The long term goal of these studies is to elucidate the molecular aspects of collagen gene expression in cultured cells treated with bleomycin, a well-studied agent that induces pulmonary fibrosis. This disease has an unknown etiology and is characterized by decreased lung compliance and a distortion of pulmonary architecture resulting from an unexplained increase in interstitial connective tissue, the principal component being collagen type I. The focus of this proposal is to examine collagen gene expression using bleomycin-treated human and bovine lung fibroblasts, smooth muscle cells and endothelial cells.
The Specific Aims are to determine: (1) if bleomycin coordinately regulates expression of collagen types I, III, IV and V genes in these cell types, (2) how collagen gene expression in these cells is modulated by growth factors, (3) the effect of mechanical strain in bleomycin-treated fibroblasts, and (4) if bleomycin treatment results in changes in alternative splicing of the primary fibronectin gene transcript. The type and quantity of matrix gene transcripts produced by cells after bleomycin treatment will be correlated with levels of proteins synthesized. Specifically, investigations will be performed using collagen types I (alpha 1 and alpha 2), III, IV (alpha 1 and alpha 2) and V (alpha 2) DNA probes as well as those coding for fibronectin and thrombospondin. RNAs will be examined by Northern, slot blot and in situ hybridization and changes in synthesis will be further evaluated by S1 nuclease and nuclear run-off transcription assays. Particular emphasis will be placed on investigation of the matrix RNAs associated with polysomes since it has previously been established that partitioning of fibroblast type I collagen mRNAs with polysomes is a feature of bleomycin-treated cells. Analysis of the type and quantity of matrix proteins synthesized will be carried out by isotopic labeling studies, and hydroxyproline and collagenase assays. Matrix proteins will be identified by Western blotting and polyacrylamide gel electrophoresis and quantitated by ELISA to determine if the protein and mRNA data coincide.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL041882-01
Application #
3359674
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1988-12-01
Project End
1994-11-30
Budget Start
1988-12-01
Budget End
1990-11-30
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University City Science Center
Department
Type
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Myers, J C; Yang, H; D'Ippolito, J A et al. (1994) The triple-helical region of human type XIX collagen consists of multiple collagenous subdomains and exhibits limited sequence homology to alpha 1(XVI). J Biol Chem 269:18549-57
Myers, J C (1993) Differential expression of type I collagen and cellular fibronectin isoforms in endothelial cell variants. Kidney Int 43:45-52
Myers, J C; Sun, M J; D'Ippolito, J A et al. (1993) Human cDNA clones transcribed from an unusually high-molecular-weight RNA encode a new collagen chain. Gene 123:211-7
Myers, J C; Kivirikko, S; Gordon, M K et al. (1992) Identification of a previously unknown human collagen chain, alpha 1(XV), characterized by extensive interruptions in the triple-helical region. Proc Natl Acad Sci U S A 89:10144-8
Huebner, K; Cannizzaro, L A; Jabs, E W et al. (1992) Chromosomal assignment of a gene encoding a new collagen type (COL15A1) to 9q21 --> q22. Genomics 14:220-4
Howard, P S; Myers, J C; Gorfien, S F et al. (1991) Progressive modulation of endothelial phenotype during in vitro blood vessel formation. Dev Biol 146:325-38
Pihlajaniemi, T; Pohjolainen, E R; Myers, J C (1990) Complete primary structure of the triple-helical region and the carboxyl-terminal domain of a new type IV collagen chain, alpha 5(IV). J Biol Chem 265:13758-66
Myers, J C; Howard, P S; Walther, S E et al. (1990) Collagen and fibronectin gene expression in cultured endothelial cells. Ann N Y Acad Sci 580:120-31
Myers, J C; Jones, T A; Pohjolainen, E R et al. (1990) Molecular cloning of alpha 5(IV) collagen and assignment of the gene to the region of the X chromosome containing the Alport syndrome locus. Am J Hum Genet 46:1024-33
Gorfien, S F; Howard, P S; Myers, J C et al. (1990) Cyclic biaxial strain of pulmonary artery endothelial cells causes an increase in cell layer-associated fibronectin. Am J Respir Cell Mol Biol 3:421-9

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