PRODUCTION OF 1,25-DIHYDROXYVITAMIN-D3 AND 24,25-DIHYDROXYVITAMIN-D3 BY GROWTH ZONE AND RESTING ZONE CHONDROCYTES IS DEPENDENT ON CELL MATURATION AND IS REGULATED BY HORMONES AND GROWTH-FACTORS

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 have been shown to promote chondrocyte proliferation and differentiation; resting zone chondrocytes respond primarily to 24,25-(OH)2D3, whereas growth zone chondrocyte respond primarily to 1,25-(OH)2D3. This study determined whether resting zone and growth zone cells produce 24,25-(OH)2D3 or 1,25-(OH)2D3; whether this production is regulated by 1,25-(OH)2D, (10(-8) M), 24,25-(OH)2D3 (10(-7) M), dexamethasone (10(-7) M), or recombinant human transforming growth factor-beta-1 (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 [H-3]25-hydroxyvitamin D3 ([H-3]25OHD3), 2% fetal bovine serum, and antibiotics. Metabolism of [H-3]25OHD3 was measured by analyzing the lipid extracts of the conditioned medium and the cell layer for [H-3]1,25OHD3, [H-3]1,25-(OH)2D3, and [H-3]24,25-(OH)2D, using flow-through scintillation spectroscopy of HPLC eluates. Chemically synthesized radioinert vitamin D3 metabolites were used as standards, and their migration was determined by absorbance at 254 nm. To ensure that the radioactive peaks were 1,25-(OH)2D3 and 24,25-(OH)2D3, the fractions were rechromatographed into three other HPLC solvent systems. Biological activity was confirmed; the addition of HPLC-purified 1,25-(OH)2D3 produced 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 [H-3]1,25-(OH)2D3 and [H-3]24,25-(OH)2D, in the conditioned medium of both types of cultures. At 24 h, the percent conversion of [H-3]25OHD, to [H-3]1,25-(OH)2D, was 5.3 +/- 1.2, and the percent conversion to [H-3]24,25-(OH)2D, was 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 [H-3]25OHD3, the percent conversion to 1,25-(OH)2D3 and 24,25-(OH)2D3 was 4.5 +/- 1.0 and 1.7 +/- 0.4, respectively. The addition of dexamethasone significantly increased the percent production of 1,25-(OH)2D, at 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-beta-1 increased the percent production of 1,25-(OH)2D3 after 1 h in resting zone cells and, after 24 h, the production of 24,25-(OH)2D, in growth zone cells. Radiolabeled 1,25-(OH)2D3 and 24,25-(OH)2D3 were not detected in the cell layer. The results of this study demonstrate that growth zone and resting zone chondrocytes can metabolize 25OHD3 to both 1,25-(OH)2D3 and 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)2D3 and 24,25-(OH)2D3 metabolites may exert an autocrine effect on chondrocytes. The results also suggest that the production of vitamin D3 by chondrocytes is regulated by hormones and factor(s); this regulation is dependent on cell maturation.