Evidence that the self-induced metabolism of 1,25-dihydroxyvitamin D-3 limits the homologous up-regulation of its receptor in rat osteosarcoma cells

Abstract

The osteoblast-like osteosarcoma cell line UMR-106 has been shown to possess high-affinity receptors for 1,25-dihydroxyvitamin D (1,25-(OH) 2D 3). Also, these cells metabolize 1,25-(OH) 2D 3 to more polar metabolites. As previously demonstrated (Pols, H.A.P., et al. (1987) Biochim. Biophys. Acta 931, 115–119) the time course of specific binding of 1,25-(OH) 2D 3 in intact UMR-106 cells was found to be characterised by (a) an ascending phase, representing association with receptor, (b) a maximum at 90–120 min and (c) a rapid descending phase, closely associated with a decrease of medium 1,25-(OH) 2D 3 due to the metabolism of the hormone. The purpose of the present study was to investigate further the self-induced metabolism of 1,25-(OH) 2D 3 in relation to the homologous up-regulation of its receptor in these cells. Inhibition of metabolism of 1,25-(OH) 2D 3 with ketoconazole resulted, after a lag-time of about 90 min, in a sharp increase of receptor accumulation. This increase in receptor level in the presence of ketoconazole was blocked by coincubation with cycloheximide and actinomycin D. Preincubation experiments with unlabeled 1,25-(OH) 2D 3 showed that the elevation of hormone binding was 1,25-(OH) 2D 3-concentration dependent (ED 50 200–300 pM). Addition of ketoconazole during these preincubations resulted in an even more pronounced accumulation of receptors, whereby the ED 50 (50–60 pM) was comparable with the dissociation constant of the 1,25-(OH) 2D 3 receptor (41.3 ± 4.3pM). In summary, these data support the concept that the self-induced metabolism of 1,25-(OH) 2D 3 has a dual effect: (1) directly, by the regulation of the cellular concentration of and, consequently, receptor occupancy by the active form of vitamin D and (2) indirectly by its ability to modulate the ligand-dependent regulation of the 1,25-(OH) 2D 3.