Received for publication, September 13, 2001, and in revised form, October 30, 2001
Platelet-derived growth factor-BB (PDGF-BB) and
basic fibroblast growth factor (bFGF) are potent growth factors active
on many cell types. The present study indicates that they directly interact in vitro. The interaction was investigated with
overlay experiments, surface plasmon resonance experiments, and
solid-phase immunoassays by immobilizing one factor or the other and by
steady-state fluorescence analysis. The interaction observed was
specific, dose-dependent, and saturable, and the
bFGF/PDGF-BB binding stoichiometry was found to be 2:1.
KD1 for the first step
equilibrium and the overall KD values were found to
be in the nanomolar and in the picomolar range, respectively. Basic
FGF/PDGF-BB interaction was strongly reduced as a function of time of
PDGF-BB proteolysis. Furthermore, docking analysis suggested that the
PDGF-BB region interacting with bFGF may overlap, at least in part,
with the PDGF-BB receptor-binding site. This hypothesis was supported
by surface plasmon resonance experiments showing that an anti-PDGF-BB antibody, known to inhibit PDGF-BB binding with its receptor, strongly
reduced bFGF/PDGF-BB interaction, whereas a control antibody was
ineffective. According to these data, the observed
bFGF·PDGF-BB complex formation might explain, at least in
part, previous observations showing that PDGF-BB chemotactic and
mitogenic activity on smooth muscle cells are strongly inhibited in the
presence of bFGF.
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INTRODUCTION |
Platelet-derived growth factor
(PDGF)1 and basic fibroblast
growth factor (bFGF) play a key role in development, wound repair, cancer growth (1, 2), and in vascular wall diseases including atherosclerosis and neointima accumulation at vascular injury sites
(3-7).
Basic FGF is produced by fibroblasts and endothelial (EC), glial, and
smooth muscle cells. PDGF is produced by platelets, monocytes, EC, and
vascular smooth muscle cells (VSMC). Both factors act on different
cells including EC, VSMC, fibroblasts, and other cells of mesenchymal
origin. Both bFGF and PDGF are reported to be increased in a variety of
conditions including tumor growth (8), thyroiditis (9), and brain
abscess (10) and to localize at the nuclear, extracellular, and
cytoplasmic (polyribosomes, endoplasmic reticulum, Golgi apparatus, and
cytoplasmic surface of plasma membrane) compartments (11-13).
PDGF is a disulfide-linked dimer (14) consisting of two polypeptides,
designated as A- and B-chain, sharing about 60% sequence homology
(15). Three dimeric forms, AA and BB homodimers and AB heterodimer,
have been described (16-18). Recently, PDGF-C and PDGF-D isoforms were
also identified (19, 20).
PDGF-BB and PDGF-AA exhibit different functional effects, depending on
the binding with two distinct cell surface tyrosine kinase receptors,
and 
. The receptors bind PDGF-AA, -AB, -BB, and -CC with
high affinity, whereas receptors bind only BB and DD dimers with
high affinity (19-22). PDGF-BB crystallographic analysis revealed that
the two chains are arranged in an antiparallel manner. Each subunit
consists of a tight cystine knot motif with two loops, loops 1 and 3, pointing in one direction and one loop, i.e. loop 2, pointing in the other directions. Due to this antiparallel arrangement,
loops 1 and 3 of one subunit are juxtaposed to loop 2 of the other.
Mutational analyses mapped the receptor-binding sites mainly within
loops 1 and 3, and within loop 2 to some extent (23-27). The dimeric
PDGF molecule thus displays two distinct receptor-binding regions, each
one formed by epitopes derived from both subunits.
Basic FGF belongs to a family of at least 22 polypeptides (28-31).
Basic FGF crystallographic analyses showed a "-trefoil" fold
consisting of three copies of a basic four-stranded antiparallel -sheet (32, 33). This growth factor acts through high affinity tyrosine kinase receptors (34) and through low affinity heparan sulfate
proteoglycan receptors (35-37). FGF receptors consist of four related
molecules with alternatively spliced isoforms, each containing a highly
conserved tyrosine kinase domain (38-40). Intracellular signaling is
initiated by receptor dimerization and receptor transphosphorylation (41-43). Furthermore, it has been suggested that bFGF activates multiple signaling pathways by also utilizing FGF receptor monomers or
multimers (44).
PDGF and bFGF may be released simultaneously and at the same site both
in vitro and in vivo (45-50). We have shown
previously that bFGF reduces in vitro the chemotactic and
mitogenic activity of PDGF-BB on VSMC (51) and that bFGF angiogenic
properties are markedly reduced by PDGF-BB (82). We therefore tested in this study whether a direct interaction between the two factors occurs,
which may underlie the observed functional inhibition. Fluorescence
titration, overlay assays, surface plasmon resonance (SPR)
measurements, and solid-phase immunoassays were carried out to evaluate
and characterize the interaction between bFGF and PDGF-BB.
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EXPERIMENTAL PROCEDURES |
Reagents--
The BIAcore instrument (BIAcoreX), sensor chips
CM5, surfactant P20, the amine coupling kit containing
N-hydroxysuccinimide, N-ethyl-N'-(3-diethylaminopropyl)carbodiimide,
and ethanolamine hydrochloride were from Amersham Biosciences AB.
Recombinant human PDGF-BB and acidic FGF (aFGF) were purchased from R & D Systems (Abingdon, UK); recombinant human bFGF, fibronectin, and
vitronectin were purchased from Invitrogen; elastase was purchased from
Worthington; bovine serum albumin fraction V (BSA) was purchased from
Sigma; blotting grade blocker nonfat dry milk was from Bio-Rad;
125I-bFGF and Hybond ECL membranes were purchased from
Amersham Biosciences AB. Antibodies used in this study include purified
mouse anti-PDGF-BB/AB monoclonal antibody (clone Sis1, PharMingen,
San Diego, CA), goat anti-human bFGF, and anti-human PDGF-BB antibodies
(R & D Systems), rabbit anti-goat IgG (H + L) horseradish
peroxidase-conjugated and rabbit anti-goat IgG (H+L) alkaline
phosphatase-conjugated antibodies (Pierce). Phenylmethylsulfonyl
fluoride (PMSF), leupeptin, DTT, and trypsin were purchased from Sigma.
Steady-state Fluorescence--
Steady-state fluorescence
titration was performed on a PerkinElmer Life Sciences 50B fluorimeter.
The excitation wavelength was set at 280 nm, and the emission
originated by both tyrosyl and tryptophyl fluorophores was followed in
the 300-360 nm range at 20 °C. In all experiments equal bandwidths
were used for both excitation and emission, and the integration time
was 1 s. To obtain information on binding constant and
stoichiometry, experiments were carried out according to the Continuous
Variation Method (52, 53). Variable amounts of the two proteins were
mixed so that the sum of their concentrations was held constant at 2 µM, throughout the experiment. The individual
contribution of the two species, which is linear with the concentration
and was estimated by distinct dilution experiments, was always
subtracted from the total fluorescence signal. Under these conditions,
the minimum fluorescence signal is recorded at the molar ratio
corresponding with the complex stoichiometric ratio (52, 53). Two types of experiments were carried out, with protein concentrations falling within the fluorescence linearity. In the first set of experiments small aliquots of 2 µM bFGF dissolved in phosphate-buffer
solution (PBS) without Ca2+ and without Mg2+,
pH 7.2, were added to 2 µM PDGF-BB dissolved in the same
buffer. In the range of molar ratios explored, i.e.
[bFGF]/[PDGF-BB] from 0 to 1.7, the trend of the fluorescence
signal was monotonic. Then a second set of experiments was carried out
by adding small aliquots of 2 µM PDGF-BB dissolved in PBS
without Ca2+ and without Mg2+, pH 7.2, to 2 µM bFGF dissolved in the same buffer with
[bFGF]/[PDGF-BB] molar ratio from 0 to 1.8. A fluorescence minimum
was observed at a PDGF-BB/bFGF molar ratio of about 0.5, suggesting a
2:1 bFGF/PDGF-BB binding stoichiometry. Fluorescence data were then
smoothed by means of the Savitzky-Golay filter implemented in the
software package Scientist by MicroMath (Salt Lake City, UT), and
nonlinear least squares analysis was performed according to the
one-step equilibrium (Reaction 1),
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Overlay Experiments--
Three hundred fifty nanograms of either
bFGF, PDGF-BB, aFGF, fibronectin, vitronectin, elastase, anti-bFGF, or
anti-PDGF-BB antibodies were spotted onto a nitrocellulose membrane,
which was then blocked with 5% milk in TPBS (0.1% Tween 20 in PBS). After washing extensively with TPBS, the membrane was
incubated with 125I-bFGF (1 µCi, 2 ml, 10 ng/ml) for
4 h at room temperature and washed extensively with TPBS.
Nitrocellulose was exposed to Kodak X-Omat AR film (Eastman Kodak Co.)
and subjected to densitometric analysis on GS 710 Calibrated Imaging
Densitometer (Bio-Rad).
SPR Experiments--
SPR assays were performed on a BIAcoreX
instrument equipped with a two-flow cell sensor chip. Basic FGF
immobilization was carried out on one flow cell, here referred to as
"sample flow cell," and the second flow cell was used as a control
cell. Basic FGF was covalently coupled to the CM5 sensor chip after
activation of the carboxymethylated dextran surface by a mixture of
0.05 M N-hydroxysuccinimide and 0.2 M
N-ethyl-N'-(3-diethylaminopropyl)carbodiimide according to a published procedure (54). The coupling reaction was
performed by injecting bFGF (80 µl, 1.25 µg/ml) diluted in 30 mM acetate buffer, pH 4.8. The residual activated groups
were blocked with 1 M ethanolamine hydrochloride, pH 8.5. Immobilized bFGF achieved about 500 resonance unit (RU) signals,
corresponding approximately to a concentration of 0.5 ng/mm2 (55). The integrity of the immobilized growth factor
was tested by injecting a polyclonal anti-bFGF antibody. All
experiments were performed using HBS (10 mM Hepes, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, pH
7.4) as running buffer and to dilute the injected factors. A flow rate
of 30 µl/min was used throughout the experiments. Soluble ligands,
namely PDGF-BB, aFGF, vitronectin, BSA, fibronectin, and PDGF-AA (30 µl), were injected for a 30-s association phase, followed by HBS flow
for a 2-min dissociation phase. The response expressed as sensorgram,
in RU versus time, was monitored at 25 °C. In the current
study, the RU response was always reported as the difference between
signals arising from the sample and the reference cell. Therefore, bulk
refractive index background and nonspecific binding of the soluble
ligands were always subtracted. Sensor chip regeneration was
successfully achieved by injecting 10 µl of 50 mM NaOH
after each injection. All injections were carried out in triplicate.
Experiments were carried out on two different sensor chips and similar
results were obtained. A low immobilization level as well as a high
flow rate (30 µl/min) and analyte concentrations suitable to limit the mass transport phenomenon were used to optimize the kinetic evaluation, according to published reports (55).
In additional experiments, PDGF-BB (10 µl, 500 nM) was
injected for a 2-min association phase, both in the presence and in the
absence of two different anti-PDGF-BB antibodies (100 µg/ml), or 0.1 mM PMSF or 0.1 mM DTT, followed by HBS flow for
a 30-s dissociation phase. A flow rate of 5 µl/min was used
throughout this experiment.
Solid-phase Immunoassay--
Basic FGF binding to PDGF-BB was
also evaluated by solid-phase immunoassays carried out as described
(56) with some modifications. Briefly, microtiter plates (Costar) were
coated by incubating 100 µl/well of PDGF-BB or heat-denatured PDGF-BB
(100 °C for 20 min) or BSA (7 µg/ml) in AC7.5 buffer (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 3 mM MgCl2, 1 mM CaCl2)
for 4 h at 4 °C. Then incubation with 3% BSA (300 µl/well)
in AC7.5 buffer was carried out overnight at 4 °C. All subsequent
operations were carried out at room temperature and by using 100 µl/well. After washing three times with AC7.5T/BSA buffer (AC7.5
containing 0.1% Tween 20 and 1 mg/ml BSA), wells were incubated with
serial dilutions of bFGF or denatured bFGF (100 °C for 20 min) for
4 h. They were washed four times as described above and incubated
with anti-bFGF or anti-PDGF-BB antibodies for 1 h. Plates were
then washed three times and incubated with rabbit anti-goat IgG (H + L)
alkaline phosphatase-conjugated antibody (1:1000 dilution) for 1 h
and washed once with AC7.5T/BSA and twice with diethanolamine buffer
(10 mM diethanolamine, 0.5 mM MgCl2). Plates were then stained with 1 mg/ml of
p-nitrophenyl phosphate in diethanolamine buffer (100 µl/well), and absorption at 405 nm (A405) was determined.
Kinetic Data Analysis--
SPR sensorgrams were analyzed by
nonlinear least squares curve fitting using BIAevaluation software
version 3.0 (Amersham Biosciences AB) and according to published
procedures (57-59). Kinetic constants were generated of the
association and dissociation curves from SPR experiments by fitting to
a single site binding model (Langmuir model) (Reaction 2).
According to this model, a single exponential fit with a
2 < 0.5 was computed. Equation 1 was used for the
dissociation phase,
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(Eq. 1)
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where Rt is the amount of bound ligand
expressed in RU at time t, and t0 is
the beginning of dissociation phase. The final dissociation rate
constant, kd, was calculated from the mean values
obtained from injections performed at least in triplicate. To analyze
the association phase, Equation 2 was employed according to
BIAtechnology Handbook,
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(Eq. 2)
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where Req is the amount of bound ligand,
expressed in RU, at equilibrium, and t0 is
the starting time of injection, and as shown in Equation 3,
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(Eq. 3)
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where C is the concentration of analyte injected over
the sensor chip surface. The association rate constant
ka was determined as the slope of a
ks versus C plot. The apparent
equilibrium dissociation constant, KD1,
was determined as the ratio of these two kinetic constants
(kd/ka).
