Glycogen Synthase Kinase-3 Is Essential for Keratinocyte Migration

Objectives: During wound healing, keratinocyte migration from wound edge is initiated by extension of lamellipodia into provisional matrix. Lamellipodia formation and keratinocyte migration depend on the activity of Rac1 GTPase. We have recently shown that protein-serine/threonine kinase inhibitor staurosporine and epidermal growth factor (EGF) induce similar long lamellipodia in cultured keratinocytes. Glycogen synthase kinase-3 (GSK-3) was required for lamellipodia extension and for staurosporine- or EGF-induced cell migration in scratch-wounded keratinocyte cultures. In the present study, we further examined the role of GSK-3 in keratinocyte migration. Methods: We studied the effect of GSK-3 inhibition on wound healing in vivo in a mouse model and in vitro in scratch-wounded keratinocyte cultures stimulated by staurosporine or EGF. The changes in expression and activation of key signaling molecules during cell migration were studied by immunoblotting. Extended lamellipodia were induced with staurosporine in cultured keratinocytes, and the effect of GSK-3 inhibitors on Rac1 localization was analyzed by immunostaining. Results: Inhibition of GSK-3 retarded re-epithelialization of experimental wounds in mice and blocked keratinocyte migration induced by staurosporine or EGF in culture. To investigate signaling pathways that are regulated by GSK-3 during keratinocyte migration, we first characterized the mechanisms of staurosporine-induced migration. Stimulation of wounded cultures by staurosporine induced expression and activation of heparin-binding EGF-like growth factor (HB-EGF), and the cell migration was totally blocked by function-blocking antibodies against the EGF receptor (EGFR) or HB-EGF. Staurosporine-induced keratinocyte migration depends, therefore, on endogenous HB-EGF signaling, and GSK-3 regulates keratinocyte migration downstream of EGFR. Treatment of cells with the GSK-3 inhibitors inhibited staurosporine-induced lamellipodia extension and prevented the accumulation of Rac1 at lamellipodia. Conclusions: GSK-3 is potentially a central regulatory molecule downstream of EGFR but upstream of Rac1 in epithelial cell migration during wound healing. Supported by CIHR (MOP-12589).