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J Biol Chem, Vol. 273, Issue 13, 7303-7310, March 27, 1998

Interaction of Heparan Sulfate from Mammary Cells with Acidic Fibroblast Growth Factor (FGF) and Basic FGF
REGULATION OF THE ACTIVITY OF BASIC FGF BY HIGH AND LOW AFFINITY BINDING SITES IN HEPARAN SULFATE*

Hassan RahmouneDagger , Hai-Lan ChenDagger , John T. Gallagher§, Philip S. RudlandDagger , and David G. FernigDagger

From the Dagger  School of Biological Sciences, Life Sciences Building, University of Liverpool, Crown Street, Liverpool L69 7ZB and the § Cancer Research Campaign Department of Medical Oncology, Christie Hospital, Wilmslow Road, Manchester M20 9BX, United Kingdom

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We have determined the relationship between the binding sites for acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and bFGF in DNA synthesis assays. The ka of the HS for aFGF fell into three groups, whereas the kd (0.0015-0.016 s-1) and the Kd (0.4-8.6 µM) formed a continuum. bFGF possessed a high affinity binding site (Kd 22-30 nM) with a fast ka (320,000-550,000 M-1 s-1), termed "fast/high," and a lower affinity site (Kd 47-320 nM) with a slower ka (35,000-150,000 M-1 s-1), termed "slow/low." Most of the species of HS possessed the latter binding site, which was able to activate bFGF in HS-deficient fibroblasts. However, the HS from the culture medium of the mammary fibroblasts and the myoepithelial-like cells possessed both a fast/high and a slow/low binding site and could not activate bFGF, although it could potentiate the growth-stimulatory activity of aFGF. Treatment of the HS possessing two binding sites for bFGF with heparitinase 1 released oligosaccharides that were able to restore the activity of bFGF in HS-deficient fibroblasts.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The two archetypal fibroblast growth factors (FGF),1 acidic FGF (aFGF; FGF-1) and basic FGF (bFGF; FGF-2), are classic examples of heparan sulfate (HS)-binding growth factors (1, 2). The FGFs thus possess two distinct types of receptors, tyrosine kinase receptors (FGFRs) and HS. The HS receptors for aFGF and bFGF have several functions. First, the interaction between these FGFs and HS stabilizes the protein with respect to spontaneous and experimentally induced denaturation as well as proteolysis (1). This is particularly relevant for aFGF, which in the absence of HS is unstable at normal physiological ionic strength, pH, and temperature. Second, the sequestration of FGFs on extracellular HS may prevent the diffusion of the growth factor within a tissue compartment or between tissue compartments, as well as allowing a local store of the growth factor to act on a restricted number of cells (3, 4). Third, the growth-stimulatory activities of the FGFs are HS-dependent. Thus, these growth factors must interact with a dual receptor system consisting of FGFRs and HS receptors, if they are to stimulate cell division or cell migration (5-7). Although the exact mechanism of the dual receptor system is a matter of debate, the requirement for both receptors is firmly established (2).

aFGF and bFGF have been implicated in regulating the development of the mammary gland of rodents and humans (Refs. 8-10; reviewed in Ref. 3). In resting ducts, bFGF is associated with the basement membrane and with the myoepithelial cell, both of which possess a large, spare capacity of HS binding sites for bFGF (11). Thus, the radius of action of the growth factor in these structures is limited by its HS receptor. However, in terminal end buds, a major site of cell proliferation between birth and puberty (12, 13), bFGF is equally distributed between the growing cells in these structures and the basement membrane, and the HS receptors for bFGF in this region of the gland do not appear to have a large excess capacity for binding to bFGF. Hence, in these growing structures, bFGF is able to diffuse more freely and is likely to be involved in the stimulation of the growth of the cells of the terminal end buds and perhaps mediate stromal-epithelial interactions (3, 11).

There has been a long standing association between alterations in both the levels of production and the overall sulfation of HS and the development of malignant mammary tumors (14, 15). In cellular models of breast cancer, analogous changes in the gross structure of HS have been observed, whereas changes in the levels of expression of the core protein of the HS proteoglycan syndecan-1 may be causally related to the acquisition of a malignant phenotype by mammary tumor cells in vitro (16-18). We have analyzed the aFGF- and bFGF-binding sites present in the HS produced by cells representative of the ductal epithelial cell, the myoepithelial cell, the stromal fibroblast, and malignant mammary tumors, by using a biosensor-based binding assay (19) and determining quantitatively the binding parameters of aFGF and bFGF for the HS from these different mammary cell types. In addition, we have measured the ability of the different species of mammary HS to activate bFGF in HS-deficient cells. The results indicate that the HS from the mammary cells possesses multiple, but independent classes of binding sites for these growth factors, and that there is a correlation between the class of the bFGF binding site and the ability of the HS to activate bFGF.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials and Cells-- Human recombinant aFGF and bFGF were prepared as described previously (20, 21). Chondroitinase ABC, Pronase, and micrococcal nucleases were obtained from Sigma (Poole, United Kingdom (UK)). Sialidase enzyme collection (EC 3.2.1.18) was from Oxford GlycoScience (Oxford, UK), and endo-beta -galactosidase (EC 3.2.1.03) and bovine pancreatic RNase were from Boehringer (Mannheim, Germany). Heparan sulfate lyase (heparitinase 1, EC 4.2.2.8) and heparin lyase (heparitinase 3, EC 4.2.2.7) were obtained from Seikagaku Co. (Tokyo, Japan).

