title: 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.
abstract: 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.
authors.affiliation: a School of Traditional Chinese Medicine , Southern Medical University , Guangzhou , China.
authors.forename: Yu-Cong
authors.initials: YC
authors.lastname: Zou
authors.affiliation: b The Third Affiliated Hospital , Guangzhou University of Traditional Chinese Medicine , Guangzhou , China.
authors.forename: Xian-Wen
authors.initials: XW
authors.lastname: Yang
authors.affiliation: a School of Traditional Chinese Medicine , Southern Medical University , Guangzhou , China.
authors.forename: Shi-Guo
authors.initials: SG
authors.lastname: Yuan
authors.affiliation: a School of Traditional Chinese Medicine , Southern Medical University , Guangzhou , China.
authors.forename: Pei
authors.initials: P
authors.lastname: Zhang
authors.affiliation: c Department of Emergency , Guang Zhou Orthopedics Hospital , Guangzhou , China.
authors.forename: Yong-Liang
authors.initials: YL
authors.lastname: Ye
authors.affiliation: a School of Traditional Chinese Medicine , Southern Medical University , Guangzhou , China.
authors.forename: Yi-Kai
authors.initials: YK
authors.lastname: Li
mesh_terms: D000328:Adult; D017209:Apoptosis; D015153:Blotting, Western; D016022:Case-Control Studies; D002453:Cell Cycle; D049109:Cell Proliferation; D002469:Cell Separation; D015536:Down-Regulation; D005260:Female; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology*; D006615:Hip / Q000473:pathology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D010012:Osteogenesis; D013167:Spondylitis, Ankylosing / Q000473:pathology*; D013194:Staining and Labeling; D014162:Transfection; D060449:Wnt Signaling Pathway
chemical_list: C501126:DKK1 protein, human; D036341:Intercellular Signaling Peptides and Proteins
keywords: Ankylosing spondylitis; dickkopf-1; fibroblasts; osteogenesis