Biosynthesis of proteoglycans by rat granulosa cells cultured in vitro.

Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and then maintained in culture. Proteoglycans were labeled using [35S]sulfate, D-[3h]glucosamine, or L-[3H]serine as precursors. 35S-labeled proteoglycans in the medium increased linearly up to 72 h after a 6- to 8-h lag period, and those in a 4 M guanidine HCl extract of the cell layer increased for about 16 h and then reached a plateau and stayed fairly constant up to 72 h. Two distinct sizes of proteoglycans were observed in the medium. The smaller (Kav = 0.60 on Sepharose CL-2B) had lower buoyant densities in dissociative gradients (rho less than 1.4 g/ml). The larger (Kav = 0.26 on Sepharose CL-2B) had high buoyant densities (recovered mainly in the bottom (D1) fraction of the dissociative gradient). More than 90% of the D1 proteoglycans contained dermatan sulfate chains (average Mr = 38,000) which yielded 84% 4-sulfated and 15% disulfated disaccharides after digestion with chondroitinase ABC. About 8% of the 35S-label in D1 was present as a heparan sulfate proteoglycan. When [3H]-glucosamine was used as a precursor, 28% of the 3H activity in the D1 proteoglycans was located in three major oligosaccharide components, two of which were similar or identical with those observed previously in D1 proteoglycans isolated from porcine follicular fluid. These results plus similar susceptibility of the labeled proteoglycans to proteolytic enzymes, especially plasmin, suggest that the granulosa cells synthesize the predominant follicular fluid proteoglycans.     

Aggregated proteoglycan synthesis in organ cultures of human nucleus pulposus.

Proteoglycans isolated under associative conditions in the presence of protease inhibitors from human nucleus pulposus contained 17% aggregate and 83% non-aggregating monomer (Kav = 0.5 on Sepharose CL-2B). Isolated aggregate after reduction and alkylation was resolved into two components (Kav = 0.15 and 0.43) on Sepharose CL-2B. Labeled proteoglycans isolated from parallel samples pulsed with [35S]sulfate and chased for up to 18 h were present largely as aggregated material (up to 78%). Reduction and alkylation of the labeled samples gave a labeled proteoglycan monomer with Kav = 0.15. Both the labeled and unlabeled chondroitin sulfate chains had the same distribution on Sepharose CL-6B and equivalent molecular weights (Mr = 2.0 x 10(3)). After chondroitinase ABC digestion, the unlabeled keratan sulfate-protein core was polydisperse with a Kav = 0.38 on Sepharose CL-4B while the labeled keratan sulfate-protein core had a Kav = 0.05. This indicates that the newly synthesized proteoglycan had a large core protein and suggests that the proteoglycans present in nucleus pulposus are originally synthesized as large molecular weight, aggregating proteoglycans.     

Isolation and characterization of proteoglycans synthesized by adult human gingival fibroblasts in vitro

The proteoglycans synthesized by fibroblasts derived from healthy human gingivae were isolated and characterized. The largest medium proteoglycan was excluded from Sepharose CL-4B but not from Sepharose CL-2B; it was recovered in the most-dense density gradient fraction and identified as a chondroitin sulfate proteoglycan. The medium contained two smaller proteoglycans; one contained predominantly chondroitin sulfate proteoglycan, while the other was comprised predominantly of dermatan sulfate proteoglycan and was quantitatively the major species. The largest proteoglycan in the cell layer fraction, excluded from both Sepharose CL-2B and Sepharose CL-4B, was found in the least-dense density gradient fraction and contained heparan sulfate and chondroitin sulfate proteoglycan. It could be further dissociated by treatment with detergent, suggesting an intimate association with cell membranes. Two other proteoglycan populations of intermediate size were identified in the cell layer extracts which contained variable proportions of heparan sulfate, dermatan sulfate, or chondroitin sulfate proteoglycan. Some small molecular weight material indicative of free glycosaminoglycan chains was also associated with the cell layer fraction. Carbohydrate analysis of the proteoglycans demonstrated the glycosaminoglycan chains to have approximate average molecular weights of 25,000. In addition, N- and O-linked oligosaccharides which were associated with the proteoglycans appeared to be sulfated in varying degrees.     

Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone

A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.     

