Abstract
A vitamin D-responsive element (VDRE) locus within the 5' region of the human osteocalcin gene promoter contains a steroid response-like half-site immediately proximal to a consensus site for the AP-1 nuclear oncogene family. In the studies described here, internal mutagenesis of the osteocalcin promoter coupled to functional assays reveal that the interaction of the vitamin D receptor is limited to the proximal region of the VDRE locus. Mutations within the distal AP-1 consensus site reduce the basal activity of the promoter but have little effect on vitamin D inducibility. The absolute level of promoter activity induced by hormone, however, is dramatically reduced in the absence of an intact AP-1 site suggesting a functional synergism between the receptor and AP-1-related proteins. In vitro receptor-DNA binding studies confirm the lack of requirement for the distal component in receptor binding. These results suggest that the osteocalcin VDRE is limited to 15 nucleotides closely juxtaposed to a distal functional AP-1 site. The close association of the two sites may lead to proto-oncogene and steroid receptor interactions that result in interesting functional consequences.
Highlights
A vitamin D-responsive element (VDRE) locus within the 5’ region of the human osteocalcin gene promoter contains a steroid response-like half-site immediately proximal to a consensus site for the AP-1 nuclear oncogene family
In the studies described here, internal mutagenesis of the osteocalcin promoter coupled to functional assays reveal that the interaction of the vitamin D receptor is limited to the proximal region of the VDRE locus
The Proximal Half of the VDRE Is Essential for Vitamin D Induction-In order to evaluate the complexity of this element within the context of its own promoter, a large osteocalcin gene fragment containing sequences -838 to +10 was introduced into Ml3 and nucleotide bases within or near the VDRE site located between
Summary
Recombinant Steroid Receptor Expression Plasmids-Human vitamin D receptor (hVDR) was expressed utilizing the p91023b vector as previously described (Baker et al, 1988; McDonnell et al, 1989b). ROS 17/2.8 cells were transfected using Polybrene by methods described by Kawai et al (1984) utilizing 10 pg of reporter plasmid and 2 fig of pSV40luciferase (de Wet et al, 1987) as internal standard. All cells were cultured in the appropriate medium containing 5% charcoal- and resin-stripped fetal calf serum and the appropriate level of hormone. D receptor utilized for DNA bandshift assays was obtained from nuclear extracts of COS-1 cells transfected with pAV-hVDR. The appropriate DNA fragment, labeled to 10’ cpm/qg, was isolated and recovered following resolution on a 4% polyacrylamide gel. The probe was methylated and incubated as described above with 50 ng of purified hVDR (>90% purity) isolated from Saccharomyces cereuisiae (McDonnell et al, 1989a) by DNA cellulose chromatography (Sone et al, 1990) and 2 Fg of untransfected. The quanine nucleotides indicated were identified through a Maxam and Gilbert sequencing reaction
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