Rat mammary (Rama 27) fibroblasts (22) and the human benign mammary (Huma) epithelial cells, Huma 123, and the Huma 109 myoepithelial-like cells were cultured as described (23, 24). The MCF-7 and ZR-75 human malignant mammary epithelial cells (25) were cultured in Dulbecco's modified Eagle's medium supplemented with 5% (v/v) fetal calf serum (Life Technologies, Inc., Paisley, Scotland), 50 ng/ml insulin, and 10 ng/ml estradiol (Sigma).

Preparation of HS-- A modification of previously described methods (26, 27) was used. Cells were subcultured into 64 15-cm diameter culture dishes (Nunc, Roskilde, Denmark), and, in four dishes, 10 µCi/ml [3H]glucosamine and 20 µCi/ml [35S]SO4 (both ICN-Flow, Thame, UK) were included in the culture medium. After 72 h, when the cells were 90% confluent, the culture medium was removed and pooled with two 5-ml PBS (137 mM NaCl, 10 mM Na2 HPO4, pH 7.2) washes of each culture dish. The cells were collected by scraping in 5 ml of 6 M urea with 0.5% (v/v) Triton X-100 and incubated in this solution overnight at 4 °C on a shaker. In the case of Rama 27 fibroblasts, two medium and two cellular samples were produced. After the removal of the culture medium and washing the cell monolayers with 5 ml of PBS, 25 ml of fresh step-down medium (SDM; DMEM with 250 µg/ml bovine serum albumin) was added to each culture dish. Twenty-four hours later, the SDM was collected as before and the cells were then detached with 2 ml of 0.5% trypsin (w/v, Sigma) in Versene (Life Technologies, Inc.) and collected by centrifugation at 3000 rpm for 10 min in 30-ml universal tubes. The cell pellet was washed with 5 ml of PBS by centrifugation as above, and the two supernatants were pooled to produce a fraction called the "trypsinate," containing trypsin-releasable HS proteoglycans. The culture dishes were scraped in 5 ml of 6 M urea, 0.5% Triton X-100 (v/v) in PBS, which was added to the cell pellet and solubilized as above.

The samples were applied to a 2.5 × 50-cm column of diethylaminoethyl (DEAE)-Sepharose Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden). Bound macromolecules were eluted with a linear gradient of NaCl (0.15-2 M NaCl in 20 mM Na2HPO4, pH 6.8) and the 3H and 35S content of aliquots of the 6-ml fractions eluting between 0.3 and 1 M NaCl, which contained proteoglycans, was determined in a Packard 1900TR scintillation counter. After exhaustive dialysis against H2O for 48 h and lyophilization, the proteoglycans were treated sequentially with: (i) chondroitinase ABC, (ii) sialidases (26), (iii) endo-beta -galactosidase and nucleases (28), and (iv) Pronase (26). The free HS chains were purified on a second DEAE-Sepharose Fast Flow column (1 × 30 cm) and eluted between 0.3 M and 1 M NaCl.

Binding Assays-- Because the HS chains were purified as peptidoglycans, these were biotinylated on the free amino group of the peptide. One hundred µg of HS in 100 µl of distilled water was incubated with 30 µl of a 50 mM solution of N-hydroxysuccinimide aminocaproate (LC) biotin (Pierce-Warriner, Chester, UK) in dimethyl sulfoxide for 72 h. Unreacted biotin was removed by fractionation on a Sephadex G-25 column (1 × 25 cm) equilibrated in distilled water, and HS chains were then lyophilized. Biotinylated HS chains were immobilized on streptavidin-derivatized surfaces as described for biotinylated heparin (19).

Binding reactions were carried out in an IAsys resonant mirror biosensor at 20 °C using three-dimensional carboxymethyl dextran and planar aminosilane surfaces (Affinity Sensors, Saxon Hill, Cambridge, UK). The negative charge of the carboxymethyl dextran surface at pH 7.2 appeared to prevent aFGF binding to HS immobilized on this surface (results not shown). Therefore, aminosilane surfaces, derivatized with streptavidin according to the manufacturer's instructions (Affinity Sensors), were used for aFGF binding assays. The bFGF-binding assays were repeated at least three times, once on an aminosilane surface and twice on a carboxymethyl dextran surface. aFGF and bFGF themselves did not bind to streptavidin-derivatized carboxymethyl dextran or aminosilane surfaces (results not shown). The distribution of the immobilized HS and of the bound aFGF and bFGF on the surface of the biosensor cuvette was inspected by examination of the resonance scan, which showed that at all times these molecules were distributed uniformly on the sensor surface and therefore were not microaggregated.

