Embryonic Chick Tendon Cells Research Articles

Inhibition of prolyl 4-hydroxylase in vitro and in vivo by members of a novel series of phenanthrolinones

Examples of a novel series of phenanthrolinones are shown to be potent competitive inhibitors of avian prolyl 4-hydroxylase, and of collagen hydroxylation, in embryonic chick tendon cells and human foreskin fibroblasts in vitro and in the oestradiol-stimulated rat uterus in vivo. Two compounds, Compound 1 (1,4-dihydrophenanthrolin-4-one-3-carboxylic acid) and Compound 5 [8-(N-butyl-N-ethylcarbamoyl)-1,4-dihydrophenathrolin-4-one-3-carboxylic acid], with comparable potencies in vivo, were chosen to investigate the effect of the inhibition of the hydroxylation of newly synthesized uterine collagen on the turnover of this protein in vivo. Inhibition of hydroxylation by more than 50% for approx. 8h following single oral doses of the compounds was associated with significant losses of radiolabelled proline and 4-hydroxyproline from collagen during this period. Progressive hydroxylation of collagen over 48h, as the inhibitory action of the compounds declined, was accompanied by a decreased loss of radiolabel from the uterine collagen. Earlier reports indicated that underhydroxylated collagen, accumulating within the endoplasmic reticulum in cells where prolyl 4-hydroxylase is inactivated, is slowly degraded, but is then rapidly hydroxylated and secreted when the activity of prolyl 4-hydroxylase is restored. Taken with the present results, this suggests that the potential use of inhibitors of prolyl 4-hydroxylase to control excessive collagen deposition in pathological fibrosis may be limited by the need to maintain continuous inhibition of collagen hydroxylation so as to facilitate intracellular degradation of the accumulated protein.

Inhibition of collagen hydroxylation by 2,7,8-trihydroxyanthraquinone in embryonic-chick tendon cells

Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. 2,7,8-Trihydroxyanthraquinone (THA) is an effective inhibitor of prolyl 4-hydroxylase by virtue of its ability to compete with the co-substrate 2-oxoglutarate (Ki = 40.3 microM). Using a simple and reproducible assay for collagen hydroxylation, we show that THA inhibits the hydroxylation of collagen in embryonic-chick tendon cells in short-term culture, with an IC50 value (concn. giving 50% inhibition) of 32 microM. In comparison, the ethyl ester of 3,4-dihydroxybenzoic acid has an IC50 value of 0.1 mM against collagen hydroxylation by chick tendon cells, whereas its Ki versus isolated prolyl 4-hydroxylase is 49 microM.

A simple fractionation of chicken egg yolk yields a protein component that stimulates cell proliferation and differentiation in primary avian tendon cells.

An easily prepared and stable protein fraction from chick egg yolk promotes cell division (40 h generation time) and expression of procollagen (60% of total protein synthesis) in primary avian tendon cells in a serum-free medium. The activity of this yolk fraction (YF) is proteinaceous as reflected by its sensitivity to protease treatment. Yolk fraction is resolved into four major components on sodium dodecyl sulfate polyacrylamide gel electrophoresis with apparent molecular weights of 82,70,42,35 (X 10(-3)). Under nondenaturing conditions, YF runs as a mixture of high molecular weight aggregates on Sephacryl G-200. We postulate that the active part of YF could be the in ovo growth promoter for embryonic chick tendon cells.

The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture

The production of [ 14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH 4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitors N-p-tosyl- l-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 ± 1.4% to 10.5 ± 2.4% with 10 μ m TLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 μ m TLCK. The incorporation of [ 14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 μg/ml leupeptin or 60 μg/ml pepstatin A. However, the production of collagen was reduced to 10.9 ± 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [ 14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 μ m TLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts.

