Platelet factor 4 disrupts the intracellular signalling cascade inducedby vascular endothelial growth factor by both KDR dependent and independent mechanisms Eric Sulpice1, Jean-Olivier
Trang 1Platelet factor 4 disrupts the intracellular signalling cascade induced
by vascular endothelial growth factor by both KDR dependent
and independent mechanisms
Eric Sulpice1, Jean-Olivier Contreres1, Julie Lacour1, Marijke Bryckaert2and Gerard Tobelem1
1
Institut des Vaisseaux et du Sang, Paris;2INSERM U348, Paris, France
The mechanism by which the CXC chemokine platelet
fac-tor 4 (PF-4) inhibits endothelial cell proliferation is unclear
The heparin-binding domains of PF-4 have been reported to
prevent vascular endothelial growth factor 165 (VEGF165)
and fibroblast growth factor 2 (FGF2) from interacting with
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 signalling
events involved in the proliferation induced by VEGF165,
which binds heparin, and by VEGF121, which does not
Activation of the VEGF receptor, KDR, and phospholipase
Cc (PLCc) was unaffected in conditions in which PF-4
inhibited VEGF121-induced DNA synthesis In contrast,
VEGF165-induced phosphorylation of KDR and PLCc was
partially inhibited by PF-4 These observations are consis-tent with PF-4 affecting the binding of VEGF165, but not that of VEGF121, to KDR PF-4 also strongly inhibited the VEGF165- and VEGF121-induced mitogen-activated protein (MAP) kinase signalling pathways comprising Raf1, MEK1/2 and ERK1/2: for VEGF165it interacts directly or upstream from Raf1; for VEGF121, it acts downstream from PLCc Finally, the mechanism by which PF-4 may inhibit the endothelial cell proliferation induced by both VEGF121 and VEGF165, involving disruption of the MAP kinase signalling pathway downstream from KDR did not seem to involve CXCR3B activation
Keywords: CXCR3B; KDR; MAP kinase; PF-4; VEGF
Angiogenesis, the formation of new capillary blood vessels,
is controlled by positive and negative regulators Tumours
secrete potent angiogenic factors, including 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 angiostatin, 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 and migration of endothelial cells
[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 of its heparin-binding domain, PF-4 retains
anti-angiogenic activity, suggesting another mechanism of
inhi-bition [16] Indeed, we recently showed that PF-4 inhibits cell proliferation by selectively 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 endothelial 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 It stimulates the proliferation, migration and differen-tiation of endothelial cells [2,18], and is involved in angio-genesis-dependent tumour progression and other diseases associated with angiogenesis, including diabetic retinopathy and rheumatoid arthritis [2,7,19] VEGF acts via the kinase insert domain-containing receptor (KDR) and Flt1 recep-tors Several lines of evidence suggest that the KDR is solely responsible for endothelial cell proliferation [20,21] Various forms of VEGF have been described [22] (VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206), all produced from a single gene by alternative splicing [23] VEGF165 possesses a heparin-binding domain necessary for full activation of KDR [24] and binding to heparan sulfates on the cell surface, whereas VEGF121 does not [25] Conse-quently, VEGF121promotes endothelial cell proliferation less efficiently than VEGF165[26] The VEGF-induced signalling pathways involved in endothelial cell proliferation have been extensively documented VEGF induces the dimerization, autophosphorylation and tyrosine kinase activity of KDRs
Correspondence toE Sulpice, Institut des Vaisseaux et du Sang, Centre
de Recherche de l’Association Claude Bernard, Hoˆpital Lariboisie`re,
8 rue Guy Patin, 75475, Paris CEDEX 10, France.
Fax: +33 1 42 82 94 73, Tel.: +33 1 45 26 21 98,
E-mail: eric_sulpice@club-internet.fr
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-3H]thymidine; VEGF, vascular endothelial growth
factor.
