EXTRACELLULAR-MATRIX BIOSYNTHESIS BY CULTURED FETAL-RAT LUNG EPITHELIAL-CELLS .1. CHARACTERIZATION OF THE CLONE AND THE MAJOR GENETIC TYPES OF COLLAGEN PRODUCED

  • 1 June 1989
    • journal article
    • research article
    • Vol. 60  (6) , 791-807
Abstract
A new cell line of fetal rat lung origin has been established using the outgrowth procedure. One clone (2G3) isolated by this procedure exhibited during early passages some of the transmission electron microscopic features (e.g., lamellar bodies) indicative of type II pneumocytes and was selected for further study. This cell line has a stable modal chromosome number of 44 and has not been found to develop tumors in athymic rodents. The clone exhibits a biphasic growth curve with an initial generation time of .apprx. 22 hours at 37.degree. C. The cultures are not contact inhibited but rather develop an organized secondary growth pattern. Initially after subculture, a monolayer is formed consisting of cells which exhibit a cobblestone appearance. After development of this monolayer, a secondary growth pattern emerges. This latter phase of growth is characterized by spindle-shaped cells displaying a pattern of organization that delimits lumina on top of the initial monolayer. At the ultrastructural level, desmosomes are observed, and concurrent with the development of the secondary growth pattern, there is the appearance of dense cytoplasmic structures which resemble lamellar bodies. Based upon the origin, growth properties, and morphologic features of the cell, this clone has been designated fetal rat lung epithelial (FRLE) cells. The collagens secreted into the culture medium and present in the cell layers of FRLE cell cultures, which have developed the secondary growth pattern, were isolated using limited pepsin digestion and differential salt fractionation. Polyacrylamide gel electrophoresis under denaturing conditions indicated that FRLE cells synthesized components corresponding to the chains present in types I, III, IV, and V collagen molecules with no major differences occurring between the profiles of cell-associated and secreted molecules. Carboxymethyl-trisacryl chromatographic analysis revealed that .apprx. 80% of the collagen synthesized was type I and the .apprx. 20% of this genetic type of collagen was recovered as the type I homotrimer. Types III, IV, and V molecules accounted for 16, 2, and 3%, respectively, of the total collagen synthesized. Additionally, the type V collagen synthesized by FRLE cells was found to have the molecular compositions .alpha.1(V).alpha.2(V).alpha.3(V) and [.alpha.1(V)]3. These observations suggest that the collagen biosynthetic profile of the fetal or immature type II cell may differ from that of the fully differentiated type II pneumocyte. Furthermore, it is proposed that cultured FRLE cells may be a useful in vitro model system for investigating the regulation of macromolecular synthesis in and the differentiation and maturation of the fetal alveolar epithelial cell.