Solid-phase immunoassay data were analyzed by nonlinear regression, by
fitting to two independent, nonequivalent sites binding model, to
evaluate the apparent equilibrium dissociation constants (KD1 and
KD2) according to the following
Reactions 3 and 4,
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This choice was consistent with the biphasic appearance of
double-reciprocal plots and was justified by the fact that a single immobilized PDGF-BB molecule shows two potential binding sites, one in
each monomer.
Basic FGF/PDGF-BB Docking--
PDGF-BB and bFGF
crystallographic structures were from the Protein Data Bank (codes 1pdg
and 2bfh, respectively). The 1pdg model consists of three chains, named
A, B, and C. A and B form a noncrystallographic disulfide-linked BB
dimer, whereas C is part of a crystallographic disulfide-linked dimer.
The docking simulation was carried out by using the A and B chains.
Both chains lack three short segments, not visible in the electron map.
The GRAMM software was used (60) to search for the best putative interaction between PDGF-BB and bFGF, by performing an exhaustive six-dimensional search through the relative molecular translations and
rotations. The simulation was performed by setting parameters suitable
for high resolution docking (mmode, generic; eta, 3.0; ro, 30; fr, 0;
crang, atom_radius; ccti, gray; crep, all; maxm, 1000; ai, 10)
according to a published report (61). The bFGF/PDGF-BB molecular
assembly with the lowest potential energy was then further evaluated to
identify residues putatively involved in the interaction.
Proteolysis of PDGF-BB--
PDGF-BB (400 ng in 50 mM
Tris-HCl, 150 mM NaCl, pH 8.0) was incubated with trypsin
(200 pg) and diluted in the same buffer at 24 °C (62, 63).
Proteolysis was allowed to proceed for different times (5, 20, 40, 80, and 160 min) and was stopped with 0.1 mM leupeptin. The
interaction of such samples with immobilized bFGF was then evaluated by
SPR according to the procedure reported above. A 2-min association
phase was followed by a 30-s dissociation phase. A flow rate of 5 µl/min was used throughout this experiment. Trypsinized PDGF-BB was
also subjected to SDS-PAGE (400 ng/lane) and transferred to a
nitrocellulose membrane for Western blot analysis and revealed with a
polyclonal anti-PDGF-BB antibody (R & D Systems).
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RESULTS |
Fluorescence Analysis--
Increasing amounts of 2 µM stock solution of PDGF-BB were added to a fixed amount
of 2 µM stock solution of bFGF, and the fluorescence was
measured at 330 nm. Then the individual contribution of bFGF and
PDGF-BB was subtracted from the total fluorescence signal, as described
under "Experimental Procedures." The resulting fluorescence was
plotted against the concentration of the added species, showing a
minimum at about 0.7 µM PDGF-BB (Fig.
1A). According to the
Continuous Variation Method used to analyze these data (52, 53), the
Job plot suggested that the formation of bFGF·PDGF-BB complex occurs
with a 2:1 binding stoichiometry, implying that both disulfide-linked
subunits of PDGF-BB are available to interact with monomeric bFGF.
Similar results were obtained collecting the signal at other
wavelengths (344 and 360 nm).
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Fig. 1.
Basic FGF·PDGF-BB complex formation by
fluorescence analysis. A, increasing amounts of PDGF-BB
were added to bFGF; the resulting fluorescence signal (expressed in
arbitrary units, AU) was plotted versus the
concentration of the added species and analyzed by nonlinear regression
to yield the one-step dissociation constant, under the condition that
F330 is linearly related to the concentration of
the ternary complex. B, the concentration of the ternary
complex, expressed in µM, was calculated by the
dissociation constant resulting from data fitting. Therefore, it
represents the population of the ternary complex at each concentration
of added PDGF-BB on the assumption that a binary intermediate complex
is scarcely populated. The experiment was carried out 3 times with
similar results. The reported data refer to a representative
experiment.
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The minimum shown by Job plots does not substantially depend on the
model chosen to treat binding data, provided the sum of concentrations
of the two interacting species is large relative to the dissociation
constant (53), as in the present case. In fact, the one-step
dissociation constant (KD) of the ternary complex
was estimated in the picomolar range (5.4×10
12
M2), and the total concentration of the two
proteins was 2 µM throughout the experiment. These values
were used to calculate the complex concentration as a function of the
total concentration of PDGF-BB (Fig. 1B).
