain further insight into the role of the SERCA we selectively stimulated SERCA in permeabilized myocytes using Fab fragments of a PLB antibody (2D12). Surprisingly, SERCA stimulation alone resulted in considerably higher wave frequencies than when mimicking ß-AR stimulation with cAMP, particularly in RyR2R420Q+/- cardiomyocytes. This may be a consequence of some protective SR Ca2+ unloading resulting from the SR Ca2+ leak via phosphorylated RyR2s in cAMP. Spark-to-spark recovery analysis suggested a remarkably higher Ca2+ release sensitivity in RyR2R420Q+/- cells, both in control and upon ß-AR stimulation. Together these findings suggest that the fine balance between SR Ca2+ loading via SERCA and the Ca2+ leak via mutated and phosphorylated RyR2s is an important determinant for the overall cellular arrhythmogenicity prevailing in the RyR2R420Q+/- myocytes.

Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation.

[Figure: see text].

Orai1 Channel Inhibition Preserves Left Ventricular Systolic Function and Normal Ca2+ Handling After Pressure Overload.

BACKGROUND: Orai1 is a critical ion channel subunit, best recognized as a mediator of store-operated Ca2+ entry (SOCE) in nonexcitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear. METHODS: To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocyte-specific expression of an ion pore-disruptive Orai1R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline (hereafter referred to as JPIII), a small-molecule Orai1 channel inhibitor suitable for in vivo delivery. RESULTS: Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. Five weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and prohypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca2+ signaling alterations (increased SOCE, decreased [Ca2+]i transients amplitude and decay rate, lower SR Ca2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from C-dnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult. CONCLUSIONS: The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress.

Specific Upregulation of TRPC1 and TRPC5 Channels by Mineralocorticoid Pathway in Adult Rat Ventricular Cardiomyocytes.

Whereas cardiac TRPC (transient receptor potential canonical) channels and the associated store-operated Ca2+ entry (SOCE) are abnormally elevated during cardiac hypertrophy and heart failure, the mechanism of this upregulation is not fully elucidated but might be related to the activation of the mineralocorticoid pathway. Using a combination of biochemical, Ca2+ imaging, and electrophysiological techniques, we determined the effect of 24-h aldosterone treatment on the TRPCs/Orai-dependent SOCE in adult rat ventricular cardiomyocytes (ARVMs). The 24-h aldosterone treatment (from 100 nM to 1 µM) enhanced depletion-induced Ca2+ entry in ARVMs, as assessed by a faster reduction of Fura-2 fluorescence decay upon the addition of Mn2+ and increased Fluo-4/AM fluorescence following Ca2+ store depletion. These effects were prevented by co-treatment with a specific mineralocorticoid receptor (MR) antagonist, RU-28318, and they are associated with the enhanced depletion-induced N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2)-sensitive macroscopic current recorded by patch-clamp experiments. Molecular screening by qRT-PCR and Western blot showed a specific upregulation of TRPC1, TRPC5, and STIM1 expression at the messenger RNA (mRNA) and protein levels upon 24-h aldosterone treatment of ARVMs, corroborated by immunostaining. Our study provides evidence that the mineralocorticoid pathway specifically promotes TRPC1/TRPC5-mediated SOCE in adult rat cardiomyocytes.

Study of Human T21 Placenta Suggests a Potential Role of Mesenchymal Spondin-2 in Placental Vascular Development.

