Tài liệu Báo cáo khoa học: Platelet factor 4 disrupts the intracellular signalling cascade induced by vascular endothelial growth factor by both KDR dependent and independent mechanisms ppt

Platelet factor 4 disrupts the intracellular signalling cascade induced
by vascular endothelial growth factor by both KDR dependent
and independent mechanisms
Eric Sulpice
1
, Jean-Olivier Contreres
1
, Julie Lacour
1
, Marijke Bryckaert
2
and Gerard Tobelem
1
1
Institut des Vaisseaux et du Sang, Paris;
2
INSERM U348, Paris, France
The mechanism by which the CXC chemokine platelet fac-
tor 4 (PF-4) inhibits endothelial cell proliferation is unclear.
The h eparin-binding domains of PF-4 have been reported to
prevent vascular endothelial growth factor 165 (VEGF
165
)
and fibroblast growth f actor 2 (FGF2) from interacting w ith
their receptors. However, other studies have suggested that
PF-4 acts via heparin-binding independent interactions.
Here, we compared the effects of PF-4 on the s ignalling
events involved in the proliferation i nduced by VEGF
165
,
which binds heparin, and by VEGF

121
, which does not.
Activation o f the VEGF receptor, KDR, and phospholipase
Cc (PLCc) was unaffected in conditions in which PF-4
inhibited VEGF
121
-induced DNA synthesis. I n contrast,
VEGF
165
-induced phosphorylation of K DR and PLCc wa s
partially inhibited by PF-4. These observations are consis-
tent with PF-4 affecting the binding of VEGF
165
, but not
that of VEGF
121
, t o KDR. P F-4 also strongly inhibited t he
VEGF
165
-andVEGF
121
-induced mitogen-activated protein
(MAP) kinase signalling pathways comprising Raf1,
MEK1/2 and E RK1/2: for VEGF
165
it interacts directly or
upstreamfromRaf1;forVEGF
121
, i t a cts downstream from
PLCc. Finally, the mechanism by which PF-4 may inhibit

the endothelial cell proliferation induced by both VEGF
121
and VEGF
165
, involving disruption of the M AP kinase
signalling pathway downstream fro m KDR did not seem to
involve C XCR3B activation.
Keywords: CXCR3B; KDR; MAP kinase; PF-4; VEGF.
Angiogenesis, the formation of new capillary blood ve ssels,
is con trolled by positive and negative regulators. Tumours
secrete potent angiogenic factors, in cluding fibroblast
growth factors (FGFs), platelet-derived growth factor B
(PDGF-B) and vascular endothelial growth factor (VEGF)
[1,2]. These factors are counterbalanced by inhibitory
molecules such as a ngiostatin, endostatin, thrombospondin,
and platelet factor-4 [3–8].
Platelet factor-4 (PF-4), a member of the CXC chemo-
kine family [9], inhibits fibroblast growth factor-2 (FGF2)-
induced proliferation a nd migration o f e ndothelial c ells
[10–14]. Various mechanisms by which PF-4 may inhibit
endothelial cell proliferation have been proposed. Via its
heparin binding property, PF-4 may inhibit FGF2-induced
FGF2-receptor activation [10,11,13,15]. However, in the
absence o f its hepar in-binding domain, PF-4 retains anti-
angiogenic activity, suggesting another mechanism of inhi-
bition [16]. I ndeed, we recently showed that PF-4 inhibits
cell p roliferation b y s electively inhibiting FGF2-induced
extracellular signal-regulated kinase (ERK) activation,
without affecting the FGF2-induced phosphatidylinositol
3-kinase activation [17]. These results strongly suggest that

PF-4 inhibits FGF2-induced end othelial cell proliferation
via an intracellular mechanism which, independently of
FGF2-induced activation of FGF2-receptors [17], leads to
ERK inhibition.
In addition to its effects on FGF2-induced proliferation,
PF-4 also inhibits the proliferation and migration of endo-
thelial cells induced by VEGF [14,15]. VEGF is the most
important angiogenic factor, and is present in diverse tumour
cells. I t s timulates the proliferation, migrati on and d ifferen-
tiation of e ndothelial cells [2,18], and is involved in angio-
genesis-dependent tumour progression and o ther diseases
associated with angiogenesis, including diabetic retinopathy
and r heumatoid arthritis [2,7,19]. VEGF a cts via the kinase
insert domain-containing receptor (KDR) and Flt1 recep-
tors. Several lines of evidence suggest that the K DR is s olely
responsible for endothelial cell p roliferation [20,21]. V arious
forms of VEGF have been described [ 22] (VEGF
121
,
VEGF
145
,VEGF
165
,VEGF
189
,andVEGF
206
), all p roduced
from a single gene by alternative splicing [23]. VEGF
165

possesses a heparin-binding domain n ecessary for f ull
activation of KDR [24] and binding to heparan sulfates on
the cell surface, whereas VEGF
121
does not [25]. Conse-
quently, VEGF
121
promotes endoth elial cellp roliferationl ess
efficiently than VEGF
165
[26]. The VEGF-induc ed signalling
pathways involved in endothelial cell proliferation have b een
extensively documented. VEGF induces the dimerization,
autophosphorylation and tyrosine kinase activity of KDRs
Correspondence to E. Sulpice, Institut des Vaisseaux et d u Sang, C entre
de Recherche de l’Association Claude Bernard, Hoˆ pital Lariboisie
`
re,
8 rue Guy Patin, 75475, Paris CEDEX 10, France.
Fax: +33 1 42 82 9 4 73, Tel.: +33 1 45 26 21 98,
E-mail:
Abbreviations: ERK, extracellular signal-regulated kinase; FGF,
fibroblast growth factor; HUVEC, human umbilical vein endothelial
cell; MAP, mitogen-activated protein; MBP, myelin basic protein;
PF-4, platelet factor 4; PDGF-B, platelet-derived growth factor B;
PI3-kinase, phosphatidyl inositol-3 kinase; PLCc, phospholipase Cc;
TdR, [methyl-
3
H]thymidine; VEGF, vascular endothelial growth
factor.

(Received 1 March 2004, re vised 1 4 May 2004, ac cepted 21 June 2004)
Eur. J. Biochem. 271, 3310–3318 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04263.x
[20,27]. Phospholipase C c (PLCc), a s ubstrate of KDR
kinase, i s then phosphorylated and activated, leading to the
activation of p rotein kinase C (PKC), followed by the serine/
threonine kinase, Raf1 and then the threonine/tyrosine
kinase, MEK1/2 (MAP kinase kinase 1/2) [28–31]. This
phosphorylation cascade u ltimately leads to a ctivation of the
mitogen-activated protein k inases (MAP kinases), also
known as extracellular signal-regulated kinases (ERK1/2),
which are essential f or VEGF-induced endoth elial cell pro-
liferation [32]. VEGF also seems to induce the phosphatidyl
inositol-3 kinase (PI3-kinase) pathway [28,33]. However,
inhibitors of PI3-kinase have no effect on VEGF-induced
MAP kinase activation and cell proliferation [29].
To distinguish between the extracellular effects of PF-4
acting on ligand/receptor activation and intracellular effects
on signalling cascades, we compared the effects of this
molecule on the signalling p athways involved in the
endothelial cell p roliferation i nduced by VEGF
165
,which
binds PF-4, a nd by VEGF
121
, which does not. In addition,
we investigated the involvement of the n ewly identified
chemokine receptor, the CXCR3B [34], in this p rocess. PF-4
inhibited the induction of human u mbilical vein endothelial
cell (HUVEC) proliferation by both VEGF
165

and
VEGF
121
.VEGF
121
-induced KDR autophosphorylation
and PLCc phosphorylation were not affected by the
presence of PF-4, whereas VEGF
165
-induced KD R a uto-
phosphorylation and PLCc phosphorylation were partially
inhibited. In contrast, PF-4 strongly inhibited the Raf1,
MEK1/2 and ERK1/2 activation s timulated by both
VEGF
165
and VEGF
121
. Thus, PF-4 inhibited the MAP
kinase pathway i ndependently of KDR activation, showin g
that PF-4 exerts inhibitory effects on VEGF
121
-induced
proliferation downstream from the receptor. Presumably
this inhibition occurs at/or upstream from Raf1 and
downstream from PLCc. We found the chemokine receptor
CXCR3B, a putative PF-4 receptor [34], in small amounts
in HUVEC. However, i t does not appear to be involved in
the inhibitory effects of P F-4 on p roliferation and MAP
kinase inhibition.
Materials and methods

