Chondrocyte differentiation is modulated by frequency and duration of cyclic compressive loading.

As part of a program of research aimed at determining the role of mechanical forces in connective tissue differentiation, we have developed a model for investigating the effects of dynamic compressive loading on chondrocyte differentiation in vitro. In the current study, we examined the influence of cyclic compressive loading of chick limb bud mesenchymal cells to a constant peak stress of 9.25 kPa during each of the first 3 days in culture. Cells embedded in agarose gel were subjected to uniaxial, cyclic compression at 0.03, 0.15, or 0.33 Hz for 2 h. In addition, load durations of 12, 54, or 120 min were evaluated while holding frequency constant at 0.33 Hz. For a 2 h duration, there was no response to loading at 0.03 Hz. A significant increase in chondrocyte differentiation was associated with loading at 0.15 Hz, and an even greater increase with loading at 0.33 Hz. Holding frequency constant at 0.33 Hz, a loading duration of 12 min elicited no response, whereas chondrocyte differentiation was enhanced by loading for either 54 or 120 min. Although not statistically significant from the 120 min response, average cartilage nodule density and glycosaminoglycan synthesis rate were highest in the 54 min duration group. This result suggests that cells may be sensitive to the level of cumulative (nonrecoverable) compressive strain, as well as to the dynamic strain history.

Proteoglycans and glycosaminoglycan fine structure in the mouse tail tendon fascicle.

The isolated mouse tail tendon fascicle, a functional and homogenous volume of tendon extracellular matrix, was utilized as an experimental system to examine the structure function relationships in tendon. Our previous work using this model system demonstrated relationships between mean collagen fibril diameter and fascicle mechanical properties in isolated tail tendon fascicles from three different groups of mice (3-week and 8-week control and 8-week Mov13 transgenic) K.A. Derwin, L.J. Soslowsky, J. Biomech. Eng. 121 (1999) 598-604. These groups of mice were chosen to obtain tendon tissues with varying collagen fibril structure and/or biochemistry, such that relationships with material properties could be investigated. To further investigate the molecular details of matrix composition and organization underlying tendon function, we report now on the preparation, characterization, and quantitation of fascicle PGs (proteoglycans) from these three groups. The chondroitin sulfate/dermatan sulfate (CS/DS)-substituted PGs, biglycan and decorin, which are the abundant proteoglycans of whole tendons, were also shown to be the predominant PGs in isolated fascicles. Furthermore, similar to the postnatal maturation changes in matrix composition previously reported for whole tendons, isolated fascicles from 8-week mice had lower CS/DS PG contents (both decorin and biglycan) and a higher collagen content than 3-week mice. In addition, CS/DS chains substituted on PGs from 8-week fascicles were shorter (based on a number average) and richer in disulfated disaccharide residues than chains from 3-week mice. Fascicles from 8-week Mov13 transgenic mice were found to contain similar amounts of total collagen and total CS/DS PG as age-matched controls, and CS/DS chain lengths and sulfation also appeared normal. However, both decorin and biglycan in Mov13 tissue migrated slightly faster on sodium dodecyl sulfate polyacrylamide gel electorphoresis (SDS-PAGE) than the corresponding species from 8-week control, and biglycan from the 8-week Mov 13 fascicles appeared to migrate as a more polydisperse band, suggesting the presence of a unique PG population in the transgenic tissue. These observations, together with our biomechanical data [Derwin and Soslowsky, 1999] suggest that compensatory pathways of extracellular matrix assembly and maturation may exist, and that tissue mechanical properties may not be simply determined by the contents of individual matrix components or collagen fibril size.

Effect of compressive loading on chondrocyte differentiation in agarose cultures of chick limb-bud cells.