(Received 1 March 2004, revised 14 May 2004, accepted 21 June 2004)
Trang 2[20,27] Phospholipase Cc (PLCc), a substrate of KDR
kinase, is then phosphorylated and activated, leading to the
activation of protein 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 ultimately leads to activation of the
mitogen-activated protein kinases (MAP kinases), also
known as extracellular signal-regulated kinases (ERK1/2),
which are essential for VEGF-induced endothelial 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 pathways involved in the
endothelial cell proliferation induced by VEGF165, which
binds PF-4, and by VEGF121, which does not In addition,
we investigated the involvement of the newly identified
chemokine receptor, the CXCR3B [34], in this process PF-4
inhibited the induction of human umbilical vein endothelial
cell (HUVEC) proliferation by both VEGF165 and
VEGF121 VEGF121-induced KDR autophosphorylation
and PLCc phosphorylation were not affected by the
presence of PF-4, whereas VEGF165-induced KDR
auto-phosphorylation and PLCc auto-phosphorylation were partially
inhibited In contrast, PF-4 strongly inhibited the Raf1,
MEK1/2 and ERK1/2 activation stimulated by both
VEGF165 and VEGF121 Thus, PF-4 inhibited the MAP
kinase pathway independently of KDR activation, showing
that PF-4 exerts inhibitory effects on VEGF121-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, it does not appear to be involved in
the inhibitory effects of PF-4 on proliferation and MAP
kinase inhibition
Materials and methods
Materials
Recombinant human PF-4 was supplied by Serbio
(Genne-villiers, France) [Methyl-3H]thymidine (TdR) was obtained
from ICN Biomedical Inc (Costa Messa, CA, USA) Cell
culture medium, fetal bovine serum, human serum and
SuperScript II Reverse Transcriptase were purchased from
Invitrogen (Cergy Pontoise, France) VEGF165, VEGF121
and anti-CXCR3 Igs (clone 49801.111) were purchased
from R & D Systems (Minneapolis, MN, USA)
Anti-ERK2, anti-KDR and nonimmune Igs were supplied by
Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA),
anti-active (pTEpY) ERK Ig by Promega (Madison, WI,
USA), anti-active MEK1/2 (phospho-Ser217/221) by Cell
Signaling Technology (Beverly, MA, USA) Anti-PLCc1,
anti-phosphotyrosine (4G10) Igs and the Raf1
immunopre-cipitation kinase cascade assay kit were obtained from
Upstate Biotechnology (Lake Placid, NY, USA)
Anti-CD-31 Ig and the isotype control were obtained from
Immu-notech (Luminy, France)
Cell culture HUVEC were isolated from human umbilical veins by collagenase digestion and were cultured in M199 medium/15 mM Hepes, supplemented with 15% (v/v) fetal bovine serum, 5% (v/v) human serum, 2 ngÆmL)1 FGF2, 2 mM glutamine, 50 IUÆmL)1 penicillin,
50 lgÆmL)1 streptomycin and 125 ngÆmL)1 amphotericin
B, in gelatin-coated flasks at 37C in an atmosphere containing 5% CO2 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 well in M199 supplemented with 15% (v/v) fetal bovine serum, 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 VEGF165or VEGF121(10 ngÆmL)1) and various concentrations of PF-4 (0–10 lgÆmL)1) and/or anti-CXCR3 or nonimmune Igs (40 lgÆmL)1) Finally, cells were incubated for 16 h with
1 lCi of [3H]TdR per dish The [3H]TdR incorporated 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 VEGF165 or VEGF121 in the presence or absence of PF-4 (10 lgÆmL)1), then lysed in RIPA buffer [17] Insoluble material was removed by centrifugation at 4C for 10 min at 14 000 g Supernatants
were incubated overnight at 4C with various antibodies recognizing KDRs (4 lgÆmL)1) or PLCc1 (6 lgÆmL)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 transferred to 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, Buckinghamshire, UK) Raf kinase assays