Materials
Recombinant human PF-4 was s upplied by Serbio (Genne-
villiers, France). [Methyl-
3
H]thymidine (TdR) was obtained
from ICN Biomedical Inc. (Costa Messa, C A, USA). Cell
culture medium, fetal bovine serum, human serum and
SuperScript II Reverse Transcriptase were purchased from
Invitrogen (Cergy Pontoise, France). V EGF
165
,VEGF
121
and anti-CXCR3 I gs (clone 49801.111) were purchased
from R & D Systems (Minneapolis, MN, USA). Anti-
ERK2, anti-KDR and nonimmune Igs w ere supplied by
Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA),
anti-active (pTEpY) ERK Ig by P romega (Madison, WI,
USA), a nti-active MEK1/2 (phospho-Ser217/221) by C ell
Signaling Technology ( Beverly, MA, USA). Anti-PLCc1,
anti-phosphotyrosine (4G10) I gs and the Raf1 immunopre-
cipitation kinase cascade assay kit were obtain ed from
Upstate Biotechnology (Lake Placid, NY, USA). Anti-CD-
31 Ig and the isotype c ontrol were obtained f rom Immu-
notech (Luminy, France).
Cell culture
HUVEC were isolated from human u mbilical veins by
collagenase digestion and were cultured in M199
medium/15 m
M
Hepes, supplemented with 15% (v/v)

fetal bovine serum, 5 % (v/v) human serum, 2 ngÆmL
)1
FGF2, 2 m
M
glutamine, 50 IUÆmL
)1
penicillin,
50 lgÆmL
)1
streptomycin and 125 ngÆmL
)1
amphotericin
B, in gelatin-coated flasks at 37 °C in an atmosphere
containing 5% CO
2
. All experiments were carried out
between passages 2 and 3. Umbilical cords were obtained
through local maternity units (Lariboisie
`
re Hospital and
Saint Isabelle Clinic) under approval, and with appro-
priate understanding and consent of the subjects.
DNA synthesis
HUVEC were seeded at 20 000 cells per w ell in M 199
supplemented with 15% (v/v) fetal bovine s erum, 5% (v/v)
human serum and 2 ngÆmL
)1
FGF2. After one day of
culture, the cells were deprived of serum for 24 h, then
cultured for a further 20 h in the presence of VEGF

165
or
VEGF
121
(10 ngÆmL
)1
) and various concentrations of PF-4
(0–10 lgÆmL
)1
) and/or anti-CXCR3 or nonimmu ne Igs
(40 lgÆmL
)1
). Finally, cells we re incubated f or 16 h with
1 lCi of [
3
H]TdR p er dish. The [
3
H]TdR i ncorporated into
the cells was counted with a liquid scintillation b-counter
(Beckman Coulter Scintillation Counter LS 6500, Fullerton,
CA, USA).
Immunoprecipitation analysis
Cells were treated with VEGF
165
or VEGF
121
in the
presence or absence of PF-4 (10 lgÆmL
)1
), then lysed in

RIPA buffer [17]. Insoluble material was removed by
centrifugation at 4 °Cfor10minat14000g. Supernatants
were incubated overnight at 4 °C with v arious antibodies
recognizing KDRs (4 lgÆmL
)1
)orPLCc1(6lgÆmL
)1
). The
antigen–antibody complexes purified with the lMACS
starting kit (Miltenyi Biotec, Bergisch Gladbach, Germany)
were separated by SDS/PAGE in 10% acrylamide gels and
transferred to nitrocellulose membranes.
Western blot analysis
Protein lysates and immunoprecipitates were separated by
SDS/PAGE in 10% acrylamide gels and t ransferred t o
nitrocellulose membranes. The membranes were probed with
antibodies against ERK-P (1 : 15 000), total ERK
(1 : 15 000), phosphotyrosine (1 : 5000), KDR (1 : 1000),
PLCc (1 : 2000), or MEK-P (1 : 1000). The membranes
were washed in Tris buffered saline, 0.1% (v/v) Tween-20
and then incubated with horseradish peroxidase-coupled
secondary antibodies. Antigen–antibody complexes were
detected with the enhanced chemiluminescence system (ECL,
Amersham Pharmacia Biotech, B uckinghamshire, UK).
Raf kinase assays
Raf1 activity was m easured using the Upstate Biotechno-
logy kit, according to the manufacturer’s instructions.
Briefly, the serine/threonine kinase, Raf1 was immunopre-
Ó FEBS 2004 Inhibition of VEGF-induced ERK activation by PF-4 (Eur. J. Biochem. 271) 3311
cipitated with an anti-Raf1 Ig coupled to protein G