Basic FGF·PDGF-BB Complex Formation Examined by Overlay
Experiments--
To detect whether specific interaction exists between
bFGF and PDGF-BB, several molecules, namely bFGF, PDGF-BB, aFGF,
fibronectin, vitronectin, elastase, anti-bFGF, and anti-PDGF-BB
antibodies, were immobilized onto a nitrocellulose membrane. The
membrane was then overlaid with 125I-bFGF (1 µCi; 10 ng/ml) and exposed to a Kodak X-Omat AR film. In these assays all
species were under native conditions. Results shown in Fig.
2 indicate a marked interaction of
labeled bFGF with immobilized bFGF, as well as with immobilized PDGF-BB
and anti-bFGF antibody. In contrast, labeled bFGF did not show any interaction with aFGF, fibronectin, vitronectin, elastase, and anti-PDGF-BB antibody. Table I shows the
mean densitometry values, expressed as percent versus bFGF,
of four different experiments. These data show that under these
conditions bFGF specifically interacts with PDGF-BB.
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Fig. 2.
Basic FGF binding with PDGF-BB, analyzed in
overlay assays. Binding of 125I-bFGF (1 µCi, 2 ml,
10 ng/ml) to 350 ng of immobilized bFGF, anti-bFGF antibody, PDGF-BB,
elastase, fibronectin, vitronectin, and anti-PDGF-BB antibody. Proteins
were spotted onto nitrocellulose filters, blocked in 5% milk, and
incubated with the soluble ligand. Nitrocellulose was then exposed to a
Kodak X-Omat AR film. The experiment was carried out 4 times with
similar results. The reported data refer to a representative
experiment.
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Table I
Overlay assay, densitometric analysis for the interaction of labeled
bFGF with different immobilized factors
Data are expressed as percent versus bFGF.
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Basic FGF/PDGF-BB Interaction Examined by SPR
Analysis--
Kinetic experiments were performed on a BIAcoreX by
immobilizing bFGF and subsequently injecting PDGF-BB as the analyte.
Immobilization of bFGF yielded ~500 RU. Fig.
3A shows the response measured
at the end of the association phase as a function of increasing PDGF-BB concentration. PDGF-BB bound the bFGF-coated chip in a
concentration-dependent manner. Association and
dissociation curves from a representative experiment performed with
five increasing concentrations of PDGF-BB (12.5-150 nM)
are shown in Fig. 3B. All sensorgrams report values obtained
after subtraction of the signal on the control flow cell. The
association phase lasted from time 0 to time 30 s and was analyzed
by nonlinear least squares curve fitting as described under
"Experimental Procedures" to yield ks values at each analyte concentration examined (57). A plot of
ks versus PDGF-BB concentration produced
a straight line (Fig. 3C) with a slope equal to the
association rate constant (ka). The
ka value for PDGF-BB binding to immobilized bFGF was
computed as (1.27 ± 0.06) × 106
s1 M1. The dissociation phase
was recorded for 2 min after the end injection and was analyzed by
nonlinear least squares curve fitting. The dissociation rate constant,
kd, was evaluated from traces obtained at ligand
saturation (Fig. 3B) as we and others previously reported
(57, 58). The kd value for PDGF-BB binding to
immobilized bFGF was calculated as (1.71 ± 0.06) × 102 s1. The apparent equilibrium
dissociation constant KD1 determined
from the ratio of the two kinetic constants
(kd/ka) was (13.5 ± 8.0) × 109 M. The experiment was
repeated in triplicate on two different chips obtaining a mean
KD1 of (23.7 ± 7.3) × 109 M.
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Fig. 3.
Sensorgrams depicting bFGF/PDGF-BB
interaction by SPR analysis. Basic FGF was immobilized onto the
chip and yielded ~500 RU, corresponding approximately to 0.5 ng/mm2. Subsequently, PDGF-BB was injected. A,
saturation curve for the binding of PDGF-BB with immobilized bFGF.
Results represent the average ± S.D. of 3 experiments.
B, sensorgrams showing the PDGF-BB binding to bFGF expressed
in RU versus time after subtraction of the control cell
signal. Left panel shows, from bottom to
top, the effects of 12.5, 25, 50, 100, and 150 nM PDGF-BB. Association and dissociation starts correspond
to the left and right arrows, respectively.
Right panel shows sensorgrams obtained at ligand saturation.
The experiment was carried out three times with similar results on two
different sensor chips. The reported data refer to a representative
experiment. C, plots of ks
versus PDGF-BB concentration. The experiment was carried out
three times on two different sensor chips with similar results. The
reported data refer to a representative experiment.
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SPR analysis was also performed to examine the interaction of aFGF,
vitronectin, BSA, and fibronectin with bFGF-coated sensor chip. Fig.
4 shows the RU response observed by
injecting 500 nM of each protein. Although PDGF-BB and
anti-bFGF antibody bound bFGF with a response of about 200 and 400 RU,
respectively, none of the other factors exhibited significant
interaction with bFGF.
