The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.     

The effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures

This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with [35S]sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of 35S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the 35S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with [35S]sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the 35S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible. Cartilage cultures maintained with retinoic acid for 1 day then switched to medium with 20% (v/v) fetal calf serum for the remainder of the culture period exhibited decreased rates of loss of 35S-labeled proteoglycans from the matrix and increased tissue hexuronate contents to levels near those observed in tissue maintained in medium with 20% (v/v) fetal calf serum throughout. Furthermore, following switching to 20% (v/v) fetal calf serum, the relative proportions of the 35S-labeled proteoglycan species remaining in the matrix of these cultures were similar to those of control cultures.     

Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage.

Proteoglycan aggregates from bovine articular cartilage have been visualized by electron microscopy of mixed proteoglycan-cytochrome c monolayers. The proteoglycan aggregates consist of proteoglycan subunits arising laterally at fairly regular intervals (20 to 30 nm) from the opposite sides of an elongated filamentous structure. The filamentous backbone in individual aggregates varies in length from 400 to 4000 nm. The individual proteoglycan subunits in the aggregate vary in length from 100 to 400 nm. However, there is no difference in the average size of the proteoglycan subunits associated with the largest or smallest aggregates. The sizes of the individual aggregates are determined mainly by the lengths of their filamentous backbones. The stoichiometry of binding of subunits to filament, calculated from the data reported here, is close to that for the binding of subunits to hyaluronic acid reported by others.     

Degradation of proteoglycan in articular cartilage

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

Inhibition of proteoglycan synthesis by hydrogen peroxide in cultured bovine articular cartilage

Oxygen-derived reactive species, generated enzymatically by the action of xanthine oxidase upon hypoxanthine, significantly inhibit proteoglycan synthesis by cultured bovine articular cartilage (Bates, E.J., Lowther, D.A. and Handley, C.J. (1984) Ann. Rheum. Dis. 43, 462-469). Here we extend these investigations and show, through the use of catalase and the specific iron chelator diethylenetriaminepentaacetic acid, that the active species involved is H2O2 and not the hydroxyl radical. Incubations of cartilage with H2O2 at concentrations of 1 X 10(-4) M and above are also inhibitory to proteoglycan synthesis. Subsequent recovery of the tissue is dependent upon the initial dose of xanthine oxidase or H2O2. Xanthine oxidase at 84 mU per incubation results in a prolonged inhibition of proteoglycan synthesis which is still apparent after 14 days in culture. Lower concentrations of xanthine oxidase (21-66 mU) are inhibitory to proteoglycan synthesis, but the tissue is able to synthesise proteoglycans at near normal rates after 3 days in culture. The inhibition of proteoglycan synthesis by 1 X 10(-4) M H2O2 is completely reversed after 5 days in culture, whereas 1 X 10(-3) M H2O2 results in a more prolonged inhibition. The synthesis of the proteoglycan core protein is inhibited, but the ability of the newly formed proteoglycans to aggregate with hyaluronic acid is unimpaired.     

The effects of trypsin treatment on proteoglycan biosynthesis by bovine articular cartilage.

The effects of mild or severe trypsin treatment of bovine articular-cartilage slices in tissue culture were studied by monitoring the incorporation of [35S]sulphate into proteoglycans. Moderate trypsin treatment caused a subsequent marked inhibition of proteoglycan biosynthesis, which was reversible with time. Analysis on Sepharose CL-2B of the proteoglycan species synthesized showed that, directly after trypsin treatment, there was a 30% increase in the synthesis of the low-Mr proteoglycan (Kav. 0.71), and the total decrease in proteoglycan biosynthesis was reflected in a decrease in the synthesis of the high-Mr proteoglycan species (Kav. 0.31). The small proteoglycan was partially characterized and shown to be a true biosynthetic product and not a breakdown product. Trypsin treatment (20 micrograms/ml per 100 mg of tissue) of cartilage slices also resulted in an increase in the glycosaminoglycan chain size of the large proteoglycan, but not of the small proteoglycan.     

Degradation of proteoglycan in articular cartilage.

