title: 1,25-Dihydroxyvitamin D(3) and extracellular inorganic phosphate activate mitogen-activated protein kinase pathway through fibroblast growth factor 23 contributing to hypertrophy and mineralization in osteoarthritic chondrocytes.
abstract: 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.
authors.affiliation: Laboratory of Cytogenetics and Molecular Genetics, Faculty of Medicine, University of Thessaly, Larissa 41110, Greece..
authors.forename: Timoklia
authors.initials: T
authors.lastname: Orfanidou
authors.forename: Konstantinos N
authors.initials: KN
authors.lastname: Malizos
authors.forename: Sokratis
authors.initials: S
authors.lastname: Varitimidis
authors.forename: Aspasia
authors.initials: A
authors.lastname: Tsezou
mesh_terms: D000368:Aged; D015153:Blotting, Western; D002114:Calcinosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002358:Cartilage, Articular / Q000378:metabolism / Q000473:pathology; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology; D005260:Female; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006984:Hypertrophy; D047468:Immunoprecipitation; D008297:Male; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010003:Osteoarthritis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010710:Phosphates / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D030181:Vitamin D Response Element / Q000235:genetics
chemical_list: C000717427:FGF23 protein, human; D010710:Phosphates; D014807:Vitamin D; D005346:Fibroblast Growth Factors; C097949:1,25-dihydroxyvitamin D; D000089703:Fibroblast Growth Factor-23; D020928:Mitogen-Activated Protein Kinases