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Co-expression of 1α-hydroxylase and vitamin D receptor in human articular chondrocytes.

BMC musculoskeletal disorders (2017-11-08)
Ann Kristin Hansen, Yngve Figenschau, Inigo Zubiaurre-Martinez

The aim was to investigate whether resident chondrocytes in human articular cartilage and in subculture express vitamin D receptor (VDR) and the enzyme that hydroxylates the prohormone 25(OH)D Cartilage from human osteoarthritic knee joints, cultured chondrocytes and cells grown in 3D spheroids were examined for the expression of VDR and 1α-hydroxylase by PCR, Western blots and immunolabelling. Receptor engagement was judged by visualizing nuclear translocation. The effects of 25(OH)D Gene expression was positive for VDR in freshly isolated cells from native cartilage, cells subcultured in monolayers and in spheroids, whereas protein expression, otherwise judged low, was apparent in monolayers. Nuclear translocation of VDR occurred upon 1α,25(OH) VDR expression in resident cartilage chondrocytes, generally considered differentiated cells, is elusive. A similar pattern applies for redifferentiated chondrocytes in spheroid cultures, whereas dedifferentiated cells, established in monolayers, stably express VDR. Both 25(OH)D

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Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose, L-glutamine, and sodium bicarbonate, without sodium pyruvate, liquid, sterile-filtered, suitable for cell culture
Trypsin-EDTA solution, 0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks′ Balanced Salt Solution with phenol red
Penicillin-Streptomycin, with 10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, 0.1 μm filtered, BioReagent, suitable for cell culture
Collagenase from Clostridium histolyticum, powder, Suitable for the digestion and isolation of physiologically active pancreatic islet cells, suitable for cell culture
TruPAGE Precast Gels, 4-12%, 10 x 10cm, 12-well
Anti-beta-Actin antibody, Rabbit monoclonal, recombinant, expressed in proprietary host, clone SP124, affinity isolated antibody