Rat Chondrosarcoma Chondrocytes Research Articles

Simvastatin promotes restoration of chondrocyte morphology and phenotype

In this study we examined whether the action of simvastatin affects re-differentiation of passaged chondrocytes and if so, whether this was mediated via changes in cholesterol or cholesterol intermediates. Bovine articular chondrocytes, of varying passage number, human knee chondrocytes and rat chondrosarcoma chondrocytes were treated with simvastatin and examined for changes in mRNA and protein expression of markers of the chondrocyte phenotype as well as changes in cell shape, proliferation and proteoglycan production. In all three models, while still in monolayer culture, simvastatin treatment alone promoted changes in phenotype and morphology indicative of re-differentiation most prominent being an increase in SOX9 mRNA and protein expression. In passaged bovine chondrocytes, simvastatin stimulated the expression of SOX9, ACAN, BMP2 and inhibited the expression of COL1 and α-smooth muscle actin. Co-treatment of chondrocytes with simvastatin plus exogenous cholesterol—conditions that had previously reversed the inhibition on CD44 shedding, did not alter the effects of simvastatin on re-differentiation. However, the co-treatment of chondrocytes with simvastatin together with other pathway intermediates, mevalonate, geranylgeranylpyrophosphate and to a lesser extent, farnesylpyrophosphate, blocked the pro-differentiation effects of simvastatin. Treatment with simvastatin stimulated expression of SOX9 and COL2a and enhanced SOX9 protein in human OA chondrocytes. The co-treatment of OA chondrocytes with mevalonate or geranylgeranylpyrophosphate, but not cholesterol, blocked the simvastatin effects. These results lead us to conclude that the blocking of critical protein prenylation events is required for the positive effects of simvastatin on the re-differentiation of chondrocytes.

A Novel Role for the BMP Antagonist Noggin in Sensitizing Cells to Non-canonical Wnt-5a/Ror2/Disheveled Pathway Activation.

Mammalian limb development is driven by the integrative input from several signaling pathways; a failure to receive or a misinterpretation of these signals results in skeletal defects. The brachydactylies, a group of overlapping inherited human hand malformation syndromes, are mainly caused by mutations in BMP signaling pathway components. Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice revealed a widening of skeletal elements in compound but not in any of the single mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-receptor specific for Wnt-5a we speculated that this phenotype might be a result of deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal elements growth and patterning. We show that Noggin potentiates activation of the Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In summary, our data demonstrate genetic interaction between Noggin and Ror2 and show that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling, a feature that in cartilage may depend on the presence of active FGF signaling. These findings indicate an unappreciated function of Noggin that will help to understand BMP and Wnt/PCP signaling pathway interactions.

CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

Hyaluronan (HA) plays an essential role in cartilage where it functions to retain aggrecan. Previous studies have suggested that aggrecan is anchored indirectly to the plasma membrane of chondrocytes via its binding to cell-associated HA. However, reagents used to test these observations such as hyaluronidase and HA oligosaccharides are short term and may have side activities that complicate interpretation. Using the CRISPR/Cas9 gene editing approach, a model system was developed by generating HA-deficient chondrocyte cell lines. HA synthase-2 (Has2)-specific single guide RNA was introduced into two different variant lines of rat chondrosarcoma chondrocytes; knockout clones were isolated and characterized. Two other members of the HA synthase gene family were expressed at very low relative copy number but showed no compensatory response in the Has2 knockouts. Wild type chondrocytes of both variants exhibited large pericellular matrices or coats extending from the plasma membrane. Addition of purified aggrecan monomer expanded the size of these coats as the proteoglycan became retained within the pericellular matrix. Has2 knockout chondrocytes lost all capacity to assemble a particle-excluding pericellular matrix and more importantly, no matrices formed around the knockout cells following the addition of purified aggrecan. When grown as pellet cultures so as to generate a bioengineered neocartilage tissue, the Has2 knockout chondrocytes assumed a tightly-compacted morphology as compared to the wild type cells. When knockout chondrocytes were transduced with Adeno-ZsGreen1-mycHas2, the cell-associated pericellular matrices were restored including the capacity to bind and incorporate additional exogenous aggrecan into the matrix. These results suggest that HA is essential for aggrecan retention and maintaining cell separation during tissue formation.

