Journal of Molecular Biology
Volume 392, Issue 2, 18 September 2009, Pages 301-318
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Syncytin-2 Plays an Important Role in the Fusion of Human Trophoblast Cells

https://doi.org/10.1016/j.jmb.2009.07.025Get rights and content

Abstract

Human endogenous retrovirus (HERV)-encoded Syncytin-1 has been suggested to play a major role in trophoblast cell fusion and thereby placenta development. However, recent studies have strongly suggested that other HERV envelope proteins could also be implicated in this process. Based on this premise, herein we compared the expression and functional implication of Syncytin-1 with the more recently described Syncytin-2 protein in various trophoblast cells. Real-time reverse transcription PCR and Western blot analyses in differentiating primary trophoblast cells first indicated a direct correlation between mRNA and protein levels of Syncytin-2 and cell fusion, while an inverse correlation for Syncytin-1 was noted. Similar reverse transcription PCR experiments and promoter studies showed that cell fusion-inducing agents in the trophoblastic BeWo cell line increased the expression of Syncytin-1 but, more importantly, augmented Syncytin-2 expression. Confocal microscopy experiments further revealed that in BeWo cells and in freshly isolated primary human trophoblast cells, Syncytin-1 was present as a cytoplasmic punctuated structure in proximity to regions of cell-to-cell contact. On the other hand, Syncytin-2 presented an inducible signal, which mainly localized to the cytoplasmic membrane. Experiments with siRNA (small interfering RNA)-transfected BeWo and primary human trophoblast cells demonstrated an important diminution in the number of cell fusion events upon repression of Syncytin-2 expression, whereas transfection experiments with Syncytin-1-specific siRNA resulted in a more modest effect. Overall, these results highlight the importance of Syncytin-2 in BeWo and primary human trophoblast cell fusion.

Introduction

Placenta development is a highly regulated process that involves the differentiation and renewal of trophoblast cells. Trophoblast cells initially differentiate either in cytotrophoblasts or in the syncytiotrophoblast layer. The syncytiotrophoblast results from the fusion of villous trophoblast cells and its renewal is assured by cytotrophoblast fusion with this syncytiotrophoblast layer.1 The syncytiotrophoblast plays a fundamental role in allowing the adequate exchange of nutrients and hormones as well as other components between the mother and the foetus.2

Formation of the syncytiotrophoblast, a process referred to as syncytialisation, has been left unexplained for several years, until recently, when a number of studies have indeed shown that envelope (Env) proteins derived from human endogenous retroviruses (HERVs) were important players in this biological process.3, 4 HERV sequences represent 8% of the human genome and are estimated to have integrated the genome of primates from 10 to 50 million years ago.5, 6 Although mostly distributed in the genome as solo long terminal repeats (LTRs), HERV sequences are also occasionally present as nearly complete or complete retroviral genome in which all or some of the retroviral genes have been deleteriously mutated.7 In fact, some of the HERV genes are still able to produce proteins, and the presence of complete viral particles has been previously described.8, 9, 10 The abnormal expression of HERV genes has also been associated with various human pathologies including breast cancer and multiple sclerosis.5, 11, 12

The function of these HERV proteins remains mostly ill-defined. However, recent evidence has suggested that envelope-like HERV proteins are crucial mediators of the fusion process involved in the syncytialisation of trophoblasts (for a review, see Ref. 13). Indeed, a first report had shown that a protein now termed Syncytin-1 played an essential role in these cellular fusion events.3, 14, 15 More importantly, a decrease in expression of Syncytin-1 has been associated with the occurrence of pregnancy-associated anomalies such as preeclampsia and hemolysis elevated liver enzymes, and low platelet syndrome.16 However, several unanswered questions remain as to the role played by Syncytin-1 in trophoblast cell fusion and it has been suggested that other HERV Env proteins could possibly be involved in this process.17, 18 The most serious candidate is termed Syncytin-2.19, 20, 21, 22, 23 Although no clear evidence has yet been presented in terms of its implication in placental syncytialisation, its exclusive expression in placental tissue (especially in cytotrophoblast cells)23 and its fusogenic capacity19, 20, 22 support the notion that this protein might be actively participating in the fusion of trophoblast cells. Recent evidence has further demonstrated that the receptor binding to the Syncytin-2 protein is also placenta-specific, as opposed to the ASCT2 receptor previously identified for Syncytin-1.15, 24, 25

Based on these recent studies, our objective was to thoroughly assess and compare the relative importance of Syncytin-1 and Syncytin-2 in trophoblast cell fusion. Using the well-characterized cell-fusion-inducible BeWo cell line and isolated primary human cytotrophoblasts, our results show that in terms of expression, cellular localisation, and function, Syncytin-2 plays a more important role in mediating cell fusion than does Syncytin-1. These data are thus indicative that Syncytin-2 strongly impacts on placental development and that trophoblast cell fusion involves more than one HERV Env protein.

Section snippets

Characterization of the human Syncytin-2 transcript

To explore and compare the functional relevance of Syncytin-2 (versus Syncytin-1) in trophoblast cell fusion, its transcript was first characterized. These analyses were performed in BeWo cells, a well-known trophoblastic cell line model, which differentiates and fuses upon treatment with agents such as forskolin and protein tyrosine phosphatase inhibitors.26 RNA from forskolin-stimulated BeWo cells was thus used to precisely map the borders of the transcript by 5′ and 3′ RACE (rapid

Discussion

Trophoblast cell differentiation is a complex process and includes important morphological changes, of which cellular fusion is an important hallmark. Recent studies had demonstrated that Syncytin-1 had a major role in this process,3, 14, 30 although the involvement of other cellular factors (including other HERV Env proteins) was proposed.18 In light of these facts and based on studies on the new Syncytin-2 protein,19, 23 our aim was to assess the role of both HERV Env proteins in BeWo and

Cell culture

The trophoblastic cell lines BeWo, Jar, and JEG-3 were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were respectively maintained in Ham's F12 (Invitrogen Canada Inc, Burlington, Canada), RPMI 1640 (Sigma-Aldrich, Oakville, Canada) and minimum essential medium (MEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 2 mM l-glutamine at 37 °C in a 5% CO2 atmosphere. Primary human trophoblasts were isolated from human placentas of

Acknowledgements

This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (B.B.). F.L.B. holds a Strategic Training Initiative in Research in Reproductive Health Sciences postdoctoral fellowship. B.B. holds a Canada Research Chair in Human Retrovirology (Tier 2). We thank Denis Flipo for his excellent technical assistance in the confocal microscopy experiments.

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