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Fig. 4.
Binding of different proteins to immobilized
bFGF analyzed by SPR. Anti-bFGF antibody (100 µg/ml), PDGF-BB
(500 nM), aFGF (500 nM), vitronectin (500 nM), BSA (500 nM), and fibronectin (500 nM) were injected onto immobilized bFGF. Different RU
responses indicate different binding of each protein to bFGF. Results
represent the average ± S.D. of 3 experiments.
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Basic FGF/PDGF-BB Interaction Examined in Solid-phase
Immunoassay--
Solid-phase immunoassays were carried out as an
alternative approach to measure the interaction between bFGF and
PDGF-BB. Different from the SPR analysis, in this experiment PDGF-BB
was the immobilized species, and bFGF was added as free in solution. Basic FGF significantly bound PDGF-BB in a
concentration-dependent manner (Fig.
5A). Nonlinear regression
analysis was carried out according to a model involving binding to two
independent nonequivalent sites, because of the biphasic appearance of
the double-reciprocal plot. This procedure yielded apparent
dissociation constants of (5.2 ± 3.0) × 109
M and (6.67 ± 5.05) × 107
M (KD1 and
KD2 respectively). The
KD1 value computed from solid-phase
immunoassay is close to the KD1 value
computed from SPR measurements indicating a good agreement between
immunoassay and SPR analysis. No binding was observed between PDGF-BB
and anti-bFGF antibody nor between denatured bFGF and PDGF-BB nor
between bFGF and denatured PDGF-BB (Fig. 5B), further
indicating that the observed bFGF/PDGF-BB interaction was specific.
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Fig. 5.
Basic FGF binding to plastic-immobilized
PDGF-BB, analyzed in solid-phase immunoassay. Solid-phase
immunoassay was performed as described under "Experimental
Procedures." Binding was detected with a polyclonal anti-bFGF
antibody and a rabbit anti-goat IgG (H + L) alkaline
phosphatase-conjugated antibody, followed by a colorimetric assay.
A, the amount of bFGF bound with PDGF-BB, expressed as
absorption at 405 nm, was plotted versus the concentration
of added bFGF. After subtracting blank values, data were smoothed by
the Savitzky-Golay algorithm and treated by nonlinear regression
analysis assuming two independent classes of nonequivalent binding
sites. The upper curve results from the additive
contributions of the theoretical curves relative to site 1 and site 2, characterized by the dissociation constants
KD1 and
KD2, respectively. B,
interaction of different soluble factors (333 nM each) to
different immobilized factors was reported as absorption at 405 nm.
Results represent the average ± S.D. of three experiments.
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Basic FGF/PDGF-BB Docking--
Different approaches can be
followed to model protein-protein interaction. In the current study,
docking simulation was carried out with the GRAMM software (60) which
allows us to predict the conformation of a two-protein complex by
performing an exhaustive six-dimensional search through the molecule
relative translations and rotations of the two molecules.
A bFGF·PDGF-BB complex corresponding to the putative energy minimum
was identified (Fig. 6A) and
was further analyzed to compare the predicted complex to published
experimental data concerning interaction sites. The PDGF-BB dimer
consists of two identical subunits, classified in the CATH data bases
(64) as a mainly -ribbon architecture. The lightly bent shape of
each subunit, as well as the side-by-side-interaction, generate a
predicted convex shape for the dimer assembly. In the predicted complex the bFGF molecule leans on the convex side of the PDGF-BB structure. Fig. 6B reports the bFGF·PDGF-BB complex, showing in
white the PDGF-BB regions known to interact with the
receptors (26). The docking simulation shows that these regions are
partially masked by bFGF in the complex. SPR analyses were then
collected to support this prediction.
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Fig. 6.
Basic FGF/PDGF-BB docking.
A, space fill representation of the complex between PDGF-BB
(blue and green chains) and bFGF (red
chain), obtained by simulated docking (see "Experimental
Procedures" for details). The simulation generated a bFGF·PDGF-BB
complex with the bFGF positioned onto the convex side of the PDGF-BB
dimer. B, comparison of docking simulation with data from
the literature. Within the predicted bFGF·PDGF-BB complex (stick
representation, colors as in A) the bFGF lies on the PDGF-BB
regions known to interact with receptors (space fill representation in
white) (26, 65).
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Binding Site Mapping on PDGF-BB--
To identify PDGF-BB regions
involved in the complex formation, SPR analysis on a bFGF-coated chip
was carried out by injecting PDGF-BB alone or in the presence of two
different anti-PDGF-BB antibodies. Specifically, a polyclonal antibody
raised against the region comprising amino acid residues 136-190 and a
monoclonal antibody raised against the 109-124 (V3) and 117-132 (V4)
segments were used (65). The results from representative experiments are shown in Fig. 7. None of the
antibodies exhibited significant affinity for the immobilized bFGF.
