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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3289-3299
Platelet Factor 4 Modulates Fibroblast Growth Factor 2 (FGF-2)
Activity and Inhibits FGF-2 Dimerization
By
Catherine Perollet,
Zhong Chao Han,
Catherine Savona,
Jacques
Philippe Caen, and
Andreas Bikfalvi
From the Growth Factor and Cell Differentiation Laboratory,
University Bordeaux; Institut des Vaisseaux et du Sang, Paris, France;
and Institute of Hematology, Chinese Academy of Medical Science,
Tianjin, China.
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ABSTRACT |
Platelet factor 4 (PF-4) inhibits angiogenesis in vitro and in vivo.
The mechanism of inhibition is poorly understood. We have investigated
the mechanism of inhibition by examining the interaction of PF-4 and
the fibroblast growth factor-2 (FGF-2)/fibroblast growth factor
receptor (FGFR) system. PF-4 inhibited the binding of FGF-2 to
high-affinity and low-affinity binding sites in murine microvascular
endothelial cells (LEII cells) and proliferation. Maximum inhibition of
binding to endothelial FGF receptors was observed at PF-4
concentrations between 5 and 10 µg/mL (half maximum inhibition at 0.6 µg/mL), and proliferation was completely inhibited at 2 µg/mL. At
this concentration, PF-4 reduced internalization of
125I-FGF-2 by threefold and delayed degradation. To gain
insight into the mechanism of inhibition, we have analyzed the
interaction of PF-4 with FGF-2/FGFR by using mutant heparan
sulfate-deficient Chinese hamster ovary (CHO) cells
transfected with the FGFR-1 cDNA (CHOm-FGFR-1) and by examining the
direct interaction with FGF-2. In the absence of heparin, PF-4
inhibited binding of 125I-FGF-2 to CHOm-FGFR-1 cells in a
concentration-dependent manner, although not completely. In the
presence of heparin, PF-4 abolished totally the stimulatory effect of
heparin. Furthermore, PF-4 complexed to FGF-2 and inhibited endogenous
or heparin-induced FGF-2 dimerization. These results indicate that PF-4
interacts with FGF-2 by complex formation, inhibiting FGF-2
dimerization, binding to FGF receptors, and internalization. This
mechanism most likely contributes to the antiangiogenic properties of
PF-4.
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INTRODUCTION |
ANGIOGENESIS INVOLVES the formation of
new blood vessels of capillary origin. This phenomenon is tightly
controled by a set of factors that include fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF).1,2
The angiogenic effects of these molecules are counterbalanced by
inhibitory molecules such as angiostatin,3,4
endostatin,5 thrombospondin-1 (TSP-1),6 the
16-kD human prolactin fragment (16-kD PRL),7 or platelet factor 4 (PF-4).8
FGF-2 mediates its biological activity by binding to specific
cell-surface receptors and heparan sulfate proteoglycans (HGSP). HSPG
contribute to the binding of FGF-2 to high-affinity receptors by
stabilizing the FGF-2/FGF receptor complex, protecting FGF-2 from
degradation, or facilitating FGF-2 oligomerization.1 Thus, angiogenesis inhibitors may impair FGF-2 activity by interfering at the
level of HSPG/FGF-2/FGF receptor interactions.
PF-4 belongs to the C-X-C chemokine family.9 This family
also includes interleukin-8 (IL-8),  -thromboglobulin,
neutrophil-activating protein, interferon-inducing protein 10 (IP-10),
and melanocyte growth-stimulating activity.9 PF-4 is a
7.8-kD protein of 70 amino acid length.10 It shares
homologies in particular with -thromboglobulin and IL-8 of 51% and
31%, respectively.9, 11 The crystal structure of human
PF-4 has been solved to a resolution of 2.4 A by molecular
replacement.12 The N-terminal residues form antiparallel
-sheet-like structures. A positively charged ring of lysine
and arginine side chains encircles the PF-4 molecule presenting
multiple potential sites for heparin binding.
PF-4 exhibits biological activity for several cell types including
megakaryocytes,13 leukocytes,14
lymphocytes,15 and endothelial cells.8,16 It
has been shown that PF-4 inhibits endothelial-cell proliferation,
migration, and angiogenesis in vitro and in vivo.8, 16 In
addition, PF-4 reduces tumor growth in vivo.17,18
Intralesional injection in mice of recombinant human PF-4 inhibited
melanoma cell or HCT 116 colon carcinoma cell growth by an
angiogenesis-dependent mechanism.17 Furthermore, virally
transduced rat glioma cells with a secretable PF-4 cDNA grew slowly in
vivo and only formed hypovascular tumors.19 This indicates
that the vasculature is the prime target for PF-4. In addition, PF-4 is
targeted in vivo to endothelial cells that undergo active
angiogenesis.20 PF-4 may also be important as a
physiological regulator of FGF activity. Indeed, platelets release
during activation an inhibitor of FGF-2 activity.21 It has
been shown that this inhibitor is identical to PF-4. Thus, PF-4 may
counteract excessive angiogenic factor activity at sites of platelet
activation.
The mechanism of PF-4 action is incompletely understood and
controversial. Sato et al22 have reported that PF-4
inhibits binding of FGF-2 to low-affinity binding sites and
high-affinity receptors in NIH 3T3 fibroblasts. Others only reported
competition of PF-4 with FGF-2 binding to low-affinity
proteoglycans.23 In agreement with the latter observation,
Luster et al24 showed that the IP-10 chemokine is
associated with cell surface proteoglycans and that IP-10 binding can
be competed by PF-4. In addition, a common heparan sulfate binding site
may also be shared with histidine-rich glycoprotein (HRGP) as this
molecule, like PF-4, displaces FGF-2 from the extracellular
matrix.23 Furthermore, Gengrinowitch et al25
reported that VEGF binding to endothelial cell VEGF receptors was
inhibited by PF-4. Finally, Gupta and Singh16 showed that
PF-4 intervenes at a specific point in the cell cycle by blocking the
progression of endothelial cells in S-phase.
