heparitin-sulfate and sodium-chlorate

Topics: Animals; Antibodies, Viral; Chlorates; Chlorocebus aethiops; Dextran Sulfate; DNA Helicases; Flavivirus; Glycosaminoglycans; Heparin; Heparitin Sulfate; Inhibitory Concentration 50; Mice; Molecular Docking Simulation; Molecular Dynamics Simulation; RNA Helicases; Serine Endopeptidases; Suramin; Vero Cells; Viral Envelope Proteins; Viral Nonstructural Proteins; Virus Internalization; Virus Replication; Zika Virus; Zika Virus Infection

Embryonic stem (ES) cells continuously decide whether to maintain pluripotency or differentiate. While exogenous leukemia inhibitory factor and BMP4 perpetuate a pluripotent state, less is known about the factors initiating differentiation. We show that heparan sulfate (HS) proteoglycans are critical coreceptors for signals inducing ES cell differentiation. Genetic targeting of NDST1 and NDST2, two enzymes required for N-sulfation of proteoglycans, blocked differentiation. This phenotype was rescued by HS presented in trans or by soluble heparin. NaClO(3) (-), which reduces sulfation of proteoglycans, potently blocked differentiation of wild-type cells. Mechanistically, N-sulfation was identified to be critical for functional autocrine fibroblast growth factor 4 (FGF4) signaling. Microarray analysis identified the pluripotency maintaining transcription factors Nanog, KLF2/4/8, Tbx3, and Tcf3 to be negatively regulated, whereas markers of differentiation such as Gbx2, Dnmt3b, FGF5, and Brachyury were induced by sulfation-dependent FGF receptor (FGFR) signaling. We show that several of these genes are heterogeneously expressed in ES cells, and that targeting of heparan sulfation or FGFR-signaling facilitated a homogenous Nanog/KLF4/Tbx3 positive ES cell state. This finding suggests that the recently discovered heterogeneous state of ES cells is regulated by HS-dependent FGFR signaling. Similarly, culturing blastocysts with NaClO(3) (-) eliminated GATA6-positive primitive endoderm progenitors generating a homogenous Nanog-positive inner cell mass. Functionally, reduction of sulfation robustly improved de novo ES cell derivation efficiency. We conclude that N-sulfated HS is required for FGF4 signaling to maintain ES cells primed for differentiation in a heterogeneous state. Inhibiting this pathway facilitates a more naïve ground state.

Topics: Amidohydrolases; Animals; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Chlorates; Embryonic Stem Cells; Flow Cytometry; Fluorescent Antibody Technique; Heparitin Sulfate; Homeodomain Proteins; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nanog Homeobox Protein; Octamer Transcription Factor-3; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sulfotransferases

Cationic antimicrobial peptides (CAPs) with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs.. Various cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB) and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated.. We show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity.. Our results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis.

Topics: Animals; Cattle; Cell Line, Tumor; Chlorates; CHO Cells; Cricetinae; Cricetulus; Drug Synergism; Heparin; Heparitin Sulfate; HT29 Cells; Humans; Lactoferrin; Lymphoma; Neoplasms; Peptide Fragments; Protein Structure, Secondary

Glycosaminoglycans (GAGs) have been implicated in the regulation of outflow resistance of aqueous humor flow through the trabecular meshwork (TM). Their role was further investigated by assessment of the effects of chlorate, an inhibitor of sulfation, and beta-xyloside, which provides a competitive nucleation point for addition of disaccharide units, in anterior segment perfusion culture.. Outflow facility was measured in perfused porcine and human anterior organ cultures treated with 20 or 50 mM sodium chlorate, or 1 mM beta-xyloside. Perturbation of extracellular matrix (ECM) components was assessed in paraffin-embedded sections by immunofluorescence and confocal microscopy. Parallel experiments were conducted on cultured TM cells.. Outflow facility increased in porcine eyes with chlorate (3-fold) and beta-xyloside (3.5-fold) treatments. In human eyes, outflow increased approximately 1.5-fold and took longer (>48 hours) to occur. By confocal microscopy, immunostaining for chondroitin and heparan sulfates was observed on edges of human TM beams in nontreated eyes, with intense staining in the juxtacanalicular tissue (JCT) region. In treated eyes, staining of beam edges was severely reduced and was instead found in plaques. Chlorate treatment resulted in a striated pattern of GAG staining in the human JCT region. Fibronectin immunostaining was altered in beta-xyloside-treated eyes, whereas in cell culture, chlorate induced formation of thick fibronectin fibrils, to which tenascin C colocalized.. Disrupting GAG chain biosynthesis increased outflow facility in perfusion culture and induced atypical ECM molecule interactions in cell culture. This study provides direct evidence of the critical role of GAG chains in regulating outflow resistance in human TM.

Topics: Aged; Aged, 80 and over; Animals; Aqueous Humor; Cells, Cultured; Chlorates; Chondroitin Sulfates; Extracellular Matrix; Female; Fibronectins; Fluorescent Antibody Technique, Indirect; Glycosaminoglycans; Glycosides; Heparitin Sulfate; Humans; Hyaluronic Acid; Male; Microscopy, Confocal; Middle Aged; Organ Culture Techniques; Swine; Tenascin; Trabecular Meshwork

Sulphated glycosaminoglycans, such as heparan sulphate, have been shown to be essential for the infectivity of many organisms. The aims of this study were to verify the role of sulphated glycosaminoglycans in chlamydial infection and to investigate whether they are present on chlamydia or chlamydial host cells. The effect of undersulphation of host cells and chlamydial elementary bodies was examined using sodium chlorate. Also studied was whether any inhibitory effect was reversible. The results strongly suggest that Chlamydia trachomatis does not produce heparan sulphate and that heparan sulphate of the host cell is necessary and sufficient to mediate chlamydial infection. The essential role played by the sulphate constituents of the host-cell glycosaminoglycan in the infectivity of LGV serovars, and to a lesser extent of serovar E, was also confirmed.

