Heparan sulfate and control of endothelial cell proliferation: increased synthesis during the S phase of the cell cycle and inhibition of thymidine incorporation induced by ortho-nitrophenyl-beta-D-xylose

The effect of xylosides on the synthesis of [35S]-sulfated glycosaminoglycans by endothelial cells in culture was investigated. Ortho-nitrophenyl-beta-D-xylose (10(-3)M) produces a dramatic enhancement on the synthesis of heparan sulfate and chondroitin sulfate secreted to the medium (20- and 100-fold, respectively). Para-nitrophenylxyloside, at the same concentration, produces an enhancement of only 37- and 3-fold of chondroitin sulfate and heparan sulfate, respectively. These differences of action seem to be related with the higher lipophilic character of ortho-nitrophenyl-xyloside. A lower enhancement of the synthesis of the two glycosaminoglycans is also observed with 2-naphtol beta-D-xylose and cis/trans-decahydro-2-naphtol beta-D-xylose. Besides stimulating the synthesis, O-nitrophenyl-beta-D-xylose as PMA [J. Cell. Biochem. 70 (1998) 563] also inhibits [3H]-thymidine incorporation by quiescent endothelial cells stimulated for growth by fetal calf serum (FCS). The combination of xylosides with PMA produced some cumulative effect. PMA stimulates the synthesis of heparan sulfate mainly at G1 phase whereas the highest enhancement of synthesis produced by the xylosides is in the S phase of the endothelial cell cycle.     

Synthesis of hyaluronate in cultured bovine articular cartilage.

The synthesis and distribution of hyaluronate and proteoglycan were studied in bovine articular cartilage in short-term explant culture with [3H]acetate and H2(35)SO4 as precursors. The incorporation of [3H]acetate into hyaluronate and sulphated glycosaminoglycans was linear with time, except that hyaluronate synthesis showed a marked lag at the beginning of the incubation. [3H]Hyaluronate represented 4-7% of the total [3H]glycosaminoglycans synthesized over a 6 h period. However, the distributions of [3H]hyaluronate and 3H-labelled sulphated glycosaminoglycans were different: about 50% of the newly synthesized [3H]hyaluronate appeared in the medium, compared with less than 5% of the 3H-labelled sulphated proteoglycans. A pulse-chase experiment revealed that the release of newly synthesized [3H]hyaluronate from cartilage was rapid. No difference was observed in the distribution of [3H]hyaluronate between medium and tissue by cartilage from either the superficial layer or the deep layer of articular cartilage. When articular cartilage was incubated with 0.4 mM-cycloheximide, proteoglycan synthesis was markedly inhibited, whereas the synthesis of hyaluronate was only partially inhibited and resulted in more of the newly synthesized hyaluronate being released into the medium. Analysis of the hydrodynamic size of [3H]hyaluronate isolated from cartilage on Sephacryl-1000 revealed one population that was eluted as a broad peak (Kav. less than 0.7), compared with two populations (Kav. greater than 0.5 and less than 0.5) appearing in the medium of cultures. These data suggest that hyaluronate is synthesized in excess of proteoglycan synthesis and that the hyaluronate that is not complexed with proteoglycans is rapidly lost from the tissue.     

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.     

