Alpha or beta human chorionic gonadotropin knockdown decrease BeWo cell fusion by down-regulating PKA and CREB activation

The aim of the present study is to delineate the role of human chorionic gonadotropin (hCG) in trophoblast fusion. In this direction, using shRNA lentiviral particles, α- and β-hCG silenced 'BeWo' cell lines were generated. Treatment of both α- and β-hCG silenced BeWo cells with either forskolin or exogenous hCG showed a significant reduction in cell fusion as compared with control shRNA treated cells. Studies by qRT-PCR, Western blotting and immunofluorescence revealed down-regulation of fusion-associated proteins such as syncytin-1 and syndecan-1 in the α- and β-hCG silenced cells. Delineation of downstream signaling pathways revealed that phosphorylation of PKA and CREB were compromised in the silenced cells whereas, no significant changes in p38MAPK and ERK1/2 phosphorylation were observed. Moreover, β-catenin activation was unaffected by either α- or β-hCG silencing. Further, inhibition of PKA by H89 inhibitor led to a significant decrease in BeWo cell fusion but had no effect on β-catenin activation suggesting the absence of non-canonical β-catenin stabilization via PKA. Interestingly, canonical activation of β-catenin was associated with the up-regulation of Wnt 10b expression. In summary, this study establishes the significance of hCG in the fusion of trophoblastic BeWo cells, but there may be additional factors involved in this process.