Analysis of regulatory regions in the COL1A1 gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression in osteoblastic cells.
The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-Pstl restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.
Pavlin
D
D
Department of Orthodontics, University of Texas Health Science Center, San Antonio 78284.
Bedalov
A
A
Kronenberg
M S
MS
Kream
B E
BE
Rowe
D W
DW
Smith
C L
CL
Pike
J W
JW
Lichtler
A C
AC
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002117:Calcitriol / Q000494:pharmacology*; D002874:Chromosome Mapping; D003094:Collagen / Q000235:genetics*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014807:Vitamin D / Q000378:metabolism; D014809:Vitamin D-Binding Protein / Q000378:metabolism
D018167:Receptors, Calcitriol; D014809:Vitamin D-Binding Protein; D014807:Vitamin D; D003094:Collagen; D002117:Calcitriol