Biosynthesis of proteoglycans by rat embryo parietal yolk sacs in organ culture

The embryonic rat parietal yolk sac has been previously shown to synthesize a number of basement membrane glycoconjugates including type IV procollagen, laminin, and entactin. In this study, parietal yolk sacs were isolated from 14.5-day rat embryos and incubated in organ culture for 4-7 h with [35S]sulfate, [3H] glucosamine, and/or 3H-labeled amino acids, and the newly synthesized proteoglycans were characterized. The major [35S]sulfate-labeled macromolecule represented approximately 90% of the medium and 80% of the tissue radioactivity. It also represented nearly 80% of the total [3H]glucosamine-labeled glycosaminoglycans. After purification by sequential ion-exchange chromatography and isopycnic CsCI density gradient ultracentrifugation, size-exclusion high-performance liquid chromatography showed a single species with an estimated Mr of 8-9 X 10(5). The intact proteoglycan did not form aggregates in the presence of exogenous hyaluronic acid or cartilage aggregates. Alkaline borohydride treatment released glycosaminoglycan chains with Mr of 2.0 X 10(4) which were susceptible to chondroitinase AC II and chondroitinase ABC digestion. Analysis by high-performance liquid chromatography of the disaccharides generated by chondroitinase ABC digestion revealed that chondroitin 6-sulfate was the predominant isomer. The uronic acid content of the glycosaminoglycans was 92% glucuronic acid and 8% iduronic acid, and the hexosamine content was 96% galactosamine and 4% glucosamine. No significant amounts of N- or O-linked oligosaccharides were detected. Deglycosylation of the proteoglycan with chondroitinase ABC in the presence of protease inhibitors revealed a protein core with an estimated Mr of 1.25-1.35 X 10(5). These results indicated that the major proteoglycan synthesized by the 14.5-day rat embryo parietal yolk sac is a high-density chondroitin sulfate containing small amounts of copolymeric dermatan sulfate. Hyaluronic acid and minor amounts of heparan sulfate proteoglycan were also detected.     

Effect of calcipenia on proteoglycan metabolism and aggregation in normal articular cartilage in vitro.

Glycosaminoglycan synthesis in normal adult dog knee cartilage cultured in medium containing 0, 0.3 MM- and 0.9 mM-Ca2+ was 52, 67 and 78%, respectively, of that in cartilage from the same joints cultured in a normal concentration of Ca2+, i.e. 1.8 mM. Pulse-chase experiments indicated that the rate of degradiation of glycosaminoglycans in cartilage cultured in the absence of Ca2+ was similar to that of glycosaminoglycans in cartilage cultured in 1.8 mM-Ca2+. Although [35S]sulphate incorporation into glycosaminoglycans was decreased in the presence of calcipenia, [3H]leucine incorporation into protein was unaffected. The average hydrodynamic size of newly synthesized proteoglycan aggregates and purified disaggregated proteoglycans from cartilage cultured in the absence of Ca2+ was similar to that of aggregates and disaggregated proteoglycans from cartilage cultured in 1.8 mM-Ca2+.     

Synthesis of aberrant glycosaminoglycans during cartilage culture in 'sulfate free' medium

Incorporation of radiolabeled sulfate into glycosaminoglycans is a widely accepted assay to measure the rate of proteoglycan synthesis. Although glycosaminoglycan synthesis is dependent on the quantity of inorganic sulfate available to proteoglycan synthesizing cells, 'sulfate free' medium is regularly used in studies regarding proteoglycan synthesis. In this study murine patellar cartilage glycosaminoglycans synthesized under 'sulfate free' conditions were compared with those synthesized at physiological sulfate concentration. Under 'sulfate free' conditions synthesis was not only decreased but low sulfated glycosaminoglycans were made that were not synthesized during incubation at physiological sulfate concentration. The use of 'sulfate free' medium should be avoided in proteoglycan synthesis studies.     

Dibutyryl cyclic AMP affects hyaluronate synthesis and macromolecular organization in normal adult articular cartilage in vitro

When normal adult dog articular cartilage was cultured in the presence of dibutyryl cyclic AMP a higher proportion than normal of newly synthesized 35S-labeled glycosaminoglycans was released from the tissue into the culture medium, although their net synthesis was not affected. In conjunction with this release of sulfated glycosaminoglycans, 24 times more [3H]glucosamine-labeled hyaluronic acid was released from the cartilage into the medium, and net hyaluronate synthesis was enhanced 3-fold. Virtually all of the newly synthesized hyaluronic acid in the medium was associated with proteoglycans. The proteoglycans in the medium of the dibutyryl cyclic AMP treated cultures were normal in hydrodynamic size and interacted normally with hyaluronic acid to form large aggregates. These results suggest that the increase in hyaluronate synthesis caused by dibutyryl cyclic AMP mayt have destabilized the interaction of proteoglycans with the collagen meshwork of the cartilage. The changes seen in normal adult articular cartilage after incubation with dibutyryl cyclic AMP, therefore, are similar to those which are observed in cartilage of osteoarthritic joints.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Control of proteoglycan synthesis. Studies on the activation of synthesis observed during culture of articular cartilages.

