Heparin and endothelial cell growth factor modulate collagen and proteoglycan production in human smooth muscle cells

The effects of heparin and endothelial cell growth factor (ECGF) on extracellular matrix production were examined in human iliac smooth muscle cells. The cells were grown in (a) medium supplemented with heparin (100 micrograms/ml) and ECGF (75 micrograms/ml), (b) medium supplemented with ECGF (75 micrograms/ml) alone, or (c) unsupplemented medium. In the presence of heparin and ECGF, collagen production was inhibited 91-95% as compared to cultures incubated with ECGF alone or without both supplemental factors. In contrast, the production of proteoglycans was elevated 2.5 fold in the presence of heparin and ECGF. Enzymatic digestion of the proteoglycans indicated that both large and small molecular weight chondroitin sulfate proteoglycans were markedly elevated, while dermatan sulfate and heparan sulfate proteoglycans were increased to a lesser extent. The results suggest that the combination of heparin and ECGF elicits potent modulation of extracellular matrix production, with divergent effects on collagen and proteoglycan synthesis.     

Proteoglycan biosynthesis in murine monocytic leukemic (M1) cells before and after differentiation

Murine monocytic leukemic (M1) cells were cultured in the presence of [3H]glucosamine and [35S]sulfate. Labeled proteoglycans were purified by anion exchange chromatography and characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with chemical and enzymatic degradation. M1 cells synthesize a single predominant species of proteoglycan which distributes almost equally between the cell and medium after 17 h labeling. The cell-associated proteoglycan has an overall size of about 135 kDa and contains three to five chondroitin sulfate chains (28-31 kDa each) attached to a chondroitinase-generated core protein of 28 kDa. The synthesis and subsequent secretion of this proteoglycan was enhanced 4-5-fold in cells induced to differentiate into macrophages. This was not a phenomenon of arrest in the G0/G1 stage of the cell cycle, since density inhibited undifferentiated cells arrested at this stage did not increase proteoglycan synthesis. The chondroitin sulfate chains contained exclusively chondroitin 4- and 6-sulfate; however, the ratio of these two disaccharides differed between the medium- and cell-associated proteoglycans, and changed during progression of the cells into a fully differentiated phenotype. Pulse-chase kinetics indicate the presence of two distinct pools of proteoglycan; one that is secreted very rapidly from the cell after a approximately 1-h lag, and a second pool that is turned over in the cell with a half-time of approximately 3.5 h. Subtle differences in the glycosylation patterns of the medium- and cell-associated species are consistent with synthesis of two pools. Papain digestion suggests that the chondroitin sulfate chains are clustered on a small protease resistant peptide. The data suggest that this proteoglycan is similar to the serglycin proteoglycan family.     

Tyrosine O-sulfate ester in proteoglycans

Tyrosine O-sulfate residues were detected in the protein core of sulfated proteoglycans. When cultured skin fibroblasts and arterial smooth muscle cells were incubated in the presence of [35S]sulfate, dermatan sulfate proteoglycan and chondroitin sulfate proteoglycan isolated from the culture medium contained tyrosine [35S]sulfate ester which accounted for 0.03%-0.82% of total 35S radioactivity incorporated into the sulfated proteoglycans. This corresponds to a tyrosine sulfation of every second (fibroblasts) and every 10th (smooth muscle cells) dermatan sulfate proteoglycan molecule. [3H]Tyrosine labeling of fibroblast dermatan sulfate proteoglycan gave a similar stoichiometry. However, the relative proportion of tyrosine [35S]sulfate in proteoglycans from arterial tissue was about 10 times higher than in that from cultured arterial cells. Pulse chase experiments with [35S]sulfate revealed that tyrosine sulfation is a late event in the biosynthesis of dermatan sulfate proteoglycan from fibroblasts and occurs immediately prior to secretion. Cultured skin fibroblasts from a patient with a progeroid variant (Kresse et al. 1987, Am. J. Hum. Gen. 41, 436-453) which exhibit a partial deficiency to synthesize dermatan sulfate proteoglycan were shown to form and to secrete a tyrosine-sulfated but glycosaminoglycan-free protein core, thus confirming a selective and independent [35S]sulfate labeling of the protein core.     

Proteoglycans synthesized in vitro by nude and normal mouse peritoneal macrophages

Peritoneal macrophages from nude mice were found to be functionally similar to 'activated' macrophages from normal mice. The objective of the present study was to characterize the proteoglycans synthesized and secreted in vitro by peritoneal macrophages isolated from nude and normal Balb/c mice and to investigate the relationship between macrophage 'activation' and changes in the proteoglycan patterns. Macrophages obtained by peritoneal lavage were seeded in Petri dishes. After 2 h incubation at 37 degrees C, the adherent cells (macrophages) were exposed to [35S]sulphate for the biosynthetic labelling of proteoglycans. After incubation, the cell and medium fractions were collected and analysed for proteoglycans and glycosaminoglycans. The glycosaminoglycans were identified and characterized by a combination of agarose gel electrophoresis and enzymatic degradation with specific mucopolysaccharidases. It was shown that 3/4 of the total 35S-labelled glycosaminoglycans were in the extracellular compartment after 24-48 h. The macrophages synthesized dermatan sulphate (68%), chondroitin sulphate (7%) and heparan sulphate (25%). Both cell and medium fractions of normal and nude mouse macrophages contained glycosaminoglycans with the same ratios, although the nude mouse macrophages synthesized 2-fold less glycosaminoglycans than the normal mouse macrophages. Lower levels of 35S-proteoglycans were also obtained from in vitro 'activated' macrophages, but the ratios of dermatan sulphate:chondroitin sulphate: heparan sulphate were altered in these cells as compared to the control. Furthermore, all the 35S-macromolecules found in the extracellular compartment of nude and normal control cells were of proteoglycan nature, in contrast to the medium fractions of 'activated' macrophages, which contain both intact proteoglycans and 'free' glycosaminoglycan chains. These results indicate that, at least as regards the proteoglycans and glycosaminoglycans, the nude mouse macrophages are not identical to the 'activated' macrophages from normal mice.     

Effects of hypoxia and hyperoxia on proteoglycan production by bovine pulmonary artery endothelial cells

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of oxygen tension on proteoglycan synthesis. Cells exposed to 3% O2 (hypoxia) for 72 h and then labeled with [35S]sulfate for 5 h accumulated significantly less [35S]proteoglycan in medium than cells exposed to 20% O2 (control). This decrease was due primarily to a reduction in heparan sulfate. Cells exposed to 80% O2 (hyperoxia) for 72 h secreted slightly more [35S]proteoglycan into medium than controls. Greater accumulation of chondroitin sulfate was responsible for the increase. The amount of cell-associated proteoglycan did not change significantly in cells cultured in 3% or 80% O2 as compared with control cells cultured in 20% O2. Proteoglycans produced by hypoxia- or hyperoxia-treated cells were found to be similar in size to proteoglycans produced by cells cultured at 20% O2. Glycosaminoglycan sulfation, as measured by ion-exchange chromatography, did not appear to change with varying oxygen tensions. Our results demonstrate that production of proteoglycans secreted by endothelial cells in culture is sensitive to oxygen tension.     

Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans.

The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with [35S]sulfate and [3H] glucosamine for 24 h and then extracted and analyzed. There was a 1.68 +/- 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of approximately 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of approximately 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 +/- 0.2-fold in media and 3.2 +/- 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.     

Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4–like heparan sulfate proteoglycan and a distinct chondroitin sulfate form

Proteoglycan accumulation by thioglycollate-elicited mouse peritoneal macrophages and a panel of murine monocyte-macrophage cell lines has been examined to determine whether these cells express plasma membrane-anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre-monocytes and is associated predominantly with the cell layer of mature macrophage-like cells. In the P388D1 cell line, the cell-associated chondroitin sulfate is largely present as a plasma membrane-anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell-associated heparan sulfate is composed of a proteoglycan fraction and protein-free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan-4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co-migrates with syndecan-4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage-like cell lines accumulate similar amounts of syndecan-4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage-like cells. Syndecan-1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells. © 1993 Wiley-Liss, Inc.     

Stimulation of sulfated-proteoglycan synthesis by forskolin in monolayer cultures of rabbit articular chondrocytes

Forskolin, a plant cardiotonic diterpene, stimulated proteoglycan biosynthesis by chondrocytes in monolayer culture. The quantitative increase in proteoglycans was dependent on the concentration of forskolin, but was relatively independent of the presence of serum. At forskolin concentrations that stimulated proteoglycan synthesis, a significant stimulation of adenylate cyclase and cAMP was also measured. The quantitative increase in proteoglycans was characterized, qualitatively, by an increased deposition of newly synthesized proteoglycan in the cell-associated fraction. An analysis of the most dense proteoglycans (fraction dA1) in the cell-associated fraction showed that more of the proteoglycans eluted in the void volume of a Sepharose CL-2B column, indicating that an increased amount of proteoglycan aggregate was synthesized in forskolin-treated cultures. The proteoglycan monomer dA1D1 secreted into the culture medium of forskolin-stimulated cultures overlapped in hydrodynamic size with that of control cultures, although cultures stimulated with forskolin and phosphodiesterase inhibitors produced even larger proteoglycans. The hydrodynamic size of 35SO4 and 3H-glucosamine-labelled glycosaminoglycans isolated from the dA1D1 fraction of the culture medium was greater in forskolin-treated chondrocytes, especially from those in which phosphodiesterase inhibitors had been added. These results indicated that forskolin, a direct activator of chondrocyte adenylate cyclase mimicked the effects of cAMP analogues on chondrocyte proteoglycan synthesis previously reported. These results implicate activation of adenylate cyclase as a regulatory event in the biosynthesis of cartilage proteoglycans, and more specifically in the production of hydrodynamically larger glycosaminoglycans.     

Enhanced sulfated-proteoglycan core protein synthesis by incubation of rabbit chondrocytes with recombinant transforming growth factor-beta 1

Rabbit articular chondrocytes were incubated with recombinant transforming-growth-factor-beta 1 (rhTGF-beta 1) and its effect on newly synthesized proteoglycan measured. rhTGF-beta 1 stimulated proteoglycan synthesis at a concentration as low as 5 ng/ml without further increases in radiosulfate incorporation up to 50 ng/ml. The quantitative increase in radiosulfate incorporation in rh-TGF-beta 1-treated chondrocytes was greater in the cell-associated culture compartment than in the medium compartment. rhTGF-beta 1 promoted an increased proteoglycan retention in the cell-associated compartment as evidenced by an increase in the t1/2 of retention from 8 h to 11 h. Specific enhanced synthesis of [35S]-methionine-labeled core proteins was seen in rh-TGF-beta 1-treated chondrocytes. rh-TGF-beta 1 increased the synthesis of the 2 core proteins derived from hydrodynamically large proteoglycans. They possessed apparent molecular weights of greater than 480 kD and 390 kD after 3-5% acrylamide gel electrophoresis. A compartmental analysis revealed that the cell-associated culture compartment contained only the larger of the 2 core proteins derived from large proteoglycans. Two other core proteins with apparent molecular weights 52 kD and 46 kD were also stimulated by rhTGF-beta 1. These results indicated that TGF-beta probably plays a significant role in stimulating proteoglycan core protein synthesis in articular chondrocytes and therefore may be an important growth factor in the restoration of cartilage extracellular matrix after injury.     

