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

• DOI: 10.3109/03008207.2015.1127916
• Publication Date: 2016-02-03T05:39:08Z
• Authors: Yu-cong Zou; Xian-wen Yang; Shi-guo Yuan; Pei Zhang; Yong-liang Ye; Yi-kai Li
• Keywords: Adult; Male; Hip; Staining and Labeling; Blotting, Western; Cell Cycle; Down-Regulation; Apoptosis; Cell Separation; Fibroblasts; Transfection; 3. Good health; 03 medical and health sciences; 0302 clinical medicine; Osteogenesis; Case-Control Studies; Humans; Intercellular Signaling Peptides and Proteins; Female; Spondylitis, Ankylosing; Wnt Signaling Pathway; Cell Proliferation

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.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.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.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.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.