Adhesiveness of human uterine epithelial RL95-2 cells to trophoblast: Rho protein regulation

Embryo implantation involves adhesion of trophoblast cells to the epithelial lining of the endometrium. Using an in-vitro model to simulate this initial interaction, we previously reported that attachment of human trophoblast-like JAR spheroids to human uterine epithelial RL95-2 cells provokes a Ca²⁺ influx in RL95-2 cells depending on apically localized integrin receptors. Here, we demonstrate that adhesiveness of RL95-2 cells for JAR spheroids, measured by a centrifugal force-based adhesion assay, is dependent on Rho GTPases, most likely RhoA. Cellular expression and distribution of RhoA were studied by fluorescence confocal microscopy, focusing on the localization of RhoA and F-actin within the adhesion sites between JAR and RL95-2 cells. Contact areas contained high amounts of RhoA and F-actin fibres near the plasma membrane. To determine whether Rho GTPases may influence JAR cell binding, we treated RL95-2 cells with Clostridium difficile toxin A, which specifically inactivates Rho GTPases. Toxin A treatment changed the subcellular distribution of endogenous RhoA in RL95-2 cells and altered RhoA and F-actin colocalization. Adhesion of JAR spheroids to RL95-2 cells treated with toxin A was largely suppressed. These data indicate that Rho GTPases, most likely RhoA, play an important role in uterine epithelial RL95-2 cells for trophoblast binding, and suggest that RhoA may be involved in local signalling cascades during early embryo implantation in vivo.