The Role Of P311 In The Transdifferentiation Of Epidermal Stem Cells To Myofibroblast-like Cells And Its Mechanism

BackgroundCutaneous wounds,caused by burns,trauma,surgery,and diabetes,are common clinical problems.How to promote wound healing without scar formation is the key to wound treatment.However,the clinical treatment effect is limited because of the unclear mechanism of wound healing and scar formation.Myofibroblasts play crucial roles during wound healing,but the wound healing process can be compromised if the myofibroblast function is not properly regulated,which can,in turn,result in excessive scarring.Conventionally,it is thought that myofibroblasts in skin wounds originated from fibroblasts in local wounds,fibrocytes in bone marrow and peripheral blood,vascular pericytes and vascular smooth muscle cells.Epidermal cells are classically assumed to regulate the fibroblast to myofibroblast differentiation by secreting transforming growth factor ?1(TGF?1),basic fibroblast growth factor(bFGF),interleukin-1?(IL-1?),tumor necrosis factor?(TNF?)and other humoral factors.In this study,we speculated that epidermal stem cells(EpSCs)could transdifferentiate into myofibroblasts or myofibroblast-like cells(MFLCs)based on the following evidence.First,the process of wound re-epithelialization resembles epithelial-mesenchymal transition(EMT).EpSCs in the periwound gradually acquired the mesenchymal features,such as loss of cell-cell junctions and apical-basal polarity,basement membrane degradation and cytoskeleton reorganization,and obtained higher motility during re-epithelialization.Second,epidermal keratinocytes,which EpSCs could differentiate into,have the potential of transdifferentiating into myofibroblasts.Several studies showed that keratinocytes in the basal epidermis could directly transdifferentiate into fibroblasts or myofibroblasts when exposed to the TGF?1,TNF?,IL-1? and fetal bovine serum.Human keratinocytes could transdifferentiate into adipocytes,smooth muscle cells and neural cells,and even undergo EMT during serial passage.Third,studies showed that epidermal cells in the periwound might be the main force source of early wound contraction,which might even occur earlier than myofibroblasts in the periwound and granulation tissue.Fourth,the cytokine and hypoxia in the wound could also induce EMT.P311,also known as neuronal regeneration related protein(NREP),Pentylenetetrazol 17(PTZ17)and neuronal protein 3.1,codes an 8-kDa cytoplasmic protein with unknown functions.P311 protein is easily degraded by the ubiquitin proteasome and matrix metalloproteinases(MMPs),and has a half-life of about 5 minutes because it contains three PEST domains and several lysine residuals.Current studies have shown that P311 participates in the nerve regeneration,alveolar development,tumor cell invasion,blood pressure homeostasis,wound healing and hypertrophic scar.Our and others' results showed that P311 was highly expressed in the EpSCs in the wound neo-epidermis,myofibroblasts in the granulation tissue,and hypertrophic scars.Furthermore,the wound healing of skin defection in the P311 knockout(KO)mice was slowed down and P311 could promote the differentiation of fibroblasts to myofibroblasts and the migration of EpSCs.Therefore,P311 played an important role in regulating myofibroblast formation and EpSC biology.P311 is also a crucial regulator of TGF?1/Smad pathway which is a key modulator of wound healing and EMT,because P311 could regulate the TGF?1 expression by regulating its auto-induction and translation.Based on the mentioned above,we hypothesized that P311 could regulate the EpSC to MFLC transdifferentiation(EpMyT)via TGF?1/Smad signaling during wound healing.This study was performed from the following three aspects: the specific role of P311 in EpMyT,the role of TGF?1/Smad signaling in P311-induced EpMyT,and the possible molecular mechanism of P311's regulation on TGF?1 expression.Methods1.The role of P311 in the transdifferentiation of EpSCs to MFLCs1.1 The in vitro role of P311 in the transdifferentiation of primary human and mouse EpSCs to MFLCs.The primary human and mouse EpSCs were isolated from human foreskins and neonatal mice by two-step digestion and differential collagen IV adherence,and cultured in serum-free medium,KSFM.The Ep SCs were then characterized by flow cytometry.EpSCs were forced to highly express P311 by using an adenovirus vector and the transfection efficiency was confirmed by Immunofluorescence(IF),flow cytometry and realtime PCR.The expression of epithelial markers(e.g.E-cadherin and ?1 integrin)and myofibroblast markers(e.g.Vimentin and ?