Topics: Animals; Cell Line; Chlamydia Infections; Chlamydia trachomatis; Chlorates; HeLa Cells; Heparitin Sulfate; Humans; Mice; Proteoglycans; Serotyping; Sulfur

Recognition events between hematopoietic progenitor cells (HPC) and bone marrow endothelial cells (BMEC) initiate homing of HPC to the bone marrow. The chemokine SDF-1 is present on BMEC and plays a crucial role in bone marrow engraftment. We studied the role of proteoglycans (PGs) on BMEC in binding and presentation of SDF-1. SDF-1 mRNA was present in three human BMEC cell lines. Competition experiments showed that 125I-SDF-1 alpha binding to the BMEC cell line 4LHBMEC was inhibited by heparins, heparan sulfate (HS) intestinal mucosa, chondroitin and dermatan sulfate (CS/DS), but not by HS bovine kidney. Pretreatment of 4LHBMEC with glycosaminoglycan (GAG)-degrading enzymes or sodium chlorate demonstrated that SDF-1 bound to both HSPGs and CS/DSPGs in a sulfation-dependent manner, as determined with an SDF-1 antibody recognizing the CXCR4-binding site. 4LHBMEC bound four-fold more SDF-1 than HUVEC. Isolated endothelial PGs did not bind SDF-1 in a filter or microplate-binding assay, suggesting the necessity of membrane association. In flow adhesion experiments, endothelial arrest of CXCR4+ KG-1 and not of CXCR4- KG-1a cells increased significantly when SDF-1 was presented on 4LHBMEC. In conclusion, SDF-1 is produced by BMEC and binds to the BMEC cell surface via HS and CS/DS-GAGs, thereby presenting its CXCR4 binding site to HPC contributing to their arrest.

Topics: Animals; Bone Marrow Cells; Cattle; Chemokine CXCL12; Chemokines, CXC; Chlorates; Chondroitin Sulfates; Dermatan Sulfate; DNA Primers; Endothelium, Vascular; Flow Cytometry; Hematopoietic Stem Cells; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Polymerase Chain Reaction; Protein Binding; Stromal Cells

The conservation of positively charged residues in the N terminus of the hepatitis C virus (HCV) envelope glycoprotein E2 suggests an interaction of the viral envelope with cell surface glycosaminoglycans. Using recombinant envelope glycoprotein E2 and virus-like particles as ligands for cellular binding, we demonstrate that cell surface heparan sulfate proteoglycans (HSPG) play an important role in mediating HCV envelope-target cell interaction. Heparin and liver-derived highly sulfated heparan sulfate but not other soluble glycosaminoglycans inhibited cellular binding and entry of virus-like particles in a dose-dependent manner. Degradation of cell surface heparan sulfate by pretreatment with heparinases resulted in a marked reduction of viral envelope protein binding. Surface plasmon resonance analysis demonstrated a high affinity interaction (KD 5.2 x 10-9 m) of E2 with heparin, a structural homologue of highly sulfated heparan sulfate. Deletion of E2 hypervariable region-1 reduced E2-heparin interaction suggesting that positively charged residues in the N-terminal E2 region play an important role in mediating E2-HSPG binding. In conclusion, our results demonstrate for the first time that cellular binding of HCV envelope requires E2-HSPG interaction. Docking of E2 to cellular HSPG may be the initial step in the interaction between HCV and the cell surface resulting in receptor-mediated entry and initiation of infection.

Topics: Cell Line; Cell Membrane; Chlorates; Dimerization; Disaccharides; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Deletion; Heparitin Sulfate; Humans; Kinetics; Ligands; Protein Binding; Protein Structure, Tertiary; Surface Plasmon Resonance; Temperature; Time Factors; Viral Envelope Proteins

We show here that the interaction between the urokinase-type plasminogen activator and its receptor, which plays a critical role in cell invasion, is regulated by heparan sulfate present on the cell surface and in the extracellular matrix. Heparan sulfate oligomers showing a composition close to the dimeric repeats of heparin (glucosamine-NSO(3)(6-OSO(3))-iduronic acid(2-OSO(3))) n = 5 and n > 5, where iduronic acid may alternate with glucuronic acid, exhibit affinity for urokinase plasminogen activator and confer specificity on urokinase/urokinase receptor interaction. Cell surface clearance of heparan sulfate reduces the affinity of such interaction with a parallel decrease of specific urokinase binding in the presence of an unaltered expression of receptor. Transfection of human urokinase plasminogen activator receptor in normal Chinese hamster ovary fibroblasts and in Chinese hamster ovary cells defective for the synthesis of sulfated glycosaminoglycans results in specific urokinase/receptor interaction only in nondefective cells. Heparan sulfate/urokinase and receptor/urokinase interactions exhibit similar K(d) values. We concluded that heparan sulfate functions as an adaptor molecule that confers specificity on urokinase/receptor binding.

Topics: Animals; Cells, Cultured; Chlorates; CHO Cells; Chromatography, Affinity; Cricetinae; Glycosaminoglycans; Heparitin Sulfate; Humans; Polysaccharide-Lyases; Proteoglycans; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Transfection; Urokinase-Type Plasminogen Activator

