The Involvement of CD146 and Its Novel Ligand Galectin-1 inApoptotic Regulation of Endothelial Cells*

Received for publication, September 11, 2012, and in revised form, December 4, 2012 Published, JBC Papers in Press, December 7, 2012, DOI 10.1074/jbc.M112.418848

Nathalie Jouve‡1,2, Nicolas Despoix‡1, Marion Espeli§, Laurent Gauthier¶, Sophie Cypowyj‡, Karim Fallague‡,Claudine Schiff§�**, Françoise Dignat-George‡ ‡‡, Frédéric Vély§�**‡‡3, and Aurélie S. Leroyer‡3,4

From the ‡Aix-Marseille Université, INSERM, UMR-S 1076, 13385 Marseille, France, the §Centre d’Immunologie de Marseille-Luminy,Aix-Marseille Université, UM2, 13288 Marseille, France, the ¶INNATE-PHARMA, 13288 Marseille, France, the �INSERM, U 1104, 13288Marseille, France, the **CNRS, UMR 7280, 13288 Marseille, France, and the ‡‡Assistance Publique – Hôpitaux de Marseille, Hôpitalde la Conception, 13385 Marseille, France

Background: CD146 is a glycosylated adhesion molecule involved in the control of vessel integrity.Results: Galectin-1 directly binds to CD146 and this interaction is involved in the control of endothelial cell apoptosis.Conclusion: Galectin-1 is identified as a novel ligand for CD146.Significance: CD146 acts as a decoy-receptor to down-regulate Galectin-1-mediated apoptosis.

CD146 is a highly glycosylated junctional adhesion molecule,expressed on human vascular endothelial cells and involved inthe control of vessel integrity. Galectin-1 is a lectin produced byvascular cells that can bindsN- andO-linked oligosaccharides ofcell membrane glycoproteins. Because both CD146 and Galec-tin-1 are involved in modulation of cell apoptosis, we hypothe-sized that Galectin-1 could interact with CD146, leading tofunctional consequences in endothelial cell apoptosis. We firstcharacterizedCD146glycosylations and showed that it ismainlycomposed of N-glycans able to establish interactions withGalectin-1. We demonstrated a sugar-dependent binding ofrecombinant CD146 to Galectin-1 using both ELISA and Bia-core assays. This interaction is direct, with aKD of 3.10�7 M, andspecific as CD146 binds to Galectin-1 and not to Galectin-2.Moreover, co-immunoprecipitation experiments showed thatGalectin-1 interacts with endogenous CD146 that is highlyexpressed by HUVEC. We observed a Galectin-1-inducedHUVEC apoptosis in a dose-dependent manner as demon-stratedbyAnnexin-V/7AADstaining. Interestingly, bothdown-regulation of CD146 cell surface expression using siRNA andantibody-mediated blockade of CD146 increase this apoptosis.Altogether, our results identify Galectin-1 as a novel ligand forCD146 and this interaction protects, in vitro, endothelial cellsagainst apoptosis induced by Galectin-1.

CD146, also known as S-Endo1, Melanoma Cell AdhesionMolecule (MCAM5 or Mel-CAM), MUC18, A32 antigen, is an

integral membrane glycoprotein of 113 kDa that belongs to theimmunoglobulin superfamily with a characteristic V-V-C2-C2-C2 domain structure (1). Using the anti-human CD146monoclonal antibody (S-Endo1) developed in our laboratory,we have previously demonstrated that CD146 is expressed onhuman vascular endothelial cells, whatever vessel caliber oranatomical region (2). CD146 is also overexpressed on sometumoral cells, activated T-cell subpopulation and pericytes(3–9). It has been previously shown that in cultured endothelialcells, CD146 is concentrated at the intercellular junctions andcontrols inter-endothelial cell cohesion, paracellular permea-bility, monocyte transmigration, and angiogenesis (10–15).Moreover, antibody-mediated cross-linking of CD146 inHUVEC (Human Umbilical Vein Endothelial Cells) inducestyrosine phosphorylation of the non-receptor tyrosine kinasep59fyn, of FAK (Focal Adhesion Kinase) and of paxillin, indi-cating a role of CD146 in the reorganization of the cytoskeleton(16). Altogether, these data identified CD146 as an adhesionmolecule of the endothelial junction. However, very few areknown about its ligands. It has been reported that CD146 func-tions as a homophilic adhesionmolecule, but this interaction isstill debated as a direct biochemical evidence for homophilicinteraction with the purified receptor is lacking (5, 17, 18).Laminin 411, also named �4�1�1 integrin, that is expressedalong the vascular endothelium, was recently discovered as aspecific ligand for CD146 (19). In this study, the authors dem-onstrated that blocking the interaction of mouse CD146 withlaminin 411 inhibits T cell adhesion (19).CD146 is a highly glycosylated protein since glycosylations

