Vitamin D-dependent rickets type II. Defective induction of 25-hydroxyvitamin D₃-24-hydroxylase by 1,25-dihydroxyvitamin D₃ in cultured skin fibroblasts

1,25(OH)₂D₃ induces 25(OH)D₃-24-hydroxylase (24-OHase) in cultured skin fibroblasts from normal subjects. We evaluated 24-OHase induction by 1,25(OH)₂D₃ in skin fibroblasts from 10 normal subjects and from four unrelated patients with hereditary resistance to 1,25(OH)₂D or vitamin D-dependent rickets type II (DD II). Fibroblasts were preincubated with varying concentrations of 1,25(OH)₂D₃ for 15 h and were then incubated with 0.5 μM [³H]25(OH)D₃ at 37°C for 30 min; lipid extracts of the cells were analyzed for [³H]24,25(OH)₂D₃ by high performance liquid chromatography and periodate oxidation. Apparent maximal [³H]24,25(OH)₂D₃ production in normal cell lines was 9 pmol/10⁶ cells per 30 min and occurred after induction with 10^-8 M 1,25(OH)₂D₃. 24-OHase induction was detectable in normal fibroblasts at ~3 x 10^-10 M 1,25(OH)₂D₃. [³H]24,25(OH)₂D₃ formation after exposure to 1,25(OH)₂D₃ was abnormal in fibroblasts from all four patients with DD II. In fibroblasts from two patients with DD II, [³H]24,25(OH)₂D₃ formation was unmeasurable (below 0.2 pmol/10⁶ cells per 30 min) at 1,25(OH)₂D₃ concentrations up to 10^-6 M. Fibroblasts from the other two patients with DD II required far higher than normal concentrations of 1,25(OH)₂D₃ for detectable [³H]24,25(OH)₂D₃ induction. In one, [³H]24,25(OH)₂D₃ production reached 2.9 pmol/10⁶ cells per 30 min at 10^-6 M 1,25(OH)₂D₃ (30% normal maximum at 10^-6 M 1,25(OH)₂D₃). In the other, [³H]24,25(OH)₂D₃ production achieved normal levels, 7.3 pmol/10⁶ cells per 30 min after 10^-6 M 1,25(OH)₂D₃. The two patients whose cells had a detectable 24-OHase induction by 1,25(OH)₂D₃ showed a calcemic response to high doses of calciferols in vivo. Our current observations correlate with these two patients' responsiveness to calciferols in vivo and suggest that their target organ defects can be partially or completely overcome with extremely high concentrations of 1,25(OH)₂D. The two patients whose cells showed no detectable 24-OHase induction in vitro failed to show a calcemic response to high doses of calciferols in vivo. In conclusion: (a) The measurement of 24-OHase induction by 1,25(OH)₂D₃ in cultured skin fibroblasts is a sensitive in vitro test for defective genes in the 1,25(OH)₂D effector pathway. (b) This assay provides a useful tool for characterizing the target tissue defects in DD II and predicting response to calciferol therapy.