Glycosaminoglycans (GAG) are a group of negatively charged molecules that have been shown to bind and directly regulate the bioactivity of growth factors and cytokines such as basic fibroblast growth factor, transforming growth factor-beta, IL-7, and interferon-gamma. The ability of GAG to interact with human IL-10 (hIL-10) and the effect of these interactions on its biologic activity were analyzed. It was demonstrated by affinity chromatography that hIL-10 binds strongly to heparin-agarose at physiological pH. Biosensor-based binding kinetic analysis indicated an equilibrium dissociation constant, K(d), of 54 nmol/L for this interaction. Human IL-10 stimulated CD16 and CD64 expression on the monocyte/macrophage population within peripheral blood mononuclear cells, with optimal concentrations between 1 and 10 ng/mL. Soluble heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate were shown to inhibit the hIL-10-induced expression of CD16 and CD64 in a concentration-dependent manner. Heparin and heparan sulfate were most effective with IC(50) values of 100 to 500 microg/mL. Considerably higher concentrations of dermatan sulfate and chondroitin 4-sulfate were required with an IC(50) of 2,000 to 5,000 microg/mL, whereas chondroitin 6-sulfate was essentially inactive. The antagonistic effect of heparin on hIL-10 activity was shown to be dependent on N-sulfation, inasmuch as de-N-sulfated heparin had little or no inhibitory effect on the IL-10- induced expression of CD16, whereas the effect of de-O-sulfated heparin was comparable to that of unmodified heparin. Furthermore, the inhibition of cell-bound proteoglycan sulfation reduced the hIL-10-mediated expression of CD16 molecules on monocytes/macrophages. Taken together, these findings support the hypothesis that soluble and cell-surface GAG and, in particular, their sulfate groups are important in binding and modulation of hIL-10 activity. (Blood. 2000;96:1879-1888)

Topics: Amino Acid Sequence; Antigens, CD; Binding, Competitive; Cells, Cultured; Chlorates; Chromatography, Affinity; Dose-Response Relationship, Drug; Flow Cytometry; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; Interleukin-10; Kinetics; Leukocytes, Mononuclear; Macrophages; Molecular Sequence Data; Monocytes; Receptors, IgG; Recombinant Proteins; Sepharose

We establish, using an ELISA approach, that recombinant human and murine IL-6 bind to an immobilized heparin-BSA complex. In the case of human IL-6, this binding is displaceable by soluble heparin, IC(50) approximately 2 microg/ml, corresponding to approximately 200 nM. This binding is specific because chondroitin sulfates B and C fail to compete, whereas chondroitin sulfate A and several heparan sulfates are weak inhibitors. Of a range of chemically modified heparins examined, the strongest competitor was the 2-O:-desulfated product, but even this showed a considerably reduced IC(50) ( approximately 30 microg/ml). The epitopes of five IL-6-specific mAbs were still accessible in heparin-bound IL-6, and the dimer formed from the association of rIL-6 with its truncated soluble receptor polypeptide, srIL-6alpha, still bound to heparin. Further analysis showed that heparin competed partially and weakly with the binding of srIL-6 to IL-6; however, it competed strongly for the binding of the rIL-6/srIL-6Ralpha dimer, to soluble glycoprotein 130. In studies of the proliferation of IL-6-sensitive Ba/F3 cells expressing glycoprotein 130, we were unable to detect any effect of either the removal of cell surface heparan sulfate, or addition of soluble heparin. By contrast, heparin was able to protect IL-6 from digestion by the bacterial endoproteinase Lys-C. Overall, our findings show that IL-6 is a heparin-binding cytokine. This interaction will tend to retain IL-6 close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine mode of activity. Moreover, this binding may serve to protect the IL-6 from proteolytic degradation.

Topics: Animals; Binding, Competitive; Cattle; Cell Division; Cell Line; Chlorates; Enzyme-Linked Immunosorbent Assay; Growth Inhibitors; Heparin; Heparitin Sulfate; Humans; Interleukin-6; Mice; Models, Molecular; Protein Binding; Receptors, Interleukin-6; Recombinant Proteins; Serum Albumin, Bovine

The murine macrophage cell line J774 was incubated with [35S]sulphate. The cell-associated 35S-labelled macromolecules were shown to be proteoglycans and glycosaminoglycans in similar amounts. The possible presence of cell-surface proteoglycans was investigated by incubating [35S]sulphate-labelled cells with trypsin for 15 min. The released material contained approx. 70% free glycosaminoglycan chains and 30% proteoglycans. The latter component was demonstrated by HNO2 treatment to contain heparan sulphate. In the total cell fraction not treated with trypsin a small but significant portion was shown to be chondroitin sulphate proteoglycan. The cell-associated glycosaminoglycans contained both chondroitin sulphate and heparan sulphate. To investigate possible biological functions of cell-surface proteoglycans in macrophages, cells were incubated with NaClO3 to inhibit sulphation of proteoglycans and beta-d-xyloside to abrogate proteoglycan expression. The uptake of oxidized 125I-tyraminylcellobiose-labelled low-density lipoprotein (125I-TC-LDL) was typically two to three times higher than that of native 125I-TC-LDL in untreated J774 cells. The cellular uptake at 37 degreesC of native 125I-TC-LDL was decreased 25% after both NaClO3 and xyloside treatment, whereas the uptake of oxidized 125I-TC-LDL was decreased 35% after both types of treatment. The mRNA levels for the scavenger receptor A-II and the LDL receptor were not affected by NaClO3 or xyloside treatment. Furthermore, fluid-phase endocytosis, measured as uptake of horseradish peroxidase, and receptor-mediated endocytosis, measured as uptake of 125I-TC-ovalbumin, were not affected by NaClO3 treatment of J774 cells. Removal of cell-surface chondroitin sulphate with chondroitinase ABC decreased only the binding of native 125I-TC-LDL, whereas removal of heparan sulphate with heparitinase decreased the binding of both oxidized and native 125I-TC-LDL. Addition of lipoprotein lipase increased the uptake of oxidized 125I-TC-LDL 1.7 times and the uptake of native 125I-TC-LDL 2.1 times. The binding of the former was more sensitive to NaClO3 treatment than the latter. The results presented support the notion that some of the uptake pathways for lipoproteins in the foam-cell-forming macrophages depend on the presence of cell-surface heparan sulphate and chondroitin sulphate.

