REGULATION OF TYPE-I COLLAGEN PRODUCTION BY INSULIN AND TRANSFORMING GROWTH-FACTOR-BETA IN HUMAN LUNG FIBROBLASTS

The effects and interaction of transforming growth factor-beta (TGF-be ta) and insulin on collagen production in human fetal lung fibroblasts was examined. Fibroblasts were labeled with [H-3]proline and collagen production was analyzed by polyacrylamide gel electrophoresis. The ad dition of insulin (2 mu g/ml) increased collagen production 5 fold and TGF-beta (5 ng/ml) increased collagen production 6 fold. The combinat ion of TGF-beta and insulin further increased type I collagen producti on (12 fold). We found that TGF-beta increased pro-alpha 1(I) collagen mRNA levels 2-3 fold, insulin increased mRNA levels by less than 2 fo ld, and the combination stimulated a 3-4 fold increase. In a nuclear r un-on assay, we found a 1.7 fold increase in the rate of transcription for the pro-alpha 1(I) collagen gene in insulin-treated cultures and a 2 fold increase in TGF-treated cultures. In fibroblasts transfected with a plasmid containing 2.4 kb of the 5' flanking sequences of the h uman pro-alpha 1(I) collagen gene, TGF-beta stimulated a 2.8 fold incr ease in promoter activity. In contrast, the addition of insulin stimul ated a small increase (less than 2 fold) in the pro-alpha 1(I) collage n promoter activity when administered alone or in combination with TGF -beta. Insulin prolonged the half-life of pro-alpha 1(I) collagen mRNA from 9.1 h to 14.3 h as assessed by treatment with actinomycin D. The insulin-induced increase in pro-alpha 1(I) collagen mRNA was blocked by the presence of cycloheximide indicating a requirement for new prot ein synthesis. These results show that the combination of TGF-beta and insulin stimulate large increases in type I collagen formation by act ing at different sites in the collagen biosynthetic pathway.