title: Effects of Wnt3A and mechanical load on cartilage chondrocyte homeostasis.
abstract: BACKGROUND
  Articular cartilage functions in withstanding mechanical loads and provides a lubricating surface for frictionless movement of joints. Osteoarthritis, characterised by cartilage degeneration, develops due to the progressive erosion of structural integrity and eventual loss of functional performance. Osteoarthritis is a multi-factorial disorder; two important risk factors are abnormal mechanical load and genetic predisposition. A single nucleotide polymorphism analysis demonstrated an association of hip osteoarthritis with an Arg324Gly substitution mutation in FrzB, a Wnt antagonist. The purpose of this study was two-fold: to assess whether mechanical stimulation modulates β-catenin signalling and catabolic gene expression in articular chondrocytes, and further to investigate whether there is an interplay of mechanical load and Wnt signalling in mediating a catabolic response.
  
  
  METHODS
  Chondrocytes were pre-stimulated with recombinant Wnt3A for 24 hours prior to the application of tensile strain (7.5%, 1 Hz) for 30 minutes. Activation of Wnt signalling, via β-catenin nuclear translocation and downstream effects including the transcriptional activation of c-jun, c-fos and Lef1, markers of chondrocyte phenotype (type II collagen (col2a1), aggrecan (acan), SOX9) and catabolic genes (MMP3, MMP13, ADAMTS-4, ADAMTS-5) were assessed.
  
  
  RESULTS
  Physiological tensile strain induced col2a1, acan and SOX9 transcription. Load-induced acan and SOX9 expression were repressed in the presence of Wnt3A. Load induced partial β-catenin nuclear translocation; there was an additive effect of load and Wnt3A on β-catenin distribution, with both extensive localisation in the nucleus and cytoplasm. Immediate early response (c-jun) and catabolic genes (MMP3, ADAMTS-4) were up-regulated in Wnt3A stimulated chondrocytes. With load and Wnt3A there was an additive up-regulation of c-fos, MMP3 and ADAMTS-4 transcription, whereas there was a synergistic interplay on c-jun, Lef1 and ADAMTS-5 transcription.
  
  
  CONCLUSIONS
  Our data suggest that load and Wnt, in combination, can repress transcription of chondrocyte matrix genes, whilst enhancing expression of catabolic mediators. Future studies will investigate the respective roles of abnormal loading and genetic predisposition in mediating cartilage degeneration.
authors.affiliation: Welsh School of Pharmacy, Redwood Building, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3NB, UK.
authors.forename: Rhian S
authors.initials: RS
authors.lastname: Thomas
authors.forename: Alan R
authors.initials: AR
authors.lastname: Clarke
authors.forename: Victor C
authors.initials: VC
authors.lastname: Duance
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Blain
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism; D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006706:Homeostasis / Q000187:drug effects* / Q000235:genetics; D051821:Lymphoid Enhancer-Binding Factor 1 / Q000235:genetics / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D013314:Stress, Mechanical; D060509:Wnt3A Protein / Q000235:genetics / Q000494:pharmacology*; D051176:beta Catenin / Q000378:metabolism
chemical_list: D024043:Collagen Type II; D051821:Lymphoid Enhancer-Binding Factor 1; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D055758:SOX9 Transcription Factor; D060509:Wnt3A Protein; D051176:beta Catenin; D051722:ADAM Proteins; D019278:Matrix Metalloproteinase 3