Downregulation of dickkopf-1 enhances the proliferation and osteogenic potential of fibroblasts isolated from ankylosing spondylitis patients via the Wnt/β-catenin signaling pathway in vitro.

BACKGROUND

Heterotopic ossification of the entheses is one of the most distinctive features in ankylosing spondylitis (AS). Fibroblasts are potential target cells for heterotopic ossification. The Wnt/β-catenin pathway and its inhibitor dickkopf-1 (DKK-1) regulate bone formation. DKK-1 expression in human AS tissues has not been documented.

OBJECTIVE

The purpose of the current study was to investigate the expression of DKK-1 in AS tissues and to elucidate its role in fibroblasts proliferation and osteogenesis in AS.

METHODS

DKK-1 expression was assessed by western blotting, real time-polymerase chain reaction (RT-PCR), and immunohistochemistry analysis of hip synovial tissues obtained from AS and control patients. Fibroblasts were isolated, cultured, and transfected with lentiviral vectors for overexpressing human DKK-1 or an shRNA for silencing DKK-1. MTS [(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium] and a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay were used to detect AS fibroblasts proliferation after transfection. The expression levels of β-catenin, phosphorylated β-catenin, c-Myc, cyclin D1, and the osteogenesis markers alkaline phosphatase (ALP), osteocalcin (OCN), and Runt-related transcription factor 2 (Runx2) were then examined by western blot analysis. Alizarin red staining (ARS) was also used to observe biomineralization activity.

RESULTS

DKK-1 was downregulated in hip synovial tissues from AS patients compared to that observed in controls. AS fibroblasts exhibited excessive proliferation, a higher growth rate, and a decreased apoptotic rate. EdU assay demonstrated that DKK-1 suppressed the growth of AS fibroblasts. Downregulation of DKK-1 decreased the phosphorylation of β-catenin and upregulated the expression of β-catenin, c-Myc, cyclin D1, and osteogenesis markers. Overexpression of DKK-1 had the opposite effect, resulting in the inhibition of the Wnt/β-catenin pathway. ARS showed an increase in biomineralization activity after the inhibition of DKK-1.

CONCLUSIONS

AS fibroblasts are characterized by an imbalance between proliferation and apoptosis. DKK-1 may play a role in switching to new bone formation in AS progression.