Type I collagen is one of a few proteins that is produced constitutively in certain cell types and in a highly regulated manner in others. This grant is designed to understand the molecular basis by which a different cell type can express the identical Alpha1(I) and Alpha2(I) collagen genes in a tissue specific manner. Using osteoblasts in organ culture, primary cultured osteoblasts, osteosarcoma cells and fibroblastic cells, the regulation of type I collagen mRNA will be ascessed using cDNA probes to Alpha1(I) and Alpha2(I) collagen mRNA and dot hybridization. Changes in the cytoplasmic levels of collagen mRNA brought on by PTH, 1,25 dihydroxyvitamin D (1,25(OH)2D3) and insulin will be explained by alteration in the nuclear transcription of the type I collagen genes and the half-life of its cytoplasmic mRNA. In situations where the mRNA change results from a difference in gene transcription, the structure of the promoter region of the gene will be examined. We plan to identify and characterize the 5' flanking region of the Alpha1(I) and Alpha2(I) collagen gene. With these probes subcloned into M13, differences in the domains of DNAse I sensitivity will be contrasted in each cell type. Detailed analysis of the 1,25(OH)2D3 binding site by a filter binding assay and footprinting technique will use purified 1,25(OH)2D3 receptor complex and defined DNA segments from the promoter region. This promoter region will be placed in a gene transfer vector derived from the bovine papilloma virus (BPV). The activity of the collagen promotor will be determined by the production of a marker protein, human metallothionein, that is a part of the BPV vector. The fusion gene will be transferred to the various cell types to determine if the promoter attains the same regulation and conformation as the actual promoter. If the two promoters are similar in the same cell, then the PBV model will be used to map the domain of the promoter or enhancer necessary for cell specific activity and hormone responsiveness. The model has the potential for isolating factors that are associated with the cell specific regulation of the promoter and for reconstituting a regulated in vitro transcription system.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR029983-07
Application #
3155733
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1981-08-01
Project End
1989-07-31
Budget Start
1987-08-01
Budget End
1989-07-31
Support Year
7
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Type
Schools of Medicine
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030
Erceg, Ivana; Tadic, Tade; Kronenberg, Mark S et al. (2003) Dlx5 regulation of mouse osteoblast differentiation mediated by avian retrovirus vector. Croat Med J 44:407-11
Tadic, Tade; Dodig, Milan; Erceg, Ivana et al. (2002) Overexpression of Dlx5 in chicken calvarial cells accelerates osteoblastic differentiation. J Bone Miner Res 17:1008-14
Tadic, T; Erceg, I; Stover, M L et al. (2001) Dlx5 induces expression of COL1A1 promoter contained in a retrovirus vector. Croat Med J 42:436-9
Dodig, M; Tadic, T; Kronenberg, M S et al. (1999) Ectopic Msx2 overexpression inhibits and Msx2 antisense stimulates calvarial osteoblast differentiation. Dev Biol 209:298-307
Bedalov, A; Salvatori, R; Dodig, M et al. (1998) 1,25-Dihydroxyvitamin D3 inhibition of col1a1 promoter expression in calvariae from neonatal transgenic mice. Biochim Biophys Acta 1398:285-93
Pan, Z; Lichtler, A C; Upholt, W B (1998) DNase I hypersensitive sites in the chromatin of the chicken Msx2 gene differ in anterior and posterior limb mesenchyme, calvarial osteoblasts and embryonic fibroblasts. Biochem Mol Biol Int 46:549-57
Walton, C M; Wu, G Y; Petruff, C A et al. (1996) A collagen enhancer-promoter construct in transgenic mice is markedly stimulated by ethanol administration. Hepatology 23:310-5
Dodig, M; Kronenberg, M S; Bedalov, A et al. (1996) Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice. J Biol Chem 271:16422-9
Kream, B E; Harrison, J R; Krebsbach, P H et al. (1995) Regulation of type I collagen gene expression in bone. Connect Tissue Res 31:261-4
Bedalov, A; Salvatori, R; Dodig, M et al. (1995) Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice. J Bone Miner Res 10:1443-51

Showing the most recent 10 out of 20 publications