MECHANISM OF BLEOMYCIN INDUCED COLLAGEN SYNTHESIS

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Bleomycin causes pulmonary fibrosis. Polysomes isolated from bleomycin-treated fibroblasts synthesize increased procollagen as compared to control polysomes, while noncollagen protein synthesis is not effected. The temporal response of cellular and polysomal synthesis of type I and type III procollagens in the lungs of bleomycin-treated chicks will be compared to the synthesis of these procollagens in bleomycin-treated chick lung fibroblasts. The contents of total cellular procollagen type I mRNAs are not altered by bleomycin treatment. However, type I procollagen mRNAs are elevated in polysomes and decreased in nuclei and in the post-polysomal cytoplasm. These data indicate a partitioning effect on type I procollagen mRNAs. The molecular mechanism(s) by which bleomycin increases polysomal procollagen synthesis will be determined. The synthesis of type I and type III procollagen mRNAs in the total cell, nuclei and their accumulation into the post-polysomal cytoplasm and polysomes will be determined. The effect of bleomycin on the nuclear quantities of procollagen hnRNAs will also be determined. Nuclei will be isolated from control and bleomycin-treated fibroblasts and transcribed in vitro. The transport of type I and type III procollagen mRNAs from nuclei and polysomes will be assessed. The effects of bleomycin on the degradation of total cellular, cytoplasmic and nuclear type I and type III procollagen mRNA sequences will be determined. To identify the mechanism(s) of increased polysomal procollagen synthesis, polysomal mRNPs will be isolated and the contents of procollagen type I and type III mRNAs will be determined. Procollagen type I and type III synthesized by these mRNPs in vitro will be determined. The salt wash of polysomes isolated from control and bleomycin-treated fibroblasts will be added to an in vitro protein synthesizing system containing control polysomes to determine the presence of a bleomycin-induced stimulating factor(s). The presence of a procollagen synthesis inhibiting factor in the control polysomes will be assessed. If such a factor(s) exists we isolate these. We will also determine if bleomycin treatment alters the secretion, accumulation and/or degradation of procollagen. This fibroblast model of bleomycin-induced lung toxicity may be then used to screen the direct effect of bleomycin and its analogues on fibroblast collagen metabolism. In addition, these studies may elucidate the molecular basis of bleomycin-induced pulmonary fibrosis.