A single binding assay consisted of adding the ligate at a known concentration in 100 µl of PBST (PBS supplemented with 0.02% (v/v) Tween 20) and then following the association reaction over a set time, usually 300 s. The cuvette was then washed three times with 200 µl of PBST, and the dissociation of bound ligate into the bulk PBST was followed over time. To remove residual bound ligate, and thus regenerate the immobilized ligand, the cuvette was washed twice with 200 µl of 2M NaCl, 10 mM Na2HPO4, pH 7.2. Binding parameters were calculated from the association and dissociation phases of the binding reactions using the non-linear curve fitting FastFit software (Affinity Sensors) provided with the instrument.

The percentage of the HS peptidoglycan chains possessing biotin coupled exclusively to the peptide moiety was also determined. The total number of binding sites for bFGF on biotinylated HS immobilized on a streptavidin-derivatized aminosilane surface was measured by repeatedly adding 3 ng/ml bFGF until saturation of the available bFGF-binding sites was observed. After removal of the bound bFGF with 2 M NaCl, beta -elimination was carried out on the immobilized HS for 16 h in 50 mM NaOH and 2 M NaBH4 at 37 °C (28) in the biosensor cuvette. The reaction was stopped with 4 M acetic acid and the supernatant removed. A 50-µl wash of the cuvette with 8 M guanidine HCl was pooled with the supernatant, which was desalted on Sephadex G-25 in H2O. The desalted HS chains were then immobilized on a fresh streptavidin-derivatized aminosilane surface and the total number of bFGF binding sites again measured. bFGF failed to bind to the cuvette washed with 8 M guanidine. Between 90% and 97% of the bFGF-binding sites were associated with HS chains possessing biotin, which was sensitive to beta -elimination, and thus linked to the peptide moiety of the peptidoglycan. The remainder of the bFGF-binding sites in the peptidoglycan chains were insensitive to beta -elimination and thus may be associated with biotin linked to the polysaccharide moiety of the peptidoglycan (results not shown).

Heparinase Digestion of HS-- HS (1 mg), prepared from the culture medium of the fibroblastic Rama 27 cells, was initially heated to 100 °C for 3 min and then incubated at 37 °C for 24 h with heparitinase 1 or heparitinase 3 at a concentration of 40 milliunits/ml in 50 mM sodium acetate buffer, pH 7.0, containing 0.1 mM calcium acetate. The enzymes were then inactivated by heating at 100 °C for 3 min and the resulting oligosaccharides desalted on a column (1 × 100 cm) of Bio-Gel P6 (Bio-Rad, Hemel Hempstead, UK) equilibrated in distilled water. Oligosaccharides in the excluded and included volumes of the column were pooled and lyophilized.

Assay for Stimulation of [3H]DNA Synthesis-- HS-deficient Rama 27 fibroblasts were prepared by a modification (29) of the method of Rapraeger et al. (7). Near-confluent Rama 27 cells in 9-cm culture dishes (Nunc) were washed twice with PBS and fresh, low sulfur Routine Medium (DMEM without SO42-, containing 20 µM methionine and 15 µM cystine (both Sigma) and supplemented with 5% (v/v) dialyzed fetal calf serum, 50 ng/ml insulin, 50 ng/ml hydrocortisone, and 15 mM NaClO3 (Fluka, Glossop, UK)) was added. After a 6 h of incubation, the cells were subcultured in this medium in 24-well dishes for assays for DNA synthesis. After 24 h, the cell monolayers were washed twice with 500 µl of PBS, and 500 µl of low sulfur SDM (DMEM without SO42-, containing 20 µM methionine and 15 µM cystine and supplemented with 250 µg/ml bovine serum albumin and 15 mM NaClO3) was added. The cells were incubated in this medium for 24 h to induce quiescence, and then the medium was replaced with 500 µl of fresh low sulfur SDM at 37 °C. bFGF and HS were added to the cells as indicated in the text. After 18 h, when S-phase DNA synthesis was maximal, 1.5 µM [3H]thymidine (0.8 µCi; ICN-Flow) was added to the cells for 1 h and the cells were then prepared for scintillation counting, exactly as described previously (30). In some experiments, control Rama 27 fibroblasts were used in DNA synthesis assays, in which case conventional DMEM was used throughout (30).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The HS chains used in this study were purified from five different mammary cell lines, representative of some of the cell types found in the normal mammary gland (Fig. 1) and in malignant mammary tumors. The ductal epithelial cell is represented by the benign Huma 123 epithelial cells, the myoepithelial cells correspond to the Huma 109 myoepithelial-like cells, and the Rama 27 fibroblasts are representative of a mammary stromal fibroblast. The MCF-7 and ZR-75 cells are malignant metastatic human mammary epithelial cells (22, 23, 25). Exhaustive digestion of the peptidoglycans with heparitinases 1-3, followed by isolation of the disaccharides on Bio-Gel P6, indicated that over 90% of the starting material was degraded to disaccharides and was thus HS (31). The composition of the disaccharides was determined in those cases where there was sufficient material. The major difference observed was a 2-fold increase in 6-O-sulfated disaccharides in the HS from the two malignant cell lines, MCF-7 and ZR-75, compared with the HS from the benign epithelial Huma 123 and the myoepithelial-like Huma 109 cell lines (31, 32).


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  		Fig. 1.   Schematic transverse section through a mammary duct showing some of the cell types found in the parenchyma and the neighboring stroma. Gray shading, stromal matrix; zigzag shading, basement membrane.

Kinetics of Binding of bFGF to HS-- The association phase of the interaction between bFGF and mammary cell HS was considerably faster than that for aFGF, whereas the dissociation phase of the reactions proceeded at similar rates (Figs. 2 and 3). Consequently, considerably lower concentrations of bFGF were used in the binding assays, and the bulk shifts observed at the start of the association and dissociation phases were quite small (Fig. 2). The interaction between bFGF with mammary cell HS exhibits a much greater complexity than found with the other HS-binding proteins we have examined,2 because some of the samples of HS possess a single binding site for bFGF, whereas other samples of HS possessed two binding sites for bFGF (Table I).


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  		Fig. 2.   Binding of bFGF to HS purified from cells. HS purified from the benign Huma 123 epithelial cells was biotinylated and immobilized on a streptavidin-carboxymethyl dextran surface. A, after the addition of bFGF to the biosensor cuvette, the binding of bFGF was followed in real time for at least 5 min. The cuvette was then quickly washed three times with 200 µl of PBST, and the dissociation of bound bFGF into 200 µl of PBST was followed over the next 2 min. Data were collected every second during the course of the experiment. B, a plot of kon against ligand concentration, the slope of which is the association rate constant, ka. The kon of bFGF for HS at each concentration of bFGF was determined using FastFit software.


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  		Fig. 3.   Binding of aFGF to HS purified from Huma 109 myoepithelial-like cells. HS was biotinylated and immobilized on a streptavidin-aminosilane surface. After the addition of aFGF to the biosensor cuvette, the binding of aFGF was followed in real time for at least 5 min. The cuvette was then quickly washed three times with 200 µl of PBST, and the dissociation of bound aFGF into 200 µl of PBST was followed over the next 2 min. Data were collected every second during the course of the experiment. Inset, a plot of kon against ligand concentration, the slope of which is the association rate constant, ka. The kon of aFGF for HS at each concentration of aFGF was determined using FastFit software.

                              
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Table I
Kinetics of bFGF binding to HS purified from mammary cells

Samples of HS Possessing a Single Binding Site for bFGF-- The HS isolated from the culture medium and the cellular fractions of the epithelial mammary cells, benign Huma 123, malignant MCF-7 and ZR-75, possessed a single, low affinity, binding site for bFGF with a ka that ranged from 49,000 to 100,000 (Table I). The kd of these binding sites for bFGF ranged from 0.0050 s-1 to 0.017 s-1. Consequently, the Kd of these binding sites for bFGF on the HS isolated from the mammary epithelial cells was between 56 nM and 240 nM (Table I). A binding site with similar bFGF-binding kinetics was also observed in the HS purified from the cellular fraction of the Huma 109 myoepithelial-like cells and of the Rama 27 fibroblasts (Table I).

Samples of HS Possessing Two Binding Sites for bFGF-- In contrast, the association phase of the binding reaction was biphasic for three samples of HS, isolated from the routine culture medium and SDM of the Rama 27 fibroblasts and the culture medium of the Huma 109 myoepithelial-like cells. Thus, these samples of HS possessed two binding sites for bFGF. The association rate constant of the fast binding site ranged from 320,000 M-1 s-1 to 520,000 M-1 s-1 (Table I). The second binding site possessed a much slower association rate constant for bFGF (ka 41,000 M-1 s-1 to 64,000 M-1 s-1) and was thus similar to the slow binding site observed in the samples of HS possessing just a single binding site for bFGF (Table I). When the dissociation phase of the binding reactions were examined, there was, however, no evidence for two sites of dissociation. Therefore, the dissociation of bFGF from both the fast and slow association sites seems to be governed by a single dissociation rate constant, ranging from 0.0095 s-1 to 0.013 s-1 (Table I) and so the fast binding site also has the highest affinity for bFGF (Kd 22 nM to 30 nM).

