Abstract
IntroductionCartilage 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.MethodsPrimary 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.ResultsChondrocyte 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.ConclusionsFGFR1 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.
Highlights
Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA)
QPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGF receptor (FGFR) isoforms expressed in human articular chondrocytes
Our results demonstrate that FGFR3 was negatively regulated by fibroblast growth factor 2 (FGF-2) at the transcriptional level through the FGFR1-extracellular signal-regulated kinase (ERK) signaling pathway in human articular chondrocytes
Summary
Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). The products of cartilage degeneration further promote matrix degradation, and stimulate the synovium to overproduce inflammatory mediators and degrading proteases, which, in turn, exacerbate cartilage matrix loss [2]. Such autocrine and paracrine loops perpetuate joint destruction, frequently resulting in irreversible disease progression. Elevated levels of pro-inflammatory cytokines, inflammatory mediators and certain growth factors potently heighten the expression of matrix-degrading enzymes Destructive proteases such as MMP-13 and ADAMTS-5 are able to cleave major components in the extracellular matrix of chondrocytes, including type II collagen and aggrecan [3,4]. In response to tissue damage, chondrocytes make attempts at matrix repair, but they often fail to restore the eroded cartilage to its original pristine hyaline state, due to multiple impairing mechanisms [5,6,7,8]
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