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Type: Thesis
Title: Regulation of the rat 25-hydroxyvitamin D3 24-hydroxylase gene promoter by 1,25(OH)₂D3 / by David Michael Kerry.
Author: Kerry, David Michael
Issue Date: 1997
School/Discipline: Dept. of Biochemistry
Abstract: The secosteroid 1,25-dihydroxyvitarnin D₃ (1,25(OH)₂D₃) is an important regulator of calcium homeostasis, cellular differentiation and proliferation. The nuclear actions of 1,25(OH)₂D₃) are mediated through the intracellular vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcription factors. The metabolic inactivation of 1,25(OH)₂D₃) through the C-24 oxidation pathway is initiated by a rnitochondrial cytochrome P450 enzyrne (CYP24). The action of CYP24 initiates the conversion of 1,25(OH)₂D₃) to water soluble metabolites. It has been well characterised that 1,25(OH)₂D₃) acts to stimulate CYP24 gene expression in a negative feedback loop. The work undertaken in this thesis was aimed at understanding at the transcriptional level how 1,25(OH)₂D₃) up-regulates CYP24 gene expression. Previous studies with the rat CYP24 promoter have identified three putative vitamin D responsive elements (VDREs), termed VDRE-I,2 and 3 in the first 298 bp of the CYP24 promoter region. To elucidate how these putative VDREs function in the context of the native promoter environment, the putative VDREs were mutated either individually or in combination by site-directed mutagenesis. The mutant promoter constructs fused to the luciferase reporter gene were then examined in transient transfection assays in the kidney and bone cell lines COS-I, nC-12 and ROS 1712.8 in the presence or absence of 1,25(OH)₂D₃). These studies have demonstrated that VDRE-I and VDRE-2 are solely responsible for the 1,25(OH)₂D₃) mediated response. VDRE-I contributes 6-fold and VDRE-2 3-fold to the overall induction of 18-fold for the native 298 bp promoter construct, thus demonstrating u 2-foId synergistic relationship between these VDREs. VDRE-3, however was unresponsive to 1,25(OH)₂D₃) even when VDRE-I and 2 were mutated. The three VDREs were also investigated at the protein-DNA level by gel-shift analysis. VDRE-1 &.2 were shown to bind a protein complex of approximately the same mobility as that which binds to the well characterised VDRE of mouse osteopontin (mSPP-1). The components of this complex were elucidated by the use of supershifting and competitive monoclonal antibodies and demonstrated to be VDR and its partner factor retinoid-X-receptor (RXR). Competition analysis revealed that VDRE-2 binds the VDR-RXR heterodimeric cornplex with a higher affinity than VDRE-1; this finding is in contrast to the transient transfection data where VDRE-1 has greater transcriptional activity. This raises the possibility of other transcription factors being involved in the 1,25(OH)₂D₃) response by enhancing VDRE-1 activity. The functionality of the characterised VDREs at more physiologically relevant concentrations of 1,25(OH)₂D₃) was determined. In COS-1 cells co-transfected with VDR, it was found that both VDREs were functional, with synergism observed over a wide range of 1,25(OH)₂D₃) concentrations 110t-10-11 M). At lower concentrations only VDRE-I was responsive to 1,25(OH)₂D₃) and similar results were obtained in ROS 1712.8 cells which express significant levels of endogenous VDR, thus demonstrating that the synergism observed may have important physiological consequences. In JTC-12 cells induction was only observed at very high concentrations of 1,25(OH)₂D₃) and may be a reflection of low VDR levels. In summary, the results establish a so far unique synergistic relationship between two VDREs which is maintained at physiological concentrations of 1,25(OH)₂D₃) with VDRE-1 the major contributor to induction, particularly at low hormone concentrations.
Dissertation Note: Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1998?
Subject: Calcium Metabolism Effect of drugs on.
Cytochrome P-450 Metabolism.
Description: Copies of author's previously published articles inserted.
Bibliography: leaves 103-119.
viii, 199, [87] leaves : ill. ; 30 cm.
Provenance: Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exception. If you are the author of this thesis and do not wish it to be made publicly available or If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.
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