Role of Small GTPase Rho in Regulating Corneal Epithelial Wound Healing

Abstract

To determine the role of small GTPase Rho and its relation with epidermal growth factor receptor (EGFR) in mediating corneal epithelial wound healing. Rho activity in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, and primary HCECs was assessed by pull-down assay followed by Western blotting. Rho functions were inhibited with specific inhibitor exoenzyme C3 (C3) and confirmed by knockdown with small interference RNA (siRNA) transfection. Effects of Rho inhibition on wound healing were determined in porcine corneal organ culture and HCEC scratch wound models. Effects of C3 on cell proliferation and focal adhesion formation were determined by BrdU incorporation assay and immunocytochemistry, respectively. Wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) induced rapid and strong RhoA activation. HB-EGF-, but not wounding-, enhanced RhoA activity was sensitive to EGFR inhibition. In corneal organ and cell culture models, C3 attenuated spontaneous and HB-EGF-induced wound closures, confirmed by delayed wound healing in cells transfected with RhoA siRNA. C3 also retarded spontaneous wound healing in the presence of hydroxyurea, a cell cycle blocker. C3 significantly reduced the number of BrdU-positive cells near the leading edge. Treatment with C3 resulted in the disruption of the cortical actin cytoskeleton and in the disappearance of paxillin-containing focal adhesion and lamellipodia. Wounding induces RhoA activation through an EGFR-independent pathway. Rho activity is required for modulating cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding.