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-beta) and insulin on collagen production in human fetal lung fibroblasts was examined. Fibroblasts were labeled with [3H]proline and collagen production was analyzed by polyacrylamide gel electrophoresis. The addition of insulin (2 micrograms/ml) increased collagen production 5 fold and TGF-beta (5 ng/ml) increased collagen production 6-fold. The combination of TGF-beta and insulin further increased type I collagen production (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 fold, and the combination stimulated a 3-4 fold increase. In a nuclear run-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 human pro-alpha 1 (I) collagen gene, TGF-beta stimulated a 2.8 fold increase in promoter activity. In contrast, the addition of insulin stimulated a small increase (less than 2 fold) in the pro-alpha 1 (I) collagen 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 protein synthesis. These results show that the combination of TGF-beta and insulin stimulate large increases in type I collagen formation by acting at different sites in the collagen biosynthetic pathway.