Raf1 activity was measured using the Upstate Biotechno-logy kit, according to the manufacturer’s instructions Briefly, the serine/threonine kinase, Raf1 was
Trang 3immunopre-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,
[32P]ATP[cP] and myelin basic protein (MBP) to
immuno-precipitated material and incubating for 30 min at 30C
[32P]MBP was quantified with a liquid scintillation
b-counter (Beckman Coulter Scintillation Counter LS
6500, Fullerton, CA, USA)
RT-PCR analysis
RT-PCR experiments were performed with 0.3 lg total
mRNA obtained from primary cultures of HUVEC, using
the SuperScript II one-step RT-PCR kit according to the
manufacturer’s instructions The following primers were
used: CXCR3B (forward) 5¢-TGCCAGGCCTTTACAC
AGC-3¢; (reverse) 5¢-TCGGCGTCATTTAGCACTTG-3¢
GAPDH (forward) 5¢-CCACCCATGGCAAATTCCAT
TCCACC-3¢
Flow cytometry
Cells were removed from culture dishes by adding 5 mM
EDTA in phosphate buffered saline and collecting the
resulting suspension We incubated 300 000 cells for 30 min
at room temperature with phycoerythrin-conjugated specific
or isotype control antibody Finally, cells were washed and
a total of 104 events were analysed on a FACScalibur
cytofluorimeter (Becton Dickinson), usingCELLQUEST
soft-ware
Results
Effect of PF-4 on the endothelial cell proliferation
induced by VEGF121and VEGF165
We first investigated the effects of VEGF165and VEGF121
on [3H]TdR incorporation into HUVEC In the presence of
VEGF165 (10 ngÆmL)1), [3H]TdR incorporation was
380 ± 33% (153 942 ± 13 401 c.p.m.) that of the control
with no growth factor (100%: 40 414 ± 2961 c.p.m.)
(Fig 1A) VEGF121 (10 ngÆmL)1) increased [3H]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 to 10 lgÆmL)1) on [3H]TdR At a PF-4 concentration of
10 lgÆmL)1, VEGF165 and VEGF121 induced DNA
syn-thesis by only 25% and 20%, respectively, of the maximum
value obtained with VEGF165 or VEGF121 alone (100%)
(Fig 1B)
These observations confirm that (a) VEGF165 and
VEGF121 promote DNA synthesis in HUVEC, with
VEGF121 being less potent than VEGF165 [26] and (b)
PF-4 inhibits the DNA synthesis induced by VEGF165and
VEGF121
PF-4 does not affect VEGF121-induced KDR
phosphorylation
We analysed the effects of PF-4 on the signalling pathways
induced by VEGF and VEGF by investigating the
effect of PF-4 on KDR activation VEGF165and VEGF121 (10 ngÆmL)1) induced significant phosphorylation of the tyrosine residues of the KDR (Fig 2A); VEGF121 had a weaker effect (48%) than VEGF165(100%) (Fig 2A,B) In the presence of PF-4 (10 lgÆmL)1), VEGF165-induced phosphorylation of the KDR was inhibited by 45%, whereas VEGF121-induced phosphorylation was unaffected (Fig 2A,B) Interestingly, the level of KDR phosphoryla-tion induced by VEGF121in the absence of PF-4 was similar
to that obtained with a combination of VEGF165 (10 ngÆmL)1) and PF-4 (10 lgÆmL)1)
PF-4 has no effect on VEGF121-induced PLCc phosphorylation
PLCc has been reported to be a downstream target of the tyrosine kinase activity of the KDR and to be involved in VEGF-induced DNA synthesis [31] PLCc phosphorylation was induced by VEGF165 (10 ngÆmL)1) and VEGF121 (10 ngÆmL)1) and the level of phosphorylation of PLCc was lower with VEGF121 (30%) than with VEGF165 (100%) (Fig 3A,B) PF-4 inhibited VEGF165-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 HUVEC were cultured with or without VEGF 165 or VEGF 121 (10 ngÆmL)1), in the presence of var-ious concentrations of PF-4 (1–10 lgÆmL)1) DNA synthesis was determined by monitoring [3H]TdR incorporation into DNA after
20 h of incubation Data are expressed as c.p.m per well in (A) or as 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.
