Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor

We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent.     

Three-dimensional culture of human meniscal cells: Extracellular matrix and proteoglycan production

Background  

The meniscus is a complex tissue whose cell biology has only recently begun to be explored. Published models rely upon initial culture in the presence of added growth factors. The aim of this study was to test a three-dimensional (3D) collagen sponge microenvironment (without added growth factors) for its ability to provide a microenvironment supportive for meniscal cell extracellular matrix (ECM) production, and to test the responsiveness of cells cultured in this manner to transforming growth factor-β (TGF-β).     

Cartilage-derived factor (CDF) I. Stimulation of proteoglycan synthesis in rat and rabbit costal chondrocytes in culture

A protease-sensitive factor was extracted from fetal bovine cartilage with 1 M guanidine hydrochloride and partially purified by gel filtration on Bio-Gel A 0.5 m and CM-Sephadex column chromatography. This cartilage-derived factor (CDF) stimulated proteoglycan synthesis in rat and rabbit costal chondrocytes in culture, as shown by increased incorporation of ³⁵SO₄^?2, [³H]-glucosamine and [³H]serine into material precipitated with cetylpyridinium chloride. In addition, CDF stimulated the synthesis of sulfated glycosaminoglycans in a dose-dependent manner. These findings suggest that CDF is involved in the control of chondrogenesis.     

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.     

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.     

Keratan sulfate proteoglycan in rabbit compact bone is bone sialoprotein II

A keratan sulfate proteoglycan was isolated under denaturing conditions from the mineral compartment of rabbit cortical bone. This small proteoglycan (Kd = 0.39 on Superose 6, Mr approximately 20,000 on sodium dodecyl sulfate gels) contained small keratan sulfate chains that were distinctly bimodal in size. The keratanase and endo-beta-galactosidase digestible glycosaminoglycan chains were O-linked to a core protein of Mr approximately 80,000. This core protein had several properties in common with the bone sialoprotein II molecule of bovine and human bone including: a closely spaced doublet band on sodium dodecyl sulfate electrophoresis gels; a high staining intensity with Stains All that was greatly diminished by neuraminidase; a significant amount of small O-linked oligosaccharides; and an amino-terminal amino acid sequence that was nearly identical to human bone sialoprotein II. (In contrast, bone sialoprotein II in human, bovine, and rat bone does not appear to have any keratan sulfate chains.) Antiserum made against the keratan sulfate proteoglycan reacted with its core protein on electrotransfers from sodium dodecyl sulfate-polyacrylamide gels.     

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.     

Tunicamycin inhibits proteoglycan synthesis in rat ovarian granulosa cells in culture

The effects of tunicamycin, an inhibitor of N-linked oligosaccharide biosynthesis, on the synthesis and turnover of proteoglycans were investigated in rat ovarian granulosa cell cultures. The synthesis of proteoglycans was inhibited (40% of the control at 1.6 micrograms/ml tunicamycin) disproportionately to that of general protein synthesis measured by [3H]serine incorporation (80% of control). Proteoglycans synthesized in the presence of tunicamycin lacked N-linked oligosaccharides but contained apparently normal O-linked oligosaccharides. The dermatan sulfate and heparan sulfate chains of the proteoglycans had the same hydrodynamic size as control when analyzed by Sepharose 6B chromatography. However, the disulfated disaccharide content of the dermatan sulfate chains was reduced by tunicamycin in a dose-dependent manner, implying that the N-linked oligosaccharides may be involved in the function of a sulfotransferase which is responsible for sulfation of the iduronic acid residues. When [35S]sulfate and [3H]glucosamine were used as labeling precursors, the ratio of 35S/3H in chondroitin 4-sulfate was reduced to approximately 50% of the control by tunicamycin, indicating that the drug reduced the supply of endogenous sugar to the UDP-N-acetylhexosamine pool. Neither transport of proteoglycans from Golgi to the cell surface nor their turnover from the cell surface (release into the medium, or internalization and subsequent intracellular degradation) was affected by the drug. Addition of mannose 6-phosphate to the culture medium did not alter the proteoglycan turnover. When granulosa cells were treated with cycloheximide, completion of proteoglycan diminished with a t1/2 of approximately 12 min, indicating the time required for depleting the core protein precursor pool. The glycosaminoglycan synthesizing capacity measured by the addition of p-nitrophenyl-beta-xyloside, however, lasted longer (t1/2 of approximately 40 min). Tunicamycin decreased the core protein precursor pool size in parallel to decreased proteoglycan synthesis, both of which were significantly greater than the inhibition of general protein synthesis. This suggests two possibilities: tunicamycin specifically inhibited the synthesis of proteoglycan core protein, or more likely a proportion of the synthesized core protein precursor (approximately 50%) did not become accessible for post-translational modifications, and was possibly routed for premature degradation.     

