Synthesis of hyaluronate in cultured bovine articular cartilage.

The synthesis and distribution of hyaluronate and proteoglycan were studied in bovine articular cartilage in short-term explant culture with [3H]acetate and H2(35)SO4 as precursors. The incorporation of [3H]acetate into hyaluronate and sulphated glycosaminoglycans was linear with time, except that hyaluronate synthesis showed a marked lag at the beginning of the incubation. [3H]Hyaluronate represented 4-7% of the total [3H]glycosaminoglycans synthesized over a 6 h period. However, the distributions of [3H]hyaluronate and 3H-labelled sulphated glycosaminoglycans were different: about 50% of the newly synthesized [3H]hyaluronate appeared in the medium, compared with less than 5% of the 3H-labelled sulphated proteoglycans. A pulse-chase experiment revealed that the release of newly synthesized [3H]hyaluronate from cartilage was rapid. No difference was observed in the distribution of [3H]hyaluronate between medium and tissue by cartilage from either the superficial layer or the deep layer of articular cartilage. When articular cartilage was incubated with 0.4 mM-cycloheximide, proteoglycan synthesis was markedly inhibited, whereas the synthesis of hyaluronate was only partially inhibited and resulted in more of the newly synthesized hyaluronate being released into the medium. Analysis of the hydrodynamic size of [3H]hyaluronate isolated from cartilage on Sephacryl-1000 revealed one population that was eluted as a broad peak (Kav. less than 0.7), compared with two populations (Kav. greater than 0.5 and less than 0.5) appearing in the medium of cultures. These data suggest that hyaluronate is synthesized in excess of proteoglycan synthesis and that the hyaluronate that is not complexed with proteoglycans is rapidly lost from the tissue.     

Effects of serum and insulin on hyaluronate synthesis by cultures of chondrocytes from the Swarm rat chondrosarcoma

Incorporation of [3H]glucosamine into hyaluronate synthesized by chondrocyte cultures was dependent on the concentration of foetal calf serum in the culture medium. [3H]Hyaluronate levels in cultures supplemented with 2% serum, or maintained without serum, were about 60 and 43%, respectively, of that in cultures maintained with 15% serum. Addition of insulin to cultures maintained with 15% serum had no significant effect on [3H]hyaluronate synthesis. Addition of the hormone to cultures maintained with 2% serum increased [3H]hyaluronate synthesis to levels either the same (1 ng insulin/ml), or greater than (100 ng insulin/ml) that in cultures maintained with 15% serum. The [3H]hyaluronate synthesized by the cultures was of very high molecular weight irrespective of the level of synthesis. [3H]Hyaluronate formed about 12% of the total [3H]glycosaminoglycan synthesized under all culture conditions. Synthesis of 35S, 3H-labelled proteoglycan was reduced, or increased, by the same relative amounts as [3H]hyaluronate, under the different culture conditions. Incorporation of [3H]glucosamine into hyaluronate by near confluent cultures of fibroblasts derived from the Swarm rat chondrosarcoma was reduced by 50% in cultures treated with 2% foetal calf serum compared to those maintained with 15% serum. [3H]Hyaluronate synthesis by fibroblast cultures treated with 2% serum was not stimulated by addition of insulin.     

Fluorescent morphological probe for hyaluronate

Hyaluronate levels change dramatically during morphogenesis of various tissues and organs. Morphological detection of the exact temporal and spatial distribution patterns of hyaluronate may help to elucidate its role in morphogenesis. Since no specific direct method for visualizing hyaluronate with the light or electron microscope is currently available, we have developed a morphological probe by exploiting the high-affinity interaction of cartilage proteoglycan with hyaluronate. The core protein of this proteoglycan consists of a region that binds specifically to hyaluronate with a high association constant, and a region to which the majority of sulfated polysaccharide chains are covalently attached. The polysaccharide chains were removed by treatment with chondroitinase ABC, and the core protein, labeled with rhodamine, was used as the probe. This fluorescent probe binds reversibly and specifically to [3H]hyaluronate in a binding assay using ammonium sulfate precipitation of the core protein. The probe has been used to visualize the cell surface hyaluronate of rat fibrosarcoma cells, 3T3 cells, and SV-40 transformed 3T3 cells, three cell types with significantly different amounts of cell surface-associated hyaluronate.     

