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CCN5 attenuates profibrotic phenotypes of fibroblasts through the Smad6-CCN2 pathway: Potential role in epidural fibrosis.

Xu H, Liu C, Sun Z, Guo X, Zhang Y, Liu M, Li P - Int. J. Mol. Med. (2015)

Bottom Line: CCN5 upregulation also reduced an increase in collagen type I, α1 (COL1A1) and total collagen concentrations.Additionally, CCN5 over-expression decreased CCN2 expression and increased Smad6 phosphorylation.Mechanism analysis revealed that blocking Smad6 signaling significantly ameliorated the inhibitory effect of CCN5 on the CCN2 levels, accompanied by the reduction in cell proliferation and collagen production.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.

ABSTRACT
Epidural fibrosis is characterized by the development of dense and thick scar tissue adjacent to the dural mater and ranked as the major contributor for post-operative pain recurrence after laminectomy or discectomy. Recently, CCN5 exhibited an inhibitory effect on connective tissue growth factor (CTGF)/CCN2 (a critical regulator for fibrotic disease)‑mediated fibrogenesis. However, its function in epidural fibrosis and the underlying mechanisms involved remain to be determined. In this study, an obvious downregulation of CCN5 was observed in scar tissues from laminectomized rats, concomitant with a marked upregulation of CCN2, suggesting a potential negative regulatory role of CCN5 in fibrogenesis. Furthermore, CCN5 overexpression notably mitigated transforming growth factor‑β1-enhanced fibroblast viability and proliferation. Of note, CCN5 upregulation inhibited the switch of fibroblasts into myofibroblasts as its overexpression abrogated the expression of the myofibroblast marker, α-smooth muscle actin (α-SMA). CCN5 upregulation also reduced an increase in collagen type I, α1 (COL1A1) and total collagen concentrations. Additionally, CCN5 over-expression decreased CCN2 expression and increased Smad6 phosphorylation. Mechanism analysis revealed that blocking Smad6 signaling significantly ameliorated the inhibitory effect of CCN5 on the CCN2 levels, accompanied by the reduction in cell proliferation and collagen production. These results confirm that CCN5 exerts an anti-fibrotic function by regulating the Smad6-CCN2 pathway, thereby indicating a potential approach for ameliorating epidural fibrosis after laminectomy.

No MeSH data available.


Related in: MedlinePlus

Effect of CCN5 on the profibrotic phenotype of fibroblasts induced by transforming growth factor-β (TGF-β1). The isolated fibroblasts were transfected or not with LV-CCN5 prior to TGF-β1 stimulation. The expression levels of α-smooth muscle actin (α-SMA), a specific marker of myofibroblasts, were analyzed by western blotting (A). mRNA (B) and protein levels (C) of CLO1A1 were asssessed. The total collagen concentration was assessed using a Sircol assay kit (D). *P<0.05.
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f4-ijmm-36-01-0123: Effect of CCN5 on the profibrotic phenotype of fibroblasts induced by transforming growth factor-β (TGF-β1). The isolated fibroblasts were transfected or not with LV-CCN5 prior to TGF-β1 stimulation. The expression levels of α-smooth muscle actin (α-SMA), a specific marker of myofibroblasts, were analyzed by western blotting (A). mRNA (B) and protein levels (C) of CLO1A1 were asssessed. The total collagen concentration was assessed using a Sircol assay kit (D). *P<0.05.

Mentions: To assess the function of CCN5 during the development of scar formation, the anti-fibrotic effect of CCN5 was examined. α-SMA is believed to be a biochemical marker of myofibroblasts transformed from fibroblasts, which result in the excessive accumulation of fibrotic tissue and subsequent scar formation and adhesion (18). Therefore, we assessed the effect of CCN5 on α-SMA and the results confirmed that CCN5 overexpression markedly decreased α-SMA levels induced by TGF-β1 (Fig. 4A), suggesting that CCN5 attenuated the transformation of fibroblasts into profibrotic myofibroblasts. COL1A1 is a major ECM protein that is able to induce collagen I formation, which is the most abundant product of fibrosis. Further analysis exhibited a notable upregulation in COL1A1 mRNA levels (Fig. 4B) and protein levels (Fig. 4C) after TGF-β1 stimulation. However, this increase was obviously attenuated when preconditioning with LV-CCN5 transfection. Simultaneously, CCN5 over expression also markedly downregulated the total collagen concentration (Fig. 4D), indicating that CCN5 antagonized TGF-β1-induced profibrotic phenotype of fibroblasts.