In this study, we sought to determine the contribution of PF-4 in the
inhibition of FGF-2 activity and its mechanism of action. In a series
of systematic studies, we show in particular that PF-4 indeed
interferes with FGF-2 binding to high-affinity receptors and inhibits
FGF-2 dimerization. The implication of this finding in the context of
antiangiogenesis is discussed.
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MATERIALS AND METHODS |
Cells
Murine lung microvascular endothelial cells (LEII cells; kindly donated
by Dr Thomas Maciag, American Red Cross, Rockville, MD) were grown in
Dulbecco's modified Eagle's medium (DMEM; GIBCO, Life Technologies,
Gaithersburg, MD) containing 10% fetal calf serum (FCS; GIBCO), 1 g/L
glucose, 1% glutamin, and 50 IU/mL penicillin/ 50 µg/mL
streptomycin at 37°C in a 5% CO2 atmosphere. Heparan sulfate-deficient chinese hamster ovary cells (CHOm-FGFR-1 cells, 745-flg; kindly donated by Dr Avner Yayon, The Weizmann Institute, Rehovot, Israel) were grown in DMEM containing 10% FCS (GIBCO), 1 g/L
glucose, and 1% nonessential amino acids at 37°C in a 5% CO2 atmosphere. These cells express less than 5% HGSP.
Binding 125I-FGF-2 to low-affinity binding sites
(extracted with 20 mmol/L HEPES, 2 mol/L NaCl, pH 7.4) was less than
3% of total specific binding (sum of specifically bound
125I-FGF-2 to high-affinity and low-affinity sites).
Cell Proliferation Experiments
Proliferation assays were performed as described.26
Briefly, cells were seeded at 20,000 cells on 3.5-cm2
dishes in complete DMEM containing 10% FCS, 1% glutamin, and antibiotics. After overnight attachment, the cells were washed once
with serum-free DMEM and test medium containing 1% FCS, and the
indicated concentrations of FGF-2 or PF-4 were added. Cells were
counted at specified days with a Coulter counter (Coultronics, Margency, France).
Binding Studies, Cross-Linking to Receptors, Internalization,
and Degradation
FGF-2 and PF-4 were labeled with 125I-Na using iodogen
(Pierce, Rockford,IL) as a coupling agent according to the
manufacturer's indications and according to Moscatelli.27
The specific activity of 125I-FGF-2 and
125I-PF-4 were 80, 000 to 200,000 cpm/ng and 35,000 to
100,000 cpm/ng, respectively. Binding experiments to high- and
low-affinity sites were performed essentially as described by
Moscatelli.27 Cells were seeded at 2.5 × 105/cm2, cultured in complete medium onto
3.5-cm2 dishes, and grown for 2 days. Cells were washed
twice with ice-cold phosphate buffer saline (PBS) before binding and
incubated with the indicated concentrations of 125I-FGF-2
or 125I-PF-4 in DMEM containing 20 mmol/L HEPES (pH 7.4),
0.15% gelatin for 2 hours at 4°C in the presence or absence of 1 µg/mL unlabeled ligand or competitors (FGF-2; PF-4; protamine sulfate
[grade III; Sigma, St Louis, MO]; VEGF 165; PDGF BB; EGF). At the end
of the incubation period, the cells were washed three times with
ice-cold PBS. 125I-FGF-2 or 125I-PF-4 was
dissociated from its cellular low-affinity binding sites by two
20-second washes with ice-cold 20 mmol/L HEPES (pH 7.4), 2 mol/L NaCl,
and from its high-affinity sites by two 20-second washes with ice-cold
20 mmol/L NaAc (pH 4.0), 2 mol/L NaCl. Bound 125I-FGF-2
was quantified using a Kontron MR 250  -counter
(Saint-Quentin-Yvelines, France). Nonspecific binding was determined by
incubating separate dishes with 125I-FGF-2 and a 100-fold
excess of unlabeled ligand. Specific binding was determined by
substracting nonspecific binding from total binding.
Cross-linking experiments of 125I-FGF-2 or
125I-PF-4 to receptors were performed and analyzed as
described by Bikfalvi et al,26 using 0.2 mmol/L
Bis(sulfosuccinyl) suberate (BS3; Pierce) in PBS as a
coupling agent. The quantity of protein used in each experiment was
normalized according to cell number or protein. Samples were run on a
7% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Autoradiographies were done against X-OMAT
AR films (Eastman Kodak, Rochester, NY) at  80°C in the presence of an intensifying screen or analyzed by PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
125I-FGF-2 internalization experiments were performed as
described by Roghani and Moscatelli.28 LEII cells were
incubated with 10 ng/mL 125I-FGF-2 with or without 2 µg/mL PF-4 at 37°C in a 5% CO2 atmosphere. At
indicated times, the cells were washed three times with ice-cold PBS,
and cell surface-bound material was extracted by washing the cells for
20 seconds twice with 2 mol/L NaCl in 20 mmol/L HEPES (pH 7.4) and
twice with 2 mol/L NaCl in 20 mmol/L acetic acid (pH 4). The amount of
internalized radioactivity was determined by solubilizing the cells
with extraction buffer containing 10% glycerol, 2% SDS, 1.6 mmol/L
EDTA in 125 mmol/L Tris-HCL (pH 6.8), and -counting. In other
experiments, aliquots of cell extracts were separated by
electrophoresis on a 15% SDS-PAGE, dried, and exposed to
autoradiography or analyzed by PhosphorImager. Furthermore, the medium
was incubated overnight at 4°C with trichloroacetic acid (TCA; 12%
final concentration) to determine TCA-soluble and precipitable
radioactivity.
