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) g ene contained a DNA element (nVDRE(hPTHrP)) homologous to a negative v itamin D response element in the human parathyroid hormone gene. It bo und 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 e lement was confirmed by the Southwestern assay combined with immunodep letion using anti-VDR monoclonal antibody, and this binding activity w as repressed by 1,25-dihydroxyvitamin D-3. Such a repression was rever sed by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitam in D-3 phosphorylates VDR to weaken its binding activity to nVDRE(hPTH rP). In electrophoretic mobility shift assay, we found anti-Ku antigen antibody specifically supershifted the MT2 nuclear protein-nVDRE(hPTH rP) complex. The nVDRE(hPTHrP)-bearing reporter plasmid produced vitam in 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 procedu re did not perturb the vitamin D response element-mediated gene stimul ation by vitamin D. These results indicate that nVDRE(hPTHrP) interact s with Ku antigen in addition to VDR to mediate gene suppression by vi tamin D.