It is well established that mechanical loading is important to homeostasis of cartilage tissue, and growing evidence suggests that it influences cartilage differentiation as well. Whereas the effect of mechanical forces on chondrocyte biosynthesis and gene expression has been vigorously investigated, the effect of the mechanical environment on chondrocyte differentiation has received little attention. The long-term objective of this research is to investigate the regulatory role of mechanical loading in cell differentiation. The goal of this study was to determine if mechanical compression could modulate chondrocyte differentiation in vitro. Stage 23/24 chick limb-bud cells, embedded in agarose gel, were subjected to either static (constant 4.5-kPa stress) or cyclic (9.0-kPa peak stress at 0.33 Hz) loading in unconfined compression during the initial phase of commitment to a phenotypic lineage. Compared with nonloaded controls, cyclic compressive loading roughly doubled the number of cartilage nodules and the amount of sulfate incorporation on day 8, whereas static compression had little effect on these two measures. Neither compression protocol significantly affected overall cell viability or the proliferation of cells within nodules. Since limb-bud mesenchymal cells were seeded directly into agarose, an assessment of cartilage nodules in the agarose reflects the proportion of the original cells that had given rise to chondrocytes. Thus, the results indicate that about twice as many mesenchymal cells were induced to enter the chondrogenic pathway by cyclic mechanical compression. The coincidence of the increase in sulfate incorporation and nodule density indicates that the primary effect of mechanical compression on mesenchymal cells was on cellular differentiation and not on their subsequent metabolism. Further studies are needed to identify the primary chondrogenic signal associated with cyclic compressive loading and to determine the mechanism by which it influences commitment to or progression through the chondrogenic lineage, or both.

NACOB presentation Keynote lecture. Cancellous bone biomechanics. North American Congress on Biomechanics.

Cancellous bone is both a biological and a mechanical structure. The interaction between these two aspects of cancellous bone is sufficiently strong that understanding the mechanical properties of the tissue is not possible without consideration of the biology. This manuscript is a mathematical expansion of a portion of the first author's Keynote lecture at the 1998 NACOB presentation. The cellular activity of cancellous bone proceeds in part by the transport of metabolites between trabecular hard tissue and marrow. The anatomical observation is that human trabeculae are seldom internally served by a blood supply, suggesting that the transport mechanisms for trabecular survival are diffusion and a collection of mechanisms for active transport of metabolites independent of blood flow. It will be demonstrated that metabolite transport by diffusion can explain two notable empirical relationships for bone: (a) the close relationship between the bone surface and the bone volume, and (b) the exponential decline in the bone volume fraction during periods of mechanical disuse. A mathematical model is also developed showing how mechanical loading can effect bone volume fraction by increasing metabolite transport between the tissue compartments.

Purification and characterization of chondroitin 4-sulfotransferase from the culture medium of a rat chondrosarcoma cell line.

Chondroitin 4-sulfotransferase, which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.

Modular arrangement of cartilage- and neural tissue-specific cis-elements in the mouse alpha2(XI) collagen promoter.

Type XI collagen, a heterotrimer specific to cartilage matrix, plays an important role in cartilage morphogenesis. We analyzed various alpha2(XI) collagen promoter-lacZ reporter gene constructs in transgenic mice to understand tissue-specific transcriptional regulation. The -530 promoter sequence was sufficient to direct reporter gene expression specifically to cartilage. Further deletion to -500 abolished reporter gene expression in cartilage but activated the expression specific to neural tissues such as brain and neural tube. An additional 47-base pair deletion resulted in random tissue expression patterns. A 24-base pair sequence from -530 to -507 of the alpha2(XI) promoter was able to switch the activity of the heterologous neurofilament light gene promoter from neural tissues to cartilage. These results suggest that the alpha2(XI) collagen gene is regulated by at least three modular elements: a basal promoter sequence distal to -453, a neural tissue-specific element (-454 to -500), and a cartilage-specific element (-501 to -530), which inhibits expression in neural tissues and induces expression in cartilage.

Chondrocyte and chondrosarcoma cell integrins with affinity for collagen type II and their response to mechanical stress.

Mechanical stress is an important regulator of chondrocyte functions but the mechanisms by which chondrocytes sense mechanical signals are unknown. Receptors for matrix molecules are likely involved in the mechanical signaling. In the first part of this study we identified integrins with affinity for the cartilage-specific collagen type II. We report that the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 isolated from bovine chondrocytes or human chondrosarcoma cells bound collagen type II as judged from affinity chromatography. The integrins alpha 3 beta 1 or alpha 9 beta 1 did not bind collagen type II-Sepharose. In the second part of the study we investigated the effect of mechanical stress on expression of matrix molecules and integrin subunits. Chondrocytes and chondrosarcoma cells, cultured on uncoated flexible silicone membranes in the presence of serum, were exposed to mechanical stress by the Flexercell system. Dynamic stimulation of chondrocytes for 3 h increased the mRNA expression of collagen type II and aggrecan as judged by Northern blotting, while the beta 1-integrin subunit was not changed. When chondrosarcoma cells were exposed to mechanical stimulation under the same conditions, mRNA expression of alpha 5 was found to increase while beta 1, alpha 2, and alpha v did not increase to significant levels. In another study the effect of mechanical stress on integrins was investigated when the cells were cultured on collagen type II-coated flex-dishes. Three hours of dynamic stress increased the mRNA expression of alpha 2-integrin subunit while the level of mRNA for integrin subunits beta 1, alpha 1, alpha 5, and alpha v showed no or small changes, indicating that matrix components may modulate the expression of integrins during mechanical stress.