CCN5 attenuates profibrotic phenotypes of fibroblasts through the Smad6-CCN2 pathway: Potential role in epidural fibrosis.

Xu H, Liu C, Sun Z, Guo X, Zhang Y, Liu M, Li P - Int. J. Mol. Med. (2015)

Effect of CCN5 on the profibrotic phenotype of fibroblasts induced by transforming growth factor-β (TGF-β1). The isolated fibroblasts were transfected or not with LV-CCN5 prior to TGF-β1 stimulation. The expression levels of α-smooth muscle actin (α-SMA), a specific marker of myofibroblasts, were analyzed by western blotting (A). mRNA (B) and protein levels (C) of CLO1A1 were asssessed. The total collagen concentration was assessed using a Sircol assay kit (D). *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494601&req=5

f4-ijmm-36-01-0123: Effect of CCN5 on the profibrotic phenotype of fibroblasts induced by transforming growth factor-β (TGF-β1). The isolated fibroblasts were transfected or not with LV-CCN5 prior to TGF-β1 stimulation. The expression levels of α-smooth muscle actin (α-SMA), a specific marker of myofibroblasts, were analyzed by western blotting (A). mRNA (B) and protein levels (C) of CLO1A1 were asssessed. The total collagen concentration was assessed using a Sircol assay kit (D). *P<0.05.
Mentions: To assess the function of CCN5 during the development of scar formation, the anti-fibrotic effect of CCN5 was examined. α-SMA is believed to be a biochemical marker of myofibroblasts transformed from fibroblasts, which result in the excessive accumulation of fibrotic tissue and subsequent scar formation and adhesion (18). Therefore, we assessed the effect of CCN5 on α-SMA and the results confirmed that CCN5 overexpression markedly decreased α-SMA levels induced by TGF-β1 (Fig. 4A), suggesting that CCN5 attenuated the transformation of fibroblasts into profibrotic myofibroblasts. COL1A1 is a major ECM protein that is able to induce collagen I formation, which is the most abundant product of fibrosis. Further analysis exhibited a notable upregulation in COL1A1 mRNA levels (Fig. 4B) and protein levels (Fig. 4C) after TGF-β1 stimulation. However, this increase was obviously attenuated when preconditioning with LV-CCN5 transfection. Simultaneously, CCN5 over expression also markedly downregulated the total collagen concentration (Fig. 4D), indicating that CCN5 antagonized TGF-β1-induced profibrotic phenotype of fibroblasts.

Bottom Line: CCN5 upregulation also reduced an increase in collagen type I, α1 (COL1A1) and total collagen concentrations.Additionally, CCN5 over-expression decreased CCN2 expression and increased Smad6 phosphorylation.Mechanism analysis revealed that blocking Smad6 signaling significantly ameliorated the inhibitory effect of CCN5 on the CCN2 levels, accompanied by the reduction in cell proliferation and collagen production.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.

ABSTRACT
Epidural fibrosis is characterized by the development of dense and thick scar tissue adjacent to the dural mater and ranked as the major contributor for post-operative pain recurrence after laminectomy or discectomy. Recently, CCN5 exhibited an inhibitory effect on connective tissue growth factor (CTGF)/CCN2 (a critical regulator for fibrotic disease)‑mediated fibrogenesis. However, its function in epidural fibrosis and the underlying mechanisms involved remain to be determined. In this study, an obvious downregulation of CCN5 was observed in scar tissues from laminectomized rats, concomitant with a marked upregulation of CCN2, suggesting a potential negative regulatory role of CCN5 in fibrogenesis. Furthermore, CCN5 overexpression notably mitigated transforming growth factor‑β1-enhanced fibroblast viability and proliferation. Of note, CCN5 upregulation inhibited the switch of fibroblasts into myofibroblasts as its overexpression abrogated the expression of the myofibroblast marker, α-smooth muscle actin (α-SMA). CCN5 upregulation also reduced an increase in collagen type I, α1 (COL1A1) and total collagen concentrations. Additionally, CCN5 over-expression decreased CCN2 expression and increased Smad6 phosphorylation. Mechanism analysis revealed that blocking Smad6 signaling significantly ameliorated the inhibitory effect of CCN5 on the CCN2 levels, accompanied by the reduction in cell proliferation and collagen production. These results confirm that CCN5 exerts an anti-fibrotic function by regulating the Smad6-CCN2 pathway, thereby indicating a potential approach for ameliorating epidural fibrosis after laminectomy.

No MeSH data available.


Related in: MedlinePlus