Syncytial fusion of human trophoblast depends on caspase 8

Differentiation of human placental villous trophoblast includes syncytial fusion of cytotrophoblast forming syncytiotrophoblast. Early stages of the apoptosis cascade were described to be involved in this differentiation process. We investigated the role of the initiator caspase 8 in syncytial fusion in vitro, cultivating placental villous explants with or without caspase 8 antisense oligonucleotides or peptide inhibitors for up to 120 h. Trophoblast fusion and differentiation were assessed by confocal microscopy, immunohistochemistry and Western blot analysis. Culture with caspase 8 antisense oligonucleotides or peptide inhibitors reduced the fusion of cytotrophoblast with the syncytiotrophoblast, and resulted in multilayered cytotrophoblast. Caspase 8 expression was suppressed by antisense oligonucleotides and caspase 8 activities were reduced by peptide inhibitors. The organic anion-transporter hOAT-4 normally expressed in the cytotrophoblast and transferred into the syncytiotrophoblast by syncytial fusion was retained in the cytotrophoblast due to lack of fusion. We conclude that expression and activity of caspase 8 is a prerequisite for differentiation and syncytial fusion of cytotrophoblast cells.     

E-cadherin in the assessment of aberrant placental cytotrophoblast turnover in pregnancies complicated by pre-eclampsia

E-cadherin is a cell–cell adhesion protein expressed in cytotrophoblasts, which is lost as they differentiate and syncytialise. We have exploited E-cadherin as a marker of cytotrophoblasts to investigate villous tissue composition in first and third trimester placentae, both in normal pregnancy and pregnancies complicated by pre-eclampsia. We have achieved this by measuring expression levels of E-cadherin at the mRNA level, using Q-PCR, and at the protein level using semi-quantitative Western blotting. We have also combined E-cadherin immunohistochemistry with morphometric analysis of area measurements to define cytotrophoblast and syncytiotrophoblast compartments. This novel use of E-cadherin has revealed a decrease in the proportion of cytotrophoblasts in villous tissue as pregnancy progresses, in the absence of changes in syncytiotrophoblast cover. Moreover, in pre-eclampsia, placental E-cadherin was raised compared to syncytiotrophoblast, suggesting either exaggerated cytotrophoblast proliferation or impaired cytotrophoblast differentiation, both alterations of potential pathogenic importance.     

Hypoxia impairs cell fusion and differentiation process in human cytotrophoblast,in vitro

During human pregnancy, the trophoblast develops from differentiation of cytotrophoblast cells into an endocrine active syncytiotrophoblast. In culture, isolated mononuclear cytotrophoblasts aggregate and then fuse to form a syncytium, reproducing the in vivo process. In this study, we examined the effect of low oxygen tension (approximately 9%, hypoxia) compared to standard conditions (approximately 19% oxygen, normoxia) on these cellular events. Under hypoxia, syncytial formation was less frequently observed, cell staining and electron microscopy revealed that cytotrophoblasts remain aggregated, with a positive proliferative cell nuclear antigen (PCNA) immunostaining. Desmoplakin and E-cadherin, both known to disappear with cytotrophoblast fusion, showed persistent expression in hypoxic cells after 3 days of culture. In contrast, the expression of actin and ezrin, two cytoskeletal proteins, was unchanged. hCG secretion and hPL expression were both decreased in hypoxic cells, reflecting a reduced syncytial formation. Thus, on day 3, the mean values for hCG secretion were 1,100 ± 155 and 289 ± 26 mlU/mL in normoxic and hypoxic conditions, respectively. The reduced cell fusion process as well as hCG secretion and hPL expression under hypoxia were reversed by reoxygenation of the cells. We conclude that under hypoxia, the formation of functional syncytiotrophoblast is impaired due to a defect in the cytotrophoblast fusion process. This may explain the observation of a higher number of cytotrophoblast cells and a reduced syncytial layer in placentas of some pathological pregnancies. © 1996 Wiley-Liss, Inc.     

IGF2 actions on trophoblast in human placenta are regulated by the insulin-like growth factor 2 receptor, which can function as both a signaling and clearance receptor

Insulin-like growth factor 2 (IGF2) enhances proliferation and survival of human first-trimester cytotrophoblasts (CTB) by signaling through the insulin-like growth factor 1 receptor (IGF1R). However, the role of the IGF2 receptor (IGF2R) in regulating trophoblast kinetics is unclear: It could act as a clearance receptor for trafficking excess ligand to lysosomes for degradation and/or directly mediate IGF2 signaling. We used an IGF2R knockdown strategy in BeWo cells and placental villous explants to investigate trophoblast proliferation and survival in response to stimulation by IGF. Both IGF1 and IGF2 significantly (P < 0.001) increased mitosis and reduced apoptosis in serum-starved BeWo cells. Small interfering RNA (siRNA)-mediated knockdown of IGF2R further enhanced IGF2-stimulated mitosis (P < 0.01), and IGF2-mediated rescue of apoptosis (P < 0.001) in these cells. Leu(27)IGF2, an IGF2 analogue that binds to IGF2R but not IGF1R, also protected IGF2R-expressing BeWo cells from apoptosis but did not increase mitosis. IGF treatment of term placental villous explants with reduced syncytial expression of IGF2R increased CTB proliferation (P < 0.001) and decreased apoptosis (P < 0.01) compared to untreated controls. Moreover, IGF2-mediated rescue of CTB apoptosis was significantly greater than that in tissue with normal IGF2R expression. Leu(27)IGF2 promoted mitogenesis and survival only in explants with intact IGF2R expression. Given that altered CTB turnover is observed in pregnancies complicated by fetal growth restriction, the development of strategies to manipulate the IGF2R signaling axis in the syncytiotrophoblast may provide a therapeutic avenue for treating this condition.     

Biochemical characterization and modulation of LH/CG-receptor during 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.     

RhoE is regulated by cyclic AMP and promotes fusion of human BeWo choriocarcinoma cells

Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. In this study we examined the role of the Rho GTPase family member RhoE in trophoblast differentiation and fusion using the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Treatment of BeWo cells with the cell permeable cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) resulted in a strong upregulation of RhoE at 24 h, coinciding with the onset of fusion. Using the protein kinase A (PKA)-specific cAMP analogue N(6)-phenyl-cAMP, and a specific inhibitor of PKA (14-22 amide, PKI), we found that upregulation of RhoE by cAMP was mediated through activation of PKA signalling. Silencing of RhoE expression by RNA interference resulted in a significant decrease in dbcAMP-induced fusion. However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, we found that RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. These results show that induction of RhoE by cAMP is mediated through PKA and promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways.     

Epidermal growth factor stimulation of trophoblast differentiation requires MAPK11/14 (p38 MAP kinase) activation

Cultured human term villous cytotrophoblasts (CT) have been reported to be nonproliferating but differentiate when exposed to epidermal growth factor (EGF). Here we show that CT differentiate into chorionic gonadotropin (beta-hCG/CGB)-expressing cells when cultured with medium alone. The addition of EGF decreases CGB secretion and prolongs production for up to 13 days. EGF stimulates the phosphorylation (activation) of the signaling intermediate p38 (MAPK11/14), and blocking phosphorylation pharmacologically with either SB203580 or SB202190 strongly inhibited spontaneous and EGF-stimulated secretion of CGB. In addition, EGF-stimulated fusion of cytotrophoblasts into syncytial units was strongly inhibited by SB203580. EGF upregulated trophoblast proliferation (measured by bromodeoxyuridine uptake) and SB203580 increased this proliferation after 5 days. In agreement with these observations, EGF and SB203580 increased expression of the G1-phase-specific gene cyclin-D1 (CCND1) and SB203580 downmodulated its inhibitor p21 (CDKN1A). When added to villous explant cultures, EGF did nothing to the pattern of CGB secretion, but addition of SB203580 prevented the normal surge in secretion during syncytial regeneration over Days 3-7. These data support the hypothesis that EGF-stimulated cytotrophoblast differentiation to syncytium requires MAPK11/14 activation, and that cytotrophoblast proliferation can be stimulated in culture by EGF and enhanced by MAPK11/14 inhibition with a consequent reduction of differentiation.     

Functional proteomics: examining the effects of hypoxia on the cytotrophoblast protein repertoire

The outcome of human pregnancy depends on the differentiation of cytotrophoblasts, specialized placental cells that physically connect the embryo/fetus to the mother. As cytotrophoblasts differentiate, they acquire tumor-like characteristics that enable them to invade the uterus. In a novel feedback loop, the increasingly higher levels of oxygen they encounter within the uterine wall influence their differentiation into vascular-like cells. Together, the invasive and cell surface properties of cytotrophoblasts enable them to form vascular connections with uterine blood vessels that divert maternal blood flow to the placenta, a critical hurdle in pregnancy. It is therefore important to understand how cytotrophoblasts respond to changes in oxygen tension. Here we used a proteomics approach, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry, to characterize the protein repertoire of first trimester human cytotrophoblasts that were maintained under standard tissue culture conditions (20% O(2)). 2-D PAGE showed a unique protein map as compared to placental fibroblasts and human JEG-3 choriocarcinoma cells. Mass spectrometry allowed the identification of 43 spots on the cytotrophoblast map. Enzymes involved in glycolysis and responses to oxidative stress, as well as the 14-3-3 signaling/adapter proteins, were particularly abundant. Hypoxia in vitro (2% O(2)) produced discrete changes in the expression of a subset of proteins in all the aforementioned functional categories. Together, these data offer new information about the early gestation cytotrophoblast protein repertoire and the generalized mechanisms the cells use to respond to changes in oxygen tension at the maternal-fetal interface.     

