1, 25-Dihydroxyvitamin D3[1, 25-(OH)2D3] and 24, 25-(OH)2D3have been shown to promote chondrocyte proliferation and differentiation; resting zone chondrocytes respond primarily to 24, 25-(OH)2D3, whereas growth zone chondrocytes respond primarily to 1, 25-(OH)2D3. This study determined whether resting zone and growth zone cells produce 24, 25-(OH)2D3or 1, 25-(OH)2D3; whether this production is regulated by 1, 25-(OH)2D3(10-8m), 24, 25-(OH)2D3(10-7m), dexametha-sone (10-7m), or recombinant human transforming growth factor-β1 (11 ng/ml); and whether the metabolites produced are biologically active. Confluent fourth passage rat costochondral growth zone or resting zone chondrocytes were cultured in Dulbecco’s Modified Eagle’s Medium containing [3H]25-hy-droxyvitamin D3([3H]250HD3), 2% fetal bovine serum, and antibiotics. Metabolism of [3H]250HD3was measured by analyzing the lipid extracts of the conditioned medium and the cell layer for [3H]1, 250HD3, [3H]1, 25-(OH)2D3, and [3H]24, 25-(OH)2D3using flow-through scintillation spectroscopy of HPLC eluates. Chemically synthesized radioinert vitamin D3metabolites were used as standards, and their migration was determined by absorbance at 254 nm. To ensure that the radioactive peaks were 1, 25-(OH)2D3and 24, 25-(OH)2D3, the fractions were re-chromatographed into three other HPLC solvent systems. Biological activity was confirmed; the addition of HPLC-purified 1, 25-(OH)2D3produced by growth zone chondrocytes elicited a dose-dependent stimulation of alkaline phosphatase specific activity in growth zone cell cultures, but had no effect on the resting zone cells. There was a time-dependent increase in both [3H]1, 25-(OH)2D3and [3H]24, 25-(OH)2D3in the conditioned medium of both types of cultures. At 24 h, the percent conversion of [3H]250HD3to [3H]1, 25-(OH)2D3was 5.3 ± 1.2, and the percent conversion to [3H]24, 25-(OH)2D3was 1.8 ± 0.4 in growth zone chondrocyte cultures. No such effect was found in cultures freeze-thawed five times or without cells. When resting zone cells were cultured with [3H]250HD3, the percent conversion to 1, 25-(OH)2D3and 24, 25-(OH)2D3was 4.5 ± 1.0 and 1.7 ± 0.4, respectively. The addition of dexamethasone significantly increased the percent production of 1, 25-(OH)2D3at 6 and 24 h and at 6 h by resting zone and growth zone cells, respectively, compared to the control values. Recombinant human transforming growth factor-β1 increased the percent production of 1, 25-(OH)2D3after 1 h in resting zone cells and, after 24 h, the production of 24, 25-(OH)2D3in growth zone cells. Radiolabeled 1, 25-(OH)2D3and 24, 25-(OH)2D3were not detected in the cell layer. The results of this study demonstrate that growth zone and resting zone chondrocytes can metabolize 250HD3to both 1, 25-(OH)2D3and 24, 25-(OH)2D3. Together with previous observations of a direct effect of the metabolites on matrix vesicle membrane enzymes, the data suggest that both 1, 25-(OH)2D3and 24, 25-(OH)2D3metabolites may exert an autocrine effect on chondrocytes. The results also suggest that the production of vitamin D3by chondrocytes is regulated by hormones and factors); this regulation is dependent on cell maturation.
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