Use of a new rat chondrosarcoma cell line to delineate a 119-base pair chondrocyte-specific enhancer element and to define active promoter segments in the mouse pro-alpha 1(II) collagen gene.

We show that a new rat chondrosarcoma (RCS) cell line established in long-term culture from the Swarm tumor displayed a stable differentiated chondrocyte-like phenotype. Indeed, these cells produced the collagen types II, IX, and XI and alcian blue-stainable cartilage-specific proteoglycans, but no type I or type III collagen. To functionally characterize their chondrocytic nature, the cells were stably transfected with a type II collagen/beta geo chimeric gene which confers essentially perfect chondrocyte-specific expression in transgenic mice. RCS cells expressed both beta-galactosidase and G418 resistance, in comparison with similarly transfected 10T1/2 and NIH/3T3 fibroblasts which did not. These cells were then used to perform a systematic deletion analysis of the first intron of the mouse type II collagen gene (Col2a1) using transient expression experiments to determine which segments stimulated expression of a luciferase reporter gene in RCS cells but not in 10T1/2 fibroblasts. Cloning of two tandem copies of a 156-base pair (bp) intron 1 fragment (+2188 to +2343) in a construction containing a 314-bp Col2a1 promoter caused an almost 200-fold increase in promoter activity in RCS cells but no increase in 10T1/2 cells. DNase I footprint analysis over this 156-bp fragment revealed two adjacent protected regions, FP1 and FP2, located in the 3'-half of this segment, but no differences were seen with nuclear extracts of RCS cells and 10T1/2 fibroblasts. Deletion of FP2 to leave a 119-bp segment decreased enhancer activity by severalfold, but RCS cell specificity was maintained. Further deletions indicated that sequences both in the 5' part of the 119-bp fragment and in FP1 were needed simultaneously for RCS cell-specific enhancer activity. A series of deletions in the promoter region of the mouse Col2a1 gene progressively reduced activity when these promoters were tested by themselves in transient expression experiments. However, these promoter deletions were all activated to a similar level in RCS cells by a 231-bp intron 1 fragment that included the 156-bp enhancer. The RCS cell-specific activity persisted even if the Col2a1 promoter was replaced by a minimal adenovirus major late promoter. This 231-bp intron 1 fragment also had strong enhancing activity in transiently transfected mouse primary chondrocytes. Our experiments establish the usefulness of RCS cells as an experimental system for studies of the control of chondrocyte-specific genes, provide an extensive delineation of segments in the Col2a1 first intron involved in chondrocyte-specific activity, and show that promoter sequences are dispensable for chondrocyte specificity.

Endochondral resorption of chick sterna in culture.

Endochondral resorption is most clearly recognized at the metaphyseal interface of the growth plate with the adjacent vasculature; however, apparently identical processes of endochondral resorption are seen in sites of primary and secondary ossification of the cartilaginous anlage of bones and in ossifying fracture callus. Recent evidence of the expression of the hypertrophic phenotype in osteoarthrotic articular cartilage suggests that endochondral resorption also may be a factor in the loss of articular cartilage in this condition. To investigate the mechanism of endochondral resorption, a model culture system was developed and characterized. The two primary centers of ossification with surrounding cartilage were dissected from embryonic chick sterna prior to (18-day-old embryos) or after (20-day-old embryos) the initiation of resorption. They were cultured either in plastic culture dishes or on chorioallantoic membranes, and resorption was detected by analysis of the loss of types II and X collagen and by histological characterization. Only sterna showing active resorption in vivo were resorbed when cultured on chorioallantoic membrane. The histological appearance of the resorption site and the specificity of resorption to the primary ossification center, seen from both the analysis of loss of collagen and histological observation, suggested that the resorption of sterna cultured on chorioallantoic membrane was similar to that observed in vivo. These studies further indicated that both vascular cells and viable chondrocytes were required for resorption. Susceptibility to resorption could be induced in resistant primary ossification centers by prior culture in the absence of vascular cells, and it is suggested that it results from the accumulation of resorption-susceptible cells and matrix as a result of continued chondrocyte development.

