Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma.

Cultured chondrocytes from the Swarm rat chondrosarcoma incorporate [35S]sulfate into proteoglycans typical of hyaline cartilage. The movement of newly synthesized proteoglycans from inside the cells into the extracellular matrix and, finally, into the culture medium was examined by measuring the distribution of 35S-labeled proteoglycans in the medium, a 4 M guanidine HCl extract of the cell layer, and in the remaining residue for a number of chase times following a 5-min pulse with [35S]sulfate. When hyaluronate oligosaccharides containing greater than or equal to 10 monosaccharides were included in the chase media, a proportion of newly synthesized proteoglycans were displaced from the matrix (4 M extract) into the culture medium. This displacement was greatest when oligomers were in the chase media between 10 and 20 min after the pulse, approximately the time when the molecules are being secreted from the cells. The proportion of link-stabilized aggregate in the medium was examined by Sepharose 2B chromatography after adding an excess of unlabeled monomer which displaces labeled monomer from complexes with hyaluronate which are not link-stabilized. The proportion of link-stabilized aggregate increased from 12% to about 70% between 12 and 120 min of chase. The presence of 40 micron hyaluronate oligosaccharides of 16 monosaccharides in the chase media retarded but did not prevent aggregate formation. Oligomers of about 50 monosaccharides, which are large enough to bind both a monomer proteoglycan and a link protein, almost completely prevented the formation of the large link-stabilized aggregates. The results suggest: (a) newly synthesized proteoglycans are not bound into link-stabilized aggregates at the time of secretion; (b) hyaluronic acid oligomers which are long enough to interact only with the hyaluronic acid-binding site of proteoglycans will retard but not prevent link-stabilized aggregation; and (c) hyaluronic acid oligomers long enough to accommodate additionally a link protein form a link-stabilized ternary complex and prevent aggregation with larger hyaluronic acid molecules.     

Cartilage proteoglycan aggregate formation. Role of link protein.

Cartilage proteoglycan aggregate formation was studied by zonal rate centrifugation in sucrose gradients. Proteoglycan aggregates, monomers and proteins could be resolved. It was shown that the optimal proportion of hyaluronic acid for proteoglycan aggregate formation was about 1% of proteoglycan dry weight. The reaggregation of dissociated proteoglycan aggregate A1 fraction was markedly concentration-dependent and even at 9 mg/ml only about 90% of the aggregates were reformed. The lowest proportion of link protein required for maximal formation of link-stabilized proteoglycan aggregates was 1.5% of proteoglycan dry weight. It was separately shown that link protein co-sedimented with the proteoglycan monomer. By competition with isolated hyaluronic acid-binding-region fragments, a proportion of the link proteins was removed from the proteoglycan monomers, indicating that the link protein binds to the hyaluronic acid-binding region of the proteoglycan monomer.     

Assembly of proteoglycan aggregates in cultures of chondrocytes from bovine tracheal cartilage.

The assembly of proteoglycan aggregates in chondrocyte cell cultures was examined in pulse-chase experiments with the use of [35S]sulphate for labelling. Rate-zonal centrifugation in linear sucrose density gradients (10-50%, w/v) was used to separate the aggregated proteoglycans from monomers and to assess the size of the newly formed aggregates. The proportion of aggregates stabilized by link protein was assessed by competition with added exogenous aggregate components. The capacity of the proteoglycans synthesized in culture to compete with exogenous nasal-cartilage proteoglycans for binding was studied in dissociation-reassociation experiments. The results were as follows. (a) The proteoglycan monomers and the hyaluronic acid are exported separately and combined extracellularly. (b) The size of the aggregates increases gradually with time as the proportion of monomers bound to hyaluronic acid increases. (c) All of the aggregates present at a particular time appear to be link-stabilized and therefore not dissociated by added excess of nasal-cartilage proteoglycan monomer or hyaluronic acid oligomers. (d) The free monomer is apparently present as a complex with link protein. The monomer-link complexes are then aggregated to the hyaluronic acid. (e) The aggregates synthesized in vitro and the nasal-cartilage aggregates differ when tested for link-stabilization by incubation at low pH. The aggregates synthesized in vitro were completely dissociated whereas the cartilage proteoglycans remained aggregated. The results obtained from dissociation-reassociation experiments performed at low pH indicate that the proteoglycan monomer synthesized in vitro does not bind the hyaluronic acid or the link protein as strongly as does the nasal-cartilage monomer.     