Trang 4lation of PLCc induced by VEGF121 was unaffected by
10 lgÆmL)1PF-4 (Fig 3A,B)
PF-4 inhibits VEGF121- and VEGF165-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 VEGF165 and VEGF121 (Fig 4A)
The level of ERK phosphorylation was higher following
VEGF165 (100%) stimulation than following VEGF121
stimulation (45%) (Fig 4A,B) The degree of ERK
phos-phorylation correlated with the mitogenic effect upon
VEGF165treatment of HUVEC In the presence of PF-4
(10 lgÆmL)1), the phosphorylation of ERK induced by
VEGF165 and VEGF121 was strongly inhibited, only
reaching 18% and 1% of maximum stimulation,
respect-ively (VEGF165alone: 100%) (Fig 4B) Thus, PF-4 acts on
the MAP kinase pathways induced by VEGF121 and
These results were confirmed by kinetic studies of ERK activation The ERK phosphorylation induced by VEGF165 and VEGF121 was maximal between 10 and 15 min of stimulation and decreased thereafter (Fig 4C,E) PF-4 strongly decreased ERK phosphorylation, to only 34% (VEGF165) and 22% (VEGF121) of maximal stimulation (Fig 4D,F)
PF-4 inhibits the VEGF121- and VEGF165-induced activation of MEK1/2 and Raf1
As ERK1/2 are phosphorylated directly and activated by MEK1/2, we investigated the phosphorylation state of these kinases in the presence of PF-4 As previously reported with ERK1/2, VEGF165 induced stronger phosphorylation of MEK1/2 (100%) than did VEGF121 (50%) (Fig 5A,B) MEK1/2 phosphorylation induced by VEGF165 and VEGF121 was strongly inhibited in the presence of PF-4 (10 lgÆmL)1) reaching, respectively, 16% and 4% of maximum stimulation (VEGF165alone: 100%) (Fig 5A,B) Thus, PF-4 inhibits the phosphorylation not only of
Fig 2 Effect of PF-4 on KDR phosphorylation induced by VEGF 165 or
VEGF 121 Serum-deprived HUVEC were incubated for 10 min with
VEGF 165 or VEGF 121 (10 ngÆmL)1) in the presence or absence of PF-4
(10 lgÆmL)1) KDR was immunoprecipitated from cell lysates and
Western blotted with an anti-phosphotyrosine Ig (A) Blots were
scanned with a laser densitometer and results are expressed as
per-centages of the maximal KDR phosphorylation obtained with
VEGF 165 (100%) (B) Values are means ± SD of three independent
experiments **P < 0.001 (Student’s t-test).
Fig 3 Effect of PF-4 on the PLCc phosphorylation induced by VEGF 165 or VEGF 121 Serum-deprived HUVEC were incubated for
10 min with VEGF 165 or VEGF 121 (10 ngÆmL)1) in the presence or absence of PF-4 (10 lgÆmL)1) PLCc was immunoprecipitated from cell lysates and Western blotted with an anti-phosphotyrosine Ig (A) Blots were scanned with a laser densitometer and results are expressed
as percentages of the maximal PLCc phosphorylation obtained with VEGF 165 (100%) (B) Values are means ± SD of three independent experiments **P < 0.001 (Student’s t-test).