-SMA)were detected by IF,realtime PCR and Western blot.Furthermore,the Collagen I and MMP2 protein level were detected by Western blot.The collagen gel contraction assay and in vitro scratch wound assay were performed to analyze the contractility and motility of EpSCs.1.2 The in vivo role of P311 in EpMyT during mouse and human burn wound healings.A thermal apparatus was used to create superficial partial-thickness burns in P311 KO and P311 WT mice.The wound healing and re-epithelialization were assessed by macroscopy and HE staining.The immunohistochemistry(IHC)was performed to detect the expression pattern of Vimentin and ?-SMA in the epidermis.The realtime PCR and Western blot were performed to detect the EpMyT-related markers in mouse burn wounds.The expression of P311,Vimentin and ?-SMA,and their co-localization in human burn wounds were also observed by IHC,serial sectioning,and IF.1.3 The effect of the wound microenvironment on P311 and ?-SMA expression in EpSCs.EpSCs were treated with IL1?,TNF?,IL6 and hypoxia,and then the P311 and ?-SMA mRNA level were detected by realtime PCR.2.P311 could regulate EpMyT via TGF?1/Smad pathway.2.1 The regulation of P311 on the TGF?1/Smad signaling.The expression of TGF?1 mRNA and protein and the secretion of TGF?1 protein in P311 KO EpSCs and P311 expressing EpSCs were detected by using realtime PCR,Western blot and ELISA,respectively.Western blot was performed to detect the effect of P311 on the expression and phosphorylation of Smad2 and Smad3 protein.2.2 The effect of T?RI/II inhibitor(LY2109761),Smad3 siRNA,and exogenous TGF?1 on the P311-induced EpMyT.The inhibitory effect of LY2109761 on the P311-induced EpMyT was confirmed by IF and Western blot,and the similar effect of Smad3 siRNA was confirmed by Western blot.The influence of exogenous TGF?1 in the transdifferentiation of P311 KO mouse EpSCs and P311 WT mouse EpSCs was detected by Western blot.3.The possible molecular mechanism of P311's regulation on the TGF?1 expression.3.1 Major stages of P311's regulation on the TGF?1 expression.The TGF?1 promoter activity assay,the bisulfite sequencing PCR(BSP),the Actinomycin D stimulation plus realtime PCR,and the untranslated region(UTR)binding assay were performed to analyze the effect of P311 on the TGF?1 promoter activity,the DNA methylation in TGF?1 promoter,the TGF?1 mRNA stability,and the TGF?1 UTR activities,respectively.3.2 The possible mechanism of P311's regulation on TGF?1 UTR activities.The information of P311 and TGF?1 protein and mRNA in all species was downloaded from the Genebank.The multiple alignment,homology analysis,and evolutionary tree analysis were performed by DNAMAN software.The length and GC content in different regions of TGF?1 mRNA were obtained by DNASTAR software.The possibility of human P311 protein binding with regulatory elements in human TGF?1 5'-UTR and 3'-UTR were predicted by the RPISeq and LncPro online tools.The possible RNA binding sites of P311 protein were predicted by the BINDN+,RNABINDRPLUS and SNBRFinder online tools.3.3 P311 might indirectly regulate the TGF?1 protein expression by e IF6.Western blot was performed to detect the effect of different P311 levels on the eIF6 expression and the influence of e IF6 knockdown and silence on TGF?1 protein expression.Results1.The in vitro role of P311 in the transdifferentiation of primary human and mouse EpSCs to MFLCs.1.1 The successful culture and transfection of primary human and mouse EpSCs.Cultured EpSCs grew in colonies and exhibited cubic shapes.And over 95% of the cultured cells were CD71—CD49f+ EpSCs.At 48 hours after transfection,over 95% of cells in P311 group and vector group expressed GFP,and the P311 mRNA expression in the P311 group was obviously elevated.1.2 P311 induces the transdifferentiation of mouse EpSCs to myofibroblast-like cells,not myofibroblasts,in vitro.After 72 hours,EpSCs began to show increased cell volumes,and a fusiform and well-spread morphology.After P311 induction,the percentage of ?