Proteoglycans in primate arteries. III. Characterization of the proteoglycans synthesized by arterial smooth muscle cells in culture

Near confluent monolayers of arterial smooth muscle cells derived from Macaca nemestrina were labeled with Na2[35S]O4 and the newly synthesized proteoglycans present in the culture medium and cell layer were extracted with either 4 M guanidine HCl (dissociative solvent) or 0.5 M guanidine HCl (associative solvent) in the presence of protease inhibitors. The proteoglycans in both compartments were further purified by cesium chloride density gradient ultracentrifugation. Two size classes of proteoglycans were observed in the medium as determined by chromatography on Sepharose CL-2B. The large population (Kav = 0.31) contained predominantly chondroitin sulfate chains with Mr = approximately 40,000. The smaller population (Kav = 0.61) contained dermatan sulfate chains of similar Mr (approximately 40,000). When tested for their ability to aggregate, only proteoglycans in the large-sized population were able to aggregate. A chondroitin sulfate containing proteoglycan with identical properties was isolated from the cell layer. In addition, the cell layer contained a dermatan sulfate component which eluted later on Sepharose CL-2B (Kav = 0.78) than the dermatan sulfate proteoglycan present in the medium. Electron microscopy of the purified proteoglycans revealed a bottlebrush structure containing a central core averaging 140 nm in length with an average of 8 to 10 side projections. The length of the side projections varied but averaged between 70 and 75 nm. Similar bottlebrush structures were observed in the intercellular matrix of the smooth muscle cell cultures after staining with Safranin 0. This culture system provides a model to investigate parameters involved in the regulation of synthesis and degradation of arterial proteoglycans.     

Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma.

Cultured chondrocytes from the Swarm rat chondrosarcoma incorporate [35S]sulfate into proteoglycans typical of hyaline cartilage. The movement of newly synthesized proteoglycans from inside the cells into the extracellular matrix and, finally, into the culture medium was examined by measuring the distribution of 35S-labeled proteoglycans in the medium, a 4 M guanidine HCl extract of the cell layer, and in the remaining residue for a number of chase times following a 5-min pulse with [35S]sulfate. When hyaluronate oligosaccharides containing greater than or equal to 10 monosaccharides were included in the chase media, a proportion of newly synthesized proteoglycans were displaced from the matrix (4 M extract) into the culture medium. This displacement was greatest when oligomers were in the chase media between 10 and 20 min after the pulse, approximately the time when the molecules are being secreted from the cells. The proportion of link-stabilized aggregate in the medium was examined by Sepharose 2B chromatography after adding an excess of unlabeled monomer which displaces labeled monomer from complexes with hyaluronate which are not link-stabilized. The proportion of link-stabilized aggregate increased from 12% to about 70% between 12 and 120 min of chase. The presence of 40 micron hyaluronate oligosaccharides of 16 monosaccharides in the chase media retarded but did not prevent aggregate formation. Oligomers of about 50 monosaccharides, which are large enough to bind both a monomer proteoglycan and a link protein, almost completely prevented the formation of the large link-stabilized aggregates. The results suggest: (a) newly synthesized proteoglycans are not bound into link-stabilized aggregates at the time of secretion; (b) hyaluronic acid oligomers which are long enough to interact only with the hyaluronic acid-binding site of proteoglycans will retard but not prevent link-stabilized aggregation; and (c) hyaluronic acid oligomers long enough to accommodate additionally a link protein form a link-stabilized ternary complex and prevent aggregation with larger hyaluronic acid molecules.     

Partial characterization of newly synthesized proteoglycans isolated from the glomerular basement membrane

Kidneys were perfused with [35S]sulfate at 4 h in vitro to radiolabel sulfated proteoglycans. Glomeruli were isolated from the labeled kidneys, and purified fractions of glomerular basement membrane (GBM) were prepared therefrom. Proteoglycans were extracted from GBM fractions by use of 4 M guanidine-HCl at 4 degrees C in the presence of protease inhibitors. The efficiency of extraction was approximately 55% based on 35S radioactivity. The extracted proteoglycans were characterized by gel-filtration chromatography (before and after degradative treatments) and by their behavior in dissociative CsCl gradients. A single peak of proteoglycans with an Mr of 130,000 (based on cartilage proteoglycan standards) was obtained on Sepharose CL-4B or CL-6B. Approximately 85% of the total proteoglycans were susceptible to nitrous acid oxidation (which degrades heparan sulfates), and approximately 15% were susceptible to digestion with chondroitinase ABC (degrades chondroitin-4 and -6 sulfates and dermatan sulfate). The released glycosaminoglycan (GAG) chains had an Mr of approximately 26,000. Density gradient centrifugation resulted in the partial separation of the extracted proteoglycans into two types with different densities: a heparan sulfate proteoglycan that was enriched in the heavier fraction (p greater than 1.43 g/ml), and a chondroitin sulfate proteoglycan that was concentrated in the lighter fractions (p less than 1.41). The results indicate that two types of proteoglycans are synthesized and incorporated into the GBM that are similar in size and consist of four to five GAG chains (based on cartilage proteoglycan standards). The chromatographic behavior of the extracted proteoglycans and the derived GAG, together with the fact that the two types of proteoglycans can be partially separated into the density gradient, suggest that the heparan sulfate and chondroitin sulfate(s) are located on different core proteins.     