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.     

Isolation and characterization of a link protein from bovine aorta proteoglycan aggregate

Proteoglycan aggregates were isolated from bovine aorta by extraction with 0.5 M guanidine hydrochloride in the presence of proteinase inhibitors and purified by isopycnic CsCl centrifugation. The bottom two-fifths (A1) of the gradient contained 30% of proteoglycans in the aggregated form. The aggregate had 14.8% protein and 20.4% hexuronic acid with hyaluronic acid, dermatan sulfate and chondroitin sulfates in a proportion of 18:18:69. A link protein-containing fraction was isolated from the bottom two-fifths by dissociative CsCl isopycnic centrifugation. The link protein that floated to the top one-fifth of the gradient was purified by chromatography on Sephadex G-200 in the presence of 4 M guanidine hydrochloride. It moved as a single band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 49 000. The amino acid composition of link protein resembled that of link protein from cartilage, but was strikingly different from that of the protein core of the proteoglycan monomer. The neutral sugar content of link protein was 3.5% of dry weight. Galactose, mannose and fucose constituted 21, 62 and 16%, respectively of the total neutral sugars. In aggregation studies the link protein was found to interact with both proteoglycan monomer and hyaluronic acid. Oligosaccharides derived from hyaluronic acid decreased the viscosity of link protein-free aggregates of proteoglycan and hyaluronic acid but not of link-stabilized aggregates, demonstrating that the link protein increases the stability of proteoglycan aggregates.     

Rabbit chondrocytes maintained in serum-free medium : I. Synthesis and secretion of hydrodynamically-small proteoglycans

The biosynthesis of sulfated proteoglycan in vitro by rabbit articular chondrocytes in first passage monolayer culture maintained in fetal bovine serum (FBS) or in serum-free conditions was compared. Neosynthesized proteoglycan in the culture medium in the most dense fraction of an associative CsCl density gradient (fraction dAl) declined with increasing time under serum-free conditions, but not when cells were maintained in the presence of serum. After one day, the major peak of incorporated 35SO4 in medium fraction dAl eluted as a retarded peak (Kav 0.28) on Sepharose CL-2B, whether cells were maintained under serum-free or serum-containing conditions. The hydrodynamic size of proteoglycan monomer fraction dAlDl obtained after one day of exposure to serum-free culture media was smaller than dAlDl from serum-containing cultures. The hydrodynamic size of dAlDl obtained from serum-free culture media became even progressively smaller after 2 and 3 days' exposure to these conditions. Hydrodynamically small sulfated proteoglycans were identified in the cell-associated dAlDl fraction as early as one day after switching chondrocytes from serum-containing to serum-free medium. Culture medium fraction dAlDl from serum-free culture medium aggregated poorly when incubated with human hyaluronic acid (HA) in the presence of bovine link protein or when dialysed against bovine nasal cartilage proteoglycan aggregate. Proteoglycan monomer from serum-containing medium reaggregated more efficiently under both conditions. No change in the size of glycosaminoglycan chains was seen in the smaller proteoglycan subpopulations, nor was there any indication of marked changes in the glycosaminoglycan types.     

Age-related decrease in the link-stability of proteoglycan aggregates formed by articular chondrocytes

Chondrocytes were isolated from the articular cartilage of rabbits aged between 6 and 50 weeks and labelled with [35S]sulphate after 48 h in monolayer culture. The percentage of the total proteoglycan monomers synthesized by each culture that were present as link-stabilized aggregates was shown to be about 83% at 6, 9 and 12 weeks, 73% at 15 weeks, 48% at 30 weeks and 32% at 50 weeks. The proliferative activity of the cells in culture also decreased markedly with the age of the donor. The results suggest that aging of chondrocytes in vivo is accompanied by a decrease in their capacity for link-protein synthesis.     

