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.     

Extracellular matrix metabolism by chondrocytes. 4. Role of glutamine in glycosaminoglycan synthesis in vitro by chondrocytes

The rate of synthesis of glycosaminoglycans by cartilage was shown to be dependent on an exogenous source of L-glutamine. In the absence of L-glutamine the tissue and cellular levels of this amino acid were rapidly depleted. The levels of nucleotide sugars and their precursors were measured after separation on Dowex 1 (formate form) in cartilage incubated with and without L-glutamine. It was found that the levels of N-acetylhexoamine 6-phosphate and UDP-N-acetylhexosamine were decreased by 27 and 40% respectively. This demonstrates that L-glutamine is required as the amido group donor in the synthesis of glucosamine 6-phosphate and that the decrease in glycosaminoglycan synthesis is due to the limitation in synthesis of UDP-N-acetylhexoamine.     

Extracellular matrix metabolism by chondrocytes. VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[3H]proline into hydroxyproline and [3H]acetate into glycosaminoglycans, was shown to be depressed by 58% and 39%, respectively, by the addition of exogenous proteoglycan at a concentration of 10 mg/ml growth media. The incorporation of L-[3H]proline into acid-insoluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity on the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-translational modification of collagen and proteoglycan.     

Extracellular matrix metabolism by chondrocytes: VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[³H]proline into hydroxyproline and [³H]acetate into glycosaminoglycans, was shown to be depressed by 59% and 39%, respectively, by the addition of exogenous proteoglycan at a concetration of 10 mg/ml growth media. The incorporation of L-[³H]proline into acid-in-soluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity of the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-traslational modification of collagen and proteoglycan.     

Extracellular matrix formation by chondrocytes in monolayer culture

In previous studies were have reported on the secretion and extracellular deposition of type II collagen and fibronectin (Dessau et al., 1978, J. Cell Biol., 79:342-355) and chondroitin sulfate proteoglycan (CSPG) (Vertel and Dorfman, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:1261-1264) in chondrocyte cultures. This study describes a combined effort to compare sequence and pattern of secretion and deposition of all three macromolecules in the same chondrocyte culture experiment. By immunofluorescence labeling experiments, we demonstrate that type II collagen, fibronectin, and CSPG reappear on the cell surface after enzymatic release of chondrocytes from embryonic chick cartilage but develop different patterns in the pericellular matrix. When chondrocytes spread on the culture dish, CSPG is deposited in the extracellular space as an amorphous mass and fibronectin forms fine, intercellular strands, whereas type II collagen disappears from the chondrocyte surface and remains absent from the extracellular space in early cultures. Only after cells in the center of chondrocyte colonies shape reassume spherical shape does the immunofluorescence reveal type II collagen in the refractile matrix characteristic of differentiated cartilage. By immunofluorescence double staining of the newly formed cartilage matrix, we demonstrate that CSPG spreads farther out into the extracellular space that type II collagen. Fibronectin finally disappears from the cartilage matrix.     

Modification of copper-catalyzed oxidation of low density lipoprotein by proteoglycans and glycosaminoglycans.

Chondroitin sulfate proteoglycans (CSPG) appear to contribute to retention of low density lipoproteins (LDL) in atherosclerotic lesions. In vitro, CSPG and glycosaminoglycans (GAG) modify LDL structure and increase its uptake by macrophages. This latter effect appears related to increased exposure of arginine- and lysine-rich segments of apoB-100. We explored whether alterations of LDL induced by human arterial CSPG and purified GAG alter the lipoprotein susceptibility to transition metals-catalyzed oxidation. Human LDL was complexed with human arterial CSPG and dissociated by raising the ionic strength. The nonaggregated, CSPG- and GAG-treated LDL was subjected to oxidation by micromolar amounts of Cu+, Cu2+, Fe2+, and Fe3+. This treatment increased LDL susceptibility to Cu2+ oxidation 3- to 5-times, as indicated by the degradation rate of phospholipids and cholesteryl esters and formation rates of dienes and thiobarbituric acid-reacting substances (TBARS). Also, human macrophages degraded the CSPG-treated, Cu2+-oxidized LDL 3- to 6-times faster than native LDL similarly treated. No enhancement of oxidation was observed with Fe2+, Fe3+, and Cu+. Quenching of the LDL intrinsic fluorescence by Cu2+ showed that heparin, CSPG, and chondroitin-6-SO4 pretreatment increased the access of Cu2+ to hydrophobic chromophores, probably tryptophan, 6- to 7-, 3- to 4-, and 2- to 3-fold, respectively. Also, the affinity constant (Ka) of LDL for Cu2+ was increased from 0.12 microM to 0.20 microM by the treatment with CSPG and GAG. These results and evaluation of the fraction of surface-accessible LDL chromophores to acrylamide quenching suggest that the increased susceptibility to oxidation may be associated with an increase in the access of Cu2+ to hydrophobic regions in LDL caused by treatment with CSPG and GAG. This effect was not detected with Cu+, Fe2+, or Fe3+. The phenomenon may contribute to acceleration of the oxidative modifications of LDL in cell culture models and in vivo.     

