Effects of prostaglandin E1 on collagen production and degradation in human fetal lung fibroblasts

We examined the effects of prostaglandin E1 on the production and degradation of collagen in human fetal lung fibroblasts. Percentage collagen production was determined by incubating confluent cultures for 6 h with [3H]proline and either [14C]glycine or [14C]leucine and measuring the relative amounts of radioactivity incorporated into collagenase-sensitive and collagenase-insensitive material. Percentage collagen degradation was determined by measuring hydroxy[14C]proline in a low-molecular-weight fraction relative to total hydroxy[14C]proline. Prostaglandin E1, when present at a concentration as low as 0.25 micrograms/ml, reduced net collagen production by a factor of one-half, from 8 +/- 2 to 4 +/- 1% (P less than 0.05). In contrast, the change in percentage degradation was relatively gradual, rising steadily from the control value of 15 +/- 2 to 33 +/- 2% at 4 micrograms/ml (P less than 0.05). The increase in degradation, while significant, could not account for the total decrease in collagen production. We conclude that prostaglandin E1 exerts its inhibitory effect on collagen production in two essentially independent ways: lowering the rate of synthesis and increasing intracellular degradation. However, the decrease in synthesis is greater than the increase in degradation.     

Ammonium chloride inhibits basal degradation of newly synthesized collagen in human fetal lung fibroblasts

The objective of this work was to characterize basal degradation of newly synthesized collagen in human fetal lung fibroblasts. Analysis of 22 separate determinations showed that in cells incubated under normal conditions, the level of intracellular degradation was normally distributed with a mean of 15.2% and a standard deviation of 2.6%. Within each experiment, however, the uncertainty (standard deviation) in determining degradation was very small, usually less than 1.5%. Consideration of the large variation between experiments and the ability of our analytic technique to detect small, but "statistically significant," differences between groups within the same experiment led us to formulate two criteria for determining whether degradation measured in cultures exposed to some agent differs in a "biologically significant" way from degradation measured in control cultures. These criteria were used to evaluate the effects of the following proteinase inhibitors on basal degradation: NH4Cl, which increases the pH of subcellular compartments that are normally acidic; and leupeptin and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), which are inhibitors of lysosomal cathepsins (B and L) that degrade collagen. NH4Cl (16 mM) lowered degradation to an extent that was both statistically and biologically significant, but neither leupeptin nor TLCK affected degradation. The effect of NH4Cl on degradation was independent of its inhibitory effects on production of collagen, protein, and ATP. These results suggest that basal degradation occurs in, or beyond, an acidic (i.e., NH4Cl-sensitive) but nonlysosomal compartment of the cell, and that NH4Cl inhibits processing within, or transport to, that compartment. This is the first report of an agent that inhibits basal degradation of newly synthesized collagen in soft tissue fibroblasts.     

Ascorbic acid stimulates collagen production without altering intracellular degradation in cultured human skin fibroblasts

The influence of ascorbic acid on intracellular degradation of collagen synthesized by cultured human-skin fibroblasts was examined. In confluent cells maintained in 0.5% serum-supplemented medium, ascorbic acid had no significant effect on collagen degradation measured with hydroxyproline as the marker. Similar results were obtained when collagen degradation was measured with the marker hydroxylysine, the cellular synthesis of which is independent of ascorbic acid. The stimulatory effects of ascorbic acid on collagen production therefore cannot be explained by a change in the rate of degradation. Ascorbic acid acts at some as yet undetermined level to increase the rate of collagen synthesis.     

Acid phosphatase activity and intracellular collagen degradation by fibroblasts in vitro

Summary Human gingival fibroblasts were cultured with collagen fibrils. The precise process of collagen phagocytosis and the relationship between acid phosphatase activity and intracellular degradation of collagen were investigated by cytochemical methods at the ultrastructural level. The collagen fibrils were first engulfed at one end by cellular processes, or the cell membrane wrapped itself around the middle of the fibrils. Collagen phagocytosis induced acid phosphatase activity in the fibroblast Golgi-endoplasmic reticulum-lysosome system. By application of the tracer lanthanum, deposits were observed in the intercellular spaces and along the fibrils being phagocytosed. At this stage, primary lysosomes were seen in close proximity to the collagen being engulfed, but no signs of fusion were observed. When the fibrils had been interiorized in whole or in part, they ultimately became enclosed within phagosomes, and no tracer was observed along the interiorized portion of the fibrils. Primary lysosomes then fused with these collagen-containing phagosomes to form phagolysosomes. Collagen degradation occurred within these bodies even though the end of a fibril might have protruded outside of the cell. These results suggest that selective and controlled phagocytosis of collagen and intracellular digestion of it may play a central role in the physiological remodeling and metabolic breakdown of the collagen of connective tissues.     

Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture

Summary In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.     

Effects of thiol protease inhibitors on intracellular degradation of exogenous beta-galactosidase in cultured human skin fibroblasts

The effects of low molecular weight (LMW) protease inhibitors of microbial origin were evaluated on the intracellular degradation of beta-galactosidase purified from Aspergillus oryzae and taken up by cultured human skin fibroblasts with beta-galactosidase deficiency. Only thiol protease inhibitors showed an effect to increase the enzyme activity. E-64, a specific inhibitor of thiol proteases, prolonged 3-fold a half life of the exogenous beta-galactosidase and when the enzyme was supplied as liposomes, the half life was prolonged 9-fold in these cells. The role of thiol proteases in the degradation of enzyme molecules was discussed.     

Lysosomal function in the degradation of defective collagen in cultured lung fibroblasts

Human fibroblasts when induced to make nonhelical , defective collagen have mechanisms for degrading up to 30% of their newly synthesized collagen intracellularly prior to secretion. To determine if at least a portion of the degradation of defective collagen occurs by lysosomes, extracts of cultured HFL-1 fibroblasts were examined for proteinases capable of degrading denatured type I [3H]procollagen. The majority of the proteolytic activity against denatured [3H]-procollagen had a pH optimum of 3.5-4; it was stimulated by dithiothreitol and inhibited 95% by leupeptin, 10% by pepstatin, and 98% by leupeptin and pepstatin together. Extracts of purified lysosomes from the fibroblasts were active in degrading denatured [3H]procollagen and were completely inhibited by leupeptin and pepstatin. To demonstrate directly that human lung fibroblasts can translocate a portion of their defective collagen to lysosomes, cultured cells were incubated with cis-4-hydroxyproline and labeled with [14C]proline to cause the cells to make nonhelical [14C]procollagen. About 3% of the total intracellular hydroxy[14C]proline was found in lysosomes. If, however, the cells were also treated with NH4Cl, an inhibitor of lysosomal function, 18% of the intracellular hydroxy[14C]proline was found in lysosomes. These results demonstrate that cultured human lung fibroblasts induced to make defective collagen are capable of shunting a portion of such collagen to their lysosomes for intracellular degradation.     

Measurement of intracellular collagen degradation

Intracellular degradation of newly synthesized collagen is quantitated by incubating fibroblasts with [¹⁴C]proline and determining the percentage of total [¹⁴C]hydroxyproline that is present in a low molecular weight fraction. Several problems make this difficult. (1) Commercial [¹⁴C]proline is often contaminated with [¹⁴C]hydroxyproline and must be purified before use. (2) Salt and [¹⁴C]proline interfere with the determination of [¹⁴C]hydroxyproline in the low molecular weight fraction and must be removed by preparative ion-exchange chromatography. (3) Epimerization of trans- to cis-hydroxyproline during acid hydrolysis is variable and must be taken into account. (4) Loss of [¹⁴C]hydroxyproline during processing varies; [³H]hydroxyproline can be used as an internal measure of recovery, even though tritium may be lost during hydrolysis. An analytic cation-exchange resin is used for the final quantitation of [¹⁴C]hydroxyproline in the low and high molecular weight fractions. With these methods, degradation of newly synthesized collagen can be determined with a precision of ± 3%.     

Regulation of intracellular protein degradation in IMR-90 human diploid fibroblasts

Human diploid fibroblasts (IMR-90) regulate their overall rates of proteolysis in response to the composition of the culture medium and the ambient temperature. The magnitude and, in some cases, the direction of the response depend on the half-lives of the cellular proteins that are radioactively labeled and the time chosen for measurements of protein degradation. Fetal calf serum, insulin, fibroblast growth factor, epidermal growth factor, and amino acids selectively regulate catabolism of long-lived proteins without affecting degradation of short-lived proteins. Fetal calf serum reduces degradative rates of long-lived proteins and is maximally effective at a concentration of 20%, but the effect of serum on proteolysis is evident only for the first 24 hr. Insulin inhibits degradation of long-lived proteins in the presence or absence of glucose and amino acids in the medium, but is maximally effective only at high concentrations (10(-5) M). Amino acid deprivation increases degradative rates of long-lived proteins for the first 6 hr, but then decreases their catabolism for the subsequent 20 hr. Lowered temperature is the only condition tested that significantly alters degradative rates of short-lived proteins. Although cells incubated at 27 degrees C have reduced rates of degradation for both short-lived and long-lived proteins compared to cells at 37 degrees C, lowered temperature reduces catabolism of long-lived proteins to a greater extent.     

