Proteoglycan biosynthesis in relation to differentiation of cord blood monocytes in vitro

Human monocytes were obtained from umbilical cord blood and cultured in vitro. By morphological criteria, the neonatal monocytes developed into macrophage-like cells in the course of 3-5 days in culture. The cells were exposed to [35S]sulphate for 24 h, either from day 0-1 or day 9-10 in vitro. The 35S-labelled macromolecules recovered were mainly associated with the medium fraction (approximately 75%) in both day 1 and day 10 cultures. These secretory macromolecules were demonstrated by the use of chondroitinase ABC-digestions to contain predominantly chondroitin sulphate proteoglycan (CSPG). [35S]galactosaminoglycan chains from day 10 cultures were more highly sulphated than the corresponding day 1 species due to the appearance of (glucuronosyl-4,6-diS-N-acetylgalactosamine) disulphated disaccharide units. The galactosaminoglycan chains in neonatal CSPG were found to increase in Mr during cultivation in vitro; from mean Mr of 20,400 to 30,200 (n = 5) in day 1 and day 10 medium proteoglycans, respectively. The corresponding Mr values for adult monocyte [35S]galactosaminoglycan chains were 21,300 and 22,800. On the basis of the concomitant changes in cellular morphology and glycosaminoglycan structure, it is concluded that neonatal monocytes, like monocytes from adults, differentiate into macrophage-like cells in vitro.     

Turnover of proteoglycans by chick lens epithelial cells in cell culture and intact lens

Chick lens epithelial cells were cultured on plastic and type IV collagen substrata, and the confluent cultures were labeled continuously with [35S]sulfate for 20 h. Intact lenses were also labeled in the same way. 35S-Proteoglycans isolated from those cultures were compared for their molecular sizes and glycosaminoglycan compositions. The results have shown that: 1) Proteoglycans synthesized by cells on type IV collagen were significantly smaller than those by cells on plastic. 2) Proteoglycans of intact lens showed a broad distribution of molecular size and contained a high proportion of chondroitin sulfate in the medium fraction compared to those of the two cell cultures. In order to explain such differences between proteoglycans from cultures, label-chase experiments with [35S]sulfate were done for proteoglycans synthesized. 35S-Proteoglycans isolated at each chase time 0, 2.5, and 17 h) were compared and the following results were found: 1) The cell layers of both "plastic" and "type IV collagen" cultures contained glycosaminoglycan species predominantly at each chase time rather than proteoglycans. 2) Changes in the glycosaminoglycan compositions of medium fractions of cell cultures were observed during the chase period; in medium of the "plastic" culture, proteoheparan sulfate increased with chase time, whereas in medium of the "type IV collagen" culture, chondroitin sulfate glycosaminoglycan (not proteoglycan) increased with chase time. 3) In intact lens culture, lens capsule fraction at every chase time contained a proteoglycan unique in molecular size, which was not found in cell culture fractions. 4) All fractions from intact lens cultures contained a higher content of chondroitin sulfate at every chase time than the respective fractions from cell cultures. These results suggest that adhesion of the cells to type IV collagen or lens capsule influences the degradation and secretion of proteoglycans. In addition, they can account partially for the above-described differences in molecular sizes and glycosaminoglycan compositions between 35S-proteoglycans from various cultures continuously labeled with [35S]sulfate.     

Modulation of the expression of chondroitin sulfate proteoglycan in stimulated human monocytes

Proteoglycan biosynthesis was studied in human monocytes and monocyte-derived macrophages (MDM) after exposure to typical activators of the monocyte/macrophage system: interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). By morphological examination, both monocytes and MDM were stimulated by these activators. Treatment with IFN-gamma resulted in a slight decrease in the expression of [35S]chondroitin sulfate proteoglycan (CSPG) in both monocytes and MDM, whereas LPS treatment increased the [35S]CSPG expression 1.8 and 2.2 times, respectively. PMA, in contrast, decreased the CSPG expression 0.4 times in monocytes, whereas MDM were stimulated to increase the biosynthesis 1.9 times. An increase in the sulfate density of the chondroitin sulfate chains was evident following differentiation of monocytes into MDM due to the expression of disulfated disaccharide units of the chondroitin sulfate E type (CS-E). However, monocytes exposed to PMA did also express disaccharides of the chondroitin sulfate E type. Furthermore, the expression of CS-E in MDM was increased 2 times following PMA treatment. An inactive phorbol ester, phorbol 12,13-diacetate, did not affect the expression of CS-E in either monocytes or MDM when compared with control cultures, suggesting that protein kinase C-dependent signal pathways may be involved in the regulation of sulfation of CSPG. Exposure to LPS or IFN-gamma did not lead to any changes in the sulfation of the chondroitin sulfate chains.     

