Exogenous glycosaminoglycans modulate extracellular and intracellular accumulation of newly synthesized glycosaminoglycans by embryonic chick skin fibroblasts

The effects of exogenous glycosaminoglycans (GAG) on newly synthesized GAG accumulation in intra- and extra-cellular compartments of 17 incubation days chick embryonic skin fibroblasts have been examined. Exogenous GAG are able to act on both total GAG synthesis and secretion by embryonic fibroblasts and on the relative concentration of individual GAG. A decline in hyaluronic acid and an increase in chondroitin sulfate plus dermatan sulfate in the cellular compartment for all GAG administered have been detected. There was also similar behaviour in the extracellular compartment after sulfated GAG administration.     

Transforming growth factor-beta induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts.

Human embryonic skin fibroblasts were pretreated with transforming growth factor-beta (TGF-beta) for 6 h and then labeled with [35S]sulphate and [3H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density-gradient centrifugation, followed by gel, ion-exchange and hydrophobic-interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains. The results showed that TGF-beta increased the production of several different 35S-labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400-500 kDa) increased 5-7-fold and a small dermatan sulphate proteoglycan (PG-S1, also termed biglycan, with a core protein of 43 kDa) increased 3-4-fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG-S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF-beta treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected. To investigate whether TGF-beta also influences the glycosaminoglycan (GAG) chain-synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF-beta-treated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF-beta treated cultures contained a larger proportion of D-glucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4- and 6-sulphation of the GAG chains. By the use of p-nitrophenyl beta-D-xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF-beta-treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF-beta-treated fibroblasts contained a larger proportion of D-glucuronosyl residues than those of the control. These novel findings indicate that TGF-beta affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAG-synthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, which can affect cell growth and migration as well as organisation of matrix fibres.     

Beta-N-acetyl-D-glucosaminidase activity in embryonic chick skin and lung tissue and cultured fibroblasts

During development the content of mesenchymal glycosaminoglycans (GAG) undergoes prominent changes, currently considered to act as regulatory signals in the epithelial-mesenchymal interactions. The factors involved in controlling GAG composition are as yet completely unknown. Lysosomal enzymes play a key role in GAG turnover. A possible mechanism for regulating GAG content could therefore be linked to developmental modulation of lysosomal glycosidases activity. We have examined the activity of the beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30; a lysosomal hydrolase cleaving glycosidic linkage of the non-reducing terminal beta-N-acetyl-D-glucosamine residues) in chick embryo skin and lung (rudiments whose GAG composition has previously been studied) at various embryonic stages. Determinations were carried out on whole organs as well as on primary cultures of fibroblasts obtained from the two rudiments. beta-N-acetyl-D-glucosaminidase activity varied greatly during development, and it was significantly different in embryonic skin and lung tissues at various incubation days. In cultured fibroblasts, the enzymatic activity varied at different incubation days correlating with the in vivo data. Developmental changes of beta-N-acetyl-D-glucosaminidase paralleled mesenchymal GAG pattern both in vivo and in vitro. Our results, therefore, support the possibility that lysosomal enzymes could be involved in the regulation of mesenchymal GAG content during development.     

Relation between hyaluronan and sulphated glycosaminoglycan synthesis and degradation in cultured embryonic fibroblasts. Effect of concanavalin A and ammonium chloride administration.

In order to evaluate the relationship between glycosaminoglycan (GAG) synthesis and degradation, the effect of NH4Cl, which inhibits lysosomal degradation, on GAG production was analysed in vitro in concanavalin A (Con A) stimulated fibroblasts from 7 and 14-day-old chick embryos. 35SO4 incorporation into total proteoglycan (PG), 3H incorporation into individual GAG classes, beta-N-acetyl-D-glucosaminidase and beta-D-glucuronidase activity were determined. The results indicate a correlation between Con A and NH4Cl effects: NH4Cl induced a reduction principally in the GAG classes most stimulated by Con A. Thus HA and DS are much more stimulated by Con A and inhibited by NH4Cl than are CS and HS.     

Adaptation of the Lyme disease spirochaete to the mammalian host environment results in enhanced glycosaminoglycan and host cell binding

The Lyme disease spirochaete, Borrelia burgdorferi, is transmitted to mammals by Ixodes ticks and can infect multiple tissues. Host cell attachment may be critical for tissue colonization, and B. burgdorferi cultivated in vitro recognizes heparin- and dermatan sulphate-related glycosaminoglycans (GAGs) on the surface of mammalian cells. To determine whether growth of the spirochaete in the mammalian host alters GAG binding, we assessed the cell attachment activities of B. burgdorferi grown in vitro or in dialysis membrane chambers implanted intraperitoneally in rats. Host-adapted B. burgdorferi exhibited approximately threefold better binding to purified heparin and dermatan sulphate and to GAGs expressed on the surface of cultured endothelial cells. Three B. burgdorferi surface proteins, Bgp, DbpA and DbpB, have been demonstrated previously to bind to GAGs or to GAG-containing molecules, and we show here that recombinant derivatives of each of these proteins were able to bind to purified heparin and dermatan sulphate. Immunofluorescent staining of in vitro-cultivated or host-adapted spirochaetes revealed that DbpA and DbpB were present on the bacterial surface at higher levels after host adaptation. Recombinant Bgp, DbpA and DbpB each partially inhibited attachment of host-adapted B. burgdorferi to cultured mammalian cells, consistent with the hypothesis that these proteins may promote attachment of B. burgdorferi during growth in the mammalian host. Nevertheless, the partial nature of this inhibition suggests that multiple pathways promote mammalian cell attachment by B. burgdorferi in vivo. Given the observed increase in cell attachment activity upon growth in the mammalian host, analysis of host-adapted bacteria will facilitate identification of the cell binding pathways used in vivo.     

Specific association of iduronic acid-rich dermatan sulphate with the extracellular matrix of human skin fibroblasts cultured on collagen gels.

Human skin fibroblasts cultured on collagen gels produced two dermatan sulphate species, one, enriched in iduronic acid residues, that bound specifically to the collagenous fibres of the gel, the other, enriched in glucuronic acid, that accumulated in the culture medium. Collagen-binding and collagen-non-binding dermatan sulphates were also produced by cells grown on plastic surfaces, but in these cultures each constituent was released into the growth medium. Net synthesis of dermatan sulphate was 3-fold higher in cells maintained on collagen gels. In contrast, heparan sulphate synthesis was not influenced by the nature of the culture surface. The concentration of heparan sulphate in surface-membrane extracts was similar for cells grown on plastic and on collagen gels, but cells cultured on collagen showed a notable increase in the content of surface-membrane dermatan sulphate. The patterns of synthesis and distribution of sulphated glycosaminoglycans observed in skin fibroblasts maintained on collagen gels may reflect differentiated cellular functions.     

Cell surface glycosaminoglycans (GAGs) of cultured chick fibroblasts : Modifications in relation to the stage of embryo development

We have investigated the changes in glycosaminoglycan (GAG) composition between cultured fibroblasts derived from 8- and 16-day chick embryos. GAG composition has been studied after [3H]glucosamine and [35S]sulfate labeling. Both the 8- and 16-day embryo fibroblasts were found to contain hyaluronic acid (HA), dermatan sulfate (DS), heparan sulfate (HS) and chondroitin sulfates (CS), the latter being the major component in 8- and 16-day cells. These four GAGs were quantified after their separation using cellulose acetate electrophoresis. The amounts of HA and CS were respectively shown to increase 2-fold and 4-fold between the 8th and 16th day of development, whereas the amounts of HS and DS resp. diminished 2.5-fold and 1.2-fold. These results show that the relative proportions of the different GAGs alter during embryo development. The fibroblasts from 8-day-old embryos detached more rapidly from the culture dishes than the cells from 16-day-old embryos when treated with trypsin. However, this difference was not directly related to the different GAG content.     

Endocytosis of a small dermatan sulphate proteoglycan. Identification of binding proteins.

Endosomal preparations from human osteosarcoma cells and from fibroblasts contain 51,000- and 26,000-Mr proteins which bind a small dermatan sulphate proteoglycan after SDS/polyacrylamide-gel electrophoresis and Western blotting. Binding can be inhibited by unlabelled proteoglycan core protein. The proteins co-precipitate with a proteoglycan core protein-antibody complex. Scatchard analysis of immobilized endosomal proteins yielded a KD of about 37 nM for the proteoglycan. In intact cells proteins of the same size can be found. They are sensitive to trypsinization. A 51,000-Mr protein is the predominant membrane protein with strong binding to immobilized dermatan sulphate proteoglycan. There are additional proteoglycan-binding proteins with Mr values of around 30,000 and 14,000 which are insensitive to trypsin treatment. In contrast with the 51,000- and 26,000-Mr proteins, they resist deoxycholate/Triton X-100 extraction several days after subcultivation.     

Glycosaminoglycans in early chick embryo

The developmental profile of glycosaminoglycans (GAGs) were examined by cellulose acetate electrophoresis and high performance liquid chromatography in the early chick embryo from late blastula (stage XIII+) to early somite developmental stages (stage HH7-9). Sulphated GAGs were present from the earliest stages. They were more abundant than the non-sulphated forms and showed stage-related changes. Chondroitin sulphate and especially dermatan sulphate appeared to be the predominant GAGs in embryos at stage XIII+. Dermatan sulphate was about three times as abundant as chondroitin sulphate at stage XII+. In contrast, embryos at the definitive streak stage (stage HH4) produced about twice as much chondroitin sulphate as dermatan sulphate. At the head process stage (stage HH5), the level of chondroitin sulphate was reduced and its relative content in the embryo was about the same as dermatan sulphate. Levels of dermatan sulphate were more than five times those of heparan sulphate from stage XIII through to stage HH5 and three times more at stage HH7-9. The 4- and 6- sulphation of chondroitin sulphate increased 14- and 10-fold respectively, from stage XIII+ to stage HH 7-9. The sulphation pattern of chondroitin sulphate had a delta(di)-4S:delta(di)-6S molar ratio ranging from 4 to 8:1 and a delta(di)-4S:delta(di)-OS molar ratio ranging from 9 to 16:1 and was developmentally regulated. Thus, chondroitin sulphate in the early chick embryo was sulphated predominately in the 4-position in all stages studied. The presence of both 4- and 6-sulphated disaccharides in chondroitin sulphate indicated that both 4 and 6 sulfotransferases were active in the early embryo. Hyaluronate and sulphated GAG content increased markedly at gastrulation when the first major cellular migrations and tissue interactions begin.     

Age related and lectin influenced changes of exoglycosidases activity in cultured chick embryonic skin fibroblasts

beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-galactosaminidase and a beta-D-galactosidase activity was determined in untreated and lectins (ConA, PNA, SBA and WGA) treated chick embryonic skin fibroblasts at two incubation stages. Activity of all three glycosidases increased between 7 and 14 incubation days. ConA and WGA affected the levels of enzymatic activity; while SBA and PNA were uneffective. We discuss these findings in relation to a possible role of glycosidases in controlling mesenchymal GAG turnover.     

Mast cell glycosaminoglycans

Mast cells contain granules packed with a mixture of proteins that are released on degranulation. The proteoglycan serglycin carries an array of glycosaminoglycan (GAG) side chains, sometimes heparin, sometimes chondroitin or dermatan sulphate. Tight packing of granule proteins is dependent on the presence of serglycin carrying these GAGs. The GAGs of mast cells were most intensively studied in the 1970s and 1980s, and though something is known about the fine structure of chondroitin sulphate and dermatan sulphate in mast cells, little is understood about the composition of the heparin/heparan sulphate chains. Recent emphasis on the analysis of mast cell heparin from different species and tissues, arising from the use of this GAG in medicine, lead to the question of whether variations within heparin structures between mast cell populations are as significant as variations in the mix of chondroitins and heparins.     

Heparan sulphate inhibition of cell proliferation induced by TGFbeta and PDGF

The effect of glycosaminoglycans (GAGs) on the proliferation of smooth muscle cells (SMC) and fibroblasts was assessed by culturing cells with or without GAGs. Porcine heparan sulphate (HS) inhibited proliferation in a dose dependent manner. At 167 mug/ml of HS this reached 88% and 72% inhibition of SMC and fibroblast growth, respectively. Pig and beef mucosal heparins also blocked proliferation, but to a lesser extent. In contrast, beef lung heparin, chondroitin sulphate, and dermatan sulphate failed to block growth factor induced proliferation. Continuous presence of HS was not required, suggesting that the inhibitory effects resulted from a direct effect on the cell rather than an interaction of the GAG with growth factors. The mechanism by which GAGs inhibit proliferation will be addressed in future studies.     

Inventory of human skin fibroblast proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulphate proteoglycans.

Heparan sulphate and chondroitin/dermatan sulphate proteoglycans of human skin fibroblasts were isolated and separated after metabolic labelling for 48 h with 35SO4(2-) and/or [3H]leucine. The proteoglycans were obtained from the culture medium, from a detergent extract of the cells and from the remaining ''matrix'', and purified by using density-gradient centrifugation, gel and ion-exchange chromatography. The core proteins of the various proteoglycans were identified by electrophoresis in SDS after enzymic removal of the glycosaminoglycan side chains. Skin fibroblasts produce a number of heparan sulphate proteoglycans, with core proteins of apparent molecular masses 350, 250, 130, 90, 70, 45 and possibly 35 kDa. The major proteoglycan is that with the largest core, and it is principally located in the matrix. A novel proteoglycan with a 250 kDa core is almost entirely secreted or shed into the culture medium. Two exclusively cell-associated proteoglycans with 90 kDa core proteins, one with heparan sulphate and another novel one with chondroitin/dermatan sulphate, were also identified. The heparan sulphate proteoglycan with the 70 kDa core was found both in the cell layer and in the medium. In a previous study [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661] it was suggested that skin fibroblasts produce a proteoglycan form of the transferrin receptor. However, the core protein of the major heparan sulphate proteoglycan now purified does not resemble this receptor, nor does it bind transferrin. The principal secreted proteoglycans are the previously described large chondroitin sulphate proteoglycan (PG-L) and the small dermatan sulphate proteoglycans (PG-S1 and PG-S2).     

Influence of Concanavalin A on 3-O-methylglucose uptake in cultured chick embryo fibroblasts. Evidence for differences related to the age of embryos

Concanavalin A (Con A) was found to inhibit hexose uptake in cultured fibroblasts derived from 8-day chick embryos and to stimulate this process in those derived from 16-day embryos. Con-A effects depended on the duration of contact with cells and lectin and were inhibited by alpha-methylmannopyrannoside. Con A was shown to mask about 70% of the hexose carriers in both 8- and 16-day embryo fibroblasts. Lectin altered the hexose uptake very rapidly. Con A only modified the Vmax of the uptake system and did not alter the Km. This indicates that either the number or mobility of hexose carriers were modified by Con-A treatment. The differential effect of lectin could be due to a modification of the hexose-carrier mobility during the embryonic differentiation of fibroblasts. Secondary effects may affect cell growth.     

D(+)catechin enhances heparan sulphate content in rat liver

Administration of (D+) catechin (100 mg/kg body wt) to rats resulted in an increase in the amount of total sulphated glycosaminoglycans (GAG) in liver. The increase was more pronounced in the case of heparan sulphate than chondroitin sulphate and dermatan sulphate. The liver slices prepared from catechin-treated rats showed a significant increase in the rate of incorporation of 35S-sulphate into GAG. Similarly there was a concentration-dependent increase in the rate of 35S-sulphate incorporation into GAG by normal liver slices in presence of catechin in vitro. Susceptibility to nitrous acid degradation and chondroitinase ABC digestion showed that more than 80% of the GAG labelled in vivo with 35S-sulphate, was heparan sulphate and about 10% chondroitin sulphate and dermatan sulphate. Gel filtration of the 35S-labelled material isolated from livers of normal and catechin-treated animals over sephacryl S-300 did not show any difference probably excluding the possibility of free GAG chains initiated on catechin or any of its metabolites in vivo. These results indicate that catechin stimulates the synthesis of sulphated GAG, particularly heparan sulphate in liver.     

A comparative study of glycosaminoglycans in cultures of human, normal and malignant glial cells.

The glycosaminoglycans (GAG) of human cultured normal glial and malignant glioma cell lines were studied using 35S-sulphate or 3H-glucosamine as markers. 35S-labelled GAG were assayed by precipitation with cetylpyridinium chloride; 3H-labelled sulphated GAG and 3H-labelled hyaluronic acid were quantitated after separation on a DEAE-cellulos column. The net production of GAG and the distribution, composition and turnover of GAG were similar in all of the normal cell lines tested, but showed a great variability in the malignant cell lines. Most of the glioma cell lines produced more hyaluronic acid and less sulphated GAG than the normal cell lines, but exceptions were noted. The GAG of the trypsin susceptible (pericellular pool of normal glial cells consisted mainly of heparan sulphate with only minor amounts of other GAG. The analogous material of most glioma cells showed hyaluronic acid as the major GAG. Material liberated by trypsin from EDTA-detached cells (membrane fraction) was enriched in heparan sulphate as compared to the entire pericellular pool. Substrate attached material (SAM) left with the plastic dish after EDTA treatment of normal cultures was rich in heparan sulphate, whereas SAM of glioma cells lacked heparan sulphate or showed greatly reduced amounts of this component. Release of newly synthesized GAG to the extracellular medium was a rapid process in the normal cells but was more or less delayed in the glioma cells. The extracellular medium of the malignant glioma cultures was consistently poor in dermatan sulphate, as compared to that of normal cultures.     

Influence of Concanavalin A on 3-O-methylglucose uptake in cultured chick embryo fibroblasts

Abstract. Concanavalin A (Con A) was found to inhibit hexose uptake in cultured fibroblasts derived from 8-day chick embryos and to stimulate this process in those derived from 16day embryos. (1) Con-A effects depended on the duration of contact with cells and lectin and were inhibited by α-methylmannopyrannoside. (2) Con A was shown to mask about 70% of the hexose carriers in both 8- and 16-day embryo fibroblasts. Lectin altered the hexose uptake very rapidly. (3) Con A only modified the V^max of the up- take system and did not alter the K^m. This indicates that either the number or mobility of hexose carriers were modified by Con-A treatment. The differential effect of lectin could be due to a modification of the hexose-carrier mobility during the embryonic differentiation of fibroblasts. Secondary effects may affect cell growth.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels.

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

A role for glycosaminoglycans in the development of collagen fibrils

Extensive data on the glycosaminoglycan (GAG) composition and the collagen fibril diameter distribution have been collected for a diverse range of connective tissues. It is shown that tissues with the smallest diameter collagen fibrils (mass-average diameter less than 60 nm) have high concentrations of hyaluronic acid and that tissues with the largest diameter collagen fibrils (mass-average diameter approximately 200 nm) have high concentrations of dermatan sulphate. It is suggested that the lateral growth of fibrils beyond a diameter of about 60 nm is inhibited by the presence of an excess of hyaluronic acid but that this inhibitory effect may be removed by an increasing concentration of chondroitin sulphate and/or dermatan sulphate. It is also postulated that high concentrations of chondroitin sulphate will inhibit fibril growth beyond a mass-average diameter of approximately 150 nm. Such an inhibition may in turn be removed by an increasing concentration of dermatan sulphate such that it becomes the dominant GAG present in the tissue.     

Glycosaminoglycan metabolism in otosclerotic bone cells

Summary— Normal and otosclerotic bone cells were cultured in vitro in serum-free medium to evaluate single glycosaminoglycan (GAG) class synthesis and secretion. Moreover, the degradative process was studied by inhibiting the lysosomal functions through the addition of ammonium chloride to the cultures, an ammine known to inhibit lysosomal degradation by neutralizing organelle activity. Otosclerotic bone cells accumulated a lower amount of GAG both in the cellular and extracellular pool compared to normal ones. The decrease was markedly higher for secreted GAG. Moreover a different pattern of single GAG class distribution was observed in the two cell types considered. In the medium of otosclerotic cells a percentage increase of hyaluronic acid (HA) and dermatan sulphate (DS) and a percentage decrease of heparan sulfate (HS) and chondroitin sulfate (CS) were observed compared to normal bone cells. Ammonium chloride had a lower effect on pathologic than on normal cells, indicating a decrease in the degradative process in otosclerotic bone cells. These results were also confirmed by the experiments on GAG uptake and degradation and by the dosage of enzymatic activity of two exoglycosidases. Since extracellular GAG composition influences bone deposition and mineralization, these data support the hypothesis that otosclerosis is the result of an error in the connective tissue matrix structure.