Cell-mediated catabolism of aggrecan. Evidence that cleavage at the "aggrecanase" site (Glu373-Ala374) is a primary event in proteolysis of the interglobular domain.

A rat chondrosarcoma cell line and primary bovine chondrocytes have been used to study cell-mediated aggrecan catabolism. Addition of 1 microM retinoic acid to chondrosarcoma cultures resulted in aggrecan proteolysis with the release of greater than 90% of the cell layer aggrecan into the medium within 4 days. NH2-terminal sequencing of chondroitin sulfate-substituted catabolic products gave a single major NH2-terminal sequence of ARGNVILTXK, initiating at Ala374. This showed that the proteinase, commonly referred to as "aggrecanase," which cleaves the Glu373-Ala374 bond of the interglobular domain of aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1990) J. Biol. Chem. 266, 8683-8685), is active in this cell system. Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also liberated during catabolism and this was characterized with three antipeptide antisera. Anti-CDAGWL was used as a general probe for G1 domain. Anti-FVDIPEN was used to specifically detect G1 domain with COOH terminus of Asn341, the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE antiserum was used to specifically detect G1 domain with COOH terminus of Gln373, the form which is the expected product of "aggrecanase"-mediated cleavage of aggrecan. Western blot analysis indicated that a single form of G1 domain of about 60 kDa was formed. G1 domain of this size reacted with both anti-CDAGWL and anti-NITEGE but not with anti-FVDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two forms of catabolic G1 domain, of about 62 and 66 kDa, were formed. Both of these forms reacted on Western blots with anti-CDAGWL and also with anti-NITEGE. It is suggested that cell-mediated catabolism of the aggrecan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu373-Ala374 bond by aggrecanase. The accumulation of G1 domain with a COOH-terminal of Glu373 shows that such aggrecanase-mediated cleavage can occur independent of the cleavage of the Asn341-Phe342 bond by matrix metalloproteinases.

Chondrocyte cells respond mechanically to compressive loads.

Many studies have illustrated the effect of mechanical loading on articular cartilage and the corresponding changes in chondrocyte metabolism, yet the mechanism through which the cells respond to loading still is unclear. The purpose of this study was to evaluate the change in shape of chondrocytes under a statically applied uniaxial compressive load. Isolated chondrocytes from rat chondrosarcoma were embedded in 2% agarose gel. Strains of 5, 10, and 15% were applied, and images of the cell were recorded from initial loading to equilibrium (15 minutes). A finite-element model was used to model the experimental setup and to estimate the mechanical properties of the chondrocyte at equilibrium. The transient behavior of the composite in the experiment was analyzed with use of a standard linear viscoelastic model. We found that all cells decreased in cross-sectional area under each of the applied compressive strains. In the finite-element model, the elasticity of the chondrocyte was similar to that of the surrounding agarose gel (4.0 kPa) and had a Poisson's ratio of 0.4. Viscoelastic analysis showed that the chondrocytes contributed a significant viscoelastic component to the behavior of the composite in comparison with the agarose gel alone. If a decrease in cell volume proportional to the decrease in cross-sectional area is assumed, the decrease observed was greater than would be predicted by a passive cellular response due to an equivalent osmotic pressure. This indicates that the chondrocyte may be altering its intracellular composition by cellular processes in response to mechanical loading.

Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix.

