Basic fibroblast growth factor (bFGF) dissociates rapidly from heparan sulfates but slowly from receptors. Implications for mechanisms of bFGF release from pericellular matrix.

The effect of heparin on the rate of binding of basic fibroblast growth factor (bFGF) to high affinity (receptor) and low affinity (heparan sulfate) binding sites on endothelial cells and CHO cells transfected with FGF receptor-1 or FGF receptor-2 was investigated. Radiolabeled bFGF bound rapidly to both high and low affinity sites on all three types of cells. Addition of 10 micrograms/ml heparin eliminated binding to low affinity sites and decreased the rate of binding to high affinity sites to about 30% of the rate observed in the absence of heparin. However, the same amount of 125I-bFGF bound to high affinity sites at equilibrium in the presence and absence of heparin. The effect of heparin on the initial rate of binding to high affinity sites was related to the log of the heparin concentration. Depletion of the cells of heparan sulfates by treatment with heparinase also decreased the initial rate of binding to high affinity receptors. These results suggest that cell-surface heparan sulfates facilitate the interaction of bFGF with its receptor by concentrating bFGF at the cell surface. Dissociation rates for receptor-bound and heparan sulfate-bound bFGF were also measured. Dissociation from low affinity sites was rapid, with a half-time of 6 min for endothelial cell heparan sulfates and 0.5 min for Chinese hamster ovary heparan sulfates. In contrast, dissociation from receptors was slow, with a half-time of 46 min for endothelial cell receptors, 2.5 h for FGF receptor-1, and 1.4 h for FGF receptor-2. These results suggest that degradative enzymes may not be needed to release bFGF from the heparan sulfates in instances where receptors and heparan sulfate-bound bFGF are in close proximity because dissociation from heparan sulfates occurs rapidly enough to allow bFGF to bind to unoccupied receptors by laws of mass action.     

Heparan sulfate primed on β-D-xylosides restores binding of basic fibroblast growth factor

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). Mutant Chinese hamster ovary cells (pgsA-745) deficient in xylosyltransferase are unable to initiate glycosaminoglycan synthesis and hence can not bind bFGF to low- and high-affinity cell surface receptors. Exposure of pgsA-745 cells to β-D-xylopyranosides containing hydrophobic aglycones resulted in restoration of bFGF binding in a manner similar to that induced by soluble heparin or by heparan sulfate (HS) normally associated with cell sulfate. Restoration of bFGF binding correlated with the ability of the β-D-xylosides to prime the synthesis of heparan sulfate. Thus, both heparan sulfate synthesis and bFGF receptor binding were induced by low concentrations (10–30 μM) of estradiol-β-D-xyloside and naphthyl-β-D-xyloside, but not by cis/trans-decahydro-2-naphthyl-β-D-xyloside, which at low concentration primes mainly chondroitin sulfate. The obligatory involvement of xyloside-primed heparan sulfate in restoration of bFGF-receptor binding was also demonstrated by its sensitivity to heparinase treatment and by the lack of restoration activity in CHO cell mutants that lack enzymatic activities required to form the repeating disaccharide unit characteristic of heparan sulfate. Xyloside-primed heparan sulfate binds to the cell surface. Restoration of bFGF receptor binding was induced by both soluble and cell bound xyloside-primed heparan sulfate and was abolished in cells that were exposed to 0.5–1.0 M NaCl prior to the bFGF binding reaction. These results indicate that heparan sulfate chains produced on xyloside primers behave like heparan sulfate chains attached to cellular core proteins in terms of affinity for bFGF and ability to function as low-affinity sites in a dual receptor mechanism characteristic of bFGF and other heparin-binding growth promoting factors.     

Basic fibroblast growth factor is internalized through both receptor-mediated and heparan sulfate-mediated mechanisms.

