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Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis.

Akhmetshina A, Palumbo K, Dees C, Bergmann C, Venalis P, Zerr P, Horn A, Kireva T, Beyer C, Zwerina J, Schneider H, Sadowski A, Riener MO, MacDougald OA, Distler O, Schett G, Distler JH - Nat Commun (2012)

Bottom Line: Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1.Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models.These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine 3 and Institute for Clinical Immunology, University of Erlangen-Nuremberg, 91054 Erlangen, Germany.

ABSTRACT
The transforming growth factor-β (TGF-β) signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of extracellular matrix in fibrotic diseases. Here we demonstrate a novel link between transforming growth factor-β and the canonical Wnt pathway. TGF-β stimulates canonical Wnt signalling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1. Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1. Activation of the canonical Wnt pathway stimulates fibroblasts in vitro and induces fibrosis in vivo. Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models. These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases.

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Activation of the canonical Wnt pathway induces fibrosis.(a) Recombinant Wnt-1 stimulated the activity of col1a2 promoter constructs (n=6). (b) Incubation with recombinant Wnt-1 increased the release of collagen protein by cultured fibroblasts in a dose-dependent manner (n=6). (c) Wnt-1 induced the expression of αSMA protein (n=5). (d) Incubation with Wnt-1 increased the mRNA levels of αSMA (n=5). (e) Wnt-1 stimulated the formation of stress fibres in cultured fibroblasts (n=6). Of note, the stimulatory effects of Wnt-1 on fibroblasts were comparable with those of TGF-β. (f) Transgenic overexpression of Wnt-10b induced dermal fibrosis in mice. The dermal thickness, the hydroxyproline content and the numbers of myofibroblasts were already increased in Wnt-10b tg mice at an age of 3 weeks and increased further with age (n ≥ 5 for all groups). Representative haematoxylin and eosin-, trichrome- and Sirius Red-stained sections of wild-type (WT) and Wnt-10b transgenic (Wnt-10b tg) mice at ages of 3, 6 and 12 weeks are shown (horizontal scale bars, 100 μm). Black vertical bars indicate the dermal thickness. * Indicates P-values of less than 0.05 as compared with mock-treated fibroblasts (a–e, analysed with the Wilcoxon signed-rank test) or compared with wild-type littermates (f, analysed using the Mann–Whitney U-test), respectively. All data are expressed as mean ± s.e.m.
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f3: Activation of the canonical Wnt pathway induces fibrosis.(a) Recombinant Wnt-1 stimulated the activity of col1a2 promoter constructs (n=6). (b) Incubation with recombinant Wnt-1 increased the release of collagen protein by cultured fibroblasts in a dose-dependent manner (n=6). (c) Wnt-1 induced the expression of αSMA protein (n=5). (d) Incubation with Wnt-1 increased the mRNA levels of αSMA (n=5). (e) Wnt-1 stimulated the formation of stress fibres in cultured fibroblasts (n=6). Of note, the stimulatory effects of Wnt-1 on fibroblasts were comparable with those of TGF-β. (f) Transgenic overexpression of Wnt-10b induced dermal fibrosis in mice. The dermal thickness, the hydroxyproline content and the numbers of myofibroblasts were already increased in Wnt-10b tg mice at an age of 3 weeks and increased further with age (n ≥ 5 for all groups). Representative haematoxylin and eosin-, trichrome- and Sirius Red-stained sections of wild-type (WT) and Wnt-10b transgenic (Wnt-10b tg) mice at ages of 3, 6 and 12 weeks are shown (horizontal scale bars, 100 μm). Black vertical bars indicate the dermal thickness. * Indicates P-values of less than 0.05 as compared with mock-treated fibroblasts (a–e, analysed with the Wilcoxon signed-rank test) or compared with wild-type littermates (f, analysed using the Mann–Whitney U-test), respectively. All data are expressed as mean ± s.e.m.

Mentions: To investigate the functional role of the canonical Wnt pathway in fibrosis, human dermal fibroblasts were incubated with Wnt-1, which we found upregulated in human fibrotic diseases. Wnt-1 stimulated the transcriptional activity in Col1a2 reporter assays (Fig. 3a). The increase in promoter activity on simultaneous stimulation with TGF-β and Wnt-1 was only slightly higher than with single stimulation with TGF-β or Wnt-1. Consistently, a dose-dependent increase in collagen release was observed in the supernatants of fibroblasts stimulated with Wnt-1 (Fig. 3b). Of note, the stimulatory effects of Wnt-1 were comparable with those of TGF-β, which is considered to belong to the most potent profibrotic mediators. Next, we analysed whether the increased release of collagen on stimulation with Wnt-1 might result from increased differentiation of resting fibroblasts into myofibroblasts. Myofibroblasts produce large amounts of extracellular matrix and have a key role in fibrotic diseases2. Incubation of resting fibroblasts with Wnt-1 increased the levels of α-smooth muscle actin (αSMA) protein and mRNA (Fig. 3c and d) and induced the formation of stress fibres (Fig. 3e), demonstrating increased differentiation of resting fibroblasts into myofibroblasts. In addition to Wnt-1, other Wnt proteins such as Wnt-3a also stimulated the release of collagen and induced differentiation of resting fibroblasts into myofibroblasts.


Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis.

