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

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.

P‐Glycoprotein (P‐gp)/ABCB1 plays a functional role in extravillous trophoblast (EVT) invasion and is decreased in the pre‐eclamptic placenta

“…It is also well known that trophoblast fusion stimulated by the protein kinase A pathway and cAMP involves the induction of GJA1 and hCG . Knockdown of their expression in the BeWo choriocarcinoma model results in an arrest of forskolin‐stimulated fusion, while overexpression of Cx43 permits Jeg‐3 cell fusion …”

“…The role of P-gp in trophoblast fusion is less surprising as a number of studies have reported the loss or down-regulation of P-gp in the spontaneous differentiation of primary CT to ST. [40][41][42] It is also well known that trophoblast fusion stimulated by the protein kinase A pathway and cAMP involves the induction of GJA1 and hCG. 43 Knockdown of their expression in the BeWo choriocarcinoma model results in an arrest of forskolin-stimulated fusion, 44,45 while overexpression of Cx43 permits Jeg-3 cell fusion. 28 As a point of speculation, beyond its well-known role in substrate distribution and bioavailability and in multidrug resistance, P-gp plays a role in cancer cell invasion, drug resistance and metastasis.…”

Relevance of Wnt10b and activation of β‐catenin/GCMa/syncytin‐1 pathway in BeWo cell fusion

“…Forskolin, a pharmacological agent, is known to induce fusion of BeWo cells by activating cAMP and its downstream pathway . We have shown previously that treatment of BeWo cells with an optimized concentration of forskolin (25 μmol/L) led to ~20‐fold ( P = .02) and ~40‐fold ( P = .003) increase in the Wnt10b transcript at 24 and 48 hour, respectively, whereas transcript levels of Wnt 3, 4, 5a, 7b, and 11 were decreased significantly within 24 hour of forskolin treatment .…”

“…Forskolin, a pharmacological agent, is known to induce fusion of BeWo cells by activating cAMP and its downstream pathway . We have shown previously that treatment of BeWo cells with an optimized concentration of forskolin (25 μmol/L) led to ~20‐fold ( P = .02) and ~40‐fold ( P = .003) increase in the Wnt10b transcript at 24 and 48 hour, respectively, whereas transcript levels of Wnt 3, 4, 5a, 7b, and 11 were decreased significantly within 24 hour of forskolin treatment . As 14 Wnts are known to be expressed in the first trimester placenta, therefore, in the present study, we investigated the expression of rest of the eight Wnts (Wnt1, Wnt2, Wnt2b, Wnt5b, Wnt6, Wnt7a, Wnt9b, and Wnt10a) on treatment with 25 μmol/L of forskolin.…”

“…However, aforementioned reports point toward the involvement of canonical Wnt signaling in trophoblast fusion, but association with specific Wnt ligand(s) is not yet established. However, previous study by our group had shown an increase in the levels of Wnt10b transcript in BeWo cells on treatment with forskolin, hinting toward a plausible involvement of Wnt10b during cAMP‐induced BeWo cell fusion. In an attempt to decipher this association, BeWo cells were knocked down for Wnt10b and studied for its effect on cell fusion induced by either forskolin or human chorionic gonadotropin (hCG) as well as hCG secretion subsequent to treatment with forskolin and to define a possible mechanism by studying β‐catenin, GCMa, and syncytin‐1 expression.…”