The in vitro cell culture age and cell density of articular chondrocytes alter sulfated-proteoglycan biosynthesis

The effect of cell culture age and concomitant changes in cell density on the biosynthesis of sulfated-proteoglycan by rabbit articular chondrocytes in secondary monolayer culture was studied. Low density (LD, 2 d), middle density (MD, 5-7 d), and high density (HD, 12-15 d) cultures demonstrated changes in cellular morphology and rates of DNA synthesis. DNA synthesis was highest at LD to MD densities, but HD cultures continued to incorporate [3H]-thymidine. LD cultures incorporated 35SO4 into sulfated-proteoglycans at a higher rate than MD or LD cultures. The qualitative nature of the sulfated-proteoglycans synthesized at the different culture ages were analyzed by assessing the distribution of incorporated 35SO4 in associative and dissociative CsCl density gradients and by elution profiles on Sepharose CL-2B. Chondrocytes deposited into the extracellular matrix (cell-associated fraction) 35SO4-labeled proteoglycan aggregate. More aggregated proteoglycan was found in the MD and HD cultures than at LD. A 35SO4-labeled aggregated proteoglycan of smaller hydrodynamic size than that found in the cell-associated fraction was secreted into the culture medium at each culture age. The proteoglycan monomer (A1D1) of young and older cultures had similar hydrodynamic sizes at all cell culture ages and cell densities. The glycosaminoglycan chains of A1D1 were hydrodynamically larger in the younger LD cultures than in the older HD cultures and consisted of only chondroitin 6 and 4 sulfate chains. A small amount of chondroitin 4,6 sulfate was detected, but no keratan sulfate was measured. The A1D2 fractions of young LD cultures contained measurable amounts of dermatan sulfate; no dermatan sulfate was found in older MD or HD cultures. These studies indicated that chondrocytes at LD synthesized a proteoglycan monomer with many of the characteristics of young immature articular cartilage of rabbits. These results also indicated that rapidly dividing chondrocytes were capable of synthesizing proteoglycans which form aggregates with hyaluronic acid. Culture age and cell density appears primarily to modulate the synthesis of glycosaminoglycan types and chain length. Whether or not these glycosaminoglycans are found on the same or different core proteins remains to be determined.     

Biosynthesis of cartilage proteoglycan and link protein by articular chondrocytes from immature and mature rabbits

Chondrocytes from immature and mature rabbits have been compared in biosynthetic studies with [3H] leucine and [35S]sulfate as precursors. The time course of incorporation of [3H]leucine into general protein, proteoglycan monomer core protein, and link protein and of [35S]sulfate into proteoglycan monomer has been examined. Proteoglycan monomer was isolated from the high buoyant density (p greater than 1.60) fractions of dissociative CsCl gradients and link protein by immunoprecipitation with antibody 8A4 followed by gel electrophoresis. Results based on the period of linear isotope incorporation showed that mature cells synthesize protein at about 40% of the rate of immature cells and both proteoglycan and link protein at about 20% of the rate of immature cells. The labeling rates obtained suggest that immature cells synthesize an approximate 1:1 molar ratio of link protein to proteoglycan monomer, and for mature cells this ratio is about 0.8:1. While cell layer retention of newly synthesized proteoglycan was markedly lower in mature relative to immature cell cultures, link protein retention was high in both immature and mature cultures; this finding provides an explanation for our previous observation (Plaas, A. H. K., and Sandy, J. D. (1984) Biochem, J. 220, 337-340) that link-free monomer accumulates in the medium of mature but not immature cultures. The link protein synthesized by both ages of cells and isolated from cell layer or medium was a single major species of apparent molecular mass 48-51 kDa. The results suggest that mature chondrocytes are less efficient than immature chondrocytes in the coordinated assembly of link-stabilized proteoglycan aggregates in this culture system.     

Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts.

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called 'link proteins' were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.     

Degradation of proteoglycan aggregate by a cartilage metalloproteinase. Evidence for the involvement of stromelysin in the generation of link protein heterogeneity in situ.

