Age-related changes in the chemical composition of bovine articular cartilage. The structure of high-density proteoglycans

Proteoglycans were extracted from the articular cartilage of foetal, calf and adult bovine metacarpal–phalangeal joints with 4m-guanidinium chloride. After extraction, the high-density proteoglycans (PG-I fractions) were prepared by sedimentation in two sequential CsCl-density-gradient procedures [Swann, Powell & Sotman (1979) J. Biol. Chem. 254, 945–954]. The PG-I fractions from foetal, calf and adult tissues accounted for 75%, 52% and 46% respectively of the extracted components. The glucosamine, galactose, N-acetylneuraminic acid and protein contents increased with age. The overall amino acid compositions of PG-I fractions were similar. Fractionation of PG-I-fraction samples on a Bio-Gel A-50m column indicated that the molecular weight decreased with age. The PG-I fractions were specifically ³H-labelled by treatment with galactose oxidase followed by reduction with NaB³H₄. The ³H radioactivity was incorporated into both galactose and galactosamine residues of different carbohydrate side chains. The elution profiles of alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples on a Sepharose 6B column showed that the molecular weights of chondroitin sulphate chains were 13500, 12000 and 10500 in foetal, calf and adult tissues respectively. Fractionation of the alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples and ³H-labelled calf and adult PG-I-fraction samples on a Bio-Gel P-10 column showed that there was an inverse relationship between the low-molecular-weight O-linked oligosaccharides and the higher-molecular-weight sialic acid-containing constituents at different ages. The oligosaccharide components of foetal, calf and adult PG-I-fraction samples represented 79%, 69% and 36% respectively of the total sialic acid content of the proteoglycans. Similarly in the ³H-labelled calf and adult samples 75% and 30% of the total radioactivity were present in the oligosaccharide components respectively. Digestion with chondroitinase AC-II and infrared analyses showed that the PG-I-fraction F and C samples contained primarily chondroitin 4-sulphate chains whereas PG-I-fraction sample A was 6-sulphated. These studies show that the major proteoglycans (PG-I fractions) in the articular cartilage of foetal, calf and adult animals differ in the content, types and structure of the chondroitin sulphate, keratan sulphate and oligosaccharide constituents. These changes in proteoglycan structure reflect the gross age-related changes in the chemical composition of the tissue.     

The structural characterization of proteoglycans of culture aortic smooth muscle cells and arterial wall of the pig

Aortic proteoglycans, from the growth medium of cultured smooth muscle cells and from sequential associative and dissociative extracts of the tissue of origin, the pig aorta, were isolated and purified by precipitation with cetylpiridinium chloride. After isopycnic CsCl gradient centrifugation under associative conditions 94% of the cell-secreted proteoglycans were recuperated in the bottom one fifth (?_av = 1.62 g/ml) fraction. In contrast 80% of the tissue proteoglycans of both extracts, fractionated into two fractions: the bottom one fifth (?_av = 1.60 g/ml) fraction and three fifths (?_av = 1.42 g/ml) fraction. Fractionated tissue proteoglycans were composed predominantly of chondroitin sulfate-dermatan sulfate (83–90%) with lower proportions of heparan sulfate (5–11%) and hyaluronic acid (3–6%) whilst cell-secreted proteoglycans showed a similar glycosaminoglycan composition but with a higher proportion of hyaluronic acid (11–13%). Sepharose 2B and C1-2B chromatography of tissue proteoglycans of high buoyant density showed the presence of only subunit proteoglycans whilst those of intermediate density contained a complex species, partially dissociable in 4 M guanidinium chloride, along with K_av 0.50 subunit species. The latter was also observed for cell-secreted proteoglycans although obtained at high buoyant density. The cell-secreted subunit proteoglycans became separated into two distinct populations when chromatographed on Sepharose 4B and C1-4B, half of which eluted in the column V_o and the rest at a K_av of 0.34.. The majority of subunit macromolecules eluted at the V_o fractions of Sepharose 6B and C1-6B columns. Unlike the major species of cartilage proteoglycans, only approx. 20% of purified arterial proteoglycans formed complexes. This proportion could be increased by only 4–7% by interaction, of a mixture of subunit cell-secreted and tissue-extracted proteoglycans, with hyaluronic acid. These results suggest that proteoglycans secreted by cultured aortic smooth muscle cells and present in the aortic tissue possess certain similar physicochemical properties and are present in the form of complex and several subunit species.     

