Extracellular matrix metabolism by chondrocytes 5. The proteoglycans and glycosaminoglycans synthesized by chondrocytes in high density cultures

Proteoglycans were extracted from the extracellular matrix of cultures of embryonic chick chondrocytes grown at high density and were purified by CsCl density gradient centrifugation. The chemical, physical and hyaluronate binding properties of the proteoglycans were similar to those observed in proteoglycans from other hyaline cartilages. Proteoglycans in the media were also purified and on analysis showed three populations of proteoglycans to be present. One population had the physical characteristics of a typical proteoglycan subunit and bound hyaluronate, the other two populations were unable to complex with hyaluronate but one had the physical characteristics of the proteoglycan subunit and the other was of smaller molecular weight. The small molecular weight appears to be a product of the enzymatic degradation of the larger molecular weight species.     

Extracellular matrix and the maintenance of the differentiated state: proteoglycans synthesized by replated chondrocytes and nonchondrocytes

It has been previously shown that undifferentiated stage 23 to 24 chick limb bud mesenchymal cells can be maintained in culture under conditions which promote chondrogenesis. As the chondrocytes mature in vitro, their proteoglycan synthesis progresses through a specific and reproducible biosynthetic program. By the eighth day of culture, the chondrocytes are making proteoglycans that are similar to proteoglycans isolated from adult animal tissues. Relative to the Day 8 proteoglycans, the proteoglycans synthesized by chick limb bud chondrocytes earlier in culture have a smaller monomer size, longer chondroitin sulfate chains, shorter keratan sulfate chains, a higher ratio of chondroitin-6-sulfate to chondroitin-4-sulfate, and a decreased ability to interact with hyaluronic acid. We have reported a procedure to remove the cells from Day 8 cultures and strip away most, if not all, of the extracellular matrix. In addition, the chondrocytes can be separated from the 40-50% nonchondrocytic cells normally found in Day 8 cultures, and the two cell populations replated separately. This report describes the analysis of the proteoglycans synthesized by replated cells; this analysis demonstrates quantitative and qualitative differences between chondrocyte and nonchondrocyte proteoglycans. The overall rate of proteoglycan synthesis is fourfold higher and the rate of synthesis of high buoyant density proteoglycans 30-fold higher for replated chondrocytes relative to nonchondrocytes. Qualitatively, more newly synthesized nonchondrocyte proteoglycans partition at lower buoyant density on CsCl equilibrium density gradients than do chondrocyte proteoglycans. Nonchondrocyte proteoglycans are of two major classes: One has a monomer size slightly smaller than that of Day 8 chondrocyte proteoglycan, but has much longer glycosaminoglycan chains. The other is considerably smaller than Day 8 chondrocyte proteoglycans, but has glycosaminoglycans of slightly larger size. In contrast, replated chondrocytes synthesize, even as soon as 4.5 hr after replating, proteoglycans that are identical to Day 8 chondrocyte proteoglycan in monomer size, in glycosaminoglycan chain size, in aggregability, and in the ratio of 6-sulfated to 4-sulfated chondroitin. Since denuding mature Day 8 chondrocytes of their extracellular matrix does not cause them to recapitulate their developmentally regulated program for the biosynthesis of proteoglycans, it is concluded that the quality of mature chondrocyte proteoglycan is not altered by the absence of extracellular matrix.     

Decreased extracellular matrix production in scurvy involves a humoral factor other than ascorbate

Our recent studies suggested that decreased collagen synthesis in bone and cartilage of scorbutic guinea pigs was not related to ascorbate-dependent proline hydroxylation. The decrease paralleled scurvy-induced weight loss and reduced proteoglycan synthesis. Those results led us to propose that the effects of ascorbate deficiency on extracellular matrix synthesis were caused by changes in humoral factors similar to those that occur in fasting. Here we present evidence for this proposal. Exposure of chick embryo chondrocytes to scorbutic guinea pig serum, in the presence of ascorbate, led to effects on extracellular matrix synthesis similar to those seen in scorbutic animals. The rates of collagen and proteoglycan synthesis were reduced to approximately 30-50% of the levels in cells cultured in normal guinea pig serum plus ascorbate, but proline hydroxylation and procollagen secretion were unaffected. Similar results were obtained with serum from fasted guinea pigs supplemented in vivo with ascorbate. The growth rate of the chondrocytes was not significantly affected by scorbutic guinea pig serum.     