Topics: Animals; Cell Line; Chlorates; Chondroitin ABC Lyase; Chondroitin Sulfates; Endocytosis; Glycosides; Heparitin Sulfate; Lipoprotein Lipase; Lipoproteins; Lipoproteins, LDL; Macrophages; Membrane Glycoproteins; Membrane Proteins; Mice; Ovalbumin; Polysaccharide-Lyases; Proteoglycans; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; RNA, Messenger; Scavenger Receptors, Class B; Trypsin

Pigeon and rabbit beta-migrating very low density lipoprotein (beta-VLDL) are similar in size and composition, yet rabbit beta-VLDL consistently stimulates greater cholesteryl ester accumulation in pigeon peritoneal macrophages than does pigeon beta-VLDL. The purpose of this study was to determine the mechanism of this difference. Pigeon beta-VLDL bound to both a high and low affinity site while rabbit beta-VLDL bound primarily to a low affinity site. The high affinity site had the characteristics of the LDL receptor. Most rabbit beta-VLDL and some pigeon beta-VLDL bound to the low affinity site that was not down-regulated by cholesterol loading. beta-VLDL binding to the low affinity site and subsequent internalization and degradation were mediated by cell surface heparan sulfate proteoglycans (HSPG). Evidence for this includes inhibition of binding and uptake by chlorate, which prevents sulfation of proteoglycans, and by treatment with heparinase but not chondroitinase ABC. beta-VLDL uptake was stimulated by lipoprotein lipase (LpL) and apolipoprotein E (apoE), both known to bind HSPGs. Uptake and degradation of beta-VLDL were not mediated by the LDL receptor or the alpha(2)MR/LRP. Thus, binding of beta-VLDL to low affinity, high capacity HSPG binding sites on pigeon macrophages appears to directly promote internalization and degradation and is largely responsible for the greater ability of rabbit beta-VLDL to stimulate cholesterol accumulation.

Topics: Animals; Apolipoproteins E; Binding, Competitive; Cells, Cultured; Chlorates; Cholesterol; Chondroitin Lyases; Columbidae; Dose-Response Relationship, Drug; Edetic Acid; Heparan Sulfate Proteoglycans; Heparin Lyase; Heparitin Sulfate; Iodine Radioisotopes; Lipoprotein Lipase; Lipoproteins, VLDL; Macrophages, Peritoneal; Membrane Proteins; Osmolar Concentration; Polysaccharide-Lyases; Proteoglycans; Rabbits; Temperature; Time Factors

The ability of extracellular matrix heparan sulfate to alter the susceptibility of human endothelial cells to S. aureus was investigated. Endothelial cells grown on extracellular matrix synthesized by S. aureus-infected endothelial cells were more susceptible to subsequent staphylococcal infection than endothelial cells grown on the extracellular matrix synthesized by untreated endothelial cells. Endothelial cells were more susceptible to S. aureus infection when 1) grown on heparitinase-treated extracellular matrix that removed heparan sulfate chains, 2) grown on extracellular matrix produced by chlorate-treated endothelial cells that reduced sulfation in the matrix heparan sulfate proteoglycans, 3) grown on heparan sulfate purified from extracellular matrix elaborated by infected endothelial cells, and 4) endothelial cells were chlorate-treated and therefore expressed desulfated cellular heparan sulfate proteoglycans. Extracellular matrix produced by S. aureus-infected endothelial cells contained heparan sulfate proteoglycans with reduced sulfation. The altered extracellular matrix with reduced sulfated heparan sulfate proteoglycans signalled the uninfected endothelial cells to produce under sulfated cellular heparan sulfate proteoglycans that increased S. aureus adherence to the endothelial cells.

Topics: Bacterial Adhesion; Cells, Cultured; Chlorates; Chromatography, Ion Exchange; Endothelium, Vascular; Extracellular Matrix; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Humans; Polysaccharide-Lyases; Proteoglycans; Staphylococcus aureus; Umbilical Cord

Tissue factor pathway inhibitor (TFPI) is a primary regulator of the initiation of blood coagulation. TFPI is internalized and degraded by HepG2 cells through the low-density-lipoprotein receptor-related protein (LRP) but also binds another molecule present on the cell surface at approx. 10-fold the abundance of LRP [Warshawsky, Broze and Schwartz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668]. When HepG2 cells are washed with heparin or dextran sulphate, a substance that binds TFPI is removed from the cell surface and can be detected in a slot-blot assay. Preincubation with trypsin destroys the reactivity of the TFPI-binding component in the slot-blot assay, suggesting that it is a protein. In addition, when the sulphation of glycosaminoglycans (GAGs) is prevented by growing the HepG2 cells in the presence of 30 mM sodium chlorate, TFPI binding is unaffected, whereas the binding of bovine lipoprotein lipase, a protein known to associate with cell-surface GAGs, falls to 50% of control levels. Dextran sulphate washes of HepG2 cells grown in sodium chlorate have an equal reactivity in slot-blot experiments to that of non-treated cells, suggesting that GAGs are not totally responsible for the binding activity observed. By using the slot blot to follow binding activity and conventional protein purification techniques, a protein species that migrates at 40 kDa after reduction was identified in the HepG2 cell wash. The binding of this protein to TFPI was confirmed with immobilized TFPI. Amino acid sequence analysis identified this protein species as a proteolytic fragment of glypican-3 (also called OCI-5), a member of the glypican family of glycosylphosphatidylinositol-anchored proteoglycans.