Proteoglycans of bovine articular cartilage. Studies of the direct interaction of link protein with hyaluronate in the absence of proteoglycan monomer

When link protein binds to hyaluronate in the absence of proteoglycan monomer a high molecular weight complex is formed. Two assay procedures have been developed to examine the formation of the complex and the rate and stoichiometry of binding of link protein to hyaluronate in the complex. In the first, the complex is isolated by differential centrifugation, and the stoichiometry of binding of link protein to hyaluronate in the sedimented complex is determined. In the second assay, which involves turbidimetry, the rate of complex formation (delta A420/min) is determined, and the amount of complex formed is determined in terms of the maximum turbidity (A420,max) attained. The effects of temperature, pH, initial total solute concentration, and the ratio by weight of link protein to hyaluronate on the amount of complex formed and on the rate of complex formation were examined. There is a linear correlation between the amount of complex formed as determined by turbidity and by differential centrifugation. Using these assays, we examined the specificity of the binding of link protein to hyaluronate and the capacity of hyaluronate oligosaccharides to competitively inhibit the binding of link protein to hyaluronate. Hyaluronate decasaccharide is the oligosaccharide of minimum size that strongly inhibits the binding of link protein to hyaluronate. Proteoglycan monomers dissociate from hyaluronate as the pH is decreased from pH 7 to pH 5. Turbidimetric studies show that the rate of binding of link protein to hyaluronate increases with decreasing pH. The binding affinity of proteoglycan monomers for hyaluronate is decreased at pH 5, whereas the binding affinity of link protein for hyaluronate is not. This difference in the effect of pH on the stability of binding of link protein to hyaluronate, compared with proteoglycan monomer, explains in part the capacity of link protein to stabilize the binding of proteoglycan monomer to hyaluronate at pH 5.     

Proteoglycans of bovine articular cartilage. The effects of divalent cations on the biochemical properties of link protein

In cartilage proteoglycan aggregates, link protein stabilizes the binding of proteoglycan monomers to hyaluronate by binding simultaneously to hyaluronate and to the G1 globular domain of proteoglycan monomer core protein. Studies reported here involving metal chelate affinity chromatography demonstrate that link protein is a metalloprotein that binds Zn2+, Ni2+, and Co2+. Zn2+ and Ni2+ decrease the solubility of link protein and result in its precipitation. However, link protein is readily soluble and functional in low ionic strength solvents from which divalent cations have been removed with Chelex 100. These observations make it possible to study the biochemical properties of link protein in low ionic strength, physiologic solvents. Studies were carried out to define the oligomeric state of link protein alone in physiologic solvents, and the transformation in oligomeric state that occurs when link protein binds hyaluronate. Sedimentation equilibrium studies demonstrate that in 0.15 M NaCl, 5 mM EDTA, 50 mM Tris, pH 7, link protein exists as a monomer-hexamer equilibrium controlled by a formation constant of 2 x 10(27) M-5, yielding a delta G' of -36 kcal/mol for the formation of the hexamer from six monomers. On binding hyaluronate oligosaccharides (HA10 or HA12), link protein dissociates to dimer. Link protein hexamer is rendered insoluble by Zn2+. Greater than 90% of the protein is precipitated by 2 mol of Zn2+/mol of link protein monomer. The binding of hyaluronate oligosaccharide by link protein strongly inhibits the precipitation of link protein by Zn2+. The link protein/hyaluronate oligosaccharide complex is completely soluble in the presence of 2 mol of Zn2+/mol of link protein. At higher molar ratios of Zn2+/link protein, the inhibitory effect of hyaluronate oligosaccharide on the precipitation of link protein is gradually overcome. Hyaluronate oligosaccharide is not dissociated from link protein by Zn2+. Hyaluronate remains bound to the link protein which is precipitated by Zn2+, or to the link protein which binds to Zn2(+)-charged iminodiacetate-Sepharose columns. Hyaluronate oligosaccharides and Zn2+ bind to different sites on link protein.     

A specific, sensitive method for the determination of hyaluronate.