Induction and prevention of chondrocyte hypertrophy in culture

Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification. At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability. Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.     

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype

The in vitro phenotype of bovine articular chondrocytes is described. Chondrocytes plated at high density in roller-bottle and dish cultures were maintained in vitro. The major matrix macromolecules, collagen and proteoglycan, synthesized by these cells were characterized during the course of the culture period. The chondrocytes synthesized mainly Type II collagen, which was found predominantly in the cell-associated matrix. The media contained a mixture of Type II and Type III collagens. Type I collagen was detectable in neither the medium nor the cell-associated matrix. The proteoglycan monomers found in media and cell-associated matrix had the same hydrodynamic sizes as monomers synthesized by cartilage slices or those extracted from adult articular cartilage. The majority of proteoglycans synthesized by the cells were found in high molecular weight aggregates which were readily recovered from the media and were extractable from cell-associated matrix with low ionic strength buffers. The results demonstrate the long-term in vitro phenotypic stability of the bovine articular chondrocytes. The advantages of the in vitro system as a model for studying the effects of external agents, such as drugs and vitamins, are discussed.     

Influence of Matricial Molecules on Growth and Differentiation of Entrapped Chondrocytes

Primary cultures of rabbit articular chondrocytes have been cultivated normally and within three-dimensional systems using different alginate matrices. The in vitro proliferation capacity of the cells immobilized in the calcium alginate beads was investigated. The growth curve showed that chondrocytes are able to grow and to divide for several days inside the beads; in parallel an increase in protein contents was also measured. The differentiated phenotype of rabbit articular chondrocytes consists of cartilage-specific proteoglycans. During serial monolayer cultures this phenotype was lost and replaced by a low level of proteoglycan synthesis. On the contrary when cultivated in beads, entrapped cells maintained their differentiated pheno-type over time; the rates of proteoglycan were similar to those of primary chondrocytes. All these parameters were tested comparatively using different substrata in monolayer cultures and in alginate gels. Assays were carried out to assess the influence of type I collagen, type IV collagen, and of fibronectine on the growth as well as on the differentiation phenotype. The encapsulation methodology is readily applicable to the culture of chondrocytes in single beads, in multiwell dishes, or to mass culture for a bioproduction of extracellular matrix components.     

Anionic glycoconjugates from differentiated and dedifferentiated cultures of bovine articular chondrocytes: Modulation by TGF-β

Summary Primary, high density bovine articular chondrocyte (BAC) cultures, stimulated with transforming growth factor-β-1, elaborated a high molecular weight anionic glycoconjugate, kDa 540, which does not contain glycosaminoglycan chains (Chan and Anastassiades, 1996). The effect of exogenously added transforming growth factor-β-1 on the elaboration of the high molecular weight glycoconjugate and of proteoglycans was studied during dedifferentiation of the chondrocytes, utilizing a serial subculture technique under anchorage-dependent conditions, up to four subcultures. The high molecular weight glycoconjugate was detected in the media of all growth-factor-stimulated chondrocyte subcultures, as well as stimulated primary cultures, but not in unstimulated primary cultures or subcultures. By contrast, a large proteoglycan, was only secreted by primary cultures and first subcultures, whether treated with transforming growth factor-β-1 or untreated. This proteoglycan contained mostly chondroitin sulfate chains, whose hydrodynamic size was increased by the addition of transforming growth factor-β-1. Further, the pattern of the proteoglycans appearing in the media of subcultures 2–4 was influenced by the addition of transforming growth factor-β-1, so that while these control subcultures elaborated both the large and small chondroitin sulfate proteoglycans, the equivalent stimulated subcultures elaborated only intermediate sized chondroitin sulfate proteoglycan(s). These results suggest that while dedifferentiation of articular chondrocytes, achieved by subculturing, strongly modulates the effect of exogenously added transforming growth factor-β-1 on the type of proteoglycan elaborated, the process of dedifferentiation does not influence the transforming-growth-factor-β-dependent synthesis of the high molecular weight anionic glycoconjugate.     

