Effects of 24R,25‐ and 1α,25‐dihydroxyvitamin D₃ on mineralizing growth plate chondrocytes

Abstract

Time- and dosage-dependent effects of 1,25(OH)(2)D(3) and 24,25(OH)(2)D(3) on primary cultures of pre- and post-confluent avian growth plate (GP) chondrocytes were examined. Cultures were grown in either a serum-containing culture medium designed to closely mimic normal GP extracellular fluid (DATP5) or a commercially available serum-free media (HL-1) frequently used for studying skeletal cells. Hoechst DNA, Lowry protein, proteoglycan (PG), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) activity and calcium and phosphate mineral deposition in the extracellular matrix were measured. In preconfluent cultures grown in DATP5, physiological levels of 24,25(OH)(2)D(3) (0.10-10 nM) increased DNA, protein, and LDH activity significantly more than did 1,25(OH)(2)D(3) (0.01-1.0 nM). However, in HL-1, the reverse was true. Determining ratios of LDH and PG to DNA, protein, and each other, revealed that 1,25(OH)(2)D(3) specifically increased PG, whereas 24,25(OH)(2)D(3) increased LDH. Post-confluent cells were generally less responsive, especially to 24,25(OH)(2)D(3). The positive anabolic effects of 24,25(OH)(2)D(3) required serum-containing GP-fluid-like culture medium. In contrast, effects of 1,25(OH)(2)D(3) were most apparent in serum-free medium, but were still significant in serum-containing media. Administered to preconfluent cells in DATP5, 1,25(OH)(2)D(3) caused rapid, powerful, dosage-dependent inhibition of Ca(2+) and Pi deposition. The lowest level tested (0.01 nM) caused >70% inhibition during the initial stages of mineral deposition; higher levels of 1,25(OH)(2)D(3) caused progressively more profound and persistent reductions. In contrast, 24,25(OH)(2)D(3) increased mineral deposition 20-50%; it required >1 week, but the effects were specific, persistent, and largely dosage-independent. From a physiological perspective, these effects can be explained as follows: 1,25(OH)(2)D(3) levels rise in hypocalcemia; it stimulates gut absorption and releases Ca(2+) from bone to correct this deficiency. We now show that 1,25(OH)(2)D(3) also conserves Ca(2+) by inhibiting mineralization. The slow anabolic effects of 24,25(OH)(2)D(3)are consistent with its production under eucalcemic conditions which enable bone formation. These findings, which implicate serum-binding proteins and accumulation of PG in modulating accessibility of the metabolites to GP chondrocytes, also help explain some discrepancies previously reported in the literature.

24R,25-Dihydroxyvitamin D3 [24R,25(OH)2D3] controls growth plate development by inhibiting apoptosis in the reserve zone and stimulating response to 1α,25(OH)2D3 in hypertrophic cells

“…Similar observations have been made in avian growth plate chondrocytes [3]. 24R,25(OH) 2 D 3 -mediated effects are maintained in mice lacking the nuclear vitamin D receptor (nVDR) [4] and are not inhibited by Ab99, a blocking antibody targeted against the 1,25-dihydroxyvitamin D3 [1␣,25(OH) 2 [5].…”

Genetic ancestry, skin reflectance and pigmentation genotypes in association with serum vitamin D metabolite balance

“…[42-52] However, in clinical trials 24,25(OH) 2 D 3 has not consistently increased bone mass or suppressed parathyroid hormone (PTH), and does not appear to influence serum concentrations of 1,25(OH) 2 D 3 and 25(OH)D 3 . [50, 53] Although CYP24A1 is the gene most highly-inducible by 1,25(OH) 2 D 3 ,[54] there is little information regarding racial/ethnic differences in CYP24A1 expression and its influence on vitamin D metabolite levels.…”