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N. Sundarraj, E. Guerriero, J. Chen, R. Eberwine, P. Kinchington; Rho Family GTPases in Regulation of the Loss in Alpha 3(IV) Collagen Expression in Activated Corneal Keratocytes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4037. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Loss in the expression of alpha 3(IV) collagen is one of the stringent phenotypic changes associated with corneal stromal keratocyte activation during wound healing. The purpose of the present study was to determine whether the members of Rho family of small GTPases, RhoA, Cdc42 or Rac, regulate b-FGF induced loss in the expression of alpha 3(IV) collagen in an in vitro tissue culture model of keratocyte activation.
Rabbit corneal keratocytes were cultured in serum free medium and infected with replication-defective inducible tet-off adenoviral vector encoding cDNAs for dominant negative(DN) HA-tagged RhoA, Cdc42 or Rac, respectively. Two days after the induction of the expression of the cDNAs in the infected cells in the medium containing b-FGF and heparin sulfate (HS), the cells were analyzed immunocytochemically using mouse anti-HA and rat anti-alpha 3(IV) collagen antibodies, and by quantitative real time RT-PCR for mRNA levels
The expression of DN-RhoA in rabbit corneal keratocytes inhibited b-FGF/HS induced microfilament assembly into stress fiber and also prevented the loss in the expression of alpha 3(IV) collagen. Downregulation of Cdc42 resulting from the overexpression of DN-Cdc42 in b-FGF/HS activated cells resulted in altered cell morphology and microfilament organization and inhibition in the loss of alpha 3(IV) collagen. Downregulation of Rac activity resulting from the overexpression of DN-Rac in the infected keratocytes, cultured in SF medium without b-FGF/HS, promoted microfilament (stress fiber) assembly but did not inhibit alpha 3(IV) collagen expression. Downregulation of Rac did not prevent FGF-induced loss in alpha 3(IV) collagen expression.
RhoA and Cdc42, members of Rho family of small GTPases, regulate a phenotypic change, namely the loss in alpha 3(IV) collagen expression, associated with corneal keratocyte activation.
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