Identification and characterization of 1,25-dihydroxyvitamin D3- responsive repressor sequences in the rat parathyroid hormone-related peptide gene

Abstract

Parathyroid hormone-related peptide (PTHRP) gene transcription is suppressed by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3. In the present report, we examined 1,25(OH)2D3- mediated repression of PTHRP expression by transfection of PTHRP promoter/reporter constructs in normal human keratinocytes and by DNA binding. We localized an element conferring 1,25(OH)2D3-mediated repression in vivo to a 47-base pair (bp) region located -1121 to -1075 from the transcriptional start site. Mobility shift analysis revealed that 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 receptor to the 47-bp sequence, whereas the addition of monoclonal antibody raised against the retinoid X receptor (RXR) further retarded the mobility of the protein-DNA complex. Consequently, the PTHRP promoter element binds a VDR·RXR heterodimer. Examination of this VDRE revealed complete sequence homology with a half-site of the human and rat osteocalcin VDRE (GGGTGA). Furthermore, mutation analysis suggests that a 16-bp domain consisting of an almost perfect repeat separated by a 3-base pair 'spacer' GGGTG-GAGAGGGGTGA 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 classical up-regulatory VDREs.