Cartilage proteoglycan aggregates were subjected to degradation by a metalloproteinase, capable of degrading proteoglycan, released from cartilage in culture. This proteinase was demonstrated to be immunologically identical with fibroblast stromelysin. An early release of hyaluronic acid-binding region and large glycosaminoglycan-attachment regions was observed. With increasing time the glycosaminoglycan-attachment regions were digested into smaller fragments and the hyaluronic acid-binding regions accumulated. The degradation of link proteins also occurred concomitantly with these events. Link proteins were converted into a component of similar size to that of the smallest native link protein component. N-Terminal sequence analysis of the three human link protein components indicated that they are all derived from the same protein core, which is closely homologous to that of the rat chondrosarcoma link protein. The two larger link proteins (Mr 48,000 and 44,000) contain the same N-terminal sequence, but they differ by the apparent presence of an N-linked oligosaccharide at residue 6 of the largest link protein component. The smallest link protein (Mr 41,000), however, has an N-terminal sequence equivalent to that commencing at residue 17 in the larger link proteins. It was found that the cartilage metalloproteinase cleaves link proteins in human neonatal cartilage proteoglycan aggregates at the His-16-Ile-17 bond, the same position at which the smallest link protein component appears to be derived naturally from the two larger link protein components. These results suggest that stromelysin secreted by chondrocytes can account for the increased accumulation of hyaluronic acid-binding regions and much of the degradation of link protein observed during aging within human articular cartilage.     

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.     

Interleukin-1-induced alterations in proteoglycan metabolism and matrix assembly

In cartilage, the large chondroitin sulfate proteoglycan exists as aggregates by interacting with link protein and hyaluronic acid. In diseases associated with cartilage degeneration, the proteoglycan does not aggregate because of a defect in the hyaluronate-binding activity. Since interleukin-1 (IL-1) is a secretory product of activated macrophages and may influence the cartilage function in joints, we studied the effects of IL-1 on the synthesis and assembly of proteoglycan by rabbit articular chondrocytes in culture. IL-1-treated cells showed a modest increase in the total proteoglycan synthesis, but also showed a more pronounced decrease in the incorporation of extracellular matrix. Affinity chromatography of the conditioned media on hyaluronic acid-Sepharose revealed that all of the proteoglycan of control cells strongly bound to hyaluronate. The IL-1-treated medium contained two fractions: one that was strongly bound to the column and a second that did not bind. The results demonstrate that the IL-1-treated cells cannot incorporate proteoglycan into the matrix partly because of a defect in the proteoglycan molecules and partly due to other mechanisms regulating proteoglycan assembly.     

Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography

Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.     

Degradation of proteoglycan in articular cartilage.

Adult rabbit articular cartilage was labelled in vivo over 48 h with [35S]sulphate and was then incubated in organ culture at pH 7.2. Approx. 65% of the tissue content of [35S]proteoglycan was released into the culture medium during the first 48 h of incubation. The average molecular size of the released proteoglycans, as assessed by fractionation on Sepharose 2B/CL and 4B/Cl, was only slightly smaller than that of the proteoglycans extracted from non-cultured cartilage with 4 M guanidine HCl. The percentage of released proteoglycans and extracted proteoglycans which formed aggregates with hyaluronic acid was approx. 25% and 75%, respectively. The results indicate that proteoglycan degradation in adult articular cartilage is initiated by a limited proteolysis of subunit core protein, with the production of non-aggregating species which diffuse readily from the tissue.     

Localization of proteoglycan monomer and link protein in the matrix of bovine articular cartilage: An immunohistochemical study