Complex Formation Between FGF-2 and PF-4
Complex formation in solution.
Ten nanograms per milliliter 125I-FGF-2 and 1µg/mL of
PF-4 were incubated in PBS with or without 0.8 mmol/L Ca++
and 0.5 mmol/L Mg++ for 1 hour at room temperature
(400-µL volume). Subsequently, 1 mmol/L BS3 (final
concentration) was added, and the samples were incubated for another 30 minutes. The reaction was stopped by adding extraction buffer (125 mmol/L Tris-Cl, pH 7.4, 10% glycerol, 1.6 mmol/L EDTA, 2% SDS, and
2% 2- mercaptoethanol) from a 5× concentrated stock solution.
Samples were then run on 12% or 15% SDS-PAGE, and the dried gels were
analyzed by PhosphorImager or autoradiography.
Immobilization of FGF-2 onto surfaces and binding experiments.
Binding of 125I-PF-4 to FGF-2- or VEGF 165-coated wells
was performed as described. Ninety-six-well enzyme-linked
immunosorbent assay (ELISA) plates were coated with 15 ng FGF-2/well in
buffer A (1 mmol/L EDTA, 20 mmol/L K2HPO4, 10 mmol/L KH2PO4, 150 mmol/L NaCl) in a volume of
50 µL. After 2 hours of incubation at room temperature, the plates
were washed five times with buffer B (10 mmol/L Tris-HCl, pH 7.2, 150 mmol/L NaCl, 0.1% Tween 20). The wells were subsequently
incubated again with buffer A, which contained an additional 0.1%
gelatin, and washed again after 1 hour with buffer B five times. The
FGF-2-coated wells were then incubated in buffer A with different
125I-PF-4 concentrations and competitors at 37°C for 1 hour. At the end of the incubation period, the wells were washed five
times with buffer B. Surface-associated 125I-PF-4 was then
extracted with 200 µL of 0.2 mol/L NaOH and counted in a -counter.
In some experiments, wells were preincubated for 1 hour with 200 ng/well (4 µg/mL) heparin in buffer A. At the end of the
preincubation period, the wells were washed twice before the addition
of 125I-PF-4. To determine nonspecific binding, separate
wells were not coated with FGF-2 and only preincubated with buffer A
before the initiation of binding. Specific binding was determined by substracting nonspecific binding from total binding.
FGF-2 Dimerization Experiments
FGF-2 dimerization was studied according to the method described by
Ornitz et al.29 Briefly, 5 ng 125I-FGF-2 and
500 ng unlabeled FGF-2 were incubated for 1 hour at room temperature
with or without the indicated concentrations of heparin and/or
PF-4 in PBS in a final volume of 45 µL. At the end of the incubation
period, 5 µL BS3 (0.1 mmol/L final concentration) was
added for another 30 minutes of incubation. The reaction was stopped by
adding SDS-sample buffer from a 5× concentrated stock solution.
The samples were boiled and run on a 12% or 15% SDS-PAGE. The gels
were analyzed by PhosphorImager or autoradiography.
For all the experiments outlined above, autoradiograms or
PhosphorImager results were analyzed by a public domain NIH Image Program developed at the US National Institutes of Health and available
from the Internet by anonymous FTP form zippy.nimh.nih.gov or by using
a Bio Profil V 6.0 scanner with Bio 1 D software (Vilber Lourmat,
Paris).
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RESULTS |
PF-4 Inhibits Binding of FGF-2 to Endothelial Cells, Internalization,
Degradation, and Biological Activity
We first performed proliferation experiments to ascertain the potency
of our PF-4 preparation. Murine lung capillary endothelial cells (LEII
cells) were incubated with PF-4 and a fixed concentration of FGF-2 (10 ng/mL), and the cell number was estimated at specified days by cell
counting. After 6 days, the cell number was increased by FGF-2
treatment alone threefold. The cell number in the presence of FGF-2 and
PF-4 (2 µg/mL) was identical to the number in unstimulated control
cells. Thus, PF-4 at 2 µg/mL inhibited completely endothelial cell
growth induced by FGF-2 (Fig 1).
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| Fig 1.
Effect of PF-4 on endothelial cell proliferation. LEII
cells were seeded at 20,000 cells/dish. After overnight attachment, the
test medium was added and cells were counted every other day for 8 days
for dishes without PF-4 and at day 6 and 8 for dishes with PF-4. ( )
1% FCS; ( ) 1% FCS + 10 ng/mL FGF-2; ( ) 1% FCS + 10 ng/mL
FGF-2 + 2 µg/mL PF-4. The figure depicts a representative experiment done in duplicates (data points as mean; standard deviation (SD) values: 0 < SD < 0.25).
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We then investigated the effect of PF-4 on the binding of FGF-2 to
endothelial cells. LEII cells were incubated with 10 ng/mL 125I-FGF-2 and increasing concentrations of PF-4 (1 to
10,000 ng/mL). Binding to low- and high-affinity sites was performed as
indicated in Materials and Methods. FGF-2 binding to low-affinity sites or high-affinity binding sites was inhibited in a
concentration-dependent manner by PF-4 (Fig
2A and B). Maximum inhibition of FGF-2 binding to low-affinity binding
sites or high-affinity sites (FGF receptors) was reached at 5 to 10 µg/mL PF-4 with half maximum inhibition at 0.72 and 0.6 µg/mL,
respectively. FGF-2 binding was not competed by any of the following
agents: human VEGF 165, platelet-derived growth factor BB (PDGF BB), or
epidermal growth factor (EGF). To ascertain the inhibitory effect of
PF-4 on FGF-2 binding, cross-linking of 125I-FGF-2 to
cell-surface receptors in the presence of PF-4 was performed (Fig 2C).
