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Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

Carthy JM, Meredith AJ, Boroomand S, Abraham T, Luo Z, Knight D, McManus BM - PLoS ONE (2015)

Bottom Line: These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation.Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice.Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: UBC James Hogg Research Centre, Institute for Heart + Lung Health, Department of Pathology and Laboratory Medicine, University of British Columbia - Providence Health Care, Vancouver, British Columbia, Canada.

ABSTRACT

Background: Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts.

Methods and results: The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2.

Conclusions: Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

No MeSH data available.


Related in: MedlinePlus

Versican increases TGF-β signaling in cultured fibroblasts.(A) Confocal imaging of contracted gels demonstrated the versican-transfected fibroblasts to have increased expression and incorporation of smooth muscle α-actin into their stress fibres (arrows, yellow in overlay). (B) The versican-transfected fibroblasts also displayed increased staining and nuclear localization of phophorylated SMAD2 in contracted collagen gels (arrows, red in overlay). (C) Representative Western blot shows increased phosphorylation of SMAD2 in cultures of versican-transfected fibroblasts (3.13 ±0.61 fold increase, p<0.05). (D) Confocal microscopy confirmed the increased SMAD2 phosphorylation and nuclear accumulation in cultures of versican-transfected fibroblasts (arrows). (Scale bars = 23.00 μm in A, 12.00 μm in B, D)
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pone.0133056.g006: Versican increases TGF-β signaling in cultured fibroblasts.(A) Confocal imaging of contracted gels demonstrated the versican-transfected fibroblasts to have increased expression and incorporation of smooth muscle α-actin into their stress fibres (arrows, yellow in overlay). (B) The versican-transfected fibroblasts also displayed increased staining and nuclear localization of phophorylated SMAD2 in contracted collagen gels (arrows, red in overlay). (C) Representative Western blot shows increased phosphorylation of SMAD2 in cultures of versican-transfected fibroblasts (3.13 ±0.61 fold increase, p<0.05). (D) Confocal microscopy confirmed the increased SMAD2 phosphorylation and nuclear accumulation in cultures of versican-transfected fibroblasts (arrows). (Scale bars = 23.00 μm in A, 12.00 μm in B, D)

Mentions: A recent report has suggested versican localizes TGF-β and increases its signaling in chondrocytes during joint formation [20]. Therefore, we hypothesized that versican may also alter the TGF-β signaling axis in cultured fibroblasts. Immunofluorescent staining demonstrated increased expression and incorporation of smooth muscle α-actin in versican-transfected fibroblasts that were cultured in type I collagen gels (Fig 6A). These cells also displayed nuclear accumulation of phosphorylated SMAD2, a direct target of TGF-β signaling, when cultured in collagen gels (Fig 6B). To confirm activation of TGF-β signaling in versican expressing fibroblasts, Western blotting was performed on cultured fibroblasts and representative blots are shown in Fig 6C. The versican expressing fibroblasts displayed increased SMAD2 phosphorylation as compared to control cells (3.13 ±0.61 fold increase, p<0.05). The increased nuclear accumulation of phosphorylated SMAD2 was again observed in 2D cultures of versican-transfected fibroblasts, confirming activation of this signaling pathway (Fig 6D).


Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

Carthy JM, Meredith AJ, Boroomand S, Abraham T, Luo Z, Knight D, McManus BM - PLoS ONE (2015)

Versican increases TGF-β signaling in cultured fibroblasts.(A) Confocal imaging of contracted gels demonstrated the versican-transfected fibroblasts to have increased expression and incorporation of smooth muscle α-actin into their stress fibres (arrows, yellow in overlay). (B) The versican-transfected fibroblasts also displayed increased staining and nuclear localization of phophorylated SMAD2 in contracted collagen gels (arrows, red in overlay). (C) Representative Western blot shows increased phosphorylation of SMAD2 in cultures of versican-transfected fibroblasts (3.13 ±0.61 fold increase, p<0.05). (D) Confocal microscopy confirmed the increased SMAD2 phosphorylation and nuclear accumulation in cultures of versican-transfected fibroblasts (arrows). (Scale bars = 23.00 μm in A, 12.00 μm in B, D)
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Related In: Results  -  Collection

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pone.0133056.g006: Versican increases TGF-β signaling in cultured fibroblasts.(A) Confocal imaging of contracted gels demonstrated the versican-transfected fibroblasts to have increased expression and incorporation of smooth muscle α-actin into their stress fibres (arrows, yellow in overlay). (B) The versican-transfected fibroblasts also displayed increased staining and nuclear localization of phophorylated SMAD2 in contracted collagen gels (arrows, red in overlay). (C) Representative Western blot shows increased phosphorylation of SMAD2 in cultures of versican-transfected fibroblasts (3.13 ±0.61 fold increase, p<0.05). (D) Confocal microscopy confirmed the increased SMAD2 phosphorylation and nuclear accumulation in cultures of versican-transfected fibroblasts (arrows). (Scale bars = 23.00 μm in A, 12.00 μm in B, D)
Mentions: A recent report has suggested versican localizes TGF-β and increases its signaling in chondrocytes during joint formation [20]. Therefore, we hypothesized that versican may also alter the TGF-β signaling axis in cultured fibroblasts. Immunofluorescent staining demonstrated increased expression and incorporation of smooth muscle α-actin in versican-transfected fibroblasts that were cultured in type I collagen gels (Fig 6A). These cells also displayed nuclear accumulation of phosphorylated SMAD2, a direct target of TGF-β signaling, when cultured in collagen gels (Fig 6B). To confirm activation of TGF-β signaling in versican expressing fibroblasts, Western blotting was performed on cultured fibroblasts and representative blots are shown in Fig 6C. The versican expressing fibroblasts displayed increased SMAD2 phosphorylation as compared to control cells (3.13 ±0.61 fold increase, p<0.05). The increased nuclear accumulation of phosphorylated SMAD2 was again observed in 2D cultures of versican-transfected fibroblasts, confirming activation of this signaling pathway (Fig 6D).

Bottom Line: These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation.Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice.Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: UBC James Hogg Research Centre, Institute for Heart + Lung Health, Department of Pathology and Laboratory Medicine, University of British Columbia - Providence Health Care, Vancouver, British Columbia, Canada.

ABSTRACT

Background: Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts.

Methods and results: The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2.

Conclusions: Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

No MeSH data available.


Related in: MedlinePlus