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Chondrocytes’ hypertrophy includes metabolic changes, matrix remodelling, proliferation and apoptosis, characteristics associated with the progression of osteoarthritis. We investigated a possible association among Runt-related transcription factor 2 (RUNX-2), SOX-9 and fibroblast growth factor (FGF)-23 mRNA expressions in articular chondrocytes in order to elucidate their contribution in the osteoarthritic hypertrophic cartilage. SOX-9, FGF-23, RUNX-2 and matrix metalloproteinase (MMP)-13 mRNA expressions were evaluated in osteoarthritic and normal chondrocytes by real-time PCR whereas MMP-13 protein expression by immunofluorescense. RUNX-2, FGF-23 and SOX-9 were down-regulated using small interfering RNA technology and transfection with liposomes. The effect of human recombinant FGF-23 (hrFGF-23) on SOX-9 and RUNX-2 expression was tested in normal chondrocytes. We found higher expression of RUNX-2 and FGF-23 and a decreased expression of SOX-9 mRNA in osteoarthritic chondrocytes compared to normal (P < 0.0001). RUNX-2 down-regulation resulted in reduced MMP-13 expression in osteoarthritic chondrocytes and inhibition of SOX-9 in increased RUNX-2 and MMP-13 mRNA expression in normal chondrocytes, whereas inhibition of FGF-23 resulted in reduced RUNX-2 mRNA expression in osteoarthritic chondrocytes (all P < 0.0001). Silencing of RUNX-2 or FGF-23 did not affect SOX-9 mRNA levels in osteoarthritic chondrocytes. Moreover simultaneous down-regulation of SOX-9 and up-regulation of FGF-23 mRNA expressions in normal chondrocytes resulted in additive up-regulation of RUNX-2 mRNA expression. Treatment of normal chondrocytes with hrFGF-23 resulted in increased RUNX-2 mRNA expression, whereas it had no effect on SOX-9 mRNA expression. We demonstrated convincing associations among RUNX-2, SOX-9 and FGF-23 in relation to MMP-13 expression in osteoarthritic chondrocytes, contributing to a better understanding of the abnormal gene expression and cartilage degeneration processes associated with osteoarthritis.

Hypertrophy and impaired mineralization are two processes closely associated with osteoarthritis (OA). 1,25-dihydroxyvitamin D(3) (1a,25(OH)(2)D(3)) and inorganic phosphate (Pi) are two important factors that are implicated in calcium and phosphate homeostasis of bone metabolism and both can be regulated by the circulating phosphaturic factor fibroblast growth factor 23 (FGF23). The objective of this study was to investigate the role of 1a,25(OH)(2)D(3) and Pi and the molecular mechanism through which they contribute to hypertrophy and mineralization in human osteoarthritic chondrocytes. For this purpose, primary human chondrocytes were obtained from articular cartilage which was collected after total knee replacement surgery in OA patients. FGF23, fibroblast growth factor receptor 1c (FGFR1c), vitamin D(3) receptor (VDR), and phosphate inorganic transporter-1 and -2 (PiT-1 and PiT-2) expression levels were evaluated and found to be significantly higher in OA chondrocytes compared with normal. In addition, we observed that the binding of FGF23 to FGFR1c was stronger in OA chondrocytes compared  with normal. Chromatin immunoprecipitation (ChIP) assay revealed, for the first time, the presence of two vitamin D response elements (VDREs) in the FGF23 promoter. Treatment of normal chondrocytes with 1a,25(OH)(2)D(3) or Pi resulted in significant up-regulation of VDR, FGF23, PiT-1, PiT-2 mRNA and protein levels, extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation and induction of hypertrophy markers collagen type X (COL10A1), osteopontin (OPN), osteocalcin (OC), catabolic markers metalloproteinase-13 (MMP-13) and the apoptotic marker caspase-9. Furthermore, VDR silencing in OA chondrocytes negatively regulated FGF23, COL10A1, OPN, OC, MMP-13 and caspase-9 expressions and ERK1/2 phosphorylation. Finally, combined VDR silencing and PiT-1, PiT-2 inhibition in OA chondrocytes resulted in additive down-regulation of FGF23 expression, ERK1/2 activation and COL10A1, OPN, OC, MMP-13 and caspase-9 expression levels. We propose that 1a,25(OH)(2)D(3) and Pi act synergistically through FGF23 signaling and ERK1/2 phosphorylation contributing to late hypertrophic events and impaired mineralization in osteoarthritic chondrocytes.

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