Placental development is particularly altered in trisomy of chromosome 21 (T21)-affected pregnancies. We previously described in T21-affected placentae an abnormal paracrine crosstalk between the villus mesenchymal core and villus trophoblasts. T21-affected placentae are known to be characterized by their hypovascularity. However, the causes of this anomaly remain not fully elucidated. Therefore, the hypothesis of an abnormal paracrine crosstalk between fetal mesenchymal core and placental endothelial cells (PLECs) was evocated. Villus mesenchymal cells from control (CMCs) and T21 placentae (T21MCs) were isolated and grown in culture to allow their characterization and collection of conditioned media for functional analyses (CMC-CM and T21MC-CM, respectively). Interestingly, PLEC proliferation and branching ability were less stimulated by T21MC-CM than by CMC-CM. Protein array analysis identified secreted proangiogenic growth factors in CMC-CM, which were reduced in T21MC-CM. Combined mass spectrometry and biochemical analysis identified spondin-2 as a factor decreased in T21MC-CM compared with CMC-CM. We found that exogenous spondin-2 stimulated PLEC proliferation and established that T21MC-CM supplemented with spondin-2 recovered conditioned media ability to induce PLEC proliferation and angiogenesis. Hence, this study demonstrates a crosstalk between villus mesenchymal and fetal endothelial cells, in which spondin-2 secreted from mesenchymal cells plays a central role in placental vascular functions. Furthermore, our results also suggest that a reduction in spondin-2 secretion may contribute to the pathogenesis of T21 placental hypovascularity.

Ezrin-anchored PKA phosphorylates serine 369 and 373 on connexin 43 to enhance gap junction assembly, communication, and cell fusion.

A limited number of human cells can fuse to form multinucleated syncytia. In the differentiation of human placenta, mononuclear cytotrophoblasts fuse to form an endocrinologically active, non-proliferative, multinucleated syncytium. This syncytium covers the placenta and manages the exchange of nutrients and gases between maternal and fetal circulation. We recently reported protein kinase A (PKA) to be part of a macromolecular signaling complex with ezrin and gap junction protein connexin 43 (Cx43) that provides cAMP-mediated control of gap junction communication. Here, we examined the associated phosphorylation events. Inhibition of PKA activity resulted in decreased Cx43 phosphorylation, which was associated with reduced trophoblast fusion and differentiation. In vitro studies using peptide arrays, together with mass spectrometry, pointed to serine 369 and 373 of Cx43 as the major PKA phosphorylation sites that increases gap junction assembly at the plasmalemma. A combination of knockdown and reconstitution experiments and gap-fluorescence loss in photobleaching assays with mutant Cx43 containing single or double phosphoserine-mimicking amino acid substitutions in putative PKA phosphorylation sites demonstrated that phosphorylation of S369 and S373 mediated gap junction communication, trophoblast differentiation, and cell fusion.

APN/CD13 is over-expressed by Psoriatic fibroblasts and is modulated by CGRP and IL-4 but not by retinoic acid treatment.

Psoriasis vulgaris is a common skin inflammatory disease characterized by recurrent flare episodes associated with scaly well-demarcated skin plaques. Skin biopsies from psoriatic patients with high PASI score (22.67 ± 8.67) and from HD were used to study APN/CD13. APN/CD13 is over-expressed in LP and nLP compare to HD skins and fibroblasts. This over-expression is positively correlated with specific enzymatic activity enhancement. However, discrepancies between APN/CD13 expression in LP and nLP prompt us to focus our study on APN/CD13 modulation. Calcitonin Gene Related Peptide (CGRP), a neuropeptide, positively modulated expression and activity of APN/CD13. CGRP consistently induced IL4 secretion, which is also involved in the increase of APN/CD13 expression and activity, which is significantly reversed using IL-4 blocking antibody. Surprisingly, retinoic acid altered the APN/CD13 enzymatic activity only in nLP fibroblasts without modification of APN/CD13 expression. APN/CD13 is over-expressed on psoriatic fibroblasts and exerted high level of activity compare to HD fibroblasts. Taken together, several factors such as CGRP and IL-4 acted on positive regulation of APN/CD13 expression and activity. This study highlighted the interest of APN/CD13 as a new potential target, which should be investigated in psoriasis.

Annexin-A5 organized in 2D-network at the plasmalemma eases human trophoblast fusion.

