title: Fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2-induced catabolic activities in human articular chondrocytes.
abstract: BACKGROUND
  Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA.
  
  
  METHODS
  Primary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity.
  
  
  RESULTS
  Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5), as well as down-regulation of aggrecan after FGF-2 stimulation. Flow cytometry, qPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGFR isoforms expressed in human articular chondrocytes. FGFR1 was activated more potently than FGFR3 upon FGF-2 stimulation. In osteoarthritic chondrocytes, FGFR3 was significantly down regulated (P < 0.05) with a concomitant increase in the FGFR1 to FGFR3 expression ratio (P < 0.05), compared to normal chondrocytes. Our results also demonstrate that FGFR3 was negatively regulated by FGF-2 at the transcriptional level through the FGFR1-ERK (extracellular signal-regulated kinase) signaling pathway in human articular chondrocytes.
  
  
  CONCLUSIONS
  FGFR1 is the major mediator with the degenerative potential in the presence of FGF-2 in human adult articular chondrocytes. FGFR1 activation by FGF-2 promotes catabolism and impedes anabolism. Disruption of the balance between FGFR1 and FGFR3 signaling ratio may contribute to the pathophysiology of OA.
authors.affiliation: Department of Biochemistry, Rush University Medical Center, 1735 W Harrison Street, Chicago, IL 60612, USA.
authors.forename: Dongyao
authors.initials: D
authors.lastname: Yan
authors.forename: Di
authors.initials: D
authors.lastname: Chen
authors.forename: Simon M
authors.initials: SM
authors.lastname: Cool
authors.forename: Andre J
authors.initials: AJ
authors.lastname: van Wijnen
authors.forename: Katalin
authors.initials: K
authors.lastname: Mikecz
authors.forename: Gillian
authors.initials: G
authors.lastname: Murphy
authors.forename: Hee-Jeong
authors.initials: HJ
authors.lastname: Im
mesh_terms: D015153:Blotting, Western; D002358:Cartilage, Articular / Q000378:metabolism*; D049109:Cell Proliferation; D002469:Cell Separation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D047468:Immunoprecipitation; D010003:Osteoarthritis / Q000378:metabolism*; D034741:RNA, Small Interfering; D060888:Real-Time Polymerase Chain Reaction; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000378:metabolism*; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering; D016222:Fibroblast Growth Factor 2; C496348:FGFR1 protein, human; C496365:FGFR3 protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1; D051498:Receptor, Fibroblast Growth Factor, Type 3; D048049:Extracellular Signal-Regulated MAP Kinases