A candidate enhancer element responsible for high-level expression of the aggrecan gene in chondrocytes

Aggrecan is the most abundant proteoglycan in cartilage. It contains a lot of negatively charged glycosaminoglycan chains along the core protein, providing a large osmotic swelling pressure within the cartilage. Therefore, the biomechanical properties of cartilage, such as its compressive load-bearing capacity, are highly dependent on the presence of abundant aggrecan in the cartilage matrix. To elucidate the transcriptional mechanism that leads to abundant expression of aggrecan by chondrocytes, we screened for enhancer elements in 130 kb of the aggrecan gene, Agc1, using a reporter assay system that we previously developed. The system is based on co-transfection of candidate enhancer elements and reporter constructs into Swarm rat chondrosarcoma chondrocytes that retain a high level of aggrecan expression. We found an element that might be involved in high-level expression of Agc1 gene in chondrocytes in vivo. The element is located 30 kb upstream of the Agc1 transcription start site.

A newly identified enhancer element responsible for type II collagen gene expression

Type II collagen is a major component of cartilage where it is present at a high concentration, which is essential for the functional maintenance of the tissue. Therefore, any fundamental understanding of the physiology of cartilage tissue must include an understanding of the mechanism that allows the high level of expression of type II collagen gene, Col2a1, by chondrocytes. To this end, we developed a new reporter assay system based on the co-transfection of candidate enhancer elements and reporter construct into Swarm rat chondrosarcoma chondrocytes that allowed their stable expression. Using this system, we screened more than 70 kb of the Col2a1 gene and found an enhancer domain that is responsible for the regulation of its expression level. The domain is localized in intron 7, and consists of an 800-bp region that contains within it a previously unidentified domain, ∼140 bp in size.

Altered Synthesis of Cartilage-Specific Proteoglycans by Mutant Human Cartilage Oligomeric Matrix Protein

BackgroundThe mechanism by which mutant cartilage oligomeric matrix protein (COMP) induces a pseudoachondroplasia phenotype remains unknown, and the reason why a mutation of a minor protein of the growth plate cartilage causes total disruption of endochondral bone formation has not yet been determined. The current study was performed to investigate the effects of mutated COMP on the synthesis of the cartilage-specific major matrix proteins of Swarm rat chondrosarcoma chondrocytes.MethodsThe Swarm rat chondrosarcoma chondrocytes transfected with a chimeric construct, which consisted of a mutant gene of human COMP and an amino acid FLAG tag sequence, were cultured in agarose gel. Formation of extracellular proteoglycan and type-II collagen by the cells was evaluated by immunohistochemical staining and measuring the 35S-sulfate incorporation.ResultsNo difference was observed for the detection of type-II collagen among the cell lines expressing mutant COMP and the control cell lines. Histochemical staining of sulfated proteoglycans with safranin-O showed that lesser amounts of proteoglycans were incorporated into the extracellular matrix of the chondrocytes transfected with the mutant gene. 35S-sulfate incorporation into the cell/matrix fractions demonstrated markedly lower radiolabel incorporation, as compared to that of the control cells.ConclusionsMutation of COMP has an important impact on the processing of proteoglycans, rather than type-II collagen, in the three-dimensional culture of Swarm rat chondrosarcoma chondrocytes.

Hyaluronan and hyaluronan-binding proteins in cartilaginous tissues.

Recent developments in the biology of hyaluronan in cartilage are reviewed. The homology between the hyaluronan-binding sites of cartilage proteoglycan and link protein is discussed. Previous reports indicate that an increased concentration of extracellular hyaluronan inhibits 35S-proteoglycan synthesis by several types of chondrocyte. We report data showing that this response varies in its reproducibility and sensitivity to low concentrations of hyaluronan in rat chondrosarcoma chondrocytes and pig laryngeal chondrocytes in suspension culture. Two newly recognized hyaluronan-binding proteins have been isolated from extracts of Swarm rat chondrosarcoma. The major protein has a molecular mass of 102 kDa and the less prominent protein a molecular mass of 91 kDa. The latter may be derived from the former. Neither protein cross-reacts with antisera against cartilage proteoglycan HABR (hyaluronan-binding region), link protein, hyaluronectin or type II collagen.

Fibroblast Growth Factors 1, 2, 17, and 19 Are the Predominant FGF Ligands Expressed in Human Fetal Growth Plate Cartilage

Fibroblast growth factors (FGF) regulate bone growth, but their expression in human cartilage is unclear. Here, we determined the expression of entire FGF family in human fetal growth plate cartilage. Using reverse transcriptase PCR, the transcripts for FGF1, 2, 5, 8-14, 16-19, and 21 were found. However, only FGF1, 2, 17, and 19 were detectable at the protein level. By immunohistochemistry, FGF17 and 19 were uniformly expressed within the growth plate. In contrast, FGF1 was found only in proliferating and hypertrophic chondrocytes whereas FGF2 localized predominantly to the resting and proliferating cartilage. In addition, only the 18 kD isoform of FGF2 was found in resting chondrocytes while proliferating chondrocytes also synthesized 22 kD and 24 kD FGF2, similar to in vitro cultivated chondrocytes. In cell growth experiments, FGF1, 2, and 17 but not FGF19 inhibited the proliferation of FGFR3-expressing rat chondrosarcoma chondrocytes (RCS) with relative potency FGF2 >> FGF1 = FGF17. We conclude that FGF1, 2, 17, and 19 are the predominant FGF ligands present in developing human cartilage that are, with the exception of FGF19, experimentally capable of inhibiting chondrocyte proliferation.

Bisindolylmaleimide I Suppresses Fibroblast Growth Factor-mediated Activation of Erk MAP Kinase in Chondrocytes by Preventing Shp2 Association with the Frs2 and Gab1 Adaptor Proteins

Fibroblast growth factors (FGFs) inhibit chondrocyte proliferation via the Erk MAP kinase pathway. Here, we explored the role of protein kinase C in FGF signaling in chondrocytes. Erk activity in FGF2-treated RCS (rat chondrosarcoma) chondrocytes or human primary chondrocytes was abolished by the protein kinase C inhibitor bisindolylmaleimide I (Bis I). Bis I inhibited FGF2-induced activation of MEK, Raf-1, and Ras members of Erk signaling module but not the FGF2-induced tyrosine phosphorylation of Frs2 or the kinase activity of FGFR3, demonstrating that it targets the Erk cascade immediately upstream of Ras. Indeed, Bis I abolished the FGF2-mediated association of Shp2 tyrosine phosphatase with Frs2 and Gab1 adaptor proteins necessary for proper Ras activation. We also determined which PKC isoform is involved in FGF2-mediated activation of Erk. When both conventional and novel PKCs expressed by RCS chondrocytes (PKCalpha, -gamma, -delta, and -epsilon) were down-regulated by phorbol ester, cells remained responsive to FGF2 with Erk activation, and this activation was sensitive to Bis I. Moreover, treatment with PKClambda/zeta pseudosubstrate lead to significant reduction of FGF2-mediated activation of Erk, suggesting involvement of an atypical PKC.

Interaction of fibroblast growth factor and C-natriuretic peptide signaling in regulation of chondrocyte proliferation and extracellular matrix homeostasis

Overexpression of C-natriuretic peptide (CNP) in cartilage partially rescues achondroplasia in the mouse. Here, we studied the interaction of fibroblast growth factor (FGF) and CNP signaling in chondrocytes. CNP antagonized FGF2-induced growth arrest of rat chondrosarcoma (RCS) chondrocytes by inhibition of the Erk mitogen activated protein kinase pathway. This effect of CNP was protein kinase G-dependent and was mimicked by the cGMP analog pCPT-cGMP. FGF2-mediated activation of both MEK and Raf-1 but not Ras or FRS2 was abolished by CNP demonstrating that CNP blocks the Erk pathway at the level of Raf-1. CNP also counteracted the FGF2-mediated degradation of RCS extracellular matrix. CNP partially antagonized FGF2-induced expression, release and activation of several matrix-remodeling molecules including matrix metalloproteinase 2 (MMP2), MMP3, MMP9, MMP10 and MMP13. In addition, CNP compensated for FGF2-mediated matrix loss by upregulation of matrix production independent of its interference with FGF signaling. We conclude that CNP utilizes both direct and indirect ways to counteract the effects of FGF signaling in a chondrocyte environment.

Gene Trap Screening for Cell Surface and Extracellular Matrix Molecules Produced by Chondrocytes

We have developed a simple and unique strategy for identifying cell surface and extracellular matrix molecules produced by chondrocytes. Our strategy comprises two methods, retrovirus-based signal sequence gene trapping and culturing of Swarm rat chondrosarcoma chondrocytes. After infection with a retrovirus vector and isolation of hygromycin-resistant clones, trapped genes could be easily identified by the 5' rapid amplification of cDNA ends (5' RACE) method. Furthermore, the treatment of isolated clones with gadolinium chloride enabled us to determine whether the trapped gene expression was dependent on the state of chondrogenic differentiation.

Activation of the ERK1/2 and p38 Mitogen-activated Protein Kinase Pathways Mediates Fibroblast Growth Factor-induced Growth Arrest of Chondrocytes

Fibroblast growth factors (FGFs) regulate long bone development by affecting the proliferation and differentiation of chondrocytes. FGF treatment inhibits the proliferation of chondrocytes both in vitro and in vivo, but the signaling pathways involved have not been clearly identified. In this report we show that both the MEK-ERK1/2 and p38 MAPK pathways, but not phospholipase C gamma or phosphatidylinositol 3-kinase, play a role in FGF-mediated growth arrest of chondrocytes. Chemical inhibitors of the MEK1/2 or the p38 MAPK pathways applied to rat chondrosarcoma (RCS) chondrocytes significantly prevented FGF-induced growth arrest. The retinoblastoma family members p107 and p130 were previously shown to be essential effectors of FGF-induced growth arrest in chondrocytes. The dephosphorylation of p107, one of the earliest events in RCS growth arrest, was significantly blocked by MEK1/2 inhibitors but not by the p38 MAPK inhibitors, whereas that of p130, which occurs later, was partially prevented both by the MEK and p38 inhibitors. Furthermore, by expressing the nerve growth factor (NGF) receptor, TrkA, and the epidermal growth factor (EGF) receptor, ErbB1, in RCS cells we show that NGF treatment of the transfected cells caused growth inhibition, whereas EGF did not. FGF- and NGF-induced growth inhibition is accompanied by a strong and sustained activation of ERK1/2 and p38 MAPK and a decrease of AKT phosphorylation, whereas EGF induces a much more transient activation of p38 and ERK1/2 and increases AKT phosphorylation. These results indicate that inhibition of chondrocyte proliferation by FGF requires both ERK1/2 and p38 MAPK signaling and also suggest that sustained activation of these pathways is required to achieve growth inhibition.

A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation.

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E–Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.

Differential Effects ofStaphylococcus aureusα-Hemolysin on the Synthesis of Hyaluronan and Chondroitin Sulfate by Rat Chondrosarcoma Chondrocytes

The plasma membranes of rat chondrosarcoma chondrocytes were permeabilized by treatment with α-hemolysin, the major toxin produced byStaphylococcus aureus,which forms small, stable, heptameric, transmembrane pores (1–2 nm in diameter) permitting influx/efflux of low-molecular-mass molecules (≤2000 Da). Treated chondrocytes were permeable to entry of trypan blue and exit of ATP. We describe the effects of α-hemolysin on the synthesis of hyaluronan (HA) and chondroitin sulfate (CS) by chondrocytes using the simple sugar [3H]glucosamine as a metabolic precursor. Chondrocytes permeabilized with α-hemolysin in serum-free media decreased intracellular ATP and synthesis of CS to ∼5% of control within 2–4 h, but synthesized HA (80% of control for 8 h; ∼65% of control at 24 h). Adding fresh medium (with or without serum) to permeabilized cells increased ATP significantly and increased HA synthesis to near initial control values. Under the same conditions, the recovery of CS synthesis approached initial levels in control but not permeabilized cells. Our model demonstrates that the biosynthesis of HA by these cellsin vitrois remarkably stable to cellular perturbations which drastically inhibit synthesis of CS on proteoglycans.

Ion exchange HPLC microanalysis of chondroitin sulfate: quantitative derivatization of chondroitin lyase digestion products with 2-aminopyridine.