Analysis of the proteoglycans synthesized by corneal explants from embryonic chicken. I. Characterization of the culture system with emphasis on stromal proteoglycan biosynthesis

Corneal explants with scleral rims were freshly prepared from day 18 chicken embryos and incubated in vitro for 3 h in the presence of various radioactive precursors. Radiolabeled proteoglycans were isolated from the stromal tissue and culture medium for analysis. Two predominant proteoglycans were identified in corneal stroma. One contains dermatan sulfate and the other contains keratan sulfate; a structural analysis of each is reported in the accompanying paper (Midura, R.J., and Hascall, V.C. (1989) J. Biol. Chem. 264, 1423-1430). A minor keratan sulfate proteoglycan distinct from the major form, a small amount of heparan sulfate proteoglycan, and some sulfated glycoproteins were also detected in stromal extracts. The biosynthesis of the dermatan sulfate proteoglycan was stable in vitro and in ovo, whereas that of the major keratan sulfate proteoglycan was stable only in ovo. Various treatments were tried to maintain a high rate of keratan sulfate synthesis with time in culture. Cooling the corneal explants to 5 degrees C was the only treatment that reduced this decline in keratan sulfate synthesis in vitro to any significant extent. Three major proteoglycans were observed in the culture medium. Two were dermatan sulfate proteoglycan and appeared to be mainly derived from the scleral tissue surrounding the corneal explant. The third proteoglycan contained keratan sulfate. It was smaller in size and lower in charge density compared to the keratan sulfate proteoglycan found in the stroma, but both appeared to have similar core protein sizes. It seems likely that this proteoglycan was synthesized in the stroma and secreted into the medium. A small amount of heparan sulfate proteoglycan and some sulfated glycoproteins were also detected in the medium.     

Chondroitin sulfate and heparan sulfate proteoglycans of PC12 pheochromocytoma cells

We have isolated and characterized the cell-associated and secreted proteoglycans synthesized by a clonal line of rat adrenal medullary PC12 pheochromocytoma cells, which have been extensively employed for the study of a wide variety of neurobiological processes. Chondroitin sulfate accounts for 70-80% of the [35S] sulfate-labeled proteoglycans present in PC12 cells and secreted into the medium. Two major chondroitin sulfate proteoglycans were detected with molecular sizes of 45,000-100,000 and 120,000-190,000, comprising 14- and 105-kDa core proteins and one or two chondroitin sulfate chains with an average molecular size of 34 kDa. In contrast to the chondroitin sulfate proteoglycans, one major heparan sulfate proteoglycan accounts for most of the remaining 20-30% of the [35S] sulfate-labeled proteoglycans present in the PC12 cells and medium. It has a molecular size of 95,000-170,000, comprising a 65-kDa core protein and two to six 16-kDa heparan sulfate chains. Both the chondroitin sulfate and heparan sulfate proteoglycans also contain O-glycosidically linked oligosaccharides (25-28% of the total oligosaccharides) and predominantly tri- and tetraantennary N-glycosidic oligosaccharides. Proteoglycans produced by the original clone of PC12 cells were compared with those of two other PC12 cell lines (B2 and F3) that differ from the original clone in morphology, adhesive properties, and response to nerve growth factor. Although the F3 cells (a mutant line derived from B2 and reported to lack a cell surface heparan sulfate proteoglycan) do not contain a large molecular size heparan sulfate proteoglycan species, there was no significant difference between the B2 and F3 cells in the percentage of total heparan sulfate released by mild trypsinization, and both the B2 and F3 cells synthesized cell-associated and secreted chondroitin sulfate and heparan sulfate proteoglycans having properties very similar to those of the original PC12 cell line but with a reversed ratio (35:65) of chondroitin sulfate to heparan sulfate.     

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.     

Stimulation of human arterial smooth muscle cell chondroitin sulfate proteoglycan synthesis by transforming growth factor-beta