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

Studies on the interaction of newly secreted proteoglycan subunits with hyaluronate in human articular cartilage

Newly secreted proteoglycans from adult human cartilage do not interact well with hyaluronate, but attain this ability with time in the extracellular matrix. The conversion process occurs in all types of cartilagenous matrix, as newborn cartilage cultures, chondrosarcoma cultures and adult chondrocyte cultures each secreted proteoglycan subunits which exhibited the delayed aggregation phenomenon. However, the rate of conversion is probably dependent upon the structure of the surrounding matrix and the cell type. In vitro, link protein appears to enhance an initial change in the hyaluronate-binding region of the newly secreted proteoglycan subunits to allows stronger interaction with hyaluronate. In a second step, which is pH- and temperature-dependent, the change becomes irreversible. Thus, in addition to its role in stabilizing the interaction of mature proteoglycan subunits with hyaluronate, link protein may also aid in promoting the conversion of the newly synthesized proteoglycan subunit to a form that is capable of strong interaction with hyaluronate.     

Force-mediated dissociation of proteoglycan aggregate in articular cartilage

Proteoglycan aggregate is the primary component in articular cartilage responsible for resisting compressive loading. It consists of a core molecule of hyaluronan and a number of side chains of aggrecan bound to hyaluronan non-covalently. The loss of aggrecan from articular cartilage is considered to be a major factor in the development of osteoarthritis. Though enzymatic digestion of aggrecan is believed to be responsible for the release of aggrecan from osteoarthritic cartilage, other mechanisms, such as direct force-mediated detachment of aggrecan from hyaluronan may also be involved. In this study, the rupture force of the single bond between hyaluronan and aggrecan in articular cartilage was directly quantified using experimental measurement and Monte Carlo simulation. Low rupture force of this bond, as determined in this study suggested a possible direct force-mediated detachment of aggrecan from proteoglycan aggregate in osteoarthritic cartilage.     

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

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

Control of proteoglycan biosynthesis. Further studies on the effect of serum on cultured bovine articular cartilage.

Proteoglycan synthesis in explant cultures of adult bovine articular cartilage is stimulated in a dose-dependent manner when the tissue is cultured in the presence of foetal-calf serum. The stimulation of proteoglycan synthesis is paralleled by a similar increase in DNA synthesis; however, when DNA synthesis is inhibited by hydroxyurea the stimulation of proteoglycan synthesis by serum remains essentially the same. The apparent half-life of the pool of proteoglycan core protein precursor was measured in freshly isolated tissue as well as in tissue cultured for 7 days in the presence and in the absence of foetal-calf serum; under all conditions the half-life was the same, suggesting that this value is independent of the net rate of proteoglycan synthesis. In the presence of actinomycin D, an inhibitor of RNA synthesis, there was a difference in the apparent half-life of the available pool of mRNA coding for proteoglycan core protein: 8.5 h for tissue maintained in the presence of serum and 3.8 h for tissue cultured in the absence of serum. It is suggested that proteoglycan synthesis is stimulated by serum factors at the level of DNA-dependent RNA synthesis. Concomitant with an increase in the rate of proteoglycan synthesis induced by the presence of serum in the culture medium, an increase in the concentrations of several glycosyltransferases involved in chondroitin sulphate synthesis was also observed.     

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.     

Effects of copper and zinc on proteoglycan metabolism in articular cartilage

Co-Cultures of porcine articular cartilage and synovium or synovial conditioned medium were used as an in vitro model to mimic inflammatory events at the cartilage/synovial junction in degenerative joint disease. This model provides a useful tool to assess the anti-inflammatory and antiarthritic properties of pharmacological agents. In this study the effects of copper and zinc on (i) PG synthesis by cartilage and (ii) synovial-induced PG depletion have been investigated. Copper sulphate at a concentration of 0.01 mM did not stimulate PG synthesis significantly in cultured cartilage explants but completely abrogated the inhibitory effects of synovial tissue in co-culture experiments. This finding was supported by the histological demonstration of copper-dependent reversal of the PG depletion in cartilage exposed to synovial conditioned medium. Zinc sulphate at 0.01 mM had no effect on PG synthesis and was unable to protect cartilage against synovialinduced PG depletion. These results reveal possible mechanisms by which copper exerts its anti-inflammatory and anti-arthritic actions.     