High molecular weight proteoglycans biosynthesized in culture by pigeon aortas

The properties of aortic proteoglycans synthesized in vitro were examined to demonstrate synthesis of intact proteoglycans by aortic tissue in culture and to compare labeling and synthetic rates of two different populations of proteoglycan. Following 3, 6, or 9 h of incubation in medium containing [³⁵S]sodium sulfate and [³H]serine, the tissue was extracted with 4.0 M guanidine hydrochloride containing protease inhibitors. Extracts were chromatographed on Sepharose CL-4B and subjected to buoyant density centrifugation under dissociative conditions. Radioactive precursors were incorporated into two major populations of aortic proteoglycan, one of high molecular weight eluting near the void volume of Sepharose CL-4B (Protooglycan I) and one of lower molecular weight (Proteoglycan II) having a K_av of 0.40–0.44. The radioactively labeled proteoglycans were localized at densities 1.50–1.56 g/ml (Preparation 1) and 1.43–1.49 g/ml (Preparation 2) following CsCl buoyant density centrifugation. Both proteoglycan populations had increased incorporation of ³⁵S and ³H over time. At all times the lower molecular weight proteoglycan had a higher specific activity (dpm ³⁵S and ³H/μg hexuronic acid). At 3, 6, and 9 h, the specific activity of Proteoglycan II was 8.2-, 6.7- and 3.0-fold higher than Proteoglycan I using ³⁵S and 13.0-, 8.1- and 2.7-fold higher using ³H, suggesting different synthetic rates for the two proteoglycans. The results illustrate synthesis of intact proteoglycans during short-term artery culture. The proteoglycan types have size and buoyant density characteristics as described for artery, but based upon changes in specific activity ratios, the two proteoglycan populations differ in rates of synthesis.     

Turnover of proteoglycans in cultures of bovine articular cartilage

Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix.     

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.     

Characterization of chondroitin sulfate isolated from trypsin-chymotrypsin digests of cartilage proteoglycans

Proteoglycans from bovine tracheal cartilage were digested with trypsin and chymotrypsin by procedures similar to those described by Mathews (Biochem. J.125, 37 (1971)). Chondroitin sulfate-peptide fragments in the digest were precipitated with cetylpyridinium chloride and subsequently fractionated on a preparative Sepharose 6B column. The fragments, which emerged from the column as a broad peak, were divided into five fractions. Rechromatography of these fractions on an analytical Sepharose 6B column indicated that they had K_av values from 0.17 (fraction 1) to 0.62 (fraction 5). The weight average molecular weight values obtained by meniscus depletion equilibrium centrifugation were 193,000, 126,000, 80,000, 46,000, and 23,000 for fractions 1 to 5, respectively. Values for the molecular weights and for the limiting viscosity numbers, [η], of the fractions were used to determine estimates for α of 0.40–0.46 and for K of 0.43–0.88 in the equation [η] = K·M_v^α. These values for α are consistent with a branched structure for the chondroitin sulfate fractions. Papain digests of each of the fractions were chromatographed on Sephadex G-200. The observed distributions of the monomer chains released by this protease were almost the same for each sample, which indicates that the individual chondroitin sulfate chains in all of the original fractions had nearly the same average molecular weights. The data in sum indicate that peptide fragments which contain from 1 to 8 polysaccharide chains are released when the proteoglycans are digested with trypsin-chymotrypsin.Analytical data indicated that all fractions contained 3–11% of their polysaccharide as keratan sulfate. This indicates either that about 50% of the keratan sulfate chains in the original proteoglycan molecules are located in close proximity to the chondroitin sulfate chains or that some peptides contain large numbers of keratan sulfate chains. Proteoglycan preparations which differed by a factor of about 6 in their ratio of chondroitin sulfate to protein yielded very similar elution patterns on Sepharose 6B after trypsin-chymotrypsin digestion.     

Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta

Cultured smooth muscle cells from pig aorta arrested in G0 phase by serum deprivation were stimulated to proliferate by replacing the medium with one containing 10% serum. Studies in DNA replication and proliferation of cells showed a relatively good synchrony: 90% of the cells were in G1 phase for 16 h after addition of serum; they entered S phase between 18 and 24 h, completed S phase and traversed G2 phase between 24 and 30–32 h; 75% of these cells multiplied after 30–32 h and the remainder were blocked at the end of G2 phase. The synthesis and secretion of sulfated proteoglycans were examined throughout a full cell cycle using metabolic labelling with [³⁵S]sulfate. Smooth muscle cells in G1 or G2 phase synthesized and secreted sulfated proteoglycans with a possible pause at the end of the G2 phase but at the beginning of the S phase and during mitosis the incorporation of [³⁵S]sulfate into these macromolecules stopped entirely. Structural characteristics of sulfated proteoglycans secreted into the medium during G1 phase and an entire cell cycle were investigated. The proportion of proteoglycan complexes and the relative hydrodynamic size of monomers and of constituent subunits of complexes were determined after chromatography on Sepharose CL-2B and CL-6B columns run under both associative and dissociative conditions. No significant differences were observed for the periods of the cell cycle that were studied:

1. 1. [³⁵S]Proteoglycan complexes represented at the end of G1 phase and of the cell cycle respectively 19 and 16% of the total [³⁵S]proteoglycans secreted into the medium.

2. 2. More than 90% of the subunits, obtained after dissociation of complexes, were characterized by a similar k_av after chromatography on Sepharose CL-2B columns eluted under dissociative conditions (k_av 0.68 at the end of G1 phase and 0.65 at the end of full cell cycle).

3. 3. About 95% of monomers synthesized at the two stages of the cell cycle were eluted at k_av 0.25 after chromatography on Sepharose CL-6B column run under associative conditions and were characterized by a similar glycosaminoglycan distribution. These results suggest that smooth muscle cells in culture liberate similar populations of proteoglycans into the medium during the G1 and G2 phases.

     

Evidence for an interaction between canine synovial cell proteoglycans and link proteins

Link proteins are glycoproteins which stabilize aggregates of proteoglycans and hyaluronic acid in cartilage. We recently identified link proteins in canine synovial cell cultures. We now find that link proteins and proteoglycans extracted from these cells under dissociative conditions sediment in the high-buoyant-density fractions of an associative cesium chloride density gradient, suggesting that link proteins interact with high-bouyant-density proteoglycans. In gradients containing [³⁵S]sulfate-labeled synovial cell extracts, 76% of the labeled sulfate and 54% of the uronic acid is found in the high-buoyant-density fractions. Under associative conditions, Sepharose 2B elution profiles of the crude synovial cell extract, synovial cell high-buoyant-density fractions, and culture medium indicate that synovial cell proteoglycans are present in monomeric form, rather than in aggregates. Synovial cell link proteins co-elute with the [³⁵S]sulfate-labeled material under the same conditions. These proteoglycans do not interact in vitro with exogenous hyaluronic acid. Dermatan sulfate, chondroitin sulfate and heparan sulfate are the major cell-associated sulfated glycosaminoglycans synthesized by cultured canine synovial cells, while hyaluronic acid is found in the culture medium. Although the proteoglycans synthesized by cultured synovial cells interact with link proteins, these data indicate that they do not interact with hyaluronic acid to form aggregates.     