Only a limited number of human cells can fuse to form a multinucleated syncytium. Cell fusion occurs as part of the differentiation of some cell types, including myotubes in muscle and osteoclasts in remodeling bone. In the differentiation of the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form endocrinologically active, non-proliferative, multinucleated syncytia. These syncytia allow the exchange of nutrients and gases between the maternal and fetal circulation. Alteration of syncytial formation during pregnancy affects fetal growth and the outcome of the pregnancy. Here, we demonstrate the role of annexin A5 (AnxA5) in syncytial formation by cellular delivery of recombinant AnxA5 and RNA interference. By a variety of co-immunoprecipitation, immunolocalization and proximity experiments, we show that a pool of AnxA5 organizes at the inner-leaflet of the plasma membrane in the vicinity of a molecular complex that includes E-Cadherin, α-Catenin and ß-Catenin, three proteins previously shown to form adherens junctions implicated in cell fusion. A combination of knockdown and reconstitution experiments with AnxA5, with or without the ability to self-assemble in 2D-arrays, demonstrate that this AnxA5 2D-network mediates E-Cadherin mobility in the plasmalemma that triggers human trophoblasts aggregation and thereby cell fusion.

[Role and regulation of cAMP signaling in human trophoblast fusion]. / Rôle de la signalisation AMPc dans la fusion des trophoblastes du placenta humain.

In the human placenta, mononuclear cytotrophoblasts fuse to form multinucleated syncytia ensuring hormonal production and nutrient exchanges between the maternal and fetal circulation. The syncytial formation is necessary for the maintenance of pregnancy and for fetal growth. The cAMP signaling pathway is the major route to trigger trophoblast fusion and its activation results in phosphorylation of specific intracellular target proteins and assembly of macromolecular protein complexes constituting the fusogenic machinery at the plasma membrane. Specificity in cAMP signaling is ensured by generation of localized pools of cAMP controlled by cAMP phosphodiesterases (PDEs) and by discrete spatial and temporal activation of protein kinase A (PKA) in supramolecular signaling clusters inside the cell organized by A-kinase-anchoring proteins (AKAPs) and by organization of signal termination by protein phosphatases (PPs). Here we summarize the current knowledge of proteins and protein macrocomplexes involved in the spatiotemporal regulation of cAMP signaling that triggers human trophoblast fusion.

Spatiotemporal regulation of cAMP signaling controls the human trophoblast fusion.

During human placentation, mononuclear cytotrophoblasts fuse to form multinucleated syncytia ensuring hormonal production and nutrient exchanges between the maternal and fetal circulation. Syncytial formation is essential for the maintenance of pregnancy and for fetal growth. The cAMP signaling pathway is the major route to trigger trophoblast fusion and its activation results in phosphorylation of specific intracellular target proteins, in transcription of fusogenic genes and assembly of macromolecular protein complexes constituting the fusogenic machinery at the plasma membrane. Specificity in cAMP signaling is ensured by generation of localized pools of cAMP controlled by cAMP phosphodiesterases (PDEs) and by discrete spatial and temporal activation of protein kinase A (PKA) in supramolecular signaling clusters inside the cell organized by A-kinase-anchoring proteins (AKAPs) and by organization of signal termination by protein phosphatases (PPs). Here we present original observations on the available components of the cAMP signaling pathway in the human placenta including PKA, PDE, and PP isoforms as well as AKAPs. We continue to discuss the current knowledge of the spatiotemporal regulation of cAMP signaling triggering trophoblast fusion.

Formaldehyde Crosses the Human Placenta and Affects Human Trophoblast Differentiation and Hormonal Functions.

