INSULIN-LIKE GROWTH-FACTOR BINDING-PROTEIN PRODUCTION AND REGULATION IN FETAL-RAT LUNG-CELLS

Insulin-like growth factor binding proteins (IGFBPs) are expressed in lung from early in gestation and may modulate IGF-stimulated fetal lung cell proliferation and/or differentiation. To begin to define IGFBP production and regulation in lung cells during development, we prepared primary cultures of 19 day gestation fetal rat lung fibroblasts and epithelial cells and identified IGFBPs secreted into medium. Ligand blot analysis of conditioned media (CM) from both cell types demonstrated IGFBP bands of approximately 39,000-45,000, 32,000, 24,000, and 22,000 M(r). These migration characteristics allowed the identification of the 39,000-45,000 M(r) bands as IGFBP-3 and the 24,000 M(r) band as IGFBP-4, while Western-immunoblot analyses localized IGFBP-2 to the 32,000 M(r) band and IGFBP-5 to the 22,000 M(r) band. Polymerase chain reaction amplification of cDNAs generated by reverse transcription of fibroblast and epithelial cell RNA using specific oligodeoxynucleotide primers for IGFBPs 1 through 6, demonstrated the presence of amplified products for IGFBP-2, -3, -4, -5, and -6. In both cell types, IGFBP-2 and -3 production was sustained during 48 h of incubation in serum-free medium, whereas IGFBP-4 abundance increased only during the first 6 to 12 h of incubation. CM from fibroblasts and epithelial cells plated at low densities contained a high abundance of IGFBP-2 per mug cellular DNA compared with cells at higher densities. In contrast, IGFBP-3 and -4 abundance normalized to cell DNA did not change with differing cell densities. In fibroblasts, IGF-I and dibutyryl cyclic adenosine monophosphate (dcAMP) each increased the abundance of the 32,000 M(r) IGFBP(s), while retinoic acid and dcAMP each increased IGFBP-4, and dexamethasone decreased IGFBP-3. Epithelial cells incubated with IGF-I increased the abundance of IGFBP-3, dcAMP and retinoic acid each increased IGFBP-4, and dexamethasone decreased IGFBP-3 and -4. These results demonstrate that multiple IGFBPs are produced by both epithelial and mesenchymal cells from fetal lung. Specific IGFBPs are regulated differently by serum withdrawal and cell density, as well as by agents that influence lung cell growth and differentiation. These findings suggest that changes in IGFBP production may influence IGF actions during fetal lung growth and development.