Evidence that the self-induced metabolism of 1,25-dihydroxyvitamin D-3 limits the homologous up-regulation of its receptor in rat osteosarcoma cells.
The osteoblast-like osteosarcoma cell line UMR-106 has been shown to possess high-affinity receptors for 1,25-dihydroxyvitamin D (1,25-(OH)2D3). Also, these cells metabolize 1,25-(OH)2D3 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)2D3 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)2D3 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)2D3 in relation to the homologous up-regulation of its receptor in these cells. Inhibition of metabolism of 1,25-(OH)2D3 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)2D3 showed that the elevation of hormone binding was 1,25-(OH)2D3-concentration dependent (ED50 200-300 pM). Addition of ketoconazole during these preincubations resulted in an even more pronounced accumulation of receptors, whereby the ED50 (50-60 pM) was comparable with the dissociation constant of the 1,25-(OH)2D3 receptor (41.3 +/- 4.3 pM). In summary, these data support the concept that the self-induced metabolism of 1,25-(OH)2D3 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)2D3.
Pols
H A
HA
Department of Internal Medicine III, Erasmus University Medical School, Rotterdam, The Netherlands.
Birkenhäger
J C
JC
Schilte
J P
JP
Visser
T J
TJ
D000818:Animals; D002117:Calcitriol / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D012516:Osteosarcoma / Q000378:metabolism*; D051381:Rats; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000378:metabolism
D003470:Culture Media; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D003609:Dactinomycin; D003513:Cycloheximide; D002117:Calcitriol; D007654:Ketoconazole