The role of proteoglycan synthesis in the development of sea urchins : II. The effect of administration of exogenous proteoglycan

The role of proteoglycan synthesis in the early development of sea urchins was studied by treating the embryos with a variety of inhibitors of proteoglycan synthesis and also with proteoglycan of exogenous origin. Developmental arrest at the blastula stage caused by p-nitrophenyl-β- -xyloside (p-NP-xyl) was cancelled by the administration of proteoglycan of exogenous origin. Proteoglycan of post-gastrular origin was effective for cancellation, but proteoglycan of blastular origin was not effective. This suggests that the hindrance of development by p-NP-xyl was due to the lack of proteoglycan synthesis at the late blastula stage. On the other hand, developmental arrest caused by 2-deoxy- -glucose (deoxy-glucose) and Na-selenate was not cancelled by the administration of proteoglycan under any condition tested. Apart from the cancelling action of proteoglycan, solely administered proteoglycan caused a blockage in development at a stage corresponding to the stage from which it was extracted, indicating that proteoglycan may be characterized by a kind of stage specificity.     

Effect of exogenous extracellular matrices on proteoglycan synthesis by cultured rabbit costal chondrocytes

We examined the effect of an extracellular matrix (ECM), produced by either bovine corneal endothelial (BCE) cells or mouse PF HR-9 teratocarcinoma cells, on the ability of rabbit costal chondrocytes to re-express their phenotype once confluent. Rabbit chondrocytes seeded at low densities and grown on plastic tissue culture dishes produced a heterogeneous cell population composed of both overtly differentiated and poorly differentiated chondrocytes, as well as fibroblastic cells. On the other hand, cultures grown on BCE-ECM- or HR-9-ECM-coated dishes reorganized into a homogeneous cartilage-like tissue composed of round cells surrounded by a refractile matrix that stained intensely with alcian green. The cell ultrastructure and that of their pericellular matrix were similar to those seen in vivo. The differentiation of chondrocyte cultures grown on the ECMs vs. plastic was reflected by a two- to three-fold increase in the maximal rate of incorporation of [35S]sulfate and [3H]glucosamine into proteoglycans. Furthermore, the ratio of 35S-labeled proteoglycans incorporated in the cell layer vs. those released into the medium was 1.5-2.5-fold higher when cultures were grown on the ECMs than on plastic. This suggests that the ECMs stimulate the incorporation of newly synthesized proteoglycans into a cartilaginous matrix. Since chondrocyte cultures grown on BCE-ECM or HR-9-ECM give rise to a homogeneous cartilage-like tissue even when seeded at low cell densities, they provide a model for the study of cell-substrate interactions that are responsible for the maintenance of the differentiated phenotype of chondrocytes.     

Increased microalbuminuria in diabetic rats is independent of angiotensin II or glomerular proteoglycan synthesis.