-SMA+ cells and Vimentin+ cells were increased,but the percentage of E-cadherin+ cells were decreased.P311 also induced the reorganization of the cytoskeleton from cortical organizations to stress fibers.The ?-SMA mRNA and protein levels in P311-expressing EpSCs were 1.56-fold(P<0.01)and 2.69-fold(P<0.05)higher than those in control EpSCs,respectively.And the vimentin protein level in the P311-expressing EpSCs was 1.72-fold higher than that in control cells(P<0.05).The protein levels of E-cadherin and ?1-integrin were 32% and 68% lower,respectively,than those in control cells(P<0.05).Furthermore,P311 promoted EpSC migration(migration index: 6.29-fold difference,P<0.01),induced collagen I and MMP2 expression(11.21-fold and 1.45-fold difference,P<0.05),but had no significant effect on contraction.1.3 P311 induces the transdifferentiation of human EpSCs to MFLCs in vitro.After P311 induction,the percentage of ?-SMA+ cells were increased(35% vs 3%,P<0.01),but the percentage of E-cadherin+ cells were decreased(22% vs 63%,P<0.01).Meanwhile,the ?-SMA protein level in P311-expressing EpSCs was 2.09-fold higher than that in control EpSCs(P<0.05).The protein levels of E-cadherin and ?1-integrin were 57.68% and 32.87% lower,respectively,than those in control cells(P<0.05).2.The in vivo role of P311 in EpMyT during mouse and human burn wound healings.2.1 Loss of P311 leads to delayed wound healing kinetics.The wound areas in P311 KO mice were significantly higher than those in P311 WT mice on days 2 and days 4 to 7 after injury.Wound re-epithelialization was clearly slower in the P311 KO mice than in the P311 WT mice on days 4 and days 7 post-injury.On days 7 post-injury,the average epidermal width in the P311 KO mice was significantly larger than that in the P311 WT mice(9.87 mm vs 6.82 mm,P<0.05),and the average neo-epidermal length in the P311 KO mice was significantly shorter than that in the P311 WT mice(1.48 mm vs 2.14 mm,P<0.05).2.2 Mesenchymal features are reduced in P311 KO mouse burn wounds.IHC showed that the expression of vimentin in the neo-epidermis of P311 KO mice was lower than that in P311 WT mice,but no specific ?-SMA expression was observed in epidermis.The mRNA levels of vimentin,TGF?1,Twist1 and Snail2 were significantly lower in burn wounds in P311 KO mice than those in P311 WT mice.The expression of ?1-integrin protein was significantly higher in burn wounds in the P311 KO mice than that in the P311 WT mice.The protein expression levels of vimentin and active and LAP-bound TGF?1 in P311 KO normal skins or burn wounds were significantly lower than those in P311 WT normal skins or burn wounds.2.3 The possible role of P311 in EpMyT during human burn wound healings.The number of epidermal cells which expressed vimentin,?-SMA or P311 was higher in human burn wounds than in normal skins.Moreover,P311+?-SMA+ cells and P311+vimentin+ cells were observed in the epidermis of human burn wounds by double staining.3.The effect of the wound microenvironment on P311 and ?-SMA mRNA expression in EpSCs.IL1? and TNF? increased P311 mRNA expression by more than 40-fold(P<0.01),and hypoxia and IL6 increased the expression of P311 by 7.58-fold(P<0.01)and 4.05-fold(P<0.05),respectively.All of them significantly increased the ?-SMA mRNA expression(P<0.05).4.The regulation of P311 on TGF?1/Smad signaling.4.1 The regulation of P311 on the TGF?1 expressionThe protein levels of LAP-TGF?1 and active TGF?1 in P311-expressing EpSCs were significantly higher by 4.21-and 6.89-fold,respectively,than those in control cells(P<0.01).The TGF?1 mRNA level in P311-expressing EpSCs was decreased to 78.81% of that in control cells(P<0.01).The concentration of total TGF?1 protein in the culture medium of P311 KO EpSCs was decreased to 51.15% of the concentration in P311 WT EpSCs(P<0.05).Meanwhile,in the P311-expressing EpSCs,the mRNA levels of T?RI and T?RII were also up-regulated by 1.72-fold(P<0.05)and 2.22-fold(P<0.01),respectively,compared to those in control cells.4.2 P311 promotes the phosphorylation of Smad2 and Smad3.The levels of the pSmad2 and pSmad3 in P311-expressing EpSCs were significantly higher by 2.51-and 2.80-fold,respectively,than those in control cells(P<0.01).Under exogenous TGF?1 stimulation,the pSmad2 and pSmad3 levels in P311 KO EpSCs were decreased to 52.17% and 65.71%,respectively,of those in P311 WT cells(P<0.05).However,total Smad2 and Smad3 were not obviously changed no matter if P311 was knocked out or elevated.5.The T?RI/II inhibitor(LY2109761)blockades the P311-induced EpMyT.After the addition of LY2109761,the phosphorylation of Smad2 and Smad3 were almost completely inhibited,and the P311-expressing cells lost their myofibroblast-like morphology and became similar to the control cells.In the P311 transfected cells,LY2109761 decreased the percentage of ?