Analysis of transcriptional regulation of the rat bone gla protein gene by 1,25-dihydroxyvitamin D3: Receptor biochemistry and gene interactions.

Abstract

The secosteroid hormone 1, 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) exerts its effects through a receptor protein which is a member of a superfamily of steroid receptor proteins. The 1,25(OH)₂D₃ receptor protein (VDR) binds both hormone and DNA with high affinity , thereby transcriptionally regulating the expression of a number of genes. A fragment of the cDNA to the human VDR containing essentially the entire open reading frame was transcribed and translated in vitro. demonstrated to exhibit The resulting protein was then the physical and functional features, i.e. molecular weight, immunoreactivity, 1,25(OH)₂D₃ binding, and DNA-cellulose binding, of the native human receptor from the T47D cell line. This validates the authenticity of the cDNA in a cell free system and provides a biochemical means of generating this rare and labile macromolecule to use in heretofore difficult structure/function studies. The gene for rat bone gla protein (BGP) was isolated and 1250 bp including 1100 bp of 5' flanking DNA were placed upstream of the human growth hormone reporter gene. Following transient transfection into the osteoblast-like rat osteosarcoma cell line, ROS 17/2.8, the BGP promoter demonstrated a low level of basal activity that was increased approximately ten-fold by addition of 10⁻⁸ M 1,25(OH)2D3. A single 249 bp fragment (-523 to -274) was sufficient to confer hormone inducibility upon both heterologous and homologous promoters. Deletion studies, complemented by evaluation with synthetic oligomers, enabled localization of the 1,25(OH)₂D₃ response element (VDRE) to within 19 bp (-456 to -438), containing an element with an imperfect direct repeat (GGTGA(N₄)GGACA) and homology to other steroid responsive elements. This enhancer element was capable of strong synergism when present in multiple copies. Gel retardation assays demonstrated that partially purified VDR from chicken or rat bound specifically and with high affinity to a DNA fragment containing the putative VDRE and this binding was perturbed by monoclonal antibodies to the VDR. surprisingly, the 249 bp fragment, when linked in an antisense orientation with respect to the BGP promoter, blocked the basal and hormone dependent gene expression. However, a 245 bp fragment 5' to the 249 bp element (-1100 to -855) restored 20-fold inducibility when linked to the first fragment in the same orientation. The presence or absence of this distal element also modulated the 1,25(OH)₂D₃ response within the milieu of the native gene. Thus, the VDRE apparently cooperates with other elements to achieve the hormonal response observed in this gene.