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

Sudha Saryu Malhotra,Satish Kumar Gupta,Pankaj Suman

doi:10.1038/srep11210

Sudha Saryu Malhotra, Satish Kumar Gupta + Show 1 more

Open Access

https://doi.org/10.1038/srep11210

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Abstract

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.

Highlights

Concomitant addition of polyclonal antibodies against human chorionic gonadotropin (hCG) suppressed fusion[14,15]
Expression of hCG starts during the course of cytotrophoblast differentiation[27]
In contrast to CTBs that undergo spontaneous in vitro fusion in medium containing fetal bovine serum, trophoblastic BeWo cells can be induced to differentiate29. cAMP activation via analogues of cAMP or inducers like forskolin, cholera toxin, Pertussis toxin, etc.[27] are used to induce fusion in vitro

Summary

Introduction

Concomitant addition of polyclonal antibodies against hCG suppressed fusion[14,15]. in trisomy 21 placentas, aberrant STB development was observed, which may be due to the presence of abnormal hCG and a decreased expression of luteinizing hormone/choriogonadotropin receptor (LHCGR)[16,17]. Taking cue from all these independent studies, we wanted to investigate whether there is a differential expression in all or some of these pathways in those trophoblastic cells which inherently produce less hCG This would reveal whether any cross communication among PKA/ p38MAPK/ ERK1/2/ β -catenin pathways exist or they function independently or may complement each other to achieve a common event of cellular fusion. To achieve these goals, BeWo cells, an established in vitro model to study trophoblast fusion[25,26] have been employed; using shRNA, α - and β -hCG-knockdown BeWo cell lines were generated. Differences in downstream signaling pathways between control and silenced cells were delineated to showcase critical molecules in hCG mediated cell fusion

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