Antagonistic Cross–Talk Among Rho GTpases Mediated By FGF–2 Through PI 3–Kinase Regulates Cell Shape Change in Corneal Endothelial Cells

Abstract

Abstract: : Purpose: Cross–talk among Rho GTPase family members (Rho, Rac and Cdc42) plays important roles in modulating and coordinating downstream cellular responses, including actin cytoskeleton reorganization and cell shape change. Our studies suggest that corneal endothelial cells (CECs) in culture have serum–induced active Rho activity, and FGF–2 causes loss of stress fiber and focal adhesions by antagonizing Rho activity. But such alterations caused by FGF–2 were completely blocked by LY294002 treatment. To further understand cross–talk among Rho GTPases mediated by FGF–2, we studied activation pathway and functional relationships among these three proteins. Methods and Results:Antithetical results were obtained: Rac, but not Rho, was greatly activated when CECs were treated with FGF–2. However, these opposite Rac and Rho activity fluctuations were completely recovered by LY294002 treatment. These data indicated that FGF–2 activates Rac, but not Rho, through PI 3–kinase. To examine how FGF–2 modulated cell shape and actin cytoskeleton organization, CECs were transfected with either constitutive active (ca) or dominant negative (dn) Rho GTPases. Cells transfected with caRho, dnRac, or dnCdc42 maintain well–organized stress fiber and polygonal cell shape. But cells transfected with either dnRho or caRac acquired elongated cell shape in which actin cytoskeleton was reorganized as cortical actin mat. Of interest, caCdc42 caused pseudopodia formation in fibroblast–like cells, suggesting that Cdc42 alone can change cell shape to a migratory phenotype. According to co–immunoprecipitation and yeast two hybridization results, Rac associates with both Cdc42 and Rho under mediation of other protein predicted guanidine exchange factor (GEF). We could detect no interaction between Cdc42 and Rho. Conclusions:Taken together, our data suggest alteration of cell shape and actin cytoskeleton are mediated by interaction among all three Rho kinases in response to FGF–2 stimulation through the action of PI 3–kinase, and Cdc42 activation is a crucial event for CECs to acquire characteristic mesenchymal phenotypes.

Keywords: cornea: endothelium • signal transduction • wound healing