Analysis of regulatory regions in the COL1A1 gene responsible for 1,25‐dihydroxyvitamin D3‐mediated transcriptional repression in osteoblastic cells

Abstract

The synthesis of type 1 colagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D₃. Earlier work from our laboratoties has indicated that vitamin D regulation is at the level of transcription, based on result from both nuclear run-off assays and functional analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloraphenicol 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 Hindlll-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. Estracts that binds specifically to this 41 bp fragment, as demonstrated by bandshift anslysis. 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 D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D₃.