Proteoglycans of mineralizing rib and epiphyseal cartilage

Rib cartilage from growing guinea pigs and epiphyseal cartilage from Beagle puppie were separated into three fractions, representing non-mineralized, low mineralized, and high mineralized, tissue, by centrifuging finely ground material in acetone/bromoform density gradients. Following extraction under dissociative conditions, the proteoglycans were fractionated by density gradient ultracentrifugation under associative and dissociative conditions.With the onset of mineralization, the cartilage lost approximately half its content of proteoglycans. The proteoglycans remaining in the calcified cartilage differed in composition and in size from those of nonmineralized tissue. With the increased mineral content of the tissues the ratios of protein to polysaccharide, of chondroitin sulfate to keratan sulfate, and of 4-sulfated to 6-sulfated chondroitin sulfate increased in the proteoglycan fraction. Furthermore, gel chromatograms indicated decreased proportions of very high molecular weight proteoglycans, in mineralized tissue.     

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.     

Studies on the hyaluronate binding properties of newly synthesized proteoglycans purified from articular chondrocyte cultures

Primary cultures of rabbit articular chondrocytes have been maintained for 10 days and labeled with [35S]sulfate, [3H]leucine, and [35S]cysteine in pulse-chase protocols to study the structure and hyaluronate binding properties of newly synthesized proteoglycan monomers. Radiolabeled monomers were purified from medium and cell-layer fractions by dissociative CsCl gradient centrifugation with bovine carrier monomer, and analyzed for hyaluronate binding affinity on Sepharose CL-2B in 0.5 M Na acetate, 0.1% Triton X-100, pH 6.8. Detergent was necessary to prevent self-association of newly synthesized monomers during chromatography. Monomers secreted during a 30-min pulse labeling with [35S]sulfate had a low affinity relative to carrier. Those molecules released into the medium during the first 12 h of chase (about 40% of the total) remained in the low affinity form whereas those retained by the cell layer rapidly acquired high affinity. In cultures where more than 90% of the preformed cell-layer proteoglycan was removed by hyaluronidase digestion before radiolabeling the newly synthesized low affinity monomers also rapidly acquired high affinity if retained in the cell layer. Cultures labeled with amino acid precursors were used to establish the purity of monomer preparations and to isolate core proteins for study. Leucine- or cysteine-labeled core proteins derived from either low or high affinity monomer preparations migrated as a single major species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with electrophoretic mobility very similar to that of core protein derived from extracted proteoglycan monomer. Purified low affinity monomers were converted to the high affinity form by treatment at pH 8.6; however, this change was prevented by guanidinium-HCl at concentrations above 0.8 M. Conversion to high affinity was also achieved by incubation of monomers in aggregate with hyaluronic acid (HA) at pH 6.8 followed by dissociative reisolation of monomer. At both pH 6.8 and 8.6 the conversion process was slow, requiring up to 48 h for the maximum increase in affinity. It is suggested that the slow increase in HA binding affinity seen during extracellular processing of proteoglycans in cartilage and chondrocyte cultures is the result of an irreversible structural change in the HA binding domain following the binding of monomer to hyaluronate. The available evidence suggests that this change involves the formation or rearrangement of disulfide bonds.     

