Effects of epidermal growth factor on collagen expression by rat kidney mesangial cells in culture.

Increased collagen production by mesangial cells plays a key role in the development and progression of glomerular sclerosis. These changes reflect in part the impact of growth factors on mesangial cells. Since mesangial cells possess receptors for epidermal growth factor (EGF) and since previous studies have documented that EGF affects collagen synthesis in other cell types, we have examined the effects of EGF on collagen biosynthesis by rat kidney mesangial (RKM) cells in culture. Exposure for 24 h to EGF did not substantially affect the growth rate of RKM cells. While the types of collagen produced by RKM cells (types I, III, IV and V) were unaltered by exposure to EGF, total collagen production was reduced ( approximately 50%). This decrease in collagen expression was not uniform for each collagen type. Type I collagen production was inhibited by approximately 50%, both type III and type IV expression were each reduced by approximately 30%, but type V collagen production was suppressed by only approximately 15%. The reduction in type I collagen synthesis was accounted for mainly by a decrease in type I homotrimer production. Since type I molecules represent approximately 95% of the total collagen produced, the decrease in overall collagen expression reflects a specific suppression by EGF on type I homotrimer production in mesangial cells. As EGF exposure resulted in a decrease in collagen production, these results suggest that the increases in synthesis and deposition of collagen observed in several glomerular diseases likely do not reflect the short-term effects of EGF on mesangial cells. Rather, these findings suggest the possibility that EGF or EGF-like growth factors may ameliorate the effects of other soluble factors that cause enhanced matrix production and deposition in renal diseases.

Characterization of the collagen phenotype of rabbit proximal tubule cells in culture.

Studies were performed to characterize the collagen phenotype of cultured rabbit proximal tubule (RPT) epithelial cells grown on plastic and on the reconstituted basement membrane preparation, Matrigel. When grown on a plastic substratum, RPT cells display a cobblestone appearance characteristic of glomerular epithelial cells. While initially forming an interlocking network of cells after subculture on Matrigel, this pattern of culture morphology rapidly develops into one characterized by isolated, organized groups of cells. Notwithstanding the effects of Matrigel on culture morphology, total cellular proliferation was reduced only 25% when RPT cells were grown on this substrate. Greater than 90% of the collagen synthesized by RPT cells grown on plastic was secreted into the culture medium. Qualitative analysis by SDS-PAGE revealed components exhibiting electrophoretic mobilities corresponding to the chains present in type IV and type I collagens. Quantitative analysis by CM-Trisacryl chromatography established that approximately 2/3 of the total collagen synthesized by RPT cells grown on plastic was type IV and approximately 1/3 type I. Quantitative analysis of the collagens produced by RPT cells grown on Matrigel again indicated the synthesis of only type IV and type I molecules but in a slightly more equal ratio of both collagen types and in the ratio of secreted to cell-associated molecules. However, the total amount of collagen synthesized by RPT cells grown on Matrigel was reduced to approximately 1% of the level synthesized by the cells grown on plastic. On plastic, approximately 3/4 of the type I collagen produced was recovered as the type I homotrimer, but on Matrigel type I homotrimers represented only approximately 55% of the total type I collagen synthesized. On Matrigel, the majority of the type IV collagen was recovered as heterotrimers containing alpha1(IV) and alpha2(IV) chains. In contrast, RTP cells grown on plastic predominantly produced type IV homotrimers containing only the alpha1(IV) chain. These data represent the initial report describing the collagens produced by nonimmortalized cultured proximal tubule cells. The finding that a significant amount of the total collagen synthesized was type IV (basement membrane) collagen, regardless of culture substrate, suggests that the RPT cells have maintained a significant degree of differentiation in culture, and thus establishes RPT cells as an appropriate model for investigating ECM changes in proximal tubule cells that occur in kidney disease. Finally, the observation that culture of RPT cells on a reconstituted basement membrane preparation results in a significant reduction in total collagen production and alterations in the molecular forms of type IV and type I molecules synthesized indicates that integrity of the tubular basement membrane may represent an important component in preventing the development of tubulointerstitial fibrosis.

