Extracellular matrix biosynthesis by cultured fetal rat lung epithelial cells. IV. Effects of chronic exposure to retinoic acid on growth, differentiation, and collagen biosynthesis.

Effects of chronic exposure to retinoic acid on a cell line (FRLE cells) established from the fetal rat lung type II alveolar epithelial cell have been studied. Chronic exposure to retinoic acid inhibited proliferation and altered the pattern of culture morphology at the light microscopic level. At the ultrastructural level, the development of lamellar body-like structures was inhibited. Although the rates of both total collagen and protein produced per cell were enhanced by chronic retinoic acid exposure, the increase in collagen production exceeded that of total protein synthesis. Chromatographic evaluation of collagen types I, III, IV, and V revealed increases in the amounts of radioactivity incorporated into each collagen type. However, the magnitudes of the increases differed for each collagen type, with the production of type IV collagen being the most enhanced. These analyses also indicated several additional effects on collagen production: 1) collagen type specific alterations in the ratio of secreted to cell associated molecules, 2) an increased ratio of type I homotrimers to type I heterotrimers, and 3) a decreased ratio of type V homotrimers to type V heterotrimers. These results indicate that chronic exposure to retinoic acid selectively affects collagen production in and differentiation of FRLE cells and establish that these changes are opposite to those observed under conditions of chronic exposure to EGF.

Extracellular matrix biosynthesis by cultured fetal rat lung epithelial cells. III. Effects of chronic exposure to epidermal growth factor on growth, differentiation, and collagen biosynthesis.

Studies have been performed to evaluate the effects of chronic exposure to epidermal growth factor (EGF) on a cell line (FRLE cells) established from the fetal rat lung type II alveolar epithelial cell. Chronic exposure to EGF enhanced proliferation and altered the culture morphology at the light microscopic level. At the ultrastructural level, chronic exposure to EGF inhibited the expression of lamellar body-like structures that occurs in the absence of EGF. Estimations of total protein and collagen production indicated that chronic exposure to EGF suppressed collagen production without significantly altering total protein synthesis. Quantitative evaluation of the genetic types of collagen (types I, III, IV, and V) produced by FRLE cells revealed decreased production of each collagen type in cultures chronically exposed to EGF. However, the magnitude of the decrease differed for each collagen type, with type III collagen synthesis being suppressed to the greatest extent. Additionally, chronic exposure to EGF resulted in enhanced secretion of types I, III, and IV collagen molecules and an increase in the ratios of type I-homotrimers to type I-heterotrimers and of type V-homotrimers to type V-heterotrimers. These findings demonstrate that chronic exposure to EGF selectively alters collagen expression in a cell line established from the fetal type II pneumocyte.

Extracellular matrix biosynthesis by cultured fetal rat lung epithelial cells. II. Effects of acute exposure to epidermal growth factor and retinoic acid on collagen biosynthesis.

The effects of acute exposure to epidermal growth factor (EGF), retinoic acid, and EGF + retinoic acid on collagen biosynthesis by fetal rat lung epithelial (FRLE) have been evaluated. Acute exposure to either EGF or retinoic acid resulted in a small increase in the amount of radioactive substrate incorporated into total proteins, but simultaneous exposure to EGF + retinoic acid increased total protein synthesis approximately 2-fold. Quantitative evaluation of the genetic types of collagen synthesized by FRLE cells (types I, III, IV, and V) revealed that each experimental condition affected collagen production in a different manner. Acute exposure to EGF selectively inhibited type III collagen production, minimally affected type I and type IV collagen synthesis, but enhanced type V collagen production. Acute exposure to retinoic acid minimally affected type III production but slightly enhanced the synthesis of type I molecules. Simultaneous acute exposure to both EGF and retinoic acid significantly increased the amounts of types I, IV, and V collagen synthesized but minimally affected type III collagen production. Evaluation of the molecular forms of type I collagen (the type I-heterotrimer-molecular composition [alpha 1 (I)]2 alpha 2(I) and the type I-homotrimer-molecular composition [alpha 1 (I)]3) synthesized under each condition revealed that EGF minimally affected the production of either molecular form of type I collagen. In contrast, acute exposure to retinoic acid in the absence or presence of EGF caused a greater than 2-fold increase in the amount of type I-homotrimers synthesized but substantially decreased the amount of type I-heterotrimers produced. These results demonstrate that alone, either EGF or retinoic acid modulate collagen synthesis by FRLE cells and that in combination, these substances exert effects on collagen production that differ from their individual actions. Thus, in addition to documenting that EGF and retinoic acid modulate collagen biosynthesis in a cell line derived from the fetal type II pneumocyte, these studies establish that FRLE cells constitute a useful in vitro model system for investigating the regulation of matrix biosynthesis and its relationship to the development of the alveolar epithelial cell.

Extracellular matrix biosynthesis by cultured fetal rat lung epithelial cells. I. Characterization of the clone and the major genetic types of collagen produced.

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 approximately 22 hours at 37 degrees 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 cells, 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 approximately 80% of the collagen synthesized was type I and that approximately 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.

Synthesis of [pro alpha 1(IV)]3 collagen molecules by cultured embryo-derived parietal yolk sac cells.

Electron immunohistochemical studies demonstrate that cultured embryo-derived parietal yolk sac (ED-PYS) carcinoma cells synthesize type IV collagen. This material has been isolated and characterized. The collagen obtained after limited pepsin digestion from the medium in which the cells are grown is composed of homogeneous components with a molecular mass of approximately 95 000 daltons. When chromatographed on (carboxymethyl)cellulose under denaturing conditions, the chains elute as acidic components slightly before the human alpha 1(I) chain and coincident with the position of elution of the pepsin-derived human alpha 1(IV) chain. This analysis indicates the presence of a single type of collagen chain in the pepsin-derived ED-PYS synthesized material. In addition, the profile of cyanogen bromide (CNBr) cleavage products obtained from the pepsin-derived ED-PYS cell collagen chains is essentially identical with that derived from the human alpha 1(IV) chain. Isolation of the medium collagen in the absence of pepsin digestion reveals the presence of two high molecular weight components equivalent in size to procollagen alpha chains. However, both high molecular weight products yield CNBr cleavage products that correspond to those obtained from the pepsin-derived alpha 1(IV) chain. The ED-PYS cell-associated collagens obtained with or without the use of pepsin contain components that are essentially identical with those isolated from the culture-medium collagen. These data provide definitive evidence for the existence of type IV collagen molecules composed solely of alpha 1(IV) procollagen chains and further document the usefulness of ED-PYS cells for investigating the biosynthesis of basement membrane components.