Topics: Amino Acid Sequence; Animals; Binding Sites; Carrier Proteins; Cattle; Cell Membrane; Chlorates; Chromatography, Affinity; Cloning, Molecular; Dextran Sulfate; Fibrinolytic Agents; Glycosaminoglycans; Glycosylphosphatidylinositols; Glypicans; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Humans; Kinetics; Lipoprotein Lipase; Lipoproteins; Molecular Sequence Data; Peptide Fragments; Proteoglycans; Recombinant Proteins; Sequence Alignment; Trypsin; Tumor Cells, Cultured

The poor ability of mammalian central nervous system (CNS) axons to regenerate has been attributed, in part, to astrocyte behavior after axonal injury. This behavior is manifested by the limited ability of astrocytes to migrate and thus repopulate the injury site. Here, the migratory behavior of astrocytes in response to injury of CNS axons in vivo was simulated in vitro using a scratch-wounded astrocytic monolayer and soluble substances derived from injured rat optic nerves. The soluble substances, applied to the scratch-wounded astrocytes, blocked their migration whereas some known wound-associated factors such as transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and heparin-binding epidermal growth factor in combination with insulin-like growth factor-1 (HB-EGF + IGF-1) stimulated intensive migration with consequent closure of the wound. Migration was not dominated by proliferating cells. Both bFGF and HB-EGF + IGF-1, but not TGF-beta 1, could overcome the blocking effect of the optic nerve-derived substances on astrocyte migration. The induced migration appeared to involve proteoglycans. It is suggestive that appropriate choice of growth factors at the appropriate postinjury period may compensate for the endogenous deficiency in glial supportive factors and/or presence of glial inhibitory factors in the CNS.

Topics: Animals; Astrocytes; Cell Movement; Cells, Cultured; Cerebral Cortex; Chlorates; Chondroitin Sulfate Proteoglycans; DNA; Drug Synergism; Glial Fibrillary Acidic Protein; Growth Substances; Heparan Sulfate Proteoglycans; Heparin Lyase; Heparitin Sulfate; Nerve Crush; Neuroglia; Optic Nerve; Polysaccharide-Lyases; Proteoglycans; Rats; Wound Healing

The MCF-7 breast cancer cells exhibit remarkable growth enhancement in response to basic fibroblast growth factor (FGF-2) stimulation in a dose dependent manner. To investigate the involvement of proteoglycans on control of FGF-2 induced proliferation, polysaccharide chains were degraded by specific enzymes. Our results showed that MCF-7 cells were unsensitive to FGF-2 after enzymatic degradation of heparin sulfate proteoglycans (HSPG) by heparinase. After metabolic inhibition of sulphation by sodium chloride, radiolabelled proteoglycans were purified and quantified by ion exchange chromatography. Sodium chlorate treatment reduced by 70% sulfation of proteoglycans. This decrease of sulphation totally inhibited FGF-2-mediated proliferation. The sulphated glycosaminoglycans which were critical in FGF-2-induced proliferation were strictly HSPG, as an addition of heparin in cell culture medium can restore FGF-2 mitogenic activity. In contrast, other glycosaminoglycans (chondroitin sulfate/hyaluronic acid) did not show any effect. These results provide clear evidence for the critical role of HSPG in FGF-2-induced proliferation on MCF-7 breast cancer cells.

Topics: Breast Neoplasms; Cell Division; Chlorates; Female; Fibroblast Growth Factor 2; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Proteoglycans; Tumor Cells, Cultured

Androgen production by adult rat Leydig cells is stimulated by pituitary LH but can also be modulated in vitro by paracrine stimulatory and inhibitory factors, many of which belong to growth factor families. Their actions are mediated through cell surface or extracellular matrix proteoglycans and the aim of this study was to determine the role of cell surface heparan sulfate proteoglycans in the regulation of testosterone secretion by adult rat Leydig cells. The presence of sodium chlorate (25 mM) and protamine sulfate (10 micrograms/ml) inhibited testosterone production by LH stimulated cells by over 50%, but had no effect on unstimulated cells. The LH responsiveness and testosterone production returned to normal after these agents were removed from the culture media. No significant difference in LH receptor numbers at the end of the culture period was seen between sodium chlorate treated and untreated cells. Testosterone production by dibutryl-cAMP stimulated Leydig cells was also inhibited by sodium chlorate. The addition of heparin inhibited testosterone production by LH stimulated cells in a dose-dependent manner, however, in unstimulated Leydig cells heparin stimulated testosterone production to up to 50% of that seen in LH stimulated cells. These data suggest that cell surface heparan sulfate proteoglycans modulate testosterone production by adult Leydig cells in vitro, and that this may involve the autocrine actions of heparin binding growth factors on the Leydig cells.

Topics: Animals; Bucladesine; Chlorates; Extracellular Matrix; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Leydig Cells; Luteinizing Hormone; Male; Protamines; Proteoglycans; Rats; Testosterone

FGF-2 activates multiple signaling pathways by a cell surface signaling complex assembled with FGF, its receptor tyrosine kinase, and heparan sulfate proteoglycan. Heparan sulfate binds to a site on the receptor and at least one site on the growth factor. Several models propose an important role for heparan sulfate not only in facilitating FGF-2 binding to its receptor tyrosine kinase but also in promoting signaling via formation of receptor dimers. Such dimers are capable of transphosphorylation of the cytoplasmic domain of the receptor, leading to the generation of phosphotyrosines that are important initiators of intracellular signaling pathways. To explore the participation of heparan sulfates in the formation of a signaling complex that activates these pathways, the binding and activity of FGF-2 on Swiss 3T3 fibroblasts and F32 lymphoid cells is examined with either native or modified forms of heparin. As shown previously, fibroblasts treated with chlorate, which inhibits the sulfation of heparan sulfate and its subsequent binding to FGF-2, display a dramatically reduced response to picomolar concentrations of FGF-2, but binding to receptors and a mitogenic response is restored by heparin. However, the restoration of high-affinity binding is seen only at an optimal concentration of heparin. Excess heparin competes for binding sites within the signaling complex such that high-affinity binding and receptor transphosphorylation are reduced. Despite this, mitogenic signaling is not diminished. A similar result is observed using heparin fragments that promote mitogenesis but not high-affinity binding. These results suggest that the high-affinity signaling complex that is necessary for stable receptor transphosphorylation differs from the signaling complex sufficient for triggering mitogenesis. We speculate that heparan sulfate in vivo participates in two hierarchies of receptor activation. In one, heparan sulfate participates in FGF-2 binding to its receptor tyrosine kinase and activation of mitogenic signaling, perhaps through monomeric receptors or the transient formation of receptor dimers. In the second hierarchy, heparan sulfate participates in the stabilization of a signaling complex that is likely to be comprised of receptor multimers that carry out effective receptor transphosphorylation. A further description of this mechanism may lead to an understanding of how heparan sulfate or its homologues can regulate specific signaling pathways within the

