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Secreted frizzled related proteins inhibit fibrosis in vitro but appear redundant in vivo.

De Langhe E, Aznar-Lopez C, De Vooght V, Vanoirbeek JA, Luyten FP, Lories RJ - Fibrogenesis Tissue Repair (2014)

Bottom Line: Both SFRP1 and FRZB inhibit the TGFβ1-induced increase of active β-catenin, but do not influence the TGFβ1-induced phosphorylation levels of SMAD3, positioning Wnt signaling activity downstream of the active TGFβ signal in lung fibroblasts, but not in alveolar epithelial cells.Although SFRP1 counteracts the effect of TGFβ1 in pulmonary cells in vitro; loss of neither SFRP1 nor FRZB alters fibrotic outcomes in the lungs in vivo.The lack of in vivo effect in the absence of specific SFRPs suggests functional redundancy within this family of Wnt antagonists.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Development and Regeneration, Laboratory of Tissue Homeostasis and Disease, Skeletal Biology and Engineering Research Center, KU Leuven, Leuven, Belgium ; Division of Rheumatology, University Hospitals Leuven, Leuven, Belgium.

ABSTRACT

Background: The pathogenesis of pulmonary fibrosis remains poorly understood. The Wnt signaling pathway regulates fibrogenesis in different organs. Here, we studied the role of two extracellular Wnt antagonists, secreted frizzled-related protein-1 (SFRP1) and frizzled-related protein (FRZB) on lung fibrosis in vitro and in vivo. For this purpose, we used an alveolar epithelial cell line and a lung fibroblast cell line, and the bleomycin-induced lung fibrosis model, respectively.

Results: During the course of bleomycin-induced lung fibrosis, Sfrp1 and Frzb expression are upregulated. Expression of Sfrp1 appears much higher than that of Frzb. In vitro, recombinant SFRP1, but not FRZB, counteracts the transforming growth factor β1 (TGFβ1)-induced upregulation of type I collagen expression both in pulmonary epithelial cells and fibroblasts. Both SFRP1 and FRZB inhibit the TGFβ1-induced increase of active β-catenin, but do not influence the TGFβ1-induced phosphorylation levels of SMAD3, positioning Wnt signaling activity downstream of the active TGFβ signal in lung fibroblasts, but not in alveolar epithelial cells. In vivo, Sfrp1 (-/-) and Frzb (-/-) mice showed identical responses to bleomycin in the lung compared to wild-type controls.

Conclusions: Although SFRP1 counteracts the effect of TGFβ1 in pulmonary cells in vitro; loss of neither SFRP1 nor FRZB alters fibrotic outcomes in the lungs in vivo. The lack of in vivo effect in the absence of specific SFRPs suggests functional redundancy within this family of Wnt antagonists.

No MeSH data available.


Related in: MedlinePlus

Downstream signaling in alveolar epithelial cells after SFRP1 and FRZB stimulation. Western blot of protein extracts from total A549 cell lysates, stimulated with TGFβ1 and SFRP1 (A) or FRZB (B), labeled with antibodies against total β-catenin and active β-catenin.
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Figure 5: Downstream signaling in alveolar epithelial cells after SFRP1 and FRZB stimulation. Western blot of protein extracts from total A549 cell lysates, stimulated with TGFβ1 and SFRP1 (A) or FRZB (B), labeled with antibodies against total β-catenin and active β-catenin.

Mentions: Activation of pulmonary fibroblasts is an important process in lung fibrosis. In TGFβ1-stimulated fibroblast MRC5 cells, SFRP1 significantly reduced TGFβ1-driven Coll1α1 expression (Figure 2A). In contrast, this effect was absent with FRZB stimulation (Figure 2B). Western blot analysis showed that TGFβ1 stimulation in MRC5 cells results in increased phosphorylation of SMAD3, but also increased active, dephosphorylated β-catenin (Figure 3). Stimulation of MRC5 cells with Wnt antagonists SFRP1 (Figure 3A) or FRZB (Figure 3B) reduces the active fraction of β-catenin. Both SFRP1 and FRZB inhibit the TGFβ1-induced increase of active β-catenin, but do not influence the TGFβ1-induced phosphorylation levels of SMAD3, positioning Wnt signaling activity downstream of the active TGFβ signal in lung fibroblasts. Epithelial-mesenchymal transition (EMT) may also contribute to fibrosis. We therefore studied the effect of recombinant SFRP1 or FRZB and TGFβ1 stimulation on alveolar epithelial cells (A549). Recombinant SFRP1 does not alter baseline E-cadherin levels, nor the TGFβ1-induced downregulation of E-cadherin in A549 cells. However, SFRP1 significantly reduced TGFβ1-induced upregulation of coll1α1 (Figure 4A). FRZB did not alter TGFβ1-induced alterations in E-cadherin or coll1α1 expression in A549 cells (Figure 4B). In contrast to our observations in lung fibroblasts, TGFβ1 does not increase active β-catenin in alveolar epithelial cells (Figure 5).


Secreted frizzled related proteins inhibit fibrosis in vitro but appear redundant in vivo.

De Langhe E, Aznar-Lopez C, De Vooght V, Vanoirbeek JA, Luyten FP, Lories RJ - Fibrogenesis Tissue Repair (2014)

Downstream signaling in alveolar epithelial cells after SFRP1 and FRZB stimulation. Western blot of protein extracts from total A549 cell lysates, stimulated with TGFβ1 and SFRP1 (A) or FRZB (B), labeled with antibodies against total β-catenin and active β-catenin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4196208&req=5

Figure 5: Downstream signaling in alveolar epithelial cells after SFRP1 and FRZB stimulation. Western blot of protein extracts from total A549 cell lysates, stimulated with TGFβ1 and SFRP1 (A) or FRZB (B), labeled with antibodies against total β-catenin and active β-catenin.
Mentions: Activation of pulmonary fibroblasts is an important process in lung fibrosis. In TGFβ1-stimulated fibroblast MRC5 cells, SFRP1 significantly reduced TGFβ1-driven Coll1α1 expression (Figure 2A). In contrast, this effect was absent with FRZB stimulation (Figure 2B). Western blot analysis showed that TGFβ1 stimulation in MRC5 cells results in increased phosphorylation of SMAD3, but also increased active, dephosphorylated β-catenin (Figure 3). Stimulation of MRC5 cells with Wnt antagonists SFRP1 (Figure 3A) or FRZB (Figure 3B) reduces the active fraction of β-catenin. Both SFRP1 and FRZB inhibit the TGFβ1-induced increase of active β-catenin, but do not influence the TGFβ1-induced phosphorylation levels of SMAD3, positioning Wnt signaling activity downstream of the active TGFβ signal in lung fibroblasts. Epithelial-mesenchymal transition (EMT) may also contribute to fibrosis. We therefore studied the effect of recombinant SFRP1 or FRZB and TGFβ1 stimulation on alveolar epithelial cells (A549). Recombinant SFRP1 does not alter baseline E-cadherin levels, nor the TGFβ1-induced downregulation of E-cadherin in A549 cells. However, SFRP1 significantly reduced TGFβ1-induced upregulation of coll1α1 (Figure 4A). FRZB did not alter TGFβ1-induced alterations in E-cadherin or coll1α1 expression in A549 cells (Figure 4B). In contrast to our observations in lung fibroblasts, TGFβ1 does not increase active β-catenin in alveolar epithelial cells (Figure 5).

Bottom Line: Both SFRP1 and FRZB inhibit the TGFβ1-induced increase of active β-catenin, but do not influence the TGFβ1-induced phosphorylation levels of SMAD3, positioning Wnt signaling activity downstream of the active TGFβ signal in lung fibroblasts, but not in alveolar epithelial cells.Although SFRP1 counteracts the effect of TGFβ1 in pulmonary cells in vitro; loss of neither SFRP1 nor FRZB alters fibrotic outcomes in the lungs in vivo.The lack of in vivo effect in the absence of specific SFRPs suggests functional redundancy within this family of Wnt antagonists.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Development and Regeneration, Laboratory of Tissue Homeostasis and Disease, Skeletal Biology and Engineering Research Center, KU Leuven, Leuven, Belgium ; Division of Rheumatology, University Hospitals Leuven, Leuven, Belgium.

ABSTRACT

Background: The pathogenesis of pulmonary fibrosis remains poorly understood. The Wnt signaling pathway regulates fibrogenesis in different organs. Here, we studied the role of two extracellular Wnt antagonists, secreted frizzled-related protein-1 (SFRP1) and frizzled-related protein (FRZB) on lung fibrosis in vitro and in vivo. For this purpose, we used an alveolar epithelial cell line and a lung fibroblast cell line, and the bleomycin-induced lung fibrosis model, respectively.

Results: During the course of bleomycin-induced lung fibrosis, Sfrp1 and Frzb expression are upregulated. Expression of Sfrp1 appears much higher than that of Frzb. In vitro, recombinant SFRP1, but not FRZB, counteracts the transforming growth factor β1 (TGFβ1)-induced upregulation of type I collagen expression both in pulmonary epithelial cells and fibroblasts. Both SFRP1 and FRZB inhibit the TGFβ1-induced increase of active β-catenin, but do not influence the TGFβ1-induced phosphorylation levels of SMAD3, positioning Wnt signaling activity downstream of the active TGFβ signal in lung fibroblasts, but not in alveolar epithelial cells. In vivo, Sfrp1 (-/-) and Frzb (-/-) mice showed identical responses to bleomycin in the lung compared to wild-type controls.

Conclusions: Although SFRP1 counteracts the effect of TGFβ1 in pulmonary cells in vitro; loss of neither SFRP1 nor FRZB alters fibrotic outcomes in the lungs in vivo. The lack of in vivo effect in the absence of specific SFRPs suggests functional redundancy within this family of Wnt antagonists.

No MeSH data available.


Related in: MedlinePlus