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FGF-2-Induced Wound Healing in Corneal Endothelial Cells Requires Cdc42 Activation and Rho Inactivation through the Phosphatidylinositol 3-Kinase Pathway

¹From the Doheny Eye Institute and the ²Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California.

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 µm/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 µm/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 µm/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.

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