ANALYSIS OF REGULATORY REGIONS IN THE COL1A1 GENE RESPONSIBLE FOR 1,25-DIHYDROXYVITAMIN D-3-MEDIATED TRANSCRIPTIONAL REPRESSION IN OSTEOBLASTIC CELLS

The synthesis of type I collagen in bone cells is inhibited by the cal cium-regulating hormone 1,25-dihydroxyvitamin D-3. Earlier work from o ur laboratories has indicated that vitamin D regulation is at the leve l 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 acetyltransfe rase reporter gene. In the present study, we investigated the molecula r basis for vitamin D-mediated transcriptional repression of the COL1A 1 gene and report the identification of a region within the COL1A1 ups tream promoter (the HindIII-Pstl restriction fragment between nucleoti des -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D-3 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 fro m cultured cells which express a high level of vitamin D receptor cont ain a hormone:receptor complex that binds specifically to this 41 bp f ragment, as demonstrated by bandshift analysis. However, deletion of t his 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 r esults indicate that a vitamin D response element similar to that desc ribed for other vitamin D responsive genes (osteocalcin and osteoponti n) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvi tamin D-3. (C) 1994 Wiley-Liss, Inc.