Phagocytic release of human leukocyte neutral proteases: Analysis of cartilage degradation products

Human polymorphonuclear leukocyte neutral proteases (HLNP) released during the process of phagocytosis of aggregated human gamma globulin were tested for their ability to degrade intact rabbit ear cartilage. Using ³⁵S-labeled cartilage as substrate, HLNP derived from 45 × 10⁷ cells released about 45% of the total incorporated ³⁵S. DE-52 chromatography of incubation supernatants revealed a single ³⁵S peak associated with minimal quantities of peptide or protein material as estimated by absorbance at OD_{230 + 280 nm}. Analytical ultracentrifugation gave a molecular weight of 51,800. Incubation of cartilage with excess α-chymotrypsin released ³⁵S-containing protein and peptide elements (M_r 79,400). Therefore, degradation, of the proteoglycans of intact cartilage by HLNP is more extensive than that noted with bovine pancreas α-chymotrypsin. The products of HLNP and α-chymotrypsin digestion of cartilage contained chondroitin sulfates A and/or C since both materials (after column chromatography) were sensitive to chrondroitinase ABC and AC digestion. Collagenolytic activity of HLNP is minimal compared to proteolytic activity as evidenced by the lack of hydroxyproline containing peptides released from cartilage during enzyme incubation. It is suggested that HNLP incubated with intact cartilage may serve as a relevant model in the search for new agents which could combat enzyme-mediated cartilage destruction.     

Identification of chondroitin sulfate E in human lung mast cells

Human lung mast cells (HLMC) enriched up to 99% purity by counter current elutriation and density gradient centrifugation were labeled with 35S-sulfate to determine cell-associated proteoglycans. The 35S-labeled proteoglycans were extracted by the addition of detergent and 4 M guanidine-HCl, and separated from unincorporated precursor by Sephadex G-50 chromatography. 35S-Proteoglycans chromatographed over Sepharose 4B with a Kav of 0.48. 35S-Glycosaminoglycans separated from the parent 35S-proteoglycans by beta-elimination and chromatographed over Sepharose 4B with a Kav of 0.63. Characterization of 35S-proteoglycans by chondroitin ABC lyase treatment revealed approximately 36% of the proteoglycan to be composed of chondroitin sulfates. Analysis by HPLC of component disaccharides liberated by chondroitin ABC lyase using an amino-cyano-substituted silica column indicated that the chondroitin sulfates consisted of the monosulfated A disaccharide (GlcUA----GaINAc4SO4) (75%) and the over-sulfated E disaccharide (GlcUA----GaINAc4,6-diSO4) (25%). Nitrous acid/heparinase-susceptible heparin proteoglycans accounted for approximately 62% of the total 35S-proteoglycans present in the HLMC. Proteoglycans remaining after exposure of the original proteoglycan extract to either heparinase or chondroitin ABC lyase were of similar size, suggesting that the majority of heparin and chondroitin sulfate glycosaminoglycans were on separate protein cores. Proteoglycans extracted from HLMC were protease insensitive. Hence, in addition to heparin proteoglycans, HLMC synthesize a hitherto unrecognized quantity of chondroitin sulfate E proteoglycans.     

Transformed mouse mammary epithelial cells synthesize undersulfated basement membrane proteoglycan

Proteoglycans deposited in the basal lamina of [14C] glucosamine-labeled normal and [3H]glucosamine-labeled transformed mouse mammary epithelial cells grown on type I-collagen gels, were extracted in 4 M guanidinium chloride and cofractionated over Sepharose CL 4B. The heparan sulfate chains carried by these proteoglycans were isolated by treatment with alkaline borohydride, protease K, chondroitinase ABC, and cetylpyridinium chloride precipitation. Heparan sulfate isolated from transformed cell cultures consistently eluted from DEAE-cellulose at lower salt concentrations and was of smaller apparent Mr when chromatographed over Sepharose CL 6B, than heparan sulfate of normal cell cultures. Experiments using doubly labeled cultures ([3H]glucosamine and [35S]sulfate) demonstrated an approximately 30% reduction in the sulfate/hexosamine ratio in heparan sulfate derived from transformed cultures. Both N- and O-sulfate were decreased. The decreased Mr and decreased sulfation of heparan sulfate upon transformation appear sufficient to explain the altered heparan sulfate/chondroitin sulfate ratios previously observed in these cells. These changes may have implications for the molecular interactions in which these proteoglycans are normally engaged during basal lamina assembly, and cause the poor basal lamina formation displayed by these transformed cells.     