Increased microalbuminuria is seen early in rats with both streptozotocin-induced and genetic (Bio-Breeding) diabetes. This study examines the roles of angiotensin II-dependent mechanism(s) and sulfation of glomerular proteoglycans in this phenomenon, as both processes have been implicated by several lines of circumstantial evidence. Anionic sites in the glomerular basement membrane, attributed to the presence of heparan sulfate, were quantitated by polyethyleneimine staining at 15, 21, and 70 days of diabetes in rats treated with streptozotocin, with or without insulin, and at 70 days in the Bio-Breeding rats. All diabetic rats developed increased microalbuminuria: control, 0.08 +/- 0.03 microgram/mL glomerular filtration rate, mean +/- SD; streptozotocin without insulin at 15 days, 0.92 +/- 0.06 microgram/mL (p < 0.05); streptozotocin with insulin at 21 days, 0.61 +/- 0.37 microgram/mL (p < 0.05 vs. control). At 70 days, both the Bio-Breeding and the streptozotocin rats sustained their microalbuminuria to the same degree (p < 0.05 vs. control). Enalapril (250 mg/L) in the drinking water of diabetic animals did not reduce the microalbuminuria. Although the polyethyleneimine-stained heparan sulfate sites decreased significantly in the streptozotocin rats, they remained unchanged in the Bio-Breeding rats. To determine the cause of reduced heparan sulfate staining, the in vitro synthesis and degree of sulfation of proteoglycans by glomeruli isolated from control and streptozotocin diabetic rat kidneys were compared. The amount of heparan sulfate synthesis and degree of sulfation were unchanged in diabetic glomeruli, although lower incorporation into the extracellular matrix and greater secretion into the medium were noted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycoprotein synthesis in explants of developing rabbit mammary gland in culture.

1. Explants of mammary glands of pregnant rabbits cultured in the absence of insulin, prolactin and cortisol incorporated [2-3H]mannose into lipid-linked mono- and oligo-saccharide and protein. 2. Inclusion of the hormones in the culture medium stimulated the incorporation of [2-3H]mannose into lipid-linked monosaccharide 4-fold, into lipid-linked oligosaccharide 4-fold and into protein 13-fold after 24 h in culture. 3. Addition of tunicamycin to the incubation medium completely inhibited the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and protein after an initial lag period of about 2h. Incorporation of this radiolabel into lipid-linked monosaccharide was increased 4-fold under these conditions. 4. Incorporation of [4,5-3H]leucine into protein was unaffected by the presence of tunicamycin. 5. Analysis of mannose-labelled protein by polyacrylamide-gel electrophoresis indicated that a major radiolabelled protein of apparent mol.wt. 65,000-70,000 was synthesized and approx. 70% of this protein appeared in the soluble fraction. 6. Glycosylation of the protein but not synthesis of its peptide backbone was sensitive to tunicamycin. 7. Possible origins of this glycoprotein synthetized when the tissue is stimulated to differentiate in culture are discussed.     

Differential effects of parathyroid hormone and somatomedin-like growth factors on the sizes of proteoglycan monomers and their synthesis in rabbit costal chondrocytes in culture

In the proteoglycans extracted from rabbit costal chondrocytes in culture, two populations of proteoglycans were distinguished by density gradient centrifugation under dissociative conditions. The major component was the faster sedimenting population (proteoglycan I), the putative 'cartilage-specific' proteoglycans, and the minor component was the slower sedimenting population (proteoglycan II). The monomeric size of proteoglycan I was closely related to the differentiation-state of chondrocytes and was a good marker of the differentiated chondrocytes. Treatment of the cultures with parathyroid hormone (PTH) induced an increase in the monomeric size of proteoglycan I. This increase was ascribed to an increase in the molecular size of the glycosaminoglycan chain in proteoglycan I. On the other hand, somatomedin-like growth factors, such as multiplication-stimulating activity (MSA) and cartilage-derived factor (CDF), did not affect the size of proteoglycan I, while they markedly stimulated the synthesis of proteoglycan I. In contrast, treatment with nonsomatomedin growth factors, such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), resulted in not only a decrease in glycosaminoglycan synthesis but also a slight decrease in size of proteoglycan I. However, synthesis and size of proteoglycan II were little affected by these agents. Thus, the present study clearly shows that PTH and somatomedin-like growth factors have differential functions in bringing about the expression of the differentiated phenotype of chondrocytes: PTH influences chain elongation and termination of glycosaminoglycans in proteoglycan I, while somatomedin-like growth factors affect primarily the synthesis and secretion of proteoglycan I.     

