Collagen prolyl hydroxylation in WI-38 fibroblast cultures: Action of hydralazine

Summary The action of hydralazine on collagen prolyl hydroxylation was studied in a cell culture system using WI-38 fibroblasts. The prolyl hydroxylation level was determined by a method involving the digestion of collagen by bacterial collagenase and the examination of specific peptides. The presence of low concentrations of hydralazine (0.2 mM) in both “young” and “old” fibroblast cultures strongly inhibited collagen prolyl hydroxylation. The degree of inhibition was greater in serum-deficient cultures. No significant improvement in the degree of hydroxylation was observed by increasing either ascorbate or iron levels in the hydralazine-containing cultures in which hydroxylation was inhibited. Some of the reported side effects of hydralazine seen in patients might be related to its inhibitory effects on mixed function oxidative (MFO) hydroxylation systems. While the ascorbate dependence of the prolyl hydroxylase system of WI-38 decreased with the “age” of the culture, hydralazine inhibition of hydroxylation was dramatic with cultures of all “ages”. This work was supported by NIH grants nos. AM15671, AM1675 and HD07376, and fellowship no. HD01998.     

Tripeptide analysis of collagen synthesized by silica-exposed fibroblast cultures

The effect of silica on collagen biosynthesis by confluent monolayers of WI-38 fibroblast cultures was examined by a more comprehensive method of analysis. The presence of the particulates had no direct effect on protein (collagen) synthesis, proline incorporation or prolyl hydroxylase activity; the latter is determined by the degree of hydroxylation. Silica, however, was highly toxic to the cells.     

Interactions between Cells and Collagen V Molecules or Single Chains Involve Distinct Mechanisms

Acid-soluble and pepsin-treated collagen V were prepared from fetal human bones or human placenta, respectively, to be tested for potential cell adhesion promoting activity. Out of 14 different collagen I-adhering cell lines, 10 showed distinct adhesion to collagen V. In all cases adhesion was followed by spreading. The activities of intact and pepsin-solubilized collagen V were similar, suggesting that the cell binding sites are restricted to the triple-helical domain of the molecules. Cell adhesion was also induced by the unfolded form of collagen V and after separation of the α chains by heparin affinity chromatography. Isolated α2(V) chains, rich in RGD sequences, were more efficient than isolated α1(V) chains. However, cell adhesion to native or denatured collagen V did not proceed by the same molecular mechanisms as shown by cell adhesion inhibition experiments. Cell adhesion to native collagen V was insensitive to the presence of RGD-containing synthetic peptides while adhesion to denatured collagen V was inhibited by the peptides. Furthermore, the results strongly suggested a major role for α1α1 and α2β1 integrins in the RGD-independent cell adhesion to native collagen V. These data indicate that collagen V is a specific adhesive substrate for different cell types. It also suggests that distinct sets of RGD-dependent and RGD-independent receptors mediate cell attachment to unfolded and native collagen V, respectively. This mechanism is shared by at least the interstitial collagens I and VI, which supports the hypothesis that when included in the triple-helical conformation of collagens, RGD sequences are either not accessible to cells or exhibit specific conformations recognized by different integrins.     

Glucosylation of galactosylhydroxylysyl residues in collagen in vitro by collagen glucosyltransferase. Inhibition by triple-helical conformation of the substrate.

Glucosylation of galactosylhydroxylysyl residues in various collagen polypeptide chains and in small peptides prepared from collagen was studied in vitro using collagen glucosyltransferase purified about 200 to 500-fold from extract prepared from chick embryos. When various denatured polypeptide or peptide chains were compared as substrates for the enzyme, no significant differences were found between citrate-soluble collagens from normal or lathyritic rats and isolated alpha1 and alpha2 chains. In contrast, gelatinized insoluble calf skin collagen, and peptides prepared from collagen and having an average molecular weight of about 500 were clearly less effective substrates as judged from their Km and V values. A marked difference was found between native and heat-denatured citrate-soluble collagen in that no synthesis of glucosylgalactosylhydroxylysine was observed with the native collagen when the reaction was studied at 30 degrees C with different times, enzyme concentrations, and substrate concentrations. When the reaction was studied as a function of temperature, little glucosylation of native collagen was observed below 37 degrees C, but there was a sharp transition in the rate of glucosylation of native collagen at temperatures above 37 degrees C, similar to that observable in the melting curve of collagen. The data suggest that triple-helical conformation of collagen prevents that glucosylation of galactosylhydroxylysyl residues.     