Topics: 3T3 Cells; Animals; Binding Sites; Cell Division; Chlorates; Cross-Linking Reagents; Fibroblast Growth Factor 2; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Humans; Lymphocytes; Mice; Oligosaccharides; Phosphorylation; Protein Binding; Proteoglycans; Receptor Protein-Tyrosine Kinases; Receptors, Fibroblast Growth Factor; Signal Transduction; Tyrosine

Tissue factor pathway inhibitor (TFPI) is mainly bound to the vessel wall and is released to circulating blood after injections of heparin. It has been suggested that the highly positively charged carboxy terminal end of heparin releasable TFPI is bound to negatively charged binding molecule(s), presumably glycosaminoglycans (GAGs), on the luminal surface of endothelial cells. The aim of the present study was to characterize this binding. Confluent monolayers of human umbilical vein endothelial cells (HUVECs) and Ea.hy926 cells were incubated with 125I-labelled recombinant TFPI (rTFPI). Two different rTFPI preparations were used in the experiments; one preparation was full-length rTFPI and one preparation was truncated at the C-terminal end (rTFPI1-161). Binding of 125I-rTFPI reached equilibrium conditions after 2 hours incubation at room temperature. Scatchard plots indicated a single class of binding sites with a mean Kd value of 164 +/- 16 nmol/L for HUVECs and a Kd value of 296 +/- 10 nmol/L for Ea.hy926 cells. The number of rTFPI binding sites per cell were approximately 1.10(7). Binding of 125I-rTFPI1-161 was non-specific. GAGs reduced binding of 125I-rTFPI in a dose-dependent manner by 50-75%. The potency of different GAGs to displace bound rTFPI was in the following order: Unfractionated heparin (UF) > low-molecular weight (LMW) heparin > hexadecasaccharides/octasaccharides/dodecasaccharides > heparan sulfate > dermatan sulfate. Treatment of the cells with heparinase III, with chondroitinase ABC lyase, or with sodium chlorate (to prevent sulfation) did not influence the binding of TFPI. We conclude that the C-terminal end is necessary for binding of TFPI to endothelial cells, but the binding is weak and does not involve GAGs.

Topics: Anticoagulants; Chlorates; Dermatan Sulfate; Endothelium, Vascular; Factor Xa Inhibitors; Fibrinolytic Agents; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; Kinetics; Lipoproteins; Time Factors

Previous studies in this laboratory have indicated that the early embryonic chick heart depends on fibroblast growth factor-2 (FGF-2; bFGF), sequentially utilized in paracrine and autocrine fashion, for its growth and development (Sugi and Lough, [1995] Dev. Biol. 168-567-574). This view emanated from immunohistochemical detection of FGF-like antigens in endoderm cells at stage 6, and later in the early myocardium at stage 9+ (Parlow et al. [1991] Dev. Biol. 146:139-147). To identify other members of the FGF family that are expressed by these cells, we have used peptide-generated antisera that specifically recognize FGFs 1 and 4. Like FGF-2, FGFs 1 and 4 were exclusively detected in the endoderm at stage 5+ and later in the myocardium, appearing as punctate cytoplasmic deposits. However, whereas FGF-2 is first detected at stage 9+, FGFs 1 and 4 did not appear until stages 11 and 15, respectively. Expression of all FGFs peaked at stages 18-24, decreasing thereafter in parallel with reduced myocardial cell proliferation. To determine these isoproteins' ability to facilitate the completion of terminal cardiac myocyte differentiation, stage 5+ precardiac mesoderm was cultured in defined medium with purified FGFs. Like FGF-2, as little as 5-10 ng/ml FGF-1 or FGF-4 supported the proliferation and differentiation of precardiac myoblasts, resulting in the formation of a vesicle containing an adherent multilayer of synchronously contractile cells. Evidence that this represented FGF receptor-mediated signaling rather than a nonspecific effect of exogenous FGF was indicated by the ability of sodium chlorate to inhibit FGF-mediated cardiogenesis. These findings are consistent with the hypothesis that, like FGF-2, FGFs 1 and 4 participate in the regulation of early heart development via paracrine and autocrine mechanisms.

Topics: Animals; Cells, Cultured; Chick Embryo; Chlorates; Dose-Response Relationship, Drug; Endoderm; Fibroblast Growth Factor 1; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Heart; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Morphogenesis; Myocardium; Proteoglycans; Proto-Oncogene Proteins; Tissue Distribution

To investigate the role of cell surface glycosaminoglycans (GAGs), including heparan sulfate (HS), on HIV-1 infection in human T cells, HIV-1 binding and infection were determined after treatment of T-cell lines and CD4+ T cells from normal peripheral blood mononuclear cells (PBMC) with GAG-degrading enzyme or a GAG metabolic sulfation inhibitor. Heparitinase I (hep I) and sodium chlorate prevented binding of HIV-1/IIIB to MT-4 cells as revealed by indirect immunofluorescence procedures, thereby inhibiting infection. Hep I was less effective in the binding inhibition of the macrophage-tropic strain HIV-1/SF162 than that of the T-cell line-tropic strain HIV-1/IIIB. The binding of HIV-1/SF162 was about 100-fold less dependent on cell surface HS than HIV-1/IIIB. Human HTLV-I positive T-cell lines expressed more HS than HTLV-I negative T-cell lines or normal CD4+ T cells when stained with anti-HS mAbs against either native or heparitinase-treated HS. With the exception of endo-beta-galactosidase (endo-beta-gal), GAG-degrading enzymes, including hep I, chondroitinase ABC (chon ABC), chondroitinase AC II (chon AC II) and keratanase, did not prevent the binding of HIV-1/IIIB to CD4+ T cells from normal PBMC. These results indicate that the cell surface HS of human T cells participates in HIV-1 infection by facilitating HIV-1/IIIB binding to MT-4 cells. In particular, the sulfation of HS chains is critical. Since the expression of cell surface HS varies among T cells, which are not consistently sensitive to hep I treatment in HIV-1 binding inhibition, other GAG-like molecules may also be involved.