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.     

Extracellular matrix metabolism by chondrocytes. 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells

The incorporation of [3H]glycine into acid-insoluble protein and of [3H]acetate into glycosaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gradients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of polyribosomes that was revered by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence of L-glutamine was also demonstrated for other avain connective tissues.     

Four classes of cell-associated proteoglycans in suspension cultures of articular-cartilage chondrocytes.

The characteristics of cell-associated proteoglycans were studied and compared with those from the medium in suspension cultures of calf articular-cartilage chondrocytes. By including hyaluronic acid or proteoglycan in the medium during [35S]sulphate labelling the proportion of cell-surface-associated proteoglycans could be decreased from 34% to about 15% of all incorporated label. A pulse-chase experiment indicated that this decrease was probably due to blocking of the reassociation with the cells of proteoglycans exported to the medium. Three peaks of [35S]sulphate-labelled proteoglycans from cell extracts and two from the medium were isolated by gel chromatography on Sephacryl S-500. These were characterized by agarose/polyacrylamide-gel electrophoresis, by SDS/polyacrylamide-gel electrophoresis of core proteins, by glycosaminoglycan composition and chain size as well as by distribution of glycosaminoglycans in proteolytic fragments. The results showed that associated with the cells were (a) large proteoglycans, typical for cartilage, apparently bound to hyaluronic acid at the cell surface, (b) an intermediate-size proteoglycan with chondroitin sulphate side chains (this proteoglycan, which had a large core protein, was only found associated with the cells and is apparently not related to the large proteoglycans), (c) a small proteoglycan with dermatan sulphate side chains with a low degree of epimerization, and (d) a somewhat smaller proteoglycan containing heparan sulphate side chains. The medium contained a large aggregating proteoglycan of similar nature to the large cell-associated proteoglycan and small proteoglycans with dermatan sulphate side chains with a higher degree of epimerization than those of the cells, i.e. containing some 20% iduronic acid.     

Extracellular matrix metabolism by chondrocytes. III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. Benzyl-beta-D-xyloside, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.     

Extracellular matrix metabolism by chondrocytes III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. $\text{Benzyl}\text{-β-}\text{D}\text{-}\text{xyloside}$, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.     

Vasopressin stimulates sulfate incorporation into proteoglycans synthesized by fetal rat chondrocytes in monolayer culture

The effects of lysine vasopressin (1–100 ng/ml) on the 24 h incorporation of [³⁵SO₄⁻] into proteoglycans synthesized by fetal rat chondrocytes in monolayer culture has been investigated. The hormone enhances sulfate incorporation into proteoglycans released in the medium and those associated with the cell layer. This enhancement was independent of cell density or stimulation of cell division by the hormone or calf serum. These observations provide evidence that the hormone stimulation of sulfate incorporation is not directly linked to hormone stimulation of cell division.     

Altered cartilage proteoglycans synthesized by chick limb bud chondrocytes cultured in serum-free defined medium

Chick high-density culture chondrocytes synthesize cartilage-specific proteoglycans with much structural similarity to the proteoglycans made by cartilage in vivo. Such cultures can be maintained in a defined medium formulated in this laboratory in which chondrogenesis occurs without the addition of serum. The proteoglycans synthesized by the chondrocytes in the presence of defined medium are of a cartilage-specific structure but differ in some aspects from the proteoglycans made in serum-containing medium. While their buoyant density, ability to aggregate with hyaluronic acid, and keratan sulfate chain size are unchanged, the proteoglycans synthesized in defined medium have altered chondroitin sulfate chains. This chondroitin sulfate is of significantly larger size and has a different sulfation pattern relative to that produced in serum-containing medium. The larger size of the chondroitin sulfate results in a larger monomer size of the defined medium proteoglycans. These differences have implications about the regulation of the structure of chondroitin sulfate proteoglycans.     

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.     

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.     

Phospholipase A(2) modification of low density lipoproteins forms small high density particles with increased affinity for proteoglycans and glycosaminoglycans.