The effect of dexamethasone on the functioning of C3 receptor sites on the surfaces of human fibroblasts

The presence of C₃ receptor sites on the cell surfaces of WI-38 fibroblasts was reported in a previous paper. Here the effect of dexamethasone sodium sulfate of C₃ receptor site function was studied. The incubation of WI-38 fibroblasts with dexamethasone sodium sulfate produces the biphasic mode of action, i.e., the growth-stimulating phase with low doses (90–230 μg/ml) and the growth-inhibiting phase with high doses (450–900 μg/ml). The function of C₃ receptor sites on WI-38 fibroblasts seems to be very stable and cannot be suppressed by the pretreatment of WI-38 fibroblasts with dexamethasone in high concentrations, where the cell growth is inhibited.     

The staining of type V collagen obtained from FBJ virus-induced osteosarcoma with concanavalin A

Type V collagen from FBJ virus-induced osteosarcoma of mice has a high content of saccharide as has been noted for type V collagens from different sources. In the present study, this collagen was found to contain significant amounts of mannose and hexosamine. Three alpha chains of this collagen were electrophoretically separated and cleaved by cyanogen bromide. The cyanogen bromide peptides, following their separation by SDS-polyacrylamide gel electrophoresis, were transferred onto nitrocellulose paper and stained with concanavalin A. Several bands derived only from alpha 3(V) stained positively, but this was inhibited by the presence of alpha-methylmannoside. Thus, at least one of these three chains appears to have an asparagine-linked oligosaccharide.     

Endothelial Matrix Assembly during Capillary Morphogenesis: Insights from Chimeric TagRFP-Fibronectin Matrix

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.     

Collagen Binding of Bifidobacterium adolescentis

In this study, 13 bifidobacterial strains were tested for their ability to adhere to immobilized extracellular matrix (ECM) proteins. Only two Bifidobacterium adolescentis strains adhered to immobilized type I and type V collagens, but not to laminin, fibronectin, and type III and IV collagens. The adhesion of B. adolescentis BB-119 to type V collagen was inhibited by type I and V collagens and gelatin, and was diminished after protease treatment of the cells. Periodate treatment of immobilized collagen and the presence of galactose inhibited the adhesion of strain BB-119 to type V collagen. Two cell surface proteins with molecular masses of 36 kDa and 52 kDa from strain BB-119 were found to bind to horseradish peroxidase-conjugated type V collagen by ligand blotting. We concluded that B. adolescentis BB-119 binds to type V collagen at galactose chains as target via these two cell surface proteins by their lectin-like activity. Received: 15 October 1996 / Accepted: 20 November 1996     

The growth of WI-38 cells in a serum-free,growth factor-free,medium with elevated calcium concentrations

Summary The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated Ca²⁺ concentrations was investigated. At 5 mM CaCl₂, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increased in cell number at saturation density over that obtained at day 1. Saturation densities were comparable when either 5 mM CaCl₂ or epidermal growth factor (1 mM CaCl₂) was used in the presence of insulin, dexamethasone and transferrin. Combining suboptimal doses of epidermal growth factor and CaCl₂ resulted in an additive effect on saturation density. Thus, nornal human diploid cells are capable of substantial growth in serum-free, hormone-free growth medium. In contrast, confluent cultures refed with the same medium are not responsive to elevated Ca²⁺ concentrations. In fact, elevated Ca²⁺ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their response to the combined treatment of insulin, transferrin and dexamethasone. This work was supported by the United States Public Health Society grants T-32, CA09171 and AG-00378. Editor's Statement This paper rigorously dissects the interplay among external Ca²⁺ concentration, cell density and specific growth factors on fibroblast growth in defined medium. Wallace L. McKeehan     

Tyrosine hydroxylase of sheep brain: some catalytic and chemical properties of the detergent-solubilized, partially purified enzyme

Abstract– Detergent-solubilized tyrosine hydroxylase from the caudate nucleus of the sheep was purified 3-fold by affinity chromatography on 3-iodotyrosine modified agarose. Supplementation of the standard assay with 1 mM Fe²⁺ resulted in only slight stimulation of the enzymic activity. The enzyme was, however, markedly inhibited by certain complexing agents specific for either Fe²⁺ or Fe³⁺. Double reciprocal plots of the kinetic data for a representative complexing agent, bathophenanthroline, showed the inhibition to be competitive with O₂ (apparent K_m 0.15 mM) and noncompetitive with either l -tyrosine or the synthetic tetrahydropterin cofactor DMPH₄ (apparent K_m's 0.12 and 0.29 mM, respectively). The combined inhibition and kinetics studies strongly suggest that brain tyrosine hydroxylase is an iron enzyme. Furthermore, the prosthetic iron very likely participates directly in catalytic function, presumably by binding molecular oxygen. The activity of ammonium sulphate-precipitated enzyme was found to be stimulated 2-fold by Fe²⁺, catalase or peroxidase, while untreated enzyme was markedly less affected by these agents. Since the only ostensible difference between the two preparations was the extensive aggregation present in the former case, the change in physical state evoked by ammonium sulphate precipitation appeared to be somehow related to this peculiar property of the enzyme.     