There are thus two distinct binding sites for bFGF in the samples of mammary HS. 1) The low affinity binding site (Kd 47-290 nM) has slow association kinetics (ka 35,000-150,000 M-1 s-1) and is termed "slow/low"; 2) the high affinity site (Kd 22-30 nM) exhibits fast association kinetics (320,000-550,000 M-1 s-1) and is termed "fast/high." It is only found in association with the slow/low binding site (Table I).

Kinetics of Binding of aFGF to HS-- Relatively high concentrations of aFGF were required to produce a signal. Consequently, the bulk shift, a result of the difference in the refractive indices of PBST and PBST containing aFGF, observed in the first 3-5 s of the association phase and in the first 3-5 s of the dissociation phase of the binding assay was quite high (Fig. 3).

Association Rate Constants-- The association rate constants of aFGF for the HS formed three groups. 1) The HS from the culture medium of malignant ZR-75 cells possessed the fastest association rate constant for aFGF, 29000 M-1 s-1 (Table II). 2) The HS isolated from the benign Huma 123 epithelial cells and their culture medium had the slowest association rate constants (870-1000 M-1 s-1, Table II). 3) The majority of the HS species, with intermediate association rate constants, ranged from 2500 M-1 s-1 to 4600 M-1 s-1 (Table II).

                              
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Table II
Kinetics of aFGF binding to immobilized HS, purified from mammary cells

The binding of 30 µM aFGF to the HS from the malignant MCF-7 cells, immobilized on an aminosilane surface, was not detectable in three separate experiments carried out on two independently prepared surfaces, although the same surfaces bound collagen 12 and bFGF (Table I).

Dissociation Rate Constants-- The kd of aFGF for the HS spanned a range from 0.0015 s-1 to 0.0016 s-1. At the slow end of this range were the HS from the Huma 109 myoepithelial-like cells and from the culture medium of the malignant epithelial MCF-7 cells. At the fast end of this range were the HS from the culture medium of the ZR-75 malignant epithelial cells and from the culture medium of the Rama 27 fibroblasts. The two extremes of kd may represent slow and fast dissociating forms of the receptor (Table II).

Affinity of aFGF for HS-- The HS isolated from the Huma 109 myoepithelial-like cells possessed the slowest kd and the HS from the culture medium of the malignant ZR-75 cells possessed the fastest ka; these two samples of HS had the highest affinity for aFGF, Kd 0.40-0.45 µM. The other samples of HS possessed a lower affinity for aFGF, Kd 0.94-8.6 µM (Table II). In all cases, only a single binding site for aFGF was observed in any particular sample of HS (Table II).

Activation of bFGF by HS-- We measured the ability of selected samples of mammary HS to activate bFGF. Samples were chosen on the basis of their bFGF-binding characteristics (Table I) and their abundance. Advantage was taken of Rama 27 fibroblasts made deficient in HS by treatment with NaClO3, in which HS-dependent growth factors such as bFGF are unable to stimulate DNA synthesis unless exogenous HS is added (29). Concentrations of HS ranging from 3 ng/ml to 30 µg/ml were tested.

HS purified from the culture medium of the Rama 27 cells possessed fast/high and slow/low binding sites (Table I). In the presence of such HS, bFGF was unable to stimulate DNA synthesis above the level observed with bFGF alone in HS-deficient fibroblasts at all concentrations of exogenously added HS (Fig. 4). A similar result was obtained with the HS from the culture medium of the myoepithelial-like Huma 109 cells, which also possesses fast/high and slow/low binding sites for bFGF (Fig. 4). However, the samples of HS with two binding sites for bFGF were not able to antagonize exogenously added heparin in HS-deficient Rama 27 cells or the endogenous HS on the surface of control Rama 27 cells (results not shown). In contrast, the HS purified from the extracellular matrix fraction of the Rama 27 fibroblasts, which possesses a single slow/low binding site for bFGF, was able to restore the growth-stimulatory effects of 3 ng/ml bFGF (Fig. 4) with the same efficiency as heparin (29). A second sample of HS with a slow/low binding site for bFGF, purified from the Huma 123 epithelial cells, also restored the activity of bFGF, although not as effectively as the HS from the extracellular matrix fraction of the Rama 27 fibroblasts (Fig. 4). The endogenous cell surface HS receptor in Rama 27 fibroblasts is of the slow/low type (purified from the trypsinate and the extracellular matrix fractions, Table I). Thus, it would appear that HS with two binding sites for bFGF, one fast/high, the other slow/low, is unable to replace such HS in this assay.


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  		Fig. 4.   Activation of bFGF by HS purified from mammary cells. Serum-starved HS-deficient Rama 27 fibroblasts were stimulated with bFGF in the presence of different species of HS (3 µg/ml). The incorporation of [3H]thymidine into DNA was determined 18 h after the initial stimulus (see "Experimental Procedures"). Results are the mean ± S.D. of triplicate determinations of one of four experiments.

Specific enzymatic cleavage of HS from the culture medium of Rama 27 cells was achieved by incubation with heparatinase 1 and heparatinase 3. Heparatinase 1 mainly cleaves the alpha 1-4 glycosidic bond between N-acetyl or N-sulfated glucosamine and glucuronic acid, present in low sulfated regions of HS, whereas heparatinase 3 cleaves the linkage between N-sulfated glucosamine and iduronate 2-sulfate, which occurs in highly sulfated regions of HS (33). When HS from the culture medium of Rama 27 cells was digested with heparitinase 1, the resulting mixture of oligosaccharides (10 µg/ml) was highly effective in restoring the growth-stimulatory activity of bFGF (Fig. 5). In contrast, the heparitinase 3-treated HS from the culture medium of the Rama 27 fibroblasts at 10 µg/ml was, like the parental polysaccharide, unable to restore the growth-stimulatory activity of bFGF (Fig. 5).


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  		Fig. 5.   Effect of heparatinase treatment of HS purified from the culture medium of Rama 27 cells on the activation of bFGF. Serum-starved HS-deficient Rama 27 fibroblasts were stimulated with 3 ng/ml bFGF in the presence of HS purified from the culture medium of Rama 27 fibroblasts that had been digested with heparitinase 1 or heparitinase 3 (see "Experimental Procedures"). White column, HS digested with heparitinase 1; gray column, HS digested with heparitinase 3; light, thinly striped column, no additions; boldly striped column, 3 ng/ml bFGF; dark, thinly striped column, 3 ng/ml bFGF and 30 ng/ml heparin. Results are the mean ± S.D. of triplicate determinations of one of two experiments.

Potentiation of aFGF by HS-- One aspect of the HS dependence of the stimulation of DNA synthesis by aFGF is the potentiation effect of the polysaccharide (1), which may be measured in normal Rama 27 fibroblasts (29). The HS from the urea/Triton extract of Rama 27 fibroblasts, and from the culture medium of Rama 27 fibroblasts and Huma 109 myoepithelial-like cells possess a single binding site for aFGF (Table II). These samples of HS are able to potentiate the activity of aFGF, although not as effectively as heparin (Fig. 6).


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  		Fig. 6.   Potentiation of the activity of aFGF by mammary cell HS. Serum-starved Rama 27 fibroblasts were stimulated with 10 ng/ml aFGF in the presence of different samples of mammary cell HS or heparin. Results are the mean ± S.D. of triplicate determinations of one of two experiments.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Binding Sites for aFGF and bFGF in HS

Two distinct binding sites for bFGF were found on the samples of mammary HS: fast/high and slow/low (Table II). The kinetics and affinity of the slow/low binding site for bFGF in the mammary HS are similar to the binding site for bFGF characterized in heparin (19). The interaction between aFGF and the HS purified from the mammary cells is characterized by three classes of association rate constant and one dissociation rate constant (Table II). The consequences of these binding kinetics are that the affinity of the samples of HS sites for aFGF may form a continuum, although the results suggest that there may be higher affinity (Kd 0.40-0.45 µM) and lower affinity (Kd 0.95-8.6 µM) forms of this receptor (Table II).

The binding sites for aFGF and bFGF in the samples of HS seem to be independent of each other, because there is no correlation between the binding of bFGF (Table I) and aFGF (Table II) to the samples of HS. For example, the HS from the malignant MCF-7 cells fails to bind detectable amounts of aFGF, yet binds bFGF. In addition, neither the ranking, in terms of association kinetics and affinity, nor the class of binding sites for bFGF in the samples of HS correspond with the results obtained with aFGF. These results thus support the hypothesis that aFGF and bFGF may recognize different structures in HS (34, 35), although the possibility remains that aFGF binds at least some of the structures recognized by bFGF, but fails to distinguish the finer features of these structures that lead to the different association kinetics observed with bFGF.

Regulation of the Growth-stimulatory Effects of bFGF and aFGF by HS

The results of the DNA synthesis assays provide some insights into the possible functions of the binding sites for aFGF and bFGF in HS. The presence of binding sites for bFGF in HS is not a guarantee that the HS is able to activate bFGF in HS-deficient fibroblasts. Thus, the HS from the culture medium of the Rama 27 fibroblasts and the Huma 109 myoepithelial-like cells possesses both fast/high and slow/low binding sites for bFGF and is unable to activate bFGF. In contrast, HS possessing a single slow/low binding site is able to activate bFGF (Fig. 4). Moreover, the HS purified from the mammary cells is clearly able to bind and activate aFGF and bFGF differentially. For example, the HS purified from the culture medium of Rama 27 fibroblasts and of Huma 109 myoepithelial-like cells has only a single class of binding site for aFGF and is able to potentiate the activity of aFGF (Fig. 6). In addition, the HS from the MCF-7 malignant epithelial cells possesses a slow/low binding site for bFGF, yet fails to bind detectable levels of aFGF. These results indicate that mammary cells are able to produce HS that may independently regulate the activities of these two closely related growth factors.

