In vitro binding of vitamin D receptor occupied by 24R,25-dihydroxyvitamin D3 to vitamin D responsive element of human osteocalcin gene.
We previously reported that 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] 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 D3 [1 alpha,25(OH)2 D3] [17]. In the present study, the interaction of 24R,25(OH)2D3-liganded VDR [24R,25(OH)2D3-VDR] with the hOC VDRE in vitro was investigated. The electrophoretic mobility shift assay (EMSA) revealed that the binding of 24R,25(OH)2D3-liganded VDR to the hOC VDRE was weak, even at concentrations of 24R,25(OH)2D3 10(5)-fold higher than 1 alpha,25(OH)2D3. 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)2D3-VDR to the VDRE was studied using hOC VDRE affinity column chromatographic assays. In the absence of NAF, the 24R,25(OH)2D3-VDR associated weakly with the VDRE compared to the 1 alpha,25(OH)2D3-liganded VDR [1 alpha,25(OH)2D3-VDR], whereas the NAF enhanced the binding of the 24R,25(OH)2D3-VDR for the VDRE. In the absence of the hOC VDRE, the binding affinity of the 24R,25(OH)2D3-VDR for the NAF was weaker than that of 1 alpha,25(OH)2D3-VDR. These results suggest that the weak interaction of the 24R,25(OH)2D3-VDR with both NAF and hOC VDRE is responsible for the weak binding of the 24R,25(OH)2D3-VDR to the VDRE detected in EMSA. In terms of VDR function, 24R,25(OH)2D3 was more potent in transactivation than in vitro binding.
Uchida
M
M
Department of Pediatrics and Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Ozonco
K
K
Pike
J W
JW
D015650:24,25-Dihydroxyvitamin D 3 / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics*; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015533:Transcriptional Activation
D009687:Nuclear Proteins; D018167:Receptors, Calcitriol; C072701:nuclear accessory protein, monkey; D015675:Osteocalcin; D015650:24,25-Dihydroxyvitamin D 3