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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

WNT and SFRP dynamics in bleomycin-induced lung fibrosis. (A) Representative images lungs from WT mice, 4 weeks after PBS or bleomycin instillation. (Hematoxylin-Eosin and Masson Trichrome staining) (B) β–catenin or goat IgG (negative control, 3rd panel) immunohistochemistry, 4 weeks after intratracheal PBS or bleomycin instillation in WT mice. (C) Total lung gene expression level of Sfrp1, Sfrp2, Frzb and Sfrp4 following bleomycin instillation (n = 4, except for bleomycin group at day 21 n = 2; data presented as mean and SEM of ΔCT values normalized to Hprt expression).
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Figure 1: WNT and SFRP dynamics in bleomycin-induced lung fibrosis. (A) Representative images lungs from WT mice, 4 weeks after PBS or bleomycin instillation. (Hematoxylin-Eosin and Masson Trichrome staining) (B) β–catenin or goat IgG (negative control, 3rd panel) immunohistochemistry, 4 weeks after intratracheal PBS or bleomycin instillation in WT mice. (C) Total lung gene expression level of Sfrp1, Sfrp2, Frzb and Sfrp4 following bleomycin instillation (n = 4, except for bleomycin group at day 21 n = 2; data presented as mean and SEM of ΔCT values normalized to Hprt expression).

Mentions: Intratracheal bleomycin instillation results in pulmonary fibrosis with excessive collagen deposition and obliteration of alveolar structures (Figure 1A). Immunohistochemistry demonstrated increased nuclear β-catenin in fibrotic zones indicating active canonical Wnt signaling, while this signal was absent in the PBS-treated lungs (Figure 1B). We used gene expression analysis of the different Sfrps to study their dynamic profile in the bleomycin-induced pulmonary fibrosis model. Sfrp1 and Sfrp2 mRNA levels were 2 log-scales more abundant than those of Frzb and Sfrp4. Sfrp5 could not be detected. Sfrp1 levels were significantly increased at all time points after bleomycin treatment but not different between time points (Figure 1C) (2-way ANOVA P = 0.0015 for bleomycin vs. PBS, P >0.05 for time and interaction). Frzb levels were significantly and consistently increased over time after bleomycin treatment (2-way ANOVA P <0.0001 for bleomycin vs. PBS, P = 0.0248 for time and P = 0.0154 for interaction). Sfrp2 and Sfrp4 levels were not different between groups or during the course of the disease, with Sfrp2 relative expression similar to Sfrp1 and Sfrp4 levels similar to baseline Frzb. We therefore further focused on SFRP1 and FRZB.


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)

WNT and SFRP dynamics in bleomycin-induced lung fibrosis. (A) Representative images lungs from WT mice, 4 weeks after PBS or bleomycin instillation. (Hematoxylin-Eosin and Masson Trichrome staining) (B) β–catenin or goat IgG (negative control, 3rd panel) immunohistochemistry, 4 weeks after intratracheal PBS or bleomycin instillation in WT mice. (C) Total lung gene expression level of Sfrp1, Sfrp2, Frzb and Sfrp4 following bleomycin instillation (n = 4, except for bleomycin group at day 21 n = 2; data presented as mean and SEM of ΔCT values normalized to Hprt expression).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: WNT and SFRP dynamics in bleomycin-induced lung fibrosis. (A) Representative images lungs from WT mice, 4 weeks after PBS or bleomycin instillation. (Hematoxylin-Eosin and Masson Trichrome staining) (B) β–catenin or goat IgG (negative control, 3rd panel) immunohistochemistry, 4 weeks after intratracheal PBS or bleomycin instillation in WT mice. (C) Total lung gene expression level of Sfrp1, Sfrp2, Frzb and Sfrp4 following bleomycin instillation (n = 4, except for bleomycin group at day 21 n = 2; data presented as mean and SEM of ΔCT values normalized to Hprt expression).
Mentions: Intratracheal bleomycin instillation results in pulmonary fibrosis with excessive collagen deposition and obliteration of alveolar structures (Figure 1A). Immunohistochemistry demonstrated increased nuclear β-catenin in fibrotic zones indicating active canonical Wnt signaling, while this signal was absent in the PBS-treated lungs (Figure 1B). We used gene expression analysis of the different Sfrps to study their dynamic profile in the bleomycin-induced pulmonary fibrosis model. Sfrp1 and Sfrp2 mRNA levels were 2 log-scales more abundant than those of Frzb and Sfrp4. Sfrp5 could not be detected. Sfrp1 levels were significantly increased at all time points after bleomycin treatment but not different between time points (Figure 1C) (2-way ANOVA P = 0.0015 for bleomycin vs. PBS, P >0.05 for time and interaction). Frzb levels were significantly and consistently increased over time after bleomycin treatment (2-way ANOVA P <0.0001 for bleomycin vs. PBS, P = 0.0248 for time and P = 0.0154 for interaction). Sfrp2 and Sfrp4 levels were not different between groups or during the course of the disease, with Sfrp2 relative expression similar to Sfrp1 and Sfrp4 levels similar to baseline Frzb. We therefore further focused on SFRP1 and FRZB.

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