Vitamin D receptor interactions with the rat parathyroid hormone gene: Synergistic effects between two negative vitamin D response elements

Vitamin D response elements (VDREs) that are required for negative regulation of rat parathyroid hormone (rPTH) gene expression have been characterized. Gel mobility shift assays using DNA restriction enzyme fragments and recombinant proteins for vitamin D and retinoic acid X receptors (VDR/RXR) revealed a sequence between -793 and -779 that bound a VDR/RXR heterodimer with high affinity (VDRE1). Furthermore, a lower affinity site (VDRE2) was detected that acted in combination with VDRE1 to bind a second VDR/RXR complex. As determined by ethylation interference analysis, the nucleotide sequence of VDRE1 consisted of GGTTCA GTG AGGTAC, which is remarkably similar to the sequence of the negative VDRE found in the chicken PTH (cPTH) gene. Using the same technique, VDRE2 was identified between positions -760 and -746 and contained the sequence AGGCTA GCC AGTTCA, Functional analysis was determined by transfection studies,with plasmid constructs that expressed the gene for chloramphenicol acetyl transferase (CAT), The ability of the VDREs to regulate gene expression was tested in their native context with the rPTH promoter as well as when positioned immediately upstream from the cPTH promoter. With either plasmid construct, exposure to 10(-8) M 1,25(OH)(2)D-3 resulted in a 60-70% decrease in CAT gene expression when both VDRE, and VDRE, were present. Examination of the individual VDREs showed that inhibition by 10-8 M 1,25(OH)(2)D-3 was only 35-40% when just VDRE, was present. By itself, VDRE, was even less effective, as significant inhibition of CAT activity (20%) was observed only in the presence of higher concentrations of 1,25(OH)(2)D-3 (10(-7) M) or when a plasmid vector that overexpressed the VDR protein was cotransfected, In conclusion, the rPTH gene contains two negative VDREs that act in concert to bind two RXR/VDR heterodimer complexes and that both VDREs are required for maximal inhibition by 1,25(OH)(2)D-3.