-SMA+ EpSCs from 39.99% to 7.93%,and increased the percentage of E-cadherin+ EpSCs from 8.37% to 43.43%(P<0.01).Meanwhile,the increases in the levels of the ?-SMA and vimentin proteins in the P311-expressing EpSCs were significantly inhibited by LY2109761,whereas the decreases in the levels of the E-cadherin and ?1-integrin proteins were significantly restored by LY2109761.Furthermore,the increased expression of collagen I and MMP2 protein in P311-expressing EpSCs were significantly inhibited by LY2109761.6.Smad3 siRNA blockades the P311-induced EpMyT.After Smad3 siRNA was added,the Smad3 protein level was decreased to 40.64% of that in P311-expressing cells(P<0.05)and the phosphorylation of Smad3 was nearly completely inhibited(P<0.01).Comparing with P311 overexpressed EpSCs and mock siRNA group,the P311 overexpressed Ep SCs with Smad3 siRNA exhibited significantly decreased ?-SMA protein and increased E-cadherin protein.7.Exogenous TGF?1 restores the EpMyT in P311 KO EpSCs.After exogenous TGF?1 was added,the level of ?-SMA was significantly increased in both P311 KO EpSCs and P311 WT EpSCs.Furthermore,the P311 KO EpSCs that were stimulated with TGF?1 contained almost the same level of ?-SMA protein as the P311 WT EpSCs that were not stimulated with TGF?1(P>0.05).8.P311 may stimulate TGF?1 expression by promoting its promoter methylation and UTR activity.P311 had no effect on the TGF?1 promoter activity,but increased the number of methylated CpG sites in the TGF?1 promoter.The TGF?1 mRNA degradation in P311-expressing EpSCs was a little less than that in control cells,but the difference was not significant(slope:-0.0805 vs-0.112,P=0.4531).P311-expressing EpSCs exhibited significantly higher activities of TGF?1 5'-UTR,3'-UTR and 3'/5'-UTR than control cells.9.The possible mechanism of P311's regulation on TGF?1 UTR activities.The identities of TGF?1 protein and Open Reading Frame(ORF)among 14 species were over 80%.However,the identities of TGF?1 5'-UTR and 3'-UTR were only 30.45% and 15.18%,respectively.The length of human and mouse TGF?1 5'-UTR were 889 bp and 867 bp,respectively,and their GC content were 71.6% and 65.9%,respectively.The length of human and mouse TGF?1 3'-UTR were 521 bp and 134 bp,respectively,and their GC content were 57.8% and 76.9%,respectively.Furthermore,the possibility of human P311 protein binding with inhibitory elements(region 63~121,422~165)and the stem loop structure(region 77~106)in TGF?1 5'-UTR,and 3'-UTR,were more than 50%.The predicted RNA binding sites of P311 protein were 29 R,31P,33 P,34K,35 E,36V,37 N,38R and39 K.10.P311 might indirectly regulate the TGF?1 protein expression by eIF6.The eIF6 protein expression was reduced in P311-expressing EpSCs and was increased in P311 KO EpSCs.Furthermore,the LAP-TGF?1 and active TGF?1 proteins were up-regulated in eIF6-knockdown EpSCs and in e IF6-silenced EpSCs.ConclusionsIn summary,this study provides the first evidence showing that P311 is a novel regulator of the potential of epidermal stem cells to transdifferentiate into myofibroblast-like cells and that this mechanism is mediated by TGF?1/Smad signaling.Moreover,P311 might stimulate TGF?1 expression by promoting TGF?1 promoter methylation at the transcriptional level and by activating TGF?1 5'/3'-UTR at the translational level.Furthermore,P311 might be induced by the cytokine and hypoxia,and the deletion of P311 resulted in delayed burn wound healing.Therefore,this study could not only show that EpSCs may be another important source of myofibroblasts during skin wound healing,but also identify a novel mechanism for cutaneous wound healing as follows: Cytokines and hypoxia-P311-TGF?1-Smad2/3-EpMyT-more MMP2 and collagen I-enhanced motility-rapid wound healing.This research enriches the biological function of P311 and the mechanism of wound healing,and implies a novel clue for the basic research and clinical treatment of wound healing.