title: Identification of signalling pathways triggered by changes in the mechanical environment in rat chondrocytes.
abstract: OBJECTIVE
  The aim of this work was to determine the pathways implicated in the mechanosensing of chondrocytes.
  
  
  METHODS
  Rat chondrocytes were cultured in collagen hydrogels of different stiffness (2-20 Pa) in normoxia and hypoxia, in monolayer and embedded inside hydrogels. First, chondrocyte were cultured on hydrogels in the presence of antibodies to block integrins. Second, custom RT-PCR array plates and western blot were used to detect changes in expression of genes implicated in downstream signalling pathways.
  
  
  RESULTS
  The results allowed us to demonstrate the mechanosensing of chondrocytes for changes in stiffness in the range of Pascals. We also identified Non-Muscle Myosin II (NMMII) and integrins α1, β1 and β3 as participants in the mechanosensing, since their blockade inhibits the sensing of the stiffness, and they are up-regulated in the process. RT-PCR arrays and western blot detected up-regulation of Paxillin, RhoA, Fos, Jun and Sox9. We detected no expression of Src in the monolayer cultures, but we found a role for this protein in 3D. The expression of HIF-1α was not modified under normoxia but was found to participate under hypoxia. Focal Adhesion Kinase (FAK), showed a direct relationship with the expression of Aggrecan in hypoxia and an inverse one in normoxia. Finally, immunofluorescence analysis located the expression of factors AP-1, Sox-9 and HIF-1α inside the cell nuclei and RhoA, Src, Paxillin and FAK close to the cytoplasmic membrane.
  
  
  CONCLUSIONS
  We determined here some of the genes that are up-regulated during the process of chondrocyte mechanosensing.
authors.affiliation: Laboratory for Orthopaedic Research, University of Navarra, School of Medicine, Pamplona, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Sanz-Ramos
authors.forename: G
authors.initials: G
authors.lastname: Mora
authors.forename: P
authors.initials: P
authors.lastname: Ripalda
authors.forename: M
authors.initials: M
authors.lastname: Vicente-Pascual
authors.forename: I
authors.initials: I
authors.lastname: Izal-Azcárate
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000378:metabolism*; D019902:Chondrocytes / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics*; D016762:Genes, fos / Q000235:genetics; D016758:Genes, jun / Q000235:genetics; D016391:Genes, src / Q000235:genetics; D006614:Hindlimb; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D016023:Integrins / Q000235:genetics / Q000378:metabolism; D024462:Myosin Type II / Q000235:genetics / Q000378:metabolism; D051419:Paxillin / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016023:Integrins; D051419:Paxillin; D055758:SOX9 Transcription Factor; D051417:Focal Adhesion Kinase 1; C496052:Ptk2 protein, rat; D024462:Myosin Type II; D020742:rhoA GTP-Binding Protein