Akhmetshina A, Palumbo K, Dees C, Bergmann C, Venalis P, Zerr P, Horn A, Kireva T, Beyer C, Zwerina J, Schneider H, Sadowski A, Riener MO, MacDougald OA, Distler O, Schett G, Distler JH - Nat Commun (2012)

Activation of the canonical Wnt pathway induces fibrosis.(a) Recombinant Wnt-1 stimulated the activity of col1a2 promoter constructs (n=6). (b) Incubation with recombinant Wnt-1 increased the release of collagen protein by cultured fibroblasts in a dose-dependent manner (n=6). (c) Wnt-1 induced the expression of αSMA protein (n=5). (d) Incubation with Wnt-1 increased the mRNA levels of αSMA (n=5). (e) Wnt-1 stimulated the formation of stress fibres in cultured fibroblasts (n=6). Of note, the stimulatory effects of Wnt-1 on fibroblasts were comparable with those of TGF-β. (f) Transgenic overexpression of Wnt-10b induced dermal fibrosis in mice. The dermal thickness, the hydroxyproline content and the numbers of myofibroblasts were already increased in Wnt-10b tg mice at an age of 3 weeks and increased further with age (n ≥ 5 for all groups). Representative haematoxylin and eosin-, trichrome- and Sirius Red-stained sections of wild-type (WT) and Wnt-10b transgenic (Wnt-10b tg) mice at ages of 3, 6 and 12 weeks are shown (horizontal scale bars, 100 μm). Black vertical bars indicate the dermal thickness. * Indicates P-values of less than 0.05 as compared with mock-treated fibroblasts (a–e, analysed with the Wilcoxon signed-rank test) or compared with wild-type littermates (f, analysed using the Mann–Whitney U-test), respectively. All data are expressed as mean ± s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f3: Activation of the canonical Wnt pathway induces fibrosis.(a) Recombinant Wnt-1 stimulated the activity of col1a2 promoter constructs (n=6). (b) Incubation with recombinant Wnt-1 increased the release of collagen protein by cultured fibroblasts in a dose-dependent manner (n=6). (c) Wnt-1 induced the expression of αSMA protein (n=5). (d) Incubation with Wnt-1 increased the mRNA levels of αSMA (n=5). (e) Wnt-1 stimulated the formation of stress fibres in cultured fibroblasts (n=6). Of note, the stimulatory effects of Wnt-1 on fibroblasts were comparable with those of TGF-β. (f) Transgenic overexpression of Wnt-10b induced dermal fibrosis in mice. The dermal thickness, the hydroxyproline content and the numbers of myofibroblasts were already increased in Wnt-10b tg mice at an age of 3 weeks and increased further with age (n ≥ 5 for all groups). Representative haematoxylin and eosin-, trichrome- and Sirius Red-stained sections of wild-type (WT) and Wnt-10b transgenic (Wnt-10b tg) mice at ages of 3, 6 and 12 weeks are shown (horizontal scale bars, 100 μm). Black vertical bars indicate the dermal thickness. * Indicates P-values of less than 0.05 as compared with mock-treated fibroblasts (a–e, analysed with the Wilcoxon signed-rank test) or compared with wild-type littermates (f, analysed using the Mann–Whitney U-test), respectively. All data are expressed as mean ± s.e.m.
Mentions: To investigate the functional role of the canonical Wnt pathway in fibrosis, human dermal fibroblasts were incubated with Wnt-1, which we found upregulated in human fibrotic diseases. Wnt-1 stimulated the transcriptional activity in Col1a2 reporter assays (Fig. 3a). The increase in promoter activity on simultaneous stimulation with TGF-β and Wnt-1 was only slightly higher than with single stimulation with TGF-β or Wnt-1. Consistently, a dose-dependent increase in collagen release was observed in the supernatants of fibroblasts stimulated with Wnt-1 (Fig. 3b). Of note, the stimulatory effects of Wnt-1 were comparable with those of TGF-β, which is considered to belong to the most potent profibrotic mediators. Next, we analysed whether the increased release of collagen on stimulation with Wnt-1 might result from increased differentiation of resting fibroblasts into myofibroblasts. Myofibroblasts produce large amounts of extracellular matrix and have a key role in fibrotic diseases2. Incubation of resting fibroblasts with Wnt-1 increased the levels of α-smooth muscle actin (αSMA) protein and mRNA (Fig. 3c and d) and induced the formation of stress fibres (Fig. 3e), demonstrating increased differentiation of resting fibroblasts into myofibroblasts. In addition to Wnt-1, other Wnt proteins such as Wnt-3a also stimulated the release of collagen and induced differentiation of resting fibroblasts into myofibroblasts.

Bottom Line: Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1.Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models.These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine 3 and Institute for Clinical Immunology, University of Erlangen-Nuremberg, 91054 Erlangen, Germany.

ABSTRACT
The transforming growth factor-β (TGF-β) signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of extracellular matrix in fibrotic diseases. Here we demonstrate a novel link between transforming growth factor-β and the canonical Wnt pathway. TGF-β stimulates canonical Wnt signalling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1. Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1. Activation of the canonical Wnt pathway stimulates fibroblasts in vitro and induces fibrosis in vivo. Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models. These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases.

Show MeSH
Related in: MedlinePlus