The relation of protein synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage.

The effect of cycloheximide on chondroitin sulphate biosynthesis was studied in bovine articular cartilage maintained in culture. Addition of 0.4 mM-cycloheximide to the culture medium was followed, over the next 4h, by a first-order decrease in the rate of incorporation of [35S]sulphate into glycosaminoglycan (half-life, t 1/2 = 32 min), which is consistent with the depletion of a pool of proteoglycan core protein. Addition of 1.0 mM-benzyl beta-D-xyloside increased the rate of incorporation of [35S]sulphate and [3H]acetate into glycosaminoglycan, but this elevated rate was also diminished by cycloheximide. It was concluded that cycloheximide exerted two effects on the tissue; not only did it inhibit the synthesis of the core protein, but it also lowered the tissue's capacity for chondroitin sulphate chain synthesis. Similar results were obtained with chick chondrocytes grown in high-density cultures. Although the exact mechanism of this secondary effect of cycloheximide is not known, it was shown that there was no detectable change in cellular ATP concentration or in the amount of three glycosyltransferases (galactosyltransferase-I, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II) involved in chondroitin sulphate chain synthesis. The sizes of the glycosaminoglycan chains formed in the presence of cycloheximide were larger than those formed in control cultures, whereas those synthesized in the presence of benzyl beta-D-xyloside were consistently smaller, irrespective of the presence of cycloheximide. These results suggest that beta-D-xylosides must be used with caution to study chondroitin sulphate biosynthesis as an event entirely independent of proteoglycan core-protein synthesis, and they also indicate a possible involvement of the core protein in the activation of the enzymes of chondroitin sulphate synthesis.     

The control of chondroitin sulphate biosynthesis and its influence on the structure of cartilage proteoglycans.

Chondroitin sulphate synthesis on proteoglycans was decreased in rat chondrosarcoma cell cultures in the presence of cycloheximide (0.1-1.0 muM) or p-nitrophenyl beta-D-xyloside (50 microM). In the presence of cycloheximide the proteoglycan monomer was of larger size, the chondroitin sulphate chains were increased in length, but a similar number of chains was attached to each proteoglycan and the size of the core protein was unaltered. In the presence of p-nitrophenyl beta-D-xyloside (50 microM), chondroitin sulphate synthesis was increased (by 60-80%), but the incorporation into proteoglycans was decreased (by 70%). The chondroitin sulphate chains were of shorter length than in control cultured and the number of chains attached to each proteoglycan was decreased. In cultures with cycloheximide or actinomycin D the synthesis of chondroitin sulphate was less inhibited on beta-xyloside than on endogenous proteoglycan. When the rate of chondroitin sulphate synthesis was decreased by lowering the temperature of cultures, the chains synthesized at 22 and 4 degrees C were much longer than at 37 degrees C, but in the presence of p-nitrophenyl beta-D-xyloside the chains were of the same length at all three temperatures. A model of chain elongation is thus proposed in which the rate of chain synthesis is determined by the concentration of xylosyl acceptor and the length of the chains is determined by the ratio of elongation activity to xylosyl-acceptor concentration.     

Effect of forskolin on synthesis of xyloside-initiated glycosaminoglycans in embryonic chick chondrocytes

The synthesis of sulfated glycosaminoglycan (GAG) chains has been studied in the presence of various concentrations of the artificial acceptor 4-methylumbelliferyl beta-D-xyloside and of forskolin. Sulfated GAG chains formed in the presence of forskolin had a smaller hydrodynamic radius than controls, as revealed by chromatography on Sepharose CL-6B. Sulfated GAGs from both control and treated cultures behaved identically when chromatographed on DEAE.     

Effect of low-density lipoproteins on the synthesis and secretion of proteoglycans by human endothelial cells in culture.

