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.     

Hyaluronan affects protein and collagen synthesis by in vitro human skin fibroblasts

Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1 microM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P < 0.05), and particularly in collagen (-40%; P < 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.     

Binding of leukotriene C4 to rat lung fibroblasts and stimulation of collagen synthesis in vitro

Arachidonate metabolites are potent biological mediators affecting multiple cellular functions. Although prostaglandins of the E series, which are products of the cyclooxygenase pathway, have been known as inhibitors or down-regulators of fibroblast proliferation and collagen synthesis, the more recently discovered products of the 5-lipoxygenase pathway have not been as extensively investigated with regard to fibroblast function. In this study, a sulfidopeptide product of the lipoxygenase pathway, leukotriene C4 (LTC4), was examined for its ability to modulate rat lung fibroblast collagen synthesis and proliferation in vitro. The data revealed the ability of LTC4 and to a lesser extent leukotriene D4 (LTD4) to stimulate collagen synthesis in a dose-dependent (10(-11)-10(-8) M) manner without affecting cellular proliferation as determined by radiolabeled thymidine incorporation; 1 nM LTC4 caused an 85% (p less than 0.02) increase above untreated controls in [3H]proline incorporation into collagenous protein in the media, which was blocked by the putative leukotriene receptor antagonist FPL55712 (10 microM) and inhibited by cycloheximide and actinomycin D. This LTC4 stimulatory effect was slightly more specific for collagen synthesis vs noncollagenous protein synthesis but was not accompanied with any change in the collagen type composition. Binding of [3H]LTC4 to these cells was specific, reversible, and saturable, with a Kd of 1.8 +/- 0.95 nM. Under equilibrium conditions, there was an estimated 2.39 X 10(4) receptors per cell. This binding was also inhibited by 10 microM FPL55712. Competitive binding studies show specificity of this binding for LTC4 relative to LTD4 and FPL55712. Furthermore, no significant conversion of LTC4 to LTD4 or leukotriene E4 was noted during the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ascorbate on collagen synthesis by lung embryonic fibroblasts

Summary Total insoluble collagen and hydroxyproline formation were examined in lung embryonic fibroblasts (IMR-90) grown in the presence or absence of added ascorbate. As expected, when the cells from both groups (+ and −ascorbate) are pulsed with [¹⁴C]proline in the presence of ascorbate, the percent hydroxylation in a 24-hr period does not vary significantly. However, there are dramatic differences in the quantity and quality of the insoluble collagen fraction produced by those cells grown for a long period of time with added ascorbate. Those cells deprived of continuous addition of ascorbate to the culture medium do not display large quantities of accumulated collagen in the cell layer fractions as measured by the hydroxyproline content, whereas the cells grown in the presence of ascorbate contain significant amounts of accumulated collagen. A new method for examining the extracellular insoluble collagen produced in cell cultures is described in these studies. With the aid of pancreatic elastase relatively pure insoluble collagen can be obtained from cells grown in culture. In those cells grown in the presence of ascorbate, the purified insoluble collagen yeilds appropriately banded fibrils when examined in the electron microscope and has an amino-acid composition that is compatible with pure collagen. On the other hand, those cells grown in the absence of ascorbate do not yield purified insoluble collagen as determined by these same criteria. The elastase procedure for the purification of insoluble collagen in cell cultures is simple, easy to use and allows one to assess additional aspects of collagen biosynthesis.     

Altered protein synthesis in ataxia--telangiectasia fibroblasts

Analysis of protein production in various strains of ataxia--telangiectasia (A--T) fibroblasts demonstrated the overproduction of a group of secreted proteins and variations in the protein characteristics of the extracellular matrix. The most prominent differences involved fibronectin, which was identified by immunochemical analysis. One- and two-dimensional gel electrophoresis demonstrated differences in the production, accumulation, and molecular weight of fibronectin on the cell surface and in the culture medium as compared to normal human fibroblasts. Three other secreted proteins with molecular weights of 185 000, 150 000, and 70 000 were also observed to be produced in excess amounts in some strains of A-T. The finding that extracellular matrix alterations are involved in the abnormal DNA synthesis and reduced cell survival in A-T cells in response to X radiation would be additional evidence for a close association between the extracellular and nuclear architecture.     

Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on collagen synthesis of human skin fibroblasts in vitro

The effect of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) on collagen metabolism by human diploid fibroblasts in confluent monolayer culture was examined. Recombinant TNF alpha reduced collagen mRNA levels 2-fold and stimulated collagenase mRNA levels 5-fold, while recombinant IFN gamma affected only collagen mRNA levels. The combination of TNF alpha (10 ng/ml) and IFN gamma (100 ng/ml) resulted in a much stronger (about 30-fold) reduction of collagen mRNA levels indicating that the two cytokines act synergistically. In contrast no such synergism was observed with respect to collagenase mRNA levels. The effect of TNF alpha and IFN gamma on collagen metabolism reported here indicates a complex interaction of different cytokines in the control of tissue remodeling that occurs during inflammation, repair, or atrophy.     