Lysosomotropic agents ammonium chloride and chloroquine inhibit both the synthesis and secretion of procollagen by freshly isolated embryonic chick tendon cells

Freshly isolated embryonic chick tendon fibroblasts were incubated in suspension culture for six hrs in the presence or absence of ammonium chloride or chloroquine. Both agents caused a marked reduction in collagen production without inhibiting non-collagen protein synthesis. The small amounts of collagen synthesized in the presence of each agent were secreted into the culture medium at rates significantly lower than exhibited by control cells. Since ammonium chloride and chloroquine have been shown to inhibit lysosomal degradation of proteins, and interfere with the fusion of membranous vesicles and receptor recycling, these results suggest a requirement for at least one of these processes in the synthesis and secretion of procollagen.

The effect of cis-4-hydroxy-L-proline on intracellular degradation of newly synthesized collagen by freshly isolated chick tendon fibroblasts.

Freshly isolated embryonic chick tendon cells were incubated for 6 hrs in suspension culture in the presence of the proline analogue, cis-4-hydroxyproline to cause the cells to synthesize collagen which was incapable of becoming triple helical. The cells were evaluated for the percentage of total protein synthesis devoted to collagen and for the percentage of newly synthesized collagen which was rapidly degraded. Collagen production in the presence of cis-4-hydroxyproline was reduced from 25% to 7% of total protein synthesis. Under normal conditions the cells degraded 8% of their newly synthesized collagen but when the cells were incubated in the presence of cis-4-hydroxyproline, 25% of their total collagen synthesized was degraded to dialyzable peptides. The enhanced degradation of nonhelical, analogue containing collagen was inhibited by inhibitors of lysosome function. These observations provide support for the concept that fibroblasts are able to recognize and degrade a portion of their newly synthesized collagen, and that defective collagen may be selectively degraded by an intracellular lysosomal process. Enhanced intracellular degradation can in part explain the decrease in collagen production by freshly isolated tendon fibroblasts incubated with cis-4-hydroxyproline.

The assembly of tetrameric prolyl hydroxylase in tendon fibroblasts from newly synthesized alpha-subunits and from preformed cross-reacting protein.

Embryonic-chick tendon cells were incubated in suspension for 4h with (14)C-labelled amino acids, cell extracts were subjected to gel filtration, and the effluent was examined by rocket immunoelectrophoresis by using antibodies specific for the beta-subunit of chick prolyl hydroxylase. Two peaks of immunoreactive protein were found. The first peak contained 40% of the immunoreactive protein eluted from the column and 100% of the enzyme activity. Polyacrylamide-slab-gel electrophoresis in sodium dodecyl sulphate of an immunoprecipitate of this peak demonstrated that it consisted of the tetrameric form of prolyl hydroxylase, subunit composition alpha(2)beta(2) where alpha and beta are non-identical subunits. Only the alpha-subunits were labelled, indicating that they were synthesized during the 4h labelling period. The beta-subunits were unlabelled, indicating that they had been synthesized before the labelling period. The second peak eluted from the gel-filtration column contained 60% of the immunoreactive protein eluted from the column and was enzymically inactive. Polyacrylamide-slab-gel electrophoresis of an immunoprecipitate of this peak indicated that it consisted of a single labelled polypeptide chain, identified as cross-reacting protein, which was related to, but not identical with, the beta-subunit of prolyl hydroxylase. Pulse-chase experiments were performed on cultured chick tendon cells to demonstrate that alpha-subunits and cross-reacting protein had half-lives of about 60h. The half-life of beta-subunits was considerably longer, and the kinetic pattern was consistent with their being derived from a labelled precursor such as cross-reacting protein. The data presented here indicate that the active tetrameric form of prolyl hydroxylase in cells is assembled from alpha-subunits which are newly synthesized, and from beta-subunits which are derived from cross-reacting protein.

Effects of granulation-tissue extracts on collagen synthesis

The effect of granulation-tissue extract on the synthesis of collagen in embryonic-chick tendon cells was found to depend on (i) the developmental phase of the granulation tissue, (ii) the concentration of the extract and (iii) the presence of diffusible components in it. The suppressing effect was maximal with concentrated extracts from 2--3-week granulation tissue (where the collagen synthesis is also maximal). The suppressing factor could be enriched by gel filtration chromatography. Cultured human synovial cells were affected like the embryonic-chick cells. When the sponge implant was imbibed with granulation-tissue extract in advance, the developing granuloma contained less collagen than the control. The stimulating effects with dilute extracts from granulation tissue were due to diffusible components. Blood serum and rheumatoid synovial fluid samples also stimulated collagen synthesis in granulation-tissue slices, less after dialysis.