Intracellular features of type II procollagen and chondroitin sulfate proteoglycan synthesis in chondrocytes

The intracellular compartments of chondrocytes involved in the synthesis and processing of type II procollagen and chondroitin sulfate proteoglycan (CSPG) monomer were investigated using simultaneous double immunofluorescence and lectin localization reactions. Type II procollagen was distributed in vesicles throughout the cytoplasm, whereas intracellular precursors of CSPG monomer were accumulated in the perinuclear cytoplasm. In this study, cytoplasmic vesicles that stained intensely with antibodies directed against CSPG monomer but did not react with type II collagen antibodies, also were observed. A monoclonal antibody, 5-D-4, that recognizes keratan sulfate determinants was used to identify the Golgi complex (the site of keratan sulfate chain elongation). Staining with 5-D-4 was restricted to the perinuclear cytoplasm. The vesicles outside the perinuclear cytoplasm that stained intensely with antibodies to CSPG monomer did not react with 5-D-4. Fluorescent lectins were used to characterize further subcellular compartments. Concanavalin A, which reacts with mannose-rich oligosaccharides, did not stain the perinuclear region, but it did stain vesicles throughout the rest of the cytoplasm. Because mannose oligosaccharides are added cotranslationally, the stained vesicles throughout the cytoplasm presumably correspond to the rough endoplasmic reticulum. Wheat germ agglutinin, which recognizes N-acetyl-D-glucosamine and sialic acid (carbohydrates added in the Golgi), stained exclusively the perinuclear cytoplasm. By several criteria (staining with the monoclonal antibody 5-D-4 and with wheat germ agglutinin), the perinuclear cytoplasm seems to correspond to the Golgi complex. The cytoplasmic vesicles that react with anti-CSPG monomer and not with anti-type II collagen contain precursors of CSPG monomer not yet modified by Golgi-mediated oligosaccharide additions (because they are not stained with wheat germ agglutinin or with the anti-keratan sulfate antibody); these vesicles may have a unique function in the processing of CSPG.     

Sulfated tyrosines of thyroglobulin are involved in thyroid hormone synthesis.

Thyroid hormone synthesis is under the control of thyrotropin (TSH), which also regulates the sulfation of tyrosines in thyroglobulin (Tg). We hypothesized that sulfated tyrosine (Tyr[S]) might be involved in the hormonogenic process, since the consensus sequence required for tyrosine sulfation to occur was observed at the hormonogenic sites. Porcine thyrocytes, cultured with TSH but without iodide in the presence of [(35)S]sulfate, secreted Tg which was subjected to in vitro hormonosynthesis with increasing concentrations of iodide. A 63% consumption of Tyr[S] (1 residue) was observed at 40 atoms of iodine incorporated into Tg, corresponding to a 40% hormonosynthesis efficiency. In addition, hyposulfated Tg secreted by cells incubated with sodium chlorate was subjected to in vitro hormonosynthesis. With 0.5 Tyr[S] residue (31% of the initial content), the efficiency of the hormonosynthesis was 29%. In comparison, when hormonosynthesis was performed by cells, with only 0.25 Tyr[S] residue (16% of the initial content), the hormonosynthesis efficiency fell to 18%. These results show that there exists a close correlation between the sulfated tyrosine content of Tg and the production of thyroid hormones.