Sepharose beads. Kinase reactions were performed in vitro
by adding inactive GST–MEK1, inactive GST–ERK2,
[
32
P]ATP[cP] and myelin basic pro tein (MBP) to immuno-
precipitated material and incubating for 30 min at 30 °C.
[
32
P]MBP was quantified with a liquid scintillation
b-c ounter (Beckman Coulter Scintillation Counter LS
6500, Fullerton, CA, USA).
RT-PCR analysis
RT-PCR e xperiments were performed with 0.3 lgtotal
mRNA obtained from primary cultures of HUVEC, using
the SuperScript I I one-step R T-PCR kit according t o the
manufacturer’s instr uctions. The following primers were
used: CXCR3B (forward) 5¢-TGCCAGGCCTTTACAC
AGC-3¢; (reverse) 5¢-TCGGCGTCATTTAGCACTTG-3¢.
GAPDH (forward) 5¢-CCACCCATGGCAAATTCCAT
GGCA-3¢; (reverse) 5¢-TCTAGACGGCAGGTCAGG
TCCACC-3¢.
Flow cytometry
Cells were removed from culture d ishes by adding 5 m
M
EDTA in phosphate buffered saline and collecting the
resulting suspension. We incubated 300 000 cells for 3 0 min
at room temperature w ith phycoerythrin-conjugated specific
or isotype c ontrol antibody. F inally, cells were washed and
a t otal of 10
4

events were analysed on a F ACScalibur
cytofluorimeter (Becton Dickinson), using
CELLQUEST
soft-
ware.
Results
Effect of PF-4 on the endothelial cell proliferation
induced by VEGF
121
and VEGF
165
We first investigated the effects of VEGF
165
and VEGF
121
on [
3
H]TdR incorporation into HUVEC. In the presence of
VEGF
165
(10 ngÆmL
)1
), [
3
H]TdR incorporation was
380 ± 33% ( 153 942 ± 13 401 c.p.m.) that o f the control
with no growth factor (100%: 40 414 ± 2961 c.p.m.)
(Fig. 1 A). VEGF
121
(10 ngÆmL

)1
)increased[
3
H]TdR
uptake to a lesser extent, to only 220 ± 7%
(89 238 ± 3164 c.p.m.) of control levels (Fig. 1A). We
then tested the effects of various concentrations of PF-4
(1 t o 10 lgÆmL
)1
)on[
3
H]TdR. At a PF-4 concentration o f
10 lgÆmL
)1
,VEGF
165
and VEGF
121
induced DNA syn-
thesis by only 25% and 2 0%, respectively, of the maximum
value obtain ed with V EGF
165
or VEGF
121
alone ( 100%)
(Fig. 1 B).
These observations confirm that (a) VEGF
165
and
VEGF

121
promote DNA synthesis i n HUVEC, with
VEGF
121
being l ess potent than VEGF
165
[26] and (b)
PF-4 inhibits the DNA synthesis induced by VEGF
165
and
VEGF
121
.
PF-4 does not affect VEGF
121
-induced KDR
phosphorylation
We analysed the effects of PF-4 on the signalling p athways
induced by VEGF
165
and VEGF
121
by investigating the
effect of PF-4 on KDR activation. VEGF
165
and V EGF
121
(10 ngÆmL
)1
) induced significant phosphorylation o f the

tyrosine re sidues of the KDR (Fig. 2A); VEGF
121
had a
weaker effect (48%) than VEGF
165
(100%) (Fig. 2A,B). In
the presence o f PF-4 (10 lgÆmL
)1
), VEGF
165
-induced
phosphorylation of t he KDR was inhibited by 45%,
whereas VEGF
121
-induced phosphorylation was unaffected
(Fig. 2A,B). Interestingly, the level of KDR phospho ryla-
tion induced by VEGF
121
in the a bsen ce of PF-4 was s imilar
to that obtained with a combination of VEGF
165
(10 ngÆmL
)1
)andPF-4(10lgÆmL
)1
).
PF-4 has no effect on VEGF
121
-induced PLCc
phosphorylation