When slices of adult rabbit articular cartilage were incubated in culture medium, the rate of incorporation of [35S]sulphate or [3H]acetate into glycosaminoglycans increased 4-8 fold during the first 5 days of incubation. Similar changes in biosynthetic activity were observed during culture of adult bovine cartilage. The activation of synthesis was not serum-dependent, but appeared to be a result of the depletion of tissue proteoglycan that occurs under these incubation conditions [Sandy, Brown & Lowther (1978) Biochim. Biophys. Acta 543, 536--544]. Thus, although complete activation was observed in serum-free medium, it was not observed if the cartilage was cultured inside dialysis tubing or in medium containing added proteoglycan subunit. The average molecular size of the proteoglycans synthesized by activated tissue was slightly larger than normal, as determined by chromatography on Sepharose CL-2B, and the average molecular size of the glycosaminoglycans synthesized by activated tissue was markedly increased over the normal. The increase in chain size was accompanied by an increase in the proportion of the chains degraded by chondroitinase ABC; these results are consistent with the preferential synthesis by activated chondrocytes of chondroitin sulphate-rich proteoglycans. The increase in glycosaminoglycan chain size was observed whether the chains were formed on endogenous core protein or on exogenous benzyl-beta-D-zyloside. An approximate 4-fold activation in culture of glycosaminoglycan synthesis on protein core was accompanied by a 1.54-fold increase in the rate of incorporation of [3H]serine into the chondroitin sulphate-linkage region of the proteoglycans. A 2.8-fold activation in culture of glycosaminoglycan synthesis on benzyl-beta-D-zyloside was accompanied by a 1.7-fold increase in the rate of incorporation of [3H]benzyl-beta-D-zyloside into glycosaminoglycans. The activation of glycosaminoglycan synthesis was, however, accompanied by no detectable change in the activity of xylosyltransferase (EC 2.4.2.26) in cell-free extracts. These results are discussed in relation to current ideas on the control of proteoglycan synthesis in cartilage.     

Structure of chondroitin sulfates analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum

The structures of chondroitin sulfate A from whale cartilage and chondroitin sulfate C from shark cartilage have been examined with the aid of the chondroitinases AC and C from Flavobacterium heparinum. The analyses of the products formed from the chondroitin sulfates by the action of the chondroitinases have shown that three types of oligosaccharides compose the structure of chondroitin sulfate A, namely, a dodeca-, hexa- and a tetra-saccharide, containing five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue, respectively. The polymer contains an average of 3 mol of each oligosaccharide per mol of chondroitin sulfate A. Each mol of chondroitin sulfate C contains an average of 5 mol of 4-sulfated disaccharide units. A tetra-saccharide containing one 4-sulfated disaccharide and one 6-sulfated disaccharide was isolated from this mucopolysaccharide by the action of the chondroitinase C, indicating that the 4-sulfated disaccharides are not linked together in one specific region but spaced in the molecule.     

Sulfate transport-deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans dependent on cysteine

We isolated 59 Chinese hamster ovary cell mutants defective in 35SO4 incorporation into glycosaminoglycans. Thirty-five mutants incorporated [6-3H]glucosamine into glycosaminoglycans normally, suggesting that they were specifically impaired in sulfate incorporation. Cell hybridization studies revealed that the 35 mutants defined a unique complementation group. Pulse-labeling one of the mutants with 35SO4 showed that it possessed a defect in a saturable, 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid-sensitive transport system required for sulfate uptake. Despite the dramatic reduction in 35SO4 incorporation, the mutant synthesized sulfated heparan and chondroitin chains. Incubation of the mutant with [35S]cysteine resulted in the formation of 35SO4, which was subsequently incorporated into the glycosaminoglycans. Similar results were obtained when wild-type cells were incubated in sulfate-free growth medium containing [35S]cysteine, and isotope dilution analysis indicated that about 15 microM of sulfate was derived from cysteine catabolism. We also found that the sulfate transport deficiency rendered the mutant resistant to 5 microM sodium chromate, whereas wild-type cells did not divide under these conditions. However, the mutant also did not proliferate in medium containing 5 microM chromate when grown in the presence of wild-type cells, suggesting that chromate was transported through cell-cell contacts. Since co-cultivating sulfate transport-deficient mutants with mutants defective in xylosyltransferase or galactosyltransferase I partially restored 35SO4 incorporation into glycosaminoglycans, intercellular sulfate transport occurred as well. Therefore, the availability of sulfate for glycosaminoglycan synthesis depends on sulfate uptake, turnover of sulfur-containing amino acids, and sulfate transport between cells.     

Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition.

The chondroitin ABC lyase digestion products of normal human femoral condyle articular cartilage and of purified aggrecan were analyzed for their mono- and nonsulfated disaccharide composition. Changes in the total tissue chemistry were most pronounced during the period from birth to 20 years of age, when the -[GlcAbeta,3GalNAc6]- disaccharide content increased from approximately 50% to 85% of the total disaccharide content and there was a concomitant decrease in the content of the 4-sulfated disaccharide. In general, the disaccharide content of the deeper layers of immature cartilage were richer in the 4-sulfated residue than the upper regions of the tissue. As the tissue aged and decreased in thickness, the disaccharide composition became more evenly 6-sulfated. The newly synthesized chondroitin sulfate chains had a similar composition to the endogenous chains and also underwent the same age and zonal changes. The monoclonal antisera 3B3(+) and 2B6(+) were used to immunolocalize the unsaturated 6- and 4-sulfated residues generated at the reducing termini of the chondroitin sulfate chains by digestion with chondroitin ABC lyase, and these analyses indicated that the sulfation pattern at this position did not necessarily reflect the internal disaccharide composition of the chains. In summary, the sulfation pattern of chondroitin sulfate disaccharides from human normal articular cartilage varies with the age of the specimen, the position (topography) on the joint surface, and the zone of cartilage analyzed. Furthermore, these changes in composition are a consequence of both extracellular, post-translational processing of the core protein of aggrecan and changes in the sulfotransferase activity of the chondrocyte.     

Turnover, change of composition with rate of cell growth and effect of phenylxyloside on synthesis and structure of cell surface sulfated glycosaminoglycans of normal and transformed cells

The synthesis of sulfated glycosaminoglycans was analysed in mouse fibroblasts during the transition from exponential growth to quiescent monolayers. 'Normal' Swiss 3T3 fibroblasts were compared with SV40 transformed 3T3, C6, ST1 and HeLa cells. p-Nitrophenyl-beta-D-xyloside, an artificial acceptor for glycosaminoglycans synthesis, was used as a probe. Exponentially growing 'normal' 3T3 cells synthesized both dermatan sulfate and chondroitin 4-sulfate, retaining the latter and releasing the former to the medium. Upon reaching quiescence these cells switched to retention of dermatan sulfate and release of chondroitin 4-sulfate. SV3T3 cells synthesized several fold less sulfated glycosaminoglycans than 'normal' 3T3. Even though SV3T3 cells are able to synthesize dermatan sulfate, they only retained chondroitin 4-sulfate, never switching to retention of dermatan sulfate. These results indicated that the transition from rapidly proliferating to resting G0 state in normal cells is accompanied by a switch from chondroitin-sulfate rich to dermatan-sulfate-rich cells. This switching was not observed with transformed cells, which are unable to enter the G0 state. Phenylxyloside caused a several fold increase in glycosaminoglycans released to the medium in both cell types, but it did not interfere with either growth rate or cell morphology. Particularly the phenylxyloside treatment led to an increase of more than 10-fold in production of dermatan and chondroitin sulfate by SV3T3, C6, ST1 and HeLa cells. This demonstrated that transformed cells have a high capacity for glycosaminoglycan synthesis. Analysis of enzymatic degradation products of glycosaminoglycans, synthesized in the presence of phenylxyloside, by normal and transformed cells, led to the finding of 4- and 6-sulfated iduronic and glucuronic acid-containing disaccharides. This result indicated that the xyloside causes the synthesis of a peculiar chondroitin sulfate/dermatan sulfate, in both normal and transformed cells.     

