In vitro binding of vitamin D receptor occupied by 24R,25-dihydroxyvitamin D-3 to vitamin D responsive element of human osteocalcin gene

We previously reported that 24R,25-dihydroxyvitamin D-3 [24R,25(OH)(2)D-3] activates the human osteocalcin gene (hOC) through vitamin D receptor (VDR) and vitamin D responsive element (VDRE) in the same manner as 1 alpha,25-dihydroxyvitamin D-3 [1 alpha,25(ON)(2)D-3] [17]. In the present study, the interaction of 24R,25(OH)(2)D-3-liganded VDR [24R,25(OH)(2)D-3-VDR] with the hOC VDRE in vitro was investigated. The electrophoretic mobility shift assay (EMSA) revealed that the binding of 24R,25(OH)(2)D-3-liganded VDR to the hOC VDRE was weak, even at concentrations of 24R,25(OH)(2)D-3 10(5)-fold higher than 1 alpha,25(OH)(2)D-3. The effect of the nuclear accessory factor (NAF), which is required for the high affinity interaction of the VDR to the VDRE, on the binding of the 24R,25(OH)(2)D-3-VDR to the VDRE was studied using hOC VDRE affinity column chromatographic assays. In the absence of NAF, the 24R,25(OH)(2)D-3-VDR associated weakly with the VDRE compared to the lu,25(OH)(2)D-3-liganded VDR [1 alpha,25(OH)(2)D-3-VDR], whereas the NAF enhanced the binding of the 24R,25(OH)(2)D-3-VDR for the VDRE. In the absence of the hOC VDRE, the binding affinity of the 24R,25(OH)(2)D-3-VDR for the NAF was weaker than that of 1 alpha,25(OH)(2)D-3-VDR. These results suggest that the weak interaction of the 24R,25(OH)(2)D-3-VDR with both NAF and hOC VDRE is responsible for the weak binding of the 24R,25(OH)(2)D-3-VDR to the VDRE detected in EMSA. In terms of VDR function, 24R,25(OH)(2)D-3 was more potent in transactivation than in vitro binding. (C) 1997 Elsevier Science Ltd.