The chorionic villus of the human placenta is the source of specific endocrine functions and nutrient exchanges. These activities are ensured by the syncytiotrophobast (ST), which bathes in maternal blood. The ST arises and regenerates throughout pregnancy by fusion of underlying cytotrophoblasts (CT). Any anomaly of ST formation or regeneration can affect pregnancy outcome and fetal growth. Because of its direct interaction with maternal blood, the ST is sensitive to drugs, pollutants and xenohormones. Ex vivo assays of perfused cotyledon show that formaldehyde, a common pollutant present in furniture, paint and plastics, can accumulate in the human placenta and cross to the fetal compartment. By means of RT-qPCR, immunoblot and immunocytochemistry experiments, we demonstrate in vitro that formaldehyde exerts endocrine toxicity on human trophoblasts, including a decrease in the production of protein hormones of pregnancy. In addition, formaldehyde exposure triggered human trophoblast fusion by upregulating syncitin-1 receptor expression (ASC-type amino-acid transporter 2: ASCT2). Moreover, we show that formaldehyde-exposed trophoblasts present an altered redox status associated with oxidative stress, and an increase in ASCT2 expression intended to compensate for this stress. Finally, we demonstrate that the adverse effects of formaldehyde on trophoblast differentiation and fusion are reversed by N-acetyl-L-cysteine (Nac), an antioxidant.

A PKA-ezrin-Cx43 signaling complex controls gap junction communication and thereby trophoblast cell fusion.

Cell fusion occurs as part of the differentiation of some cell types, including myotubes in muscle and osteoclasts in remodeling bone. In the human placenta, mononuclear cytotrophoblasts in a human chorionic gonadotropin (hCG)-driven process fuse to form multinucleated syncytia that allow the exchange of nutrients and gases between the maternal and fetal circulation. Experiments in which protein kinase A (PKA) is displaced from A-kinase anchoring proteins (AKAPs), or in which specific AKAPs are depleted by siRNA-mediated knockdown, point to ezrin as a scaffold required for hCG-, cAMP- and PKA-mediated regulation of the fusion process. By a variety of immunoprecipitation and immunolocalization experiments, we show that ezrin directs PKA to a molecular complex of connexin 43 (Cx43, also known as GJA1) and zona occludens-1 (ZO-1, also known as TJP1). A combination of knockdown experiments and reconstitution with ezrin or Cx43 with or without the ability to bind to its interaction partner or to PKA demonstrate that ezrin-mediated coordination of the localization of PKA and Cx43 is necessary for discrete control of Cx43 phosphorylation and hCG-stimulated gap junction communication that triggers cell fusion in cytotrophoblasts.

Mesenchymal activin-A overcomes defective human trisomy 21 trophoblast fusion.

Placental development is markedly abnormal in trisomy 21 (T21) pregnancies. We hypothesized that abnormal paracrine cross talk between the fetal mesenchymal core and the trophoblast might be involved in the defect of syncytiotrophoblast formation and function. In a large series of primary cultured human cytotrophoblasts isolated from second-trimester control (n = 44) and T21 placentae (n = 71), abnormal trophoblast fusion and differentiation was observed in more than 90% of T21 cases. We then isolated and cultured villous mesenchymal cells from control (n = 10) and T21 placentae (n = 8) and confirmed their fetal origin. Conditioned medium of control mesenchymal cells overcame the abnormal trophoblast fusion of T21 cytotrophoblasts by activating the TGFß signaling pathway, as shown by the phosphospecific protein microarray analysis and the use of TGFß signaling pathway antagonists. Using protein arrays, we further analyzed the cytokines present in the conditioned medium from control and T21 mesenchymal cells. Activin-A was identified as strongly secreted by cells from both sources, but at a significantly (P < 0.01) lower level in the case of T21 mesenchymal cells. Recombinant activin-A stimulated T21 trophoblast fusion. Blocking activin-A antibody inhibited the fusion induced by conditioned medium and exogenous activin-A. Furthermore, follistatin, an activin-A binding protein largely secreted by T21 mesenchymal cells, inhibited the conditioned medium fusogenic activity. These results show that the defective trophoblast fusion and differentiation associated with T21 can be overcome in vitro and reveal the key role of the fetal mesenchymal core in human trophoblast differentiation.

Expression in Escherichia coli and purification of human recombinant connexin-43, a four-pass transmembrane protein.