PLCc has b een reported to be a downstream t arget of the
tyrosine kinase activity of the KDR and to be involved in
VEGF-induced DNA synthesis [31]. P LCc phosphorylation
was induced by VEGF
165
(10 ngÆmL
)1
)andVEGF
121
(10 ngÆmL
)1
) and th e level o f phosphorylation o f PLCc was
lower with VEGF
121
(30%)thanwithVEGF
165
(100%)
(Fig. 3A,B). PF-4 inhibited VEGF
165
-induced PLCc phos-
phorylation by 66% (Fig. 3B). In contrast, the phosphory-
Fig. 1. PF-4 inhibits the DNA synthesis induced by VEGF
121
and
VEGF
165
in HUVEC. Serum -deprived HU VEC w er e cu ltured w ith o r
without VEGF
165
or VEGF

121
(10 ngÆmL
)1
), in the presen ce of var-
ious concentrations of PF-4 (1–10 lgÆmL
)1
). DNA synthesis was
determined by monitoring [
3
H]TdR in corporation into DN A after
20 h of incubatio n. Data are expressed as c.p.m. p er well in (A) or a s a
percentage of the maximal incorporation obtained with VEGF
165
(––)
and VEGF
121
(- - -) ( B). Values are means ± SD of four independent
experiments performed in triplicate.
3312 E. Sulpice et al. (Eur. J. Biochem. 271) Ó FEBS 2004
lation of PLCc induced by VEGF
121
was unaffected by
10 lgÆmL
)1
PF-4 (Fig. 3 A,B).
PF-4 inhibits VEGF
121
- and VEGF
165
-induced MAP kinase

pathway activation
We then investigated the effect of PF-4 on the ERK
activation necessary for VEGF-induced proliferation of
HUVEC [30,32]. In the absence of PF-4, ERK phosphory-
lation was induced by VEGF
165
and V EGF
121
(Fig. 4 A).
The level of ERK phosphorylation was higher followin g
VEGF
165
(100%) stimulation than following VEGF
121
stimulation (45%) (Fig. 4A,B). The degree o f ERK phos-
phorylation correlated with the mitogenic effect upon
VEGF
165
treatment o f HUVEC. I n the presence of PF-4
(10 lgÆmL
)1
), the phosphorylation of ERK induced by
VEGF
165
and VEGF
121
was s trongly inh ibited, only
reaching 18% and 1 % of maximum stimulation, respect-
ively (VEGF
165

alone: 100%) (Fig. 4B). Thus, PF-4 acts on
the MAP kinase pathways induced by VEGF
121
and
VEGF
165
.
These r esults were confirme d b y kinetic studies of ERK
activation. The E RK phosphorylation induced by VEGF
165
and VEGF
121
was maximal between 10 and 15 min of
stimulation and decreased thereafter (Fig. 4C,E). PF-4
strongly decreased ERK phosphorylation, to only 34%
(VEGF
165
)and22%(VEGF
121
) of maximal stimulation
(Fig. 4 D,F).
PF-4 inhibits the VEGF
121
- and VEGF
165
-induced
activation of MEK1/2 and Raf1
As ERK1/2 are phosphorylated directly and activated by
MEK1/2, we in vestigated the phosphorylation state of these
kinases in the presence of PF-4. A s previously reported w ith

ERK1/2, VEGF
165
induced stronger phosphorylation of
MEK1/2 (100%) than did VEGF
121
(50%) (Fig. 5A,B).
MEK1/2 phosphorylation induced by VEGF
165
and
VEGF
121
was strongly inhibited in the presence of PF-4
(10 lgÆmL
)1
) reaching, respective ly, 16% and 4% of
maximum stimulation (VEGF
165
alone: 100%) (Fig. 5A,B).
Thus, PF-4 inhibits the phosphorylation not only of
Fig. 2. Effec t o f P F-4 o n K DR phos phorylation induced by VEGF
165
or
VEGF
121
. Serum-deprived HUVEC were incubated for 10 min w ith
VEGF
165
or VEGF
121
(10 n gÆmL

)1
) in the presen ce or absence of PF-4
(10 lgÆmL
)1
). KDR w as immunoprecipitated from cell lysates and
Western blotted with an anti-phosphotyrosine Ig (A). Blots were
scannedwithalaserdensitometer and results are expr essed as per-
centages of the maximal KDR phosphorylation obtained with
VEGF
165
(100%) (B). Values are means ± S D of three independent
experiments. ** P < 0.001 (Student’s t-test).
Fig. 3. Effec t of P F-4 o n the PLC c phosphorylation induced by
VEGF
165
or VEGF
121
. Serum-deprived HUVEC were incubated for
10 min with V EGF
165
or VEGF
121
(10 ngÆmL
)1
) in the presence or
absence of PF-4 (10 lgÆmL
)1
). PLCc was immunoprecipitated from
cell lysates a nd Western blotted w ith an anti-phosphotyrosine Ig (A).
Blots were scanned with a laser densitometer and results are e xpressed