Effect of vanadate on cartilage-matrix proteoglycan synthesis in rabbit costal chondrocyte cultures

The effect of vanadate on proteoglycan synthesis by cultured rabbit costal chondrocytes was examined. Rabbit chondrocytes were seeded at low densities and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of 4 microM vanadate to the culture medium induced a morphologic differentiation of the fibroblastic cells to spherical chondrocytes, and increased by two- to threefold incorporation of [35S]sulfate and [3H]glucosamine into large, chondroitin sulfate proteoglycans. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, in that chemical analyses showed increases in the accumulation of macromolecules containing hexuronic acid and hexosamine in vanadate-maintained cultures. However, vanadate had only a marginal effect on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant material. These results provide evidence that vanadate selectively stimulates the synthesis of proteoglycans characteristically found in cartilage by rabbit costal chondrocyte cultures.     

Characterization of a human eosinophil proteoglycan, and augmentation of its biosynthesis and size by interleukin 3, interleukin 5, and granulocyte/macrophage colony stimulating factor

Human eosinophils were cultured for up to 7 days in enriched medium in the absence or presence of recombinant human interleukin (IL) 3, mouse IL 5, or recombinant human granulocyte/macrophage colony stimulating factor (GM-CSF) and then were radiolabeled with [35S]sulfate to characterize their cell-associated proteoglycans. Freshly isolated eosinophils that were not exposed to any of these cytokines synthesized Mr approximately 80,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 80,000 glycosaminoglycans. RNA blot analysis of total eosinophil RNA, probed with a cDNA that encodes a proteoglycan peptide core of the promyelocytic leukemia HL-60 cell, revealed that the mRNA which encodes the analogous molecule in eosinophils was approximately 1.3 kilobases, like that in HL-60 cells. When eosinophils were cultured for 1 day or longer in the presence of 10 pM IL 3, 1 pM IL 5, or 10 pM GM-CSF, the rates of [35S]sulfate incorporation were increased approximately 2-fold, and the cells synthesized Mr approximately 300,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 30,000 35S-labeled glycosaminoglycans. Approximately 93% of the 35S-labeled glycosaminoglycans bound to the proteoglycans synthesized by noncytokine- and cytokine-treated eosinophils were susceptible to degradation by chondroitinase ABC. As assessed by high performance liquid chromatography, 6-16% of these chondroitinase ABC-generated 35S-labeled disaccharides were disulfated disaccharides derived from chondroitin sulfate E; the remainder were monosulfated disaccharides derived from chondroitin sulfate A. Utilizing GM-CSF as a model of the cytokines, it was demonstrated that the GM-CSF-treated cells synthesized larger glycosaminoglycans onto beta-D-xyloside than the noncytokine-treated cells. Thus, IL 3, IL 5, and GM-CSF induce human eosinophils to augment proteoglycan biosynthesis by increasing the size of the newly synthesized proteoglycans and their individual chondroitin sulfate chains.     

Biosynthesis of glycosaminoglycans in peritoneal macrophages from the guinea pig

The majority of glycosaminoglycans synthezied in peritoneal macrophages from the guinea pig in vitro were secreted into culture medium. The secreted glycosaminoglycans were reduced in size with alkali treatment, indicating that the glycosaminoglycanas existed in the form of proteoglycans. After the glycosaminoglycans were digested with chondroitinase AC and ABC, the high voltage paper electrophoretic analysis and the descending paper chromatographic analysis indicated the presence of a considerable amount of unsaturated disulfated disaccharides. Based on the enzymatic assay with chondro-4- and 6-sulfatase, the positions of sulfation in the disulfated disaccharide have been identified as the 4- and 6-position of N-acetylgalactosamine, Moreover, the results of the ion-exchange chromatography and the chondroitinase AC and ABC digestion indicate that ΔDi-diS_E derived from dermatan sulfate. This suggests that peritoneal macrophages are capable of synthesizing oversulfated proteodermatan sulfate as main component. The proportion of synthesized oversulfated dermatan sulfate to the total glycosaminoglycans was independent of the incubation time, and the distribution of oversulfated dermatan sulfate in cell and incubation medium also did not change. After exposure of macrophages to Escherichia coli for 15 min, the incorporation of [³⁵S]sulfate and [³H]glucosamine into the glycosaminoglycans was increased by about 40% with no significant change in the proportion of synthesized oversulfated dermatan sulfate, but the relese of glycosaminoglycans into the culture medium remains essentially unchanged. The difference of the existence of oversulfated dermatan sulfate is not yet understood.     

