Certain lung injuries induce large increases in connective tissue content, particularly collagen, resulting in fibrosis. During injury, cells are exposed to effector substances such as transforming growth factor-beta (TGF-beta), that regulate production of matrix components. This project focuses on establishing the mechanisms involved during the stimulation of collagen transcription by TGF-beta. Our preliminary work demonstrates that TGF-beta stimulates lung fibroblasts to accumulate type I collagen and alpha(I) mRNA. TGF-beta increases alpha(I) transcription by a unique protein complex binding to a cis-regulatory region termed TAE. This element is located 1.6 kb upstream from the transcriptional start site. Co-transfection with double stranded dsTAE abrogates the TGF-beta response. When TAE is mutated within the promoter, the basal activity increases to TGF-beta stimulated levels suggesting that TAE is a repressor element. TAE acts as a TGF-beta response element cloned upstream of a collagen basal (224 bp) promoter, but not when cloned upstream thymidine kinase (TK) promoter. In addition, transgenic animal studies with mice containing the collagen 3.6 kb promoter upstream of chloramphenicol acetyl transferase gene (CAB demonstrate that subset of fibroblasts have increased CAT expression during fibrosis or injections with TGF-beta. Therefore, other transcriptional regulatory regions are important for collagen gene expression in other cells. Indeed, data suggests there may be a response element in the first intron. We hypothesize that, during TGF-beta stimulation of fibroblasts, proteins are modified, enter the nucleus an bind to the TAE sequence in the alpha(I) collagen promoter releasing repression of transcription through prot binding to proximal cis-acting sites. In vivo certain myofibroblast populations respond similarly to TGF-beta whereas others seem to require, additional regulatory regions to open chromatin structure and/or for stimulation during acute stages preceding fibrosis.
The specific aims of this proposal are to 1. perform site directed mutagenesis of rat alpha(I) proximal Nor and intron to establish critical bases for TGF-beta response 2: characterize the proteins binding to TAE and analyze the TGF-beta response mechanism. We have recently cloned potential TAE binding proteins. The mrnA expression in cells and lung tissue will be determined. Full length clones will be expressed to determine function. We will identify the proteins associated with TAE. We will detect the presence of phosphorylation changes in TAE binding proteins and determine if transcription factors are translocated into nucleus during TGF-beta stimulation. 3: determine whether transgenic animals with mutated collagen promoters with and without other regulatory regions increase CAT transcription during bleomycin induced fibrosis 4: characterize further the inhibition of TGF-beta response by dsTAE oligonucleotides. Continue evaluating the extent of inhibition t TAE to assess the use of dsTAE as an antifibrotic agent.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Specialized Center (P50)
Project #
5P50HL056386-05
Application #
6411237
Study Section
Project Start
2001-01-22
Project End
2002-06-30
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
5
Fiscal Year
2001
Total Cost
$308,594
Indirect Cost
Name
Boston University
Department
Type
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Akuthota, Praveen; Capron, Kelsey; Weller, Peter F (2014) Eosinophil purification from peripheral blood. Methods Mol Biol 1178:13-20
Bandeira-Melo, Christianne; Weller, Peter F (2005) Mechanisms of eosinophil cytokine release. Mem Inst Oswaldo Cruz 100 Suppl 1:73-81
Segel, Michael J; Izbicki, Gabriel; Cohen, Pazit Y et al. (2003) Role of interferon-gamma in the evolution of murine bleomycin lung fibrosis. Am J Physiol Lung Cell Mol Physiol 285:L1255-62
Lucey, Edgar C; Goldstein, Ronald H; Breuer, Raphael et al. (2003) Retinoic acid does not affect alveolar septation in adult FVB mice with elastase-induced emphysema. Respiration 70:200-5
Subramaniam, Meera; Sugiyama, Kumiya; Coy, David H et al. (2003) Bombesin-like peptides and mast cell responses: relevance to bronchopulmonary dysplasia? Am J Respir Crit Care Med 168:601-11
Izbicki, Gabriel; Or, Reuven; Christensen, Thomas G et al. (2002) Bleomycin-induced lung fibrosis in IL-4-overexpressing and knockout mice. Am J Physiol Lung Cell Mol Physiol 283:L1110-6
Bandeira-Melo, Christianne; Hall, John C; Penrose, John F et al. (2002) Cysteinyl leukotrienes induce IL-4 release from cord blood-derived human eosinophils. J Allergy Clin Immunol 109:975-9
Segel, M J; Or, R; Tzurel, A et al. (2001) All-trans-retinoic acid (ATRA) is of no benefit in bleomycin-induced lung injury. Pulm Pharmacol Ther 14:403-7
O'Regan, A W; Hayden, J M; Body, S et al. (2001) Abnormal pulmonary granuloma formation in osteopontin-deficient mice. Am J Respir Crit Care Med 164:2243-7
Berkman, N; Kremer, S; Or, R et al. (2001) Human recombinant interferon-alpha2a and interferon-alphaA/D have different effects on bleomycin-induced lung injury. Respiration 68:169-77

Showing the most recent 10 out of 45 publications