Salt Concentration Determines 1,25-Dihydroxyvitamin D3Dependency of Vitamin D Receptor–Retinoid X Receptor–Vitamin D-Responsive Element Complex Formation

The electrophoretic mobility shift assay was used to determinein vitroformation of the vitamin D receptor–retinoid X receptor β (VDR–RXRβ) heterodimer complex on vitamin D-response elements (VDREs) from rat osteocalcin, mouse osteopontin, rat 25-hydroxyvitamin D324-hydroxylase, and human parathyroid hormone (PTH) genes. Baculovirus-expressed rat VDR was used as VDR and the binding reactions were performed at salt concentrations ranging from 50 to 170 mmKCl. Without ligand, optimum complex formation was observed at 50 mmKCl and markedly decreased with increasing KCl for all VDREs. In the presence of 1,25-dihydroxyvitamin D3, optimum complex formation occurred between 110 and 130 mmKCl for positive (enhancer) VDREs. At low salt concentrations (50–70 mmKCl), 1,25-dihydroxyvitamin D3did not increase complex formation and actually caused a slight decrease. However, above 90 mmKCl, 1,25-dihydroxyvitamin D3markedly increased complex formation and at 150–170 mmKCl, a concentration that presumably mimics physiologic nuclear levels, 1,25-dihydroxyvitamin D3appeared to be required for complex formation. With the suppressivecis-acting sequence, i.e., PTH–VDRE, optimum detection of VDR complexes in the presence of 1,25-dihydroxyvitamin D3occurred at a lower salt concentration (90–110 mmKCl). Moreover, no specific complexes were formed at high salt concentrations, even when 1,25-dihydroxyvitamin D3was added. Thus, when analyzing an effect of ligand on VDR–RXR–VDRE complex formation, it is essential that the reaction be carried out with a range of salt concentrations. Further, 1,25-dihydroxyvitamin D3appears to be required for formation of the VDR–RXRβ–VDRE complex at salt concentrations approaching physiological.