Characterization of a novel collagen chain in human placenta and its relation to AB collagen.

A novel collagen chain, termed alpha C, has been isolated from human placenta by limited pepsin digestion. The collagen containing the alpha C chain copurifies with placental AB collagen during selective salt precipitation but is virtually absent from fetal birth membranes, which contain relatively larger amounts of AB. Both native AB and alpha C-containing collagens are resistant to human skin collagenase under conditions that support cleavage of type I by greater than 90%. The alpha C chain was separated from alpha B by phosphocellulose chromatography and subsequently from alpha P by chromatography on CM-cellulose. Its amino acid composition is distinct from alpha A and alha B although all three chains posses compositional features in common; the carbohydrate content of the alpha C chain was intermediate between those of alpha A and alpha B. Analysis by NaDodSO4-polyacrylamide gel electrophoresis of peptides produced by CNBr cleavage and by limited digestion with the enzyme mast cell protease indicated different and unique products for the alpha A, alpha B, and alpha C chains. The data support the existence of another collagen chain which is related to the alpha A and alpha B chains but which is structurally unique. The proteins containing these chains may in turn comprise a subfamily of collagen isotypes which represents a divergence from and/or specialization of the type IV basement membrane collagens.     

The degradation rates of type I, II, and III collagens by tadpole collagenase

The degradation rates of type I, II, and III collagens by tadpole collagenase were studied by measuring the viscosity of the solution and the contents of alpha chains and alpha A chains of collagen, using SDS-polyacrylamide gel electrophoresis followed by densitometric analysis of the separated peptide bands. An empirical parameter was derived from the viscosity, and was shown to change in parallel with the content of alpha chains upon incubation with tadpole collagenase almost to the stage of complete digestion of collagen. Linear plots of parameters reflecting the concentration of intact collagen molecules against time were obtained, indicating the degradation to be pseudo-first order. The first-order rate constants for the degradation of Type I, II, and III collagens with tadpole collagenase at 30, 25, and 20 degrees C gave activation energies of 60 kcal/mol for Type III collagen and 40 kcal/mol for Type I and II collagens. There appeared to be a dependency of the degradation rates on the conformation of the collagen molecules (which is affected by temperature).     

Physico-chemical characterisation and molecular organisation of the collagen from the skin of an air-breathing fish (Ophiocephalus striatus)

Collagen has been prepared from the skin of an air-breathing Indian fish(Ophiocephalus striatus) by extraction with cold 0.5M acetic acid and purification by alternate precipitation with NaCl and dialysis against 0.02M Na₂HPO₄. The purified collagen was characterised with respect to physico-chemical properties, amino acid composition and chromatography of the denatured collagen. The fish collagen has a higher shrinkage temperature and denaturation temperature compared to that of the allied teleosts living in exclusively aquatic medium. These differences could possibly be reflections of the response to the rigours of the environment. As found for other vertebrate collagens, the fish collagen contains two kinds of single chains the α₁ and α₂ chains as revealed by sodium dodecyl sulphate-polyacrylamidegel electrophoresis and carboxymethylcellulose chromatography.     

Carnitine uptake and fatty acid utilization by diploid cells aging in culture

Uptake of carnitine by cultured human fetal lung flbroblasts (WI-38 and IMR-90) and by smooth muscle cells from calf aorta and from human uterus was found to be temperature dependent and saturable. IMR-90 cells showed an apparent K_m of 6–8 μM and a V of 21–28 pmol/h/10⁶ cells for l-carnitine. Transport was abolished by N-ethylmaleimide and was inhibited variably by octanoyl-d-carnitine, d-carnitine, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Although WI-38 and IMR-90 cells accumulate lipids as they age in culture, they take up carnitine as rapidly as do smooth muscle cells of aorta and uterus that do not exhibit such accumulation. Comparison of the rates of carnitine uptake by IMR-90 fibroblasts during the logarithmic phase of growth shows no difference between “young” and “old” cultures. In contrast, when confluent or postconfluent monolayers were compared and uptake expressed as a function of cell number, cells grown from late passages took up carnitine more rapidly than did cells grown from early passages. However, when account was taken of cell size, and carnitine expressed as a function of cell volume, the differences in carnitine uptake between early and late passages were no longer apparent for the confluent or postconfluent monolayers examined. Moreover, late passage fibroblasts took up and oxidized radioactive palmitate at least as rapidly as did cells from early passages. Our results suggest that accumulation of lipid in aging fibroblasts is not due to decreased carnitine uptake or fatty acid oxidation.     

