Tài liệu Báo cáo khoa học: Platelet factor 4 disrupts the intracellular signalling cascade induced

Platelet 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 Bryckaert2 and Gerard Tobelem1

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 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 VEGF165 it 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 presentin 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 proliferationless

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: [email protected]

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, revised 14 May 2004, accepted 21 June 2004)

Eur. J. Biochem. 271, 3310–3318 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04263.x