title: DNA methylation regulates sclerostin (SOST) expression in osteoarthritic chondrocytes by bone morphogenetic protein 2 (BMP-2) induced changes in Smads binding affinity to the CpG region of SOST promoter.
abstract: BACKGROUND
  Sclerostin (SOST), a soluble antagonist of Wnt signaling, is expressed in chondrocytes and contributes to chondrocytes' hypertrophic differentiation; however its role in osteoarthritis (OA) pathogenesis is not well known. Based on our previous findings on the interaction between Wnt/β-catenin pathway and BMP-2 in OA, we aimed to investigate the role of DNA methylation and BMP-2 on SOST's expression in OA chondrocytes.
  
  
  METHODS
  SOST mRNA and protein expression levels were investigated using real-time polymerase chain reaction (PCR) and Western blot, respectively. The methylation status of SOST promoter was analysed using methylation-specific PCR (MSP), quantitative methylation-specific PCR (qMSP) and bisulfite sequencing analysis. The effect of BMP-2 and 5'-Aza-2-deoxycytidine (5-AzadC) on SOST's expression levels were investigated and Smad1/5/8 binding to SOST promoter was assessed by Chromatin Immunoprecipitation (ChΙP).
  
  
  RESULTS
  We observed that SOST's expression was upregulated in OA chondrocytes compared to normal. Moreover, we found that the CpG region of SOST promoter was hypomethylated in OA chondrocytes and 5-AzadC treatment in normal chondrocytes resulted in decreased SOST methylation, whereas its expression was upregulated. BMP-2 treatment in 5-AzadC-treated normal chondrocytes resulted in SOST upregulation, which was mediated through Smad 1/5/8 binding on the CpG region of SOST promoter.
  
  
  CONCLUSIONS
  We report novel findings that DNA methylation regulates SOST's expression in OA, by changing Smad 1/5/8 binding affinity to SOST promoter, providing evidence that changes in DNA methylation pattern could underlie changes in genes' expression observed in OA.
authors.affiliation: Laboratory of Cytogenetics and Molecular Genetics, University of Thessaly, Faculty of Medicine, Biopolis, Larissa, 41500, Greece. ioanna_papathanasiou@yahoo.gr.
authors.forename: Ioanna
authors.initials: I
authors.lastname: Papathanasiou
authors.affiliation: Laboratory of Cytogenetics and Molecular Genetics, University of Thessaly, Faculty of Medicine, Biopolis, Larissa, 41500, Greece. fotini.kostopoulou@gmail.com.
authors.forename: Fotini
authors.initials: F
authors.lastname: Kostopoulou
authors.affiliation: Department of Orthopaedic Surgery, University of Thessaly, Faculty of Medicine, Biopolis, Larissa, 41500, Greece. malizos@med.uth.gr.
authors.forename: Konstantinos N
authors.initials: KN
authors.lastname: Malizos
authors.affiliation: Laboratory of Cytogenetics and Molecular Genetics, University of Thessaly, Faculty of Medicine, Biopolis, Larissa, 41500, Greece. atsezou@med.uth.gr.|Department of Biology, University of Thessaly, Faculty of Medicine, Biopolis, Larissa, 41500, Greece. atsezou@med.uth.gr.
authors.forename: Aspasia
authors.initials: A
authors.lastname: Tsezou
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000368:Aged; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis* / Q000235:genetics; D019902:Chondrocytes / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; C422903:SOST protein, human; D051785:Smad Proteins