J. Russell et al., Vitamin D receptor interactions with the rat parathyroid hormone gene: Synergistic effects between two negative vitamin D response elements, J BONE MIN, 14(11), 1999, pp. 1828-1837
Vitamin D response elements (VDREs) that are required for negative regulati
on of rat parathyroid hormone (rPTH) gene expression have been characterize
d. Gel mobility shift assays using DNA restriction enzyme fragments and rec
ombinant proteins for vitamin D and retinoic acid X receptors (VDR/RXR) rev
ealed a sequence between -793 and -779 that bound a VDR/RXR heterodimer wit
h high affinity (VDRE1). Furthermore, a lower affinity site (VDRE2) was det
ected that acted in combination with VDRE1 to bind a second VDR/RXR complex
. As determined by ethylation interference analysis, the nucleotide sequenc
e of VDRE1 consisted of GGTTCA GTG AGGTAC, which is remarkably similar to t
he sequence of the negative VDRE found in the chicken PTH (cPTH) gene. Usin
g the same technique, VDRE2 was identified between positions -760 and -746
and contained the sequence AGGCTA GCC AGTTCA, Functional analysis was deter
mined by transfection studies,with plasmid constructs that expressed the ge
ne for chloramphenicol acetyl transferase (CAT), The ability of the VDREs t
o regulate gene expression was tested in their native context with the rPTH
promoter as well as when positioned immediately upstream from the cPTH pro
moter. With either plasmid construct, exposure to 10(-8) M 1,25(OH)(2)D-3 r
esulted in a 60-70% decrease in CAT gene expression when both VDRE, and VDR
E, 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 i
tself, VDRE, was even less effective, as significant inhibition of CAT acti
vity (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 negat
ive 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.