Effects of transforming growth factor-beta on collagen synthesis by normal rat kidney epithelial cells.

The effects of transforming growth factor-beta (TGF-beta) on the growth of and collagen production by NRK52E cells, a clonal line established from normal rat kidney epithelial cells, have been characterized. NRK52E cells were grown in the absence or presence of TGF-beta for 4 days followed by incubation for 24 hours in serum-free medium containing [3H]proline. The secreted and cell-associated collagens produced by control and experimental cultures were isolated by limited pepsin digestion and differential salt fractionation. TGF-beta inhibited proliferation by about 50% but did not affect overall culture morphology. Both protein and collagen synthesis were increased in experimental cultures, but the increase in total collagen production exceeded that of total protein synthesis. Although NRK52E cells grown in the presence of TGF-beta continued to produce the same types of collagen (types I, III, IV, and V), their relative amounts were changed. In the experimental cultures, type I collagen production was increased eightfold, types III and V collagen levels were increased two-fold, but type IV production was only slightly enhanced. In addition to increasing total collagen production by about fivefold, TGF-beta increased the ratio of type I to type III about threefold but minimally affected the ratio of secreted to cell-associated molecules. These findings establish that TGF-beta specifically affects collagen production in NRK52E cells and that these alterations differ in many ways from the affects of epidermal growth factor. Because TGF-beta increased total collagen expression, these results provide additional evidence implicating this growth factor as a positive mediator of matrix accumulation in renal disease.

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.

Effects of transforming growth factor-beta on collagen synthesis by fetal rat lung epithelial cells.

Studies were performed to characterize the effects of acute and chronic exposure to transforming growth factor-beta (TGF-beta) on collagen biosynthesis by fetal rat lung epithelial (FRLE) cells, a cell line established from the fetal rat lung alveolar epithelial cell. Neither condition of exposure to TGF-beta stimulated cell growth, but both conditions increased total protein synthesis. Quantitative evaluation by carboxymethyl-Trisacryl chromatography revealed that FRLE cells synthesized types I, III, IV, and V collagen under all circumstances. Acute and chronic exposure to TGF-beta increased total collagen production approximately 50% and 300%, respectively, with the increases in total collagen production exceeding those of total protein synthesis. In addition, these analyses indicated that the production of types I and III molecules was stimulated to a greater extent than was the synthesis of types IV and V molecules. Both experimental conditions increased the ratio of secreted to cell-associated molecules for types I and III molecules, decreased this ratio for type IV collagen, but minimally affected the culture distribution of type V collagen. Additionally, both conditions of exposure to TGF-beta were found to increase the proportion of the homotrimeric forms of types I and V molecules relative to their heterotrimeric counterparts. Thus, these studies establish that TGF-beta selectively and type-specifically alters collagen production without affecting growth in an epithelial cell line of fetal rat lung origin.

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.

Effects of epidermal growth factor on collagen synthesis by an epithelioid cell line derived from normal rat kidney.

Studies were performed to characterize the effects of both acute and chronic exposure to epidermal growth factor (EGF) on collagen biosynthesis by NRK52E cells, an epithelioid line established from normal rat kidney. Both conditions of exposure to EGF stimulated cell growth. Qualitative analysis revealed that NRK52E cells continued to synthesize types I, III, IV, and V collagens under all circumstances. However, both acute and chronic exposure to EGF increased two- to fourfold the amounts of collagenous proteins secreted by NRK52E cells, but decreased by 40 to 80% the amounts of collagenous material derived from the cell layers. Quantitative evaluation of type I and type III molecules confirmed that both conditions of exposure to EGF resulted in substantial increases in the amounts of these types of collagen secreted by the cells. However, analysis of the cell-associated types I and III molecules revealed that, while prolonged exposure to EGF significantly decreased the production of both types of collagen, acute exposure minimally affected type I collagen levels, but decreased type III production greater than 40%. Thus, conditions of acute exposure resulted in a slight increase in the total amount of type I collagen produced, but a slight decrease in the amount of type III collagen synthesized. In contrast, chronic exposure to EGF decreased the total amount of both collagen types synthesized, with type III being decreased to a greater extent (approximately 70%) than was type I (approximately 50%). Thus, the ratio of type III to type I collagen produced by NRK52E cells was decreased under both conditions of exposure to EGF. Therefore, these studies establish that EGF can modify matrix production in an epithelioid line of renal origin.

