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

Toshihide Nishishita,Tomoki Okazaki,Toshio Ishikawa,Tetsuya Igarashi,Keishi Hata,Etsuro Ogata,Toshiro Fujita

doi:10.1074/jbc.273.18.10901

Abstract

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.

Highlights

The mechanism by which steroid/thyroid nuclear hormone receptors activate gene transcription has been extensively studied [1,2,3,4,5,6,7,8]
We found that the human parathyroid hormone-related peptide gene contained a DNA element homologous to a negative vitamin D response element in the human parathyroid hormone gene
The 1/4-diluted anti-vitamin D receptor (VDR) antibody weakened this complex, it did not supershift the complex nor did its 1/20 dilution affected the formation of the complex, which was in contrast to the case of VDRE found in the mouse osteopontin gene (VDREmop) and MT2 nuclear proteins. These results suggest that nVDREhPTHrP-MT2 protein complex contained VDR but not retinoic acid X receptor (RXR)␣, and VDR might be included in this complex in a manner different from the authentic VDR-RXR␣ heterodimer formation

Summary

Introduction

The mechanism by which steroid/thyroid nuclear hormone receptors activate gene transcription has been extensively studied [1,2,3,4,5,6,7,8]. Among them there have been so many lines of solid evidence showing that vitamin D receptor (VDR), thyroid hormone receptor, and retinoic acid receptor employ a common machinery to exert their ligand-dependent specific effects; all of them utilize a retinoic acid X receptor (RXR) in common as a partner of a heterodimer [2, 4] This mechanism seems to be confined only to gene stimulation but not to gene repression. The mechanism of negative gene regulation by VDR and vitamin D is only partially understood [15,16,17,18,19,20,21] In this case, an active form of vitamin D, 1,25-dihydroxyvitamin D3, can be considered an end product of parathyroid hormone (PTH) action, and this metabolite, in turn, inhibits the synthesis of PTH mRNA to keep the blood calcium level constant. We examined the molecular mechanism of negative regulation of the PTHrP gene by vitamin D in MT2 cells

Methods

Results

Conclusion