Anoxia-induced changes in purine nucleoside metabolism of in vitro aged human fibroblasts

Inosine deriving from the metabolism of adenosine or inosine monophosphate (IMP) in the fibroblast provides the substrate for xanthine oxidase and is, therefore, an important source of toxic oxygen free radicals. With well-oxygenated medium, adenosine release appears to be greater for aged than young fibroblasts. In that the adenosine release by young cells is enhanced by reduced oxygenation, the effect anoxic stress on the release of the purine nucleosides adenosine and inosine by low-passage (PDL 23-26; young) vs. high-passage (PDL 43-51; aged) human lung fibroblasts (IMR-90) was studied. Cultures of confluent fibroblasts were incubated for 16 hr under normoxic (NF) or anoxic (AF) atmospheres. The release of adenosine and inosine was determined by HPLC at 0, 3, 6 and 24 hr after termination of the 16-hr period. Immediately following anoxia (time 0), adenosine release by young AF was 29% greater than for young NF, whereas both the youn     

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.     

Alkaline phosphatase activation and collagen synthesis in human skin fibroblasts in culture

It has been noted in regenerating wounds that alkaline phosphatase activity in fibroblasts reaches a maximum when the collagen production is greatest. Tissue culture studies were carried out to show that prednisolone phosphate, while increasing the specific activity of alkaline phosphatase in human diploid skin fibroblasts, did not affect accumulation of collagen-hydroxyproline in monolayers or media. Addition of sodium ascorbate, resulted in rapid accumulation of hydroxyproline in the culture over a 13-day interval, while alkaline phosphatase activity increased only slightly over the last 6 days. When prednisolone and ascorbate were added in combination, alkaline phosphatase activity was significantly increased: but accumulation of hydroxyproline was no greater than in cultures to which ascorbate alone was added. Activation of alkaline phosphatase induced by prednisolone phosphate does not appear to be directly related to the biosynthesis of collagen in human skin fibroblasts in tissue culture.     

Changes in collagen synthesis by human bone marrow fibroblasts with progressive subcultivation

Bone marrow fibroblasts from normal and leukemic patients were used to investigate the relationship between serial subcultivation and changes in collagen synthesis. A regime was established to generate subcultures up to 35 cumulative population doublings (CPDs) in normal cells and to 9 CPDs in leukemic cells. In both types of cells, collagen synthesis decreased as subcultivation progressed. In normal cells, collagen synthesis was reduced to 10% of the original levels at 18 CPDs and in leukemic cells at 8 CPDs. In normal fibroblasts, collagen synthesis was more profoundly affected than overall protein synthesis by subcultivation. In acute lymphoblastic leukemia-derived fibroblasts, the decrease in collagen synthesis paralleled that of total protein.     

Tumor necrosis factor inhibits collagen and fibronectin synthesis in human dermal fibroblasts

Tumor necrosis factor (TNF) caused inhibition of collagen production by confluent cultures of human dermal fibroblasts in a dose-dependent manner. Concomitant increase of prostaglandin E2 release was observed as a result of TNF-induced cell activation. However, a blockade of the cyclooxygenase pathway of arachidonate metabolism by indomethacin did not abrogate the inhibitory effect of TNF on collagen synthesis, suggesting that this effect could be independent of prostaglandin metabolism. Gel electrophoresis of the newly synthesized macromolecules from the culture media showed that both type I and type III collagens as well as fibronectin were affected by the inhibition. Electrophoresis of cell layer-associated proteins demonstrated that the reduction in amounts of collagen and fibronectin in the medium did not result from an intracellular accumulation of these macromolecules. Production of procollagens was reduced in parallel to that of collagens, suggesting that the effect of TNF is exerted before the processing steps of procollagens. These results clearly show that TNF could play a role in modulation of matrix deposition by fibroblasts during inflammatory processes.     

Functional diversity of lysyl hydroxylase 2 in collagen synthesis of human dermal fibroblasts

The pathogenesis of fibrosis, especially involving post-translational modifications of collagen, is poorly understood. Lysyl hydroxylase 2 (long) (LH2 (long)) is thought to play a pivotal role in fibrosis by directing the collagen cross-link pattern. Here we show that LH2 (long) exerts a bimodal function on collagen synthesis in human dermal fibroblasts. Adenoviral-mediated overexpression of LH2 (long) resulted in a mRNA increase of collagen α1(I) but not of fibronectin and fibrillin-1. This was accompanied by a higher mRNA level of prolyl-4-hydroxylase but not of other ER proteins (Bip, Hsp47, LH1, LH3). The collagen mRNA increase led to an elevated collagen synthesis, which was higher in the fraction of extracellularly deposited, cell-associated collagen than in the medium. The cross-link pattern of cell-associated collagen showed an increase of the hydroxylysine-aldehyde-derived cross-link dihydroxylysinonorleucine and a decrease of the lysine-aldehyde-derived component hydroxylysinonorleucine. The helical lysyl hydroxylation of the procollagen molecule was unaltered. The increase of collagen synthesis in fibroblasts overexpressing LH2 (long) was independent from cross-linking as it was also observed in the presence of β-aminopropionitril, a cross-linking inhibitor. Together our data identify LH2 (long) as a bifunctional protein and underscores its potential role in the pathogenesis of fibrosis.     

Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel

Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contact the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reduction of pro alpha 1 (I), pro alpha 2 (I), and pro alpha 1 (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix.