SALT CONCENTRATION DETERMINES 1,25-DIHYDROXYVITAMIN D-3 DEPENDENCY OFVITAMIN-D-RECEPTOR RETINOID-X-RECEPTOR VITAMIN-D-RESPONSIVE ELEMENT COMPLEX-FORMATION

The electrophoretic mobility shift assay was used to determine in vitr o formation of the vitamin D receptor-retinoid X receptor beta (VDR-RX R beta) heterodimer complex on vitamin D-response elements (VDREs) fro m rat osteocalcin, mouse osteopontin, rat 25-hydroxyvitamin D-3 24-hyd roxylase, and human parathyroid hormone (PTH) genes. Baculovirus-expre ssed rat VDR was used as VDR and the binding reactions were performed at salt concentrations ranging from 50 to 170 mM KCl. Without ligand, optimum complex formation was observed at 50 mM KCl and markedly decre ased with increasing KCl for all VDREs. In the presence of 1,25-dihydr oxyvitamin D-3 optimum complex formation occurred between 110 and 130 mM KCl for positive (enhancer) VDREs. At low salt concentrations (50-7 0 mM KCl), 1,25-dihydroxyvitamin D-3 did not increase complex formatio n and actually caused a slight decrease. However, above 90 mM KCl, 1,2 5-dihydroxyvitamin D-3 markedly increased complex formation and at 150 -170 mM KCl, a concentration that presumably mimics physiologic nuclea r levels, 1,25-dihydroxyvitamin D-3 appeared to be required for comple x formation. With the suppressive cis-acting sequence, i.e., PTH-VDRE, optimum detection of VDR complexes in the presence of 1,25-dihydroxyv itamin D-3 occurred at a lower salt concentration (90-110 mM KCl). Mor eover, no specific complexes were formed at high salt concentrations, even when 1,25-dihydroxyvitamin D-3 was added. Thus, when analyzing an effect of ligand on VDR-RXR-VDRE complex formation, it is essential t hat the reaction be carried out with a range of salt concentrations. F urther, 1,25-dihydroxyvitamin D-3 appears to be required for formation of the VDR-RXR beta-VDRE complex at salt concentrations approaching p hysiological. (C) 1997 Academic Press.