Production of fibrolast-pneumocyte-like factor by fetal rabbit lung fibroblasts: Isolation and effects of it and related factors on fetal type II cells in vitro

Abstract

Fetal lung fibroblasts interact with type II epithelial cells, inducing their maturation. This interaction arises by secretion of factors which alter fetal type II cell function. To analyze these factors, conditioned medium (CM) was produced by exposing serum-free minimum essential medium, with [ 35S]methionine (5μCi/ml), to confluent cultures of fetal rabbit lung fibroblasts. This medium was tested for ability to stimulate [ 3H]choline incorporation by fetal type II cells and subsequently fractioned on molecular weight filtration columns P60 (2.5cm × 90cm; NMW cutoff, 60kd; 1 M acetic acid) and A1.5m (2.5cm × 90cm; NMW cutoff, 1,500kd; Tris-buffered saline) and a hydroxyapatite column (HT) (1.5cm × 30cm; NaCl and 0.01 − 0.03M phosphate). Crude medium stimulated choline incorporation into phosphatidylcholine. [ 35S]methionine was resolved in void volume material of apparent molecular weight of 6000 daltons on the P60 filtration column. Filtration on the A1.5m column showed two major fractions with radiolabel incorporation. Each of these was resolved into two subfractions on HT chromatography. The high molecular mass fraction contained material which stimulated [ 3H]choline incorporation by fetal type II cells. The low molecular mass fraction tended to inhibit [ 3H]choline incorporation. The second subfractions of both the first and second primary fractions inhibited [ 3H]thymidine incorporation into DNA by fetal type II cells. SDS-PAGE electrophoresis and autoradiography showed that under reducing conditions, each peak contained several proteins. However few of these displayed radioactivity. These results indicate that protein factors produced by fetal lung fibroblasts may be involved in regulating both differentiation and replication of fetal type II cells.