Identification and characterization of 1,25-dihydroxyvitamin D3-responsive repressor sequences in the rat parathyroid hormone-related peptide gene.
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" 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 classical up-regulatory VDREs.
Kremer
R
R
Department of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada.
Sebag
M
M
Champigny
C
C
Meerovitch
K
K
Hendy
G N
GN
White
J
J
Goltzman
D
D
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002117:Calcitriol / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured; D002645:Chickens; D002522:Chlorocebus aethiops; D017931:DNA Primers; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D044162:Parathyroid Hormone-Related Protein; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D017931:DNA Primers; C470121:PTHLH protein, human; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D002117:Calcitriol