PDGF-BB interaction with bFGF measured in the presence of the
polyclonal antibody was slightly reduced as compared with PDGF-BB alone
(367 versus 395 RU). Interestingly, the monoclonal antibody,
which is known to inhibit PDGF-BB receptor binding (26), strongly
decreased the RU response (66 versus 395 RU), suggesting
that the PDGF-BB region interacting with bFGF overlaps, at least in
part, a PDGF-BB receptor-binding site.
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Fig. 7.
Basic FGF/PDGF-BB interaction in the presence
of anti-PDGF-BB antibodies, analyzed by SPR. PDGF-BB binding to
immobilized bFGF was measured in the presence and in the absence of a
polyclonal or a monoclonal anti-PDGF-BB antibody (100 µg/ml). Equal
amounts of each antibody alone showed no interaction with bFGF. The
experiment was carried out three times with similar results. The
reported data refer to a representative experiment.
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Similar SPR experiments were performed to study the bFGF/PDGF-BB
interaction in the presence of an alkylating and a reducing agent, PMSF
and DTT, respectively. Fig. 8 shows the
RU responses observed in a representative experiment. PMSF did not
affect bFGF/PDGF-BB interaction, whereas DTT significantly inhibited
the interaction, up to 70%. These data suggest that
PMSF-dependent covalent modification of serine residues
does not influence the bFGF/PDGF-BB interaction, whereas the integrity
of PDGF-BB disulfide-bridges is required for the binding to bFGF.
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 8.
PMSF and DTT dependence of PDGF-BB binding to
immobilized bFGF, analyzed by SPR. PDGF-BB binding with
immobilized bFGF was tested in the presence of 0.1 mM PMSF
or 0.1 mM DTT. Asterisk indicates significant
difference as compared with PDGF-BB alone (p < 0.01).
Results represent the average ± S.D. of three experiments.
|
|
Interaction of Trypsinized PDGF-BB with bFGF Examined by SPR
Analysis--
To test further the specificity of the bFGF/PDGF-BB
interaction, trypsinized PDGF-BB was injected onto immobilized bFGF,
and the interaction was analyzed by SPR. Fig.
9A shows that, under these
conditions, PDGF-BB lost the ability to be recognized by the specific
antibody in Western blot, progressively as a function of time of
trypsin treatment. Fig. 9B shows the RU response, measured 20 s after the end of association phase, observed by injecting PDGF-BB trypsinized at various times onto immobilized bFGF. Under these
conditions trypsin alone did not show any interaction with immobilized
bFGF (data not shown), whereas binding of PDGF-BB to bFGF strongly
decreased as function of time of proteolysis, reaching a plateau after
about 30 min.
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 9.
PDGF-BB proteolysis and analysis of
trypsinized PDGF-BB binding with bFGF, by SPR. A,
PDGF-BB (400 ng) was incubated at 24 °C without trypsin (1st
lane) or with trypsin (200 pg) for 5, 20, 40, 80, and 160 min
(2nd to 6th lanes). The reaction was stopped with
0.1 mM leupeptin. Samples were denatured and analyzed by
SDS-PAGE and Western blot and revealed with a polyclonal anti-PDGF-BB
antibody. The reported data refer to a representative experiment.
B, trypsinized PDGF-BB binding with immobilized bFGF was
measured as function of time of proteolysis. Results represent the
average ± S.D. of four experiments.
|
|
| |
DISCUSSION |
In the present study we demonstrate for the first time that a
direct interaction between human bFGF and human PDGF-BB occurs in
vitro. The bFGF/PDGF-BB interaction has been observed in
solid-phase-based assays, i.e. SPR, solid-phase immunoassay,
and overlay experiments, by immobilizing either one factor or the
other, as well as in a liquid-phase-based assay, i.e. in
steady-state fluorescence experiments. The data obtained consistently
show that the interaction is specific, dose-dependent, and
saturable. Kinetic experiments carried out in SPR and solid-phase
immunoassays consistently indicate a KD1
value, corresponding to the first step of the binding equilibrium,
falling in the low nanomolar range (23.7 and 5.2 nM
respectively), and fluorescence experiments indicate the
KD value of the whole equilibrium in the picomolar
range. The reported KD values for PDGF-BB and bFGF
interaction with the corresponding receptors are in the low nanomolar
ranges (66-69), suggesting that the bFGF·PDGF-BB complex formation,
under certain conditions, may alter interaction with the corresponding
receptors, thus modulating their activity. The hypothesis that the
complex itself may alter binding with the bFGF and PDGF-BB receptors is
currently under investigation. Findings of the present study should be
discussed in light of our previously reported data (51) indicating that PDGF-BB chemotactic and mitogenic activity on primary smooth muscle cells are markedly inhibited in the presence of bFGF. We also collected
data indicating that a reciprocal inhibitory effect is present, since
bFGF activity on endothelial cells is markedly inhibited by PDGF-BB
both in vitro and in vivo.2 These
data demonstrate that the simultaneous presence of these molecules
leads to a marked functional impairment of either factors. Basic
FGF·PDGF-BB complex formation may represent the mechanism underlying,
at least in part, the observed functional interference, although the
direct link is still under evaluation. They also indicate a novel
mechanism to modulate activity of these factors. As recently pointed
out, protein-protein interactions are essential for almost all
biological processes and homo- and hetero-dimerization may
induce subtle changes in monomer protein concentration, therefore influencing protein activity (70).