The presence of a lipoprotein profile with abundance of small, dense low density lipoproteins (LDL), low levels of high density lipoproteins (HDL), and elevated levels of triglyceride-rich very low density lipoproteins is associated with an increased risk for coronary heart disease. The atherogenicity of small, dense LDL is believed to be one of the main reasons for this association. This particle contains less phospholipids (PL) and unesterified cholesterol than large LDL, and the apoB-100 appears to occupy a more extensive area at its surface. Although there are experiments that suggest a metabolic pathway leading to the overproduction of small, dense LDL, no clear molecular model exists to explain its association with atherogenesis. A current hypothesis is that small, dense LDL, because of its higher affinity for proteoglycans, is entrapped in the intima extracellular matrix and is more susceptible to oxidative modifications than large LDL. Here we describe how a specific reduction of approximately 50% of the PL of a normal buoyant LDL by immobilized phospholipase A(2) (PLA(2)) (EC 3.1.1.4) produces smaller and denser particles without inducing significant lipoprotein aggregation (<5%). These smaller LDL particles display a higher tendency to form nonsoluble complexes with proteoglycans and glycosaminoglycans than the parent LDL. Binding parameters of LDL and glycosaminoglycans and proteoglycans produced by human arterial smooth muscle cells were measured at near to physiological conditions. The PLA(2)-modified LDL has about 2 times higher affinity for the sulfated polysaccharides than control LDL. In addition, incubation of human plasma in the presence of PLA(2) generated smaller LDL and HDL particles compared with the control plasma incubated without PLA(2). These in vitro results indicate that the reduction of surface PL characteristic of small, dense LDL subfractions, besides contributing to its small size and density, may enhance its tendency to be retained by proteoglycans.     

Structure and metabolism of multiple heparan sulphate proteoglycans synthesized by the isolated rat glomerulus.

Metabolism of biosynthetically [35S]sulphate-labelled heparan sulphate proteoglycan (HSPG) was studied in the isolated glomerulus. Chromatography and electrophoresis resolved HS into 5 components, designated HS1a, HS1b, and HS2 to HS4 in order of increasing Kd. Both HS1a (250 kDa) and HS1b (130 kDa) are present in the glomerular basement membrane and have glycosaminoglycan chains of 25-45 kDa. Chemical analysis of glycosaminoglycan chains indicated a similar content of 50% N-sulphation and 30% 6-O-sulphation on the hexosamine residues of all HSs, with the remaining 20% of sulphate likely at the 2-O-position of uronic acid residues. By pulse-chase analysis, the basement-membrane fraction was found to have a half-life of residency in the glomerulus of 37 h. Both HS1a and HS1b are mainly released intact into the medium and are not further broken down in that compartment. In contrast, HS2 is almost completely released into the medium immediately after synthesis and is not normally recovered from the tissue. It is a 90-kDa HSPG with a hydrophobic core protein and glycosaminoglycan chains similar in size to those of HS1. In addition to these larger PGs, HS3 and HS4 represent glycosaminoglycan chains with little or no core protein. HS1a, HS1b and HS2 were iodinated and deglycosylated. Each has a 30-kDa core protein in addition to 18 kDa of chondroitinase ABC- and nitrous-acid-resistant O-linked carbohydrate. This suggests the possibility of a single core protein with variable glycosylation and destination. HS1a has 5-6 glycosaminoglycan chains, HS1b 2-3 and HS2 1-2. We propose that basement-membrane HSPG (HS1a and HS1b) and a related, underglycosylated secreted HSPG (HS2) are the major HSPGs synthesized by the isolated glomerulus. Other molecular species may represent discrete steps in the turnover of basement-membrane HSPG.     

Extracellular matrix synthesis by articular chondrocytes and synovial fibroblasts in long-term monolayer culture

Synthesis of collagen and proteoglycan by rabbit articular chondrocytes and synovial fibroblasts has been studied over a 12-week period in primary monolayer culture. Chondrocytes, but not fibroblasts, accumulate large quantities of proteoglycan over the culture period studied. Radiolabeling studies with [35S]sulfate have shown that the major proteoglycan synthesized by cultured chondrocytes is similar to the proteoglycan of cartilage matrix. Chondrocytes also synthesize a smaller dermatan sulfate proteoglycan, which is apparently the only proteoglycan species produced by synovial fibroblasts. Collagen synthesis was studied by radiolabeling with [3H]proline. Cultured chondrocytes produce mainly Type II collagen, with lesser amounts of Type I, whereas synovial fibroblasts produce Type I collagen and some low molecular weight collagenous species. Therefore, long-term monolayer culture permits the production of extensive chondroid matrix by chondrocytes, but not fibroblasts.     

Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).