Abstract

The implantation stage of pregnancy is characterized by invasion of cytotrophoblast cells into the endometrial cells of the uterus. The invasive potential of cytotrophoblasts cells is influenced by changes in integrin-Focal adhesion kinase (FAK) signalling complexes. Previous studies have suggested Epithelial Membrane Protein 2 (EMP2), a GAS3 family tetraspan protein, may modulate the activity of integrin-FAK complexes. In this study, we assessed how changes in EMP2 expression in cytotrophoblasts cells influence integrin-FAK expression, and addressed the effect this has on the invasive properties of the cytotrophoblast cells. To test our hypothesis that EMP2 expression influences cytotrophoblast cell invasion, JAR human choriocarcinoma cytotrophoblast cell lines were generated to express varying levels of EMP2. EMP2 expression in these cells was validated by Western blot. The cell lines were subsequently examined for expression of FAK, α2- integrin, α6- integrin and β3- integrin using flow cytometry and Western blot. EMP2 expression in human cytotrophoblast cells was also detected using immunohistochemistry. Cell adhesion assays using the extracellular matrix proteins Laminin, Fibronectin and Collagen, were employed to elucidate potential differences in adhesion in EMP2-modulated JAR cells. Finally, transwell invasion assays were performed to determine whether changes in EMP2 expression could modulate the invasive ability of JAR cytotrophoblast cells. EMP2 expression was detected in JAR and human cytotrophoblast cells. By Western blot, we demonstrate that increasing EMP2 expression increases FAK expression. Flow cytometry shows that an increase EMP2 expression results in an increase in β3- integrin, and reduced EMP2 levels decrease β3- integrin expression. Interestingly, increasing EMP2 reduced the expression of α6- integrin and a decrease in EMP2 expression increased the expression of α6- integrin. Changes in EMP2 however did not alter α2- integrin expression. Overexpression of EMP2 increased cell adhesion onto extracellular matrix proteins, namely Fibronectin. Finally, the invasion properties of JAR cytotrophoblast cells were enhanced by EMP2 overexpression, and a reduction in EMP2 levels resulted in a decrease in cytotrophoblast invasion. These findings suggest a physiologic role for EMP2 in trophoblast cell biology during implantation. This may be relevant to diseases associated with abnormal invasion of cytotrophoblast cells into the endometrium, such as Preeclampsia.