Sulfated glycosaminoglycans such as chondroitin sulfate are composed of three structural domains, a linkage oligosaccharide, connecting the chain to the core protein, a variably sulfated disaccharide repeat structure within the chain and a nonreducing terminal, and these domains may confer specific functions on particular chain populations. We report here a new and highly sensitive method for the detection and quantitation of all nonreducing terminal residues and internal disaccharides obtained by chondroitinase ABC or ACII digestion of aggrecan chondroitin sulfate. The procedure involves a quantitative reductive animation of the reducing ends of sulfated mono- and disaccharide chondroitinase products with 2-aminopyridine and boranedimethylamine. All derivatized saccharides can be separated and quantitated by fluorescence in a single chromatographic step on an AS4A anion exchange column, eluted with a gradient (0-500 nM) of sodium trifluoroacetate. The reproducibility and stability of the derivatisation, together with the sensitivity of the chromatography system, allowed for routine quantitation in the range of 3-500 pmol of reducing group (corresponding to about 1.5-250 ng of disaccharide or 0.75-125 ng of monosaccharide). Moreover, the fluorescence yield (fluorescence area units per pmol of reducing group) was virtually identical for all saccharides analyzed. Application of this method to an analysis of aggrecan purified from calf epiphyseal cartilage and from rat chondrosarcoma chondrocyte cultures allowed a precise identification and quantitation of the internal disaccharides and the nonreducing terminal structures, together with an estimation of the number average molecular weight of CS chains in these aggrecan preparations.

Effects of brefeldin A on aggrecan core protein synthesis and maturation in rat chondrosarcoma cells.

In this paper, the effects of the fungal metabolite, brefeldin A, on the synthesis and maturation of aggrecan core protein precursor were studied in rat chondrosarcoma chondrocytes. The aggrecan core protein precursor was partially identified in total protein pools isolated from cell extracts based on its selective cleavage at a single site by the restriction protease factor Xa. During a 2-h labeling period with [3H]serine as precursor, brefeldin A inhibited the synthesis of mature aggrecan from its aggrecan core protein precursor consistent with an inhibition of chondroitin sulfate chain elongation and sulfation as described in the accompanying paper (Calabro, A., and Hascall, V. C. (1994) J. Biol Chem. 269, 22764-22770). This inhibition is presumably the result of the disruption of vesicular transport by brefeldin A, which isolates the aggrecan core protein precursor at the level of the trans-Golgi cisternae from the enzymes for chondroitin sulfate chain elongation and sulfation located in the trans-Golgi network. Brefeldin A also inhibited the exocytosis of all radiolabeled secretory proteins from the cell layer into the medium compartment, which is also consistent with the disruption of vesicular transport attributed to this metabolite. Although total protein synthesis was inhibited by 12% in the presence of brefeldin A, the aggrecan core protein precursor accumulated within the cell layer indicating that the inhibition of chondroitin sulfate synthesis by brefeldin A was not the result of a lack of aggrecan core protein precursor. When the brefeldin A block was removed and cultures chased in the presence of cycloheximide to prevent new protein synthesis, vesicular transport through the cell was re-established and chondroitin sulfate chains were added to a large proportion of the aggrecan core protein precursor that had accumulated during the brefeldin A block. These results suggest that the machinery for chondroitin sulfate synthesis and for protein exocytosis, that were disrupted by brefeldin A treatment, recover after removal of the brefeldin A, even in the presence of cycloheximide, and that the structures involved in these processes reassemble from previously existing proteins. Interestingly, two other proteins with the same relative abundance as the aggrecan core protein precursor were observed. An approximately 210-kDa protein with the characteristics of the fibronectin subunit, and an unidentified approximately 150-kDa protein which was efficiently cleaved by the protease Xa enzyme.