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 type I homotrimer collagen molecules by cultured human lung adenocarcinoma cells.

Studies have been performed to evaluate both the relative amounts and molecular forms of the collagens synthesized by a new cell line (HU1) established from a human lung adenocarcinoma. The collagens secreted into the culture medium and extracted from the cell layers of cultured HU1 cells were isolated after limited pepsin digestion and differential salt fractionation. More than 70% of the collagen synthesized by HU1 cells was secreted into the culture medium rather than remaining in the cell layer. Polyacrylamide gel electrophoresis under denaturing conditions of the collagens indicated the presence of components with properties corresponding to those of the chains present in the types I homotrimer, III, IV, and V collagens. Carboxymethyl-trisacryl chromatographic analysis revealed that approximately 90% of the total collagen synthesized by HU1 cells corresponded to the type I homotrimer and that the cells did not synthesize the alpha 2(I) collagen chain. Of the remaining collagen, types III, IV, and V molecules represented 6, 1, and 4%, respectively, of the total produced. These data establish the relative proportions of the collagens synthesized by cultured HU1 cells and represent one of the initial documentations of a cell line established from a carcinoma of pulmonary origin that synthesizes type I homotrimer molecules. Furthermore, these findings suggest that HU1 cells may be a useful model for investigating the molecular basis of alterations in collagen biosynthesis associated with neoplasia.

The effects of tissue pretreatment and pepsin levels on the isolation of collagens from human placenta.

A comparative study has been undertaken to ascertain the effects of different tissue pretreatment procedures on the recovery of the major genetic types of collagen from human placenta. Essentially the same recovery of types I, III, IV and V collagen was obtained from placenta which was directly processed, from placenta which was stored at -70 degrees C after washing, and from dried acetone extracts of this tissue. Each collagen type isolated from the treated tissue preparations displayed properties consistent with those exhibited by its counterpart obtained from fresh tissue which was directly processed. Furthermore, it was observed that while the amount of types I and III collagen recovered was directly proportional to the level of pepsin employed, the recovery of types IV and V collagen was inversely related to this isolation parameter. These results establish that human placenta can be either stored frozen or as a dry acetone extract without affecting either the yield or the profile of the different genetic types of collagen recovered and that the amounts of different genetic types of collagen recovered can be modulated by the amount of pepsin employed in the isolation.

The cytotoxins of cobra venoms. Isolation and partial characterization.

Eight basic proteins which lyse virus-transformed mouse fibroblasts in culture have been isolated from the venoms of six Asian Naja naja subspecies. These cytotoxins appear to represent an homologous series of proteins, all within the molecular weight range of 7000-8000. They have been divided into three arbitrary types on the basis of amino acid composition, electrophoretic mobilities and elution order upon ion-exchange chromatography. The rate at which the toxins effect cell lysis: (1) appears to be a function of the basicity of each toxin; (2) is dependent upon toxin concentration; (3) is temperature dependent; and (4) is inhibited by heparin sulfate. In view of the physical changes, which the cell undergoes during lysis and of the various factors which affect the action of these proteins, it is proposed that interaction of membrane receptors with the toxin, leading to alteration of cell membrane structure, is the principal event which ultimately leads to the disruption of the cell.