Effects And Mechanisms Of Wnt/β-catenin Signal Pathway On TGF-β1Induced Phenotype Change Of Dermal Fibroblasts Into Myofibroblasts

【Background】Hypertrophic scar is a fibro-proliferative disorder of wound healingafter skin tissue injuries. Myofibroblasts, which trans-differentiated fromfibroblasts, are main effectors contributing to hypertrophic scar formation.TGF-β1is the key stimulus in inducing the fibroblasts-to-myofibroblaststransition. There are crosstalk effects between TGF-β1and Wnt signalingin the regulation of various physiological and pathological processesbesides through Smads, which are canonical mediators of the TGF-β1signal. Wnt signaling is an important pathway in regulating thedevelopment of organs and tissues, and the differentiation of stem cells. Itis reported that Wnt signal might be involved in the wound healing, fibrotic diseases and the activation of pro-fibrotic cells. However, there istissue or organ-dependent effects of Wnt signaling on the above processes.The specific effects of Wnt on the dermal fibroblasts transition and themolecular regulation mechanism between Wnt and TGF-β1are stillunclear.【Purposes】1. To elucidate the expression and regulation mechanism of Wntsignal pathway components in the hypertrophic scars and scar tissuederived fibroblasts.2. To verify the specific effects and mechanism of Wnt signaling onthe TGF-β1induced dermal myofibroblasts transition.【Contents】1. Measure the expression levels of Wnt signaling components in thehypertrophic scars and the scar tissues derived fibroblasts.2. The myofibroblasts transition is induced by TGF-β1stimulationand the mRNA expression profile of Wnts family was determined byreal-time PCR.3. Detect the expression levels of molecular markers ofmyofibroblasts transition using real-time PCR, immunoblot andimmunocytochemistry after the activation of Wnt signal by its activatorSB216763. Observe the contraction function of the fibroblasts byFibroblasts populated collagen lattices assays.4. Construction of β-catenin over-expression vector; synthesis of the siRNA pair targeting β-catenin.5. Detect the expression levels of the myofibroblasts markers byreal-time PCR, immunoblot and immunocytochemistry after transfectionof β-catenin over-expression vector or siRNA.6. Measure the expression levels of α-SMA, COLⅠ and COLⅢ inhypertrophic scar tissue derived fibroblasts after SB216763andover-expression of β-catenin by real-time PCR and immunoblot; Measurethe expression levels of TGF-β1, Smad3and Smad7after SB216763treatment.7. Investigate the effects of Wnt4on the expression levels of α-SMA,COLⅠ and COLⅢ induced by TGF-β1and the autocrine effects ofTGF-β1.【Results】1. Compared with normal skin tissues, the expression levels of Wnt2,Wnt3a, Wnt4and Wnt10b decreased significantly in hypertrophic scartissues. Similarly, the expression levels of Wnt2, Wnt3a, Wnt4andWnt10b were also down-regulated in hypertrophic scar derived fibroblastscompared with those from normal skin tissues.2. After TGF-β1stimulation, the expression of Wnt2, Wnt3a, Wnt4and Wnt10b mRNA increased significantly. The expression of β-cateninmRNA did not change. However, the protein level of β-catenin showed agradual increase after TGF-β1stimulation.3. The TGF-β1induction of α-SMA, COLⅠ and COLⅢ wereinhibited when the SB216763, activator of Wnt/β-catenin, was applied in combination with TGF-β1. The immunocytochemistry showed that thenumber of α-SMA-positive myofibroblasts decreased after SB216763treatment. The fibroblasts populated collagen lattices assays also showedthat the contractility of fibroblasts was inhibited by SB216763.4. The β-catenin over-expression vector and siRNA were constructedor synthesized successfully.5. Over-expression of β-catenin inhibited the TGF-β1inducedexpression of α-SMA, COLⅠ and COLⅢ. The number of α-SMApositive myofibroblasts decreased after over-expression of β-catenin asshown by immunocytochemistry. When the expression of β-catenin wasdown-regulated by siRNA, the TGF-β1induced expression of α-SMA,COLⅠ and COLⅢ showed a further increase.6. The expression levels of α-SMA, COL Ⅰ and COL Ⅲ inhypertrophic scar derived fibroblasts were down-regulated by SB216763treatment and over-expression of β-catenin. The expression levels ofTGF-β1and Smad3were down-regulated by SB216763, but theexpression level of Smad7did not change.7. Wnt4inhibited the expression of α-SMA, COLⅠ and COLⅢinduced by TGF-β1and also negatively regulated the autocrine effect ofTGF-β1. Meanwhile, Wnt4could reverse the myofibroblasts transition.【Conclusion】1. During the dermal fibroblasts transition, TGF-β1could activate theWnt/β-catenin signaling pathway and β-catenin at the protein levelthrough up-regulating the Wnt2, Wnt4and Wnt10b. 2. Activation of Wnt/β-catenin could inhibit the TGF-β1induceddermal fibroblasts transition by forming a negative feedback loop.3. Activation of Wnt/β-catenin could inhibit the expression levels ofα-SMA, COLⅠ and COLⅢ by hypertrophic scar derived fibroblasts.4. The inhibitory effects of Wnt/β-catenin on TGF-β1signalingcould be associated with its negative regulation on the TGF-β1andSmad3expression, as well as on the autocrine effects of TGF-β1...