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

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.

Figure 1

Expression of CCN5 and CCN2 in scar tissues after laminectomy. To determine the expression levels of CCN5 and CCN2, 24 healthy male Lewis rats (12 weeks old) were subjected to laminectomy. Then, the scar and surrounding normal tissues were collected. The expression levels of CCN5 and CCN2 were detected by RT-PCR (A). The corresponding protein levels were also evaluated by western blotting (B). ^*P<0.05 vs. normal tissues.

Figure 2

CCN5 transfection enhances CCN5 expression in primary fibroblasts. Primary fibroblasts were obtained from the tail skin of rats and cultured in DMEM medium. Lentivirus plasmid pWPT-GFP was introduced to construct the recombinant pWPT-CCN5 plasmids, following packaging with vectors of pCMV-VSV-G and pCMV-dR8.91. The corresponding transfection effect of CCN5 mRNA (A) and protein levels (B) was assessed individually with RT-PCR and western blotting. ^*P<0.05.

Figure 3

The function of CCN5 on fibroblast cell proliferation and viability. To assess the effect of CCN5 on cell proliferation and viability in response to the transforming growth factor-β (TGF-β) (10 ng/ml) for 12 h, cells were seeded in 24-well plates and treated with LV-CCN5, or vector transfection. Then, 20 µl MTT reagent was added for 6 h to determine the roles of CCN5 overexpression in cell viability (A). Furthermore, the corresponding effects of CCN5 on cell proliferation were analyzed by addition of [³H]-thymidine (B). ^*P<0.05.

Figure 4

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.

Figure 5

CCN5 overexpression attenuates CCN2 levels. Following transfection with the recombinant CCN5, the cells were stimulated with transforming growth factor-β (TGF-β1). The expression levels of CCN2 mRNA (A) and protein (B) were detected by RT-PCR and western blotting, respectively. ^*P<0.05.

Figure 6

CCN5 functions in TGF-β1-induced proliferation and profibrotic phenotype through the Smad6-CCN2 pathway. To investigate the underlying mechanism, the effect of CCN5 on Smad6 signaling was determined (A). After silencing Smad6 levels by preconditioning with its specific siRNA, the expression levels of CCN2 were measured (B). The function of Smad6 in CCN5-triggered inhibitory effects on fibroblast proliferation (C) was ascertained by [³H]-TdR. The corresponding effect on collagen contents was also determined (D). ^*P<0.05.