Based on the hypothesis that an important component of the fibrotic responses in lung fibrosis is an increased transcription of the type I and III collagen genes, this proposal seeks to determine the role of the cis-acting elements within these genes, and the function of the cellular transacting factors, which control the expression of these genes in fibroblasts. The proposed studies will first focus on the enhancer segment of the mouse alpha2(I) collagen gene, delineate its active sites, identify and purify the factors which bond to these sites, determine their function by several approaches including in vitro transcription, also generate cDNAs and specific antibodies for these factors. The properties of these factors will then be compared with those which interact with similar elements in the alpha1(I) and alpha(III) collagen genes. The second part of this proposal is based on the above studies and also on ongoing work in our laboratory that focuses on the promoter elements of the these genes and their regulation by growth factors (such as TGF-beta) and products of oncogenes. A mouse model will be used in which lung fibrosis as well as type I and III collagen synthesis in lung fibroblasts are induced by bleomycin. Since lung fibroblasts from bleomycin-treated animals lose their phenotype of increased type I collagen synthesis in culture, transgenic mice will be generated using a type I collagen promoter-enhancer-CAT chimeric gene. If, as expected, the transgene responds to bleomycin as the endogenous type I collagen genes, transgenic mice with mutations in specific cis-acting elements of the promoter-enhancer to type I collagen genes will be tested to see whether a specific mutation abolishes the response of the transgene to bleomycin. If such mutation is also known to inhibit the binding of a specific DNA binding protein, this will suggest that this DNA binding protein plays a role in the bleomycin-induced fibrotic response. If in addition this mutation fails to respond to a particular growth factor or other effector in DNA transfection experiments, a role for this growth factor in lung fibrosis will be suggested. In studies on biopsy samples and fibroblasts obtained from patients with lung fibrosis those specific cellular DNA binding factors most likely to be responsible for the fibrotic response will be examined using reagents developed in the first part of this proposal.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL041264-05
Application #
3358911
Study Section
Special Emphasis Panel (SRC (17))
Project Start
1988-07-01
Project End
1993-04-30
Budget Start
1992-05-08
Budget End
1993-04-30
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Texas MD Anderson Cancer Center
Department
Type
Other Domestic Higher Education
DUNS #
001910777
City
Houston
State
TX
Country
United States
Zip Code
77030
Nakashima, Kazuhisa; de Crombrugghe, Benoit (2003) Transcriptional mechanisms in osteoblast differentiation and bone formation. Trends Genet 19:458-66
Hasegawa, T; Takeuchi, A; Miyaishi, O et al. (1997) Cloning and characterization of a transcription factor that binds to the proximal promoters of the two mouse type I collagen genes. J Biol Chem 272:4915-23
Hasegawa, T; Zhou, X; Garrett, L A et al. (1996) Evidence for three major transcription activation elements in the proximal mouse proalpha2(I) collagen promoter. Nucleic Acids Res 24:3253-60
Bou-Gharios, G; Garrett, L A; Rossert, J et al. (1996) A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice. J Cell Biol 134:1333-44
Garrett-Sinha, L A; Eberspaecher, H; Seldin, M F et al. (1996) A gene for a novel zinc-finger protein expressed in differentiated epithelial cells and transiently in certain mesenchymal cells. J Biol Chem 271:31384-90
Rossert, J A; Chen, S S; Eberspaecher, H et al. (1996) Identification of a minimal sequence of the mouse pro-alpha 1(I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts. Proc Natl Acad Sci U S A 93:1027-31
Rossert, J; Eberspaecher, H; de Crombrugghe, B (1995) Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice. J Cell Biol 129:1421-32
Niederreither, K; D'Souza, R; Metsaranta, M et al. (1995) Coordinate patterns of expression of type I and III collagens during mouse development. Matrix Biol 14:705-13
D'Souza, R N; Niederreither, K; de Crombrugghe, B (1993) Osteoblast-specific expression of the alpha 2(I) collagen promoter in transgenic mice: correlation with the distribution of TGF-beta 1. J Bone Miner Res 8:1127-36
Hatamochi, A; de Crombrugghe, B; Krieg, T (1993) Purification of a novel factor which binds to the mouse alpha 2 (I) collagen promoter. FEBS Lett 327:325-31

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