Abstract
Purpose: :
Rho, Rac and Cdc42 GTPases regulate organization of actin cytoskeleton and therefore are involved in the regulation of cell adhesion, migration and contraction of activated stromal cells during corneal wound healing. We had previously shown that RhoA regulates several TGF-beta1 and FGF-2 -mediated phenotypic characteristics of the activated stromal cells. In the present study we evaluated the dowonstream effects of Rac in activated keratocytes.
Methods: :
: Keratocytes isolated from rabbit corneal stroma, plated in a serum free (SF) medium, were treated with FGF-2/heparin or TGF-beta1 in the presence or absence of Rac inhibitor (NSC23766). In separate experiments keratocytes were infected with replication-defective inducible Tet-off adenoviral vectors encoding cDNAs for dominant negative (DN) or constitutively active (CA) HA-tagged Rac and the expression of the recombinant proteins was induced during the activation of keratocytes. Specific phenotypic changes were analyzed by immunocytochemistry and western blotting, and the relative abundance of specific mRNAs was estimated by quantitative RT-PCR
Results: :
Immunocytochemical and western blot analyses indicated that Rac inhibition decreased tenascin-C levels in keratocytes activated with TGF-beta1 and FGF-2 suggesting that Rac promotes tenascin-C expression. This was confirmed by demonstrating increased expression of Tenascin-C in adenoviral infected cells expressing CA-Rac, and decrease expression in the cells expressing DN-Rac. Rac inhibition during TGF-beta1 and FGF-2 activation inhibited G1/S progression as evident from the growth curve and reduced number of Ki67 (a proliferative nuclear antigen) positive cells. The levels of alpha-smooth muscle actin mRNA and protein were upregulated when Rac was inhibited during the activation of keratocytes to myofibroblasts.
Conclusions: :
Upregulation of tenascin-C and downregulation of RhoA and alpha-smooth muscle actin (which promote contractile phenotype) by Rac signaling in activated stromal cells may be a mechanism to promote rapid migration of the keratocytes to the wounded region.
Keywords: cornea: basic science • cornea: stroma and keratocytes • wound healing