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

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

The in vitro cell culture age and cell density of articular chondrocytes alter sulfated-proteoglycan biosynthesis

The effect of cell culture age and concomitant changes in cell density on the biosynthesis of sulfated-proteoglycan by rabbit articular chondrocytes in secondary monolayer culture was studied. Low density (LD, 2 d), middle density (MD, 5-7 d), and high density (HD, 12-15 d) cultures demonstrated changes in cellular morphology and rates of DNA synthesis. DNA synthesis was highest at LD to MD densities, but HD cultures continued to incorporate [3H]-thymidine. LD cultures incorporated 35SO4 into sulfated-proteoglycans at a higher rate than MD or LD cultures. The qualitative nature of the sulfated-proteoglycans synthesized at the different culture ages were analyzed by assessing the distribution of incorporated 35SO4 in associative and dissociative CsCl density gradients and by elution profiles on Sepharose CL-2B. Chondrocytes deposited into the extracellular matrix (cell-associated fraction) 35SO4-labeled proteoglycan aggregate. More aggregated proteoglycan was found in the MD and HD cultures than at LD. A 35SO4-labeled aggregated proteoglycan of smaller hydrodynamic size than that found in the cell-associated fraction was secreted into the culture medium at each culture age. The proteoglycan monomer (A1D1) of young and older cultures had similar hydrodynamic sizes at all cell culture ages and cell densities. The glycosaminoglycan chains of A1D1 were hydrodynamically larger in the younger LD cultures than in the older HD cultures and consisted of only chondroitin 6 and 4 sulfate chains. A small amount of chondroitin 4,6 sulfate was detected, but no keratan sulfate was measured. The A1D2 fractions of young LD cultures contained measurable amounts of dermatan sulfate; no dermatan sulfate was found in older MD or HD cultures. These studies indicated that chondrocytes at LD synthesized a proteoglycan monomer with many of the characteristics of young immature articular cartilage of rabbits. These results also indicated that rapidly dividing chondrocytes were capable of synthesizing proteoglycans which form aggregates with hyaluronic acid. Culture age and cell density appears primarily to modulate the synthesis of glycosaminoglycan types and chain length. Whether or not these glycosaminoglycans are found on the same or different core proteins remains to be determined.     

Stimulation of sulfated-proteoglycan synthesis by forskolin in monolayer cultures of rabbit articular chondrocytes

Forskolin, a plant cardiotonic diterpene, stimulated proteoglycan biosynthesis by chondrocytes in monolayer culture. The quantitative increase in proteoglycans was dependent on the concentration of forskolin, but was relatively independent of the presence of serum. At forskolin concentrations that stimulated proteoglycan synthesis, a significant stimulation of adenylate cyclase and cAMP was also measured. The quantitative increase in proteoglycans was characterized, qualitatively, by an increased deposition of newly synthesized proteoglycan in the cell-associated fraction. An analysis of the most dense proteoglycans (fraction dA1) in the cell-associated fraction showed that more of the proteoglycans eluted in the void volume of a Sepharose CL-2B column, indicating that an increased amount of proteoglycan aggregate was synthesized in forskolin-treated cultures. The proteoglycan monomer dA1D1 secreted into the culture medium of forskolin-stimulated cultures overlapped in hydrodynamic size with that of control cultures, although cultures stimulated with forskolin and phosphodiesterase inhibitors produced even larger proteoglycans. The hydrodynamic size of 35SO4 and 3H-glucosamine-labelled glycosaminoglycans isolated from the dA1D1 fraction of the culture medium was greater in forskolin-treated chondrocytes, especially from those in which phosphodiesterase inhibitors had been added. These results indicated that forskolin, a direct activator of chondrocyte adenylate cyclase mimicked the effects of cAMP analogues on chondrocyte proteoglycan synthesis previously reported. These results implicate activation of adenylate cyclase as a regulatory event in the biosynthesis of cartilage proteoglycans, and more specifically in the production of hydrodynamically larger glycosaminoglycans.     

A study of the interaction between cartilage proteoglycan and link protein.