Pleiotrophin inhibits chondrocyte proliferation and stimulates proteoglycan synthesis in mature bovine cartilage

Pleiotrophin (PTN) is a secreted heparin-binding, developmentally regulated protein that is found in abundance in fetal, but not mature, cartilage. SDS-page and glycosaminoglycan (GAG) analysis of sulfate-radiolabeled proteoglycans isolated from the medium of mature cultured chondrocytes treated with PTN showed a threefold increase in the levels of proteoglycan synthesis. In contrast, in cultures of fetal chondrocytes, no changes in proteoglycan synthesis were observed. Thymidine incorporation experiments showed a dose-dependent decrease in proliferation of treated cells compared with control cultures, suggesting that pleiotrophin had an inhibitory effect on growth of chondrocytes. Neither FGF or heparin reversed the inhibitory effect of PTN. Capillary electrophoresis of chondroitinase ABC-digested proteoglycans isolated from mature chondrocytes showed 2-4-fold increases in the amounts of the 4S- and 6S-substituted GAG chains for the PTN-treated chondrocytes. Northern analysis showed a twofold upregulation in the mRNA levels of biglycan and collagen type II, but no difference in the message levels for decorin and aggrecan. These results establish that PTN inhibits cell proliferation, while stimulating the synthesis of proteoglycans in mature chondrocytes in vitro, suggesting that PTN may act directly or indirectly to regulate growth and proteoglycan synthesis in the developing matrix of fetal cartilage.     

Immunological characterization of basement membrane types of heparan sulfate proteoglycan

Antibodies were raised against a small high-density and a large low-density form of heparan sulfate proteoglycan from a basement membrane-producing mouse tumor and were characterized by radioimmunoassays, immunoprecipitation and immunohistological methods. Antigenicity was due to the protein cores and included epitopes unique to the low density form as well as some shared by both proteoglycans. The antibodies did not cross-react with other basement membrane proteins or with chondroitin sulfate proteoglycans from interstitial connective tissues. The heparan sulfate proteoglycans occurred ubiquitously in embryonic and adult basement membranes and could be initially detected at the 2-4 cell stage of mouse embryonic development. Low levels were also found in serum. Biosynthetic studies demonstrated identical or similar proteoglycans in cultures of normal and carcinoembryonic cells and in organ cultures of fetal tissues. They could be distinguished from liver cell membrane heparan sulfate proteoglycan, indicating that the basement membrane types of proteoglycans represent a unique class of extracellular matrix proteins.     

Stimulation by glucocorticoid of the synthesis of cartilage-matrix proteoglycans produced by rabbit costal chondrocytes in vitro

The effect of glucocorticoids on sulfated proteoglycan synthesis by rabbit costal chondrocyte cultures exposed to serum-free conditions has been examined. Low density cultures of rabbit costal chondrocytes were maintained on dishes coated with extracellular matrix produced by bovine corneal endothelial cells and exposed to a 9:1 mixture (v/v) of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with transferrin, high density lipoproteins, fibroblast growth factor, and insulin (Medium A). Chondrocytes maintained in the presence of Medium A supplemented with 10(-7) M hydrocortisone reorganized, at confluence, into a homogeneous cartilage-like tissue composed of round cells surrounded by a refractile matrix in which abundant thin collagen fibrils characteristic of type II collagen were observed. The cell ultrastructure and fibrils of the pericellular matrix were similar to those seen in vivo. In contrast, cells maintained in the presence of Medium A alone, once they reached confluence, formed a fibroblastic multilayer and produced thick collagen bundles. The level of 35SO4(2-) incorporated into large cartilage-specific proteoglycans in glucocorticoid-supplemented cultures was 33-fold higher than that of glucocorticoid-free cultures. The level of 35SO4(2-) incorporated into small ubiquitous proteoglycans was only 4-fold higher than that of glucocorticoid-free cultures. On the other hand, the level of [3H]glucosamine incorporated into hyaluronate in glucocorticoid-supplemented cultures was 4.5-fold lower than that of glucocorticoid-free cultures. Within 24 h of their addition to confluent cultures, hydrocortisone or dexamethasone markedly stimulated proteoglycan synthesis. This effect was not mimicked by androgens, estrogens, progesterone, or an inactive form of glucocorticoids such as deoxycorticosterone. This suggests that glucocorticoids have a direct and specific stimulatory effect on cartilage-specific proteoglycan synthesis and are essential for the maintenance of this synthesis in low density chondrocyte cultures.     

