Wnt4 increases the thickness of the epidermis in burn wounds by activating canonical Wnt signalling and decreasing the cell junctions between epidermal cells

Abstract

Background Burn wound healing is a complex process and the role of Wnt ligands varies in this process. Whether and how Wnt4 functions in burn wound healing is not well understood. In this study, we aim to reveal the effects and potential mechanisms of Wnt4 in burn wound healing. Methods First, the expression of Wnt4 during burn wound healing was determined by immunofluorescence, Western blotting and qPCR. Then, Wnt4 was overexpressed in burn wounds. The healing rate and healing quality were analysed by gross photography and haematoxyline and eosin staining. Collagen secretion was observed by Masson staining. Vessel formation and fibroblast distribution were observed by immunostaining. Next, Wnt4 was knocked down in HaCaT cells. The migration of HaCaT cells was analysed by scratch healing and transwell assays. Next, the expression of β-catenin was detected by Western blotting and immunofluorescence. The binding of Frizzled2 and Wnt4 was detected by coimmunoprecipitation and immunofluorescence. Finally, the molecular changes induced by Wnt4 were analysed by RNA sequencing, immunofluorescence, Western blotting and qPCR in HaCaT cells and burn wound healing tissues. Results The expression of Wnt4 was enhanced in burn wound skin. Overexpression of Wnt4 in burn wound skin increased the thickness of epidermis. Collagen secretion, vessel formation and fibroblast distribution were not significantly impacted by Wnt4 overexpression. When Wnt4 was knocked down in HaCaT cells, the ratio of proliferating cells decreased, the ratio of apoptotic cells increased and the ratio of the healing area in the scratch healing assay to the number of migrated cells in the transwell assay decreased. The nuclear translocation of β-catenin decreased in shRNA of Wnt4 mediated by lentivirus-treated HaCaT cells and increased in Wnt4-overexpressing epidermal cells. RNA-sequencing analysis revealed that cell junction-related signalling pathways were significantly impacted by Wnt4 knockdown. The expression of the cell junction proteins was decreased by the overexpression of Wnt4. Conclusions Wnt4 promoted the migration of epidermal cells. Overexpression of Wnt4 increased the thickness of the burn wound. A potential mechanism for this effect is that Wnt4 binds with Frizzled2 and increases the nuclear translocation of β-catenin, thus activating the canonical Wnt signalling pathway and decreasing the cell junction between epidermal cells.