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: 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- 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. 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