The results of the binding assays and DNA synthesis assays may thus provide an explanation for the observation in other systems, including a hormone-insensitive breast cancer cell line (36) and neural development (37), that HS can regulate independently the activity of bFGF and, under some circumstances, aFGF.

Implications of the FGF Binding Sites for the Structure of Mammary Cell HS

bFGF-- The binding structures for bFGF in HS have been extensively characterized. In a study with HS purified from the culture medium of human fetal skin fibroblasts (27), three binding structures for bFGF were defined as saccharide fragments named Oligo-H, Oligo-M, and Oligo-L, which have high, medium, and low affinity for bFGF as determined by the concentration of NaCl required to dissociate the bFGF-oligosaccharide complex. A key feature of the Oligo-H tetradecasaccharide is the presence of a core of a five disaccharide repeat of N-sulfated glucosamine and 2-O-sulfated iduronate. The length of this internal repeat is progressively shorter in the two lower affinity oligosaccharides, Oligo-M and Oligo-L, which, however, possess a greater content of 6-O-sulfated glucosamine. Therefore, the presence of the 6-O-sulfate does not contribute to the binding of bFGF. Subsequent studies have supported both the key role played by N-sulfated glucosamine and 2-O-sulfated iduronate in mediating the binding of bFGF to HS and the absence of a role in this interaction for 6-O-sulfated glucosamine (34, 38-43). However, although 6-O sulfation is not needed for binding to bFGF, it may be required for potentiation of bFGF activity, possibly because it tethers the HS·bFGF complex to the FGFR (35).

The elucidation of the relationship between the two binding sites for bFGF identified in the present study and the binding structures found in previous studies will require the formal identification of the structures represented by the fast/high and the slow/low binding sites. The results of the heparatinase 1 digestions do, however, give some clues as to the structural basis of the bFGF-binding and growth-stimulatory properties of the two binding sites in mammary cell HS. HS possessing just a slow/low binding site can activate bFGF, but when this binding site is accompanied in the HS by the fast/high binding site, such HS is no longer able to activate bFGF (Fig. 4). One explanation for this observation is that there is a physical link between the two binding sites, i.e. the binding sites are on the same HS chain or are on two chains that remain linked by a small, Pronase-resistant peptide. In support of this contention are the results of the heparatinase 1 digestion of HS purified from the culture medium of the Rama 27 fibroblasts, which will cleave the HS chains in low sulfated domains, enriched in N-acetyl glucosamine, thus releasing sulfated domains (S-domains) as oligosaccharides. The ability of the heparatinase 1-treated HS to activate bFGF could therefore be the result of the separation of the fast/high and slow/low binding sites, implying that, when together, the two sites somehow bind bFGF in a manner that interferes with the recognition of the FGFR kinase receptor. Interestingly, a recent study on glycosaminoglycans structurally related to HS suggests that that low affinity binding of bFGF to glycosaminoglycans may be important for the delivery of growth-stimulatory signals by bFGF to cells (44).

aFGF-- The binding structure of aFGF in HS has not been established at the same level of detail as that of bFGF. It is known that sulfated regions of heparin, which are enriched in 6-O sulfate groups on glucosamine, are important for the interaction of aFGF (34, 41). However, the structural basis for the different association and dissociation rate constants of aFGF for the mammary cell HS remain to be determined. The necessity of 6-O-sulfate groups for the binding of aFGF is distinct from the requirements of bFGF but similar to HGF/SF, which also requires 6-O-sulfate groups for binding to HS (45). However, the patterns of binding of aFGF (Table II) and HGF/SF2 to the different samples of HS do not always correlate. Thus, the binding sites for HGF/SF and aFGF in HS may be structurally distinct, despite both proteins showing a requirement for the presence of 6-O sulfate groups in HS.

Biological Implications of the Binding Sites for bFGF and aFGF in HS

The five cell lines used as a source of HS in the present study represent a model of some of the cells found in the normal mammary gland (Fig. 1) and in malignant tumors. Thus, the purified HS chains may possess functions that are representative of those normally expressed by the analogous cells in vivo.

The two binding sites for bFGF may contribute to the regulation of the transport and the biological activity of the growth factor. In the rodent and human mammary gland, the major sites of synthesis of bFGF are the intermediate cells of the terminal end buds, the myoepithelial cells of resting ducts, with a contribution from stromal cells, which may include fibroblasts (9, 46). There is a relationship between the bFGF-binding sites and the cell type producing the HS (Table I). Rama 27 fibroblasts and Huma 109 myoepithelial-like cells both secrete HS with fast/high and slow/low bFGF binding sites, which is unable to activate bFGF. In vivo, the analogous cells deposit many of the components of the basement membrane (3). Therefore, if HS of this type were present in vivo in the stromal matrix and basement membrane, the diffusion of the bFGF produced by the myoepithelial and stromal cells would be directional, toward the basement membrane, which would act as a sink for bFGF. Using immunocytochemical techniques, it has been suggested that such a sink exists in a number of tissues, (4, 47), including resting ducts in the rat mammary gland (11).