A sensitive and specific assay for hyaluronate was devised. Hyaluronate contained in biological mixtures was digested with a commercially available microbial hyaluronate lyase. The β-Δ4,5-eneglucopyranuronic acid residues contained at the nonreducing termini of the resulting oligosaccharides were oxidized with periodic acid to yield, among other products, formyl pyruvic acid. The latter compound reacted with thiobarbituric acid to yield a chromophore with an absorption maximum at 549 nm. Optimal conditions for quantitative assay of hyaluronate are described.     

Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (Mr,e) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with [35S]methionine label. The three protein species isolated by hyaluronate affinity chromatography (Mr,e 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of 3H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The apparent dissociation constant of HABP for hyaluronate was approximately 10(-8) M, and kinetic analyses showed these binding interactions were complex and of a positive cooperative nature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of proteoglycan aggregates in rat chondrosarcoma chondrocyte cultures treated with tunicamycin

Proteoglycan monomer and link protein isolated from the Swarm rat chondrosarcoma both contain glycosylamine-linked oligosaccharides. In monomer, these N-linked oligosaccharides are concentrated in a region of the protein core which interacts specifically with both hyaluronate and link protein to form proteoglycan aggregates present in cartilage matrix. Chondrocyte cultures were treated with tunicamycin to inhibit synthesis of the N-linked oligosaccharides, and the ability of the deficient proteoglycan and link protein to form aggregates was studied. Cultures were pretreated with tunicamycin for 3 h and then labeled with either [3H]mannose, [3H]glucosamine, [3H]serine, or with [35S]sulfate for 6 h in the presence of tunicamycin. Formation of link protein-stabilized proteoglycan aggregates in the culture medium was inhibited by up to 40% when the cells were treated with 3 micrograms of tunicamycin/ml, a concentration which inhibited 3H incorporation with mannose as a precursor by about 90%, but by only 15% with glucosamine as a precursor. When exogenous proteoglycan aggregate was added to the culture medium, however, it was found that both endogenous monomer and link protein synthesized in the presence of tunicamycin were fully able to form link-stabilized aggregates. This suggests that glycosylamine-linked oligosaccharides on monomer and on link protein are not necessary for their specific interactions with hyaluronate and with each other. Further, although tunicamycin did not inhibit net synthesis of hyaluronate, transfer of hyaluronate from the cell layer to the culture medium was retarded. This phenomenon accounted for most if not all of the decrease in the amount of proteoglycan which formed aggregates in the medium of cultures treated with tunicamycin.     

Tunicamycin partially delays release of newly synthesized hyaluronate from Swarm rat chondrosarcoma chondrocytes

Tunicamycin (5-100 micrograms/ml) inhibits total [3H]hyaluronate synthesis in cultures of Swarm rat chondrosarcoma chondrocytes by approx. 15%. In agreement with previous results (Lohmander, L.S., Fellini, S.K., Kimura, J.H., Stevens, R.L. and Hascall, V.C. (1983) J. Biol. Chem. 258, 12280-12286) the relative decrease in [3H]hyaluronate radioactivity in the culture medium was greater than in the cell layer. Treated cultures show a concentration-related decrease in the proportion of medium 35S-labelled proteoglycans forming 'natural aggregates'. Pulse-chase experiments in cultures pretreated with tunicamycin (100 micrograms/ml, 13 h) showed that 30-40% of the total [3H]hyaluronate synthesized is released more slowly from these chondrocytes than from control culture chondrocytes. Release of some hyaluronate molecules may be delayed by 6 h or more. After a 24 h chase period almost all the [3H]hyaluronate is released from the cells. The proportion of 35S-labelled proteoglycans present as aggregates in the 24 h chase medium (57%) remained depressed compared to controls (81%), although the monomers could form aggregates if exogenous hyaluronate was added. Hyaluronate synthesized in the presence of tunicamycin has the same hydrodynamic size as control culture hyaluronate, as assessed by its sedimentation profile in CsSO4 gradients and its chromatographic profile on a dissociative Sephacryl S-1000 column.     