INTERMITTENT DNA SYNTHESIS AND PERIODIC EXPRESSION OF ENZYME ACTIVITY IN THE CELL CYCLE OF WI-38

Synchronous cultures of WI-38 were obtained using an automated system for detachment and partitioning of mitotic cells which operates without the use of inhibitors, altered medium, or lowered temperatures. The generation time in synchronous WI-38 is 19.5 h and the duration of S phase when determined from the percentage of labeled metaphase cells or nuclei is 12 h. DNA replication in WI-38 occurs in three temporally distinct and rapid bursts separated by intervals of greatly reduced synthesis within what is nominally described as the DNA synthetic (S) period. Lactate dehydrogenase (LDH) displayed maxima in G₁ between 2 and 4 h and again at 10 and 16 h. Peaks in LDH activity were coordinated with DNA replication in a fashion similar to that reported for diploid Chinese hamster cells. Oscillations in LDH activity are more pronounced in normal diploid fibroblasts than in established and neoplastic lines.     

The biosynthesis of cartilage type collagen during limb regeneration in the larval salamander

In order to determine the relationships between the biosynthesis of cell-specific products and the morphological and cytological appearance of cells, the synthesis of cartilage type collagen was examined during different stages of regeneration of larval amphibian limbs. We have found that during blastemal formation, chondrocytes cease their synthesis of detectible levels of cartilage type collagen. This was accomplished by analyzing the radioactively labeled collagens synthesized in short-term culture by pieces of limbs containing a cross section of all limb tissues present, and comparing these collagens to the collagens synthesized by blastemas from corresponding limbs. The labeled collagens were extracted, purified, and analyzed by chromatography on carboxymethyl cellulose columns. Whereas all of the pieces of limbs analyzed, either before regeneration was initiated or after redifferentiation of cartilage had begun, synthesized clearly detectible levels of cartilage type collagen, none of the blastemas produced detectible levels of this cartilage-specific molecule. Thus, it seems that the normal production of (α1)₃, cartilage type collagen, is inhibited during the blastemal stage of amphibian limb regeneration.     

A new method of iodinating collagens for use in radioimmunoassay.

Purified collagens from a variety of species were iodinated to a high specific activity with the N-hydroxysuccinimide ester of I¹²⁵-labeled p-hydroxyphenyl propionic acid (Bolton-Hunter reagent). Labeling had no effect on the immunoreactivity of the collagen as determined by hemagglutination inhibition. This compound presumably acylates the abundant ?-amino groups of lysyl and hydroxylysyl residues in the collagen molecule. Using this method it is possible to label pepsin-extracted collagen from which the terminal nonhelical extensions containing tyrosine have been cleaved. The application of Bolton-Hunter-labeled collagens to radioimmunoassay of affinity-purfied antibodies against collagen is demonstrated.     

Procollagen and collagen produced by a teratocarcinoma-derived cell line,TSD4: Evidence for a new molecular form of collagen

Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel electrophoresis of the highly purified procollagen fraction demonstrated that the fraction is composed of θ chains (150,000 daltons), pro α chains (130,000 daltons), and α chains (100,000 daltons). Limited pepsin digestion of this fraction yielded a single species of collagen molecules having a chain composition (α1)₃, as did collagen isolated from the cell layer. Each α1 chain appears to be slightly larger than α1 chains from calf or human type I and type III collagen. Amino acid analysis and cyanogen bromide peptide profiles of pepsin-treated TSD4 collagen demonstrated significant differences from those of other collagens (II, III, IV) of the type α1(X)₃, although similar to that of the α1 chain of type I collagen, [α1(I)]₂α2. Taken together, acrylamide gel electrophoresis, amino acid composition, electron microscopy, and cyanogen bromide peptide analysis indicate that this material represents a new molecular species of collagen not previously characterized, probably related to [α1(I)]₃.