Coutifaris C, Kao L C, Sehdev H M, Chin U, Babalola G O, Blaschuk O W, Strauss J F
Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia 19104.
Development. 1991 Nov;113(3):767-77. doi: 10.1242/dev.113.3.767.
The morphologic and functional differentiation of human trophoblast cells culminates in the formation of the terminally differentiated multinucleated syncytial trophoblast. In culture, isolated mononuclear cytotrophoblasts aggregate and then fuse to form syncytia, recapitulating the in vivo process. In the present studies, we investigated the expression of the Ca(2+)-dependent cell adhesion molecule (CAM), E-cadherin, during the morphologic differentiation of trophoblastic cells. Cytotrophoblasts were isolated from human chorionic villi, and JEG-3 and BeWo choriocarcinoma cells, cytotrophoblastic cell lines which under standard culture conditions are not fusion competent, were obtained by dispersion of ongoing cultures. Cultures were terminated at timed intervals and E-cadherin was analyzed by immunocytochemistry and electron microscopy using specific antibodies. In addition, E-cadherin expression was investigated by western and northern blotting. During the aggregation of cytotrophoblasts, E-cadherin was localized on the cell surface at points of cell-cell contact and could not be demonstrated following cellular fusion. In contrast, it remained on the surface of aggregated JEG-3 and BeWo cells throughout the duration of culture. Western blot analysis revealed a time-dependent increase in E-cadherin (120 x 10(3) Mr) which coincided with maximal aggregate formation at 24 h in both normal cytotrophoblasts and JEG-3 cells. A marked reduction of E-cadherin in fusing cytotrophoblasts was subsequently observed as syncytial trophoblasts became the predominant cellular form in culture. In agreement with the immunohistochemical observations, there was no change in E-cadherin levels in the non-fusing JEG-3 cells. Northern blotting demonstrated a significant reduction in the 4.5 kb transcript in fusion-competent cells over the 96 h of culture. Exposure of the normally non-fusing BeWo cells to 1.5 mM 8-bromo cyclic AMP induced cellular fusion and syncytium formation. This process was accompanied by a disappearance of E-cadherin from the cell surface as assessed by immunocytochemistry and western blotting and a parallel reduction in the abundance of the E-cadherin mRNA. Immunoneutralization experiments using an antiserum directed against the extracellular domain of cadherins inhibited syncytium formation in normal trophoblasts compared to an antiserum against the E-cadherin cytoplasmic tail, which had no effect upon aggregation and fusion of these cells. We conclude that E-cadherin exists in a dynamic state in fusion-competent cytotrophoblasts and that down regulation of its gene expression coincides with cellular fusion. In addition, this process appears to be cyclic AMP-mediated in BeWo choriocarcinoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)
人滋养层细胞的形态和功能分化最终形成终末分化的多核合体滋养层。在培养中，分离的单核细胞滋养层细胞聚集然后融合形成合体滋养层，重现体内过程。在本研究中，我们调查了Ca(2+)依赖的细胞粘附分子（CAM）E-钙粘蛋白在滋养层细胞形态分化过程中的表达。从人绒毛膜绒毛中分离出细胞滋养层细胞，通过传代培养获得JEG-3和BeWo绒毛膜癌细胞系，这些细胞滋养层细胞系在标准培养条件下不具备融合能力。在不同时间间隔终止培养，使用特异性抗体通过免疫细胞化学和电子显微镜分析E-钙粘蛋白。此外，通过蛋白质印迹法和Northern印迹法研究E-钙粘蛋白的表达。在细胞滋养层细胞聚集过程中，E-钙粘蛋白定位于细胞间接触点的细胞表面，细胞融合后则无法检测到。相反，在整个培养过程中，它仍保留在聚集的JEG-3和BeWo细胞表面。蛋白质印迹分析显示E-钙粘蛋白（120×10(3) Mr）呈时间依赖性增加，这与正常细胞滋养层细胞和JEG-3细胞在24小时时最大聚集体形成相吻合。随后观察到，随着合体滋养层细胞成为培养中主要的细胞形式，正在融合的细胞滋养层细胞中E-钙粘蛋白显著减少。与免疫组织化学观察结果一致，未融合的JEG-3细胞中E-钙粘蛋白水平没有变化。Northern印迹显示，在96小时的培养过程中，有融合能力的细胞中4.5 kb转录本显著减少。将通常不融合的BeWo细胞暴露于1.5 mM 8-溴环磷酸腺苷可诱导细胞融合和合体滋养层形成。通过免疫细胞化学和蛋白质印迹评估，这一过程伴随着E-钙粘蛋白从细胞表面消失，同时E-钙粘蛋白mRNA丰度平行降低。与针对E-钙粘蛋白胞质尾的抗血清相比，使用针对钙粘蛋白胞外结构域的抗血清进行免疫中和实验可抑制正常滋养层细胞中的合体滋养层形成，而针对E-钙粘蛋白胞质尾的抗血清对这些细胞的聚集和融合没有影响。我们得出结论，E-钙粘蛋白在有融合能力的细胞滋养层细胞中处于动态状态，其基因表达的下调与细胞融合同时发生。此外，在BeWo绒毛膜癌细胞中，这一过程似乎是由环磷酸腺苷介导的。（摘要截短至400字）
