MOLECULAR EVENTS IN COLLAGEN GENE RESPONSE TO VITAMIN D

  • Pavlin, Dubravko (PI)

Project: Research project

Project Details

Description

Molecular mechanisms controlling type I collagen gene expression
are highly complex in that, besides the cell-specific expression of
basal levels, this gene is controlled by calcitropic hormones (1,25-
dihydroxyvitamin D3 and parathyroid hormone) in some cell types
but not others. The proposed experiments are based on the preliminary data from
the laboratories of applicant's sponsor and advisor, indicating that
1,25-D inhibits type I collagen synthesis in bone cells at the level
of transcription. The model system proposed in this study
includes rat osteosarcoma (ROS 17/2.8) cells transfected either
transiently with chimeric plasmids containing marker gene, or
permanently with bovine papilloma virus (BPV), both carrying
cloned alpha-1(I) regulatory genomic sequences. Since
preliminary data showed that the -3.6 kb alpha-1(I) genomic
fragment induced high basal expression of marker gene and 1,25-
D-dependent inhibition of the collagen promoter, the first specific
aim of these experiments is to determine whether the recently
cloned alpha-1(I) first intron sequences have an additive efect on
basal expression and/or 1,25-D-dependent regulation of the 3.6 kb
fragment. Furthermore, deletion analysis will be used to identify
the smallest fragments (cis-active elements) within the -3.6 kb
and the first intron sequences responsible for strong basal
expression and inhibition by 1,25-D. This cis-active fragments
will also be joined to a constitutive promoter, such as thymidine
kinase (TK), in order to demonstrate their role in conferring high
basal activity and hormone responsivity. Finally, trans-acting
nuclear factors from untreated and 1,25-D-treated ROS 17/2.8
cells that bind to a specific DNA sequences and presumably
regulate alpha-1(I) promoter, will be detected both in vitro, using
gel retardation and exonuclease protection assays in crude nuclear
extracts, and in vivo, using BPV vector and exonuclease
protection assay in stable transfection experiments.
StatusFinished
Effective start/end date6/1/885/31/93

Funding

  • National Institutes of Health
  • National Institutes of Health: $22,715.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Dentistry(all)

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