account for about 35% (1) of its apparent molecular weight,suggesting that this protein could interact with sugar-bindingproteins such as lectins. Interestingly, Galectin-1 is a 14 kDalectin widely expressed in mammalian organism that is pro-duced by vascular, interstitial, epithelial, and immune cells (20).This lectin is known to specifically bind galactoside derivativesof glycoproteins on cell membranes (21). Indeed, variousmem-brane glycoconjugates have been identified as binding partnersof Galectin-1 such as �1 integrins, CD2, CD3, CD4, CD43,CD45, and GM1 ganglioside. In addition, Galectin-1 binds to a

* This work was supported by institutional resources provided by Aix-Mar-seille University, INSERM and by a grant from the Fondation de France.

1 Both authors contributed equally to this work.2 Recipient of Doctoral funds from the Region PACA and is supported by

Inserm, Region Funding.3 Both authors contributed equally to this work.4 To whom correspondence should be addressed: INSERM UMR-S 1076, Fac-

ulté de Pharmacie, 27 bd Jean Moulin, 13385 MARSEILLE cedex 5. Tel.:334-9183-5687; Fax: 334-9183-5602; E-mail: aurelie.leroyer@univ-amu.fr.

5 The abbreviations used are: MCAM, melanoma cell adhesion molecule;HUVEC, human umbilical vein endothelial cells; FAK, focal adhesion kinase;EDC/NHS (N-ethyl-N�-3-dimethylaminopropyl) carbodiimidehydrochlo-ride and N-hydroxysuccinimide; SPR, surface plasmon resonance.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 4, pp. 2571–2579, January 25, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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number of extracellular-matrix components in a dose-depen-dent and �-galactoside-dependent manner. For example,laminin and fibronectin, which are highlyN-glycosylated, inter-act with Galectin-1 (22). Because Galectin-1 is a divalent lectin,it can also form complexes with receptor partners participatingin cell-matrix recognition. Thus, Galectin-1 is a multifunc-tional protein involved in a variety of biological activities suchas cell-matrix interactions, immune response, inflammatoryresponse, tumorigenesis, and apoptosis of both tumor andimmune cells (23, 24).Galectin-1 and CD146 showed intriguing common biologi-

cal functions. First, Galectin-1 expression knock-down in thezebrafishmodel results in impaired vascular network formation(25) whereas suppression of CD146 expression in the samemodel results in poorly developed intersomitic vessels (26),suggesting that both Galectin-1 and CD146 are involved in vas-cular development in this model. Second, tumor growth inGalectin-1-null mice is markedly impaired because of insuffi-cient tumor angiogenesis (25). In a similar way, tumor growthwas inhibited inmice following the administration ofmonoclo-nal antibody targeting CD146, and this effect was accompaniedby a reduction of tumor blood vessel density (27). In addition,Galectin-1 is involved inmelanoma progression (28, 29) as wellas CD146, which level of expression was correlated with anadvance tumor stage in melanoma (30). Third, Galectin-1 wasdescribed to activate VEGFR-2 signaling (31, 32), and it hasbeen recently shown that VEGFR-2 phosphorylation is depen-dent of CD146 (33). Fourth, as well as CD146, Galectin-1showed altered expression and localization in activated andtumor endothelial cells (34). Finally, it has been previouslyshown that endothelial cells secreteGalectin-1 upon inflamma-tory conditions (34). Moreover, Galectin-1 can induce apopto-sis of T lymphocytes and can regulate bcl-2 expression (35, 36).Along the same line, CD146 expression is both up-regulated atthe membrane and secreted after TNF� treatment (12), and itsoverexpression leads to activation of survival and anti-apop-totic pathways (37). These data combined with the strong gly-cosylation of CD146 prompted us to look for a direct interac-tion between endothelial CD146 and Galectin-1 protein as wellas to determine its potential role in endothelial cell functions.Thus, we hypothesized that Galectin-1 could interact withCD146 and that the binding of Galectin-1 to CD146 mightmodulate endothelial cell apoptosis.

EXPERIMENTAL PROCEDURES

Cells—HUVEC were obtained from different donors andprepared according to a previously described protocol (38).This work has been approved by the local ethical committee N°2006-A006-16-45 Assistance Publique Hôpitaux de Marseille.Written parental informed consents have been obtained. Theywere cultured on 0,2% gelatin, in EBM-2 (Endothelial BasalMedium) (Lonza, CC3156) supplemented with EGM-2 (Endo-thelial Growth Medium) (Lonza, CC4176). When divided,HUVEC were detached using trypsin incubation during 2 min,followed by addition of fetal calf serum, which permits trypsinneutralization.

Transfection of CD146 Complementary DNA in FibroblasticCell Lines—CD146 transfected fibroblasts were obtained aspreviously described (11). Briefly, CD146 cDNAwas inserted inthe EcoRI sites of the mammalian expression vector PCI-neo.The fibroblastic L929 cell line was cultured in Dulbecco modi-fied Eagle’smedium-HamF12 supplementedwith 10%FCS andwas transfected using Lipofectamine (Invitrogen). The trans-fected cells were selected by resistance to geneticin (800mg/ml;Invitrogen) and stable clones were obtained by dilution and3-fold subcloning. Cells were further selected on the basis ofCD146 expression determined by Western blotting and flowcytometry using S-Endo1 anti-CD146 antibody.CD146/Galectin-1 Immunofluorescence Staining—CD146-

transfected fibroblasts or endothelial progenitor cells were cul-tured on 0.2% gelatin-coated culture slide (BD, 354118). Cellswere stimulated with 20 ng/ml of TNF-� for 20 h and washedbriefly with cold PBS before incubation for 15 min in PBS con-taining 3% paraformaldehyde. After incubation with recombi-nant human Galectin-1 (5 �g/ml), cells were rinsed with PBSand permeabilized with 0.2% Triton in PBS for 5 min. Stainingwas performed using mouse antibody directed against humanCD146, (1:200, S-Endo1), rabbit anti-Galectin-1 antiserum(1:100). Cells were washed with cold PBS and incubated witheither Alexa Fluor 488-labeled goat anti-mouse (1:500) forCD146 immunostaining or Alexa Fluor 546-labeled goat anti-rabbit (1:500) for Galectin-1. Mounted slides were then ana-lyzed using LSM 510 Zeiss confocal microscope.Enzyme-linked Immunoadsorbent Assay (ELISA)—Human

CD146-Fc was obtained as previously described for the mouseCD146-Fc (39). Briefly, CD146-Fc fusion protein contains theextracellular part of human CD146 fused to the Fc moiety of ahuman IgG1. The coding sequence of human extracellularCD146 was obtained by PCR using specific primers (forward5�-GCTAGCGGAGCAGCCTGCGCCTGAG-3�, reverse 5�-GGATCCCGCCGGCTCTCCGGCTCCG-3�) and cloned inpCDM7 vector. Recombinant CD146-Fc protein was obtainedafter transient transfection of the COS-7 eukaryotic cell lineand was purified as previously described (39). Quality andpurity of CD146-Fc were assessed using Coomassie Blue andWestern blotting with anti-CD146 antibodies (7A4 clone Bio-cytex) and HRP-coupled donkey anti-human-Ig antibodies(BD).Human His-tagged Galectin-1 and Galectin-2 proteins were

obtained and purified as previously described (40). Recombi-nant human ICOS Ligand Fc (Inducible T-cell COStimulator)was provided by Jacques Nunes (UMR 891, Marseille, France)and used as a negative control. 10 �g/ml of CD146-Fc proteinswere coated on ELISA plate (VWR, 1315801) overnight in PBSat 4 °C. After BSA (bovine serum albumin) saturation, distinctconcentrations of His-tagged Galectin-1 and Galectin-2 pro-teins were incubated for 1 h at room temperature. Interactionswere revealed by an incubation with a monoclonal antibodydirected against His tag (BD, cat 552565) followed by an incu-bation with donkey anti-mouse IgG-HRP (1:1000, Jackson Lab-oratories). Revelation was performed by addition of TMB(Tetramethylbenzidine) (Sigma, T0440-100-1). In competitionexperiments using sugars, galectin incubation was performedin the presence of 0.2 M of either maltose or lactose.

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Surface Plasmon Resonance Assay—Surface plasmon reso-nance measurements were performed on a Biacore T100 appa-ratus (Biacore GE Healthcare) at 25 °C in HBS-EP� buffer (10mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.0005% sur-factant P20). In all Biacore experiments, HBS-EP� buffer (Bia-core GE Healthcare) served as running buffer and sensorgramswere analyzed with Biacore T100 evaluation software. CD146-and Ligand Fc proteins were immobilized covalently to car-boxyl groups in the dextran layer on a Sensor Chip CM5. Thesensor chip surface was activated with EDC/NHS (N-ethyl-N�-3-dimethylaminopropyl) carbodiimidehydrochloride andN-hydroxysuccinimide (Biacore GEHealthcare). Proteins werediluted at 10 �g/ml in coupling buffer (10 mM acetate, pH 5.2)and injected until the appropriate immobilization level wasreached (i.e. 600 to 800 Resonance Units RU). Deactivation ofthe remaining activated groups was performed using 100 mM

ethanolamine pH 8 (Biacore GE Healthcare). Then, a solutionof Galectin-1 (1.7 �M) was injected for 2 min throughCD146-Fc and ICOS Ligand-Fc channels.For steady state experiments, serial dilutions from 4 nM to 2

�M of soluble Galectin-1 were injected for 6 min at a constantflow rate of 40 �l/min on dextran layers containing immobi-lizedCD146 recombinant proteins and allowed to dissociate for1min before regeneration by a 8 s injection of 500mMNaCl and10 mM NaOH buffer. The flow cell containing immobilizedICOS Ligand-Fc proteins was used as a negative control forblank subtraction.The resulting sensorgrams were analyzed by global fitting

using the appropriate model. In this model, the equilibriumdissociation constant KD is obtained by calculating the slopefrom a pseudo-Scatchard plotting of Req versus Req/C. Thecurves show the specific signal obtained after subtraction of thebackground (obtained using immobilized ICOS Ligand-Fc).For solution inhibition experiments, Galectin-1 proteins, at aconstant concentration of 50 �g/ml, were pre-incubated withincreasing concentrations of lactose or maltose (from 0 to 50mM, Sigma Aldrich) and injected for 2 min at a flow rate of 10�l/min onto the CD146 chips. After each cycle, sensorchipswere regenerated by 8 s injection of 500 mM NaCl and 10 mM

NaOH buffer at flow rate of 40 �l/min.Analysis of CD146 Glycosylations—Deglycosylation experi-

ments were performed using PNGase (New England Biolabs,P0704L), neuraminidase (New England Biolabs, P0720S) or�-N-acetyl galactosaminidase (New England Biolabs, P0734S).Briefly, 5 �g of CD146-Fc was heated 10 min at 100 °C withdenaturing buffer and further submitted to PNGase in G7buffer and Nonidet P-40 during 2 h at 37 °C. Either neuramini-dase or galactosaminidase was directly incubated with 5 �g ofprotein 2 h at 37 °C.Deglycosylated proteins were separated in 4–12% precise

protein SDS-PAGE (Perbio Science, #25204). CD146 wasdetected by Western blot using anti-CD146 mAb (Biocytex,7A4 clone; 1:1000) followed by HRP-conjugated anti-mouseIgG mAb (1:10000 Jackson Laboratories) and revealed by ECLPlus reagent (GE Healthcare, RPN 2132).Assessment of Endothelial Cell Apoptosis—Endothelial cell

apoptosis was determined byAnnexin-V/7AAD-positive stain-ing. HUVECwere plated in 6-well plates and incubated at 37 °C

in a 5% CO2 atmosphere. All experiments were performedbetween passages 2 and 6. When cells reached 70% of conflu-ence, they were stimulated with Galectin-1 during 24 h.Annexin-V/7AAD co-staining was rapidly performed afterHUVEC detachment, with the Annexin-V-FITC/7AAD kitfrom Beckman Coulter (cat PNIM 3614). Positive cells wereanalyzed on a FC500 flow cytometer (Beckman Coulter). Apo-ptosis was also evaluated with a cell death detection ELISA kit(Roche Applied Science), according to the manufacturer’sinstructions.Neutralizing anti-CD146 (S-Endo1 clone, Biocytex, cat 5050)

monoclonal antibody and its corresponding isotypic control(Biocytex) were used at the final concentration of 5 �g/ml inthis assay. The caspase-family inhibitor Z-VAD-FMK (Biovi-sion, 1010–100) was used at the concentration of 2 �M.Ribonucleic Acid (RNA)-mediated Silencing of CD146 in

HUVEC—The RNA interference treatment was performedusing subconfluent HUVEC cultured in antibiotic-free serumsupplementedmedium.Transfection of siRNAdirected againstCD146 (20 �M, Invitrogen, HSS181039) or control siRNA (20�M, Invitrogen, 12935–300) was performed by nucleofection(kit HUVEC old, Lonza, VPB-1492) according to the manufac-turer’s instructions. Transfection efficiency was assessed bymonitoring CD146 expression by flow cytometry analysis usingS-Endo1 anti-CD146 mAb or isotype-matched control mAb(Biocytex, 5050) and by Western blot as previously described.Briefly, for Western blot analysis, cells were washed in PBS,submitted to trypsin action, and extracted with 500 �l of ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris HCl, pH 7.4, 2.4mMEDTA, 1%Nonidet P-40, 0.5mMphenylmethylsulfonyl flu-oride) for 30 min at 4 °C. After centrifugation (12,000 � g, 10min, 4 °C) to eliminate cell debris and nuclei, protein extractswere submitted to 7.5% NuPage SDS-polyacrylamide gel elec-trophoresis (Invitrogen) and transferred onto nitrocellulosemembrane (Invitrogen).Statistical Analysis—Data are expressed as mean � S.E.M.

Student tests andMannWhitney tests for independent sampleswere used. Statistical analysis was performed with Prism soft-ware for Windows (SPSS Software). Differences were consid-ered significant at p � 0.05.

RESULTS

Galectin-1 Interacts with CD146 in Endothelial Cells—Toinvestigate whether Galectin-1 interacts with endothelialCD146, we first performed immunoprecipitation of Galectin-1from endothelial cells with a specific rabbit anti-Galectin-1serum. Blotting of the resulting precipitate showed the interac-tion of Galectin-1 with CD146 in HUVEC (Fig. 1A). Pre-im-mune rabbit serum, used as negative control (IP control, Fig.1A), was not able to precipitate CD146 and Galectin-1 fromendothelial cells. We further performed several immunofluo-rescence analyses by confocal microscopy to detect a colocal-ization of CD146 and Galectin-1. Once secreted, Galectin-1binds rapidly to the surfaceofmanycell surfaceproteinsand to theextracellularmatrix. This featuremakes difficult the identificationof a specific colocalization of CD146 andGalectin-1. Neverthelessa partial colocalization of CD146 and Galectin-1 was observedwhen CD146-transfected fibroblastic cell line was exposed to

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exogenous Galectin-1 (Fig. 1B). To test whether endogenousGalectin-1couldalso colocalizewithendothelialCD146, endothe-lial cells were stimulated with TNF-�, described to induce Galec-tin-1 secretion. We also detected some colocalization areas usingendothelial progenitor cells (Fig. 1C).The Interaction of Galectin-1 with CD146 Is Direct and

Specific—To further determine whether the Galectin-1/CD146interaction is direct, we performed biochemical assays usingCD146-Fc (Fc-taggedCD146 extracellular domain) and solubleGalectin-1 (His-tagged Galectin-1). CD146-Fc was purifiedfrom supernatant of transiently transfected COS-7 cells. Coo-massie Blue staining after SDS-PAGE separation showed thatpurity of the protein was more than 90% (Fig. 2A, left panel).Both CD146 and human-Fc moieties are detected by Westernblot (Fig. 2A, right panel).Using ELISA assay, the interaction between CD146 and

Galectin-1 was detected in a dose-dependent way, whereas nobinding was observed with ICOS Ligand-Fc-coated plates, usedas a negative control (Fig. 2B). ELISA assays demonstrated thatGalectin-1 binds to CD146, whereas Galectin-2, which is struc-turally closely related to Galectin-1, does not (Fig. 2B). These

data suggest a specific interaction between CD146 and Galec-tin-1, as Galectin proteins were expressed and purified accord-ing to the same protocol.The surface plasmon resonance (SPR) study confirmed a

direct interaction between CD146 and Galectin-1, whereas nobinding was observed with ICOS Ligand-Fc (Fig. 2C, upperpanel). Serial concentrations of Galectin-1 were used in SPRassay (Fig. 2C,middle panel) to determine the equilibrium dis-sociation constant. These data were plotted under a pseudo-Scatchard graph in which the slope of the line corresponds tothe KD. The dissociation constant at the equilibrium (KD) wasequal to 3.10�7 M (Fig. 2C, lower panel).The Interaction of Galectin-1 with CD146 Is Dependent of

GlycosylationMotifs—CD146 is a highly glycosylated protein asglycosylations account for about 35% of its apparent molecularweight. Moreover, Galectin-1 is able to establish bivalent ormultivalent interactions with N- and O-glycans. Thus, we ana-lyzed the glycosylation status of CD146-Fc protein. We inves-tigated the potential shift of molecular weight of CD146-Fcexposed to either neuraminidase or glycosidases. A shift ofCD146 molecular weight was observed using N-glycosidase

FIGURE 1. Galectin-1 interacts with cells expressing CD146. A, co-immunoprecipitation of CD146 with Galectin-1 in endothelial cells. Rabbit antiserumdirected against Galectin-1 (IP Gal-1) was used to precipitate specific proteins in HUVEC. Western blot was revealed using either 7A4 anti-CD146 Mab orGalectin-1 antiserum. Pre-immune rabbit anti-serum was used as a control of the immunoprecipitation (IP control). B, colocalization of recombinant Galectin-1and CD146 in CD146-transfected fibroblasts. Immunofluorescence staining of Galectin-1 (green, Gal-1, upper left picture), CD146 (red, CD146, lower left picture),differential interference contrast (DIC, upper right picture) and merged of Gal-1 and CD146 staining (merged, lower right picture). Staining was performed afterincubation with recombinant Galectin-1 protein (5 �g/ml). Yellow staining on merged picture represents a colocalization of the two proteins. C, colocalizationof endogenous Galectin-1 and CD146 in endothelial cells. Immunofluorescence staining of Galectin-1 (green, Gal-1, middle pictures), CD146 (red, CD146, leftpictures), and merged of Gal-1 and CD146 staining (merged, right pictures). Staining was performed on sub-confluent endothelial progenitor cells. Arrow showsa co-localization of the two proteins.

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whereas no changewas observed using either neuraminidase orO-glycosidase (Fig. 3A). These data show that CD146-Fc pro-tein is mainly N-glycosylated protein.Galectin-1 can bind to its cognate ligands by regular interac-

tion with �-galactoside sugars or by direct protein-proteininteraction (40, 41). SPR and ELISA studies were performed inthe presence of lactose, which is a competitor of Galectin-1binding to �-galactosides. A dose-dependent inhibition ofGalectin-1 binding to CD146-Fc was observed, whereas non-competing maltose had no effect (Fig. 3B, ELISA assay and Fig.3C, SPR assay). These results indicate that glycosylation motifsare key players of the interaction.CD146 Plays a Protective Role in Galectin-1-induced Endo-

thelial Cell Apoptosis—To further investigate whether theirinteraction could affect endothelial cell apoptosis, we per-formed 7AAD/Annexin-V staining, used as markers for cellintegrity and phosphatidylserine surface exposure. Apoptosisof HUVEC was increased after exposure to increasing concen-trations of Galectin-1 (0, 2, 10, and 50 �g/ml), whereas Galec-tin-2 had a dose dependent effect even but in a lower extend(Fig. 4A). In addition, the Galectin-1 apoptotic effect on endo-thelial cells was confirmed using histones release quantifica-tion. HUVEC exposed to 5 and 10 �g/ml of Galectin-1 releasedcytoplasmic histones 2.78 � 0.69 and 4.42 � 0.19 times morethan unstimulated HUVEC (control) (Fig. 4B).To address the role of CD146 in Galectin-1-induced apopto-

sis, we did a specific siRNA silencing of CD146 using nucleo-

fection of HUVEC. CD146-silenced HUVEC no longerexpressed CD146 as demonstrated by the absence of CD146staining, using bothWestern blot and flow cytometry, on trans-fected HUVEC (Fig. 4C). When CD146 was absent, HUVECwere more susceptible to apoptosis upon Galectin-1 treatment(Fig. 4D, left panel). This effect was dependent of theGalectin-1concentration. Quantification of endothelial apoptotic cellsafter exposure to 20 �g/ml of Galectin-1 during 24 h showed asignificant increase of apoptosis in CD146-silenced HUVEC(3.34 � 0.17-fold/cont, n � 9) when compared with controlsiRNA-transfected HUVEC (1.95 � 0.24-fold/cont, n � 5; p �0.001) (Fig. 4D, right panel).We further examined the contribution ofCD146 inGalectin-

1-induced endothelial apoptosis using blocking anti-CD146antibody (S-Endo1 clone, 5 �g/ml) and its corresponding IgGisotypic control, at the same concentration. Endothelial cellswere treated with Galectin-1 (20 �g/ml) alone or after previousexposure to anti-CD146 antibody. Incubation of HUVEC withthe anti-CD146 antibody, before exposure toGalectin-1, signif-icantly increased by 1.60� 0.34-fold (n� 5 each; p� 0.032) thepercentage of Annexin-V/7AAD positive endothelial cells (Fig.4E), whereas the addition of anti-CD146 alone did not affectendothelial apoptosis.We further investigated the mechanisms by which CD146/

Galectin-1 interactionmodulates endothelial cell apoptosis. Todetermine whether this effect is caspase-dependent, the sameexperiment was performed using the caspase-family inhibitor

FIGURE 2. Galectin-1 directly interacts with CD146. A, quality and purity of CD146-Fc were assessed using Coomassie Blue staining and Western blotting. Leftpanel, in SDS-PAGE assay, 10, 5, and 1 �g of recombinant proteins were loaded in lanes 1, 2, and 3, respectively. Right panel, 10 ng of recombinant Fc wereseparated on SDS-PAGE and blotted with anti-CD146 (7A4 clone) antibody (lane 1) or anti-human Ig (lane 2). B, ELISA analysis of Galectin-1 interactions withimmobilized CD146-Fc and irrelevant ICOS Ligand-Fc (ICOSL-Fc) proteins. Increasing concentrations of Galectin-1 were tested (from 1 to 50 �g/ml) andGalectin-2 was used as a control for binding specificity. C, SPR analysis of the interaction between CD146 and Galectin-1. Upper panel, Galectin-1 (1,7 �M) wasinjected for 2 min through CD146-Fc (black line) and ICOSL-Fc (dashed line) channels. Middle panel, serial concentrations of Galectin-1 (4 nM to 2 �M) wereinjected through both channels. Lower panel, value of the dissociation constant at the equilibrium is obtained by calculating the slope (�Kd) from a pseudo-Scatchard plotting of Req versus Req/C. The curves show the specific signal obtained after subtraction of the background.

