Activation of Wnt/β-catenin signalling is required for TGF-β/Smad2/3 signalling during myofibroblast proliferation

Abstract

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β-catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt-mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor-β (TGF-β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α-SMA and collagen I) and the TGF-β signalling pathway that include smad2/3 and its phosphorylated form p-smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β-catenin revealed epithelial-mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β-catenin in regulation of the signalling network, which acts to counteract autocrine TGF-β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1-smad2/3 signalling through Wnt/β-catenin contribute to lung fibrosis.

Keywords: Smad2/3; Wnt/β-catenin; myofibroblast proliferation; pulmonary fibrosis.

Figure 1

Immunohistochemical analysis of E‐cadherin, α‐SMA and β‐catenin in pulmonary fibrosis rat model. Representative images of lung biopsy specimens obtained from rats with pulmonary fibrosis and healthy control subjects. Top two lines, arrows indicate E‐cadherin‐positive alveolar epithelial cell just in normal lung tissues and α‐SMA‐positive fibroblast‐like cells in the pulmonary alveolus in bleomycin‐injured rats. Last line, arrows indicate nuclear β‐catenin‐positive fibroblast‐like cells in the pulmonary alveolus. n = 5 bleomycin‐ and saline‐treated mice. Original magnification ×400.

Figure 2

Western blot was used to examine the protein expression of α‐SMA in HELF (A) and A549 (B) cells exposed to the indicated doses of TGF‐β1 for 24 hrs.

Figure 3

Effect of ICG‐001 exposure on the expression of vimentin, α‐SMA and collagen I in HELF cells. (A and B) Marker protein of fibroblast‐to‐myofibroblast transition vimentin, α‐SMA and collagen I overexpression in HELF cells caused by TGF‐β1 for 24 hrs. (C) Total protein of β‐catenin and (D) positive‐β‐catenin protein in the nuclear was tested by Western blot. Data are shown as means ± S.D., (n = 3). **P < 0.01, compared with control group. ##P < 0.01, compared with model group.

Figure 4

Effect of ICG‐001 exposure on the expression of E‐cad, vimentin, α‐SMA and collagen I in A549 cells. (A and B), Marker protein of epithelial–mesenchymal transition E‐cadherin vimentin, α‐SMA and collagen I overexpression in A549 cells caused by TGF‐β1 for 24 hrs. (C) Total protein of β‐catenin and (D) positive‐β‐catenin protein in the nuclear was tested by Western blot. Data are shown as means ± S.D., (n = 3). **P < 0.01, compared with control group. ##P < 0.01, compared with model group.

Figure 5

TGF‐β1‐induced fibrotic reaction is Smad dependent. ICG‐001 inhibits TGF‐β1‐induced Smad2/3 expression fibroblast induction and EMT, and representative Western blot and quantitative analysis of total Smad2/3 in lysate from HELF (A) and A549 (B) cells treated with ICG‐001. GAPDH or β‐actin is used as a loading control (n = 3; **P < 0.01 compared with control group; ##P < 0.01 compared with TGF‐β1 in the absence of β‐catenin siRNA). C) Representative Western blot and quantitative analysis of total β‐catenin in lysate from A549 and HELF cells transfected with β‐catenin or control siRNA followed by TGF‐β1treatment for 24 hrs. The expression of β‐catenin was detected by Western blots. β‐actin is used as a loading control (n = 3; **P < 0.01 compared with control group; ##P < 0.01 compared with TGF‐β1 in the absence of β‐catenin siRNA). Representative Western blot and quantitative analysis of α‐SMA, E‐cadherin, p‐Smad2/3 and Smad2/3 in lysate from A549 (D) and HELF (E) cells transfected with β‐catenin or control siRNA followed by TGF‐β1treatment for 24 hrs. β‐actin is used as a loading control (n = 3; **P < 0.01 compared with control group; ##P < 0.01 compared with TGF‐β1 in the absence of β‐catenin siRNA).

Figure 6

β‐catenin promotes the fibroblast‐to‐myofibroblast transition and EMT through the activation of the TGF‐β1/Smad2/3 signalling pathway. In general, TGF‐β/Smad2/3 signalling is initiated with TGF‐β‐induced phosphorylation of the cytoplasmic signalling molecules Smad2/3. The phospho‐Smad2/3 (p‐Smad2/3) combines with β‐catenin and translocates to the nucleus, where it will modulate the transcription of several target genes by binding with cyclic AMP‐responsive element‐binding protein (CREB)‐binding protein (CBP) to DNA sequences. Our findings suggest that β‐catenin promote both basal and TGF‐β1‐induced phosphorylation of Smad2/3.