Regulation of Type I Collagen Production by Insulin and Transforming Growth Factor-β in Human Lung Fibroblasts

Abstract

The effects and interaction of transforming growth factor-β (TGF-β) 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 μg/ml) increased collagen production 5 fold and TGF-β (5 ng/ml) increased collagen production 6 fold. The combination of TGF-β and insulin further increased type I collagen production (12 fold). We found that TGF-β increased pro-α1(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-α1(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-α1(I) collagen gene, TGF-β 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-α1(I) collagen promoter activity when administered alone or in combination with TGF-β. Insulin prolonged the half-life of pro-α1(I) collagen mRNA from 9.1 h to 14.3 h as assessed by treatment with actinomycin D. The insulin-induced increase in pro-α1(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-β and insulin stimulate large increases in type I collagen formation by acting at different sites in the collagen biosynthetic pathway.

Key Words:

• Insulin
• TGF-β
• type I collagen