The binding sites for aFGF in mammary cell HS are weaker than those of bFGF (Table II) or HGF/SF.2 aFGF is an unstable protein under physical conditions similar to those found in the extracellular space (1). At high concentrations, mammary cell HS, which binds aFGF, also protects aFGF from denaturation (Fig. 6; Ref. 29). Therefore, the concentration and spatial distribution of aFGF binding sites in the HS of the mammary gland may not only provide a path for diffusion of the growth factor but also may dictate the limits of aFGF activity.

aFGF and bFGF are expressed ectopically by human malignant mammary tumors (8) and are likely to contribute to the growth of the tumors directly by activating the endogenous FGFRs on the malignant cells, and indirectly by promoting angiogenesis (3). The absence of the non-stimulatory two-site (fast/high)/(slow/low) HS receptor for bFGF on the malignant cells (Table II) supports a role for bFGF in the growth of malignant tumors. In addition, because malignant cells must produce proteases and heparanases to invade the surrounding stroma (48), these cells are likely to be able to release bFGF stored on the (fast/high)/(slow/low) receptor of the basement membrane and stromal matrix.

The HS purified from the malignant ZR-75 cells binds aFGF less efficiently than the corresponding HS purified form the culture medium of these cells, whereas the HS from the MCF-7 cells fails to bind aFGF (Table II). These results suggest that it may be important for the growth and survival of malignant tumors for the cells to favor the diffusion of aFGF away from the tumor into the surrounding stroma, perhaps to stimulate angiogenesis.

    FOOTNOTES

* This work was supported by the Cancer and Polio Research Fund, the Cancer Research Campaign, the Medical Research Council, the Mizutani Foundation for Glycoscience, and the North West Cancer Research Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-151-794-4388; Fax: 44-151-794-4349; E-mail: dgfernig{at}liv.ac.uk.

1 The abbreviations used are: FGF, fibroblast growth factor; aFGF (FGF-1), acidic FGF; bFGF (FGF-2), basic FGF; DMEM, Dulbecco's modified Eagle's medium; FGFR, fibroblast growth factor receptor; HGF/SF, hepatocyte growth factor/scatter factor; HS, heparan sulfate; Huma, human mammary; PBS, phosphate-buffered saline; PBST, PBS with Tween 20; Rama, rat mammary; SDM, step-down medium.

2 H. Rahmoune, H.-L. Chen, J. T. Gallagher, P. S. Rudland, and D. G. Fernig, submitted for publication.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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N. Sergeant, M. Lyon, P. S. Rudland, D. G. Fernig, and M. Delehedde
Stimulation of DNA Synthesis and Cell Proliferation of Human Mammary Myoepithelial-like Cells by Hepatocyte Growth Factor/Scatter Factor Depends on Heparan Sulfate Proteoglycans and Sustained Phosphorylation of Mitogen-activated Protein Kinases p42/44
J. Biol. Chem., May 26, 2000; 275(22): 17094 - 17099.
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V. Nurcombe, C. E. Smart, H. Chipperfield, S. M. Cool, B. Boilly, and H. Hondermarck
The Proliferative and Migratory Activities of Breast Cancer Cells Can Be Differentially Regulated by Heparan Sulfates
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M. Delehedde, M. Seve, N. Sergeant, I. Wartelle, M. Lyon, P. S. Rudland, and D. G. Fernig
Fibroblast Growth Factor-2 Stimulation of p42/44MAPK Phosphorylation and Ikappa B Degradation Is Regulated by Heparan Sulfate/Heparin in Rat Mammary Fibroblasts
J. Biol. Chem., October 20, 2000; 275(43): 33905 - 33910.
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C. L. R. Merry, S. L. Bullock, D. C. Swan, A. C. Backen, M. Lyon, R. S. P. Beddington, V. A. Wilson, and J. T. Gallagher
The Molecular Phenotype of Heparan Sulfate in the Hs2st-/- Mutant Mouse
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J. Kreuger, M. Salmivirta, L. Sturiale, G. Gimenez-Gallego, and U. Lindahl
Sequence Analysis of Heparan Sulfate Epitopes with Graded Affinities for Fibroblast Growth Factors 1 and 2
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O. Ostrovsky, B. Berman, J. Gallagher, B. Mulloy, D. G. Fernig, M. Delehedde, and D. Ron
Differential Effects of Heparin Saccharides on the Formation of Specific Fibroblast Growth Factor (FGF) and FGF Receptor Complexes
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