as percentages of the maximal PLCc pho sph orylation o btained wit h
VEGF
165
(100%) (B). Values are means ± S D of three independent
experiments. **P < 0 .001 (Student’s t-test).
Ó FEBS 2004 Inhibition of VEGF-induced ERK activation by PF-4 (Eur. J. Biochem. 271) 3313
ERK1/2, but also of MEK1/2, i nduced by VEGF
121
and
VEGF
165
.
We investigated the effect of P F-4 on Raf1 kinase, which
is responsible directly for MEK1/2 phosphorylation. We
found that t he Raf1 activity induced by VEGF
165
and
VEGF
121
was strongly inhibited by PF-4 (10 lgÆmL
)1
)
(Fig. 5C). The inhibition was similar for VEGF
165
-and
VEGF
121
-induced Raf1 activities.
CXCR3 blocking antibody had no effect on PF-4 activity
The results described above suggest that PF-4 affected the

VEGF
165
and VEGF
121
-induced MAP kinase pathway
and proliferation b y an intracellular mechanism involving
the modulation of R af1 activity. The i nhibition of the
MAP kinase pathway by an intracellular mechanism
induced by PF-4 suggests that this chemokine may induce
angiostatic activity via a specific receptor. Recent data
have suggested that PF-4 can bind a newly cloned
chemokine receptor isoform named CXCR3B [34]. W e
therefore studied the involvement of this receptor in the
inhibition, by PF-4, of VEGF-indu ced MAP kinase
activation and proliferation of HUVEC. W e t ested f or
CXCR3B mRNA in HUVEC by RT-PCR. We d etected
CXCR3B mRNA in HUVEC and in skeletal muscle, used
as a positive control [34] (Fig. 6A). However, FACS
analysis, using an antibody that recognizes both C XCR3A
and CXCR3B, indicated that only 10% of HUVEC cells
were positive (Fig. 6B); all HUVEC cells expressed CD-31
(Fig. 6B). Despite few cells expressing this receptor on
their surface, we investigated whether CXCR3B mediated
the antiangiogenic e ffects of PF-4 in our model. An
antibody block ing CXCR3 [34], was unable to reverse the
inhibitory effects of PF-4 (5 lgÆmL
)1
) o n proliferation o r
MAP kinase activity (Fig. 6C,D), suggesting that in our
model, PF-4 does not act through this receptor (CXCR3).

Fig.4. EffectofPF-4onVEGF
165
- and VEGF
121
-induced ERK activation. Serum-deprived HU VEC we re incub ated fo r 10 min with VEGF
165
or
VEGF
121
(10 ngÆmL
)1
) in the presence or absence of PF-4 (10 lgÆmL
)1
) (A,B) or for v arious periods of t ime with VEGF
165
or VEGF
121
(10 ngÆmL
)1
)intheabsence(––inD,F)orpresence( inD,F)ofPF-4(10lgÆmL
)1
) (C,D,E,F). Cell l ysates we re analys ed by Western b lotting,
using polyclonal a ntibodies against ERK-P and total E RK. Blots were sc anned with a la ser densitometer and re sults are ex pressed as p ercentages of
the maximal ERK phosphorylation i nduced by VEGF
165
(B,D) or VEGF
121
(F). Values are means ± S D of three independent experiments.
**P < 0.001 (S tudent’s t-test).
3314 E. Sulpice et al. (Eur. J. Biochem. 271) Ó FEBS 2004

Discussion
We recently showed that the antiangiogenic chemokine,
PF-4, inhibits FGF2-induced cell proliferation via an
intracellular mechanism [17]. In t his study, we investigated
the e ffect of PF-4 on another angiogenic f actor o f prime
importance, VEGF, and compared the mechanisms by
which PF-4 i nhibits the DNA synthesis induced by
VEGF
165
and VEGF
121
.
The DNA synthesis induced by VEGF
165
and VEGF
121
was strongly inhibited b y PF-4 (10 lgÆmL
)1
) in HUVEC.
Previous work showed that PF-4 efficiently inhibits the
binding of VEGF
165
to its receptor, but not that of
VEGF
121
[26]. Thus, PF-4 may d isrupt the KDR-mediated
signal transduction induced by VEGF
121
by means of an
unknown m echanism that does not involve t he disruption of

VEGF
121
binding [26]. We find that PF-4 acts downstream
from receptor activation under conditions of VEGF
121
stimulation. In contrast, PF-4 also acts at the receptor level
for VEGF
165
. Indeed, the level o f tyrosine phosphorylation
of the KDR and o f PLCc decreased s ignificantly (45% a nd
66%, respectively) following the addition of PF-4
(10 lgÆmL
)1
). This is consistent with partial inhibition of
the binding of VEGF
165
to its receptor [26]. How ever, the
levels of tyrosine phosphorylation of the K DR and PLCc
were not affected by PF-4 in conditions of VEGF
121
stimulation. Thus, PF-4 disrupts KDR-mediated signal
transduction at a postreceptor level fo llowing VEGF
121
stimulation.
We investigated at which step VEGF
165
-andVEGF
121
-
induced intracellular signalling is a target of PF-4 inhibition.

Activation of the MAP kinases, ERK1/2, is important for
the proliferation of HUVEC [31]. We therefore focused on
the effect of PF-4 on the kinases involved in the signalling
pathways leading to ERK1/2 stimulation. The level of
phosphorylation o f Raf1, MEK1/2 and ERK1/2 induced
by both growth factors, VEGF
165
and VEGF
121
,was
strongly decreased by P F-4. Thus, PF-4 a cts directly on or
upstream f rom Raf1 a nd downstream from PLCc in the
signalling cascade ind uced by VEGF
121
. This m echanism
may b e also involved i n the inhibition of VEGF
165
-induced
ERK activation. Indeed, P F-4 only partially inhibited the
phosphorylation of KDR and PLCc whereas the phos-
phorylation of Raf1, MEK1/2 and ERK1/2 a ctivity was
almost abolished.
How P F-4 r egulates the activation of the MAP k inase
pathway downstream from the KDR is currently under
investigation. PKC and Raf1, both s timulated by VEGF and
downstream from PLCc, m ay be involved [28,29]. PKC is
involved in MAP kinase activation by VEGF [29,31,35] but
not by FGF2 [36–38]. As PF-4 inhibits both V EGF- and
FGF2-induced MAP kinase phosphorylation [17], PF-4 may
act on a target common to the FGF2 a nd VEGF signalling

pathways. T hus, PKC do es not seem to be a good candidate.
Raf1 is a key signalling molecule for both VEGF a nd
FGF2. It is a serine/threonine kinase, regulated by
phosphorylation of s erine and tyrosine residues [ 39–43].
Ser259 is the main inhibitory site of Raf1, but the
Fig.5. EffectofPF-4onVEGF
165
-andVEGF
121
-induced MEK1/2 and Raf1 a ctivation. Serum- deprive d HU VEC w ere i nc ubated f or 10 min with
VEGF
165
or VEG F
121
(10 ngÆmL
)1
) in the prese nce or absence of PF-4 (10 lgÆmL
)1
). Cell lysates were analysed b y Western blotting, using
polyclonal antibodi es against M EK1/2-P and tota l MEK (A). B lots were scanned with a laser densitometer and r esults are expressed as percentages
of the maximal MEK phosphorylation induced by VEGF
165
(B). Serum-deprived HUVEC were incubated for 8 min with VEGF
165
or VEGF
121
(10 ngÆmL
)1
) in the presence or absence of PF-4 (10 lgÆmL
)1