Cross-linked material was detected after SDS-PAGE and autoradiography.
The intensity of the cross-linked material was decreased threefold
(34% of control; Fig 2C, lane 1) in the presence of PF-4 as estimated
by NIH Image Program analysis.
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| Fig 2.
(A through C) Effect of PF-4 on 125I-FGF-2
binding to endothelial cells and cross-linking. LEII cells (500,000 cells/dish) were incubated at 4°C with 10 ng/mL
125I-FGF-2 and increasing concentrations of PF-4 in the
absence or presence of 1 µg/mL unlabeled FGF-2.
125I-FGF-2 bound to high-affinity (A) or
low-affinity binding sites (B) was determined as indicated in Materials
and Methods. Nonspecific binding is indicated in black bars. Specific
binding (ng/106 cells) is shown as inset. (C) Cross-linking
of 125I-FGF-2 to receptors in the presence or
absence of 10 µg/mL PF-4 or unlabeled ligand. (A and B)
Representative experiment done in duplicates (data points as mean; SD
values: [A] 0 < SD < 0.8; [A, inset] 0 < SD < 0.03; [B] 0.13 < SD < 0.8).
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We next studied the effect of PF-4 on internalization and degradation
of FGF-2. To measure FGF-2 internalization, we incubated LEII cells
with 10 ng/mL 125I-FGF-2 in the presence or absence of 2 µg/mL PF-4 at 37°C over a 24-hour period. Analysis of
membrane-bound and intracellular 125I-FGF-2 was performed
as indicated in Materials and Methods. As depicted in
Fig 3, long-term internalization of
125I-FGF-2 was strongly inhibited by PF-4. In the absence
of PF-4, maximum internalization was 0.209 ng
125I-FGF-2/106 cells. This value decreased to
0.102 ng 125I-FGF-2/106 cells when the
experiment was performed in the presence of PF-4. The initial rate of
internalization after 1 hour was reduced approximately threefold (R2 = 0.037 ng/106 cells/h) in the presence of PF-4 when compared
with control (R1 = 0.116 ng/106 cells/h). When the medium
was precipitated with trichloro-acetic acid (TCA) and analyzed for
TCA-soluble and precipitable radioactivity, we found that PF-4
decreased moderately the amount of the TCA-soluble radioactivity,
although the kinetics of release were similar (data not shown). In
addition, we examined the patterns of the 125I-FGF-2
degradation fragments in cell extracts (data not shown). Three
degradation fragments of 15 kD, 10 kD, and 6 kD were detected. In the
absence of PF-4, 125I-FGF-2 was almost completely
converted into the 15-kD, 10-kD, and 6 kD form at 8 hours. In the
presence of PF-4, the appearance of the 125I-FGF-2
degradation fragments was delayed and the conversion of the 18-kD FGF-2
into the 15-kD, 10-kD, and 6-kD forms was only observed after 12 hours.
This indicated that the conversion of 18-kD FGF-2 into lower molecular
weight FGF-2 forms is delayed by PF-4.
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| Fig 3.
Internalization of 125I-FGF-2 in the
presence of PF-4. For internalization experiments, LEII cells (500,000 cells/dish) were incubated at 37°C with 10 ng/mL
125I-FGF-2 in the presence () or absence () of 2 µg/mL PF-4 or 1 µg/mL unlabeled ligand. At the indicated time
points, cell surface-bound and internalized 125I-FGF-2
were determined as indicated in Materials and Methods. The figure
depicts a representative experiment done in duplicates (data points as
mean; SD values: 0 < SD < 0.03 ).
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Effect of PF-4 on FGF-2 Binding in Cells Deficient of Heparan
Sulfates and Expressing FGFR-1
To assess more accurately the interaction of PF-4 with the FGF-2/FGF
receptor system, we chose to study the interaction of PF-4 and FGF-2
with FGFR-1. We used mutant CHO cells deficient of heparan sulfates
that express FGFR-1 (CHOm-FGFR-1). These cells have been used for the
analysis of heparin requirement in FGF-2 binding.28-31 We
investigated the effect of PF-4 on 125I-FGF-2 binding in
the absence and presence of heparin in this cell type. In the absence
of heparin, PF-4 inhibited FGF-2 binding in a concentration-dependent
manner (Fig 4A). Half maximum inhibition was reached at a PF-4 concentration of 0.85 µg/mL and maximum inhibition at 5 to 10 µg/mL. Maximum inhibitory PF-4 concentrations reduced 125I-FGF-2 binding to approximately 50%. The
effect of PF-4 on heparin-induced 125I-FGF-2 was
investigated in two ways. First, when binding was performed in the
presence of increasing heparin concentrations (1 to 1,000 ng/mL),
125I-FGF-2 binding was totally blocked at all heparin
concentrations by 10 µg/mL of PF-4 (Fig 4B). Second, in the presence
of a fixed concentration of heparin (50 ng/mL) and increasing
concentrations of PF-4 (0.01 to 5 µg/mL), PF-4 abolished the effect
of heparin on 125I-FGF-2 binding at 1 µg/mL with a half
maximum inhibition at 0.4 µg/mL (Fig 4C). Finally, cross-linking of
125I-FGF-2 to FGFR-1 was inhibited by PF-4 in the absence
or presence of 50 ng/mL heparin (Fig 4D). Heparin by itself increased
the intensity of the cross-linked material approximately twofold as estimated by the NIH imager program. In the absence of heparin, PF-4
decreased 125I-FGF-2 binding twofold (40% of unstimulated
control). Furthermore, the stimulatory effect of heparin on the
cross-linking of 125I-FGF-2 to FGFR-1 was totally
abrogated by PF-4.
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| Fig 4.
