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 24 h, coinciding with the onset of fusion. Using the protein kinase A (PKA)-specific cAMP analogue N(6)-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.

Figure 1. Effect of cyclic AMP on RhoE expression and fusion in BeWo cells.

BeWo cells were treated with or without 1mM dbcAMP and studied at the indicated times. Cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (A) and densitometric analysis of blots (B). Cells were fixed, immunostained for desmosomal protein (green) and counterstained with Hoechst 33258 (blue) (C) and cell fusion was quantified (D) as described in Materials & Methods. Results are presented as mean ± SEM for three separate experiments. *p<0.05, **p<0.01 compared with control (determined by ANOVA).

Figure 2. Attenuation of RhoE expression in response to cyclic AMP in JEG-3 cells.

BeWo or JEG-3 cells were treated with 1mM dbcAMP. At the indicated times cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (A) with densitometric analysis of RhoE expression normalised to β-actin expression (B). Cells were fixed, immunostained for desmosomal protein (C) and cell fusion was quantified (D) as described in Materials & Methods. Results are presented as mean ± SEM for three separate experiments. *p<0.05, **p<0.01 compared with BeWo cells (determined by ANOVA).

Figure 3. Upregulation of RhoE by cyclic AMP is mediated through protein kinase A.

BeWo cells were treated with 1mM dbcAMP or PKA-selective cAMP analogue (Phe) for 24h. Cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (A) with densitometric analysis of RhoE expression normalised to β-actin expression (B). BeWo cells were pretreated with the specific PKA inhibitor PKI or vehicle for 1h then treated with 1mM dbcAMP in the presence or absence of PKI. After 24h cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (C) with densitometric analysis of RhoE expression normalised to β-actin expression (D). Representative blots from three separate experiments are shown.

Figure 4. Effect of RhoE knockdown on BeWo cell fusion and differentiation.

BeWo cells were transfected with 50nM RhoE siRNA or a non-silencing control then treated with 1mM dbcAMP and studied at the time points indicated. Cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (A). Cells were also fixed and immunostained for desmosomal protein and cell fusion quantified (B) as described in Materials & Methods. Cell lysates were also studied for expression of PLAP, β-hCG and β-actin by immunoblotting (C) with densitometric analysis normalised to β-actin expression (D). Results are presented as mean ± SEM for four separate experiments. *p<0.05 compared with non-silencing control (determined by ANOVA).

Figure 5. Effect of hypoxia on cAMP-induced RhoE expression.

BeWo cells were cultured at 20% O₂ (normoxia) or 1% O₂ (hypoxia) for 24h, then treated with 1mM dbcAMP at 20% or 1% O₂ for a further 24h. Cell lysates were made and RhoE and β-actin expression was assessed by immunoblotting (A) with densitometric analysis of RhoE expression normalised to β-actin expression (B). A representative blot from three separate experiments is shown.

Figure 6. RhoE is expressed in primary human villous cytotrophoblasts.

Lysates were prepared from BeWo cells or freshly isolated primary human villous cytotrophoblasts (three separate preparations) and assessed for RhoE and β-actin expression by immunoblotting.