Topics: beta-Galactosidase; CD4-Positive T-Lymphocytes; Cell Line; Cell Membrane; Chlorates; Chondroitin Lyases; Fluorescent Antibody Technique, Indirect; Glycosaminoglycans; Glycoside Hydrolases; Heparitin Sulfate; HIV-1; Humans; Polysaccharide-Lyases; T-Lymphocytes

Vitronectin, a 75-kDa plasma protein is also found in the extracellular matrix, where it is believed to promote cell adhesion and migration. In addition to its role in adhesion, matrix vitronectin is also believed to function as an opsonin promoting the clearance of thrombin-serpin complexes from the matrix. Vitronectin is cleared from the matrix by receptor-mediated endocytosis followed by lysosomal degradation, suggesting that cells can regulate the levels of vitronectin present in the matrix. However, the mechanism by which plasma vitronectin associates with the extracellular matrix remains unclear. Studies were conducted to define the binding site(s) for vitronectin in fibroblast cell layers. Sodium chlorate, a competitive inhibitor of proteoglycan sulfation, produced a dose-dependent decrease in both binding and degradation of vitronectin. This inhibition was reversible in that removal of chlorate returned both binding and degradation of vitronectin to near control levels within 24 h. The binding of vitronectin to cell layers was not dependent on cells because vitronectin bound directly to isolated matrix. Isolated matrices prepared from cell layers treated with sodium chlorate also exhibited a dose-dependent decrease in vitronectin binding, consistent with the binding site for vitronectin in the matrix being sulfated proteoglycans. Binding and degradation of vitronectin were also sensitive to the addition of exogenous heparin, suggesting that the heparin binding domain of vitronectin was mediating binding to the matrix. Incubating fibroblast monolayers with heparinase III resulted in a 40% decrease in binding and degradation of vitronectin. Taken together, the above findings suggest that vitronectin's binding to the matrix and its subsequent degradation are dependent on heparan sulfate proteoglycans.

Topics: Binding Sites; Cells, Cultured; Chlorates; Extracellular Matrix; Fibroblasts; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Humans; Male; Proteoglycans; Vitronectin

IGFBP-3 and IGFBP-5, but not the other 4 IGF binding proteins, specifically bound to endothelial cell (EC) monolayers. Charged compounds, such as heparin and heparan sulfate, competed for this binding. Of the 6 IGFBPs, IGFBP-3 and IGFBP-5 had the greatest heparin affinity. Peptides of 18 amino acids were synthesized, corresponding to a common basic region of IGFBP-3 (P3), IGFBP-5 and IGFBP-6 (P6) which contained a heparin binding sequence. P3 and P6 inhibited IGFBP-3 and -5 binding to endothelial cell monolayers and the peptides bound directly to EC extracellular matrix. This suggested that the C-terminal basic segment of IGFBP-3/-5 is important for the association of the binding protein with the EC monolayer.

Topics: Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Carrier Proteins; Cattle; Chlorates; Chromatography; Endothelium, Vascular; Extracellular Matrix; Glycosaminoglycans; Glycosylation; Heparin; Heparin Lyase; Heparitin Sulfate; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth Factor Binding Proteins; Molecular Sequence Data; Peptide Fragments; Polysaccharide-Lyases; Protamines; Sulfates

Human HeLa adenocarcinoma cells did not respond to basic fibroblast growth factor (bFGF or FGF-2) but did bind the same amount of bFGF as responsive Chinese hamster lung fibroblasts (CCL 39). Heparinase II treatment of HeLa and CCL 39 cells resulted in a decrease of bFGF binding by 96 and 57%, respectively, indicating that heparan sulfate molecules were involved in bFGF binding. On HeLa cells, bFGF bound to a single family of low-affinity sites. Cross-linking experiments of 125I-bFGF to HeLa cells yielded several labeled complexes. Cell-associated 125I-bFGF was internalized in both cell types either by high-affinity receptors and heparitinase-sensitive sites in CCL 39 cells or by heparitinase-sensitive binding sites only in HeLa cells. The binding of bFGF to nonresponsive HeLa cells and its internalization via a family of heparitinase-sensitive binding sites might illustrate other functions of bFGF unrelated to cell proliferation.

Topics: Animals; Binding Sites; Blotting, Northern; Cattle; Cell Division; Cell Line; Chlorates; Cricetinae; DNA; Fibroblast Growth Factor 2; Fibroblasts; HeLa Cells; Heparitin Sulfate; Humans; Kinetics; Lung; Polysaccharide-Lyases; Recombinant Proteins

Amphiregulin, a member of the epidermal growth factor family with heparin binding affinity, functions as a natural regulator of keratinocyte growth. Autocrine signaling by amphiregulin and the effects of exogenous recombinant cytokine were studied in serum-free cultures of human neonatal keratinocytes. A metabolic inhibitor of proteoglycan sulfation was used to assess the role of cellular heparin-like glycosaminoglycans in amphiregulin-dependent growth. Keratinocytes plated at > 10(3) cells/cm2 grew in an autocrine manner in the absence of exogenous epidermal growth factor or amphiregulin. Incubation of keratinocytes with an amphiregulin-blocking antibody indicated that approximately 70% of autocrine growth is mediated by endogenous amphiregulin. Proliferation potential in the presence of recombinant human amphiregulin was dose dependent and saturable and above approximately 1 ng/ml was comparable to that achieved with similar concentrations of epidermal growth factor. Sodium chlorate, which blocks glycosaminoglycan sulfation, reversibly inhibited epidermal growth factor-dependent proliferation by 42%, exogenous amphiregulin-dependent proliferation by 75%, and autocrine growth by 95%; concurrent incubation with 1-100 micrograms/ml heparin partially reversed this inhibition. Exogenous heparin in the absence of chlorate, however, nearly completely inhibited growth under autocrine conditions and in the presence of recombinant amphiregulin. Structure-function studies indicate that the polymerization level, high sulfate group density, and possibly iduronic acid content of heparin-like moieties correlate with their inhibitory activity. Collectively, these observations indicate that amphiregulin is the major autocrine factor for keratinocytes and demonstrate that exogenous amphiregulin is an effective growth promoting factor with molar potency similar to that of epidermal growth factor. Autocrine and paracrine signaling by amphiregulin may require cellular heparin-like glycosaminoglycans, presumably as matrix or membrane proteoglycans, whereas soluble glycosaminoglycans inhibit signaling, possibly by competing for cytokine binding.

