β-catenin is a central mediator of pro-fibrotic Wnt signaling in systemic sclerosis

Abstract

Objectives: Pathologic fibroblast activation drives fibrosis of the skin and internal organs in patients with systemic sclerosis (SSc). β-catenin is an integral part of adherens junctions and a central component of canonical Wnt signaling. Here, the authors addressed the role of β-catenin in fibroblasts for the development of SSc dermal fibrosis.

Methods: Nuclear accumulation of β-catenin in fibroblasts was assessed by triple staining for β-catenin, prolyl-4-hydroxylase-β and 4',6-diamidino-2-phenylindole (DAPI). The expression of Wnt proteins in the skin was analysed by real-time PCR and immunohistochemistry. Mice with fibroblast-specific stabilisation or fibroblast-specific depletion were used to evaluate the role of β-catenin in fibrosis.

Results: The auhors found significantly increased nuclear levels of β-catenin in fibroblasts in SSc skin compared to fibroblasts in the skin of healthy individuals. The accumulation of β-catenin resulted from increased expression of Wnt-1 and Wnt-10b in SSc. The authors further showed that the nuclear accumulation of β-catenin has direct implications for the development of fibrosis: Mice with fibroblast-specific stabilisation of β-catenin rapidly developed fibrosis within 2 weeks with dermal thickening, accumulation of collagen and differentiation of resting fibroblasts into myofibroblasts. By contrast, fibroblast-specific deletion of β-catenin significantly reduced bleomycin-induced dermal fibrosis.

Conclusions: The present study findings identify β-catenin as a key player of fibroblast activation and tissue fibrosis in SSc. Although further translational studies are necessary to test the efficacy and tolerability of β-catenin/Wnt inhibition in SSc, the present findings may have clinical implications, because selective inhibitors of β-catenin/Wnt signaling have recently entered clinical trials.

Figure 1

β-catenin is stabilised in fibroblasts in SSc skin. (A) Immunofluorescence images show staining against chromatin (blue), β-catenin (green), and prolyl-4-hydroxylase-β (red) as well as overlay of these three stains in skin sections from patients with SSc and healthy individuals. Images are in 200- and 1000-fold magnifications. (B) Quantification of nuclei that stained for β-catenin, DAPI and prolyl-4-hydroxylase divided by the total number of DAPI and prolyl-4-hydroxylase-positive fibroblastic cells. Values are expressed in per cent of β-catenin-positive nuclei.

Figure 2

Stabilisation of β-catenin in dermal fibroblasts is induced by increased expression of canonical Wnt ligands. (A) mRNA expression profiles of all canonical Wnt ligands in skin from patients with SSc and healthy individuals. mRNA levels of the Wnt ligands are illustrated in relation to the corresponding β-actin values (internal control). Percentage of skin samples that expressed specific Wnt ligands are illustrated below the graph. (B) Immunohistochemistry demonstrated increased expression of Wnt-1 and Wnt-10b but not of Wnt-4 in skin sections of SSc patients. Representative images are shown at 200-fold magnification. (C) Increased mRNA levels of axin-2 in the skin of SSc patients. The relative expression of axin-2 in the SSc samples compared to control samples is expressed as x-fold increase.

Figure 3

Development of progressive dermal fibrosis in ΔEx3 mice with fibroblast-specific stabilisation of β-catenin. Cre-mediated recombination resulting in fibroblast-specific activation of β-catenin was induced by tamoxifen injection (n=10). Mice of the same genotype injected with oil served as controls (n=7). (A) Trichrome staining with blue staining for collagens. Pictures are shown at 100-fold magnification. (B) Skin thickening as determined by H and E staining. Values are expressed in relation to skin thickness of non-activated, oil-treated mice 2 weeks after mock treatment. White bars show non-activated, oil-treated ΔEx3 mice and grey bars represent skin thickness of activated, tamoxifen-treated ΔEx3 mice. (C) Hydroxyproline content in non-activated (white bars) and activated (grey bars) ΔEx3 mice. Values are expressed in relation to hydroxyproline content of non-activated, oil-treated mice at week 2. (D) α-SMA positive myofibroblasts in the skin from non-activated (white bars) and activated (grey bars) ΔEx3 mice. Values are expressed in relation to α-SMA counts of non-activated, oil-treated mice 2 weeks after Cre activation.

Figure 4

Prevention of bleomycin-induced experimental dermal fibrosis in Ctnnb1 mice with fibroblast-specific depletion of β-catenin. Cre-mediated recombination was activated by tamoxifen injection. Mice of the same genotype injected with oil were used as controls. For all groups n=6. (A) Trichrome staining with blue staining for collagens (100-fold magnification). (B) Skin thickening as determined by H and E staining. Values are expressed in relation to skin thickness of non-activated (oil), NaCl-treated mice. (C) Hydroxyproline content expressed in relation to hydroxyproline content of non-activated (oil), NaCl-treated mice. (D) α-SMA positive myofibroblasts expressed in relation to α-SMA counts in non-activated (oil), NaCl-treated mice.