(A through D) Effect of PF-4 on the binding of
125I-FGF-2 to receptors in cells deficient of heparan
sulfates and expressing FGFR-1 (CHOm-FGFR-1), and cross-linking.
CHOm-FGFR-1 cells (500,000 cells/dish) were incubated with 10 ng/mL
125I-FGF-2 with or without PF-4, 50 ng/mL heparin, or 1 µg/mL unlabeled ligand. Binding of 125I-FGF-2 to
receptors or cross-linking was performed as indicated in Materials and
Methods. (A) Effect of increasing concentrations of PF-4 on
high-affinity binding of 125I-FGF-2 to receptors in the
absence of heparin. (B) Effect of increasing concentrations of heparin
on 125I-FGF-2 binding in the presence ( ) or
absence () of 10 µg/mL PF-4. (C) Effect of increasing
concentrations of PF-4 on 125I-FGF-2 binding in the
presence or absence of 50 ng/mL heparin. (D) Cross-linking of
125I-FGF-2 to receptors in the presence or absence of 10 µg/mL PF-4 or heparin. The differences in the amounts of
125I-FGF-2 bound between the different experiments reflect
clonal variability in FGFR-1 expression. (A through C)
Representative experiments done in duplicates (data points as mean; SD
values: [A] 0 < SD < 0.04; [B] 0.01 < SD < 0.3;
[C] 0.005 < SD < 0.09).
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We compared the effect of PF-4 on 125I-FGF-2 binding in
CHOm-FGFR-1 cells with that of protamine sulfate. Protamine sulfate at maximum inhibitory concentrations (20 to 100 µg/mL) decreased the
binding of 125I-FGF-2 to FGF receptors in CHOm-FGFR-1
cells by approximately 80% in the absence of heparin. In addition, the
inducing effect of heparin on 125I-FGF-2 binding was also
inhibited by protamine sulfate (data not shown).
Taken together, these results indicate that PF-4 significantly inhibits
FGF-2 binding to FGFR-1 in the absence of heparin. In addition, PF-4
also abrogates the inducing effect of heparin on FGF-2 binding.
Binding of PF-4 to Endothelial Cells and Cells Deficient of
Heparan Sulfates and Expressing FGFR-1
To investigate whether PF-4 by itself is able to bind to endothelial
cells or CHOm-FGFR-1 cells, we performed binding experiments with
125I-PF-4 (Fig 5).
125I-PF-4 bound to high- or low-affinity binding sites was
extracted as indicated in Materials and Methods. As indicated in Fig 5, 125I-PF-4 significantly bound to low-affinity binding
sites (up to 0.72 ng/106 cells) but only very weakly to
high-affinity binding sites (0.015 to 0.03 ng/106 cells).
In addition, no significant binding to CHOm-FGFR-1 cells could be
detected. Furthermore, when 125I-PF-4 was cross-linked to
LEII or CHOm-FGFR-1 cells, no cross-linked material was visible (data
not shown). These results indicate that PF-4 binds significantly to
low-affinity binding proteoglycans but not to high-affinity FGFR-1. In
addition, specific high-affinity PF-4 receptors were not detected in
LEII or CHOm-FGFR-1 cells.
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| Fig 5.
Binding of 125I-PF-4 to endothelial cells
and cells deficient of heparan sulfates and expressing FGFR-1. LEII
cells or CHOm-FGFR-1 cells (500,000 cells/dish) were incubated with
125I-PF-4 in the presence or absence of unlabeled ligand
and binding to high-affinity (HA, cell surface-bound
125I-PF-4 extracted with 2 mol/L NaCl buffer at pH 4) or
low-affinity sites (LA, cell surface-bound 125I-PF-4
extracted with 2 mol/L NaCl buffer at pH 7.4) were analyzed as
indicated in Materials and Methods. The binding of the figure depicts
representative experiments done in duplicates (data points as mean + SD).
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PF-4 Complexes With FGF-2
We next set out to examine whether PF-4 complexes with FGF-2. We
examined this by performing cross-linking experiments of FGF-2 and PF-4
and by directly studying the interaction of these molecules immobilized
onto surfaces.
125I-FGF-2 was incubated with PF-4 at room temperature for
20 minutes and cross-linked by using BS3 as a cross-linking
agent (Fig 6A). The cross-linked material was subsequently analyzed by SDS-PAGE and autoradiography or
PhosphorImager. In the presence of the cross-linking agent, two bands
of 36 kD and of 25 kD were observed. The 36-kD band corresponded to
endogenous FGF-2 dimers and was visible in the absence of PF-4,
although weakly under our experimental conditions. With 10 ng/mL
125I-FGF-2 and 1 µg/mL PF-4 in solution, an additional
25-kD band was revealed by autoradiography. Under the experimental
conditions, scanning analysis indicated relative intensities of the 25 kD or 36 kD of 20% and 8%, respectively (72% for the remaining 18-kD FGF-2 band). To study the effect of divalent cations, the cross-linking reactions were performed in the absence and presence of 0.8 mmol/L Ca++ and 0.5 mmol/L Mg++. Divalent cations did
not influence the appearance or the intensity of the 25-kD cross-linked
material. As a control, the cross-linking reaction was performed with
bovine serum albumin (BSA) instead of PF-4. Complex formation was not
observed between BSA and 125I-FGF-2, even at 10 µg/mL
BSA.
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| Fig 6.
(A through C) Complex formation of PF-4 with FGF-2. (A)
Cross-linking experiments in solution. One microgram of PF-4 and 10 ng
125I-FGF-2 were incubated in 400 µL PBS in the presence
or absence of 0.8 mmol/L Ca++ and 0.5 mmol/L
Mg++ for 1 hour. Subsequently, 1 mmol/L BS3
was added for another 30 minutes. At the end of the incubation period,
extraction buffer was added and aliquots were loaded onto a 12%
SDS-PAGE. The dried gel was analyzed by PhosphorImager or autoradiography. (B) Binding of 125I-PF-4 to cell surface
immobilized FGF-2. Fifteen nanograms per 50 microliters of FGF-2 was
adsorbed onto the surface of a 96-well ELISA plate and incubated with
different concentrations of 125I-PF-4. Binding was
performed and analyzed as indicated in Materials and Methods. Scatchard
plot is shown as inset. The figure depicts an representative experiment
done in triplicates (data points as mean; SD values: 24 < SD < 780). (C) Competion of 125PF-4 binding to FGF-2. Wells were
coated with FGF-2 at 15 ng/50 µL. Some of the wells were preincubated
with 200 ng heparin. Plates were washed twice with buffer A to remove
unbound heparin. Others wells received buffer alone. Ten nanograms
125I-PF-4, with or without 5 µg PF-4, 5 µg FGF-2, or 50 ng heparin, was added to the wells. Binding was performed and analyzed
as indicated in Materials and Methods. The figure depicts a
representative experiment done in triplicates (data points as mean + SD). 100% corresponds to 7,000 cpm.
|
|
We next set out to examine the direct interaction of PF-4 and FGF-2
using a solid-phase binding assay (Fig 6B). Ninety-six-well ELISA
plates were coated with 15 ng FGF-2, and binding of
125I-PF-4 was performed as indicated in Materials and
Methods. When increasing concentrations of 125I-PF-4 were
added to the wells, concentration-dependent binding to FGF-2 was
observed. The affinity for PF-4 binding to FGF-2 calculated from the
data above was high with an estimated kd of 3.7 × 10 8 mol/L. We next investigated the competition of
direct binding between FGF-2 and PF-4. PF-4, FGF-2, and heparin were
tested for competing with binding of 125I-PF-4 to FGF-2
(Fig 6C). The experiments were peformed under two conditions. In one
set of experiments, the wells were coated with 15 ng FGF-2 and
preincubated with 200 ng/well (4 µg/mL) heparin. In another set of
experiments, preincubation was done with buffer only. When
preincubation was done with buffer only, 125I-PF-4 binding
was strongly inhibited by 5 µg FGF-2 (21% of control), 5 µg PF-4
(5% of control), and 50 ng heparin (10% of control). In
heparin-preincubated wells, 125I-PF-4 bound still to
FGF-2-coated wells, and binding was also inhibited by these
competitors. The competion of the binding by FGF-2 was slightly less
effective in heparin-preincubated wells (31% of control). This may
suggest that the heparin binding domain of FGF-2 is only partially
involved in the association between PF-4 and FGF-2 and that other
domains are also implicated.
The association of PF-4 with FGF-2 was not specific for FGF-2 alone,
because PF-4 was able to bind surface immobilized VEGF 165 (data not
shown).25 However, as already reported, PF-4 did not bind
to insulin, transferrin, or VEGF 121.25 Furthermore, binding of PF-4 to BSA was not observed (data not shown).
PF-4 Inhibits FGF-2 Dimerization
We next investigated the effect of PF-4 on FGF-2 dimerization
(Fig 7). The dimerization experiments were
performed according to the technique described by Ornitz et
al.29 In this procedure, 125I-FGF-2 and
unlabeled FGF-2 are incubated together to enhance the dimerization
signal. As seen in Fig 7A, not only was the appearance of FGF-2 dimers
stimulated by heparin with this method, but so was that of FGF-2
multimeric complexes. When 5 ng 125I-FGF-2 and 500 ng
unlabeled FGF-2 were incubated with increasing heparin concentrations
and a fixed PF-4 (1 µg) concentration (50 µL incubation volume),
FGF-2 dimerization was strongly inhibited. The intensity of the 36-kD
band as estimated by the NIH Image Program was reduced by PF-4
threefold to fourfold (32% and 25% of control) at all heparin
concentrations. In addition, the high molecular weight complex that
represents most likely FGF-2 multimers was also totally inhibited by
PF-4. Moreover, in the presence of PF-4 additional cross-linked
material of 25-kD size was visible. This signal possibly corresponds to
FGF-2/PF-4 heterodimeric complexes. The specificity of the
cross-linking reaction was assessed by incubating labeled FGF-2,
unlabeled FGF-2, and heparin with increasing concentrations of BSA (100 ng/mL to 4 mg/mL) and then performing the cross-linking reaction. Even
at very high BSA (4 mg/mL) concentrations, no effect on FGF-2
dimerization and no complex formation between BSA and FGF-2 was
observed (data not shown).
View larger version (59K):
[in this window]
[in a new window]
| Fig 7.
(A and B) Effect of PF-4 on FGF-2 dimerization. Five
nanograms 125I-FGF-2 and 500 ng unlabeled FGF-2 were
incubated in 45 µL PBS with or without heparin and specified PF-4
concentration. After 1 hour of incubation, 5 µL cross-linking reagent
was added and the samples were incubated for further 30 minutes. At the
end of the incubation period, extraction buffer was added and the samples were boiled and loaded on a 12% SDS-PAGE. The dried gels were
analyzed by autoradiography. (A) Effect of increasing heparin concentrations (ng) on FGF-2 dimer formation in the presence of 1 µg
PF-4. (B) Concentration dependency of the effect of PF-4 (µg) on
FGF-2 dimerization in the absence (left panel) or presence (right
panel) of 50 ng heparin.