Interactions between extracellular matrix components and proteoglycans released from monocytesin vitro

³⁵S-labelled chondroitin sulfate proteoglycans isolated from conditioned media of cultured human monocytes (day 1in vitro) and monocyte-derived macrophages (day 6in vitro) were chromatographed on columns of immobilized fibronectin and collagen, respectively. The elution profiles prior to and after alkali treatment were compared with those of standards chondroitin 4-sulfate and chondroitin sulfate E and heparin. The day 6³⁵S-proteoglycans have a higher sulfate density than the day 1 species, but this difference did not affect the elution profiles after chromatography on collagen-Sepharose, whereas the day 6 proteoglycans bound more firmly than the day 1 fraction to fibronectin-Sepharose. The elution patterns obtained for these distinct proteoglycans closely resembled those of heparin and oversulfated chondroitin sulfate E standards, and clearly demonstrated the importance of sulfate density both for the affinity to fibronectin and collagen. Neither day 1 nor day 6³⁵S-proteoglycans were found to interact with hyaluronate.Abbreviations used CSPG chondroitin sulfate proteoglycan - GAG glycosaminoglycan - CS chondroitin sulfate - CS-E chondroitin 4,6 disulfate - MDM monocyte-derived macrophages     

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

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

Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate

A differentiated population of cells with metachromatically staining granules and surface IgE receptors was obtained from mouse bone marrow cultured for 2 weeks in the presence of conditioned medium derived from concanavalin A-stimulated splenocytes. The cells were found to incorporate large amounts of [35S]sulfate into an intracellular 35S-labeled proteoglycan of Mr approximately 200,000 containing a maximum of seven glycosaminoglycan side chains (Mr = 25,000). After chondroitinase ABC treatment of density gradient-purified [3H] serine-labeled proteoglycan, the resulting core was Mr approximately 26,000 as assessed by gel filtration. Two-dimensional cellulose acetate electrophoresis of beta-eliminated 35S-labeled glycosaminoglycan revealed a single type of glycosaminoglycan that migrated at the position of oversulfated chondroitin sulfate E from squid cartilage. Chondroitinase ABC degradation of the 35S-labeled glycosaminoglycan yielded two cleavage products in approximately equal molar amounts which co-migrated in both descending paper chromatography and high voltage paper electrophoresis with a monosulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and a disulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-6-di-O-sulfo-D-galactose. The release of some free [35S]sulfate from the oversulfated disaccharide with either chondro-4-sulfatase or chondro-6-sulfatase and the complete desulfation by their combined action established that the oversulfated disaccharide contained N-acetylgalactosamine-4,6-disulfate. The 35S]labeled proteoglycan of these unique IgE receptor-bearing and histamine-containing cells, therefore, is composed of chondroitin sulfate E rather than heparin glycosaminoglycan, and thus is the first identification of such an intracellular localized proteoglycan in a mammalian cell.     

Divergent regulation of proteoglycan and glycosaminoglycan free chain expression in human keratinocytes and melanocytes

Summary Keratinocytes and melanocytes, which together form units of structure and function within human epidermis, are known to differ in expression of autocrine growth factors, particularly those with heparin binding affinity. Because such cytokines could be regulated by the endogenous heparinlike glycosaminoglycan, heparan sulfate, proteoglycan synthesis was compared between human keratinocytes and melanocytes cultured from a common donor. Following steady-state isotopic labeling under conditions of active growth (low density cultures) and growth inhibition (high density cultures), the sulfated polymers were isolated from conditioned media and cell extracts. We found that keratinocytes produced substantially more sulfated glycosaminoglycans than did the melanocytes. There was no evidence for hyaluronic acid synthesis by the melanocytes. The majority of [³⁵S]-sulfate labeling was in the heparan sulfates of the keratinocytes and in the chondroitin sulfates of the melanocytes. During the transition from active growth to growth inhibition, there was increased heparan sulfate proteoglycan and free chain synthesis by keratinocytes but not by melanocytes, and chondroitin sulfate proteoglycan production declined in both cell lineages. The differences may reflect divergent evolution as each cell type came to exploit those complex polysaccharides in different ways to regulate molecular pathways of growth and differentiation. The coupling of growth inhibition with augmented synthesis of heparan sulfates observed for the keratinocytes suggests a regulatory role in growth factor signaling in that cell type.     

Proteoglycans in normal and neoplastic monocytes

35S proteoglycans produced by normal and neoplastic (U-937) monocytes after a 20-h pulse with [35S]sulfate in vitro have been isolated and compared. Both cell types produce exclusively chondroitin sulfate proteoglycan (CSPG), which are released into the medium and are not contained within the cells. The neoplastic cell-derived molecules were much larger in molecular size, due to the substitution of galactosaminoglycan chains, with an approximate Mr of 60,000. The corresponding chains in monocyte CSPG had an Mr of approx. 20,000. The latter chains were also found to be more sulfated than their neoplastic counterparts.     

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.     

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.     

Secreted and cellular proteochondroitin sulfates of a human B lymphoblastoid cell line contain different protein cores.

Proteoglycans of the human B lymphoblastoid cell line LICR-LON-HMy2 were metabolically labeled with [35S]sulfate. High-density fractions of 35S-labeled material separated by CsCl gradient ultracentrifugation were further purified by anion exchange chromatography and gel filtration. Two proteoglycans, isolated from cell lysates and culture supernatants, were characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with enzymatic degradation. Treatment with chondroitinase AC completely degraded the glycosaminoglycan moiety of the proteoglycans. Three to 4 chondroitin sulfate chains (average molecular mass = 26 kDa) were estimated for each of the two proteoglycans. Differences between the proteochondroitin sulfates (CSPG) were observed in the content of N-linked oligosaccharides. After chondroitinase AC treatment the resulting band in SDS-PAGE of the secreted CSPG was sensitive to treatment with endoglycosidase F (Endo F) which further reduced the molecular mass from 30 to 21.5 kDa, whereas the band of the cellular CSPG after chondroitinase AC treatment (molecular mass = 30 kDa) remained resistant to Endo F treatment. The composition of amino acids was different in the protein cores, suggesting differences in the primary structure. Both CSPG contained a high percentage of glycine and serine. For both CSPG a molecular mass of approximately 135 kDa was deduced from the hydrodynamic sizes of the glycosaminoglycan chains obtained after alkaline/borohydride treatment and the migration of the protein/oligosaccharide complexes in SDS-PAGE. 75% of all [35S]sulfate-labeled molecules were found in the culture supernatant and 25% in the cellular fraction. 35S-Labeled material in the culture supernatant consisted exclusively of intact CSPG, whereas 35S-Labeled molecules in the cellular preparation consisted largely of free chondroitin sulfate chains. Only 8.3% of the cellular material, isolated from the microsomal fraction, was intact CSPG. In pulse-chase experiments maximal secretion of CSPG was found after 4 h, comprising approximately 40% of totally synthesized CSPG. From these experiments we tentatively conclude that a small proportion of CSPG synthesized by LICR-LON-HMy2 cells is membrane-associated, a larger portion is secreted, and another portion is intracellularly degraded.     

The expression of proteoglycans in phorbol ester induced U-937 cells

U-937 monoblastic cells were differentiated into macrophage-like cells in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Control cells and differentiated cells were labeled with³⁵S-sulfate and were both found to produce exclusively chondroitin sulfate proteoglycan. No differences in glycosaminoglycan structure or macromolecular properties of the proteoglycans produced in the two different cell systems could be observed. However, the differentiated cells were found to have a lower capacity for chondroitin sulfate proteoglycan synthesis, both under ordinary experimental conditions, and when exposed to stimulators of glycosaminoglycan biosynthesis such as -d-xylosides.Abbreviations SDS sodium dodecyl sulfate - TPA 12-O-tetradecanoylphorbol-13-acetate - PG proteoglycan - GAG glycoaminoglycan - CS chondroitin sulfate - CSPG chondroitin sulfate proteoglycan - NASDAE naphthol AS-D acetate esterase     

Cellular distribution of the Ia-associated chondroitin sulfate proteoglycan

The Ia-associated chondroitin sulfate proteoglycan (CSPG) found in anti-Ia and anti-invariant chain immunoprecipitates was originally detected in [35S] sulfate-labeled extracts derived from unseparated populations of splenocytes. To determine whether the CSPG was produced only by a subpopulation of spleen cells, we examined various cell populations for their ability to produce the CSPG. We found that B lymphocytes were the predominant source of CSPG in the spleen. The synthesis of the Ia-associated CSPG in spleen cell cultures was not diminished by the depletion of T cells or adherent cells. Moreover, the CSPG was readily detected in lysates derived from the Lyb-5- B cell subsets of xid mice, splenocytes from athymic (nude) mice, and in vitro B cell hybridomas. Peritoneal exudate macrophages from indomethacin-treated mice were also found to be capable of producing the CSPG. In all of the studies performed to date, no dissociation of the synthesis of the CSPG from the synthesis of Ia was observed in any cell type. We therefore tentatively conclude that all cells that synthesize conventional Ia molecules also synthesize the CSPG. Finally, we have been able to use anion exchange chromatography to prepare proteoglycan-enriched fractions to isolate the CSPG. This purification step has allowed us to convincingly demonstrate that the CSPG can be labeled with amino acids, and is a necessary step for detecting amino acid-labeled CSPG. This purification step method was used in the accompanying report to begin a quantitative examination of the Ia/CSPG complex, to monitor the kinetics of CSPG synthesis and association with Ia, and to determine its subcellular localization.     

Effect ofin vitro differentiation on proteoglycan structure in cultured human monocytes

Parellel toin vitro differentiation of human monocytes into macrophage-like cells, the cells change their synthesis of glycosaminoglycans from chondroitin 4-sulfate to highly sulfated chondroitin sulfate, containing 4,6-disulfatedN-acetylgalactosamine units [Kolsetet al. (1983) Biochem J 210:661–67]. After exposure of monocyte cultures to [³⁵S]sulfate for 24h either from the onset of cultivation, prior to differentiation, or from day 4, after differentiation,³⁵S-macromolecules from medium and cell-layer were isolated and characterized. The cell-layer of day 5 cultures contained both proteoglycans and free polysaccharide chains, while the³⁵S-macromolecules present in the cell-layer of day 1 cultures and in medium of both monocytes and macrophage-like cells were almost exclusively of proteoglycan nature. Proteoglycans produced by macrophage-like cells were of larger size than the monocyte proteoglycans, most likely due to an increased polysaccharide chain length. These proteoglycans, in contrast to the monocyte-derived species, also showed affinity for fibronectin at physiological ionic strength.     

Inhibition of transmembrane calcium influx induces decrease in proteoglycan synthesis in immature rat sertoli cells

Beyond increased cAMP synthesis, calcium influx has been involved in signal transduction triggered by the gonadotropin follicle‐stimulating hormone (FSH), the main regulator of Sertoli cells functions. In order to delineate a possible involvement of calcium in the regulation of proteoglycan synthesis, we have examined the effect of low‐voltage‐activated calcium channel blocker verapamil on both [³⁵S]‐sulfate and [³H]‐glucosamine incorporation into proteoglycan molecules neosynthesized by cultured Sertoli cells from 20‐day‐old rats. Verapamil induced a dose‐ and time‐dependent decrease in labeling of both secreted and cell‐associated proteoglycans, as determined by quantitative solid‐phase assay. This effect was mimicked by the addition of the calcium chelator EGTA, suggesting that verapamil effect resulted from the inhibition of transmembrane calcium influx. The decrease in apparent proteoglycan synthesis appeared to be attributable primarily to a lowering of the glycanation process, as shown by experiments using an exogenous acceptor for glycosaminoglycan synthesis. Moreover, verapamil induced a decrease in relative proportion of heparan sulfate proteoglycans in the cell layer. Pulse‐chase kinetics demonstrated that verapamil also altered proteoglycan catabolism, leading to glycosaminoglycan retention in the cell layer and inhibiting the proteoglycan desulfation step. We conclude that intracellular calcium is essential to maintain Sertoli cell proteoglycan expression and could thus be involved in the repression of Sertoli cell cAMP‐dependent syntheses such as estradiol production. J. Cell. Biochem. 76:322–331, 1999. © 1999 Wiley‐Liss, Inc.     