Trang 5ERK1/2, but also of MEK1/2, induced by VEGF121 and
VEGF165
We investigated the effect of PF-4 on Raf1 kinase, which
is responsible directly for MEK1/2 phosphorylation We
found that the Raf1 activity induced by VEGF165 and
VEGF121 was strongly inhibited by PF-4 (10 lgÆmL)1)
(Fig 5C) The inhibition was similar for VEGF165- and
VEGF121-induced Raf1 activities
CXCR3 blocking antibody had no effect on PF-4 activity
The results described above suggest that PF-4 affected the
VEGF165 and VEGF121-induced MAP kinase pathway
and proliferation by an intracellular mechanism involving
the modulation of Raf1 activity The inhibition 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] We therefore studied the involvement of this receptor in the inhibition, by PF-4, of VEGF-induced MAP kinase activation and proliferation of HUVEC We tested for CXCR3BmRNA in HUVEC by RT-PCR We detected CXCR3BmRNA in HUVEC and in skeletal muscle, used
as a positive control [34] (Fig 6A) However, FACS analysis, using an antibody that recognizes both CXCR3A 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 effects of PF-4 in our model An antibody blocking CXCR3 [34], was unable to reverse the inhibitory effects of PF-4 (5 lgÆmL)1) on proliferation or MAP kinase activity (Fig 6C,D), suggesting that in our model, PF-4 does not act through this receptor (CXCR3)
Fig 4 Effect of PF-4 on VEGF 165 - and VEGF 121 -induced ERK activation Serum-deprived HUVEC were incubated for 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 various periods of time with VEGF 165 or VEGF 121
(10 ngÆmL)1) in the absence (–– in D,F) or presence (- - - in D,F) of PF-4 (10 lgÆmL)1) (C,D,E,F) Cell lysates were analysed by Western blotting, using polyclonal antibodies against ERK-P and total ERK Blots were scanned with a laser densitometer and results are expressed as percentages of the maximal ERK phosphorylation induced by VEGF 165 (B,D) or VEGF 121 (F) Values are means ± SD of three independent experiments.
**P < 0.001 (Student’s t-test).
Trang 6We recently showed that the antiangiogenic chemokine,
PF-4, inhibits FGF2-induced cell proliferation via an
intracellular mechanism [17] In this study, we investigated
the effect of PF-4 on another angiogenic factor of prime
importance, VEGF, and compared the mechanisms by
which PF-4 inhibits the DNA synthesis induced by
VEGF165and VEGF121
The DNA synthesis induced by VEGF165and VEGF121
was strongly inhibited by PF-4 (10 lgÆmL)1) in HUVEC
Previous work showed that PF-4 efficiently inhibits the
binding of VEGF165 to its receptor, but not that of
VEGF121[26] Thus, PF-4 may disrupt the KDR-mediated
signal transduction induced by VEGF121 by means of an
unknown mechanism that does not involve the disruption of
VEGF121binding [26] We find that PF-4 acts downstream
from receptor activation under conditions of VEGF121
stimulation In contrast, PF-4 also acts at the receptor level
for VEGF165 Indeed, the level of tyrosine phosphorylation
of the KDR and of PLCc decreased significantly (45% and
66%, respectively) following the addition of PF-4
(10 lgÆmL)1) This is consistent with partial inhibition of
the binding of VEGF165to its receptor [26] However, the
levels of tyrosine phosphorylation of the KDR and PLCc
were not affected by PF-4 in conditions of VEGF121
stimulation Thus, PF-4 disrupts KDR-mediated signal
transduction at a postreceptor level following VEGF121
stimulation
We investigated at which step VEGF165- and VEGF121 -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 of Raf1, MEK1/2 and ERK1/2 induced
by both growth factors, VEGF165 and VEGF121, was strongly decreased by PF-4 Thus, PF-4 acts directly on or upstream from Raf1 and downstream from PLCc in the signalling cascade induced by VEGF121 This mechanism may be also involved in the inhibition of VEGF165-induced ERK activation Indeed, PF-4 only partially inhibited the phosphorylation of KDR and PLCc whereas the phos-phorylation of Raf1, MEK1/2 and ERK1/2 activity was almost abolished