Absence of keratan sulphate from skeletal tissues of mouse and rat.

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.     

Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures

The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of [35S]sulfate and [3H]glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on [35S]sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on [35S]sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased [3H]thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.     

Stimulation of proteoglycan synthesis in chick embryo sternum by serum and L-3,5,3'-triiodothyronine.

Incorporation of sulfate into alcian blue-precipitable glycosaminoglycan of 12-day-old chick embryo sterna is stimulated by addition, separately or together, of normal human serum and physiological concentrations of thyroid hormones (Audhya, T.K., and Gibson, K.D. (1975) Proc. Natl. Acad, Sci. U. S. A. 72, 604--608). We present evidence that this stimulation is due to increased synthesis of at least one proteoglycan, with minor alterations in the size and chemical composition of the glycosaminoglycans. Pulse-chase experiments showed no detectable loss of label during the chase, in control sterna or sterna incubated with serum and L-3,5,3'-triiodothyronine; thus, all incorporation was the result of synthesis of glycosaminoglycans. In double-label experiments, with 35SO4(2-) and [3H]acetate, the molar ratio of 3H and 35S incorporated into glycosaminoglycans was changed little, if at all, by addition of serum or triiodothyronine or both, at concentrations which increased incorporation up to 2-fold. Glycosaminoglycans isolated from these and other incubations gave similar elution patterns from agarose columns, and identical electrophoretic patterns on cellulose acetate. Digestion with chondroitinase ABC (chondroitin ABC lyase; EC 4.2.2.4.) showed that incorporation was into chondroitin sulfate and possibly hyaluronic acid, and that the proportions of non-sulfated, 4-sulfated, and 6-sulfated disaccharide units differed little between stimulated and unstimulated sterna. Incorporation of [3H]serine into glycosaminoglycans from papain digest of sterna paralleled incorporation of 35SO4(2-), and indicated a number average molecular weight between 21,000 and 25,000 for the newly synthesized chondroitin sulfate. This value was confirmed by gel filtration chromatography, which also showed that the average molecular weight of the newly synthesized chondroitin sulfate decreased up to 15% under conditions of 2-fold stimulation. Proteoglycans were extracted from sterna incubated with [3H]serine and 35SO4(2-) and analyzed by isopycinic centrifugation in CsCl and by zone sedimentation in a sucrose gradient. A major proteoglycan fraction could be separated by either method. Incorporation of both isotopes into this proteoglycan fraction, and into glycosaminoglycans isolated after papain digestion, was stimulated in a coordinate manner. Almost identical results were obtained with both separation techniques. The results indicate that the synthesis of the major proteoglycan, and probably also of a minor one, is stimulated by serum and triiodothyronine.     

Change in the synthesis of glycosaminoglycans by fibrotic vitreous induced by erythrocytes

Vitreous fibrosis was induced in rabbit eyes by intravitreal injection of erythrocytes. The fibrotic vitreous removed from experimental animals were then incubated with [³H]glucosamine at 37°C for 24 h. The newly synthesized ³H-labeled glycosaminoglycans were isolated by 4 M guanidium hydrochloride extraction followed by pronase digestion. The ³H-labeled glycosaminoglycans were then characterized by gel filtration column chromatography and by specific enzymatic degradation, i.e., hyaluronidase, chondroitinase AC, and/or chondroitinase ABC. The disaccharides derived from chondroitinase ABC degradation were identified by thin-layer chromatography. We previously demonstrated that 91% of the total glycosaminoglycan synthesized by normal vitreous was hyaluronic acid. Our present results indicate that in the fibrotic vitreous, the synthesis of hyaluronic acid was decreased to 26%, whereas the synthesis of chondroitin sulfate increased to 59% of the total newly synthesized glycosaminoglycans. These results suggest that cells present in fibrotic vitreous resemble fibroblasts with respect to their activities in glycosaminoglycans synthesis.