Role of small GTPase Rho in regulating corneal epithelial wound healing.
OBJECTIVE
To determine the role of small GTPase Rho and its relation with epidermal growth factor receptor (EGFR) in mediating corneal epithelial wound healing.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Yin
Jia
J
Department of Ophthalmology, Kresge Eye Institute, Wayne State UniversitySchool of Medicine, Detroit, Michigan 48201, USA.
Lu
Jinshuang
J
Yu
Fu-Shin X
FS
D036002:ADP Ribose Transferases / Q000235:genetics; D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000235:genetics; D001973:Bromodeoxyuridine; D002460:Cell Line; D002465:Cell Movement; D049109:Cell Proliferation; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D009924:Organ Culture Techniques; D051419:Paxillin / Q000378:metabolism; D025941:Protein Interaction Mapping; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014162:Transfection; D014945:Wound Healing / Q000502:physiology*; D020741:rho GTP-Binding Proteins / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000199:Actins; D051419:Paxillin; D034741:RNA, Small Interfering; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D066246:ErbB Receptors; D001905:Botulinum Toxins; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D001973:Bromodeoxyuridine