Identification and Characterization of 1,25-Dihydroxyvitamin D3-responsive Repressor Sequences in the Rat Parathyroid Hormone-related Peptide Gene

• Published in: The Journal of biological chemistry 1996-07, Vol.271 (27), p.16310-16316
• Main Authors: Kremer, Richard, Sebag, Michael, Champigny, Céline, Meerovitch, Karen, Hendy, Geoffrey N., White, John, Goltzman, David
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Binding Sites; Binding, Competitive; Calcitriol - pharmacology; Cell Line; Cells, Cultured; Chickens; Chlorocebus aethiops; DNA Primers; Glutathione Transferase - biosynthesis; Humans; Keratinocytes - cytology; Keratinocytes - metabolism; Kinetics; Mice; Molecular Sequence Data; Parathyroid Hormone-Related Protein; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Biosynthesis; Proteins - genetics; Rats; Recombinant Fusion Proteins - biosynthesis; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Sequence Homology, Nucleic Acid; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.27.16310

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”▪▪GAG▪ 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.