Production of cartilage-typic proteoglycans in cultures of chondrocytes from elastic cartilage

Chondrocytes from rabbit ear cartilage were isolated and cultured as monolayers in Ham's F-12 medium. The proteoglycans synthesized by short-term cultures formed a high proportion of aggregates and contained chrondroitin-4- and -6-sulfate in a 2:1 proportion. Dermatan sulfate was not present. The average molecular weight of the chondroitin sulfate was about 20,000. Keratan sulfate with an average molecular weight of about 6000 could be isolated from the proteoglycan monomers. Rabbit ear chondrocytes in culture thus produced proteoglycans comparable to those isolated from hyaline cartilage. Culture for longer periods and plating at lower density caused a decrease in the proportion of aggregated proteoglycans. Primary cultures continued to synthesize aggregated proteoglycans for at least 2 weeks, while subdivision of the cultures caused a shift toward the production of small-sized “ubiquitous proteoglycans.” The synthesis of proteoglycan aggregates could, however, be partly restored by transfer of the monolayer cells to a suspension culture.     

Differential effects of parathyroid hormone and somatomedin-like growth factors on the sizes of proteoglycan monomers and their synthesis in rabbit costal chondrocytes in culture

In the proteoglycans extracted from rabbit costal chondrocytes in culture, two populations of proteoglycans were distinguished by density gradient centrifugation under dissociative conditions. The major component was the faster sedimenting population (proteoglycan I), the putative 'cartilage-specific' proteoglycans, and the minor component was the slower sedimenting population (proteoglycan II). The monomeric size of proteoglycan I was closely related to the differentiation-state of chondrocytes and was a good marker of the differentiated chondrocytes. Treatment of the cultures with parathyroid hormone (PTH) induced an increase in the monomeric size of proteoglycan I. This increase was ascribed to an increase in the molecular size of the glycosaminoglycan chain in proteoglycan I. On the other hand, somatomedin-like growth factors, such as multiplication-stimulating activity (MSA) and cartilage-derived factor (CDF), did not affect the size of proteoglycan I, while they markedly stimulated the synthesis of proteoglycan I. In contrast, treatment with nonsomatomedin growth factors, such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), resulted in not only a decrease in glycosaminoglycan synthesis but also a slight decrease in size of proteoglycan I. However, synthesis and size of proteoglycan II were little affected by these agents. Thus, the present study clearly shows that PTH and somatomedin-like growth factors have differential functions in bringing about the expression of the differentiated phenotype of chondrocytes: PTH influences chain elongation and termination of glycosaminoglycans in proteoglycan I, while somatomedin-like growth factors affect primarily the synthesis and secretion of proteoglycan I.     

Modulation of keratan sulfate synthesis on lumican by the action of cytokines on human articular chondrocytes.