). Raf1 activity was quantified after Raf1 immunop recipitation, by means of an in vitro
kinase assay. Raf1 specific activity i s expressed as relative activity (C). Values a re m eans ± SD of three i ndepe ndent e xperim ents. *P <0.01;
**P < 0.001 (Student’s t-test).
Ó FEBS 2004 Inhibition of VEGF-induced ERK activation by PF-4 (Eur. J. Biochem. 271) 3315
phosphorylation of this r esidue is not affected by PF-4
(data not shown ). Thus, it i s unclear how PF-4 affects
Raf1 activity in HUVEC. Increases in cAMP levels and
the activation of the cAMP-dependent protein kinase A
(PKA) may be involved [44]. Indeed, P KA inhibits the
MAP k inase pathway by blocking Raf1 activity in many
cell systems [45–47]. Moreover, PF-4 increases cAMP
levels in human microvascular endothelial cells (HMEC-1
cell line) transfected with a construct encoding a new
chemokine isoform receptor – CXCR3B – the only seven-
transmembrane chemokine receptor able to bind PF-4
with high affinity [34]. Alternative splicing of t he CXCR3
mRNA gives rise to two different chemokine receptors:
CXCR3A and CXCR3B [34]. However, only 10% of
HUVEC expressed CXCR3 (CXCR3A plus CXCR3B)
on the cell surface in serum deprivation conditions. We
evaluated the involvement of CXCR3 in the inhibitory
effect of PF-4, u sing a blocking antibody [34]. U nlike for
ACHN cells under the same conditions [34], we were
unable to reverse the inhibitory effect of PF-4 on the
MAP kinase pathway and on HUVEC proliferation.
Similar r esults were obtained with lower concentrations of
Fig. 6. Effec t of CXCR3-blocking antibody on PF-4-induced proliferation and MAP kinase inhibition. Amplification of the CXCR3B mRNA in
HUVEC and skeletal muscle by RT -PCR (A). Flow cyto metry analysi s of C XCR3 expressio n in HU VEC. Staining of cells with the CXCR3
antibody (clone 498011) (grey), with the a nti-CD-31 Ig (––) and w ith the co ntrol isotype (- - -) ( B). R esults are r ep resentative o f f our i ndep endent
experiments. Serum-deprived HUVEC were cultured with VEGF

165
or VEGF
121
(10 n gÆmL
)1
), in the presence or absence of 5 lgÆmL
)1
of PF-4
and 40 lgÆmL
)1
of CXCR3 blocking antibody or nonimmune IgG. DNA synthesis was determined by [
3
H]TdR incorporation into DNA after 20 h
of incu bation. D ata a re e xpressed a s a percen tage of the maximal inc orporatio n obtaine d w ith VE GF
165
(100%) (C) or VEGF
121
(D). Values are
means ± SD of thre e in dependent experiments performed in triplicate. Serum-deprived HUVEC were incubated for 10 min with VEGF
165
or
VEGF
121
(10 ngÆmL
)1
) in the presence or absence of P F-4 (5 lgÆmL
)1
) and CXCR3-blocking antibody or nonimmune I gG (40 lgÆmL
)1
). Cell

lysates were analysed b y Western blotting. Blots were scanned with a laserdensitometerandresultsareexpressedaspercentagesofthemaximal
ERK phosphorylation induced by VE GF
165
(C) or V EGF
121
(D). Results a re representative of three i ndepe ndent experiments.
3316 E. Sulpice et al. (Eur. J. Biochem. 271) Ó FEBS 2004
PF-4 (0.5 to 5 lgÆmL
)1
) a nd various concentrations (5 to
40 lgÆmL
)1
) of blocking a ntibody (data not shown). This
absence of effect could be explained by the restricted
expression of CXCR3 in HUVEC: FACS analysis indi-
cates that 100% of ACHN cells express C XCR3 on their
surface [34], whereas only 10% of HUVEC were positive.
Further experiments will be required to fully determine the
role of CXCR3B in HUVEC, nevertheless, our findings
suggest that t his c hemokine receptor isoform is p robably
not central to PF-4 induced angiostatic activity in our
model. Most chemokines bind and activate different
chemokine receptor isoforms [48–50], and it would be
valuable to determine which bind PF-4 and are expressed
in HUVEC. S tudies o f cAM P modu lation in HUVEC
upon PF-4 stimulation, and its possible e ffect on Raf1
inhibition may also be i nformative.
In conclusion, this report is the first to show that the
signal transduction pathways of two isoforms of VEGF
(VEGF

121
and V EGF
165
)mayberegulatedbyPF-4ata
postreceptor level. These results, and those for the F GF2
signalling pathway, suggest that a specific mechanism of
inhibition is triggered by PF-4, blocking MAP kinase
pathway activation. The ability o f PF-4 to abolish the
proliferation of endothelial cells induced by the two major
angiogenic growth factors s ecreted by tumours – VEGF and
FGF2 – may be useful for the development of treatments
based on the inhibition of angiogenesis. Any such therap y
would however, require a better understanding of the
mechanism underlying this effect.
Acknowledgements
We wou ld like to thank the maternity units of Hoˆ pital Laribo isie
`
re a nd
Clinique Saint Isabelle for providing the umbilical cords. This work was
supported by I VS and grants f rom l’Association pour l a Recherche sur le
Cancer and from LiguecontreleCancer(contract numbers 5820 and
7566).
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