|
|
We then investigated the effect of increasing PF-4 concentrations on
FGF-2 dimer formation. In the absence of heparin, increasing amounts of
PF-4 (10 to 1,000 ng) gradually reduced the appearance of the 36-kD
band. PF-4 at 1 µg inhibited significantly the intensity of the 36-kD
band. In parrallel, the 25-kD band was visible at PF-4 amounts of 0.1 µg and higher. Similarily, in the presence of heparin, increasing
PF-4 amounts also inhibited the intensity of the 36-kD band with the
appearance of the 25-kD band at 0.5 µg and higher.
| |
DISCUSSION |
We undertook a systematic study to clarify the inhibitory mechanism of
PF-4 on FGF-2 activity in endothelial cells. The results reported
herein indicate that PF-4 interferes with FGF-2 and FGF receptors by
inhibiting FGF dimer formation, binding, internalization, and
degradation. This is based on the following observations: (1) PF-4
inhibited binding to low-affinity binding sites and to high-affinity
FGF receptors in endothelial cells and biological activity, (2) PF-4
inhibited FGF-2 internalization and delayed degradation, (3) PF-4
inhibited FGF-2 binding in CHOm-FGFR-1 in the absence of heparin, (4)
PF-4 abrogated the stimulatory effect of heparin on FGF-2 binding in
CHOm-FGFR-1 cells, (5) FGF-2 complexes with PF-4, and (6) PF-4
inhibited residual and heparin-induced FGF-2 dimerization.
PF-4 inhibited high- and low-affinity binding of FGF-2 to murine
capillary endothelial cells. These data are reinforced by cross-linking
of 125I-FGF-2 to FGF receptors that showed a strong
decrease in the intensity of the cross-linked material when the
experiment is performed in the presence of PF-4. This is in agreement
with the results reported by Sato et al,22 who also
reported inhibition of FGF-2 binding to high- and low-affinity binding
sites in murine 3T3 fibroblasts. In contrast to these observations,
others only described competition of FGF-2 binding to low-affinity
proteoglycans.23 The reasons for these differences are not
known at the present time. The nature of the low-affinity binding
proteoglycan has recently been investigated. Luster et al24
have reported that PF-4 but not other members of the chemokine family
inhibited IP-10 binding to proteoglycans. Thus, these data may indicate
a common proteoglycan binding site for IP-10 and PF-4. We found that
PF-4 inhibited endothelial cell proliferation at maximum concentrations of 2 µg/mL. This is in agreement with the data of Gupta and Singh et
al,16 who also reported inhibition within this PF-4
concentration range. These authors used bovine retinal microvascular
endothelial cells but also endothelial cells from large vessels (human
aorta, fetal bovine heart, and human umbilical cord) in their
experiments. The concentration of PF-4 required for half maximum
inhibition was in the range of 1 to 3 µg/mL for these different cell
types. Furthermore, these concentrations are physiologically
significant because platelets may generate locally in vivo very high
PF-4 concentrations ranging between 2.5 to 25 µg/mL.32
PF-4 concentrations that inhibited maximally FGF-2 proliferation
decreased FGF-2 internalization by threefold and delayed the appearance
of FGF-2 degradation fragments. This indicates that in addition to
impairment of FGF-2 binding, internalization of FGF-2 is strongly
inhibited by PF-4, and this at a PF-4 concentration that did not
completely inhibit FGF-2 binding to LEII cells.
To explain the inhibitory effect of PF-4 on FGF-2 binding and activity,
several mechanisms may be considered. First, PF-4 may only compete with
FGF-2 for the availability of heparan sulfates that have been shown to
be important in FGF-2 activity. Second, PF-4 may directly bind FGF-2
receptors and compete for binding at a receptor level. Third, PF-4 may
complex to FGF-2 and inhibit FGF-2 dimer formation. We therefore
undertook a systematic study to analyze the mechanism of inhibition by
PF-4.
In a first series of experiments we used CHO cells deficient of heparan
sulfates and transfected with FGFR-1. We observed an inhibitory effect
of increasing PF-4 concentrations on FGF-2 binding with maximum
inhibition between 5 and 10 µg/mL, and this in the absence of
heparin. When binding was performed in the presence of heparin, PF-4
completely abrogated the stimulatory effect of heparin on FGF-2
binding. Two conclusions may be drawn from these data. First, FGF-2 is
clearly able to bind significantly to this cell type in the absence of
heparin. This observation is in agreement with Roghani et
al33 who also found significant FGF-2 binding to
CHOm-FGFR-1 cells in the absence of heparin. Second, the inhibitory action of PF-4 certainly involves more than only an antiheparin effect
as PF-4 already impedes FGF-2 binding in the absence of heparin. This
may also suggest that, in addition to the heparin binding domain, other
PF-4 domains are also implicated.
None of the studies reported so far was able to identify a specific
cell-surface receptor for PF-4.9 PF-4 only bound to IL-8
receptors in leukocytes when modified at the
N-terminus.14,34 Furthermore, IP-10 binding to endothelial
cells can be competed by PF-4, but not by other members of the
chemokine family.24 These binding sites are dependent on
the presence of cell surface HGSP. Accordingly, we only detected
significant low-affinity binding of PF-4 to endothelial cells.
Furthermore, cell surface receptors for PF-4 could not be detected by
cross-linking of 125I-PF-4 to endothelial cells. Finally,
there was clearly no direct interaction between PF-4 and FGFR-1 because
125I-PF-4 failed to bind to CHOm-FGFR-1 cells. Thus,
these results indicate that PF-4 does not inhibit FGF-2 binding by
directly associating with FGFR-1 and blocking access of the ligand to
FGF receptors.
FGF-2 is able to associate, besides with cell surface FGF receptors,
with a number of molecules including soluble or matrix-bound FGF
receptors35; proteoglycans,36 including
perlecan,37 glypican I,38 glypican III,39 and ryudocan40; and
 2-macroglobulin.41 We found that PF-4
associated with FGF-2. When 125I-FGF-2 was incubated with
PF-4 in solution, a complex of 25 kD was detected by cross-linking.