Subcellular localization of hyaluronate synthetase in oligodendroglioma cells

In order to provide some insight into the mechanism of hyaluronate synthesis, the subcellular localization of the synthetase system for hyaluronate was determined in eukaryotic cells. The mouse oligodendroglioma cell line G26-24, which produces copious amounts of hyaluronate in culture, was chosen as a system for these studies. Protease treatment and homogenization of cells followed by hyaluronate synthetase assay suggested that nucleotide-binding sites and trypsin-sensitive synthetase sites were not exposed at the outer membrane surface. Protease treatment following homogenization did result in decreased activity. Membrane fragments, prepared by gentle homogenization in iso- and hypotonic buffers, were subjected to differential centrifugation followed by several continuous and discontinuous sucrose equilibrium and velocity gradient systems. Hyaluronate synthetase activity co-fractionated with a plasma membrane marker in all systems, including those in which Golgi markers were separable. Treatment of intact cells in culture with several hyaluronidases resulted in a marked stimulation of cell-free synthetase activity. The stimulated activity was also found exclusively in plasma membrane-enriched fractions.     

Membrane-associated hyaluronate-binding activity of chondrosarcoma chondrocytes

The association of hyaluronate with the surface of chondrocytes was examined by several approaches using primary cultures of chondrocytes derived from the Swarm rat chondrosarcoma. In culture, chondrosarcoma chondrocytes produced large pericellular coats, which can be visualized by particle exclusion, and which can be removed by Streptomyces hyaluronidase. Exposure of chondrocytes, which had been metabolically labelled with 3H-acetate, to exogenous hyaluronate or to Streptomyces hyaluronidase resulted in the release of 36-38% of the endogenous, labelled chondroitin sulfate from the cell layer into the incubation solution. These results imply that at least 37% of the cell layer chondroitin sulfate proteoglycan is retained there by an interaction with hyaluronate. Thus membranes were prepared from cultured chondrocytes and examined for sites which bind 3H-hyaluronate. Binding was observed and found to be saturable, specific for hyaluronate, of high affinity (Kd = approximately 10(-10) M), and destroyed by treating the membranes with trypsin. The 3H-hyaluronate-binding activity was inhibited competitively by hyaluronate decasaccharides but not by hexasaccharides or octasaccharides, indicating that the binding sites recognize a sequence of hyaluronate composed of five disaccharide repeats. The binding activity was partially purified from a detergent extract of chondrocyte membranes by ion exchange chromatography on DEAE-cellulose, followed by affinity chromatography on wheat germ agglutinin-agarose. Analysis of the partially purified binding activity by SDS-PAGE revealed five protein bands of 48,000-66,000 daltons in silver-stained gels. SDS-PAGE followed by Western blotting and exposure to monoclonal antibodies which recognize epitopes present in link protein and in the hyaluronate-binding region of cartilage proteoglycan revealed no immunoreactive protein bands in the partially purified material. We conclude that one mechanism by which hyaluronate associates with the chondrocyte surface may be via interaction with a membrane-bound hyaluronate-binding protein which is distinct from link protein and proteoglycan.     

Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate.

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.     

Interactions of cartilage proteoglycans with hyaluronate. The role of the hyaluronate acetamido groups.

Hyaluronate oligomers were treated with anhydrous hydrazine in the presence of hydrazine sulfate to remove the N-acetyl groups. Complete deacetylation could not be achieved without extensive degradation of the oligosaccharide chain. Partially deacetylated oligomers exhibited decreased inhibition of cartilage proteoglycan-hyaluronate interaction as compared to the unreacted starting material; re-N-acetylation by reaction with acetic anhydride restored the inhibitory activity to a great extent. When the hydrazine-treated oligosaccharides were reacted with other acyl anhydrides, the inhibitory potency was restored to an extent which was inversely related to the size of the acyl group. Thus, for maximal interaction between hyaluronate and proteoglycan, the glucosamine residue of hyaluronate must be N-acylated with a minimally sized acyl group.     

