title: Src and fibroblast growth factor 2 independently regulate signaling and gene expression induced by experimental injury to intact articular cartilage.
abstract: OBJECTIVE
  To investigate whether cartilage injury activates protein tyrosine kinases distinct from fibroblast growth factor (FGF)-related signaling, and whether they contribute to injury-induced gene responses.
  
  
  METHODS
  Phosphokinases and protein tyrosine phosphorylation were assayed by Western blotting of cartilage lysates. Immunoprecipitation and Western blotting with 4G10 antibody and immunoprecipitation kinase assay were carried out. Tyrosine-phosphorylated proteins on silver-stained gels of injured cartilage lysates were identified by mass spectrometry. Messenger RNA induction in cartilage explants was assessed by quantitative reverse transcriptase-polymerase chain reaction.
  
  
  RESULTS
  Protein tyrosine phosphorylation occurred within seconds of injury to the surface of intact articular cartilage, as did activation of MAPKs and IKK. Activation did not reoccur upon reinjury of cultured explants. The prominent tyrosine-phosphorylated proteins focal adhesion kinase, paxillin, and cortactin were identified as substrates of Src family kinases. The Src family kinase inhibitor PP2 blocked injury-induced tyrosine phosphorylation. It did not prevent activation of the MAPKs and IKK but differentially inhibited 8 of 10 inflammatory response genes that were induced by injury. In contrast, FGF signaling blockade with PD173074 reduced all MAPK and IKK activation by ∼50% and inhibited a different subset of genes but had no effect on Src-like signaling.
  
  
  CONCLUSIONS
  Injury to the surface of intact articular cartilage activates Src-like kinases as well as MAPKs and IKK (implying NF-κB activation). FGF-2 contributes to MAPK/IKK activation but not to Src-like signaling, suggesting that the latter is a parallel pathway that also regulates the injury-induced inflammatory gene response.
authors.affiliation: Kennedy Institute of Rheumatology, University of Oxford, 65 Aspenlea Road, London W6 8LH, UK. fiona.watt@kennedy.ox.ac.uk
authors.forename: Fiona E
authors.initials: FE
authors.lastname: Watt
authors.forename: Heba M
authors.initials: HM
authors.lastname: Ismail
authors.forename: Athanasios
authors.initials: A
authors.lastname: Didangelos
authors.forename: Matthew
authors.initials: M
authors.lastname: Peirce
authors.forename: Tonia L
authors.initials: TL
authors.lastname: Vincent
authors.forename: Robin
authors.initials: R
authors.lastname: Wait
authors.forename: Jeremy
authors.initials: J
authors.lastname: Saklatvala
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000187:drug effects / Q000293:injuries / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011743:Pyrimidines / Q000494:pharmacology; D017468:Receptors, Fibroblast Growth Factor / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013552:Swine; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D016328:NF-kappa B; C115711:PD 173074; D011743:Pyrimidines; D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases