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of Ets-1 Transcription FactorCCL2 Regulates Angiogenesis via Activation

Mostarica-Stojkovic and Anuska V. AndjelkovicSvetlana M. Stamatovic, Richard F. Keep, Marija

http://www.jimmunol.org/content/177/4/2651doi: 10.4049/jimmunol.177.4.2651

2006; 177:2651-2661; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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CCL2 Regulates Angiogenesis via Activation of Ets-1Transcription Factor1

Svetlana M. Stamatovic,* Richard F. Keep,*† Marija Mostarica-Stojkovic,§ andAnuska V. Andjelkovic2*‡

Although recent studies have suggested that CC chemokine CCL2 may directly affect the angiogenesis, the signaling eventsinvolved in such regulation remain to be determined. This study investigated a potential signal mechanism involved in CCL2-induced angiogenesis. Our in vitro and in vivo (hemangioma model of angiogenesis) experiments confirmed earlier findings thatCCL2 can induce angiogenesis directly. Using a gene array analysis, CCL2 was found to induce expression of several angiogenicfactors in brain endothelial cells. Among the most prominent was an up-regulation in Ets-1 transcription factor. CCL2 induceda significant increase in Ets-1 mRNA and protein expression as well as Ets-1 DNA-binding activity. Importantly, Ets-1 antisenseoligonucleotide markedly abrogated in vitro CCL2-induced angiogenesis, suggesting that Ets-1 is critically involved in this process.Activation of Ets-1 by CCL2 further regulated some of Ets-1 target molecules including �3 integrins. CCL2 induced significantup-regulation of �3 mRNA and protein expression, and this effect of CCL2 was prevented by the Ets-1 antisense oligonucleotide.The functional regulation of Ets-1 activity by CCL2 was dependent on ERK-1/2 cascade. Inhibition of ERK1/2 activity by PD98509prevented CCL2-induced increases in Ets-1 DNA-binding activity and Ets-1 mRNA expression. Based on these findings, we suggestthat Ets-1 transcription factor plays a critical role in CCL2 actions on brain endothelial cells and CCL2-inducedangiogenesis. The Journal of Immunology, 2006, 177: 2651–2661.

A ngiogenesis, the formation of new blood vessels frompreexisting blood vessels, takes place in many physio-logical and pathological conditions such as embryo de-

velopment, ovulation, wound healing, rheumatoid arthritis, dia-betic proliferative retinopathy, and tumorigenesis (1–5).Angiogenesis consists of series of highly ordered and tightly reg-ulated events including degradation of the existing basement mem-brane, chemotaxis, proliferation, and capillary tube formation (6).Many factors have been found to influence angiogenesis. Somehave a stimulatory effect on angiogenesis, including vascular en-dothelial growth factor (VEGF),3 basic fibroblast growth factor(bFGF), angiopoietin-1, ELR� CXC chemokines IL-8, NAP-2,ENA-78 and GRO (7–14). Others, such as endostatin, angiostatin,and ELR� chemokines IFN-�-inducible protein and monokine in-duced by IFN-�, inhibit angiogenesis (13–14).

Recently, the CC chemokine MCP-1 (CCL2/MCP-1/JE) hasbeen added to the growing list of angiogenic modulators (15–18).Best known for its role in modulating inflammatory responses byinducing monocyte/macrophage recruitment to sites of inflamma-tion, CCL2 also has a distinct role in angiogenesis. The molecularmechanisms by which CCL2 regulates angiogenesis have still to

be fully elucidated. Until very recently, it was generally acceptedthat CCL2 indirectly stimulates angiogenesis via its chemoattrac-tant effects on monocytes/macrophages, which in turn may releasedirect-acting angiogenic factors (15, 16, 18). However, a few re-cent studies have suggested that CCL2 may also exert direct an-giogenic effects (17). Support for this hypothesis comes from thefact that endothelial cells express the CCL2 receptor CCR2(19–21).

The current study was aimed at identifying critical intracellularsignaling molecules that might be involved in CCL2-induced an-giogenesis. Attention was focused on Ets-1 because this transcrip-tional factor is believed to be involved in regulating angiogenesis.

Materials and MethodsAll procedures were performed in strict accordance with the National In-stitute of Health’s Guide for the Care and Use of Laboratory Animals andwere approved by the Institutional Animal Care and Use Committee ofUniversity of Michigan.

Cells

Brain endothelial cell line (bEnd.3) was purchased from American TypeCulture Collection. Cells were grown in medium containing DMEM, 10%heat-inactivated FBS, 1� antibiotic/antimycotic, and 2 mM glutamine (allpurchased from Invitrogen Life Technologies). Cells from 22 to 25 pas-sages were used for all experiments. Primary cell cultures of brain micro-vascular endothelial cells were prepared from CCR2 knockout mice orwild-type mice by a method already established in our laboratory and de-scribed in detail previously (21).

In vitro angiogenesis assay

This assay was performed using previously described methods (22, 23).Bovine fibrinogen (at final concentration of 2.5 mg/ml; Sigma-Aldrich)was dissolved in DMEM supplemented with aprotinin (200 �g/ml; Sigma-Aldrich) and dispensed into 12-well tissue culture plates. Polymerizationwas induced by addition of thrombin (25 U/ml; Sigma-Aldrich) for 1 h at37°C. After that, bEnd.3 cells were seeded onto the fibrin gel at 5 �104/well and allowed to attach for up to 2 h. The medium was then care-fully removed, and fibrin solution, mixed with test agent(s), was added tothe cells. Fresh medium with matched test agent(s) was added on top of thegenerated fibrin gel overlay, and tube formation was assessed 24 h later.

*Department of Neurosurgery, †Molecular and Integrative Physiology and ‡Pathol-ogy, University of Michigan, Medical School, Ann Arbor, MI 48109; and §Instituteof Microbiology and Immunology, School of Medicine, University of Belgrade, Bel-grade, Serbia and Montenegro

Received for publication December 29, 2005. Accepted for publication May 23, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grant NS 044907 (to A.V.A.) from the National In-stitutes of Health.2 Address correspondence and reprint requests to Dr. Anuska V. Andjelkovic, De-partment of Neurosurgery and Pathology, University of Michigan, MI 48109. E-mailaddress: anuskaa@umich.edu3 Abbreviations used in this paper: VEGF, vascular endothelial growth factor; bFGF,basic fibroblast growth factor; uPA, urokinase.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

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The total length of tubular structures was calculated in five different areasusing Image J software (National Institutes of Health) (23).

For blocking experiments, bEnd.3 cells were first incubated in the se-rum-free medium containing PD 98059 (20 �M; Calbiochem) for 1 h ortreated with antisense or sense oligonucleotides as described below. Theywere then trypsinized and plated onto the fibrin gels.

Hemangioma formation in nu/nu mice

Male nude mice (nu/nu; provided by The Jackson Laboratory), 10–12 wkof age, were used. Mice were anesthetized with i.p. ketamine/xylazine (100mg/kg, 5 mg/kg). They were then inoculated with 0.5 ml of bEnd.3 cellsuspension at 5 � 106 cells/ml s.c. in the right femoral flank. Over 3 wk,hemangioma size was measured with caliper three times per week, andhemangioma weight (in grams) was estimated by the following formula:length � width2/2. Experiments were terminated when hemangiomaweight reached �2 g (3 wk postinoculation). The hemangiomas were ex-cised, weighed, photographed, frozen in OCT-embedding medium, andprocessed for H&E staining and immunohistochemistry. Hemangioma tis-sue was also taken for RT-PCR analysis.

To establish the contribution of CCL2 to hemangioma growth and an-giogenesis, inhibition studies were performed where (nu/nu) mice bearingbEnd.3 cells received either CCL2 antisense (5�-AAGCGTGACAGAGACCTGCATAGTCGTGG-3�) phosphorothioate oligonucleotide (5 �Meach; Oligos Etc.) into the peritumor (hemangioma) area or a monoclonalanti-CCL2-neutralizing Ab (25 �g/ml/mouse; R&D Systems) i.p. at days 1,6, 9, 12, 21 after inoculation. Controls received either sense phosphoro-thioate oligonucleotide (5�-CCACCACTATGCAGGTCTCTGTCACGTTT-3�, 5 �M; Oligos Etc.) or vehicle (PBS).

Proliferation assay

Briefly, bEnd.3 cells were cultured in complete medium at a density of 1 �105 cells/ml on 96-well microplates. After 12 h, the medium was removedand replaced with DMEM containing 0.5% FBS plus murine recombinantCCL2 or VEGF (PreproTech) at different concentrations (12.5–400 ng/ml)for 16 h. Medium alone was added as a negative control. bEnd.3 cellproliferation was measured using a colorimetric BrdU incorporation assay(Cell Proliferation ELISA; Roche) according to the manufacturer’sinstructions.

Chemotaxis assay

Chemotaxis assays were performed in BD BioCoat Fibronectin Cell Cul-ture Inserts (BD Biosciences). Briefly, bEnd.3 cells or CCR2�/� mousebrain microvascular endothelial cells (5 � 104 cells in 250 �l of DMEMcontaining 0.5% FBS) were added to the 3.0-�m insert of the chemotaxisplates. Serial 2-fold dilutions of recombinant mouse CCL2 (range 0.8 to400 ng/ml) in DMEM containing 0.5% FBS was added to the wells. VEGF(10 ng/ml) or medium alone was used as positive and negative controls.The cells were labeled with Calcein-AM (Molecular Probes) and allowedto migrate across a membrane insert for 22 h. The intensity of fluorescencewas measured with a fluorescence plate reader (Applied Biosystems; Cyto-Flour 4000 plate reader) configured to read at excitation/emission wave-lengths of 485/530 nm. The number of migrated cells was estimated fromstandard curve.

For blocking experiments, in addition to CCL2, medium was supple-mented with 10 �g/ml neutralizing goat IgG anti-mouse CCL2 Ab (or thenonspecific isotype-matched control Ab; both obtained from R&DSystems).

cDNA array

At different times after CCL2 treatment (0–16 h), bEnd.3 cells were har-vested, and total RNA was prepared using TRIzol reagents (Invitrogen LifeTechnologies). The procedure for biotinylated cDNA probe synthesis wasdone using the AmpoLabeling-LPR kit (SuperArray Bioscience) accordingto the manufacturer’s instructions. The resulting cDNA probe was hybrid-ized to GEArray Q Series mouse angiogenesis gene array (SuperArrayBioscience) according to the manufacturer’s instructions. The relative ex-pression level of each gene was analyzed using a software package pro-vided by SuperArray (SuperArray Bioscience).

EMSA

Nuclear extracts were prepared from bEnd.3 cells using a Nuclear ExtractKit (Active Motif) according to the manufacturer’s instructions. The pro-tein concentration was determined using the Bio-Rad protein assay. EMSAwas performed with the Panomics EMSA kit (Panomics) according to man-ufacturer’s protocol. In brief, Ets-1-binding reactions were conducted with5 �g of nuclear proteins and 10 ng of biotinylated double-stranded oligo-

nucleotide probe containing the DNA-binding motif for Ets-1 (provided byPanomics). For competition experiments and supershift assays, excess ofthe corresponding cold (unlabelled) probe or ant-Ets-1 Ab (2 �g/ml; SantaCruz Biotechnology) was added to the binding reactions and incubated for5 min before addition of the labeled probes. Reactions were subjected toelectrophoresis on 6% polyacrylamide gel in 0.5� Tris-borate-ethylenediamine tetraacetic acid (TBE) buffer followed by electroblotting onto ny-lon membranes (Biodyne B membrane) and UV cross-linking. Biotinylatedoligonucleotides were then detected by probing with streptavidin conju-gated to HRP and visualized by ECL and autoradiography.

Transfection

Ets-1 antisense and sense phosphorothioate oligonucleotides were pur-chased from Oligos Etc. The sequences used were as follows: sense, 5�-ATGAAGGCGGCCGTCGATCT-3�; and antisense, 5�-AGATCGACGGCCGCCTTCAT-3�. Briefly, when bEnd.3 cell culture reached �90%confluence, the cells were washed once with serum-reduced OptiMEM Imedium. The transfection of oligonucleotides was then performed withOptiMEM I medium containing 500 nM oligonucleotides and 12 �g/mllipofectamine (Invitrogen Life Technologies). Cells were incubated withthe transfection medium for 4 h at 37oC, 5% CO2, before changing back tonormal medium. After an additional incubation for 20 h, cells were used inblocking experiments. Efficiency of transfection was established by West-ern blot analysis and EMSA.

Western blotting

Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris(pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate,0.1% SDS, and 2 mM sodium orthovanadate) containing protease inhibi-tors (10 �g/ml leupeptin, 10 �g/ml aprotinin, 1 mM EDTA, and 1 mMPMSF). The protein content was determined using Pierce protein assay kit.Equal amounts of protein were electrophoretically separated by SDS-PAGE on 7.5 or 10% gels and transferred to Trans-Blot nitrocellulosemembrane (Bio-Rad). Immunoblotting was performed using rabbit anti-Ets-1 Ab (Santa Cruz Biotechnology), anti- Ets-1 phospho-(T38) Ab(Novus Biological), anti-phospho-ERK1/2 Ab (Cell Signaling Technol-ogy), or hamster anti-mouse CD61 (integrin �3 chain) Ab (BD Bio-sciences). Immunoreactive proteins were visualized using an ECL detec-tion kit (Pierce). Autoradiographic images underwent semiquantitativedensitometry analysis using National Institutes of Health image softwarepackage (version 1.63). After probing for pERK1/2 or phospho-Ets-1 toexamine ERK activation status or level of phosphorylated Ets-1, immuno-blots were stripped using Restore Western Blot Stripping Buffer (PierceBiotechnology) and reprobed with rabbit anti-ERK1/2 Ab (Cell SignalingTechnology) or anti-Ets-1 Ab.

MAPK assays

In vitro MAPK assay was performed using a MAPK assay kit (UpstateBiotechnology) according to the manufacturer’s recommendations.

Immunofluorescence microscopy and histopathology

Immunofluorescence staining was performed on 6-�m-thick cryosectionsof bEnd.3 cell-induced hemangiomas. The sections were preincubated inblocking solution (PBS containing 2% BSA and 0.5% Tween 20) for 1 hand then incubated with goat anti-mouse CCL2 Ab (R&D Systems), ratanti-mouse CD31 Ab (BD Biosciences), or rat anti-mouse F4/80 Ab (Se-rotec). After overnight incubation at 4°C, the sections were washed andincubated with secondary Ab conjugated to FITC or Texas Red for 1 h atroom temperature. Sections were then analyzed by confocal microscopy(Zeiss; LSM 510). Some sections from each hemangioma were staineddirectly with H&E for standard histological examination.

RT-PCR

Total RNA was isolated from bEnd.3 cells or hemangioma using theTRIzol reagent (Invitrogen Life Technologies). Briefly, 2 �g from eachsample was reverse-transcribed into cDNA using an Invitrogen Life Tech-nologies cDNA Synthesis Kit, following the manufacturer’s protocol. Ets-1cDNA was amplified as a 510-bp fragment by PCR with the forwardprimer 5� -TACCCTTCCGTCATTCTCC-3� and reverse primer 5�- TTTTTCCTCTTTCCCCATC-3�. �3-integrin cDNA was amplified as 430 bpwith the forward primer 5�-GGGGACTGCCTGTGTGACTC-3� and re-verse primer 5�-CTTTTCGGTCGTGGATGGTG-3. Samples were stan-dardized using primers specific to cDNA encoding mouse �-actin. A totalof 40 cycles for Ets-1 and 30 cycles for �3 integrin were applied. The PCRcycles included 1-min denaturation at 94°C, 1-min annealing at 55°C, and

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1-min extension at 72°C, except for the first cycle, which had 2-min de-naturation, and the last cycle with 10-min elongation. The PCR productswere resolved using electrophoresis on a 2% agarose gel in 1� TBE buffer(Tris-HCl/EDTA/boric acid; pH 8). The gel was stained with ethidiumbromide and photographed.

ELISA

Supernatants collected from in vitro angiogenic assays in fibrin gel wereassayed for VEGF level by ELISA, using the mouse VEGF Quantikine kit(R&D Systems).

Statistics

Statistical analyses were performed using commercially available software(Stat-View; SAS Institute). One-way ANOVA was used to compare themean responses among the experimental groups. The Dunnett’s test wasused to determine significance between groups.

ResultsCCL2 as potent angiogenic factor

To examine the potential angiogenic effect of CCL2, the expres-sion and effect of CCL2 on hemangioma development in vivo wasexamined. Hemangiomas offer a unique model to study angiogen-esis. They are a primary tumor of microvasculature in which an-giogenesis is initially excessive followed by inhibition and regres-sion of the newly formed blood vessels. Injection of 5 � 106

Polyoma middle T-transformed endothelial cells (bEnd.3 cells) s.c.in the right femoral flank of nu/nu mice was sufficient to inducehemangioma formation over 21 days. As shown in Fig. 1A, thehemangiomas were cystic vascular structures with high levels ofCCL2 in infiltrated macrophages (F4/80� cells) and endothelialcells (CD31� cells). To establish that CCL2 plays a significant rolein hemangioma formation, on days 1, 6, 9, 12, and 21 after bEnd.3injection mice received neutralizing anti-CCL2 Ab or CCL2 anti-sense phosphorothioate oligonucleotide. Inhibition of CCL2 activ-ity/synthesis markedly reduced hemangioma size (Fig. 1B). Thus,CCL2 can exert a potent effect on hemangioma formation andangiogenesis.

The effect of CCL2 on angiogenesis was further examined invitro using a fibrin gel angiogenesis assay and bEnd.3 cells. CCL2significantly enhanced the formation of tube-like structures com-pared with controls (vehicle-treated cells; Fig. 2). The angiogenicresponse of bEnd 3 cells to CCL2 was dose dependent. After 24-hstimulation with CCL2, the greatest increase in tube-like formationwas observed at the concentration of 100 ng/ml. This increase in

tube formation was comparable to that observed after stimulationwith VEGF, a known potent angiogenic factor (Fig. 2).

If CCL2 acts as a direct angiogenic factor, then it is expectedthat endothelial cells possess CCR2, the receptor for CCL2. Asalready stated, CCL2 exerts its biological effects on endothelialcells through CCR2 (19–21). Fig. 3A shows that there is a low,constitutive expression of the CCL2 and CCR2 transcripts inbEnd.3 cells. However, CCL2 treatment of bEnd.3 cells for 4 hinduced a significant increase in CCR2 expression. This increase inCCR2 expression was comparable to that observed after stimula-tion of bEnd 3 cells with TNF-� (10 ng/ml).

Analyzing the hemangioma tissue, we also found a significantamount of CCL2 and CCR2 mRNA expression, the level of whichwas diminished by applying CCL2 antisense phosphorothioate oli-gonucleotide (Fig. 3A).

Conditioned media were collected from bEnd 3 cells seeded onthe fibrin gel after 0–24 h of treatment with CCL2 and analyzedfor VEGF by ELISA. We found that the amount of VEGF was notsignificantly different between control (nontreated bEnd3 cells)and CCL-2-treated samples (data not shown).

CCL2 also induced proliferation of bEnd.3 cell in a concentra-tion-dependent manner (Fig. 3B). Quantification of incorporatedBrdU in bEnd.3 cells indicated peak proliferation at CCL2 con-centration of 50 ng/ml ( p � 0.01 vs control). This proliferativeresponse was comparable to that observed with VEGF (50 ng/ml).This strongly suggests that CCL2 has a potent mitogenic effect onbEnd.3 cells. The proliferative effect of CCL2 was blocked in thepresence of CCL2-neutralizing Ab, and it was also CCR2 depen-dent. In experiments where brain endothelial cells were preparedfrom mice genotype CCR2�/�, CCL2 was not able to induce aproliferative response (Fig. 3B).

Stimulation of endothelial cell motility is a common feature ofangiogenic factors. As shown in Fig. 3C, CCL2 also displays che-motactic activity for endothelial cells. CCL2 induced directionalbEnd.3 cell migration (chemotaxis) in a concentration-dependentmanner (0.78–400 ng/ml). Maximal migration was obtained with100 ng/ml CCL2. CCL2-induced bEnd.3 cell chemotaxis was sim-ilar to that found with VEGF (Fig. 3C). Inhibiting CCL2 with ananti-CCL2-neutralizing Ab completely blocked CCL2-enhancedcell migration. In addition, brain endothelial cell migration towardthe CCL2 was CCR2 dependent. In the range of concentrationstested (0.78–400 ng/ml), CCL2 did not elicit migration of brain

FIGURE 1. CCL2 contributes to angiogenesis invivo and induces angiogenesis in vitro. A, Hemangio-mas formed after inoculation of bEnd.3 cells into theright flank of a nu/nu mouse (see Material and Methodsfor details) were stained with H&E or used for fluores-cence immunohistochemistry using anti-CCL2, anti-F4/80 and anti-CD31 Abs. CCL2 was highly expressedin cystic walls of hemangioma, mostly in F4/80 positivemacrophages and CD31 positive endothelial cells. Scalebars, 10 �m. B, Effect of inhibiting CCL2 activity usingneutralizing Ab to CCL2 (25 �g/ml, i.p.) or CCL2 an-tisense phosphorothioate oligonucleotide (5 �M) onhemangioma growth. Photographs show hemangiomasat day 21. The graph shows the time course of heman-gioma weight. Values are mean � SD; n � 6; �, p �0.01; ��, p � 0.001 vs nontreated bEnd.3 cells.

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endothelial cells prepared from CCR2�/� mice. Thus, the chemo-tactic response of brain endothelial cells to CCL2 stimulation islinked to the expression of CCR2 in these cells.

Taken together, these results indicate that CCL2 may act as apotent direct angiogenic factor. In subsequent experiments to in-vestigate the mechanisms underlying such angiogenic activity, 100ng/ml CCL2 was used because this induced the peak angiogenicresponse.

Mechanisms of CCL2-induced angiogenesis

To identify potential mechanisms of CCL2-induced angiogenesis,a series of gene array experiments were performed to examinetranscript abundance in endothelial cells exposed to CCL2. TableI shows changes in angiogenic factor RNA expression after treat-ment with CCL2 for 4 and 16 h. Values in the columns for everylisted gene represent expression as a percentage of an internal con-trol (�-actin expression) present on every membrane. In addition,the expression of certain genes in cells treated with CCL2 for 4 and16 h is compared with control (untreated) cells in the column la-beled percentage of fold increase. Only genes where expressionwas increased 1.5-fold or greater were taken as up-regulated genes.From the table, it is evident that CCL2 induced expression of thefollowing: 1) transcription factors closely associated with angio-genesis (Ets-1, SMAD); 2) growth factors and their receptors(TGF�, TGF�, ephrin B2, ephrin B4, as well as receptors for theVEGF-KDR, neuropilin, angiopoietin Tie1, Tie2, TGF�R1,TGF�R2; and 3) adhesion molecules, particularly integrins sub-units �5, �v, and �3 (CD61), then PECAM-1 and VCAM-1; and 4)matrix proteins, proteases, and protease inhibitors (collagen 18a1and fibronectin, Adamts1, urokinase, MMP9, thrombospondin 1,plasminogen inhibitor 1) and other factors (NOS3, cox1, andcox2).

To elucidate possible mechanisms that might be involved inCCL2-induced angiogenesis, we correlated up-regulated geneswith specific transcription factor expression. The results showedthat the Ets-1 transcription factor was highly expressed after CCL2treatment as were some of the genes that are Ets-1 dependent, suchas integrins �3 subunits (CD61), �v subunits, urokinase (uPA),MMP9, the VEGF receptor KDR, and the angiopoietin receptors

Tie1, Tie2. Due to this, further studies focused on the activity ofEts-1 during CCL2-induced angiogenesis.

CCL2-induced angiogenesis through activation of Ets-1transcription factor

To begin to investigate whether Ets-1 plays a critical role in CCL2-induced angiogenesis, Ets-1 mRNA and protein expression as wellas Ets-1 activation (phosphorylation) and Ets-1 DNA-binding ac-tivity were analyzed during treatment of bEnd.3 cells with CCL2.Cells were exposed to 100 ng/ml CCL2 for the varying time pe-riods (1–16 h). CCL2 induced significant up-regulation of Ets-1mRNA within 2 h. This up-regulation peaked at 4 h and persistedup to 16 h (Fig. 4A). Up-regulation of Ets-1 mRNA was followedby increased Ets-1 protein expression (peak at 8 h; Fig. 4A). Be-sides up-regulation of Ets-1 mRNA and proteins, CCL2 also in-duced brief phosphorylation Ets-1 (on the threonine 38 residue)observed here in time period 0–4 h (Fig. 4B). This change inphosphorylation status is considered as a trigger for transfer ofEts-1 to nucleus and binding to specific DNA sequences (24).Therefore, we analyzed nuclear extracts for Ets-1-binding activity.As shown Fig. 4C, treatment of bEnd.3 cells with CCL2 inducedan increased Ets-1-binding activity over time period 0–4 h. Spec-ificity of the Ets-1-binding activity was tested by 1) competitionwith homologous DNA and 2) by supershift assay, and thosestudies verified that CCL2 induced specific Ets-1-binding activity(Fig. 4C).

To confirm that Ets-1 transcription factor plays a critical role inCCL2-induced angiogenesis, bEnd.3 cells were transfected withEts-1 sense, mismatched, or antisense phosphorothioate oligonu-cleotides. To determine transfection efficiencies, phosphorothioateoligonucleotides were end-labeled with FITC and transfected intobEnd.3 cells. Homogenous staining of the nuclei with pronouncedaccumulation in the nucleoli was noted after 6 h in 50% of bEnd.3cells, and this was used as the transfection time (data not shown).In addition, to test whether antisense oligonucleotide effectivelyblocked the DNA-binding activity of Ets-1, gel mobility shift as-says were performed, which showed that binding activity of Ets-1was reduced (50%) in bEnd.3 cells transfected with antisenseoligonucleotides (data not shown).

FIGURE 2. Effect of CCL2 on tube formation using an in vitro angiogenesis assay in fibrin gels. Media were supplemented with different CCL2concentrations or VEGF or no growth factor (control). Tube formation was observed after 24 h and total tubular length was calculated (�m). The graphshows average tubular length from five independent experiments, with each well being measured in five randomly selected areas. Values are mean � SD;��, p � 0.001 vs control. Scale bar, 100 �m

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Reducing Ets-1 activity with antisense oligonucleotide signifi-cantly blocked CCL2-induced angiogenesis in the fibrin gel assaycompared with cells where Ets-1 was not blocked or bEnd.3 cellstransfected with sense Ets-1 oligonucleotide (Fig. 4C). These setsof experiments indicate that Ets-1 could be a critical transcriptionfactor for CCL2-induced angiogenesis.

CCL2-regulated Ets-1 target molecule �3 integrin

Ets-1 transcription factors are denoted as important angiogenicswitch molecules that change quiescent endothelial cells to an an-giogenic phenotype (24–26). One of the factors regulated by Ets-1is �3 integrin, a critical factor in the process of angiogenesis. Ourinitial gene array analysis showed an up-regulation of this integrinin bEnd.3 cells after CCL2 treatment (Table I). Further analysiswith PCR and Western blot demonstrated that CCL2 induced anincrease in �3-integrin mRNA and protein expression between 4and 16 h (Fig. 5, A and B). This expression was regulated at thetranscription and translation levels. Inhibition of transcription byactinomycin B or inhibition of translation by cyclohexamide sig-nificantly decreased �3-integrin mRNA and protein expression, re-spectively (data not shown).

Blocking Ets-1 activity with Ets-1 antisense oligonucleotide sig-nificantly reduced integrin �3 mRNA and protein expression afterCCL2 treatment. In contrast, transfection of bEnd.3 cells withsense Ets-1 oligonucleotide did not diminish �3 mRNA and pro-tein expression. Thus, increased expression of �3 integrin withCCL2 treatment results from Ets-1 activation, and CCL2 probablyexerts its angiogenic activity through �3-integrin function.

CCL2 activates Ets-1 transcription factors through ERK1/2activation

Phosphorylation of threonine 38 residues on Ets-1 can play a rolein mediating its transcriptional activity in response to different fac-tors (26–28). To investigate the role of phosphorylation in CCL2-induced Ets-1 activation, we examined whether CCL2 induces ac-tivation of ERK1/2 in bEnd.3 cells. Then, through the inactivationof ERK1/2 activity, the contribution of ERK1/2 in CCL2-inducedEts-1 activation was evaluated. As shown in Fig. 6A, CCL2 treat-ment of bEnd.3 cells induced activation of ERK1/2, with the peakactivity after 10 min based on an in vitro kinase assay and byWestern blot analysis for phosphorylated ERK1/2. Inhibition of

FIGURE 3. A, Expression of CCL2 and CCR2 mRNA in control bEnd.3 cells and hemangioma tissue with and without CCL2 antisense or sensephosphorothioate oligonucleotide treatment. bEnd.3 cells possess mRNA for CCL2 and its receptor, CCR2, under resting conditions. Increased expressionof CCR2 was found in bEnd.3 cells during a treatment with CCL2 (100 ng/ml) or in the presence of TNF-� (10 ng/ml). High expression of CCL2 and CCR2mRNA was found in hemangiomas and this was reduced by treatment with CCL2 antisense but not CCL2 sense treatment. Values from semiquantitativedensitometric analysis represent means � SD from five independent experiments. B, CCL2 exerted a mitogenic effect on bEnd.3 cells in vitro. Cells werestimulated with CCL2 or VEGF at the indicated concentrations and allowed to proliferate for 16 h. BrdU incorporation was measured for the last 4 h. Totest specificity of the CCL2 effect, CCL2-induced proliferation was also examined in the presence of a neutralizing CCL2 Ab (CCL2Ab) or a specific isotypeAb (goat IgG). The receptor dependence of CCL2-induced proliferation was examined by performing the proliferation assay on brain endothelial cellsprepared from mice phenotype CCR2�/�. Data represent mean � SD from five independent experiments. �, p 0.05; ��, p � 0.001. C, CCL2 increasesbEnd.3 cell migration through Transwell filters. Calcein-AM-labeled bEnd.3 cells were layered on the top of Transwell filters. A chemotactic gradient wasestablished by adding varying concentrations of CCL2 or VEGF (25 ng/ml) to the lower chamber and cell migration measured after 22 h. In controls, nogrowth factor was added. To test specificity, CCL2-induced migration was measured in the presence of a neutralizing CCL2 Ab (CCL2Ab) or a specificisotype Ab (goat IgG). The receptor dependence of CCL2-induced migration was examined by performing the assay on brain endothelial cells preparedfrom mice phenotype CCR2�/�. Values represent means � SD from five independent experiments. ��, p � 0.001 vs nontreated controls.

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Table I. Angiogenic factor RNA expression

Genebank Gene Name Control 4 hrx-Fold

Increase 16 hrx-Fold

Increase

NM_009621 Adamts1 1.02 97.13 (95)1 83.6 (82)1NM_013906 Adamts8 1.38U83509 Angiopoietin-1 2.88 0.198 (14)2NM_007426 Angiopoietin2 0.565U22516 AngiogeninNM_007643 CD36 0.93 11.98 (13)1NM_009868 Cadherin 5 84.78 100 1.2 83.6 (1)NM_007693 Vasostatin/c Hromogranin A 10.3 92.62 (9)1 48.49 (4.7)1NM_009929 Procollagen, Type XVIII, �1 2.28 0.4 (6)2 44 (19)1M13926 G-CSF 14.46 0.94 (15)2NM_010217 Tissue factor 99.43 100 (1) 80.95 (0.8)NM_007901 Edg1 14.38 88.27 (6.1)1 83.63 (6)1NM_007909 Ephrin A2 0.063 -NM_010109 Ephrin A receptor 0.37 7.88 (21)1 3.5 (9.4)1NM_010111 Ephrin B2 92.91 100 (1) 83.5 (0.9)NM_010113 EGF 15.45 (15.4)1NM_007912 EGFR 0.029NM_007932 Endoglin 36.27 99.17 (2.7)1 83.5 (2.3)1NM_010144 Ephrin B4 9.27 57.41 (6.2)1 80.9 (9)1U71126 crb-2 1.76 0.21 (8.4)1 35.92 (20.5)1NM_011808 c-cts1 39.68 99.82 (2.5)1 83.63 (2)1NM_010168 Prothrombin kringle-1U67610 aFGF 1.4 (1.4)NM_030614 FGF16M30644 bFGFM30642 FGF4 5.57 1.11 (5)2 (5.5)2M92416 FGF6 3.78 (3.8)2 (3.8)2U58503 FGF7/KGF 8.11 16.93 (2.1)1 (8)2M33760 FGFR1 (FLG) 100 100 (1) 83.63 (0.8)M81342 FGFR3 33.15 4.42 (7.5)2 10.84 (3)2NM_008011 FGFR4 3.83 (3.8)1 5.12 (5.1)1D89628 VEGF-D/FIGFX59397 KDR 4.99 89.87 (18)1 83.6 (17)1L07297 VEGFR 100 100 (1) 83.63 (0.8)M18194 Fn1 23.84 14.71 (1.6)2 79.3 (3.3)1J04596 Gro1 0.28 0.015 (0.05)X84046 HGF 2.11 7.52 (3.6)1 3.63 (1.7)1NM_010431 Hif1a 88.92 99.84 (1.1) 83.63 (1)M31885 ID1 0.3 (0.3) 17.2 (17)1NM_008321 ID3NM_010502 IFN�1 - 19.72 (19.7)1NM_010510 IFN-b1 1.01 2.31 (2.3)1K00083 IFNr 3.37 0.79 (4.3)1NM_010512 IGF-1 0.604 (0.6) 83.6 (84)1NM_010548 IL-10NM_016780 CD61 41.08 (41)1 83.6 (84)1NM_008539 Madh1 3.37 (3.4)1 83.63 (84)1NM_010784 MidkineNM_008610 Gelatinase ANM_013599 Gelatinase B 0.17 (0.2) 8.89 (9)1NM_031195 SR-A 12.46 (12.5)1NM_008713 NOS3 14.77 86.69 (5.9)1 83 (5.7)1NM_008737 Neuropilin 35.59 (35.6)1 81.1 (81)1M29464 PDGF � 0.028 (0.03)AF162784 PDGF� 0.65 (0.65)NM_011058 PDGFR�NM_008809 PDGFR� 15.37 (15.4)1NM_008816 PECAM1 16.82 100 (6)1 83.63 (4.9)1AB017491 PF4 4.12 (4)1 1.4NM_008827 Placental growth factor 77.26 (77)1 83.63 (84)1X02389 PLAU 96.84 (97)1 83.63 (84)1NM_008969 PTGS1 7.75 (7.8)1 70 (70)1NM_011198 Cox-2 0.477 4.13 (8.7)1 25.5 (53.5)1D90225 Pleiotrophin 0.28 (0.3)NM_019765 Restin 0.06 100 (100)1 76.7 (77)1NM_011333 Scya2 1.15 1.15 39.34 (40)1NM_009257 MaspinM33960 PAI-1 8.07 29.95 (4)1 42.12 (5)1X16490 PAI-2AF017057 PEDF 1.21 1.2 47.05 (47)1NM_009242 SPARC 76.32 100 1.3 83.63 (1.1)1NM_009263 Osteopontin 1.86 23.64 (12.7)1 50.5 (27.2)1

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ERK1/2 activity with PD98059 significantly reduced DNA-bind-ing activity of Ets-1 in bEnd.3 cells stimulated by CCL2 as well asphosphorylation of Ets-1 (Fig. 6B). However, PD98059 also re-duced CCL2-induced up-regulation of Ets-1 mRNA and protein,indicating that ERK1/2 may modulate activation of Ets-1 by mod-ifying Ets-1 turnover as well as through phosphorylation.

DiscussionCCL2 has been considered as a major factor in facilitating angio-genesis through its recruitment of macrophages to sites of woundinjury or peritumor areas and the subsequent release of angiogenicfactors by those macrophages (15–18, 29). However, some recentstudies have indicated that CCL2 might also have direct effects onangiogenesis (17, 30). The current study supports this latter con-cept with evidence that CCL2 promotes some steps of the angio-genic process (endothelial cell proliferation, migration, and tubuleformation). This study also helps to elucidate the molecular mech-anisms underlying CCL2-induced angiogenesis. In particular, itindicates the following: 1) Ets-1 transcription factor is a criticalfactor of CCL2-induced angiogenesis; 2) via activation of Ets-1,CCL2 regulates expression of other crucial angiogenic factors,such as integrin �3 subunits; and 3) CCL2 regulates Ets-1 activa-tion through MAPK (ERK1/2).

Before discussing these results and their implications further, itis necessary to clarify the use of polyoma middle T-transformedbrain endothelial cells (bEnd.3 cells) in our study and the impor-tance of the angiogenic effect of CCL2 on these cells. Our resultsindicate that End.3 cells are a good source for developing heman-giomas. Hemangiomas represent a powerful model to study in vivoangiogenesis for the several reasons: the angiogenic process is ex-tremely potent; the rapid proliferation hemangioma is associatedwith macrophage infiltration, indicating an important role for themicroenvironment in angiogenesis; and hemangiomas are com-monly encountered in humans, providing important clinical rele-vance. Inoculation with polyoma middle T-transformed endothe-lial cells is also considered as a good autocrine model ofangiogenesis and hemangioma (31, 32). We felt that using bEnd.3cells to evaluate CCL2-induced angiogenesis manner would haveseveral advantages. 1) Due to the fact that the hemangiomas highlyexpress CCL2, investigating the angiogenic effects of CCL2 would

give us additional information about hemangioma evolution. 2)Phenotypically, bEnd.3 cells are very similar to brain endothelialcells. 3) The cells can be used to study the process of angiogenesisin vitro. 4) The cells can be pharmacologically and geneticallymanipulated in vitro for later injection, providing a unique modelwith which to study the influence of tumor cell-derived signals thatregulate angiogenesis.

CCL2 is known to participate in angiogenic events under manyconditions (15–18). For example, monocyte recruitment by CCL2is a critical event in the neovascularization that occurs in chronicinflammatory conditions such as rheumatoid arthritis, psoriasis,atherosclerosis, and different types of tumors (29–31, 33–38). De-spite an established correlation between CCL2 levels, infiltratingmacrophages, and angiogenesis, several models of angiogenesisindicate that CCL2 can promote an angiogenic phenotype in en-dothelial cells by a direct endothelial effect as well as indirectly viarecruiting macrophages. The possibility of such a direct effect issupported by the fact that endothelial cells are known to expressCCR2, the sole receptor for CCL2 (19–21). Although our RT-PCRanalysis showed that bEnd.3 cells express low levels of CCR2mRNA under resting conditions, the fact that CCL2 in vitro reg-ulated bEnd.3 cell migration in a dose-dependent manner as wellthe fact that adding anti-neutralizing CCL2 Ab diminishes CCL2-induced chemotaxis of bEnd.3 cells, support findings about pres-ence of functional active CCR2. We believe that low level of ex-pression of CCR2 mRNA on bEnd.3 cells under resting conditionsmight be the result of specific in vitro conditions that have alreadybeen shown to be critical for down-regulation of CCR2 in mono-cytes/macrophages (39).

The molecular mechanisms underlying chemokine-induced an-giogenesis have not been extensively investigated. Only a fewstudies have indicated that chemokines (particularly CXCL8) canswitch endothelial cells to an angiogenic phenotype, regulatingseveral angiogenic factors such as MMP1 and MMP9 (40, 41). Ourgene array analysis, for the first time, offers a detailed analysis ofthe angiogenic factors regulated by CCL2, offering insight intohow this chemokine can directly regulate angiogenesis. Our resultsindicate that Ets-1 transcription factor is a critical factor in thatprocess.

Table I. Continued

Genebank Gene Name Control 4 hrx-Fold

Increase 16 hrx-Fold

Increase

D13738 Tic-2 0.9 2.83 (3.14)1 48.64 (54)1U65016 TGF-�M13177 TGF�1 34.49 (34.5)1 82.3 (82.3)1X57413 TGF �2 11.21 (11)1 7.31 (7.3)1M32745 TGF�3 1.46 (1.5)1 15.39 (15.4)1D28526 TGF�R1(ALK-5) 6.09 (6.1)1 83.18 (83.2)1NM_009371 TGF�R2 0.198 86.99 (439)1 83.63 (422)1AF039601 [beta] glycan 4.12 2.96 (1.5)2 78.94 (19)1M87276 THBS1 1.68 10.84 (6.4)1 16.78 (10)1L07803 THBS2L24434 TIMP2AF102887 THBS3X73960 Tie1 24.86 (25)1 78.99 (79)1NM_011593 TIMP1 1.06 (1) 50.96 (51)1Mm181969 TIMP2 9.66 79.96 (8.3)1 83.63 (9)1NM_011607 Tenascin C 1.63 0.969 (0.6) 66.94 (41)1NM_013693 TNF � 0.866M84487 VCAM-1 0.09 4.99 (55)1 8.49 (94)1M95200 VEGF 0.6 0.6U48800 VEGF-B 0.67 (0.67) 0.7 (0.7)U73620 VEGF-C 0.05 (0.05)

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The proto-oncogene c-Ets-1 encodes the prototypic member of anovel family of transcription factors, the Ets proteins. Ets transcriptionfactors bind via an 80-aa C-terminal domain to a GGA (A/T) con-sensus sequence called the Ets binding site or PEA3 element (24, 27),and it is presented in the promoters of many genes involved in cellularproliferation, differentiation, development, hematopoiesis, apoptosis,metastasis, tissue remodeling, and angiogenesis (24, 27). For exam-ple, there are Ets binding sites in the genes for collagenase-1 (MMP-1), stromelysin-1 (MMP-3), MMP9, uPA, VEGFR1 (Flt1), VEGFR2(KDR), and integrin chain expression (�3, �v, and �4) that controltheir transcription (25, 26, 42–44). In vivo, Ets-1 expression has beenassociated with new blood vessel formation under both physio-logical and pathophysiological conditions, such as chronic in-flammatory reactions and tumor-associated angiogenesis (28,45, 46). Several angiogenic factors like VEGF, angiotensin II,TGF�, and acidic fibroblast growth factor use Ets-1 to regulateangiogenesis (46 – 49). Our study indicates that CCL2 directlyregulates angiogenesis through activation of Ets-1 transcriptionfactor and up-regulation of Ets-1-regulated angiogenic mole-

cules. In this manner, CCL2 regulated processes associated withbrain endothelial cells proliferation, migration, and tubular for-mation (tubule formation was reduced 95% after treatment withEts-1 antisense oligonucleotide). These data support a physio-logical role for Ets-1 in initiation and or/propagation of sprout-ing and capillary formation.

Among the Ets-1-dependent genes activated during CCL2-in-duced angiogenesis, �3 subunits integrins (CD61) may have animportant role (25, 26, 44). Our study clearly shows that CCL2,through Ets-1 activation, regulated �3 expression (mRNA and pro-tein) on bEnd.3 cells. These results strongly suggest that CCL2may contribute to angiogenesis by inducing synthesis and expres-sion of �3 integrin in endothelial cells. We cannot, however, ex-clude the possibility that CCL2, like VEGF, might also act syn-ergistically with �3 integrin in regulating the complex process ofangiogenesis and that this might further enhance the effects ofCCL2 on microvascular formation (50).

CCL2 may regulate Ets-1 activation at several levels. Phosphor-ylation of Ets-1 on the threonine 38 positions by ERK1/2 strongly

FIGURE 4. CCL2-induced activation of Ets-1 transcription factor. A, CCL2 induced increased expression of Ets-1 mRNA and protein in bEnd.3 cells.bEnd.3 cells were treated with murine recombinant CCL2 (100 ng/ml) for 0–16 h. Representative examples of PCR and Western blot for CCL2 are shown.Levels of Ets-1 mRNA and protein were expressed as a ratio to those of a housekeeping gene, �-actin. B, CCL2 also induced phosphorylation of Ets-1 onthe threonine 38 position evaluated here by Western blot analysis. C, Ets-1 DNA binding activity was evaluated by EMSA or Supershift assay. Nuclearextracts were prepared from control bEnd.3 cells or cells treated with CCL2 (100 ng/ml) in the presence or absence of homologue DNA (competitor) wereseparated on 6% gels. In the Supershift assay, polyclonal anti-Ets-1 Ab (2 �g/ml) was added. D, Reducing Ets-1 activity using phosphorothioated antisenseEts-1oligonucleotide significantly diminished CCL2-induced tube formation in bEnd.3 cells. Sense Ets-1 phosphorothioated oligonucleotide sequence hadno effect. Scale bar, 100 �m. Values represent means � SD from three independent experiments. ��, p � 0.001 vs CCL2-treated cells.

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increases Ets-1 DNA-binding activity (51, 52). Thus, hepatocytegrowth factor/scatter factor induces scattering and morphogenesisof epithelial cells through phosphorylation and activation of Ets-1as the result of MAPK pathway activation (53, 54). In addition, astudy by Watanabe et al. (47) on retinal endothelial cells showedthat VEGF and hypoxia-induced Ets-1 mRNA expression and thatthis was diminished after MEK/ERK1/2 blockade. That study in-dicated two very important points: 1) MEK/ERK1/2 induced phos-phorylation of Ets-1 and enhanced its DNA-binding activity tocertain genes; and 2) phosphorylated Ets-1 could bind to its ownpromoter and regulate its own presence and activity in cells via thisfeedback mechanism (47). These increases in Ets-1 mRNA levelwere considered to be associated with the transformation of brainendothelial cells into the angiogenic phenotype. Our results concurwith these findings. CCL2 induced increased binding activity ofEts-1 through activation of ERK1/2 pathway, and inhibition of thissignal (through the specific inhibitor PD 98059) diminished Ets-1activity and expression of �3 integrin. At the same time, CCL2also induced an increase in Ets-1 mRNA and protein levels, andthis event was also ERK1/2 dependent. ERK1/2 activation is crit-ical for the induction of an angiogenic phenotype in bEnd.3 cellsby CCL2.

Although our study has focused on CCL2 regulation of angio-genesis via Ets-1, a recent study by Zhan et al. (46) found thatEts-1 could regulate CCL2 expression in smooth muscle cells dur-ing inflammatory vascular remodeling. If this is also true in brainendothelial cells, CCL2 up-regulation may result in Ets-1 activa-tion and further CCL2 up-regulation in an autocrine fashion. Thispossibility requires further investigation.

What is the significance of the findings in the current study? In

our opinion, this study offers new insight into the mechanismsunderlying angiogenesis and pathological disorders associatedwith abnormal angiogenesis. It also opens new possibilities forantiangiogenic therapy. In terms of mechanism, this study providesfor the first time a piece of evidence on how chemokines (CCL2,in particular) can directly regulate angiogenesis and expression ofangiogenic factors. Considering that some other chemokines (e.g.,CXCL8) also regulate Ets-1-dependent molecules (MMP2 andMMP9) via interaction with their own endothelial receptors, wewould like to underscore the fact that the described pattern ofmodulation of angiogenesis by CCL2 may also apply to other che-mokines with angiogenic properties (e.g., CXCL8 or CXCL12)and may be potentially unique for chemokine action. In addition,we would also like to highlight that, in our opinion, CCL2 is amodulator of angiogenic processes, enhancing this process eitherthrough direct action on endothelial cells or by modulating theactivity of other essential angiogenic factors (e.g., VEGF). CCL2obviously has a complex and bidirectional relationship withVEGF, and their activity should be considered as a synchronizedcooperative activity of two cytokines/growth angiogenic factors. Interms of therapy, understanding the mechanism of action of dif-ferent angiogenic factors may enable multiple approaches (andcombination approaches) to block angiogenesis. The manipulationof chemokine function may have merit as a new therapeutic ap-proach. Although further experimental validation is needed, thesefindings open up a new direction for future development of ther-apeutic strategies in treating angiogenesis and angiogenic-relateddisorders such as hemangioma.

DisclosuresThe authors have no financial conflict of interest.

FIGURE 5. Effect of CCL2 on �3 integrin mRNAand protein expression in bEnd.3 cells. CCL2 inducedsignificant up-regulation of �3 integrin mRNA (A; PCR)and protein expression (B; Western blot) in bEnd.3 cellsover 4 to 16 h. Values are expressed relative to thehousekeeping gene, �-actin, and represent means � SDfrom three independent experiments. ��, p � 0.001 vscontrol. Blocking Ets-1 activity using phosphorothio-ated antisense Ets-1 oligonucleotide significantly dimin-ished Ets-1 mRNA (C; PCR) and protein expression (D;Western blot) during treatment with CCL2. As a control,bEnd.3 cells were transfected with phosphorothioatedEts-1 sense oligonucleotide. Values represent means �SD from three independent experiments; ��, p � 0.001vs control.

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FIGURE 6. Participation of ERK1/2 in Ets-1 activation by CCL2. A,ERK1/2 phosphorylation (Western blot) and activity (MAPK assay) wereexamined in bEnd.3 cells treated with CCL2 (100 ng/ml) for 1 h. Robustphosphorylation and ERK1/2 activation was found by 10 min and persistedfor 30 min. Values represent means � SD from three independent exper-iments. ��, p � 0.001 vs control (nontreated) cells. B, Inhibition of ERK1/2activation (using inhibitor PD98059) greatly diminished CCL2-inducedEts-1 binding activity as evaluated by EMSA. Treatment with Ets-1 anti-sense oligonucleotide also blocked Ets-1 binding activity. C, As well asdiminishing Ets-1 binding activity, PD98059 also significantly reducedCCL2-induced Ets-1 mRNA, protein expression in bEnd.3 cells and phos-phorylation of Ets-1 threonine 38 residues. Values represent means � SDfrom three independent experiments and are expressed as a ratio to thehousekeeping gene, �-actin. ��, p � 0.001 vs CCL2-treated cells.

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