Topics: Amphiregulin; Antibodies; Cell Division; Cell Line; Chlorates; Culture Media, Serum-Free; Cytokines; Dose-Response Relationship, Drug; EGF Family of Proteins; Epidermal Growth Factor; Glycoproteins; Glycosaminoglycans; Growth Substances; Heparin; Heparitin Sulfate; Humans; Intercellular Signaling Peptides and Proteins; Keratinocytes; Recombinant Proteins

In response to androgen stimuli, SC-3 cells cloned from mouse mammary carcinoma (Shionogi carcinoma 115) secrete heparin-binding growth factor, which is able to bind to fibroblast growth factor (FGF) receptor, ultimately resulting in SC-3 cell proliferation. A role for glycosaminoglycan in the process of this androgen-induced autocrine loop was examined. When quiescent SC-3 cells were stimulated with testosterone, enhanced DNA synthesis was demonstrated even at the early phase (12-24 h) of stimulation. During this early period, autocrine growth-promoting activity was found to be associated with cell surface and extracellular matrix, but not to be present in the conditioned medium. This bound form of growth-promoting activity was able to be extracted with 2 M NaCl (pH 7.5) and absorbed onto a heparin-Sepharose affinity column, from which it was eluted at a concentration of 1.1-1.3 M NaCl. Extracted growth factor, whose activity was partially blocked by anti-basic FGF antibody, up-regulated the expression of FGF receptor-1 mRNA. These characteristics were similar to those of a soluble form of SC-3-derived growth factor previously reported from our laboratory. Androgen-induced enhancement of DNA synthesis was inhibited by simultaneous treatment of SC-3 cells with 10 mIU/ml heparitinase or 25 mM sodium chlorate (an inhibitor of phosphoadenosine sulfate synthesis). However, chlorate treatment did not affect the synthesis and distribution of androgen-induced growth factor, suggesting that the response of chlorate-treated cells to this growth factor was impaired. These results indicate that heparan sulfate has important roles in concentrating androgen-induced heparin-binding growth factor on or very close to the cell surface and in potentiating its bioactivity.

Topics: Animals; Cell Division; Chlorates; Chromatography, Affinity; Clone Cells; DNA Replication; DNA, Neoplasm; Glycosaminoglycans; Growth Substances; Heparitin Sulfate; Kinetics; Mammary Neoplasms, Experimental; Mice; Polysaccharide-Lyases; Sulfates; Sulfur Radioisotopes; Testosterone; Thymidine; Time Factors; Tritium; Tumor Cells, Cultured

Basic fibroblast growth factor (bFGF) binds to heparan sulfate proteoglycans at the cell surface and to receptors with tyrosine kinase activity. Prevention of binding between cell surface heparan sulfate and bFGF (i) substantially reduces binding of fibroblast growth factor to its cell-surface receptors, (ii) blocks the ability of bFGF to support the growth of Swiss 3T3 fibroblasts, and (iii) induces terminal differentiation of MM14 skeletal muscle cells, which is normally repressed by fibroblast growth factor. These results indicate that cell surface heparan sulfate is directly involved in bFGF cell signaling.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Chlorates; Fibroblast Growth Factor 2; Fibroblasts; Heparitin Sulfate; In Vitro Techniques; Mice; Muscles; Polysaccharide-Lyases; Protein Binding; Receptors, Cell Surface; Structure-Activity Relationship

Enzymatic deglycosylation of plasma membrane proteoglycans and metabolic inhibition of glycosaminoglycan sulfation were employed as complementary methods to evaluate the effects of reduced cell surface content of functionally intact proteoglycans on the proliferative potential of cells. A Flavobacter heparinum extract, possessing multiple glycosaminoglycan substrate specificities, markedly inhibited the time-dependent expansion of BALB/c 3T3 fibroblast and human squamous cell carcinoma monolayers in culture and concurrently reduced the proportion of subconfluent cell populations in S-cell cycle phase by DNA flow-cytometry analysis. This antiproliferative effect was partially reproduced by lyases with heparan sulfate or chondroitin sulfate monospecificity, alone and in combination. The observed lability of heparan sulfate lyases I and II in serum-containing medium possibly hampered full reproduction of the effects of the multifunctional reagent. Growth inhibition of comparable magnitude was observed when glycosaminoglycan sulfation was metabolically blocked with sodium chlorate. The chlorate anion had its expected effect of substantially reducing sulfated glycosaminoglycan synthesis by the cells. Following release from serum deprivation, analysis of the progression of synchronized cell populations past the G1 restriction point suggested that in situ digestion with the glycosaminoglycan lyases limited, but did not delay, the numbers of cells entering S phase. These data support the hypothesis that plasma membrane proteoglycans mediate some of the cell-growth-promoting effects of serum factors via their glycosaminoglycan side chains.

Topics: Animals; Cell Cycle; Cell Division; Cell Line; Cell Membrane; Chlorates; Glycosaminoglycans; Glycosylation; Heparitin Sulfate; Mice; Proteoglycans; Sulfates