Basic fibroblast growth factor (bFGF) was internalized at a rapid rate by Chinese hamster ovary (CHO) cells that do not express significant numbers of high affinity receptors for bFGF as well as CHO cells that have been transfected with cDNA encoding FGF receptor-1 or FGF receptor-2. Internalization of bFGF was completely blocked by the addition of 10 micrograms/ml heparin in the parental CHO cells but only partially inhibited in cells expressing transfected FGF receptors. Bovine aortic endothelial cells also exhibit heparin-sensitive and heparin-resistant internalization of bFGF. The internalization of bFGF through the heparin-resistant pathway in CHO cells was efficiently competed by addition of unlabeled bFGF, was proportional to the number of receptors expressed, and approached saturation, suggesting that the heparin-resistant internalization was due to high affinity receptors. Internalization of bFGF through the heparin-sensitive pathway was not efficiently competed by unlabeled bFGF and did not approach saturation at concentrations of bFGF up to 50 ng/ml, properties similar to the interaction of bFGF with low affinity heparan sulfate binding sites on the cell surface. Internalization of bFGF in CHO cells not expressing FGF receptors was inhibited by heparin, heparan sulfate, and dermatan sulfate, the same glycosaminoglycans that block binding to cell-surface heparin sulfates. Internalization of bFGF in the parental CHO cells was inhibited at the same concentrations of heparin that block binding to cell-surface heparan sulfates. Finally, inhibition of the sulfation of CHO cell heparan sulfates by the addition of chlorate or digestion of CHO cell heparan sulfates with heparinase inhibited bFGF internalization in the parental CHO cells. These results demonstrate that bFGF can be internalized through a direct interaction with cell-surface heparan sulfates. Thus, there are two pathways for internalization of bFGF: high affinity receptor-mediated and heparan sulfate-mediated.     

High and low affinity binding sites for basic fibroblast growth factor on cultured cells: absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells

Scatchard analysis of binding of 125I-basic fibroblast growth factor (FGF) to baby hamster kidney (BHK) cells revealed the presence of two binding sites: a high affinity site with KD of 20 pM and 80,000 sites per cell and a low affinity site with KD of about 2 nM and 600,000 sites per cell. The binding to the two sites could be separated by first washing the cells with 2 M NaCl at pH 7.5 which released the low affinity binding and then extracting the cells with 0.5% Triton X-100 to recover the 125I-basic FGF bound to high affinity sites. The binding to the high affinity site was acid sensitive, suggesting that it represented binding to the receptor. Binding to the low affinity site could be competed strongly by heparin and less strongly by heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. Treatment of BHK cells with heparinase abolished 62% of the low affinity binding, suggesting that the low affinity binding represented binding to cell-associated, heparin-like molecules. A variety of other cell types, including bovine capillary endothelial (BCE) cells, also demonstrated both low and high affinity binding sites. To test whether the low affinity binding might play a role in the basic FGF stimulation of plasminogen activator (PA) production by BCE cells, heparin was added to BCE cultures at concentrations which totally blocked binding of 125I-basic FGF to the low affinity sites. Addition of the heparin did not diminish the increased PA production induced by basic FGF. This suggests that the low affinity binding has no direct role in the stimulation of PA production in BCE cells.     

Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.     

Specific binding of basic fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor

The binding of iodinated basic fibroblast growth factor (bFGF) to low-density heparan sulfate proteoglycan purified from the Engelbreth Holm Swarm (EHS) sarcoma was investigated using different techniques. The tumor clearly contained bFGF, the level being comparable to that found in other tissues such as human or bovine brain. 125I bFGF strongly bound to the basement membrane-like matrix of EHS frozen sections as revealed by autoradiography. Iodinated bFGF bound to purified heparan sulfate proteoglycan but not to laminin or collagen type IV, three components isolated from the same tumor. In contrast, acidic fibroblast growth factor (aFGF) displayed negligible binding to heparan sulfate proteoglycan. Binding of bFGF to frozen sections and to purified proteoglycan could be strongly inhibited by heparin and was displaced by an excess of unlabeled factor and completely suppressed after heparitinase and heparinase treatments. Binding was a function of the salt concentration and was abolished at 0.6 M NaCl. Scatchard analysis indicated the affinity site had a Kd of about 30 nM, a value 10-15 higher than that recently reported by Moscatelli (J. Cell. Physiol., 131:123-130, 1987) in the case of the low-affinity binding sites present on the surface of baby hamster kidney (BHK) cells.     

Laminarin sulfate mimics the effects of heparin on smooth muscle cell proliferation and basic fibroblast growth factor-receptor binding and mitogenic activity

Heparin and heparin-like molecules may function, apart from their effect on hemostasis, as regulators of cell growth and neovascularization. We investigated whether similar effects are exerted by laminarin sulfate, an unrelated polysulfated saccharide isolated from the cell wall of seaweed and composed of chemically O-sulfated b?-(1,3)-linked glucose residues. Laminarin sulfate exhibits about 30% of the anticoagulant activity of heparin and is effective therapeutically in the prevention and treatment of cerebrovascular diseases. We characterized the effect of laminarin sulfate on interaction of the heparin-binding angiogenic factor, basic fibroblast growth factor (bFGF), with a naturally produced subendothelial extracellular matrix (ECM) and with cell surface receptor sites. Laminarin sulfate (1-2 m?g/ml) inhibited the binding of bFGF to ECM and to the surface of vascular smooth muscle cells (SMC) in a manner similar to that observed with heparin. Likewise, laminarin sulfate efficiently displaced both ECM-and cell-bound bFGF at concentrations as low as 1 m?g/ml. Both laminarin sulfate and heparin efficiently induced restoration of bFGF receptor binding in xylosyltransferase-deficient CHO cell mutants defective in initiation of glycosaminoglycan synthesis. Moreover, laminarin sulfate elicited bFGF receptor activation and mitogenic response in heparan sulfate(HS)-deficient, cytokine-dependent lymphoid cells. These results indicate that laminarin sulfate effectively replaced the need for heparin and HS in the induction of bFGF receptor binding and signaling. In other experiments, laminarin sulfate was found to inhibit the proliferation of vascular SMC in a manner similar to that observed with heparin. These effects of laminarin sulfate may have potential clinical applications in diverse situations such as wound healing, angiogenesis, and atherosclerosis. © 1995 Wiley-Liss, Inc.     

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.     

Elastase-mediated release of heparan sulfate proteoglycans from pulmonary fibroblast cultures. A mechanism for basic fibroblast growth factor (bFGF) release and attenuation of bfgf binding following elastase-induced injury.

We have investigated elastase-mediated alterations in the expression of basic fibroblast growth factor (bFGF) receptors and proteoglycan co-receptors and characterized the subsequent effects on bFGF receptor binding profiles. For these studies, pulmonary fibroblast cultures were treated with porcine pancreatic elastase, and elastase-mediated changes in bFGF receptor expression and binding profiles were assessed. Quantitation of [(35)S]sulfate-labeled proteoglycan and total glycosaminoglycan release from fibroblast matrices indicated that elastase treatment released sulfated proteoglycan from the cell surface in a time- and dose-dependent fashion that correlated strongly with elastase-mediated bFGF release. Ligand binding studies indicated that elastase treatment decreased total binding of (125)I-bFGF to the cell surface and affected both fibroblast growth factor receptor and heparan sulfate proteoglycan (HSPG) binding sites. Western blot analyses indicated that elastase treatment did not release significant amounts of fibroblast growth factor receptor protein. These findings indicate that elastase-mediated HSPG release from fibroblast matrices reduces the effective affinity of bFGF for its receptor. Collectively, these studies suggest that HSPG co-receptors are important mediators of the pulmonary fibroblast response to elastase treatment and that bFGF, HSPG, and other elastase-released entities play an important role in the response of the lung to chronic injury.     

Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor.

The role of low affinity, heparin-like binding sites for basic fibroblast growth factor (bFGF) was investigated in CHO cells mutant in their metabolism of glycosaminoglycans. Heparan sulfate-deficient mutants transfected to express a cloned mouse FGF receptor cDNA are not able to bind bFGF. It is demonstrated that free heparin and heparan sulfate can reconstitute a low affinity receptor that is, in turn, required for the high affinity binding of bFGF. These studies suggest that the low affinity receptor is an accessory molecule required for binding of bFGF to the high affinity site. Such an obligatory interaction of low and high affinity FGF receptors suggests a physiological role for heparin-like, low affinity receptors and constitutes a novel mechanism for the regulation of growth factor-receptor interactions.     

Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: Implications for potentiation of growth factor action by heparin

Heparan sulfate proteoglycans on the cell surface act as low affinity binding sites for acidic and basic fibroblast growth factor (FGF) [Moscatelli (1887): J Cell Physiol 131:123–130] and play an important role in the interaction of FGF with the FGF receptor (FGFR). In this study, several aspects of the interaction of FGFs with cell surface heparan sulfate proteoglycans were examined. Reciprocal cross blocking studies demonstrated that acidic FGF (aFGF) and basic FGF (bFGF) bind to identical or closely associated heparan sulfate motifs on BALB/c 3T3 cell surface heparan sulfate proteoglycans. However, the binding affinity of the two growth factros for these heparan sulfate proteoglycans differs considerably, competition binding data indicating that aFGF has a 4.7-fold lower affinity than bFGF for 3T3 heparan sulfate proteoglycan. Subsequent studies of dissociation kinetics demonstrated that bFGF dissociates form the FGFR at least 10-fold slower than aFGF, whereas, following removal of cell surface heparan sulfate proteoplycan. Subsequent studies of dissociation kinetic demonstrated that bFGF dissociates from the FGFR at least 10-fold slwer than aFGF, whereas, following removal of cell surface heparan sulfate proteoglycans by heparinase treatment, the dissociation rate of both FGFs is similar and rapid. These results support the concept that cell surface heparan sulfate proteoglycans stabilize the interactio fo FGF with FGFR, possibly by the formatin of a ternary complex. © Wiley-Liss, Inc.     

Transforming growth factor beta 1 stimulates the production of basic fibroblast growth factor binding proteoglycans in Balb/c3T3 cells.

Basic fibroblast growth factor (bFGF) binds to cell surface receptors and to heparin sulfate proteoglycans. Heparan sulfate binding may limit bFGF degradation and be an obligatory step for bFGF cell interaction. Transforming growth factor-beta 1 (TGF-beta 1) is a potent regulator of proteoglycan production and composition. The possibility that TGF-beta 1 synergistically regulates bFGF activity by altering bFGF-proteoglycan interactions was investigated. TGF-beta 1 increased 125I-bFGF binding to the extracellular matrix (ECM) of Balb/c3T3 cells 2-4-fold by increasing the number of bFGF binding sites. Increased bFGF binding correlated with a 2-5-fold increase in the production of sulfated proteoglycans, including heparan sulfate proteoglycans. TGF-beta 1 selectively stimulated production of high molecular mass proteoglycans (190-300 kDa) in conditioned medium and stimulated all proteoglycans in ECM. 125I-bFGF bound to TGF-beta 1 induced proteoglycans immobilized onto cationic nylon filters. Furthermore, ECM isolated from TGF-beta 1-treated cells incorporated more mitogenically active bFGF than native ECM. The mitogenic potential of the ECM was significantly reduced by treatment with heparinase. These results suggest that the ability of TGF-beta 1 to stimulate binding of bFGF to ECM, increase ECM heparan sulfate proteoglycan, and potentiate the mitogenic activity of bFGF are linked. Thus one aspect of TGF-beta 1/bFGF synergy may involve modulation of the ECM.     

Stimulation of fibroblast growth factor receptor-1 occupancy and signaling by cell surface-associated syndecans and glypican

The formation of distinctive basic FGF-heparan sulfate complexes is essential for the binding of bFGF to its cognate receptor. In previous experiments, cell-surface heparan sulfate proteoglycans extracted from human lung fibroblasts could not be shown to promote high affinity binding of bFGF when added to heparan sulfate-deficient cells that express FGF receptor-1 (FGFR1) (Aviezer, D., D. Hecht, M. Safran, M. Eisinger, G. David, and A. Yayon. 1994. Cell 79:1005-1013). In alternative tests to establish whether cell-surface proteoglycans can support the formation of the required complexes, K562 cells were first transfected with the IIIc splice variant of FGFR1 and then transfected with constructs coding for either syndecan-1, syndecan-2, syndecan-4 or glypican, or with an antisense syndecan-4 construct. Cells cotransfected with receptor and proteoglycan showed a two- to three- fold increase in neutral salt-resistant specific 125I-bFGF binding in comparison to cells transfected with only receptor or cells cotransfected with receptor and anti-syndecan-4. Exogenous heparin enhanced the specific binding and affinity cross-linking of 125I-bFGF to FGFR1 in receptor transfectants that were not cotransfected with proteoglycan, but had no effect on this binding and decreased the yield of bFGFR cross-links in cells that were cotransfected with proteoglycan. Receptor-transfectant cells showed a decrease in glycophorin A expression when exposed to bFGF. This suppression was dose-dependent and obtained at significantly lower concentrations of bFGF in proteoglycan-cotransfected cells. Finally, complementary cell- free binding assays indicated that the affinity of 125I-bFGF for an immobilized FGFR1 ectodomain was increased threefold when the syndecan- 4 ectodomain was coimmobilized with receptor. Equimolar amounts of soluble syndecan-4 ectodomain, in contrast, had no effect on this binding. We conclude that, at least in K562 cells, syndecans and glypican can support bFGF-FGFR1 interactions and signaling, and that cell-surface association may augment their effectiveness.     

Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4

Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.     

Staphylococci Bind Heparin-Binding Host Growth Factors

Staphylococcus aureus, which mediated binding to heparan sulfate, and also strains of coagulase-negative staphylococci (CNS) adhered in high numbers to polymers with end-point attached heparin. A characteristic feature of several cell growth factors is strong affinity for heparin. In the present study, binding of the ¹²⁵I-labeled heparin-binding growth factors (HBGF), acidic and basic fibroblast growth factor (aFGF, bFGF), and platelet-derived growth factor (PDGF) by S. aureus and CNS strains was examined. Staphylococcal strains used in this study bind bFGF and PDGF, but not aFGF. The binding of bFGF and PDGF was time dependent, influenced by pH and ionic strength for S. aureus Cowan 1. Preincubation of staphylococcal cells with unlabeled bFGF enhanced bFGF binding, but heparin, protamine sulfate, poly-L-lysine, and suramin were potent inhibitors of ¹²⁵I-bFGF binding to cells of S. aureus Cowan 1. Glycosaminoglycans of comparable size (chondroitin sulfate), other polysulfated polymers (λ-carrageenan, fucoidan), and some polysulfated polysaccharides (dextran sulfate, pentosan polysulfate) inhibited binding of both GFs to various extents. The partial inhibition of binding of both GFs after protease and periodate treatments indicates that both proteinaceous and other carbohydrate moieties participate in the binding. A lysozyme cell surface extract and bacterial lysates of S. aureus Cowan 1 competitively inhibited binding of ¹²⁵I-bFGF and ¹²⁵I-PDGF. These results suggest that staphylococci have the ability to bind two of the HBGFs, bFGF and PDGF, but not aFGF, via more than one cell structure. These binding structures seem to be exposed on the cell surface and deeply anchored in the cytoplasmic membrane as well.     

Effect of chlorate on the sulfation of lipoprotein lipase and heparan sulfate proteoglycans. Sulfation of heparan sulfate proteoglycans affects lipoprotein lipase degradation

In avian-cultured adipocytes 76% of the newly synthesized lipoprotein lipase is degraded before release into the medium (Cupp, M., Bensadoun, A., and Melford, K. (1987) J. Biol. Chem. 262, 6383-6388). The same group (Cisar, L. A., Hoogewerf, A. J., Cupp, M., Rapport, C. A., and Bensadoun, A. (1989) J. Biol. Chem. 264, 1767-1774) has proposed that the interaction of lipoprotein lipase with a class of cell surface heparan sulfate proteoglycans is necessary for degradation to occur. To test further this hypothesis, the binding capacity of the plasma membrane for the lipase was decreased by inhibiting the sulfation of glycosaminoglycans with sodium chlorate, an inhibitor of sulfate adenyltransferase. Chlorate decreased sulfate incorporation into trypsin-releasable heparan sulfate proteoglycans to 20% of control levels. The amount of uronic acid in the trypsin-releasable heparan sulfate proteoglycans remained constant. Therefore, chlorate decreased sulfation density on heparan sulfate chains by approximately 5-fold. In the same fractions, chlorate increased the median heparan sulfate Mr measured on Sephacryl S-300. Chlorate decreased the maximum binding of 125I-lipoprotein lipase to adipocytes by 4-fold, but no significant effects on the affinity constants were observed. Chlorate increased lipoprotein lipase secretion in a dose-dependent relationship up to 30 mM. Utilizing a pulse-chase protocol, it was shown that lipase synthesis in control and chlorate-treated cells was not significantly different and that the increased secretion could be accounted for by a decreased lipoprotein lipase degradation rate. In control cells 77 +/- 11% of the synthesized enzyme was degraded whereas in chlorate-treated cells degradation was reduced to 42 +/- 9% of the synthesized amount. The present study shows that decreased sulfation of heparan sulfate proteoglycans decreases the maximum binding of the lipase for the adipocyte cell surface. Consistent with the model that binding of lipoprotein lipase to cell surface heparan sulfate is required for lipase degradation, degradation is reduced in chlorate-treated cultures. In this report it is also shown that chlorate inhibits lipoprotein lipase sulfation and that desulfation of the enzyme has no effect on its catalytic efficiency or on its binding to cultured adipocytes.     

Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.     

Characterization of heparin-binding growth-associated factor receptor on NIH 3T3 cells.

Scatchard plot analysis of the binding of 125I-labeled heparin binding cell growth-associated factor (125I-HBGAF) to NIH 3T3 cells revealed a single class of high affinity receptors (-5000/cell) with kd of -0.6 nM. 125I-HBGAF was covalently cross-linked to the cell surface receptor on NIH 3T3 cells with disuccinimidyl suberate (DSS). Two 125I-HBGAF-cross-linked complexes of 170 kDa and 142 kDa were observed on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The 125I-HBGAF-cross-linked complex formation was completely abolished in the presence of greater than or equal to 100-fold excess of unlabeled HBGAF but not PDGF, EGF, aFGF, bFGF, or insulin. 125I-HBGAF appeared to undergo rapid internalization and relatively slow degradation following binding to the HBGAF receptor on NIH 3T3 cells. These results suggest that NIH 3T3 cells express a high affinity HBGAF receptor which shows two different estimated molecular masses of -155 kDa and -127 kDa. This high affinity HBGAF receptor was also found to express in other cell types.     

Gekko-sulfated glycopeptide inhibits tumor angiogenesis by targeting basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) is a therapeutic target of anti-angiogenesis. Here, we report that a novel sulfated glycopeptide derived from Gekko swinhonis Guenther (GSPP), an anticancer drug in traditional Chinese medicine, inhibits tumor angiogenesis by targeting bFGF. GSPP significantly decreased the production of bFGF in hepatoma cells by suppressing early growth response-1. GSPP inhibited the release of bFGF from extracellular matrix by blocking heparanase enzymatic activity. Moreover, GSPP competitively inhibited bFGF binding to heparin/heparan sulfate via direct binding to bFGF. Importantly, GSPP abrogated the bFGF-stimulated proliferation and migration of endothelial cells, whereas it had no inhibitory effect on endothelial cells in the absence of bFGF. Further study revealed that GSPP prevented bFGF-induced neovascularization and inhibited tumor angiogenesis and tumor growth in a xenograft mouse model. These results demonstrate that GSPP inhibits tumor angiogenesis by blocking bFGF production, release from the extracellular matrix, and binding to its low affinity receptor, heparin/heparan sulfate.     

Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells

Follistatin, an activin-binding protein secreted by cultured rat granulosa cells, was shown to associate with the cell surface by affinity labeling with 125I-activin. Addition of follistatin to the cultured cells demonstrated a typical ligand-binding saturation curve, suggesting that granulosa cells have a specific binding site for follistatin. This binding was markedly inhibited by heparin and heparan sulfate, but not by chondroitin sulfates A and C, keratan sulfate, and dermatan sulfate. When granulosa cells were treated with glycosaminoglycan-degrading enzymes before or after addition of follistatin to the cultures, heparinase and heparitinase treatments resulted in significant suppression of the binding, whereas treatment with chondroitinase ABC had no effect. A competition study of the binding using heparin derivatives demonstrated that follistatin seemed to recognize O-sulfate groups in the heparin molecule. Heparitinase-treated granulosa cells exhibited almost full responsiveness to activin, indicating that the enzyme treatment had no effect on activin and receptor interaction. These results suggest that follistatin/activin-binding protein binds to heparan sulfate side chains of proteoglycans on the granulosa cell surface to regulate the various actions of activin.