Effect of Flightless I Expression on Epidermal Stem Cell Niche During Wound Repair

Objective: Activation of epidermal stem cells (EpSCs) from their quiescent niche is an integral component of wound reepithelialization and involves Wnt/beta-catenin (beta-Cat) signaling and remodeling of the actin cytoskeleton. The aim of this study was to investigate the effect of Flightless I (Flii), a cytoskeletal protein and inhibitor of wound healing, on EpSC activation during wound repair. Approach: Genetically modified Flii mice (Flii knockdown: Flii(+/-), wild type: WT, Flii overexpressing: Flii(Tg/Tg)) received two incisional wounds along the lateral axis of the dorsal skin. Indicators of EpSC activation (epidermal growth factor receptor 1 [EGFR1], leucine-rich repeats and immunoglobulin-like domains-1 [Lrig1], K14), Wnt/beta-Cat signaling (Lgr6, Flap2, beta-Cat, and axis inhibition protein 2 [Axin2]), and cell proliferation (proliferating cell nuclear antigen [PCNA]) were assessed using immunohistochemistry. beta-Cat stabilization was examined using western blotting with cell cycling and differentiation of isolated CD34(+)ITGA6(high) EpSCs examined using real time-quantitative polymerase chain reaction after treatment with wound-conditioned media. Results: Flii(+/-) led to increased numbers of activated EpSCs expressing PCNA, elevated EGFR1, and decreased Lrig1. EpSCs in Flii(+/-) hair follicle niches adjacent to the wounds also showed expression of Wnt-activation markers including increased beta-Cat and Lgr6, and decreased Axin2. EpSCs (CD34(+)ITGA6(high)) isolated from Flii(+/-) unwounded skin showed elevated expression of cell-cycling genes including Delta Np63, filaggrin (Fila), involucrin (Invo), cyclin D1 (Ccnd1), and cell-division cycle protein-20 (Cdc20); and elevated Delta Np63 and Invo after treatment with wound-conditioned media compared with WT and Flii(Tg/Tg) counterparts. Innovation: Flii was identified as an inhibitor of EpSC activation that may explain its negative effects on wound reepithelialization. Conclusion: Flii may inhibit EpSC activation by interrupting Wnt/beta-Cat signaling. Strategies that reduce Flii may increase activation of EpSCs and promote reepithelialization of wounds.