The ability of chondrocytes from calf articular cartilage to synthesize and assemble a mechanically functional cartilage-like extracellular matrix was quantified in high cell density (approximately 10(7) cells/ml) agarose gel culture. The time evolution of chondrocyte proliferation, proteoglycan synthesis and loss to the media, and total deposition of glycosaminoglycan (GAG)-containing matrix within agarose gels was characterized during 10 weeks in culture. To assess whether the matrix deposited within the agarose gel was mechanically and electromechanically functional, we measured in parallel cultures the time evolution of dynamic mechanical stiffness and oscillatory streaming potential in uniaxial confined compression, and determined the intrinsic equilibrium modulus, hydraulic permeability, and electrokinetic coupling coefficient of the developing cultures. Biosynthetic rates were initially high, but by 1 month had fallen to a level similar to that found in the parent calf articular cartilage from which the cells were extracted. The majority of the newly synthesized proteoglycans remained in the gel. Histological sections showed matrix rich in proteoglycans and collagen fibrils developing around individual cells. The equilibrium modulus, dynamic stiffness, and oscillatory streaming potential rose to many times (>5x) their initial values at the start of the culture; the hydraulic permeability decreased to a fraction (approximately 1/10) that of the cell-laden porous agarose at the beginning of the culture. By day 35 of culture, DNA concentration (cell density), GAG concentration, stiffness, and streaming potential were all approximately 25% that of calf articular cartilage. The frequency dependence of the dynamic stiffness and potential was similar to that of calf articular cartilage. Together, these results suggested the formation of a mechanically functional matrix.

Heterogeneity of keratan sulfate substituted on human chondrocytic large proteoglycans.

Newly synthesized 35S-labeled chondrocytic keratan sulfate chains were generated by chondrocytes of human chondrosarcoma cell line 105KC and were analyzed for heterogeneity of regional substitution, hydrodynamic size, and charge density. After isolation of the high density large chondrocytic proteoglycans and sequential digestions with chondroitinase ABC, L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin, and alpha-chymotrypsin, followed by Superose 6 chromatography, two populations of keratan sulfate-containing proteoglycan fragments were identified and pooled separately. Keratan sulfate chains from each of the regions were compared after release by Pronase digestion, and differences in substitution patterns were observed; keratan sulfate chains of greater polydispersity, as well as a population of larger hydrodynamic size, were present in only one of the two regions. Alkaline/borohydride treatment confirmed both the existence of a population of uniquely large keratan sulfate chains and its restriction to a single region of proteoglycan fragments. In addition to heterogeneity of hydrodynamic size, the keratan sulfate chains exhibited regional heterogeneity of charge density and hence, of sulfation patterns. Analysis by Mono Q chromatography identified distinct groups of keratan sulfate that segregated by charge density and whose proportionate composition differed between the proteoglycan regions. Furthermore, the most highly charged species were unique to a single region and encompassed the chains of larger hydrodynamic size. This suggests that there may be regional heterogeneity of keratan sulfate chains substituted along a single class of proteoglycans and identifies a novel population of large, highly sulfated chondrocytic keratan sulfate chains.

Synthesis of chondrocytic keratan sulphate-containing proteoglycans by human chondrosarcoma cells in long-term cell culture.

Keratan sulphate is an integral component of the large aggregating proteoglycans of mature human articular cartilage. The keratan sulphate content of chondrocytic proteoglycans increases during maturation, and it is a useful marker of mature-type chondrocytic proteoglycans. Ordinarily, in cell culture, chondrocytes from non-neoplastic tissues dedifferentiate, diminish or cease to synthesize aggregating proteoglycans with the same amount of keratan sulphate as those formed in vivo, and do not maintain their in vivo phenotype. In tissue culture, this down-regulation of synthesis of keratan sulphate is irreversible. The study of the metabolism of mature human chondrocytes has been hampered by the absence of stable models. We report a cell-line, 105KC, derived from a human chondrosarcoma, that has maintained a stable proteoglycan phenotype during more than three years of culture. Analysis with immunofluorescence suggested that 105KC cells continued to synthesize keratan sulphate in long-term culture. Biochemical analysis demonstrated that 105KC cells maintained the production of chondrocytic large-aggregating proteoglycans and that keratan sulphate composed 13 per cent of their glycosaminoglycan content. To our knowledge, 105KC represents the first model to have maintained the post-fetal chondrocytic proteoglycan phenotype in stable culture. This study documents the feasibility of the development of mature chondrocytic cell-lines and sheds light on the biological characteristics of chondrosarcomas.

The effects of long term monolayer culture on the proteoglycan phenotype of a clonal population of mature human malignant chondrocytes.

