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Self-induction of 1,25-dihydroxyvitamin D3 metabolism limits receptor occupancy and target tissue responsiveness
1National Animal Disease Center, United States Department of Agriculture, Ames, Iowa 50010.
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.
Insights
Self-induced metabolism of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) limits its receptor (VDR) binding and cell responsiveness. Inhibiting this metabolism enhances VDR occupancy and vitamin D
Area of Science:
- Endocrinology and Molecular Biology
- Vitamin D Metabolism and Signaling
Background:
- The vitamin D receptor (VDR) plays a crucial role in mediating the effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3).
- Understanding VDR regulation is essential for comprehending vitamin D's biological activity and therapeutic potential.
Purpose of the Study:
- To investigate whether self-induced metabolism of 1,25-(OH)2D3 affects VDR occupancy, total VDR levels, and target cell responsiveness.
- To elucidate the role of cellular metabolism in regulating vitamin D signaling.
Main Methods:
- Comparison of whole-cell VDR binding assays with cytosol VDR binding assays.
- Scatchard analysis to determine VDR levels and binding affinity (Kd).
- Assessment of 1,25-(OH)2D3 metabolism using competitive inhibitors.
Main Results:
- Whole-cell assays showed apparent VDR down-regulation due to 1,25-(OH)2D3 degradation, contrasting with cytosol assays indicating VDR up-regulation.
- Scatchard analysis confirmed increased total VDR in cytosol assays but decreased VDR in whole-cell assays.
- Metabolism inhibitors reversed apparent VDR down-regulation and amplified cellular responses to 1,25-(OH)2D3.
Conclusions:
- Cellular metabolism of 1,25-(OH)2D3 leads to its degradation, causing apparent down-regulation of VDR and reduced target cell responsiveness.
- Self-induced metabolism acts as a regulatory mechanism controlling VDR occupancy and the biopotency of 1,25-(OH)2D3.
- Inhibiting 1,25-(OH)2D3 metabolism enhances VDR signaling and cellular responses.