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 (nVDREhPTHrP) 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 Xalpha receptor (RXRalpha) 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 D3. Such a repression was reversed by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitamin D3 phosphorylates VDR to weaken its binding activity to nVDREhPTHrP. In electrophoretic mobility shift assay, we found anti-Ku antigen antibody specifically supershifted the MT2 nuclear proteinnVDREhPTHrP complex. The nVDREhPTHrP-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 nVDREhPTHrP interacts with Ku antigen in addition to VDR to mediate gene suppression by vitamin D.
Nishishita
T
T
Endocrine Genetics and Hypertension Unit, 4th Department of Internal Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo 112, Japan.
Okazaki
T
T
Ishikawa
T
T
Igarashi
T
T
Hata
K
K
Ogata
E
E
Fujita
T
T
D034961:Antigens, Nuclear; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000235:genetics; D044162:Parathyroid Hormone-Related Protein; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000235:genetics*; D018167:Receptors, Calcitriol / Q000378:metabolism*; D014807:Vitamin D / Q000378:metabolism* / Q000494:pharmacology
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D018167:Receptors, Calcitriol; D014807:Vitamin D; D004247:DNA; D010744:Phosphoric Monoester Hydrolases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen