Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.     

Proteoglycan biosynthesis by chick embryo retina glial-like cells

In this report we present biochemical evidence that purified cultures of chick embryo retina glial-like cells actively synthesize heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans as well as hyaluronic acid. Glial-like cell cultures were metabolically labeled with [3H]glucosamine and 35SO4, and the medium, cell layer, and substratum-bound fractions were analyzed separately. Proteoglycans were characterized according to charge, apparent molecular size, and glycosaminoglycan (GAG) composition and were found to be differentially distributed among the cellular compartments. HS was the predominant GAG overall and was the major species found in the cell layer and substratum-bound fractions. CS/DS was also present in each fraction and comprised the largest proportion of GAGs in the medium. The major GAG-containing material resolved into three different size classes. The first, found in the cell layer and substratum-bound fractions, contained both CS/DS and HS and was of large size. A second, intermediately sized class with a higher CS/DS:HS ratio was found in the medium. The smallest class was found in the cell layer fraction and comprised HS, most likely present as free GAG chains. In addition, each fraction contained hyaluronic acid. Characteristics of these macromolecules differ from those produced by purified cultures of chick embryo retina neurons and photoreceptors in terms of size, compartmental distribution, and presence of hyaluronic acid.     

Effects of wheat germ agglutinin and concanavalin A on the accumulation of glycosaminoglycans in pericellular matrix of human dermal fibroblasts. A comparison with insulin.

The effect of insulin, wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (ConA) on [3H]glucosamine incorporation into pericellular glycosaminoglycans (GAGs) was investigated in two lines of cultured human dermal fibroblasts. Insulin and WGA stimulated [3H]glucosamine incorporation into hyaluronic acid (HA) and heparan sulphate (HS) without any alteration of chondroitin sulphate (CS) and dermatan sulphate (DS) contents. ConA increased [3H]glucosamine incorporation into HS, CS and DS, but had no effect on [3Hglucosamine incorporation into HA. PNA affected neither the content, nor the composition of GAGs. In contrast to PNA, ConA and WGA stimulated glycolysis and demonstrated an evident antiproliferative effect on dermal fibroblasts. Thus, both the insulin-like action of WGA and ConA on cultured dermal fibroblasts and the differences between the effects of lectins on modulation of GAGs synthesis appear to be determined by their chemical structure.     

Production of proteoglycans by human lung fibroblasts (IMR-90) maintained in a low concentration of serum.

Maintenance of fibroblasts in 0.5% serum results in viable but non-proliferative cells that may be analogous to fibroblasts in vivo. The synthesis of proteoglycans by human embryo lung fibroblasts in Eagle's minimal essential medium with 0.5% newborn-bovine serum or with 10% serum has been compared. A similar amount of [35S]sulphate-labelled glycosaminoglycan per cell was secreted by fibroblasts in 10% or 0.5% serum. 35SO42-incorporation into sulphated glycosaminoglycans was enhanced in 0.5% serum when expressed per mg of cell protein, but [3H]glucosamine incorporation was decreased. The charge density of these glycosaminoglycans was not changed as determined by ion-exchange chromatography. It was concluded that decreased protein/ cell resulted in an apparent increase in 35S-labelled glycosaminoglycan synthesis/mg of cell protein, whereas decreased uptake of [3H]glucosamine resulted in a decrease in their glucosamine labelling. The proteoglycans secreted by fibroblasts in 0.5% serum were similar in glycosaminoglycan composition, chain length and buoyant density to the dermatan sulphate proteoglycan, which is the major secreted component of cells in 10% serum. Larger heparan sulphate and chondroitin sulphate proteoglycans, which comprise about 40% of the total secreted proteoglycans of cultures in 10% serum, were greatly diminished in the medium of cultures in 0.5% serum. The proteoglycan profile of medium from density-inhibited cultures in 10% serum resembles that of proliferating cultures, indicating that lack of proliferation was not responsible for the alteration. The dermatan sulphate proteoglycan, participating in extracellular matrix structure, may be the primary tissue product of lung fibroblasts in vivo.     

Hypoxia modifies the effect of PDGF on glycosaminoglycan synthesis by primary human lung cells

Hypoxia, a consequence of interstitial lung diseases, may lead to secondary pulmonary hypertension and pulmonary vascular remodeling. Hypoxia induces activation and proliferation of lung cells and enhances the deposition of extracellular matrix including glycosaminoglycans (GAGs). To elucidate the cell biological mechanisms underlying the development of secondary pulmonary hypertension, we studied the effect of hypoxia on GAG synthesis by human lung cells. GAG synthesis was measured by incorporation of [(3)H]glucosamine; GAGs were isolated, purified, and characterized with GAG-degrading enzymes. Fibroblasts and vascular smooth muscle cells (VSMCs) synthesized hyaluronic acid, heparan sulfate, and chondroitin sulfates, whereas dermatan sulfate was found only in fibroblasts. Hypoxia did not influence the size or charge of the individual GAGs. However, hypoxia inhibited platelet-derived growth factor-induced [(3)H]glucosamine incorporation in secreted GAGs, especially hyaluronic acid, in VSMCs. In contrast, it stimulated GAG secretion, specifically heparan sulfate, by fibroblasts. Our results indicate that hypoxia induces modifications in GAG synthesis by human lung VSMCs and fibroblasts that may be correlated to pathophysiological manifestations in lung diseases causing hypoxia.     

Enhanced channelling of sulphate through a rapidly exchangeable sulphate pool in response to stimulated glycosaminoglycan synthesis in pancreatic epithelial cells.

The ability of cells to decorate glycosaminoglycans (GAGs) with sulphate in highly specific patterns is important to extracellular matrix biogenesis and placing appropriate glycosulphated ligands on the cell surface. We have examined sulphate metabolism in two pancreatic duct epithelial cell lines - PANC-1 and CFPAC-1 (derived from a cystic fibrosis patient) with a view to understanding how pancreatic cells utilise intracellular sulphate. [35S]Sulphate uptake was rapid and reached near steady state levels within 10 min. However, the intracellular specific activity of [35S]sulphate for PANC-1 and CFPAC-1 reached only 35 and 10%, respectively, of the medium specific activity at 10 min. Therefore, sulphate appears to reside within two compartments; a rapidly exchangeable sulphate pool (RESP) and a slowly exchangeable sulphate pool (SESP). Reducing chloride in the medium, increased the specific activity of [35S]sulphate within cells and increased the size of the inorganic sulphate pool, suggesting that the RESP was enlarged. Sulphate pools were not different in size between the two cell lines in physiological NaCl. Increasing the size of the sulphate pool had no effect on [35S]sulphate:[3H]glucosamine ratios incorporated into glycosaminoglycans (GAGs); however, stimulating the synthesis of GAGs with 4-methylumbelliferyl-beta-d-xyloside, stably elevated [35S]:[3H] ratios. This was due to higher [35S]sulphate incorporation. [35S]Cysteine contributed less than 0.1% of the cells' sulphate requirements. We conclude that in the face of elevated demand for sulphate, pancreatic cells appear to channel a greater proportion through the RESP.     

Glycosaminoglycan-dependent and -independent inhibition of neurite outgrowth by agrin

Agrin is a proteoglycan that can inhibit neurite outgrowth from multiple neuronal types when present as a substrate. Agrin's neurite inhibitory activity is confined to the N-terminal segment of the protein (agrin N150), which contains heparan sulfate (HS) and chondroitin sulfate (CS) side chains. We have examined the activities of various purified recombinant agrin fragments and their glycosaminoglycan (GAG) side chains in neurite outgrowth inhibition. Inhibitory activity was tested using dissociated chick ciliary ganglion neurons or dorsal root ganglion explants growing on laminin or N-cadherin. Initial experiments demonstrated that agrin N150 lacking GAG chains inhibited neurite outgrowth. Both halves of N150, each containing HS and/or CS side chains, could also inhibit neurite growth. Experiments using agrin fragments in which the GAG acceptor residues were mutated, or using agrin fragments purified from cells deficient in GAG synthesis, demonstrated that inhibition by the N-terminal portion of N150 requires GAGs, but that inhibition from the C-terminal part of N150 does not. Thus, the core protein or other types of glycosylation are important for inhibition from the more C-terminal region. Our results suggest that there are two distinct mechanisms for neurite outgrowth inhibition by agrin, one that is GAG-dependent and one that is GAG-independent.     

Monensin inhibits synthesis of proteoglycan, but not of hyaluronate, in chondrocytes

Monensin (10nm-1mum) inhibited the incorporation of [(35)S]sulphate and [(3)H]glucosamine into proteoglycans by rat chondrosarcoma cells, but the incorporation of [(3)H]glucosamine into hyaluronate was unaffected. The results suggest that hyaluronate synthesis occurs in a cell compartment separate from chondroitin sulphate synthesis.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

Concanavalin A affects glycosaminoglycans cellular and extracellular accumulation in cultured embryonic fibroblasts

Incorporation of 3H glucosamine and 35SO4 into glycosaminoglycans and proteoglycans produced and secreted by 7, 11 and 14 day chick embryo fibroblasts in vitro after concanavalin A treatment has been determined. Lectin differently affects 3H and 35SO4 incorporation. It enhances 3H labelled GAG accumulation in both cellular and extracellular compartments. Total incorporation of 35SO4 remains unchanged whereas the intracellular one is stimulated and the extracellular is reduced. All the effects are more relevant in the early stages of development. HA and PG cellular and extracellular accumulation seems to be independently regulated.     

Domains of neuronal heparan sulphate proteoglycans involved in neurite growth on laminin

Summary A single neuronal cell assay of neurite growth was utilized to determine types and domains of neuronal proteoglycans involved in neurite growth on laminin. Perturbations of biosynthesis and processing, enzymatic digestion with specific lyases, and competition with glycosaminoglycan side chains produced complementary data consistent with a molecular model implicating glycosaminoglycan (GAG) residues of heparan sulphate proteoglycans (HSPGs) in neurite growth. The observations suggest that HSPGs promote neurite growth on laminin by bridging between binding domains for HSPGs on laminin and on the neuronal cell surface, and that the bridge is tethered at both ends by noncovalent interactions between the binding domains and GAG side chains. Sulphation of the GAGs of HSPGs appears to be critical to the tethering and/or neurite growth-promoting activity of neuronal HSPGs.     

Isolation of 35S- and 3H-labelled proteoglycans from cultures of human embryonic skin fibroblasts.

35SO42- - and [3H]-leucine-labelled proteoglycans were isolated from the medium of a fibroblast culture, from an EDTA extract of the monolayer, and from consecutive dithiothreitol and guanidine hydrochloride extracts of the cells. Proteoglycans of different sizes were isolated from the extracts by gel chromatography on Sepharose 4B. In the medium and the EDTA extract the largest proteoglycans contained only 35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparan [35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparin [35S]sulphate. The galactosaminoglycan-containing proteoglycans of the various extracts were separated into a larger component, containing chondroitin sulphate-like side chains, and a smaller component, containing dermatan sulphate. The larger proteoglycan of the medium showed reversible association-dissociation behaviour when chromatographed on Sepharose CL2B in phosphate-buffered saline and 4M-guanidine hydrochloride respectively. This property remained after removal of extraneous proteins by CsCl-density-gradient centrifugation in guanidine hydrochloride. The association was markedly increased by the addition of high-molecular-weight hyaluronic acid.     

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.     

Inhibition of branching morphogenesis and alteration of glycosaminoglycan biosynthesis in salivary glands treated with β-d-xyloside

The role of glycosaminoglycans (GAGs) in the branching morphogenesis of embryonic mouse salivary glands was investigated by culturing the glands in the presence of xylose derivatives which stimulate synthesis of the xyloselinked classes of GAGs. Branching morphogenesis is inhibited severely, but reversibly, by 0.5–1.0 mM π-nitrophenyl-β-d-xylopyranoside and the inhibition correlates with a stimulation of incorporation of [³H]glucosamine (1.8-fold) and [³⁵S]sulfate (almost 3-fold) into GAGs. The effect of β-xyloside on accumulation of newly synthesized GAG also occurs in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the production of free GAG chains rather than proteoglycan-associated GAGs is being stimulated. The xyloside effects apparently do not result from general cytotoxicity of the derivatives, since similar concentrations of the α-anomer do not alter salivary branching or GAG synthesis, the rudiments resume morphogenesis when returned to control medium, and the effect on GAG synthesis is stimulatory rather than inhibitory. The study suggests that GAG biosynthesis plays an important role in salivary development, and that xylosides provide useful probes for characterizing the molecular events controlling branching morphogenesis.     

Glycosaminoglycan variants in the C2 muscle cell line

Using a replica technique, we have isolated and characterized five genetic variants of the C2 mouse muscle cell line that are defective in incorporation of radiolabeled sulfate into glycosaminoglycans (GAGs). The variants incorporate free sulfate into GAGs at 5-20% of wild-type levels. None of the variants is defective in sulfate transport across the cell membrane, and in no case could the deficit in incorporation of sulfate be reversed by addition of an artificial initiator of GAG biosynthesis, p-nitrophenyl beta-D-xyloside. Analysis of the incorporation of [3H]glucosamine into GAGs by the variants revealed three different patterns: one variant incorporated [3H]glucosamine at the wild-type level; one, S27, at a severely reduced level; and three at intermediate levels. Four of the five variants showed marked deficits in their ability to differentiate and fuse. The remaining variant, S27, formed multinucleated myotubes and expressed acetylcholine receptor with a normal time course. Differentiation of the first four variants could not be restored by addition of exogenous GAGs or extracellular matrix. Because of the important roles that GAGs and proteoglycans are thought to play in the differentiation of muscle, these genetic variants should serve as useful tools in functional analyses of these molecules.     

Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone

A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.     

Influence of collagen gel substratum on response to low-density lipoprotein by cultured human skin fibroblasts

The effect of low-density lipoprotein (LDL) on accumulation of glycosaminoglycans (GAG) was compared in cultures of human skin fibroblasts on a conventional plastic substratum and in a native type I collagen gel. The 24-h incorporation of [3H]glucosamine and Na2(35)SO4 into GAG secreted into the medium or associated with the substratum and cell surface (SCA) was measured in cells at subconfluent densities. When cells were grown on plastic, 13-25% of the labeled GAG was in the SCA pool. Cells cultured within a collagen gel matrix incorporated three times more [3H]glucosamine and up to five times more [35S]sulfate into this pool. The addition of LDL (300 micrograms protein/mL) to the medium increased the level of total GAG incorporation of [3H]glucosamine by 40-50% and of [35S]sulfate by 15-20% on both substrata. For cells on plastic the relative increase in the medium and SCA pool was similar, whereas for cells in collagen gel the response to LDL was twice as great in the SCA pool as in the medium. The distribution of GAG types was unaffected by LDL; hyaluronic acid remained the principal GAG in the media pools of both substrata, heparan sulfate remained the main SCA GAG in cultures on plastic, and dermatan sulfate remained the dominant GAG in the SCA pool of collagen gel cultures. LDL degradation was measured at intervals up to 48 h after the addition of 125I-labeled LDL. The rate of accumulation of degraded LDL products was lower in collagen gel cultures, but the final levels achieved were the same in the two substrata. Concentrations of total cell cholesterol were similar, although the increases in free cholesterol induced by LDL were 26% greater in cells within collagen gel than in those on plastic. We conclude that fibroblasts grown within a collagen gel, as compared with those on a plastic substratum, (i) accumulate more GAG that remain attached to the substratum and cell surface; (ii) respond to LDL with a similar degree of increase in GAG accumulation, but more of the increase is found in the substratum and cell surface compartment; and (iii) accumulate more intracellular free cholesterol in response to LDL.     

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).     

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.     

Incorporation of radioactive precursors into glycosaminoglycans by rat muscle fibroblasts exposed to a solubilized rat bone matrix fraction

Confluent cultures of rat muscle fibroblastic cells respond by increased glycosaminoglycan (GAG) synthesis when cultured in medium containing a solubilized bone matrix fraction (SBM) at a concentration of 100 micrograms/ml. The metabolism of the GAG associated with the cell pellet, the cell surface and the tissue culture medium fractions was studied, in the presence and absence of SBM, by measuring the incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the isolated GAG. Net synthesis of hyaluronic acid and of chondroitin sulfate in the medium fraction increased more rapidly in cultures containing SBM compared to controls, and the accumulation of labelled GAG in the medium of the treated cultures was approximately linear with respect to the length of incubation. The addition of SBM also resulted in increased incorporation of 3H and of 35S into the GAG of the cell surface and cell pellet fractions. In these fractions, stimulation of incorporation of radioactivity occurred in two waves: an early, relatively minor increase and a later relatively major increase. The relatively major stimulation of radioactivity into the GAG of the cell surface fraction occurred between 24 and 48 h and was independent of any apparent effect of serum.