P311 induces the transdifferentiation of epidermal stem cells to myofibroblast-like cells by stimulating transforming growth factor β1 expression.

BACKGROUND: Epithelial to mesenchymal transition, especially to myofibroblasts, plays an important role in wound healing, fibrosis, and carcinogenesis. Epidermal stem cells (EpSCs) are responsible for epidermal renewal and wound re-epithelialization. However, it remains unclear whether and how EpSCs transdifferentiate into myofibroblasts or myofibroblast-like cells (MFLCs). Here, we provide the first evidence showing that P311 induces EpSC to MFLC transdifferentiation (EpMyT) via TGFβ1/Smad signaling.

METHODS: Wound healing and mesenchymal features were observed in the P311 KO and P311 WT mouse model of superficial second-degree burns. After the primary human or mouse EpSCs were forced to highly express P311 using an adenoviral vector, EpMyT was observed by immunofluorescence, real-time PCR, and western blot. The activity of TGFβ1 and Smad2/3 in EpSCs with different P311 levels was observed by western blot. The TβRI/II inhibitor LY2109761 and Smad3 siRNA were applied to block the EpMyT in P311-overexpressing EpSCs and exogenous TGFβ1 was to restore the EpMyT in P311 KO EpSCs. Furthermore, the mechanism of P311 regulating TGFβ1 was investigated by bisulfite sequencing PCR, luciferase activity assay, and real-time PCR.

RESULTS: P311 KO mouse wounds showed delayed re-epithelialization and reduced mesenchymal features. The human or mouse EpSCs with overexpressed P311 exhibited fusiform morphological changes, upregulated expression of myofibroblast markers (α-SMA and vimentin), and downregulated expression of EpSC markers (β1-integrin and E-cadherin). P311-expressing EpSCs showed decreased TGFβ1 mRNA and increased TGFβ1 protein, TβRI/II mRNA, and activated Smad2/3. Moreover, LY2109761 and Smad3 siRNA reversed P311-induced EpMyT. Under the stimulation of exogenous TGFβ1, the phosphorylation of Smad2 and Smad3 in P311 KO EpSCs was significantly lower than that in P311 WT EpSCs and the EpMyT in P311 KO EpSCs was restored. Furthermore, P311 enhanced the methylation of TGFβ1 promoter and increased activities of TGFβ1 5'/3' untranslated regions (UTRs) to stimulate TGFβ1 expression. P311(+)α-SMA(+) cells and P311(+)vimentin(+) cells were observed in the epidermis of human burn wounds. Also, P311 was upregulated by IL-1β, IL-6, TNFα, and hypoxia.

CONCLUSIONS: P311 is a novel TGFβ1/Smad signaling-mediated regulator of transdifferentiation in EpSCs during cutaneous wound healing. Furthermore, P311 might stimulate TGFβ1 expression by promoting TGFβ1 promoter methylation and by activating the TGFβ1 5'/3' UTR.