Bleomycin regulation of transforming growth factor-beta mRNA in rat lung fibroblasts.
Pulmonary fibrosis is a well-known toxic response to bleomycin treatment. Here we demonstrate the direct effects of bleomycin on lung fibroblasts that resulted in a marked increase of collagen synthesis as compared with total noncollagen protein synthesis. Bleomycin treatment of rat lung fibroblast cultures resulted in an increase of total cellular transforming growth factor-beta (TGF-beta) mRNA and increased secretion of TGF-beta protein into the conditioned media. beta 2-Microglobulin was measured as an mRNA that did not increase with bleomycin treatment. The bleomycin-induced increase of TGF-beta mRNA was decreased by cells cultured in the presence of either cycloheximide, an inhibitor of protein synthesis, or 2-mercapto-1-(beta-4-pyridethyl) benzimidazole, an inhibitor of RNA synthesis. To assess the mechanism underlying increased steady-state mRNA levels, the nuclear fraction was isolated from bleomycin-treated cells and the TGF-beta transcripts were determined. Transcription of TGF-beta mRNA was increased 12 h after bleomycin treatment, whereas the transcription of type I procollagen, type III procollagen, and beta-actin mRNAs were increased after 48 h of bleomycin treatment. beta 2-Microglobulin mRNA synthesis was not increased within this time frame. These results suggest bleomycin regulation of TGF-beta at both the mRNA and protein levels. Rats lung fibroblasts were separated by cell sorting into two subpopulations. One population of fibroblasts demonstrated increased procollagen type I mRNAs, whereas fibroblasts in the other population had increased procollagen type III mRNA. Following bleomycin treatment, TGF-beta mRNA was shown to be located more prominently in those fibroblasts that contain primarily collagen type I mRNAs.
Department of Biochemistry, College of Medicine, University of Vermont, Burlington 05405.
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Breen
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Shull
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Burne
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Absher
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Kelley
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Phan
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Cutroneo
D000818:Animals; D001761:Bleomycin / Q000494:pharmacology*; D015152:Blotting, Northern; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D008168:Lung / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011347:Procollagen / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism
D011347:Procollagen; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D001613:beta 2-Microglobulin; D001761:Bleomycin; D003094:Collagen