RhoE Is Regulated by Cyclic AMP and Promotes Fusion of Human BeWo Choriocarcinoma Cells

Abstract

Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. In this study we examined the role of the Rho GTPase family member RhoE in trophoblast differentiation and fusion using the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Treatment of BeWo cells with the cell permeable cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) resulted in a strong upregulation of RhoE at 24h, coinciding with the onset of fusion. Using the protein kinase A (PKA)-specific cAMP analogue N6-phenyl-cAMP, and a specific inhibitor of PKA (14–22 amide, PKI), we found that upregulation of RhoE by cAMP was mediated through activation of PKA signalling. Silencing of RhoE expression by RNA interference resulted in a significant decrease in dbcAMP-induced fusion. However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, we found that RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. These results show that induction of RhoE by cAMP is mediated through PKA and promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways.

Pathophysiological Functions of Rnd 3/ RhoE

“…In an in vitro model of villous cytotrophoblast fusion, Collett et al treated BeWo choriocarcinoma cells with a cell permeable cyclic AMP analogue, dibutyryl cyclic AMP (dbcAMP). As a result, there was a strong upregulation of Rnd3 24h after dbcAMP treatment, coinciding with the observation of the onset of cell fusion (28). Liebig with colleagues (77) also observed that RND3 protein levels were specifically and transiently upregulated upon basal keratinocyte differentiation, while RND3 depletion induced hyperproliferation and delayed initiation of keratinocyte differentiation.…”

The cAMP-responsive element binding protein (CREB) transcription factor regulates furin expression during human trophoblast syncytialization

“…The significance of the cAMP/PKA signaling pathway has been proven in regulating the transcription and post-translational acetylation, dephosphorylation and desumoylation of glial cells missing-1 (GCM1), which subsequently regulates syncytin expression and controls human trophoblast cell differentiation along the syncytialization pathway as the master transcription factor [43,44,54e56]. Moreover, the cAMP/PKA pathway also regulates transcriptional activities of many genes in the human trophoblast fusogenic machinery, including CYP17 [57], old astrocyte specifically induced substance (OASIS) [43], human placental growth factor (PlGF) [58] and the Rho GTPase family member RhoE [45], as well as the production and secretion of hCG [43,59] and progesterone [60] by the syncytiotrophoblast.…”

“…Several signaling pathways have been reported to be involved during trophoblast cell fusion, including PKA [43][44][45], ERK1/2 [43,46] and p38 MAPK [43,46,47] pathways. To identify upstream signaling pathways that drive the upregulation of furin during FSKinduced fusion of BeWo cells, different inhibitors of these signaling pathways were administrated upon FSK treatment.…”

Cell Signaling Pathways Involved During Invasion and Syncytialization of Trophoblast Cells

“…CBP‐GCMa complex, in turn, leads to transactivation of fusogenic protein syncytin‐1 . There are further evidences that suggest the role of RhoE, a member of Rho GTPase family in trophoblast fusion downstream of cAMP/PKA signaling (Fig. ).…”