Trophoblast Cell Fusion and Differentiation Are Mediated by Both the Protein Kinase C and A Pathways
Figure 1
PMA induced cell fusion, DYSF expression, and activation of PKC in BeWo cells while 4αPMA was inactive.
(A) BeWo cells were treated with 0.25% DMSO (solvent control, CTRL) or with PMA (1, 10, 100, 1000 nM) or 4αPMA (1, 10, 100, 1000 nM) for 72 h. Cell lysates were generated and immunoblots were probed with anti-DYSF. Each lane received equal amounts of protein and detection of GAPDH served as an additional loading control. Up-regulation of DYSF occurred with all concentrations of PMA tested. On the other hand, none of the concentrations of 4αPMA tested induced DYSF expression. (B) Immunofluorescence analysis of BeWo cells treated with 0.25% DMSO (controls), 10 nM PMA, or 10 nM 4αPMA for 72 h. The cells were then fixed and subsequently double-labeled for detection of DYSF (red) and E-cadherin (green). Nuclei were labeled with DAPI. While there can be a low level of spontaneous fusion in control cells (in our hands this ranges from about 4 to 9%), most cells are not fused and have at their borders intact E-cadherin labeling. Moreover, DYSF labeling was not detectable in non-fused BeWo cells. However, treatment of BeWo cells with 10 nM PMA for 72 h led to increased levels of cell fusion as indicated by the breakdown of E-cadherin labeling and the expression of DYSF in fused cells. When BeWo cells were treated with 10 nM 4αPMA for 72 h there was no detectable increase in cell fusion or DYSF expression. Arrows indicate areas enlarged and placed in insets. Bar = 50 µm. (C, D) Activation of PKC with PMA but not with 4αPMA. The electrophoretic mobility of BeWo cell PKC was monitored using a phospho-PKC (pan) β II (C) and phospho-PKC δ(Ser 643) (D) antibodies. (C) In control cells (DMSO treated), two bands were detected with this antibody indicating a basal level of PKC (pan) β II phosphorylation. Following treatment with PMA, there was a change in the mobility of the lower band over the time course of this experiment (15, 30, 60, and 120 min). This change in mobility was not observed following 4αPMA treatment. The distances between the top and bottom bands were measured on the original x-ray films; these distances are indicated in the measured distances (mm). Phospho-PKC (pan) antibody detects both α and β isoforms, thus the upper band was labeled α/β. (D) In control cells (DMSO treated), this antibody detected a basal level of PKC δ phosphorylation. Following treatment with PMA, there was a change in the mobility of this band over the time course of this experiment (15, 30, 60, and 120 min). This change in mobility was not observed following 4αPMA treatment. (E) Phospho-PKC (pan) β II was translocated from soluble to particulate fraction with PMA treatment. In control cells (DMSO treated), two bands were detected in soluble fraction and only lower band was in particulate fraction with phospho-PKC (pan) βII. Following treatment with 100 nM PMA for 1 h, phospho- PKC (pan) β II was translocated to particulate fraction and there was a change in the mobility of the lower band as described in Figure 1C. Results are representative of three independent experiments.