Using monospecific antisera and immunofluorescence microscopy, proteoglycan monomer (PG), and link proteins were demonstrated throughout the extracellular matrix of bovine articular cartilage. A narrow band of strong pericellular staining was usually observed for both molecules, indicating a pericellular concentration of proteoglycan monomer: this conclusion was supported by dye-binding studies. Whereas PG was evenly distributed throughout the remaining matrix, more link protein was detectable in interterritorial sites in middle and deep zones. Well-defined zones of weaker territorial staining for link protein stained strongest for chondroitin sulfate. Trypsin treatment of cartilage resulted in a loss of most of the PG staining, but some selective retention of link protein, particularly around chondrocytes in the superficial zone at and near the articular surface. This residual staining was largely removed if sections were fixed after chondroitinase treatment. After extraction of cartilage with 4M guanidine hydrochloride, only PG remained and this was concentrated in the superficial zone. These observations are shown to support the concept of aggregation of PG and link protein with hyaluronic acid (HA) in cartilage matrix, and the binding of PG and link protein to HA, which is attached to the chondrocyte surface. Culture of cartilage depleted of PG and link protein by trypsin demonstrated that individual chondrocytes can secrete both PG and link proteins and that the organization of cartilage matrix can be regenerated in part over a period of 4 days.     

Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures

The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of [35S]sulfate and [3H]glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on [35S]sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on [35S]sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased [3H]thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.     

Studies on the hyaluronate binding properties of newly synthesized proteoglycans purified from articular chondrocyte cultures

Primary cultures of rabbit articular chondrocytes have been maintained for 10 days and labeled with [35S]sulfate, [3H]leucine, and [35S]cysteine in pulse-chase protocols to study the structure and hyaluronate binding properties of newly synthesized proteoglycan monomers. Radiolabeled monomers were purified from medium and cell-layer fractions by dissociative CsCl gradient centrifugation with bovine carrier monomer, and analyzed for hyaluronate binding affinity on Sepharose CL-2B in 0.5 M Na acetate, 0.1% Triton X-100, pH 6.8. Detergent was necessary to prevent self-association of newly synthesized monomers during chromatography. Monomers secreted during a 30-min pulse labeling with [35S]sulfate had a low affinity relative to carrier. Those molecules released into the medium during the first 12 h of chase (about 40% of the total) remained in the low affinity form whereas those retained by the cell layer rapidly acquired high affinity. In cultures where more than 90% of the preformed cell-layer proteoglycan was removed by hyaluronidase digestion before radiolabeling the newly synthesized low affinity monomers also rapidly acquired high affinity if retained in the cell layer. Cultures labeled with amino acid precursors were used to establish the purity of monomer preparations and to isolate core proteins for study. Leucine- or cysteine-labeled core proteins derived from either low or high affinity monomer preparations migrated as a single major species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with electrophoretic mobility very similar to that of core protein derived from extracted proteoglycan monomer. Purified low affinity monomers were converted to the high affinity form by treatment at pH 8.6; however, this change was prevented by guanidinium-HCl at concentrations above 0.8 M. Conversion to high affinity was also achieved by incubation of monomers in aggregate with hyaluronic acid (HA) at pH 6.8 followed by dissociative reisolation of monomer. At both pH 6.8 and 8.6 the conversion process was slow, requiring up to 48 h for the maximum increase in affinity. It is suggested that the slow increase in HA binding affinity seen during extracellular processing of proteoglycans in cartilage and chondrocyte cultures is the result of an irreversible structural change in the HA binding domain following the binding of monomer to hyaluronate. The available evidence suggests that this change involves the formation or rearrangement of disulfide bonds.     

Synthesis of a "free" form of hyaluronic acid by articular chondrocytes in monolayer culture

Primary and first passage rabbit chondrocyte cultures synthesized a "free" form of hyaluronic acid (HA-f) previously characterized in rabbit cartilage. HA-f was isolated from the [3H] glcN/35SO4-labelled cell-associated-fraction (CAF) and from the culture medium by successive equilibrium centrifugations in Cs2SO4/CsCl/Cs2SO4 under low salt conditions. The culture medium HA-f appeared in the void volume of Sepharose CL-2B eluted with low salt, (0.5M sodium acetate), and was susceptible to digestion with Streptomyces hyaluronidase. HA-f aggregated purified rabbit cartilage proteoglycan monomer. These results indicated that HA-f probably subserves hyaluronic acid already complexed with proteoglycan monomer. Newly synthesized HA-f may be required for the continual formation of proteoglycan aggregates.     

Passive loss of proteoglycan from articular cartilage explants

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.