Synthesis of 25-Hydroxyvitamin D₃-26,23-Lactone but not 24,25-Dihydroxyvitamin D₃ from 25-Hydroxyvitamin D₃ in Opossum Kidney Cells Treated with 1α,25-Dihydroxyvitamin D₃

Abstract

The pathway from 25-hydroxyvitamin D₃ (25[OH]D₃) to 25-hydroxyvitamin D₃-26,23-lactone (25[OH]D₃-26,23-lactone) is important in the renal degradation of vitamin D metabolites. Our objective was to investigate the regulation of 25(OH)D₃ metabolism in the opossum kidney (OK) cell line which has been used for studying the actions of the calcium-regulating hormones. While the untreated OK cells did not produce 25(OH)D₃ metabolites, the synthesis of the lactone was increased dose-dependently by 20 h pretreatment with 1α,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃), peaked at 10^-7 M 1,25(OH)₂D₃, and decreased with higher concentrations. Little production of 24,25-dihydroxyvitamin D₃ (24,25[OH]₂D₃) was observed after any dose of 1,25(OH)₂D₃. The increase in the production of lactone induced by 10^-7 M 1,25(OH)₂D₃ was detectable at 2 h, peaked between 4 and 24 h, and decreased gradually until 48 h. Its induction was inhibited by actinomycin D and cycloheximide, suggesting that de novo protein synthesis is required for such induction. After treatment with 10^-7 M 1,25(OH)₂D₃ for 20 h, 24-hydroxylase (24-OHase) mRNA was not detected in OK cells, but was found evident in COS-7 cells (a renal cell line). Moreover, the COS-7 cells treated with 10^-7 M 1,25(OH)₂D₃ metabolized 25(OH)D₃ to 24,25(OH)₂D₃. These results indicate that 1,25(OH)₂D₃ stimulates the synthesis of 25(OH)D₃-26,23-lactone in a dose- and time-dependent manner, but does not induce the gene expression or activity of 24-OHase in OK cells.

1α,25-Dihydroxyvitamin D₃ - 24,25-Dihydroxyvitamin D₃ - 25-Hydroxyvitamin D₃-26,23-Lactone - 25-Hydroxyvitamin D₃-24-Hydroxylase - Opossum Kidney Cell