Glycoproteins from experimental granulation tissue and their effects on collagen synthesis in embryonic chick tendon cells

Buffer-soluble and pronase-liberated glycoproteins from experimental granulation tissue were fractionated by gel filtration and DEAE-cellulose chromatography. The age of the granuloma was reflected in the gel filtration pattern. Two glycoproteins were isolated, purified to homogeneity and analyzed for their carbohydrate and amino acid compositions. The collagen synthesis in embryonic chick tendon cells was measured in the presence of these fractions, which were found to be inhibiting even at 10 −6 M. These glycoproteins may be significant in the feedback regulation of the development of granulation tissue and fibrosis.

The subcellular fractionation of embryonic chick tendon and cartilage cells: a re-examination.

A re-examination of the subcellular fractions obtained from matrix-free chick tendon and cartilage cells has been made since the discovery that three out of four of the micrographs of chick tendon microsomal fractions published in an earlier paper from this laboratory were not authentic. The present studies demonstrate that by using the procedures previously reported it is possible to isolate microsomal and submicrosomal fractions from tendon and cartilage cells which exhibit typical morphology when examined by electron microscopy. These observations are consistent with our original biochemical characterization of subcellular fractions, which we know to be valid. Other publications from this laboratory in which these fractionation procedures have been applied to studies of collagen biosynthesis are in no way compromised, and indeed, most of our data have been confirmed by several other laboratories.

Dependence of Collagen Synthesis by Embryonic Chick Tendon Cells on the Extracellular Concentrations of Glutamine

An agent in rheumatoid synovial fluid which stimulates collagen synthesis by embryonic chick tendon cells was identified as glutamine. The stimulating effect was observed only at a subnormal concentration of proline (0.02 mM). The concentration of glutamine in rheumatoid synovial fluid is about 2.5 mM but it falls rapidly during storage even at -20 degrees C. Collagen synthesis is shown to depend on the extracellular concentrations of glutamine and proline in addition to any connective tissue activating factors.

Isolation and partial characterization of polyribosomal messenger ribonucleoproteins from embryonic-chick tendon cells [proceedings

Isolation and partial characterization of polyribosomal messenger ribonucleoproteins from embryonic-chick tendon cells [proceedings

Connective tissue activation by synovial fluids and synovial-tissue extracts of rheumatoid arthritis patients.

Prompted by Castor's investigations (4) on a connective-tissue-activating peptide (CTAP) we investigated the stimulative effect of synovial fluids and synovial-tissue extracts on the synthesis of collagen by incubated embryonic-chick tendon cells. The stimulative effect was greater with synovial-tissue extracts from more severe cases than with samples from patients having milder forms of the disease. There was no correlation between the stimulation by synovial fluids and synovial-tissue extracts from the same patient. The stimulative activity was lost at dialysis. A slight stimulation in the incorporation of glucosamine was also observed. Treatment of the patients with gold, chloroquine or steroids decreased the stimulating capacity. These effects seem to depend on factors different from those described by Castor. The differerences in the stimulant activity of samples from various groups cannot be entirely due to glutamine, which is one of the limiting nutrients of the embryonic-chick tendon cells.

Application of Granulation–Tissue Slices and Matrix–Free Tendon Cells in the Screening of Connective Tissue–Active Indoles

Abstract Four new synthetic indoles have been found to suppress the synthesis of collagen both in incubated granuloma–slices and in detached embryonic–chick tendon cells as well as to decrease the secretion of collagen to the extracellular space, the latter of which was also inhibited by a membrane–active detergent. The effects of serotonin and indomethacin were studied as a background and data are presented on the association of serotonin to plasma membrane fractions. The synthesis of collagen in vitro is suggested as a useful test in the preliminary screening of potential antirheumatic drugs.

The route of secretion of procollagen. The influence of alphaalpha'-bipyridyl, colchicine and antimycin A on the secretory process in embryonic-chick tendon and cartilage cells.

