title: Self-induction of 1,25-dihydroxyvitamin D3 metabolism limits receptor occupancy and target tissue responsiveness.
abstract: Whole cell 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptor (VDR) binding assays, which measure VDR in the presence of the metabolic machinery of the cell, were used in conjunction with a cytosol binding assay for VDR to determine if self-induced metabolism of 1,25-(OH)2D3 limits VDR occupancy, total VDR levels, and target cell responsiveness. Treatment of cells with 0.5 nM 1,25-(OH)2[3H]D3 for 16 h results in up-regulation of total cell VDR from 82 to 170 fmol/mg protein as measured in a cytosol binding assay. Conversely, whole cell binding assays of VDR showed a 1,25-(OH)2D3-mediated apparent down-regulation of VDR from 90 to 40 fmol/mg protein. Scatchard analysis using the cytosol binding assay demonstrated that 1,25-(OH)2D3 treatment increased total cell VDR from 93 to 154 fmol/mg protein. In contrast, Scatchard analysis with the whole cell binding assay demonstrated that 1,25-(OH)2D3 treatment resulted in reduction in total cell VDR from 100 to 64 fmol/mg protein. Initial Kd estimates with the whole cell binding assay suggested that 1,25-(OH)2D3 treatment resulted in a reduction in VDR Kd from 0.6 to 6.2 nM. This apparent reduction in the affinity of VDR for 1,25-(OH)2D3 was due to degradation of free 1,25-(OH)2[3H]D3 which occurred during whole cell saturation assay. Competitive inhibitors of 1,25-(OH)2D3 metabolism were found to reverse the apparent receptor down-regulation observed in whole cell binding assays of treated cells. In addition, the presence of competitive inhibitors amplified responses of cells to 1,25-(OH)2[3H]D3 treatment as measured by an increased occupancy of VDR by 1,25-(OH)2[3H]D3 and increased up-regulation of VDR over that observed without metabolism inhibitors. These data demonstrate that self-induced target tissue deactivation of 1,25-(OH)2D3 regulates 1,25-(OH)2D3 occupancy of VDR and ultimately the biopotency of 1,25-(OH)2D3 in target cells.
authors.affiliation: National Animal Disease Center, United States Department of Agriculture, Ames, Iowa 50010.
authors.forename: T A
authors.initials: TA
authors.lastname: Reinhardt
authors.forename: R L
authors.initials: RL
authors.lastname: Horst
mesh_terms: D001667:Binding, Competitive; D001943:Breast Neoplasms; D002117:Calcitriol / Q000378:metabolism*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005260:Female; D006706:Homeostasis; D006801:Humans; D007700:Kinetics; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism*
chemical_list: D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D002117:Calcitriol