Frictional response of bovine articular cartilage under creep loading following proteoglycan digestion with chondroitinase ABC

The specific aim of this study was to investigate the effect of chondroitinase ABC treatment on the frictional response of bovine articular cartilage against glass, under creep loading. The hypothesis is that chondroitinase ABC treatment increases the friction coefficient of bovine articular cartilage under creep. Articular cartilage samples (n = 12) harvested from two bovine knee joints (1-3 months old) were divided into a control group (intact specimens) and a treated group (chondroitinase ABC digestion), and tested in unconfined compression with simultaneous continuous sliding (+/- 4 mm at 1 mm/s) under a constant applied stress of 0.5 MPa, for 2500 s. The time-dependent response of the friction coefficient was measured. With increasing duration of loading, treated samples exhibited a significantly higher friction coefficient than control samples as assessed by the equilibrium value (treated: micro(eq) = 0.19 +/- 0.02; control: micro(eq) = 0.12 +/- 0.03; p = 0.002), though the coefficient achieved immediately upon loading did not increase significantly (treated: micro(min) = 0.0053 +/- 0.0025; control: micro(min) = 0.037 +/- 0.0013; p = 0.19). Our results demonstrate that removal of the cartilage glycosaminoglycans using chondroitinase ABC significantly increases the overall time-dependent friction coefficient of articular cartilage. These findings strengthen the motivation for developing chondroprotective strategies by increasing cartilage chondroitin sulfate content in osteoarthritic joints.     

Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage.

The addition of foetal calf serum to explant cultures of adult bovine articular cartilage is known to stimulate proteoglycan synthesis in a dose-dependent manner. We have now shown the activity in serum responsible for this effect to be heat- and acid-stable, to be associated with a high-Mr complex in normal serum but converted to a low-Mr form under acid conditions. The activity has an apparent Mr approximately 10,000 and isoelectric points similar to those reported for insulin-like growth factors (IGFs). Addition of a monoclonal antibody against insulin-like growth factor-I (IGF-I) prevented foetal calf serum from stimulating proteoglycan synthesis. Physiological concentrations of recombinant IGF-I or pharmacological levels of insulin when added to cartilage cultures mimicked the proteoglycan-stimulatory activity of serum. IGF-I appeared to act by increasing the rate of proteoglycan synthesis and did not change the nature of the proteoglycan synthesized nor the rate of proteoglycan catabolism by the tissue, suggesting that IGF-I may be important in the regulation of proteoglycan metabolism in adult articular cartilage. Furthermore, IGF-I can replace foetal calf serum in the culture medium, thereby allowing the use of a fully-defined medium which will maintain the synthesis and tissue levels of proteoglycan in adult articular cartilage explants for up to 5 days.     

Age-related effects of TGF-beta on proteoglycan synthesis in equine articular cartilage

The synthesis of proteoglycans was measured in normal equine articular cartilage of ages 9 months to 20 years and the effect of TGF-beta1 on this activity was investigated. The rate of incorporation of [(35)S]Na(2)SO(4) decreased with age as did the responsiveness of the tissue to the growth factor. The enhanced synthesis of proteoglycan induced at all ages by TGF-beta1 was down-regulated by IL-1 beta and retinoic acid. The expression of mRNA for TGF-beta1, 2, and 3 was also measured, and although the level of TGF-beta1 was highest at all ages, the expression of each growth factor decreased with age.     

The N-terminal sequence of the large proteoglycan of articular cartilage

A peptide with hyaluronic acid-binding properties was isolated from trypsin digests of bovine articular cartilage proteoglycan aggregate. This peptide originated from the N-terminus of the proteoglycan core protein, retained its function of forming complexes with hyaluronate and link protein and contained at least one keratan sulfate chain. Amino acid sequence data demonstrated that the first six amino acid residues of the N-terminus of bovine articular cartilage proteoglycan core protein differed from the same region from the rat chondrosarcoma proteoglycan. Further sequence data indicate areas of considerable sequence homology in the hyaluronic acid-binding regions of proteoglycans from the two species.     

Extractable proteoglycan from human femoral-head articular cartilage.

Proteoglycans were prepared from human femoral-head articular cartilage by using either guanidinium hydrochloride or MgCl2 as extractant, followed by density-gradient centrifugation. The proteoglycan subunit had a particle weight of 2.6 x 10(6), with a radius of gyration, RG, of 68.5 nm in 150 mM-NaCl/20 mM-sodium phosphate buffer, pH 7.0. The proteoglycan aggregate had a particle weight of 3.7 x 10(6) (RG 84 nm) for guanidinium hydrochloride extracts and 8.7 x 10(6) (RG 118 nm) for MgCl2 extracts in the same buffer. The addition of excess of high-molecular-weight hyaluronate did not significantly alter the particle size of the aggregate. The small increase in size probably reflects a rapid equilibrium between hyaluronate and proteoglycan monomers, and is not due to proteolytic cleavage producing non-aggregating units. Experiments that support the rapid-interaction hypothesis include analytical ultracentrifugation and column chromatography. This interaction does not appear to be pressure-sensitive at 20 degrees C, but is sensitive to temperature variation near the physiological range.     

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

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