We studied the effect of low-density lipoproteins (LDL) on the synthesis and secretion of proteoglycans by cultured human umbilical-vein endothelial cells. Confluent cultures were incubated with [35S]sulphate or [3H]glucosamine in lipoprotein-deficient serum in the presence and in the absence (control) of LDL (100-400 micrograms/ml), and metabolically labelled proteoglycans in culture medium and cell layer were analysed. LDL increased accumulation of labelled proteoglycans in medium and cell fractions up to a concentration of 200 micrograms/ml. At this concentration of LDL the accumulations of proteoglycans in medium and cell layer were 65% and 32% respectively above control for 35S-labelled proteoglycans, and 55% and 28% respectively above control for 3H-labelled proteoglycans. At concentrations above this LDL was found to depress the accumulation of proteoglycans in medium and cell layer. Gel filtration on Sepharose CL-4B showed that in both control and LDL-treated cultures the cell layer contained a large (Kav. = 0) and a small (Kav. = 0.35) heparan sulphate proteoglycan, whereas the culture medium contained a large heparan sulphate proteoglycan (Kav. = 0) and a smaller isomeric chondroitin sulphate proteoglycan (control, Kav. = 0.35; LDL-treated, Kav. = 0.17). The relative increase in hydrodynamic size of the isomeric chondroitin sulphate proteoglycan (Mr 150,000 compared with 90,000) in the medium of cultures exposed to LDL was partly attributable to the larger size of the glycosaminoglycan side chains (Mr 39,000 compared with 21,000). The isomeric chondroitin sulphate proteoglycan in LDL-treated culture was relatively enriched in chondroitin 6-sulphate compared with that in control cultures (39% compared with 29%). Pulse-chase studies showed that LDL treatment did not alter the turnover rate of proteoglycans as compared with controls, implying that the elevation in proteoglycan accumulation in LDL-treated cultures was due to enhanced synthesis. These results demonstrate that LDL can modulate proteoglycan synthesis by cultured vascular endothelial cells, resulting in the secretion of a larger isomeric chondroitin sulphate proteoglycan enriched in chondroitin 6-sulphate.     

Initiation of altered heparan sulphate on beta-D-xyloside in rat hepatocytes

Inhibition of protein synthesis by cycloheximide 10(-3)M reduced the incorporation of [35S]sulphate into heparan sulphate to about 5% of untreated hepatocytes. Addition of rho-nitrophenyl beta-D-xyloside could partially revert this inhibitory effect. The sulphated material isolated from the cell layer or secretions of hepatocytes grown in presence of cycloheximide and rho-nitrophenyl beta-D-xyloside were shown to be mostly free heparan sulphate chains not bound to core protein. Covalent association of beta-xylosides to the heparan sulphates was demonstrated for heparan sulphate synthetized in the presence of [35S]sulphate, cycloheximide and the fluorogenic 4-methylumbelliferyl beta-D-xyloside. Beta-Xylosides served as an initiator of heparan sulphate chain synthesis in rat hepatocytes only in the absence of protein synthesis. Heparan sulphates primed on artificial beta-xylosides are slightly smaller in molecular size and are more sulphated than chains linked to core protein.     

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

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

Articular cartilage cultured with catabolin (pig interleukin 1) synthesizes a decreased number of normal proteoglycan molecules.

A homogeneous preparation of catabolin from pig leucocytes caused a reversible dose-dependent (0.01-1 nM) decrease in the synthesis of proteoglycan in slices of pig articular cartilage cultured in serum-free medium. The monomers that were synthesized and secreted in the presence of catabolin had the same average hydrodynamic size and ability to aggregate as the controls, and the core protein was substituted with the same number of glycosaminoglycan chains. The chains were the same average length and charge as normal and were sulphated to the same extent as the controls. Newly synthesized extracellular proteoglycan was not preferentially degraded. A 2-3-fold increase in glycosaminoglycan synthesis occurred in control and catabolin-treated cartilage in the presence of beta-D-xyloside (1 mM), more than 80% being secreted into the medium as free chains. Decreased incorporation of sulphate was not reversed in the presence of lysosomal-enzyme inhibitors, and there was no evidence in pulse-chase experiments of increased intracellular degradation of glycosaminoglycan chains before secretion. It is concluded that catabolin-treated cartilage synthesizes a smaller number of normal proteoglycan molecules.     

Transforming growth factor-beta induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts.

Human embryonic skin fibroblasts were pretreated with transforming growth factor-beta (TGF-beta) for 6 h and then labeled with [35S]sulphate and [3H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density-gradient centrifugation, followed by gel, ion-exchange and hydrophobic-interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains. The results showed that TGF-beta increased the production of several different 35S-labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400-500 kDa) increased 5-7-fold and a small dermatan sulphate proteoglycan (PG-S1, also termed biglycan, with a core protein of 43 kDa) increased 3-4-fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG-S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF-beta treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected. To investigate whether TGF-beta also influences the glycosaminoglycan (GAG) chain-synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF-beta-treated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF-beta treated cultures contained a larger proportion of D-glucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4- and 6-sulphation of the GAG chains. By the use of p-nitrophenyl beta-D-xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF-beta-treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF-beta-treated fibroblasts contained a larger proportion of D-glucuronosyl residues than those of the control. These novel findings indicate that TGF-beta affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAG-synthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, which can affect cell growth and migration as well as organisation of matrix fibres.     