We have recently shown, using a well-defined in vitro model, that connexin 43 (Cx43) is directly involved in human cytotrophoblastic cell fusion into a multinucleated syncytiotrophoblast. Cx43 appears to interact with partner proteins within a fusogenic complex, in a multi factorial and dynamic process. This fusogenic complex remains to be characterized and constituent proteins need to be identified. In order to identify proteins interacting with the entire Cx43 molecule (extracellular, transmembrane and intracellular domains), we produced and purified full-length recombinant Cx43 fused to glutathione S-transferase (GST-Cx43) and used it as "bait" in GST pull-down experiments. Cx43 cDNA was first cloned into the pDEST15 vector in order to construct a GST-fusion protein, using the Gateway system. The fusion protein GST-Cx43 was then expressed in Escherichia coli strain BL21-AI™ and purified by glutathione-affinity chromatography. The purified fusion protein exhibited the expected size of 70 kDa on SDS-PAGE, western blot and GST activity. A GST pull-down assay was used to show the capacity of the full-length recombinant protein to interact with known partners. Our results suggest that this method has the capacity to produce sufficient full-length recombinant protein for investigations aimed at identifying Cx43 partner proteins.

Human trophoblast in trisomy 21: a model for cell-cell fusion dynamic investigation.

Trophoblastic cell fusion is one essential step of the human trophoblast differentiation leading to formation of the syncytiotrophoblast, site of the numerous placental functions. This process is multifactorial and finely regulated. Using the physiological model of primary culture of trophoblastic cells isolated from human placenta, we have identified different membrane proteins directly involved in trophoblastic cell fusion: connexin 43, ZO-1 and recently syncytins. These fusogenic membrane retroviral envelop glycoproteins: syncytin-1 (encoded by the HERV-W gene) and syncytin-2 (encoded by the FRD gene) and their receptors are major factors involved in human placental development. Disturbances of syncytiotrophoblast formation are observed in trisomy 21-affected placentas. Overexpression of the copper/zinc superoxide dismutase (SOD-1), encoded by chromosome 21 as well as an abnormal hCG signaling are implicated in the defect of syncytiotrophoblast formation. This abnormal trophoblast fusion and differentiation in trisomy 21-affected placenta is reversible in vitro by different ways.

ZO-1 is involved in trophoblastic cell differentiation in human placenta.

Trophoblastic cell-cell fusion is an essential event required during human placental development. Several membrane proteins have been described to be directly involved in this process, including connexin 43 (Cx43), syncytin 1 (Herv-W env), and syncytin 2 (Herv-FRD env glycoprotein). Recently, zona occludens (ZO) proteins (peripheral membrane proteins associated with tight junctions, adherens junctions, and gap junctions) were shown to be involved in mouse placental development. Moreover, zona occludens 1 (ZO-1) was localized mainly at the intercellular boundaries between human trophoblastic cells. Therefore the role of ZO-1 in the dynamic process of human trophoblastic cell-cell fusion was investigated using primary trophoblastic cells in culture. In vitro as in situ, ZO-1 was localized mainly at the intercellular boundaries between trophoblastic cells where its expression substantially decreased during differentiation and during fusion. At the same time, Cx43 was localized at the interface of trophoblastic cells and its expression increased during differentiation. To determine a functional role for ZO-1 during trophoblast differentiation, small interfering RNA (siRNA) was used to knock down ZO-1 expression. Cytotrophoblasts treated with ZO-1 siRNA fused poorly, but interestingly, decreased Cx43 expression without altering the functionality of trophoblastic cell-cell communication as measured by relative permeability time constant determined using gap-FRAP experiments. Because kinetics of Cx43 and ZO-1 proteins show a mirror image, a potential association of these two proteins was investigated. By using coimmunoprecipitation experiments, a physical interaction between ZO-1 and Cx43 was demonstrated. These results demonstrate that a decrease in ZO-1 expression reduces human trophoblast cell-cell fusion and differentiation.