title: Rho GTPases and regulation of cell migration and polarization in human corneal epithelial cells.
abstract: OBJECTIVE
  Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration.
  
  
  METHODS
  Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector.
  
  
  RESULTS
  Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding.
  
  
  CONCLUSIONS
  Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells.
authors.affiliation: Ocular Surface Research Group, Singapore Eye Research Institute, Singapore, Singapore, Singapore.
authors.forename: Aihua
authors.initials: A
authors.lastname: Hou
authors.forename: Li Xian
authors.initials: LX
authors.lastname: Toh
authors.forename: Kah Hui
authors.initials: KH
authors.lastname: Gan
authors.forename: Khee Jin Ryan
authors.initials: KJ
authors.lastname: Lee
authors.forename: Edward
authors.initials: E
authors.lastname: Manser
authors.forename: Louis
authors.initials: L
authors.lastname: Tong
mesh_terms: D002465:Cell Movement; D016764:Cell Polarity; D019573:Epithelium, Corneal / Q000166:cytology*; D020868:Gene Silencing; D006801:Humans; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D020764:cdc42 GTP-Binding Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D054462:p21-Activated Kinases / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; C116367:PAK4 protein, human; D054462:p21-Activated Kinases; C417188:RHOJ protein, human; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins