Abstract
Purpose:
Wnt5a activates β-catenin-independent pathways for regulation of cellular functions that play crucial roles in wound healing process in cornea including cell migration and polarity. Elucidation of Wnt5a signaling may help identify potential therapeutic targets for enhanced corneal wound healing; however, Wnt5a signaling in corneal endothelial cells (CEC) has not been well characterized.
Methods:
Expression and/or activation of Frizzled-5, Cdc42, Rac1 were analyzed by immunoblotting. Scratch-induced directional migration assay was employed to measure migratory rates. Activation of Cdc42 and Rac1 were determined by GTP pull-down assay. RhoA activity was measured using RhoA specific G-LISA assay kit.
Results:
Stimulation of human CECs with Wnt5a alone resulted in 50% enhancement in cell migration as measured by scratch induced migration assay and this effect could be completely blocked by ML141 (Cdc42 inhibitor). Co-treatment of both Wnt5a and Y27632 (inhibitor of Rho-associated kinase) resulted in a 62% enhancement of migration. The synergistic effects of Wnt5a and Y27632 on human CECs migration were partially blocked by ML141 and this, in turn, was completely abolished by treatment with both ML141 and RhoA activator. Under these conditions, activation of both Cdc42 and Rac1 and inactivation of RhoA were observed in the Wnt5a treated cells. We further confirmed that activated Cdc42 negatively regulates RhoA activity and this regulation plays important role in endothelial migration.
Conclusions:
These findings suggest that Wnt5A mediated migration in human CEC is mediated by activation of Cdc42 and inactivation of RhoA.
Keywords: 481 cornea: endothelium •
765 wound healing •
714 signal transduction