Stimulation of glycosaminoglycan production in murine tumors

Three types of murine tumors, B-16 melanoma, A-10 carcinoma, and S-180 sarcoma, were shown to contain elevated glycosaminoglycan (GAG) concentrations in vivo as compared to normal muscle or subcutaneous tissue. Hyaluronate was especially concentrated in the A-10 carcinoma, which contained approximately six times more hyaluronate than subcutaneous tissue and 18 times more than muscle. In all three tumors, chondroitin sulfates, especially chondroitin-4-sulfate, were present in higher concentrations than in the normal tissues. In culture, however, all three tumor cell lines produced less than 5% as much GAG as mouse fibroblasts, when measured by incorporation of [3H] acetate or by chemical analysis. Varying the culture passage number or the medium composition, ie, glucose, serum, and insulin concentrations, had little effect on GAG synthesis by the tumor cells. The low GAG levels in the tumor cell cultures were not due to hyaluronidase activity in their media. In an attempt to mimic possible host-tumor cell interactions that could account for the elevated GAG levels in vivo, tumor cells were cocultured with fibroblasts, but no stimulation above the amount made by the tumor cells alone plus that by the fibroblasts alone was observed. Conditioned media from the tumor cells, either dialyzed or not against fresh complete medium, had no effect on fibroblast GAG synthesis. Tumor extracts, however, were found to stimulate synthesis of hyaluronate by fibroblasts. Stimulation by extracts of A-10 carcinoma was greater than and additive to that of serum. The above results strongly suggest that GAG production in these tumors is in part regulated by host-tumor interactions.     

Chondrocyte-mediated depletion of articular cartilage proteoglycans in vitro.

The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The 'clipped' monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.     

Pericellular coat of chick embryo chondrocytes: structural role of hyaluronate

Chondrocytes produce large pericellular coats in vitro that can be visualized by the exclusion of particles, e.g., fixed erythrocytes, and that are removed by treatment with Streptomyces hyaluronidase, which is specific for hyaluronate. In this study, we examined the kinetics of formation of these coats and the relationship of hyaluronate and proteoglycan to coat structure. Chondrocytes were isolated from chick tibia cartilage by collagenase-trypsin digestion and were characterized by their morphology and by their synthesis of both type II collagen and high molecular weight proteoglycans. The degree of spreading of the chondrocytes and the size of the coats were quantitated at various times subsequent to seeding by tracing phase-contrast photomicrographs of the cultures. After seeding, the chondrocytes attached themselves to the tissue culture dish and exhibited coats within 4 h. The coats reached a maximum size after 3-4 d and subsequently decreased over the next 2-3 d. Subcultured chondrocytes produced a large coat only if passaged before 4 d. Both primary and first passage cells, with or without coats, produced type II collagen but not type I collagen as determined by enzyme-linked immunosorbent assay. Treatment with Streptomyces hyaluronidase (1.0 mU/ml, 15 min), which completely removed the coat, released 58% of the chondroitin sulfate but only 9% of the proteins associated with the cell surface. The proteins released by hyaluronidase were not digestible by bacterial collagenase. Monensin and cycloheximide (0.01-10 microM, 48 h) caused a dose-dependent decrease in coat size that was linearly correlated to synthesis of cell surface hyaluronate (r = 0.98) but not chondroitin sulfate (r = 0.2). We conclude that the coat surrounding chondrocytes is dependent on hyaluronate for its structure and that hyaluronate retains a large proportion of the proteoglycan in the coat.     

Adhesion to extracellular materials by neural crest cells at the stage of initial migration

Summary Trunk-level neural anlagen bearing neural crest cells at the stage of initiation of migration were isolated from chick embryos and explanted in serum-free medium onto glass substrates which had previously been treated with extracellular materials. After 0.5–2 h incubation, the expiants were dislodged with a stream of culture medium and the substrate examined for adherent crest cells. Crest cells adhered to collagen gels, and adhered to and spread on adsorbed fibronectin; antiserum to fibronectin prevented adhesion to fibronectin but not to collagen gels. Air-dried collagen gels and collagen solutions were less adhesive, the adhesivity declining with longer drying time and lower collagen concentration. Crest cells adhered poorly to dried gelatin and not at all to adsorbed collagen. Fibronectin increased the adhesion to dried collagen and gelatin. Pretreatment of collagen gels with hyaluronate retarded adhesion. Hyaluronate pretreatment also retarded adhesion to adsorbed fibronectin but only when adsorbed collagen was also present. Pretreatment of collagen gels with the proteoglycan monomer from bovine nasal cartilage had no effect of the adhesion of crest cells, but the proteoglycan almost completely inhibited adhesion to adsorbed fibronectin, but only when absorbed collagen was also present. The results are discussed in terms of the control of migration of neural crest cells by extracellular materials.     

