1,25-dihydroxyvitamin D3 down-regulation of PHEX gene expression is mediated by apparent repression of a 110 kDa transfactor that binds to a polyadenine element in the promoter.
The PHEX gene encodes an endopeptidase expressed in osteoblasts that inactivates an uncharacterized peptide hormone, phosphatonin, which suppresses bone mineralization as well as renal phosphate reabsorption and vitamin D bioactivation. We demonstrate that 1alpha-25-dihydroxyvitamin D (1,25(OH)2D3), the, active renal vitamin D metabolite, decreases PHEX mRNA in the rat osteoblastic cell line, UMR-106, as well as in mouse calvaria. Promoter/reporter construct analysis of the murine PHEX gene in transfected UMR-106 cells localized the repressive effect of 1,25(OH)2D3 to the -133 to -74 bp region, and gel mobility shift experiments revealed that 1,25(OH)2D3 treatment of the cells diminished the binding of a nuclear protein(s) to a stretch of 17 adenines from bp -116 to -100 in the proximal PHEX promoter. Either overexpression of a dominant-negative vitamin D receptor (VDR) or deletion of this sequence of 17 A-T base pairs abolished the repressive effect of 1,25(OH)2D3 by attenuating basal promoter activity, indicating that this region mediates the 1,25(OH)2D3 response and is involved in basal transcription. South-western blot analysis and DNA affinity purification show that an unidentified 110 kDa nuclear protein binds to the poly(A) element. Because 1,25(OH)2D3-liganded VDR neither binds to the polyadenine region of the PHEX promoter nor directly influences the association of the 110 kDa transfactor, we conclude that 1,25(OH)2D3 indirectly decreases PHEX expression via VDR-mediated repression (or modification) of this novel transactivator. Thus, we have identified a cis-element required for PHEX gene transcription that participates in negative feedback control of PHEX expression and thereby modulates the actions of phosphatonin.
Hines
Eric R
ER
Department of Pediatrics, College of Medicine, Steele Memorial Children's Research Center, University of Arizona, Tucson, Arizona 85724, USA.
Kolek
Olga I
OI
Jones
Marci D
MD
Serey
Samantha H
SH
Sirjani
Nafisseh B
NB
Kiela
Pawel R
PR
Jurutka
Peter W
PW
Haussler
Mark R
MR
Collins
James F
JF
Ghishan
Fayez K
FK
D000225:Adenine / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D001842:Bone and Bones / Q000378:metabolism; D002117:Calcitriol / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation; D017353:Gene Deletion; D005799:Genes, Dominant; D006728:Hormones / Q000737:chemistry; D008024:Ligands; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; D011061:Poly A; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012509:Sarcoma / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D006728:Hormones; D008024:Ligands; D011506:Proteins; D012333:RNA, Messenger; D011061:Poly A; D004247:DNA; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; C506232:Phex protein, mouse; C506229:Phex protein, rat; D002117:Calcitriol; D000225:Adenine