Inactivation of unbound rat liver glucocorticoid receptor by N-alkylmaleimides at sub-zero temperatures

Biosynthetically radiolabelled heparan sulphate proteoglycans have been isolated from the growth medium and the cell lysate of a human neuroblastoma cell line (CHP100). Chromatography on Sepharose CL-4B identified two heparan sulphate proteoglycans in the medium (Kav 0.220 and 0.389), whereas in the cell lysate the major proteoglycan species were more heterogenous and of a smaller overall molecular size (Kav 0.407) than the medium-derived counterparts. Chromatography on Sepharose CL-6B of free heparan sulphate glycosaminoglycan chains showed that the majority of cell-layer-derived material heparan sulphate 2, Kav = 0.509) was smaller than medium heparan sulphates (heparan sulphate 1 and heparan sulphate 2, Kav 0.230 and 0.317). Analysis of the patterns of polymer sulphation by nitrous acid treatment, gel chromatography and high-voltage electrophoresis established that in each heparan sulphate fraction there was on average 1.1 sulphate residues per disaccharide with an N:O sulphate ratio of 1.1. Heparan sulphate in the medium had a high proportion of di-O-sulphated disaccharides in regions of the chain with repeat disaccharide sequences of structure GlcA-GlcNSO3, whereas cell-associated material was enriched in di-O-sulphated tetrasaccharides of alternating sequences GlcA-GlcNAc-GlcA-GlcNSO3. The identification of several populations of heparan sulphate proteoglycans differing in molecular size and glycosaminoglycan fine structure may reflect the functional diversity of this family of macromolecules in the nervous system.     

Proteoglycans synthesized by cultured mouse osteoblasts

Proteoglycan synthesis in nonmineralizing osteoblast cultures was investigated. Cultures were labeled with [35S]sulfate or [3H]serine, and proteoglycans were extracted from medium and cell layer with 4 M guanidine HCl. Labeled material was subjected to Sepharose CL-4B and DEAE-Sephacel chromatography and polyacrylamide gel electrophoresis. The size and composition of the glycosaminoglycan chains and the protein core size were determined. Two proteoglycan populations were isolated by Sepharose CL-4B chromatography: a minor excluded species with chondroitin sulfate chains of apparent Mr 25,000 and a smaller population (Kav = 0.43) accounting for 80% of the total labeled material. This small population resolved into two species by polyacrylamide gel electrophoresis. Both species contain dermatan sulfate chains of apparent Mr 40,000 and a core protein with Mr 45,000 on sodium dodecyl sulfate gels. With the exception of their glycosaminoglycan composition these species appear similar to those extracted from bone. In addition, high molecular weight hyaluronic acid and glycosaminoglycan peptides were found in cell extracts.     

The synthesis of proteoglycans by human T lymphocytes

We have examined the proteoglycans produced by highly-purified cultures of human T-lymphocytes. The proteoglycans were metabolically labelled with [35S]sulphate and analysed in cellular and medium fractions using DEAE-cellulose chromatography, gel filtration and specific enzymatic and chemical degradations. The results showed that the T cells synthesized a relatively homogeneous, proteinase-resistant chondroitin 4-sulphate proteoglycan that accumulated in the culture medium during a 48 h incubation period. The cellular fraction contained a significant amount of free chondroitin sulphate chains that were not secreted into the medium. These polysaccharides were formed by intracellular degradation of proteoglycan in a chloroquine-sensitive process, indicating a requirement for an acidic environment. In contrast to chondroitin sulphate derived from proteoglycan, chondroitin sulphates synthesized on the exogenous primer, beta-D-xyloside, were mainly secreted by the cells. beta-D-Xylosides caused an 8-fold stimulation in the synthesis of chondroitin sulphate, but decreased the synthesis of proteoglycan by about 50%. These proteoglycans contained shorter chondroitin sulphate chains than their normal counterparts. The results indicate that although proteoglycans are mainly secretory components in human T-cell cultures, a specific metabolic step leads to the intracellular accumulation of free glycosaminoglycans. Separate functions are likely to be associated with the intracellular and secretory pools of chondroitin sulphate.     

Detergent-solubilized proteoglycans in rat testicular Sertoli cells

Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products.     

Heparan sulfate proteoglycan synthesis and metabolism by mouse uterine epithelial cells cultured in vitro

Characterization and metabolism of heparan sulfate glycosaminoglycans and proteoglycans (HSPGs) synthesized by primary cultures of mouse uterine epithelial cells are reported. HSPGs were detected in both the medium and in the cell-associated fraction, whereas glycosaminoglycans containing little or no protein (free glycosaminoglycans) were found primarily in the cell-associated fraction. The cell-associated HSPGs were relatively large (Kav = 0.1 on Superose 12), had a buoyant density in cesium chloride gradients of 1.45-1.55 g/ml, and contained heparan sulfate chains that fell into two size classes, exhibiting Kav values on Superose 12 of 0.2-0.5 and 0.7-0.8, respectively. The free glycosaminoglycan chains displayed a Kav on Superose 12 of 0.6-0.7. The secreted HSPGs were smaller (median Kav on Superose 12 of 0.28) than the cell-associated HSPGs. More than 90% of the cell-associated HSPGs contained hydrophobic portions, as evidenced by their ability to bind to octyl-Sepharose. In contrast, only 10-15% of the secreted HSPGs bound to octyl-Sepharose. HSPGs were detected at both apical and basal cell surfaces/extracellular matrices by indirect immunofluorescence in vitro and in utero and by accessibility to external proteases in vitro. It was estimated that 60-70% of the total cell-associated HSPGs were exposed at the cell surface. The HSPGs released from the cell surface by proteases were slightly smaller than the intact HSPGs and lacked the hydrophobic properties of the latter. These observations suggested that the cell surface HSPGs contain a small, hydrophobic domain that functions in the attachment of HSPGs to cells. The free glycosaminoglycans appeared to be primarily intracellular and were not secreted. The cell-associated HSPGs turned over rapidly (t1/2 = 1.5 h) and appeared to be the precursors to the free glycosaminoglycans. Metabolic turnover of the free glycosaminoglycan pool was a relatively slow process (t1/2 = 10-12 h).(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen.

Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.     

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

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

Biosynthesis of heparan sulfate proteoglycans of developing chick breast skeletal muscle in vitro

Previous studies have reported an increase in heparan sulfate glycosaminoglycan (HSGAG) during skeletal muscle differentiation in culture. We have investigated this phenomenon further in relation to the heparan sulfate proteoglycans (HSPG) produced by myogenic cultures. Pulse-chase analysis indicated an approx. 3-fold increase in heparan sulfate synthesis in myotube cultures over that in proliferating or aligning myoblast cultures. Muscle fibroblast culture heparan sulfate synthesis was higher than that of myoblasts but was lower than myotubes. The turnover rates appeared to be the same for all stages of development, with a t1/2 of approx. 5 h. Enrichment for heparan sulfate by Sepharose CL-4B and DEAE-Sephacel chromatography indicated an increase in the hydrodynamic size of the proteoglycan produced by myotubes over that from myoblasts, with a shift in Kav from 0.14-0.19 to 0.07. Fibroblasts synthesized the smallest proteoglycan, with a Kav of 0.22. All of the proteoglycans contained similar sized glycosaminoglycan chains with an estimated molecular weight of 30,000-40,000. Localization of the heparan sulfate proteoglycan in myotube cultures by trypsin sensitivity indicated much of the intact proteoglycan to be closely associated with the cell surface, while internalized material appeared in a degraded form.     

Effect of cycloheximide, beta-D-xylosides and beta-D-galactosides on heparin biosynthesis in mouse mastocytoma.

Heparin biosynthesis has been investigated with mouse mastocytoma in vitro. Minced tumour tissue catalysed the incorporation of [35S]sulphate and [3H]glucosamine into heparin and to a smaller extent into chondroitin sulphate. Addition of cycloheximide caused an inhibition (greater than 80%) of incorporation of each labelled precursor into both polysaccharides. Addition of benzyl beta-D-xyloside relieved the inhibition of incorporation into chondroitin sulphate and restored it to more than threefold that of the control incubation. The effect of beta-D-xyloside on incorporation into heparin was less marked although a consistent small increase of incorporation into this polysaccharide was observed. beta-D-Xyloside did, however, cause a marked incorporation of 35S and 3H labels into material of low molecular weight, which appeared to comprise heparin-like fragments. It is proposed that these fragments arise through a breakdown of the usual process of heparin biosynthesis.     

