Purpose: Acquisition of elongated cells with pseudopodia is observed when corneal endothelial cells (CECs) are simultaneously treated with basic fibroblast growth factor (FGF)-2 and RhoA inhibitors. This study was designed to determine whether these phenotypes are migratory and whether Cdc42 activation and RhoA inactivation are involved in cell migration.
Methods: A scratch-induced directional migration assay was used to measure migratory rates. Activation of Cdc42 was determined by GTP pull-down assay. Transfection was performed using constitutively active (ca) or dominant negative (dn) Rho guanosine triphosphatase (GTPase) vectors.
Results: Stimulation with basic FGF-2 alone resulted in a 43% recovery of the wound area, whereas CECs treated with FGF-2 and Y27632 (inhibitor of Rho-associated kinase) achieved an 84% recovery of the wound area with a fast migratory speed (0.72 microm/min). The synergistic effects of FGF-2 and Y27632 were completely blocked by LY294002 (PI 3-kinase inhibitor). Under these conditions, activation of PI 3-kinase and Cdc42 were observed in the migratory cells. The involvement of activated Cdc42 and inactivated Rho in endothelial migration was determined by transfecting CECs with ca- or dnRho GTPase vectors. A high migratory rate (0.52 microm/min) was seen in CECs expressing caCdc42, whereas endothelial migration was completely inhibited in CECs expressing caRho. When cells expressing caCdc42 were treated with FGF-2, migration reached the maximum rate (0.69 microm/min), similar to that observed in cells treated with FGF-2 and Y27632.
Conclusions: These findings suggest that endothelial migration is induced by activated Cdc42 and inactivated Rho via PI 3-kinase after FGF-2 stimulation and that Cdc42 activation is crucial for CECs to acquire the characteristic migratory phenotypes.