Both bFGF and PDGF-BB are potent angiogenic factors (71) and are
expressed in vivo under physiologic conditions (1). Despite
this, angiogenesis is not observed under normal conditions, and
neo-angiogenesis processes are activated only under specific physiologic stimuli, such as the menstrual cycle, and in pathologic conditions such as wound healing, cancer growth, diabetic retinopathy, and ischemia. Mechanisms able to modulate these and/or other growth factors are under thorough investigation (72-74). Their activity is
reported to be regulated by the expression level of the corresponding receptors (66, 75), by controlling their active folding (23) as well as
heparin-binding features (76, 77). PDGF-BB activity is also modulated
by its binding to 2-macroglobulin (78), and bFGF
activity is also modulated by its binding with perlecan, sialoglycolipids, and thrombospondin-1 (79-81). An additional
mechanism possibly playing a role to modulate the activity of PDGF-BB
and bFGF, reported in the present study, is the direct interaction of
these growth factors.
Experiments carried out in the presence of PMSF suggest that serine
residues are not involved in the bFGF/PDGF-BB interaction. In contrast,
reducing disulfide bonds of PDGF-BB with DTT almost completely
abolished its interaction with immobilized bFGF (Fig. 6), suggesting
that the integrity of disulfide bonds is required. This observation was
further supported by the docking analysis, which identified the V4
region, containing two cysteine residues, as putatively involved in the
interacting region.
Although further studies with bFGF and PDGF-BB mutants are necessary to
definitely map the interacting sites, data collected in the present
study suggest that the PDGF-BB regions interacting with bFGF may
overlap a receptor-binding site. In fact, a monoclonal antibody
recognizing V3-V4 regions of PDGF-BB, able to inhibit its binding to
the receptor (65), almost abolished bFGF/PDGF-BB interaction (Fig. 5).
This observation indicates that the interacting region may overlap, at
least in part, the PDGF-BB receptor-binding site and is further
supported by a docking analysis, suggesting that conditions improving
the bFGF·PDGF-BB complex formation may alter, at least in part, the
PDGF-BB ability to bind its receptors (51). Further investigation is
needed to definitely conclude that bFGF/PDGF-BB binding would account
for the inhibitory effect observed (51, 82).
In conclusion, bFGF·PDGF-BB complex formation may represent a novel
mechanism to modulate the activity of both growth factors.
We thank Dr. L. Ambrosone at the Dipartimento
di Scienze e Tecnologie Agro-Alimentari, Ambientali e Microbiologiche,
Università del Molise, Campobasso, for helpful discussion on
binding equilibria. We also thank Dr. T. C. Petrucci at
Laboratorio di Biologia Cellulare, Istituto Superiore di Sanità,
Roma, and Prof. P. De Santis, Dipartimento di Chemistry,
Università "La Sapienza", Roma, for the helpful comments
during the preparation of this manuscript. We also gratefully thank
Dipartimento di Istologia ed Embriologia Medica, Univeristà "La
Sapienza," Roma, for the use of the fluorimeter apparatus.
The abbreviations used are:
PDGF, platelet-derived growth factor;
bFGF, basic fibroblast growth factor;
EC, endothelial cells;
VSMC, vascular smooth muscle cells;
SPR, surface
plasmon resonance;
BSA, bovine serum albumin;
PMSF, phenylmethylsulfonyl fluoride;
DTT, dithiothreitol;
PBS, phosphate
buffer solution;
RU, resonance units;
aFGF, acidic FGF.
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TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Laboratorio di
Patologia Vascolare, Istituto Dermopatico dell'Immacolata, via dei
Monti di Creta 104, 00167 Roma, Italy. Tel.: 39-06-66462431 or
39-06-66462433; Fax: 39-06-66-46-24-30; E-mail:
a.facchiano@idi.it.
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