Transforming growth factor-beta 1 stimulates synthesis of proteoglycan aggregates in calf articular cartilage organ cultures

Previous work showed that transforming growth factor-beta 1 (TGF-beta 1), added alone to bovine cartilage organ cultures, stimulated [35S]sulfate incorporation into macromolecular material but did not investigate the fidelity of the stimulated system to maintain synthesis of cartilage-type proteoglycans. This paper provides evidence that chondrocytes synthesize the appropriate proteoglycan matrix under TGF-beta 1 stimulation: (i) there is a coordinated increase in hyaluronic acid and proteoglycan monomer synthesis, (ii) link-stable proteoglycan aggregates are assembled, (ii) the hybrid chondroitin sulfate/keratan sulfate monomeric species is synthesized, and (iv) there is an increase in protein core synthesis. Some variation in glycosylation patterns was observed when proteoglycans synthesized under TGF-beta 1 stimulation were compared to those synthesized under basal conditions. Thus comparing TGF-beta 1 to basal samples respectively, the monomers were larger (Kav on Sepharose CL-2B = 0.29 vs 0.41), the chondroitin sulfate chains were longer by approximately 3.5 kDa, the percentage of total glycosaminoglycan in keratan sulfate increased slightly from approximately 4% (basal) to approximately 6%, and the unsulfated disaccharide decreased from 28% (basal) to 12%. All of these variations are in the direction of a more anionic proteoglycan. Since the ability of proteoglycans to confer resiliency to the cartilage matrix is directly related to their anionic nature, these changes would presumably have a beneficial effect on tissue function.     

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.     

Preparative electrophoresis on agarose submerged gels of two aggregating proteoglycan monomers from articular cartilage

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.     

In vitro production of proteoglycans in the articular-epiphyseal cartilage of growing pigs

The failure of cartilage mineralization in osteochondrotic cartilage may be due to an impaired proteoglycan production. Thein vitro production of proteoglycans was therefore studied in the joint cartilage of growing pigs, aged 9–18 weeks, after incubation of cartilage samples with³⁵S-sulfate. Cartilage was obtained from different areas of the femoral condyles and samples from these areas were further divided into three layers, where the superficial layer contains articular cartilage and the basal layers consist of growth cartilage. There was no significant difference in the overall amount of³⁵S-proteoglycans synthesized in different areas of the condyles. However, the total production of³⁵S-proteoglycans per mg tissue was highest in the basal layer in all areas. This was not due to a larger number of cells; the superficial layer contained more DNA per mg tissue than the basal layer. Gel chromatography on Sepharose CL-2B of the cartilage extracts, which resulted in the separation of large proteoglycans (K _av 0.4) from proteoglycans of small hydrodynamic size (K _av 0.8), showed that the relative amount of large proteoglycans increased with the distance from the articular surface. Again, no difference in the relative amounts of large and small proteoglycans were found when cartilage from different areas were compared. Osteochondrotic cartilage was detected in the pigs aged 12–18 weeks. In areas where osteochondrotic cartilage were present, the total production of³⁵S-proteoglycans was lowered and the relative amount of large proteoglycans was less than that found in the adjoining areas devoid of osteochondrotic lesions. The data available indicate that the higher relative amount of small proteoglycans in the osteochondrotic cartilage was partly caused by degradation of the large proteoglycans (aggrecan).     

Acquisition of hyaluronate-binding affinity in vivo by newly synthesized cartilage proteoglycans.

