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

Abstract

BackgroundEpithelial 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.MethodsWound 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.ResultsP311 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.ConclusionsP311 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0421-1) contains supplementary material, which is available to authorized users.

P311 Deficiency Leads to Attenuated Angiogenesis in Cutaneous Wound Healing

“…Microvascular endothelial cells are the principal parenchymal cells participating in wound angiogenesis (Tonnesen et al, 2000 ) that involves a phenotypic alteration of endothelial cells, directed migration, and various mitogenic stimuli (Li et al, 2003 ). Our previous studies demonstrated that P311 was a crucial factor in wound healing (Li et al, 2016 ; Yao et al, 2017 ). However, the possible role of P311 in cutaneous wounds angiogenesis was still unknown.…”

“…Following firstly identified to expressed in the embryonic brain in mice by Studler (Studler et al, 1993 ), P311 was found to express in almost all kinds of cells, like motoneurons (Fujitani et al, 2004 ), kidney tubular epithelial cells (Yao et al, 2015 ), glioblastomas (McDonough et al, 2005 ), epidermal stem cells (Li et al, 2016 ), fibroblast (Tan et al, 2010 ; Cheng et al, 2017 ), smooth muscle cells (Badri et al, 2013 ), which implied wide biological functions of P311. Actually, P311 has been shown to promote the nerve (Fujitani et al, 2004 ) and lung regeneration (Zhao et al, 2006 ), glioma invasion (McDonough et al, 2005 ), and to induce myofibroblast differentiation (Pan et al, 2002 ; Tan et al, 2010 ; Li et al, 2016 ), cell migration (McDonough et al, 2005 ; Yao et al, 2017 ). Moreover, a decreased vascular smooth muscle cell contractility, hypotonic blood vessels, and vascular hypotension was found in P311 knockout mice compared with the wild type (Badri et al, 2013 ), and even a changed behavioral responses in learning and memory (Taylor et al, 2008 ).…”

“…More recently, we found that P311 could increased the activity of Rho A and Rac1 (Yao et al, 2017 ), which are critical signal transducers for inducing the formation of lamellipodia, filopodia, invadopodia, and blebs during cell migration (Sadok and Marshall, 2014 ). In addition, P311 was identified as a inducer of EpMyT $(Epidermal stem cell transdifferentiate into myofibroblasts) through TGFβ1/Smad signaling (Li et al, 2016 ).…”

FG-4592 Accelerates Cutaneous Wound Healing by Epidermal Stem Cell Activation via HIF-1α Stabilization

“…Mouse EpSCs were isolated from newborn (aged 0–2 days) mouse skin, according to a method described previously [1]. Briefly, the mouse skin tissues were incubated in 0.5% dispase II in 0.9% NaCl solution overnight at 4°C.…”

“…Cutaneous wounds resulting from conditions such as trauma, burns, surgery, and diabetes are common clinical problems [1]. However, how wound healing is effectively promoted, as well as how chronic refractory wounds are treated, is not well understood.…”