Collagen glucosyl- and galactosyltransferases of cultured human fetal lung fibroblasts

Collagen-galactosyltransferase and collagen-glucosyltransferase activities have been studied in cultured human fetal lung WI-38 and IMR-90 diploid fibroblasts. These enzymes functioned in concert to synthesize glucosylgalactosylhydroxylysine units as found naturally in collagens, basement membranes, and certain serum glycoproteins. The transferases used UDP-Gal and UDP-Glc as glycose donors, collagens and collagen-derived peptides or glycopeptides as glycose acceptors, and worked best in the presence of manganese as a required divalent cation. Two pH optima, between pH 6 and 6.5 and between pH 7.5 and 8, were noted for each type of transferase, and these optima, particularly in the case of glucosyltransferase, were evident regardless of size of acceptor employed in the assay. About 35% of the total activity of each enzyme was found in the soluble fractions of cell homogenates, and, of the particulate fraction activities, about 50% could be released by mild sonication or by treatment with Triton X-100. Assessment of transferase activities as a function of cellular aging in culture revealed that significant decreases in enzyme levels occurred as the cell approached senescence (late Phase II), and these effects were reversed when cells attained senescence (Phase III). Addition of ascorbic acid to young cultures, under conditions known to increase endogenous collagenpeptide hydroxylation, caused no effects on the activities of the glycosyltransferases toward exogenous acceptors. These results suggested that the activities of collagen-hydroxylases and glycosyltransferase might not be coordinately regulated, and that, regardless of the hydroxylation events, glycosylation of the peptide might be limited to a specific fraction of hydroxylysine residues during the post-translational modification of collagen.     

Prolyl-tRNA-based rates of protein and collagen synthesis in human lung fibroblasts.

Chondrocyte cultures were established from foetal bovine tracheal cartilage and maintained in Ham's F12 medium with or without 10% (v/v) foetal calf serum. The proteoglycans were isolated and characterized. (1) The proteoglycans from cultures both with and without serum distributed in associative or dissociative CsCl gradients like proteoglycans from cartilage tissue. (2) The amino acid composition, protein contents and glucosamine/galactosamine ratios were grossly identical with those of the tissue derived proteoglycans. (3) Sedimentation coefficients (s⁰) for the monomers were 21.0S and 22.7S from cultures without and with serum respectively. The s⁰ values obtained for aggregates were 72.3S and 93.2S respectively. The limiting viscosity numbers [η] were 248ml/g and 298ml/g respectively. These data corresponded well to those obtained for the tissue-derived proteoglycans. (4) The sizes of the core proteins and chondroitin sulphate chains respectively were the same for both types of cell-culture proteoglycans and similar to those of the tissue proteoglycans. Both the keratan sulphate-rich region and the hyaluronic acid-binding region were identified. The latter, however, was not resistant to limit digestion with trypsin, in contrast with the fragment derived from the bovine nasal cartilage. (5) About 70% of the cell-culture proteoglycans chromatographed in the void volume on a Sepharose 2B column, whereas reduced and alkylated samples (monomers) chromatographed completely included in the column. The two link proteins present in A1 preparations of cartilage proteoglycans were also present in A1 preparations of cell-culture proteoglycans. (6) A minor portion (10%) of the ³⁵S-labelled proteoglycans in the cultures was associated with the cells. Reduced and alkylated samples were larger compared with the monomers in the medium, and chromatographed partly (25%) excluded on the Sepharose 2B column. A larger proportion (50%) of the non-reduced samples chromatographed in the void volume of the column.     

Altered rates of collagen synthesis in in vitro aged human lung fibroblasts

Summary Absolute rates of protein and collagen synthesis based on prolyl-tRNA as the precursor were determined in two age groups of IMR-90 human lung fibroblasts. Compared with midrange fibroblasts [population doubling level (PDL)=20 to 30] aged fibroblasts (PDL>40) were larger in siz in terms of protein and RNA per cell, generally proliferated more slowly, exhibited different steady state [³H]proline transfer RNA (tRNA) precursor pool specific radioactivities, synthesized collagen at a substantially lower rate, and exhibited a reduction in the percent commitment to collagen synthesis. Total protein synthetic rates were reduced slightly in aged versus midrange fibroblasts but the difference was not statistically significant. Proliferative capacity (PDL/wk) correlated better with these changes than cumulative PDL. Cell size (protein/cell) was the variable that had the highest correlation with the reduction in collagen synthesis observed in human lung fibroblasts. Thus, an important differentiated function of human lung fibroblasts, collagen synthesis, is greatly diminished in vitro in large, slowly dividing fibroblasts. This study was supported in part by Grants PHS HL 14212 (Pulmonary SCOR) from the National Institutes of Health and by a postdoctoral research fellowship (J. N. H.) from the American Lung Association.     

Effects of HEMA on type I collagen protein in human gingival fibroblasts

The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized adhesive systems due to degradation processes or the incomplete polymerization of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental adhesives. Cytotoxic effects due to high concentrations of HEMA have already been investigated, but the influence of minor toxic concentrations on specific proteins such as type I collagen has not been studied in depth. The objective of this project was to study the effect of minor toxic concentrations of HEMA on human gingival fibroblasts (HGFs), investigating modification in cell morphology, cell viability, and the influence on type I collagen protein. Primary lines of human gingival fibroblasts were exposed to 3 mmol/L HEMA for different periods of time (24 h, 72 h, 96 h). The cell vitality was determined by MTT assay, and high-resolution scanning electron microscopy analysis was performed to evaluate differences in cell morphology before and after treatment. The presence and localization of type I collagen was determined by immunofluorescence in HGFs treated with HEMA for the same period of time. The vitality of the cells decreased after 72 h of exposure. The HGFs grown in monolayer and observed by field emission in-lens scanning electron microscopy demonstrated a preserved surface morphology after 24 h of treatment, while they showed an altered morphology after 96 h of treatment. Immunofluorescence demonstrated a reduction of type I collagen due to HEMA exposure after 96 h. From these results, we conclude that low concentrations of HEMA can significantly alter the morphology of human gingival fibroblasts and interfere with the presence of type I collagen protein.     

Modulation of collagen production by fibroblasts. Effects of chronic exposure to agonists that increase intracellular cyclic AMP.

Cultured human lung fibroblasts were evaluated for their responsiveness to isoprenaline (isoproterenol) or prostaglandin E2 before and after chronic incubation with the agonist. Cells incubated for 6 h with either agonist were suppressed in terms of collagen production and exhibited increased intracellular cyclic AMP. Cells incubated for 72 h with the agonist and then re-challenged for 6 h with the same agonist did not demonstrate suppressed collagen production or increased cyclic AMP. Cells incubated for 72 h with isoprenaline still responded to prostaglandin E2 when challenged for 6 h; however, when the order of agonist exposure was reversed, cells incubated with prostaglandin E2 did not respond to a challenge by isoprenaline. If cells were allowed to recover for 48 h without the agonist after a 72 h chronic incubation, they recovered their responsiveness to the agonist. The results indicate that, although cultured fibroblasts may become desensitized to one agonist, they may retain their sensitivity to a second agonist and chronic suppression of collagen production may be achieved by alternate exposure to isoprenaline and prostaglandin E2.     

A novel model system for characterization of phagosomal maturation, acidification, and intracellular collagen degradation in fibroblasts

Intracellular collagen degradation by fibroblasts is an important but poorly understood pathway for the physiological remodeling of mature connective tissues. The objective of this study was to determine whether gingival fibroblasts that express endogenous alpha(2)beta(1) integrin, the collagen receptor, would exhibit the cellular machinery required for phagosomal maturation and collagen degradation. There was a time-dependent increase of collagen bead internalization and a time-dependent decrease of bead-associated alpha(2)beta(1) integrin after initial bead binding. beta-Actin and gelsolin associated transiently with beads (0-30 min) followed by LAMP-2 (60-240 min) and cathepsin B (30-240 min). Cytochalasin D prevented phagosome formation and also prevented the sequential fusion of early endosomes with lysosomes. Collagen bead-associated pH was progressively reduced from 7.25 to 5.4, which was contemporaneous with progressive increases in degradation of bead-associated collagen (30-120 min). Concanamycin blocked acidification of phagolysosomes and collagen degradation but not phagosome maturation. Phagosomal acidification was partly dependent on elevated intracellular calcium. These studies demonstrate that the cellular machinery required for intracellular collagen degradation in fibroblasts closely resembles the vacuolar system in macrophages.