Articular cartilage chondrocytes maintain biosynthetic heterogeneity in cell culture, but undergo irreversible dedifferentiation of their proteoglycan phenotype, as defined by keratan sulfate content. A recently described cell line of malignant human chondrocytes, 105KC, was the first to maintain a differentiated keratan sulfate-proteoglycan phenotype in long-term culture. A clone of 105KC, labeled KC2H3, is currently described and represents a distinct and metabolically more homogeneous population of mature chondrocytes than 105KC. KC2H3 cells universally express keratan sulfate biosynthesis, as defined by indirect immunofluorescence. In addition, KC2H3 expresses a more mature proteoglycan phenotype than 105KC, as demonstrated by the keratan sulfate content: 24% of glycosaminoglycan content of the aggregating proteoglycans of KC2H3 versus 13% for 105KC. Further reported are the effects of long term monolayer culture on the proteoglycan phenotype expressed by KC2H3. After more than 16 months in continuous monolayer, KC2H3 cells remained morphologically indistinguishable from those maintained in suspension alternating with monolayer. In addition, the proteoglycan phenotype remained mature, without a tendency towards dedifferentiation. The flattened morphology adopted by chondrocytes while in monolayer has been considered a stimulus of dedifferentiation; the present study is the first to examine the direct effects of physical state on a homogeneous and stable population of chondrocytes.

Biochemical characterization of long-term culture of the Swarm rat chondrosarcoma chondrocytes in agarose.

Difficulty in maintaining phenotypic stability of the Swarm rat chondrosarcoma in long-term monolayer cultures has prompted investigation of alternative conditions that would enable extended maintenance of these cells, permitting use of the tumor as a model system for the long-term study of proteoglycan metabolism. Morphological analysis of the growth of the chondrosarcoma chondrocytes in agarose has shown stability of the culture over a 20 day period with respect to the ability of the cells to proliferate and synthesize an Alcian blue-positive extracellular matrix. The present study confirms these findings through analysis of the growth characteristics of the culture and the pattern of proteoglycan and collagen synthesis. The chondrocytes actively synthesize a proteoglycan-rich matrix at a rate dependent on the initial plating density and concentration of serum in the culture medium. These factors similarly affect the proliferative capabilities of the culture as demonstrated by the growth curves obtained at different culture conditions. During 20 days in culture, the cells synthesize an aggregating chondroitin sulfate proteoglycan and collagen type II, typical of cartilage and this chondrosarcoma. In addition, small molecular weight proteoglycans were found to be present at concentrations of up to 10% of the total proteoglycan population. Degradative rates are slow, the proteoglycan half-life is about 30 days, but can be enhanced with retinol, reducing the half-life to 2 days.

Xylosyl transfer to the core protein precursor of the rat chondrosarcoma proteoglycan.

Rat chondrosarcoma chondrocytes were labeled with [3H]serine or [3H]mannose as a precursor. Intracellular proteoglycan core protein precursor was purified from cell lysates by immunoprecipitation with polyclonal antibodies against the hyaluronic acid-binding region, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core precursor was eluted from the gels and treated with alkaline borohydride in order to convert serine residues substituted with xylose or N-acetylgalactosamine to alanine (or with alkaline sulfite to convert them to cysteic acid). After acid hydrolysis, the proportions of labeled serine and alanine (or cysteic acid) were determined by high performance liquid chromatography, and the results were compared with those obtained for the completed proteoglycan molecules isolated from the same cultures. In the completed proteoglycans, about 55% of the serine residues were substituted with xylose or N-acetylgalactosamine, while the corresponding figure for the intracellular precursor molecules was less than 5%. These results indicate, in agreement with our previous kinetic data, that the major part of the xylosyl transfer to the chondrosarcoma proteoglycan core protein precursor must occur late in the processing sequence, i.e. after about 85% of its intracellular lifetime and no more than 7 min before the addition of the rest of the chondroitin sulfate chain. The ratio of [3H]mannose to [3H]fucose in the core precursor was about 19, while that for the complete proteoglycan was about 2. This indicates the presence of high mannose, N-linked oligosaccharides on the core protein precursor which are converted to the complex forms on the completed proteoglycan. These data provide further support that the core precursor resides mainly in the pre-Golgi compartment and that xylosylation occurs mainly in a Golgi compartment.

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.