We have studied the hyaluronate-binding properties of aggregating cartilage proteoglycans synthesized in vivo by immature (6-week), mature (25-week) and aged (75-week) rabbits. Precursor isotope (35SO4) was given by intra-articular injection and articular cartilage was removed from rabbits after periods ranging from 1.5 h to 168 h. Proteoglycans were extracted with 4 M-guanidinium/HCl and monomers were isolated by CsCl gradient centrifugation under dissociative conditions. The percentages of both radiolabelled and total tissue monomers with a high affinity for hyaluronate [that is, capable of forming aggregates on Sepharose CL-2B in the presence of 0.8% (w/w) hyaluronate] were then determined. For all samples about 30% of the tissue monomers were high-affinity; however, less than 5% of the radiolabelled monomers were high-affinity at 1.5 h after injection, and this figure increased gradually with time in vivo. The increase was rapid in immature rabbits, such that after 24 h, about 30% of the radiolabelled monomers were high-affinity; on the other hand for mature and aged rabbits the increase was markedly slower such that 30% high-affinity was attained only after about 72 h. The results show that aggregating cartilage proteoglycans are secreted in vivo in a 'precursor' form with a low affinity for hyaluronate, and suggest that conversion of these monomers to a form with a higher binding affinity occurs with a half-time of about 12 h in immature cartilages but greater than 24 h in mature cartilages. The possible relationship of these findings to the process of proteoglycan aggregation in vivo is discussed.     

Effect of insulin on the altered production of proteoglycans in rib cartilage of experimentally diabetic rats

Costal cartilage from experimentally diabetic rats, labeled in vivo or in vitro with [35S]sulfate, was shown to incorporate less label into proteoglycans than cartilage from nondiabetic rats. Analyses of guanidine HCl cartilage extracts by gel chromatography on Sepharose CL-2B showed two major peaks at Kav approximately 0.4 and 0.8 (peaks I and II, respectively). Cartilage extracts from the diabetic rats contained predominantly peak II proteoglycans, while 60 and 55%, respectively, of the total 35S-labeled proteoglycans extracted from control cartilage labeled in vivo and in vitro with [35S]sulfate were present in peak I. After insulin treatment of the diabetic rats, the relative amount of peak I 35S-labeled proteoglycans synthesized in vivo was increased to 70%. The overall in vivo incorporation of [35S]sulfate into proteoglycans was also stimulated in diabetic rats treated with insulin to levels above those found for control rats. Thus, diabetes-induced changes in the biosynthesis of rat costal cartilage proteoglycans may be alleviated by normalization of the diabetic state by insulin treatment. However, addition of insulin (10(-5)-10(-9) M) to the culture medium did not affect the amount of 35S-labeled proteoglycans synthesized in vitro or the relative amounts of peak I proteoglycans produced by control or diabetic cartilage, suggesting that insulin does not have a direct effect on proteoglycan production. Moreover, no decrease in the amount of 35S-labeled proteoglycans produced was found when glucose at high concentrations was present in the culture medium. However, the presence of rat serum resulted in an increase in the amount of 35S-labeled proteoglycans produced by both control and diabetic cartilage, demonstrating that the cartilage explants were metabolically responsive to stimulatory factors.     

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

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

Characterization of the tissue-specific proteoglycans synthesized by chondrocytes from nanomelic chick embryos.

Cartilage from the avian mutant nanomelia has been reported to synthesize cartilage-specific proteoglycans, PGS(SC)-I, at 1-2% of normal values [McKeown & Goetinck (1979) Dev. Biol. 71, 203-215]. Proteoglycans were endogenously labelled with [35S]sulphate and extracted from cartilage in 4 M-guanidine hydrochloride and chromatographed on controlled-pore glass 1400. PGS(SC)-I was obtained from the void volume of these columns. Dissociative sucrose-density-gradient analysis revealed a greater than normal polydispersity in the nanomelic PGS(SC)-I. Fractions from both the controlled-pore glass 1400 void volume and sucrose gradients were tested for their ability to bind specific antibody against cartilage proteoglycan monomer. In all instances, binding of normal fractions was greater than 90%, whereas binding to nanomelic fractions ranged from 20 to 65%. Chromatography of PGS(SC)-I on controlled-pore glass 2500 resulted in 70% of the normal and 25% of the mutant proteoglycans eluting as aggregates. Chondroitin sulphate chains from mutant PGS(SC)-I appeared slightly larger than normal when chromatographed on controlled-pore glass 500. In addition, PGS(SC)-I from nanomelic cartilage is more susceptible to proteolysis in vitro than the PGS(SC)-I from normal cartilage. This evidence suggests that the small amount of cartilage-specific proteoglycan synthesized by nanomelic cartilage is not normal.     

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.