The interaction between proteoglycan and link protein extracted from bovine articular cartilage (15-18-month-old animals) was investigated in 0.5 M-guanidinium chloride. The proteoglycans, radiolabelled as the aggregate (A1 fraction), were fractionated by two 'dissociative' density-gradient centrifugations (A1D1D1) followed by a rate-zonal centrifugation (S1) to yield an A1D1D1S1 preparation. At least 65% of these proteoglycans were able to bind to hyaluronate, but only 52% were able to bind to link protein as assessed by chromatography on Sepharose CL-2B. Over 80% of the [3H]link-protein preparation, radiolabelled as the aggregate, was able to interact with proteoglycan as assessed by chromatography on Sepharose CL-4B. Equilibrium-boundary-centrifugation studies performed at low link-protein concentrations (2.42 x 10(-9) M-5.93 x 10(-8) M) were analysed by Scatchard-type plots and indicated a Kd of 1.5 x 10(-8) M and a stoichiometry, n = 0.56, i.e. approx. 56% of those proteoglycans capable of binding to link protein had a strong site for link protein if a 1:1 stoichiometry were assumed. However, experiments performed at higher link-protein concentrations (3.5 x 10(-7) M and 8 x 10(-7) M) yielded stoichiometry values which were link-protein-concentration-dependent. Non-equilibrium binding studies using chromatography on Sepharose CL-2B and rate-zonal centrifugation yielded apparent stoichiometries between 0.6 and 7.5 link-protein molecules per proteoglycan monomer as a function of increasing link-protein concentration. It was concluded that a proportion of the proteoglycan molecules had a strong site for binding a single link protein (Kd 1.5 x 10(-8) M) and that at high link-protein concentrations a weaker, open-ended, process of link-protein self-association nucleated upon the strong link-protein-proteoglycan complex occurred. Hyaluronate oligosaccharides appeared to abolish a proportion of this self-association (as observed by Bonnet, Dunham & Hardingham [(1985) Biochem. J. 228, 77-85] in a study of link-protein-hyaluronate-oligosaccharide interactions) so as to leave a link protein:proteoglycan stoichiometry of 2. It is not clear whether this second link-protein molecule binds directly to the proteoglycan or to the first link protein.     

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.     

Maturation-related differences in the structure and composition of proteoglycans synthesized by chondrocytes from bovine articular cartilage

Calf (2-3-month-old) and steer (approximately 18-month-old) bovine articular chondrocytes were isolated and cultured as high density monolayers. The proteoglycans synthesized on day 5 during a 15-h period of labeling with [35S]sulfate or [3H]glucosamine were isolated and characterized. The majority (greater than 70%) of the newly synthesized proteoglycans were found in the medium. When viewed in the electron microscope, medium-derived proteoglycans of high buoyant density were longer in calf than in steer. The medium and extracts of the cell layer were pooled and the radiolabeled proteoglycans were fractionated by isopycnic density gradient centrifugation performed under dissociative conditions. The low buoyant density fraction contained, in both calf and steer, small-sized nonaggregating proteoglycans containing chondroitin sulfate. The high buoyant density fraction contained greater than 90% of the newly synthesized proteoglycans. The majority were able to interact with hyaluronic acid to form aggregates. Calf high buoyant density fraction proteoglycans were larger, had longer chondroitin sulfate chains and lower ratios of keratan sulfate chains/chondroitin sulfate chains than steer high buoyant density fraction proteoglycans. These maturation-related differences are typical of those present in the proteoglycans of the calf and steer cartilage matrix from which the chondrocytes were isolated. Experiments with beta-D-xylosides showed that steer cultures had the capacity to synthesize twice as many chondroitin sulfate chains/cell as calf cultures. At each xyloside concentration used, chondroitin sulfate chains were longer in calf than steer. At both ages, chain size decreased with increase in rate of synthesis; the relationship between chain size and rate of synthesis was, however, quite different at the two ages. The results of these studies suggest that articular chondrocytes have an inherent program that determines the quality of proteoglycans synthesized at different ages.     

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

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

Selective aggregation of proteoglycans with hyaluronic acid.

Conditions were established to separate proteoglycan aggregate (AH1) from a bovine nasal septum extract by associative rate zonal sedimentation on a NaCl gradient. The AH1 has a higher protein content than the mixed aggregate-monomer (A1) isolated by conventional associative CsCl density gradient centrifugation from a portion of the same extract. The same associative rate zonal conditions separated the A1 fraction into aggregated AH1 containing hyaluronic acid and nonaggregated proteoglycan monomer (N1) essentially free of hyaluronic acid. The AH1 fraction is richer in protein and keratin sulfate than is N1. Dissociative rate zonal sedimentation of A1 under conditions which totally sedimented most of the disaggregated monomer (AH1-D1) and the nonaggregated monomer N1 separated a less sedimentable protein and keratan sulfate-rich proteoglycan monomer (AH1-D2). Chromatography on Sepharose 2B under dissociative conditions demonstrated that the nonaggregated N1 monomer is intermediate in size between the disaggregated monomers AH1-D1 and AH1-D2. N1 has a buoyant density higher than AH1 and is practically equivalent to AH1-D1. All are dense fractions so that separation by CsCl density gradient equilibration is not feasible.     

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.