Oversulfated chondroitin sulfate proteoglycan in cultured human peritoneal macrophages

Human peritoneal macrophages were cultured in vitro and labeled with [35S]-sulfate. Both on day 1 and day 6 in culture the cells were found to synthesize exclusively chondroitin sulfate proteoglycan, the main part (70%) being associated with the medium after a 20 hour pulse. The glycosaminoglycan chains were found to be oversulfated both after 1 and 6 days in culture, due to the presence of disulfated disaccharide units.     

Effects of platelet-derived growth factor and transforming growth factor-beta 1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells

Platelet-derived growth factor (PDGF) and transforming growth factor-beta 1 (TGF-beta 1) increase [35S]sulfate incorporation into proteoglycan (PG) by monkey arterial smooth muscle cells but have opposite effects on cell proliferation. The combination of these two growth regulatory peptides has an additive effect on PG synthesis but no effects on cell proliferation. The time course of sulfate incorporation after stimulation indicates that both growth factors cause maximal incorporation of sulfate into glycosaminoglycan chains by 12-18 h. The PG that is most affected is a large CSPG (Mr approximately 1.2 x 10(6)) which can be immunoprecipitated by an antibody against versican, a large CSPG synthesized by human skin fibroblasts. The hydrodynamic size of this molecule increases after PDGF and TGF-beta 1 stimulation, but the size of the core glycoprotein (Mr approximately 450,000) remains the same. Treatment with either growth factor leads to an increase in the amount of core glycoprotein for this PG. This increase correlates with an increase in the steady state level of mRNA identified by hybridization to a cDNA encoding versican. The two growth factors also increase the glycosaminoglycan chain length of this PG accounting for the greater hydrodynamic size of the molecule after stimulation. In contrast, PDGF and not TGF-beta 1 changes the composition of the glycosaminoglycan chains attached to this PG by doubling the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate. These results indicate that although both of these growth factors increase the net synthesis of a large versican like CSPG, they differ in their effects on the structure of the glycosaminoglycan chains. These post-translational modifications may relate to the growth state of the cells.     

Induction of chondroitin sulfate proteoglycan synthesis and secretion in lymphocytes and monocytes

The ability of mononuclear leukocytes to synthesize and secrete proteoglycans was evaluated. Using radiolabeling with H2 35SO4, it is shown that peripheral blood mononuclear cells (PBMC) and their major subpopulations (B cells, T cells, and monocytes), as well as mouse spleen cells, all secreted easily detectable proteoglycan. After 24-h labeling periods, 90% of macromolecular 35S could be detected in culture media. This material was primarily (greater than 95%) chondroitin-4-sulfate proteoglycan (CSPG). Production and secretion of CSPG could be stimulated more than 200% in PBMC and 300% in T cell populations by high concentrations of concanavalin A and phorbol 12- myristate-13-acetate; lipopolysaccharide induced a small (twofold) but reproducible increase in CSPG secretion by adherent mononuclear leukocytes. The CSPG secreted by PBMC was relatively small in size compared to chondrocyte CSPG (130,000 daltons vs. 2-4 million daltons) but possessed similar sizes of glycosaminoglycan chains and greater solubility in low ionic strength solutions. This sulfated polyanion, which was produced endogenously by leukocytes and was actively secreted, might function as a co-mediator or "second messenger" in certain immune responses.     

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.