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).     

Proliferation-dependent changes of proteoglycan metabolism in arterial smooth muscle cells

Cultured arterial smooth muscle cells synthesize and secrete two types of sulfated proteoglycans designated as proteoglycan A and proteoglycan B. Proteoglycan A has been characterized as chondroitin sulfate-rich, whereas proteoglycan B was found to be dermatan sulfate-rich [Schmidt, A. & Buddecke, E. (1985) Eur. J. Biochem. 153, 260-273]. During the logarithmic growth phase, arterial smooth muscle cells incorporated about 3 times more [35S]sulfate into the total proteoglycans secreted into the culture medium than did non-dividing cells. When arterial smooth muscle cells stopped proliferating the ratio of [35S]proteoglycan A/B increased. No differences were detected in the respective molecular and chemical characteristics of purified proteoglycans A and B isolated from both proliferating and non-dividing cells. Regardless of the growth phase proteoglycan A had a molecular mass of about 280 kDa and contained 8-9 chondroitin sulfate-rich side chains. Proteoglycan B had a molecular mass of about 180 kDa and contained 6-7 dermatan sulfate-rich side chains. The [35S]methionine-labelled protein cores of proteoglycan A and B had a molecular mass of about 48 kDa, but were distinguishable by their specific reactions to monospecific antibodies. Proliferating cells endocytosed proteoglycan B at a rate up to 100% higher than that of non-dividing cells. In all growth phases proteoglycan A was endocytosed at a 10-fold lower rate than proteoglycan B.     

Cell-free translation of mRNA encoding an arterial smooth muscle cell proteoglycan core protein

The size and immunological reactivity of the primary gene products of a small non-aggregating dermatan sulfate proteoglycan from bovine and monkey arterial smooth muscle cells were examined after cell-free translation of mRNA. Antisera against the dermatan sulfate proteoglycans from bovine articular cartilage, DSPG II [Rosenberg et al. J. Biol. Chem. 260, 6304 (1985)] and human skin fibroblasts [Glossl et al. J. Biol. Chem. 259, 14144 (1984)] were used to show that the unmodified smooth muscle precursor core protein was immunologically related to both the cartilage and fibroblast core proteins. The size of the precursor core proteins within each species was identical regardless of the tissue source. Comparison of the precursor core proteins synthesized by primate and bovine cells revealed that the bovine core proteins were approximately 1500 Da larger than the primate core proteins as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A similar size difference was observed when the mature core proteins of monkey smooth muscle cells and bovine articular chondrocytes were compared after removal of the glycosaminoglycan chains. These results indicate that arterial smooth muscle cells synthesize a dermatan sulfate proteoglycan whose core protein is similar to, if not the same as, the cartilage and fibroblast dermatan sulfate proteoglycan core proteins. These core proteins may be encoded by the same gene that has diverged in size during speciation.     

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.     

Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII.

Sulfated tyrosine residues within recombinant human factor VIII were identified by [35S]sulfate biosynthetic labeling of Chinese hamster ovary cells which express human recombinant factor VIII. Alkaline hydrolysis of purified [35S]sulfate-labeled factor VIII showed that greater than 95% of the [35S]sulfate was incorporated into tyrosine. [3H]Tyrosine and [35S]sulfate double labeling was used to quantify the presence of 6 mol of tyrosine sulfate per mole of factor VIII. Amino acid sequence analysis of thrombin and tryptic peptides isolated from [35S]sulfate-labeled factor VIII demonstrated tyrosine sulfate at residue 346 in the factor VIII heavy chain and at residues 1664 and 1680 in the factor VIII light chain. In addition, the carboxyl-terminal half of the A2 domain contained three tyrosine sulfate residues, likely at positions 718, 719, and 723. Interestingly, all sites of tyrosine sulfation border thrombin cleavage sites. The functional importance of tyrosine sulfation was examined by treatment of cells expressing factor VIII with sodium chlorate, a potent inhibitor of tyrosine sulfation. Increasing concentrations of sodium chlorate inhibited sulfate incorporation into factor VIII without affecting its synthesis and/or secretion. However, factor VIII secreted in the presence of sodium chlorate exhibited a 5-fold reduction in procoagulant activity, although the protein was susceptible to thrombin cleavage. These results suggest that tyrosine sulfation is required for full factor VIII activity and may affect the interaction of factor VIII with other components of the coagulation cascade.     

Sulfation of two tyrosine-residues in human complement S-protein (vitronectin)

Human S-protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human hepatoma-derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine-labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S-protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S-protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S-protein. Examination of the S-protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S-protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S-protein may stabilize the conformation of S-protein in the absence of thrombin-antithrombin III complexes and may play a role in its binding to thrombin-antithrombin III complexes during coagulation.     

Degradation of endocytosed dermatan sulfate proteoglycan in human fibroblasts

Endocytosis and subsequent degradation of iduronic acid-rich small dermatan sulfate proteoglycan from fibroblast secretions were studied in human fibroblasts. Upon endocytosis of [3H]leucine- and [35S]sulfate-labeled proteoglycan release of free leucine was 10 to 15 times more rapid than that of inorganic sulfate. Within approximately 3 h a steady state was approached between transport of proteoglycan to the compartment of core protein degradation and release of free leucine. No such steady state could be found with respect to the dermatan sulfate chains. In the presence of benzyloxycarbonyl-Phe-Ala-diazomethylketone or of other SH-protease inhibitors the degradation of the protein moiety of endocytosed proteoglycan was much less inhibited than the degradation of the polysaccharide chain. Benzyloxycarbonyl-Phe-Ala-diazomethylketone did not affect the degradation of dermatan sulfate chains taken up by fluid phase endocytosis and the activities of all known dermatan sulfate-degrading enzymes. Percoll gradient centrifugation indicated that also in the presence of the protease inhibitor the partially degraded proteoglycan accumulated in dense lysosomes. The isolation of intracellular dermatan sulfate peptides and molecular size determinations of endocytosed dermatan sulfate proteoglycan supported the conclusion that a critical proteolytic step is required before the dermatan sulfate chain becomes accessible to hydrolytic enzymes.     

Heparan sulfate depletion within pulmonary fibroblasts: implications for elastogenesis and repair

We investigated the role of sulfated proteoglycans in regulating extracellular matrix (ECM) deposition in pulmonary fibroblast cultures. Fibroblast cultures were subject to pharmacologic and enzymatic interventions to modify sulfated proteoglycan levels. Native and proteoglycan-depleted fibroblasts were treated with porcine pancreatic elastase at 2-4-day intervals and the elastase-mediated release of fibroblast growth factor 2 (FGF-2) and glycosaminoglycans was determined. Elastase treatment released significantly less FGF-2 and glycosaminoglycans (GAG) from PG-depleted fibroblasts with respect to native cells. Equilibrium ligand binding studies indicated that 125I FGF-2 binding at both cell surface receptor and heparan sulfate proteoglycan sites was reduced to different extents based on the method of proteoglycan depletion. Quantitation of elastin protein and message levels indicated that biological sulfation is required for the proper incorporation of tropoelastin into the extracellular matrix. These results suggest that sulfated proteoglycans play a central role in modulating pulmonary fibroblast extracellular matrix composition and are important mediators of elastolytic injury.     

Proteoglycans in primate arteries. III. Characterization of the proteoglycans synthesized by arterial smooth muscle cells in culture

Near confluent monolayers of arterial smooth muscle cells derived from Macaca nemestrina were labeled with Na2[35S]O4 and the newly synthesized proteoglycans present in the culture medium and cell layer were extracted with either 4 M guanidine HCl (dissociative solvent) or 0.5 M guanidine HCl (associative solvent) in the presence of protease inhibitors. The proteoglycans in both compartments were further purified by cesium chloride density gradient ultracentrifugation. Two size classes of proteoglycans were observed in the medium as determined by chromatography on Sepharose CL-2B. The large population (Kav = 0.31) contained predominantly chondroitin sulfate chains with Mr = approximately 40,000. The smaller population (Kav = 0.61) contained dermatan sulfate chains of similar Mr (approximately 40,000). When tested for their ability to aggregate, only proteoglycans in the large-sized population were able to aggregate. A chondroitin sulfate containing proteoglycan with identical properties was isolated from the cell layer. In addition, the cell layer contained a dermatan sulfate component which eluted later on Sepharose CL-2B (Kav = 0.78) than the dermatan sulfate proteoglycan present in the medium. Electron microscopy of the purified proteoglycans revealed a bottlebrush structure containing a central core averaging 140 nm in length with an average of 8 to 10 side projections. The length of the side projections varied but averaged between 70 and 75 nm. Similar bottlebrush structures were observed in the intercellular matrix of the smooth muscle cell cultures after staining with Safranin 0. This culture system provides a model to investigate parameters involved in the regulation of synthesis and degradation of arterial proteoglycans.     

Identification of chick corneal keratan sulfate proteoglycan precursor protein in whole corneas and in cultured corneal fibroblasts.

The precursor protein to the chick corneal keratan sulfate proteoglycan was identified by immunoprecipitation with antiserum to its core protein from lysates of [35S]methionine-pulsed corneas and corneal fibroblasts in cell culture. Antiserum to the keratan sulfate proteoglycan immunoprecipitated a doublet of Mr 52,000 and 50,000 and minor amounts of a Mr 40,000 protein from pulsed corneas. Pulse-chase experiments, which permitted the conversion of the precursor proteins to proteoglycans and digestion of the glycosaminoglycans on immunoprecipitated proteoglycans with keratanase or chondroitinase ABC, showed that the Mr 52,000-50,000 doublet was converted to a keratan sulfate proteoglycan and the Mr 40,000 protein was converted to a chondroitin sulfate proteoglycan. Chick corneal fibroblasts in cell culture primarily produced the smaller (Mr50,000) precursor protein, and in the presence of tunicamycin the precursor protein size was reduced to Mr35,000, which indicates that the core protein contains approximately five N-linked oligosaccharides. Pulse-chase experiments with corneal fibroblasts in culture showed that the precursor protein was processed and secreted into the medium. However, its sensitivity to endo-beta-galactosidase and resistance to keratanase indicate that the precursor protein was converted to a glycoprotein with large oligosaccharides and not to a proteoglycan. This suggests that, although the precursor protein for the proteoglycan is produced in cultured corneal fibroblasts, the sulfation enzymes for keratan sulfate may be absent.     

Inventory of human skin fibroblast proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulphate proteoglycans.

Heparan sulphate and chondroitin/dermatan sulphate proteoglycans of human skin fibroblasts were isolated and separated after metabolic labelling for 48 h with 35SO4(2-) and/or [3H]leucine. The proteoglycans were obtained from the culture medium, from a detergent extract of the cells and from the remaining ''matrix'', and purified by using density-gradient centrifugation, gel and ion-exchange chromatography. The core proteins of the various proteoglycans were identified by electrophoresis in SDS after enzymic removal of the glycosaminoglycan side chains. Skin fibroblasts produce a number of heparan sulphate proteoglycans, with core proteins of apparent molecular masses 350, 250, 130, 90, 70, 45 and possibly 35 kDa. The major proteoglycan is that with the largest core, and it is principally located in the matrix. A novel proteoglycan with a 250 kDa core is almost entirely secreted or shed into the culture medium. Two exclusively cell-associated proteoglycans with 90 kDa core proteins, one with heparan sulphate and another novel one with chondroitin/dermatan sulphate, were also identified. The heparan sulphate proteoglycan with the 70 kDa core was found both in the cell layer and in the medium. In a previous study [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661] it was suggested that skin fibroblasts produce a proteoglycan form of the transferrin receptor. However, the core protein of the major heparan sulphate proteoglycan now purified does not resemble this receptor, nor does it bind transferrin. The principal secreted proteoglycans are the previously described large chondroitin sulphate proteoglycan (PG-L) and the small dermatan sulphate proteoglycans (PG-S1 and PG-S2).     

Occurrence of the HNK-1 epitope (3-sulfoglucuronic acid) in PC12 pheochromocytoma cells, chromaffin granule membranes, and chondroitin sulfate proteoglycans

After biosynthetic labeling of sulfated glycoproteins in rat and goldfish brain and PC12 pheochromocytoma cells with sodium [35S]sulfate, it was observed that all of the bands reactive with the HNK-1 antibody on immunoblots of sodium dodecyl sulfate-polyacrylamide gels corresponded with sulfate-labeled proteins detected by fluorography. These results support data from other studies, which indicate that the HNK-1 epitope is a 3-sulfo-glucuronic acid residue. In addition to its presence in a wide range of nervous tissue glycoproteins, the HNK-1 epitope was also detected in chromaffin granule membranes, chondroitinase ABC, and in chondroitin sulfate proteoglycans of brain, cartilage, and chondrosarcoma. However, it is not present in the heparan sulfate proteoglycan of brain, or in either of two chondroitin sulfate/dermatan sulfate proteoglycans in the chromaffin granule matrix.     

Increased activity of chondroitin sulfate-synthesizing enzymes during proliferation of arterial smooth muscle cells

Cultured arterial smooth muscle cells incorporate [35S]sulfate into the extracellular chondroitin sulfate/dermatan sulfate containing proteoglycans at a higher rate in the phase of logarithmic growth than do non-dividing cells. The cell growth-dependent decrease in 35S incorporation with increasing cell density is accompanied by a decrease in the activity of chondroitin sulfate-synthesizing enzymes. The specific activity of xylosyl transferase, N-acetylgalactosaminyl transferase I and chondroitin sulfotransferase declines as the cells proceed from low to high densities. The corresponding correlation coefficients are 0.86, 0.91 and 0.89. The ratio of C-6OH/C-4OH sulfation of chondroitin shows a cell proliferation-dependent decrease indicating an inverse correlation of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase activity. The observed changes in the expression of enzyme activities are thought to have some implications in the pathogenesis of arteriosclerosis, the initial stages of which are characterized by proliferation of arterial smooth muscle cells.     

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.     

Activation of heparin cofactor II by fibroblasts and vascular smooth muscle cells

Inhibition of thrombin by heparin cofactor II (HCII) is accelerated by dermatan sulfate, heparan sulfate, and heparin. Purified HCII or defibrinated plasma was incubated with washed confluent cell monolayers, 125I-thrombin was added, and the rate of formation of covalent 125I-thrombin-inhibitor complexes was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Fibroblasts and porcine aortic smooth muscle cells accelerated inhibition of thrombin by HCII 2.3-7.5-fold but had no effect on other thrombin inhibitors in plasma. Human umbilical vein endothelial cells and mouse macrophage-derived cells did not accelerate the thrombin-HCII reaction. IMR-90 normal human fetal lung fibroblasts treated with heparinase or heparitinase accelerated the thrombin-HCII reaction to the same degree as untreated cells. In contrast, treatment with chondroitinase ABC almost totally abolished the ability of these cells to activate HCII while chondroitinase AC had little or no effect, suggesting that dermatan sulfate was responsible for the activity observed. [35S]Sulfate-labeled proteoglycans were isolated from IMR-90 fibroblast monolayers and conditioned medium and fractionated into two peaks on Sepharose CL-2B. The lower Mr proteoglycans contained 74-76% dermatan sulfate and were 11-25 times more active with HCII than the higher Mr proteoglycans which contained 68-97% heparan sulfate. The activity of the lower Mr proteoglycans decreased 70-90% by degradation of the dermatan sulfate component with chondroitinase ABC. These results confirm that dermatan sulfate proteoglycans are primarily responsible for activation of HCII by IMR-90 fibroblasts. We suggest that HCII may inhibit thrombin when plasma is exposed to vascular smooth muscle cells or fibroblasts.     

Proteoglycans synthesized by cultured fibroblasts derived from normal and inflamed human gingiva

Summary The in vitro proliferations rates and ptoteoglycans synthesized by adult human gingival fibroblasts derived from six age- and sex-matched donors of healthy and chronically inflamed gingiva were analyzed. Fibroblasts from inflamed gingiva demonstrated a slower growth rate than cells from healthy tissue. The rate of incorporation of [³⁵S]sulfate into cell layer-associated proteoglycans and the release of these macromolecules into the culture medium did not differ appreciably between the two groups of cells. Similarly, no detectable differences in the overall charge of the proteoglycans synthesized by normal and inflamed gingival fibroblasts, as assessed by their elution from DEAE-Sephacel, were noted. However, sepharose CL-4B chromatography revealed that the medium-associated proteoglycans made by the inflamed tissue fibroblasts were depleted in one species of chondroitin sulfate proteoglycans and contained more dermatan sulfate than did control cells. In addition, the intracellular proteoglycan pool was found to be greatly diminished in the inflamed tissue fibroblast cell layers. Glycosaminoglycan analysis of the proteoglycans confirmed these observations. Compared to normal gingival fibroblasts, the inflamed tissue fibroblasts released less heparan sulfate into the medium. Additionally, increased levels of dermatan sulfate and depleted amounts of chondroitin sulfate in the medium of inflamed gingival cells were noted. The observed changes were stable through several transfers in culture and indicate that chronically inflamed tissue may contain fibroblasts mainfesting a heritable phenotype differing from fibroblasts in normal connective tissue. P. Mark Bartold was supported by a C. J. Martin Fellowship for the National Health and Medical Research Council of Australia. This work was also supported by grants DE-03301 and DE-02600 from the National Institutes of Health, Bethesda, MD.     

Tyrosine sulfation: a post-translational modification of proteins destined for secretion?

Protein sulfation was studied in germ-free rats by prolonged in vivo labeling with [35S]sulfate. Specific sets of sulfated proteins were observed in all tissues examined, in leucocytes, and in blood plasma. No protein sulfation was detected in erythrocytes. Analysis of the type of sulfate linkage showed that sulfated proteins secreted into the plasma contained predominantly tyrosine sulfate, whereas sulfated proteins found in tissues contained largely carbohydrate sulfate. This implies some kind of selection concerning the intracellular processing, secretion, turnover or re-uptake of sulfated proteins which is responsible for the enrichment of tyrosine-sulfated proteins in the plasma.     

Interaction of small dermatan sulfate proteoglycan from fibroblasts with fibronectin

Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4 degrees C, DS-PG core protein was uniformly distributed over the cell surface. At 37 degrees C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength. Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides containing either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.     

Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.