Insulin-like Growth Factor I Regulation of Swarm Rat Chondrosarcoma Chondrocytes in Culture

Insulin-like growth factor I (IGF-I) is anabolic for chondrocytes and is thought to be important in regulating such normal cartilaginous tissues as the epiphyseal growth plate. In the present studies, we have investigated the role of IGF-I in the regulation of neoplastic cartilage. Chondrocytes cultured from a transplantable rat chondrosarcoma were analyzed for responsiveness to IGF-I with respect to DNA and glycosaminoglycan synthesis as determined by labeling with radioactive thymidine and sulfate, respectively. Stimulation of [3H]thymidine and [35S]sulfate incorporation by IGF-I was two to four times that in serum-free controls, with half-maximal stimulation at 1 × 10-9M. The efficacy of IGF-I was approximately one-half of that of serum in stimulating [3H]thymidine incorporation and was comparable to that of serum for [35S]sulfate incorporation. When Swarm rat chondrosarcoma chondrocytes were cultured in the presence of IGF-I and exposed to graded concentrations of anti-IGF-I antibody, [3H]thymidine incorporation and [35S]sulfate incorporation were attenuated in a dose-dependent fashion to 29 and 25% of antibody-free controls, respectively. Nonspecific antibody not raised against IGF-I was not inhibitory. These observations suggest that the majority of IGF-I action on these cells is susceptible to immunoinhibition. To estimate the contribution of IGF-I to the regulation of these cells by serum, Swarm rat chondrosarcoma chondrocytes were cultured with graded concentrations of either calf serum or fetal calf serum in the presence of anti-IGF-I antibody, nonspecific antibody, or no other additives. Specific antibody attenuated the effect of calf serum on both [3H]thymidine and [35S]sulfate incorporation with overall inhibition of 52% (P < 0.01) and 48% (P < 0.001), respectively. Nonspecific antibody superimposed small, variably stimulatory or inhibitory effects on those of calf serum. When chondrosarcoma chondrocytes were incubated with fetal calf serum, anti-IGF-I antibody exerted a minimal inhibitory effect, reducing both [3H]thymidine and [35S]sulfate incorporation by less than 25%. The immunoinhibition of both pre- and postnatal serum could be overcome in a dose-dependent fashion by increasing serum concentrations. These results suggest that the factors influencing Swarm rat chondrosarcoma chondrocytes may be developmentally regulated and that the contribution of IGF-I to the action of serum increases between fetal and postnatal life. These data support the hypothesis that chondrosarcoma is a somatomedin-responsive neoplasm and suggest that this tumor may be susceptible to interventions directed toward mechanisms that block insulin-like growth factor action.

Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis.

Several studies have suggested that chondrocytes must have the capacity to internalize and degrade extracellular hyaluronan. In the present study we show direct evidence that hyaluronan is, in fact, endocytosed by chondrocytes and that the endocytosis is mediated via cell surface CD44/hyaluronan receptors. Cultures of bovine articular chondrocytes as well as rat chondrosarcoma chondrocytes were incubated with either fluorescein- or 3H-labeled hyaluronan. Intense binding and accumulation of labeled hyaluronan was visualized by fluorescence microscopy or bright-field/dark-field microscopy following autoradiography. Cell surface hyaluronan was removed with either trypsin or Streptomyces hyaluronidase in order to distinguish and quantify intracellular endocytosed hyaluronan. Labeled hyaluronan was visualized within small discrete intracellular vesicles distributed throughout the cytoplasm. Binding and endocytosis of fluorescein- or 3H-labeled hyaluronan was totally blocked by the addition of excess unlabeled hyaluronan or hyaluronan hexasaccharides, competitive inhibitors of hyaluronan/hyaluronan receptor interactions. Binding and endocytosis was also blocked by the addition of anti-CD44 monoclonal antibodies. Characterization of endocytosed 3H-labeled hyaluronan demonstrated that a significant portion of the hyaluronan was degraded by both the bovine articular and rat chondrosarcoma chondrocytes. Interestingly, a higher proportion of bound hyaluronan was internalized by the bovine chondrocytes. Therefore, hyaluronan receptor-mediated endocytosis and degradation of hyaluronan may provide a critical link to the maintenance and homeostasis of cartilage tissue.

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.

Intermediary metabolism in the Swarm rat chondrosarcoma chondrocyte

The rat chondrosarcoma chondrocyte has the dual capacity to metabolize glucose (mainly via glycolysis) and glutamine (via an oxidative pathway). Glutamine metabolism, unlike that of glucose, is unable to sustain intracellular ATP concentrations. Glutamine consumption by the chondrosarcoma chondrocyte, however, is significantly in excess of its utilization as an amide-group donor in hexosamine synthesis, implying a novel and major role in cell metabolism.