Size-dependent hyaluronate degradation by cultured cells

Hyaluronate degradation was examined in cultures of vascular wall cells (bovine aortic endothelial cells, rat aortic smooth muscle cells) and in nonvascular cells (chick embryo fibroblasts). The three cell types examined all produced hyaluronidase activity in culture which had a strict acidic pH requirement for activity. This suggested that the enzyme was active only within an acidic intracellular compartment and therefore that hyaluronate degradation occurred at an intracellular site. This was supported by the observation that the presence of hyaluronidase activity alone was not sufficient to ensure degradation of extracellular hyaluronate. Rather, the key limiting factor in this process appeared to be hyaluronate internalization, and this was found to be hyaluronate size-dependent and to a degree, cell-specific. The relationship of these results to morphogenesis and tissue remodeling is discussed.     

Chondroitin sulfate proteoglycan (PG-M-like proteoglycan) is involved in the binding of hyaluronic acid to cellular fibronectin

Preparations of cellular fibronectin from chick embryonic fibroblasts have previously been shown to have hyaluronate-binding activity. However, gel filtration and CsCl isopycnic centrifugation of fibronectin preparations showed that the binding activity was associated with molecules with a density and a molecular weight higher than those of fibronectin. An immunoprecipitation assay using antibodies to the chondroitin sulfate proteoglycan (PG-M) from the mesenchyme of chick embryo limb bud showed that the hyaluronate-binding activity of fibronectin preparations was precipitable with this antibody. The immunoprecipitation analyses also showed that fibronectin preparations as well as conditioned culture medium and extracts of chick embryonic fibroblasts contained a chondroitin sulfate proteoglycan, the protein-enriched core molecules from which were identical to those from PG-M with respect to electrophoretic mobility and immunological reactivity. This proteoglycan was purified from conditioned culture medium and extracts of fibroblasts by dissociative CsCl isopycnic centrifugation. The proteoglycans from medium or extracts gave core derivatives with electrophoretic mobility identical to those from PG-M, and they had equal hyaluronate-binding activities. These results, taken together, suggest that most, if not all, of the hyaluronate-binding activity in preparations of chick cellular fibronectin is due to a proteoglycan identical to PG-M. This proteoglycan was also found to bind directly to fibronectin and to type I collagen, but not to laminin or type IV collagen. It is possible that the fibroblast proteoglycan mediates interactions between hyaluronate, fibronectin, and type I collagen, thereby participating in formation of the pericellular matrix of fibroblasts.     

High-uptake and low-uptake forms of proteoglycans secreted by arterial smooth muscle cells

Cultured arterial smooth muscle cells synthesize and secrete two types of sulfated proteoglycans, designated as proteoglycan A and B, into the culture medium. They are isolated as immunologically distinct monomers with relative molecular masses of 280 X 10(3) and 180 X 10(3) and are characterized as chondroitin-sulfate-rich (A) and dermatan-sulfate-rich (B) proteoglycans. Both proteoglycan A and B were labelled with [35S]sulfate and used for studies of endocytosis. Uptake of proteoglycan B by arterial smooth muscle cells shows saturable kinetics. At saturation (500 microM) one cell may endocytose up to 1.5 X 10(6) proteoglycan B molecules/h. Proteoglycan A is internalized at a 10-fold lower rate. No saturation kinetics were observed at high proteoglycan A concentrations (500 microM). Endocytosis of proteoglycan B in the presence of an excess of proteoglycan A and vice versa suggest that proteoglycan A and B do not compete for the same receptor site. Free hyaluronate or chondroitin sulfate do not inhibit the uptake of proteoglycan B or A. The results suggest that proteoglycan B is internalized by arterial smooth muscle cells via a high-affinity receptor-mediated process, whereas proteoglycan A is taken up by fluid endocytosis and/or by low-affinity endocytotic processes.