I. Embryonic-chick tendon cells were pulse-labelled for 4 min with [14C]proline and the 14C-labelled polypeptides were chased with unlabelled proline for up to 30 min. Isolation of subcellular fractions during the chase period and their subsequent analysis for bacterial collagenase-susceptible 14C-labelled peptides demonstrated the transfer of procollagen polypeptides from rough to smooth microsomal fractions and thence to the extracellular medium. Parallel analyses of Golgi-enriched fractions indicated the involvement of this organelle in the secretory pathway of procollagen. Sodium dodecylsulphate/polyacrylamide-gel electrophoresis of the 14C-labelled polypeptides present in the Golgi-enriched fractions demonstrated that the procollagen polypeptides were all present as disulphide-linked pro-gamma components. 2. When similar kinetic studies of the intracellular transport of procollagen were conducted with embryonic-chick cartilage cells almost identical results were obtained, but the rate of translocation of cartilage procollagen was significantly slower than that observed for tendon procollagen. 3. When hydroxylation of procollagen polypeptides was inhibited by alphaalpha'-bipyridyl, the nascent polypeptides accumulated in the rough microsomal fraction. 4. When cells were pulse-labelled for 4min with [14C)proline and the label was chased in the presence of colchicine, secretion of procollagen was inhibited and an intracellular accumulation of procollagen 14C-labelled polypeptides was observed in the Golgi-enriched fractions. 5. The energy-dependence of the intracellular transport of procollagen was demonstrated in experiments in which antimycin A was found to inhibit the transfer of procollagen polypeptides from rough to smooth endoplasmic reticulum. 6. It is concluded that procollagen follows the classical route of secretion taken by other extracellular proteins.

Further Characterization of Procollagen Messenger Ribonucleic Acid from Embryonic Chick Tendon Cells

The isolation and characterization of specific mammalian mRNA species, e.g. those for globin, immunoglobulin and crystallin, have been facilitated by the existence of cell types, e.g. reticulocytes, myeloma cells and lens cells, in which protein synthesis is devoted predominantly to the synthesis of these individual proteins (for review see Brawerman, 1974). Fibroblasts isolated from embryonic chick tendons by enzymic digestion (Dehm & Prockop, 1971) provide an ideal system with which to commence studies on procollagen mRNA, since procollagen synthesis accounts for the major protein-synthetic activity of these cells and the absence of any matrix avoids the possibility of non-specific RNA-protein aggregation which can occur when intact connective tissue is used. Preliminary studies with these cells (Harwood et al., 1974) have indicated that their major mRNA species is of a size appropriate to presumptive procollagen mRNA, and we have been able to demonstrate that mRNA extracted from these tendon cells can direct in a wheat-germ cell-free system the synthesis of polypeptides having the characteristics of Type I pro-a chains (Harwood et al., 19750). Procollagen, like other proteins for export, is synthesized on membrane-bound polyribosomes, and procollagen mRNA activity, as assessed by the amount of [CIproline incorporated into collagenase-susceptible peptides, is associated almost exclusively with these subcellular components (Harwood et a ~ . , 19756). Consequently, the mRNA isolated from membrane-bound ribosomes has been used to characterize further the molecular size and poly(A) content of the procollagen messenger. Tendon cells were incubated with [3H]uridine for 2 hat 37°C in modified Krebs medium containing 0.04pg of actinomycin D/ml (Harwood et al., 1974) and free and membrane-bound ribosomes isolated by the method of Blobel & Potter (1967). RNA was extracted from the bound ribosomes by the procedures of Lee et al. (1971) and then subjected to electrophoretic analysis under conditions in which (a) the RNA retains its secondary structure (Loening, 1967) and (6) the RNA is denatured by treatment with formamide (Staynov et al., 1972; Pinder et al., 1974). Analyses of extracts from membrane-bound ribosomes under non-denaturing conditions revealed that, when stringent precautions were taken toavoidribonucleaseattack during isolation and processing, the major RNA species labelled under these conditions migrated with an apparent molecular weight of 1.65 x lo6. Similar analyses carried out in the presence of formamide resulted in the [3H]RNA again moving on electrophoresis in the region of a standard of28 S rRNA and having a molecular weight of approx. 1.7 x lo6. This observation suggests that procollagen mRNA, unlike the majority of messengers studied to date, has a high degree of secondary structure. This conclusion is consistent with the predicted base composition of procollagen mRNA, which must code for a polypeptide rich in glycine (over 300 residues/mol) and the imino acids proline plus hydroxyproline (over 200 residues/mol). Since the codons for glycine and proline contain at least two residues of G and C per triplet respectively, procollagen mRNA will have unusually high contents of G and C residues and as a consequence may assume a highly folded conformation. That the RNA species described above was indeed procollagen mRNA was confirmed by using procedures similar to those reported by Berns et al. (1974) and Rosen et al. (1975). RNA from bound ribosomes was electrophoresed in polyacrylamide gels in the presence of formamide and the RNA was extracted from 1 mm slices by homogenization in 0.1 M-sodium acetate buffer, pH5.0, at 4°C. Approx. 15% of the RNA was recovered from the gel and after precipitation with 2 vol. of ethanol at -20°C the RNA was redissolved in water and assayed for procollagen mRNA activity (Harwood et al.,

Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [(14)C]lysine were assayed for hydroxy[(14)C]lysine and hydroxy[(14)C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[(14)C]lysine and glucosylgalactosylhydroxy[(14)C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [(14)C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. (14)C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5'-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.

Effects of antirheumatic drugs on sponge-induced granulation tissue, rheumatoid synovial tissue, matrix-free tendon cells and fibroblast plasma membranes in vitro

Slices of sponge-induced, mature granulation tissue were incubated in the presence of antirheumatic drugs (sodium acetylsalicylate, indomethacin and phenylbutazone at concentrations of 10 −7–10 −2M) and labelled precursors of nucleic acids, proteins and acid mucopolysaccharides. Analogous experiments were made with sliced human synovial tissue from rheumatoid patients and with matrix-free embryonic chick tendon cells. The synthetic functions were suppressed by 10 −4–10 −3M indomethacin and phenylbutazone but only by 10 −2m acetylsalicylic acid. At low concentrations (10 −5–10 −6M) there was an increase in the radioactivity in proteins during the early period of the incubation but this was reversed later. Indomethacin and phenylbutazone affected the production of collagen at two points: (i) the synthesis of the peptide chain and. more notably, (ii) the secretion of collagen to the extracellular space. Indomethacin affected the final content of cellular collagen only slightly. Preliminary experiments on the effect of indomethacin on the activities of enzymes in plasma membranes prepared from granulation tissue showed some effect on leucyl-β-naphthylamidase and Na +, K + -activated Mg 2+- adenosine triphosphatase. The use of such systems for the screening of new connective tissue-active drugs is discussed. The secretion of collagen from matrix-free embryonic tendon cells is the most sensitive target for indomethacin in vitro.

Plasma membranes from experimental granulation tissue.

1. A procedure was developed for the preparation of plasma membranes from experimental granulation tissue of the rat without the addition of enzymes. The yield is better than 20% and the purification at least tenfold. 2. Values are given for the activities of 5'-nucleotidase, Na-+, k-+-activated Mg-2+dependent adenosine triphosphatase and leucine beta-naphthylamidase, for lipid composition, and for the gel-electrophoretic patterns of proteins and glycoporteins in the membrane preparations. 3. The plasma membranes from the mature granulation tissue contain proportionally more protein in the lipid phase, but the specific activities of 5'-nucleotidase and Na-+,K-+-activated Mg-2+-dependent adenosine triphosphatase are smaller than in the proliferating tissue. Certain differences were repeatedly observed in the gel-electrophoretic patterns of the developmental phases. 4. The plasma membranes from the granulation tissue were compared with those from rat peritoneal macrophages and from embryonic-chick tendon cells.

The Association of Collagen Galactosyl- and Glucosyl-Transferases with Subcellular Fractions of Embryonic Chick Tendon Cells

The Association of Collagen Galactosyl- and Glucosyl-Transferases with Subcellular Fractions of Embryonic Chick Tendon Cells