This may correspond to an FGF-2/PF-4 heterodimer of one molecule FGF-2
and a PF-4 monomer. Higher molecular weight complexes were not detected
on 12% PAGE with this method. Solid phase-binding assays indicated
concentration-dependent binding of 125I-PF-4 to FGF-2 that
occured with high affinity. 125I-PF-4 binding to
immobilized FGF-2 could be competed by FGF-2, heparin, and PF-4.
Furthermore, when the plates were preincubated with 4 µg/mL heparin,
125I-PF-4 still associated to surface immobilized FGF-2.
In addition, FGF-2 competed a little less with 125I-PF-4
binding, albeit significantly. This may suggest that in addition to the
heparin-binding domains, other FGF-2 domains may be involved in the
interaction with PF-4.
In the present model of FGF receptor activation, FGF-2 dimer formation
is required to induce FGF receptor dimerization and activation.42 It has been reported that heparin is
important for FGF-2 dimer formation. However, the magnitude of the
heparin effect is still a matter of debate.29-31,33,42-44
We found that PF-4 inhibited residual (endogenous) and heparin-induced
FGF-2 dimer formation. The formation of FGF-2 oligomers was also
abrogated by PF-4. This correlates with the inhibition of FGF-2 binding in LEII and CHOm-FGFR-1 cells and of biological activity. This is also
in agreement with recent nuclear magnetic resonance (NMR) studies that
indicate that FGF receptor activation absolutely requires the formation
of FGF-2 dimers.43 It is to note that we observed residual
binding of FGF-2 to FGFR-1 in CHOm-FGFR-1 cells at PF-4 concentrations
that almost completely inhibited FGF-2 dimerization. This fraction is
approximately 50% of total binding in absence of heparin and of
approximately 25% of heparin-induced binding to FGFR-1. This suggests
that binding but not receptor activation is, to a some extent, FGF-2
dimer-independent.
What are the domains implicated in the interaction between PF-4 and
FGF-2? PF-4 contains several domains potentially implicated in its
activity. The C-X-C motif is located at the PF-4 N-terminus. It has
been reported that cleavage of this sequence that retains the peptide
17-70 will dramatically increase the inhibitory activity of
PF-4.45 PF-4 17-70 may occur as a natural peptide in
leucocytes produced by a leucocyte elastase.45 The major
heparin-binding domain is localized at the C-terminus between amino
acid 58-70. Maione et al46 have modified this sequence to
eliminate the lysine residues needed for heparin binding but to retain
the amphipatic -helical structure of the carboxyterminus. These
modified molecules retained full antiangiogenic activity. Another study
suggested that a loop containing Arg-20, Arg-22, His-23, and Thr-25, as well as Lys-46 and Arg-49, is also involved in heparin
binding.47 Site-directed mutagenesis and heparin binding
indicates that the arginines residues are especially important. We are
currently examining the FGF-2 and PF-4 domains implicated in the
interaction between these two molecules. Preliminary data indicate that
the heparin-binding domain in FGF-2 or PF-4 is not the only one
implicated and is alone insufficient to account for the biological
effects of PF-4 toward FGF-2.
In the light of the results reported here, the following model of PF-4
inhibition is suggested. Spontaneous FGF-2 dimer formation or
heparin-induced FGF-2 dimer formation is inhibited by PF-4. The
inhibition of FGF-2 dimerization may result from complex formation of
FGF-2 with PF-4 that empedes the successful association of two FGF-2
molecules. This in turn inhibits FGF receptor dimerization, FGF-2
dimer-dependent binding, receptor activation, and internalization. A
number of molecules, including endogenous angiogenesis inhibitors, are
able to interfere with FGF-2 binding to receptors and biological activity.48,49 It is possible that several of these are
acting by a similar extracellular mechanism providing a trap for
angiogenic molecules, no longer allowing successful receptor
activation. During the preparation of this manuscript, Taraboletti et
al50 reported binding of TSP-1 to FGF-2. More
interestingly, Miao et al51 showed that a synthetic
heparin-mimicking nonsulfated polyanionic aromatic compound (RG-13577)
of 5-kD size inhibited FGF-2 activity and dimerization. Our data
indicate that this mechanism also applies to endogenous angiogenesis
inhibitors.
| |
FOOTNOTES |
Submitted October 9, 1997;
accepted December 11, 1997.
Supported by grants from la Ligue Nationale Contre le Cancer,
l'Association pour la Recherche sur le Cancer (ARC), la Région Aquitaine, le Pôle Médicament Aquitaine, la Fondation
GEFLUC, la Fondation Simone et Cino del Duca, and le MSR (to A.B.).
Address reprint requests to Andreas Bikfalvi, MD, PhD, Laboratoire des
Facteurs de Croissance et de la Differenciation Cellulaire, Bâtiment de Recherche Biologie Animale, Avenue des
Facultés, 33405 Talence, France.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr David Moscatelli (New York University Medical
Center, New York) and Dr Sophie Javerzat (Growth Factor and Cell
Differentiation Laboratory, University Bordeaux I) for critically
reading of the manuscript, Dr Daniel B. Rifkin (Department of Cell
Biology, New York University Medical Center) for the gift of
recombinant FGF-2, Dr Thomas Maciag (American Red Cross, Rockville, MD)
for providing LEII cells, Dr Avner Yayon (The Weizmann Institute, Rehovot, Israel) for providing CHOm-FGFR-1 cells, Jean Amiral (Serbio,
Les Ulis, France) for providing recombinant human PF-4, and Dr Jean
Plouet (CNRS, Toulouse) for providing recombinant VEGF. The authors
also thank Jean-François Comps for his help in the preparation of
the figures.
| |
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Abstract
Introduction
Abstract