Summary Human platelet-derived transforming growth factor-beta (TGF-beta) is a cell-type specific promotor of proteoglycan synthesis in human adult arterial cells. Cultured human adult arterial smooth muscle cells synthesized chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans, and the percent composition of these three proteoglycan subclasses varied to some extent from cell strain to cell strain. However, TGF-beta consistently stimulated the synthesis of chondroitin sulfate proteoglycan. Both chondroitin 4- and chondroitin 6-sulfate were stimulated by TGF-beta to the same extent. TGF-beta had no stimulatory effect on either class of [³⁵S]sulfate-labeled proteoglycans which appeared in an approximately 1:1 and 2:1 ratio of heparan sulfate to dermatan sulfate of the medium and cell layers, respectively, of arterial endothelial cells. Human adult arterial endothelial cells synthesized little or no chondroitin sulfate proteoglycan. Pulse-chase labeling revealed that the appearance of smooth muscle cell proteoglycans into the medium over a 36-h period equaled the disappearance of labeled proteoglycans from the cell layer, independent of TGF-beta. Inhibitors of RNA synthesis blocked TGF-beta-stimulated proteoglycan synthesis in the smooth muscle cells. The incorporation of [³⁵S]methionine into chondroitin sulfate proteoglycan core proteins was stimulated by TGF-beta. Taken together, the results presented indicate that TGF-beta stimulates chondroitin sulfate proteoglycan synthesis in human adult arterial smooth muscle cells by promoting the core protein synthesis. Supported in part by grants from the Public Health Service, U.S. Department of Health and Human Services, Washington, DC (CA 37589 and HL 33842), RJR Nabisco, Inc., and Chang Gung Biomedical Research Foundation (CMRP 291).     

Biosynthesis and secretion of dermatan sulphate proteoglycans in cultures of human skin fibroblasts.

Fibroblasts in culture were incubated with [3H]leucine and [35S]sulphate for 1-24 h. A large glucuronic acid-rich and a small iduronic acid-rich dermatan sulphate proteoglycan were isolated with the use of isopycnic density-gradient centrifugation, ion-exchange and gel chromatography. After 3 h the accumulation in the cell layer of the small proteoglycan reached a steady state, whereas the large one continued to increase, albeit more slowly. In the medium both proteoglycans accumulated 'linearly', although the large one appeared somewhat later than the small one. The composition of the polysaccharide chains and the size of the protein cores did not vary during the experiment. The two proteoglycans were synthesized at approximately similar rates, but were distributed differently in the culture. The small proteoglycan was mainly confined to the medium, whereas the large one was found in the medium as well as in a cell-associated pool. There was an intracellular accumulation of iduronic acid-rich dermatan sulphate as free polysaccharides.     

Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture

Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [3H]-amino acids and [35S]sulfate. Two heparan-35SO4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan-35SO4 proteoglycan eluted with the column void volume and at a Kav of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan-35SO4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B Kav 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan-35SO4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan-35SO4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan-35SO4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan-35SO4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan-35SO4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B Kav 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963-970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans.     

Serglycin is the major secreted proteoglycan in macrophages and has a role in the regulation of macrophage tumor necrosis factor-alpha secretion in response to lipopolysaccharide

It has recently been shown that serglycin is essential for maturation of mast cell secretory granules. However, serglycin is expressed also by other cell types, and in this study we addressed the role of serglycin in macrophages. Adherent cells were prepared from murine peritoneal cell populations and from spleens, and analyzed for proteoglycan synthesis by biosynthetic labeling with [35S]sulfate. Conditioned media from serglycin-/- peritoneal macrophages and adherent spleen cells displayed a 65-80% reduction of 35S-labeled proteoglycans, compared with corresponding material from serglycin+/+ cells, indicating that serglycin is the dominant secretory proteoglycan in macrophages of these origins. In contrast, the levels of intracellular proteoglycans were similar in serglycin+/+ and serglycin-/- cells, suggesting that serglycin is not stored intracellularly to a major extent in macrophages. This is in contrast to mast cells, in which serglycin is predominantly stored intracellularly. Transmission electron microscopy revealed that the absence of serglycin did not cause any major morphological effects on peritoneal macrophages, in contrast to dramatic defects in intracellular storage vesicles in peritoneal mast cells. Several secretory products were not found to be affected by the lack of serglycin. However, the secretion of tumor necrosis factor-alpha in response to lipopolysaccharide stimulation was markedly higher in serglycin-/- cultures than in those of serglycin+/+. The present report thus demonstrates that serglycin is the major proteoglycan secreted by peritoneal macrophages and suggests that the macrophage serglycin may have a role in regulating secretion of tumor necrosis factor-alpha.     

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

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