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) mRNA expression and localization and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) in human placenta of the first, second, and third trimester

The cellular localization of protein tyrosine phosphatase 51 (PTPIP51) and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) was studied in human placentae of different gestational stages. The expression of PTPIP51 protein and mRNA was observed in the syncytiotrophoblast and cytotrophoblast layer of placentae from the first, second, and third trimesters. In contrast, PTP1B expression was restricted to the syncytiotrophoblast during all gestational stages. Cells of the cytotrophoblasts and parts of the syncytiotrophoblasts expressing high amounts of PTPIP51 were found to execute apoptosis as shown by TdT-mediated dUTP-biotin nick end labeling assay, cytokeratin 18f, and caspase 3 expression. PTPIP51 could also be traced in the endothelium and smooth muscle cells of placental arterial and venous vessels, identified by double immunostainings with antibodies directed against van Willebrand factor and alpha-smooth muscle actin. Some of these cells showing a high PTPIP51 reactivity were Ki67 positive, indicating proliferation. Additionally, a small population of placental CD14-positive macrophages and mesenchymal cells within the villous stroma were detected as PTPIP51 positive. Our data suggest that both proteins, PTPIP51 and PTP1B, play a role in differentiation and apoptosis of the cytotrophoblast and syncytiotrophoblast, respectively. Moreover, PTPIP51 may also serve as a cellular signaling partner in angiogenesis and vascular remodeling.     

Direct involvement of HERV-W Env glycoprotein in human trophoblast cell fusion and differentiation

We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation.     

Regulated expression of cadherin-11 in human extravillous cytotrophoblasts undergoing aggregation and fusion in response to transforming growth factor beta 1

Transforming growth factor beta 1 is believed to be a key regulator of extravillous cytotrophoblast invasion during the first trimester of pregnancy. In addition, this growth factor has been shown to regulate cellular differentiation and fusion in cultured extravillous cytotrophoblasts. To date, the cellular mechanisms by which transforming growth factor beta 1 promotes these developmental processes remain poorly understood. Recent studies indicate that the expression of the novel cadherin subtype, known as cadherin-11, is associated with the terminal differentiation and fusion of villous cytotrophoblasts isolated from the human term placenta and human myoblasts in vitro. In this study, cadherin-11 mRNA and protein expression were examined in primary cultures of human extravillous cytotrophoblasts cultured in the presence of increasing concentrations of transforming growth factor beta 1 using northern and western blot analysis, respectively. Transforming growth factor beta 1 was shown to increase cadherin-11 mRNA and protein expression in these cultured extravillous cytotrophoblasts in a dose-dependent manner. Cadherin-11 was further localized to the large cellular aggregates and multinucleated cells that formed in response to increasing concentrations of transforming growth factor beta 1 using immunocytochemistry. Collectively, these observations suggest that the morphogenetic effects of transforming growth factor beta 1 on cultured extravillous cytotrophoblasts are mediated, at least in part, by an increase in cadherin-11 expression. This study not only adds to the understanding of the cellular mechanisms by which transforming growth factor beta 1 promotes trophoblast differentiation and fusion but provides useful insight into the cell biology of the cadherins.     

Choriocarcinoma cells increase the number of differentiating human cytotrophoblasts through an in vitro interaction

The human placenta arises from the zygote through single cell intermediates called cytotrophoblasts that in turn give rise to a syncytium. In culture, mononucleated cytotrophoblasts exhibit little, if any, cell division but are converted to multinucleated cells. Choriocarcinoma, the malignant tumor of placenta trophoblast, comprises a mixed population of dividing cellular intermediates that resemble cytotrophoblasts but are less differentiated. Because the choriocarcinoma intermediates arise from dividing cells, the tumor may contain one or more cell types in abundance not present in the population of isolated placental cells. To study placental differentiation through cell-cell interaction, choriocarcinoma cell lines were co-cultured with placenta-derived cytotrophoblasts, and placental hormone biosynthesis, as a marker of differentiation was examined. We reasoned that intermediates formed by the tumor might interact with and complement those intermediates in the placenta-derived cytotrophoblast population. Co-culturing either the JAr or JEG choriocarcinoma cell lines with cytotrophoblasts elevated the synthesis of the chorionic gonadotropin alpha and beta subunits 10-20 fold, and human placental lactogen 5-fold. The effect was specific for these trophoblast-derived cells, since comparable quantities of Chinese hamster ovary or HeLa cells did not affect the placental cytotrophoblast culture. Further experiments suggested that the source of enhanced synthesis was the cytotrophoblasts. We propose that an interaction between cytotrophoblasts and choriocarcinoma cells occurs, which results in an increased number of differentiating cytotrophoblasts. Such co-cultures may represent a model system for examining choriocarcinoma cell interaction with normal cells, a process known to occur in vivo. The data are also consistent with the hypothesis that the regulated chorionic gonadotropin production in the placenta is determined by interaction among trophoblast cells at different stages of differentiation.     