1,25-Dihydroxyvitamin D3 inhibits synthesis and enhances degradation of proteoglycans in osteoblastic cells

The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), an active form of vitamin D3, on the metabolism of proteoglycans by an osteoblastic cell line MC3T3-E1 were studied. Cells metabolically labeled with [35S]sulfate and/or [3H]glucosamine synthesized large and small dermatan sulfate proteoglycans and heparan sulfate proteoglycan. The incorporation of [35S]sulfate into proteoglycans for 1 h was reduced by 1,25-(OH)2D3 in a dose-dependent manner with a maximum reduction of 40% obtained at 10(-8)M 1,25-(OH)2D3. This effect was observed for all the proteoglycans with the decrease for the large dermatan sulfate proteoglycan most prominent. Treatment with 1,25-(OH)2D3 did not influence the degree of sulfation nor the molecular size of the glycosaminoglycan chains. Thus, the change in the incorporation of [35S] sulfate reflects net change in the synthesis of proteoglycans. When cells were treated with beta-D-xyloside, 1,25-(OH)2D3 also inhibited net synthesis of dermatan sulfate glycosaminoglycan chains on this exogenous substrate suggesting that it decreases the capacity of the cells for glycosaminoglycan synthesis. The incorporation of [3H]glucosamine into hyaluronic acid was also inhibited up to 70% by 10(-8) M 1,25-(OH)2D3. Treatment with 24,25-dihydroxyvitamin D3 did not cause significant changes in the proteoglycan synthesis. Degradation of proteoglycans associated with the cell layer was enhanced by treatment with 1,25-(OH)2D3 at 10(-8) M. Proteoglycans exogenously added to the culture were also degraded with a cell-mediated process which was stimulated by treatment with 10(-8) M 1,25-(OH)2D3. These results demonstrate that 1,25-(OH)2D3 reduces the synthesis and stimulates the degradation of proteoglycans in osteoblastic cells in culture.     

The effect of beta-D-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors.

Incorporation of [35S]]sulphate, [3H]glucose and [3H]serine into glycosaminoglycans and proteoglycans of embryonic-chicken sternum was measured in vitro in incubation medium containing 4-methylumbelliferyl beta-D-xyloside or p-nitrophenyl beta-D-xyloside at low concentrations, and in the absence of inhibitors of protein synthesis. Incorporation of sulphate was decreased by 80% in incubations in which 1mM-4-methylumbelliferyl beta-xyloside or 2.5 mM-p-nitrophenyl beta-xyloside was present; under these conditions, serum factors stimulated incorporation to only a small extent. When the concentration of the xyloside was decreased tenfold, incorporation of sulphate was inhibited by 60-70%, but when normal human serum or L-3,3',5-tri-iodothyronine or both were also added to the incubation medium, incorporation was markedly stimulated. Experiments in which [35S]sulphate and [3H]glucose were incorporated simultaneously, and enzymic analysis of glycosaminoglycans formed in such experiments, indicated that chondroitin sulphate formed in the presence of 0.1 mM-4-methylumbelliferyl beta-xyloside contained 30-40% less sulphate than did chondrotin sulphate synthesized in the absence of xylosides. Similar experiments, with [3H]serine instead of [3H]glucose, suggested also a 20-30% decrease in chain length of the chondroitin sulphate; this was confirmed by direct gel filtration of labelled glycosaminoglycans on a calibrated column. Incorporation of [3H]glucose or [3H]serine was stimulated by serum and tri-iodothyronine in parallel with incorporation of sulphate. The changes seen in the total chondroitin sulphate were mirrored in the major proteoglycan fraction, purified by isopycnic centrifugation of salt-extracted proteoglycans. The labelling pattern of chondroitin sulphate from this proteoglycan indicated that decreased sulphation of chondroitin sulphate was largely due to the inferior ability of short polysaccharide chains to accept sulphate, with some direct interference with transfer of sulphate to all chains. The results also suggested that the action of serum factors on synthesis of proteochondroitin sulphate is exercised at the level of either protein synthesis or transport to the sites of initiation of polysaccharide synthesis.     

Developmental changes in sulphation of chondroitin sulphate proteoglycan during myogenesis of human muscle cultures

The synthesis and secretion of chondroitin sulphate proteoglycan (CSPG) was examined in human muscle cultures during myogenesis prior to myoblast fusion and following myotube formation. Results from this study demonstrate that the major CSPG secreted into the medium had a Kav of 0.15 on Sephacryl 500 (exclusion limit of 10(7) Da) and contained predominantly unsulphated residues in mononucleated cell cultures but these became increasingly sulphated in postfusion cultures. Fibroblasts synthesised small amounts of a smaller molecular weight CSPG indicating that the Kav 0.15 proteoglycan is solely synthesised by cells of the myogenic lineage. These findings illustrate that sulphation of CSPG is developmentally regulated during myogenesis of human muscle cells grown under differentiating conditions.