IDENTIFICATION AND CHARACTERIZATION OF 1,25-DIHYDROXYVITAMIN D-3-RESPONSIVE REPRESSOR SEQUENCES IN THE RAT PARATHYROID HORMONE-RELATED PEPTIDE GENE

Parathyroid hormone-related peptide (PTHRP) gene transcription is supp ressed by 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3), the active metab olite of vitamin D-3. In the present report, we examined 1,25(OH)(2)D- 3-mediated repression of PTHRP expression by transfection of PTHRP pro moter/reporter constructs in normal human keratinocytes and by DNA bin ding, We localized an element conferring 1,25(OH)(2)D-3-mediated repre ssion in vivo to a 47-base pair (bp) region located -1121 to -1075 fro m the transcriptional start site, Mobility shift analysis revealed tha t this vitamin D response element (VDRE) forms DNA-protein complexes. The addition of a monoclonal antibody that recognizes the DNA binding region of the vitamin D receptor (VDR) attenuated binding of the recep tor to the 47-bp sequence, whereas the addition of monoclonal antibody raised against the retinoid X receptor (RXR) further retarded the mob ility of the protein-DNA complex. Consequently, the PTHRP promoter ele ment binds a VDR-RXR heterodimer, Examination of this VDRE revealed co mplete sequence homology with a half-site of the human and rat osteoca lcin VDRE: (GGGTGA). Furthermore, mutation analysis suggests that a 16 -bp domain consisting of an almost perfect repeat separated by a 3-bas e pair ''spacer'' GGGTGGAGAGGGGTGA is responsible for the DNA-protein interaction within this 47-bp sequence. Our results therefore indicate the existence of an inhibitory VDRE within the PTHRP promoter that is similar in sequence composition and cellular factor requirement to cl assical upregulatory VDREs.