The IGF axis and placental function. a mini review

It is well known that the insulin-like growth factor (IGF) axis is an important regulator of foetal growth and in recent years, it has been suggested that the ligands IGF-I and IGF-II may, in part, mediate this effect by promoting proper placental development and function. In other tissues, IGF effects on metabolism, proliferation and differentiation are primarily mediated via IGF binding protein-regulated interaction of IGFs with the type 1 IGF receptor and therefore here, we review the placental expression and postulated role, of each of the IGF axis components and discuss the cellular mechanisms through which these effects are exerted.     

Differential Effects of Sodium Butyrate and Lithium Chloride on Rhesus Monkey Trophoblast Differentiation

Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O₂) and differentiation proceeds along an extravillous pathway (giving rise to invasive extravillous cytotrophoblasts) and a villous pathway (giving rise to multinucleated syncytiotrophoblast). Interstitial extravillous cytotrophoblasts invade the decidua, while endovascular extravillous cytotrophoblasts are involved in re-modelling uterine spiral arteries. We tested the idea that sodium butyrate (an epigenetic modulator) induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/β-catenin pathway. The results show that syncytiotrophoblast formation was increased by butyrate, accompanied by nuclear accumulation of β-catenin, and increased expression of EnvV2 and galectin-1 (two factors thought to be involved in trophoblast fusion). Surprisingly, the expression of GCM1 and syncytin-2 was not affected by sodium butyrate. When trophoblasts were incubated with lithium chloride, a GSK3 inhibitor that mimics Wnt activation, nuclear accumulation of β-catenin also occurred but differentiation into syncytiotrophoblast was not observed. Instead the cells differentiated to mononucleated spindle-shaped cells and showed molecular and behavioral characteristics of endovascular trophoblasts. Another highly specific inhibitor of GSK3, CHIR99021, failed to induce endovascular trophoblast characteristics. These observations suggest that activation of the Wnt/β-catenin pathway correlates with both trophoblast differentiation pathways, but that additional factors determine specific cell fate decisions. Other experiments suggested that the differential effects of sodium butyrate and lithium chloride might be explained by their effects on TNFα production. The results provide valuable tools to manipulate trophoblast differentiation in vitro and to better understand the differentiation pathways that occur during early gestation.     

Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle inhibitors p21, p27 and p57 in human first trimester placenta and deciduas

Although placental development and implantation depend on the coordination of trophoblast proliferation, differentiation and invasion, little is known about the cell cycle regulators that govern the control of these events. The hypothesis that the coordinated expression of cell cycle progression and inhibition factors will determine whether cytotrophoblasts proliferate or undergo cell cycle arrest or cell cycle exit allowing subsequent differentiation was tested. The cell cycle promotors cyclin A, cyclin B1, PCNA, Ki67 and the cell cycle inhibitors p21, p27 and p57 were immunolocalized in tissue sections of first trimester pregnancies (weeks 6 and 9–12). Double staining with cytokeratin 7 allowed unambiguous identification of extravillous cytotrophoblast (EVT) in the decidua. Villous cytotrophoblasts were immunolabelled for Ki67 and cyclin A but only few were stained with anti-cyclin B1. The syncytiotrophoblast was devoid of immunoreactivity for any of the cell cycle progression factors. It expressed especially p21, whereas p27 and p57 were predominantly found in villous cytotrophoblasts. PCNA, Ki67, cyclin A and cyclin B1 were immunolocalized in proximal and distal EVTs of anchoring villi and in EVT which had invaded the upper decidual segments. All EVTs strongly expressed p27 and p57, but not p21. These data clearly suggest different functions for p21, p27 and p57 in placental development with distinct roles for p21 and p57 in syncytiotrophoblast and EVT differentiation, respectively. p27 appears to be involved in both the processes. The results may also challenge the concept of differential mitotic activity in the proximal and distal parts of the first trimester cytotrophoblast cell column, but more functional studies are clearly needed. The presence of p27 and p57 in EVT cells, which invade the deciduas deeply, may account for the loss of mitogenic potential of these cells.