A negative vitamin D response DNA element in the human parathyroid hormone-related peptide gene binds to vitamin D receptor along with Ku antigen to mediate negative gene regulation by vitamin D

We found that the human parathyroid hormone-related peptide (hPTHrP) gene contained a DNA element (nVDRE(hPTHrP)) homologous to a negative vitamin D response element in the human parathyroid hormone gene. It bound to vitamin D receptor (VDR) but not retinoic acid X alpha receptor (RXR alpha) in the human T cell line MT2 cells. VDR binding to this element was confirmed by the Southwestern assay combined with immunodepletion using anti-VDR monoclonal antibody, and this binding activity was repressed by 1,25-dihydroxyvitamin D-3. Such a repression was reversed by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitamin D-3 phosphorylates VDR to weaken its binding activity to nVDRE(hPTHrP). In electrophoretic mobility shift assay, we found anti-Ku antigen antibody specifically supershifted the MT2 nuclear protein-nVDRE(hPTHrP) complex. The nVDRE(hPTHrP)-bearing reporter plasmid produced vitamin D-dependent inhibition of the reporter activity in MT2 cells, which was markedly masked by the introduction of the Ku antigen expression vector in the antisense orientation. On the other hand, such a procedure did not perturb the vitamin D response element-mediated gene stimulation by vitamin D. These results indicate that nVDRE(hPTHrP) interacts with Ku antigen in addition to VDR to mediate gene suppression by vitamin D.