Abstract
PurposeEpithelial 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.MethodsRho 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.ResultsCdc42, 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.ConclusionRho 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.
Highlights
The cornea epithelium ensures appropriate light transmission and refraction for clear vision and protects the eye against environmental insults, physical trauma, and chemical injury [1,2,3]
Human corneal epithelial Large T antigen (HCET) cells were cultured in Dulbecco’s minimum essential medium (DMEM)/F12 supplemented with 5% fetal bovine serum (FBS) at 37°C in 5%
We used RT-PCR to exam the expression of human Rho proteins in immortalized HCET cells (Figure 1A) using HeLa cells as a positive control for a number of the primer pairs (Figure 1B)
Summary
The cornea epithelium ensures appropriate light transmission and refraction for clear vision and protects the eye against environmental insults, physical trauma, and chemical injury [1,2,3]. Corneal epithelial cells express a variety of proteins not found in other epithelial cells such as keratin 12 [4]. The differentiation of these cells is related to the migration process, since ‘progenitor’ cells migrate centripetally from the limbus towards the center. For these reasons, the molecular control of corneal epithelial cell migration deserves further study, especially for processes that are known to be cell type specific. Rho-family proteins are needed for cell adhesion complex assembly which requires actomysoin contractility [5,6]. The protein Cdc is needed to direct cell polarization in many cell types [5,8] and acts through its kinase effector myotonicdystrophy-kinase-related CDC42-binding kinase (MRCK) to control the actomyosin filaments in the lamellipodia [9,10]
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