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.
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