How PF-4 regulates the activation of the MAP kinase pathway downstream from the KDR is currently under investigation PKC and Raf1, both stimulated by VEGF and downstream from PLCc, may 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 VEGF- and FGF2-induced MAP kinase phosphorylation [17], PF-4 may act on a target common to the FGF2 and VEGF signalling pathways Thus, PKC does not seem to be a good candidate Raf1 is a key signalling molecule for both VEGF and FGF2 It is a serine/threonine kinase, regulated by phosphorylation of serine and tyrosine residues [39–43] Ser259 is the main inhibitory site of Raf1, but the
Fig 5 Effect of PF-4 on VEGF 165 - and VEGF 121 -induced MEK1/2 and Raf1 activation Serum-deprived HUVEC were incubated for 10 min with VEGF 165 or VEGF 121 (10 ngÆmL)1) in the presence or absence of PF-4 (10 lgÆmL)1) Cell lysates were analysed by Western blotting, using polyclonal antibodies against MEK1/2-P and total MEK (A) Blots were scanned with a laser densitometer and results 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 immunoprecipitation, by means of an in vitro kinase assay Raf1 specific activity is expressed as relative activity (C) Values are means ± SD of three independent experiments *P < 0.01;
**P < 0.001 (Student’s t-test).
Trang 7phosphorylation of this residue is not affected by PF-4
(data not shown) Thus, it is 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, PKA inhibits the
MAP kinase 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 the 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, using a blocking antibody [34] Unlike 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 results were obtained with lower concentrations of
Fig 6 Effect 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 cytometry analysis of CXCR3 expression in HUVEC Staining of cells with the CXCR3 antibody (clone 498011) (grey), with the anti-CD-31 Ig (––) and with the control isotype (- - -) (B) Results are representative of four independent experiments Serum-deprived HUVEC were cultured with VEGF 165 or VEGF 121 (10 ngÆmL)1), in the presence or absence of 5 lgÆmL)1of PF-4 and 40 lgÆmL)1of CXCR3 blocking antibody or nonimmune IgG DNA synthesis was determined by [ 3 H]TdR incorporation into DNA after 20 h
of incubation Data are expressed as a percentage of the maximal incorporation obtained with VEGF 165 (100%) (C) or VEGF 121 (D) Values are means ± SD of three independent 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 PF-4 (5 lgÆmL)1) and CXCR3-blocking antibody or nonimmune IgG (40 lgÆmL)1) Cell lysates were analysed by Western blotting Blots were scanned with a laser densitometer and results are expressed as percentages of the maximal ERK phosphorylation induced by VEGF 165 (C) or VEGF 121 (D) Results are representative of three independent experiments.
Trang 8PF-4 (0.5 to 5 lgÆmL)1) and various concentrations (5 to
40 lgÆmL)1) of blocking antibody (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 CXCR3 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 this chemokine receptor isoform is probably
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 Studies of cAMP modulation in HUVEC
upon PF-4 stimulation, and its possible effect on Raf1
inhibition may also be informative
In conclusion, this report is the first to show that the
signal transduction pathways of two isoforms of VEGF
(VEGF121 and VEGF165) may be regulated by PF-4 at a
postreceptor level These results, and those for the FGF2
signalling pathway, suggest that a specific mechanism of
inhibition is triggered by PF-4, blocking MAP kinase
pathway activation The ability of PF-4 to abolish the
proliferation of endothelial cells induced by the two major
angiogenic growth factors secreted by tumours – VEGF and
FGF2 – may be useful for the development of treatments
based on the inhibition of angiogenesis Any such therapy
would however, require a better understanding of the
mechanism underlying this effect
Acknowledgements
We would like to thank the maternity units of Hoˆpital Lariboisie`re and
Clinique Saint Isabelle for providing the umbilical cords This work was
supported by IVS and grants from l’Association pour la Recherche sur le
Cancer and from Ligue contre le Cancer (contract numbers 5820 and
7566).
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