Syncytin-2 Plays an Important Role in the Fusion of Human Trophoblast Cells Amandine Vargas, Julie Moreau, Sébastien Landry, Frédérique LeBellego, Chirine Toufaily, Éric Rassart, Julie Lafond and Benoit Barbeau⁎ Département des Sciences Biologiques, Université du Québec à Montréal, Montréal, Québec, Canada H2X 3X8 Centre de Recherche BioMed, Université du Québec à Montréal, Montréal, Québec, Canada H2X 3X8 Received 26 February 2009; received in revised form 29 June 2009; accepted 7 July 2009 Available online 16 July 2009 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. © 2009 Elsevier Ltd. All rights reserved. Edited by J. Karn Keywords: trophoblast cells; Syncytin-1; Syncytin-2; cell fusion Introduction Placenta development is a highly regulated pro- cess that involves the differentiation and renewal of trophoblast cells. Trophoblast cells initially differ- entiate either in cytotrophoblasts or in the syncytio- trophoblast 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 syncytiotropho- blast 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 *Corresponding author. Département des Sciences Biologiques, Université du Québec à Montréal, SB-R860, 2080 St-Urbain, Montreal, Québec, Canada H2X 3X8. E-mail address: barbeau.benoit@uqam.ca. Abbreviations used: Env, envelope; HERV, human endogenous retrovirus; LTR, long terminal repeat; RACE, rapid amplification of cDNA ends; ORF, open reading frame; GADPH, glyceraldehyde-3- phosphate dehydrogenase; siRNA, small interfering RNA; FBS, fetal bovine serum; PBS, phosphate-buffered saline; HPRT-1, hypoxanthine phosphoribosyl transferase 1; PI, propidium iodide; HRP, horseradish peroxidase. doi:10.1016/j.jmb.2009.07.025 J. Mol. Biol. (2009) 392, 301–318 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.