Type 1 collagen is the major structural protein in bone, and it's high level synthesis is an important characteristic of the osteoblast phenotype. We have evidence that transcription of the alpha1 (I) collagen gene is regulated differently in osteoblasts than in other collagen producing cell types. We believe that by investigating the DNA sequences and transcription factors that are involved in regulating the gene in osteoblasts that we will achieve a greater fundamental understanding of osteoblast differentiation, which is critical to our understanding of many bone disorders.
The specific aims of this grant are: A. To functionally characterize the DNA sequences and clone the proteins which bind to a 49 bp region between -1719 and -1670 bp in the rat alpha1(1) collagen gene which has been shown to be necessary for significant expression of the gene in bone. This will be carried out by analysis of site specific mutations in transgenic animals, analysis of DNA-protein interactions using gel mobility shifts, cloning transcription factors, and analyzing their functions using expression vectors and antisense nucleic acids. B. To evaluate the possible role of the region between -2297 bp and -3521 bp of the alpha1(1) promoter in transcription during normal bone development using immunohistochemistry and in situ hybridization. If we find that this region plays a role in transcription of the gene during some phase of the osteoblast life cycle, we will analyze the DNA sequences and transcription factors responsible for this regulation by in vitro analysis of DNA-protein interactions, in vitro mutagenesis, transfection into cultured bone cells, and analysis of select mutations in transgenic mice. C. To determine the regions of the alpha1(1) gene necessary to direct high level transcription in bone and other collagen producing cells in transgenic animals and by transfection of cultured cells. D. The second basic goal of this grant and the fourth specific aim is to determine the mechanism of the 1,25 dihydroxyvitamin D (vitamin D) inhibition of the alpha1(1) gene in bone cells. This will be accomplished by testing mutated promoter constructs for vitamin D response in transfected cells and transgenic mice, by analyzing the binding of the vitamin D receptor to fragments of the alpha1(I) promoter, and by evaluating possible interactions of the vitamin D receptor with factors involved in basal transcription of the gene.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR029983-11A1
Application #
2078671
Study Section
Special Emphasis Panel (ZRG4-OBM-2 (06))
Project Start
1981-08-01
Project End
1999-07-31
Budget Start
1995-08-01
Budget End
1996-07-31
Support Year
11
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Pediatrics
Type
Schools of Medicine
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030
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