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Differential effects of 20-epi vitamin D analogs on the vitamin D receptor homodimer
N J Koszewski1, T A Reinhardt, R L Horst
1University of Kentucky Medical Center, Department of Internal Medicine, Lexington, USA.
Abstract:
Vitamin D analogs have received increased attention because of their possible therapeutic benefits in treating osteoporosis and various proliferative disorders. Several analogs were examined for their effects on DNA binding of the vitamin D receptor (VDR) homodimer complex with the murine osteopontin vitamin D response element. All of the tested analogs increased complex binding by recombinant human VDR in the electrophoretic mobility shift assay and notable differences in mobility of these complexes were observed. A panel of C-terminal anti-VDR antisera were screened for their ability to interact with analog-bound VDR homodimer complexes or as a heterodimer complex with recombinant human retinoid X receptor alpha (rhRXR alpha). Like calcitriol, analog-bound heterodimer complexes were largely resistant to interaction with these antisera; however, striking differences were observed with the various antisera in an analogous homodimer binding experiment. KH1060 and CB1093, analogs with 20-epi conformations, produced homodimer complexes that were 3- to 6-fold more resistant to supershifting with Ab180 compared with the hormone or EB1089. Chymotrypsin digestion in combination with Western blotting using a C-terminal anti-VDR antiserum revealed similar digestion patterns for all ligands. However, KH1060- and CB1093-bound VDR complexes were more resistant to digestion than either calcitriol or EB1089. Finally, the ability of these compounds to yield stable homodimer complexes was assessed by challenging preformed homodimer with the exogenous addition of rhRXR alpha extracts. Although new heterodimer complexes appeared in a time-dependent fashion, the preformed homodimer complexes exhibited stable binding throughout the time course of the experiment. The results indicate that VDR homodimers are targets of vitamin D analogs with differential effects on C-terminal protein conformation that may partially explain the varied biological responses of these compounds.
Insights
Vitamin D analogs affect vitamin D receptor (VDR) homodimer binding and conformation. Specific analogs like KH1060 and CB1093 show increased resistance to antibody interaction and digestion, influencing therapeutic potential.
Area of Science:
- Molecular biology
- Endocrinology
- Pharmacology
Background:
- Vitamin D analogs are investigated for treating osteoporosis and proliferative disorders.
- Understanding vitamin D receptor (VDR) interactions is crucial for analog development.
Purpose of the Study:
- To examine the effects of vitamin D analogs on VDR homodimer DNA binding.
- To investigate how analogs influence VDR conformation and interaction with retinoid X receptor alpha (RXR alpha).
Main Methods:
- Electrophoretic mobility shift assay (EMSA) to assess DNA binding.
- Western blotting with specific antisera to analyze VDR-antigen interactions.
- Chymotrypsin digestion to evaluate protein stability.
Main Results:
- All tested analogs enhanced VDR-DNA complex binding, with varying mobility shifts.
- KH1060 and CB1093 analogs induced VDR homodimer complexes more resistant to antibody supershifting and digestion.
- VDR homodimer complexes demonstrated stable binding, even with the addition of rhRXR alpha.
Conclusions:
- Vitamin D receptor homodimers are direct targets of vitamin D analogs.
- Analogs differentially affect VDR C-terminal protein conformation, potentially explaining varied biological responses.
- Specific analogs like KH1060 and CB1093 exhibit unique conformational effects on VDR.