VITAMIN D HORMONE MECHANISM OF ACTION IN CULTURED CELLS

Project: Research project

Project Details

Description

This application proposes the continuation of our investigation of the
molecular and biological functions of the vitamin D hormone (1,25(OH)2D3)
in cultured cell systems. There will be a new focus on 1,25(OH)2D3
action in osteoblast-like bone cells and epithelial cells of the
intestine, where vitamin D exerts its major effects upon bone remodeling
and mineral absorption, respectively. In the last grant period we
accomplished the molecular cloning, overexpression and immunocytochemical
localization of the human vitamin D receptor (hVDR), and elucidated the
vitamin D response element (VDRE) in the rat osteocalcin gene. These
findings will be extended to probe the phosphorylation of hVDR in
relation to its ability to bind to the VDRE and enhance transcription of
an osteocalcin promoter/reporter construct in transfected cells.
Site-directed mutagenesis will be employed to determine the residues in
hVDR that are phosphorylated and stimulation of the relevant protein
kinases in intact cells will be effected to test in vivo significance.
Next, hVDR will be overexpressed in the insect baculovirus system and in
E. coli to generate sufficient quantities for biochemical study.
Expressed hVDR will be purified for study of phosphorylation by protein
kinase C and casein kinase II, in vitro. Phosphopeptide mapping of hVDR
overexpressed in COS-7 cells will be used to elucidate the site of
1,25(OH)2D3-dependent, hVDR phosphorylation. Preliminary data that a
nuclear receptor auxiliary factor (RAF) is required for high-affinity VDR
binding to the VDRE will be pursued by characterizing, purifying and
cloning the major RAF in CV-1 cells. RAF will be cloned by screening an
expression vector library with purified hVDR/[32p]VDRE. Ultimately, the
above components will be reassembled as purified factors in an in vitro
transcription system designed to determine the mechanism whereby
1,25(OH)2D3 stimulates the tissue specific expression of osteocalcin.
Finally, a molecular comparison of 1,25(OH)2D3/VDR action on osteocalcin
will be made with the effect on calbindin-D28k (CaBP) induction in
cultured embryonic duodena. Because it is inhibited by cycloheximide,
CaBP regulation by 1,25(OH)2D3 may be unique in that an intermediary
induced protein may bind to a non-VDRE enhancer in the CaBP promoter
and/or stabilization of CaBP mRNA may occur. In toto, these experiments
should define the precise signal transduction pathways whereby vitamin D
controls the expression of crucial 'bone and intestinal genes, and these
results may provide clues to the molecular basis of clinical vitamin D
resistance as well as to the role of vitamin D in the prevention of
osteoporosis.
StatusFinished
Effective start/end date7/1/8411/30/12

Funding

  • National Institutes of Health
  • National Institutes of Health: $230,613.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $321,453.00
  • National Institutes of Health: $29,925.00
  • National Institutes of Health: $284,160.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $281,318.00
  • National Institutes of Health: $284,160.00
  • National Institutes of Health: $299,754.00
  • National Institutes of Health
  • National Institutes of Health: $148,757.00
  • National Institutes of Health: $303,000.00
  • National Institutes of Health
  • National Institutes of Health: $249,428.00
  • National Institutes of Health: $331,096.00
  • National Institutes of Health: $312,090.00
  • National Institutes of Health: $209,697.00
  • National Institutes of Health: $341,028.00

ASJC

  • Medicine(all)

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