Adult human articular chondrocytes were used to investigate why keratan sulfate/polylactosamine chains are deficient on the lumican residing in the matrix of adult articular cartilage, whereas they are present on the lumican residing in the matrix of juvenile cartilage. Under serum-free conditions with either monolayer cultures, agarose cultures, or micromass cultures, the adult chondrocytes synthesized a form of lumican possessing keratan sulfate/polylactosamine chains. Thus, the adult chondrocytes are capable of producing a proteoglycan form of lumican and this appears to be the default synthesis preference. The micromass culture system proved useful for demonstrating that growth factors/cytokines present in the extracellular milieu are capable of influencing the structure of the keratan sulfate/polylactosamine chains on the secreted lumican. Of particular note was the ability of IL-1beta to promote the secretion of a form of lumican deficient in keratan sulfate/polylactosamine chains, whereas with bFGF, IGF-1 and TGFbeta keratan sulfate/polylactosamine chains were present, though their size or degree of substitution varied. Thus, growth factors/cytokines are able to modulate the molecular form of lumican. Furthermore, additional studies showed that this modulation was not due to the degradation of keratan sulfate/polylactosamine chains following proteoglycan secretion, but represented a direct effect on synthesis.     

Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and × collagen and matrix mineralization

In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type X collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hypertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentiation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or X collagen antibodies revealed that type X collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and X collagen, indicating further differentiation of chondrocytes to a new type of 'post-hypertrophic' cell. This cell type, descending from a type X collagen producing chondrocyte, is different from the previously described 'dedifferentiated' or 'modulated' types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type X collagen synthesis. First calcium deposits were detected by alizarine red S staining in type X collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type X collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis.     

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.     

Differing in vitro biology of equine,ovine, porcine and human articular chondrocytes derived from the knee joint: an immunomorphological study

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.     

Synthesis of small proteoglycans substituted with keratan sulfate by rabbit articular chondrocytes

35S-Labeled proteoglycans produced by chondrocytes from immature and mature rabbits were fractionated on associative CsCl gradients. In all cultures, greater than 85% of the incorporated radioactivity was present in the A1 fraction (rho 1.60) as chondroitin sulfate/keratin sulfate-substituted aggregating proteoglycan monomer; the remainder was present in small proteoglycans in the A2, A3, and A4 fractions of low buoyant densities (rho 1.53, 1.45, 1.37, respectively). Detailed glycosaminoglycan analysis of the A2, A3, and A4 fractions showed dermatan sulfate-rich species were present throughout. However, in both immature and mature cultures, 30-45% of the glycosaminoglycans in the A3/A4 combined fractions were present as keratan sulfate, as shown by insensitivity to digestion with chondroitinase ABC, specific digestion with endo-beta-galactosidase, and reactivity with antibody 5D4. Immature and mature chondrocytes synthesized very similar amounts of the low buoyant density keratan sulfate proteoglycan on a per cell basis. Moreover, 51 and 37% of the total keratan sulfate produced by immature and mature chondrocytes, respectively, were present in the low buoyant density proteoglycan. Pulse-chase experiments indicated that the low buoyant density keratan sulfate was not derived from the large aggregating proteoglycan by proteolysis in the extracellular space. The small keratan sulfate proteoglycans appear to be present as a species distinct from the small dermatan sulfate proteoglycans in these cultures in that they can be separated on Q-Sepharose chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent size (40-60 kDa), composition, and heterogeneity of the keratan sulfate proteoglycans suggest that they may be related to the small keratan sulfate proteoglycans of cornea.     

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.     

Effect of vanadate on cartilage-matrix proteoglycan synthesis in rabbit costal chondrocyte cultures

The effect of vanadate on proteoglycan synthesis by cultured rabbit costal chondrocytes was examined. Rabbit chondrocytes were seeded at low densities 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 4 microM vanadate to the culture medium induced a morphologic differentiation of the fibroblastic cells to spherical chondrocytes, and increased by two- to threefold incorporation of [35S]sulfate and [3H]glucosamine into large, chondroitin sulfate proteoglycans. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, in that chemical analyses showed increases in the accumulation of macromolecules containing hexuronic acid and hexosamine in vanadate-maintained cultures. However, vanadate had only a marginal effect on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant material. These results provide evidence that vanadate selectively stimulates the synthesis of proteoglycans characteristically found in cartilage by rabbit costal chondrocyte cultures.