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Z-VAD-FMK.No significant effect onGalectin-1-induced apo-ptosis was detected in this setting (data not shown).

DISCUSSION

The present study identifies Galectin-1 as a novel ligand forCD146 and demonstrates that this interaction is involved in thecontrol of endothelial cell apoptosis. We demonstrated thatCD146 specifically interacts with Galectin-1 as no binding wasdetected with Galectin-2. Using real-time analysis by SPR, wedetermined a KD of 3.10�7 M for this interaction, consistentwith reported affinity ofGalectin-1 interaction to pre-BCR (41).It is known that Galectin-1 can bind either in a sugar-depen-dent or independent way to their ligands (22). We showed thatCD146-Fc protein is sialylated and mainly N-glycosylated andthat the lactose inhibits the interaction. Altogether, theseresults demonstrated that CD146 interacts with Galectin-1 in asugar-dependent manner.Many of the proposed regulatory effects of Galectin-1

appeared to involve reversible binding to receptors with affini-ties in the low�M range (42–45). In addition, Galectin-1 exhib-its unique biochemical properties, which make its functionalanalysis even more complex. This protein contains unpairedcysteine residues in the CRD that, in the absence of carbohy-drate binding activity, can form intramolecular disulfide bondsand thereby diminishes its known biological functions (22).Thus, glycan recognition partially protects Galectin-1 fromoxidative inactivation and enhances Galectin-1 dimerization(46). Indeed, Galectin-1 exists as a mixture of monomers anddimers at physiological concentrations (45, 47). Therefore,

the different functions of this lectin can be due to 1) themonomeric or dimeric form of this protein (45, 47–50), 2)the influence of oxidative versus reducing microenviron-ments (46), 3) the engagement of Galectin-1 with ligands(51), and 4) the in vivo levels of Galectin-1 in physiologicaland pathological concentrations.In this study, we showed that high levels of exogenous Galec-

tin-1 displays, in vitro, a pro-apoptotic effect on endothelialcells (micromolar range concentration), consistent with thepro-apoptotic effect of Galectin-1 described on other celltypes. Exogenous Galectin-1 has been previously describedto have a biphasic effect on the growth of distinct cell typesincluding endothelial cells. Whereas low concentrations(nanomolar range) induced cell proliferation, high concen-trations (micromolar range, equivalent to 20 �g/ml) ofGalectin-1 seemed to have inhibitory effects (48, 52–57).Previous studies have shown that endothelial cells secreteGalectin-1 at the ng/ml level upon inflammatory conditions(56). Nevertheless, once secreted, Galectin-1 rapidly binds tothe surface of the secreting or neighboring cells or to com-ponents of extracellular matrix. As Galectin-1 remainsbound to cell or extracellular matrix glycoconjugates, deter-mination of the local concentration of Galectin-1 is basicallyimpossible. Thus, we could hypothesize that, during highinflammatory situation, endothelial cells can be locallyexposed to high concentration of Galectin-1.Beside its role in endothelial cell permeability and angiogen-

esis, CD146 overexpression has also been associated with sur-

FIGURE 3. The interaction of Galectin-1 with CD146 is dependent of glycosylation motifs. A, Western blot analysis of CD146-Fc submitted to severalglycosidase or neuraminidase treatments. A shift of CD146 molecular weight was observed using N-glycosidase. No change was detected using eitherneuraminidase or O-glycosidase treatments. B, ELISA analysis of the sugar-dependant binding of Galectin-1 to CD146. ELISA analysis of Galectin-1 interactionswith immobilized recombinant CD146 proteins in the presence of either 0.2 M lactose or 0.2 M maltose. Galectin-2 was used as a control for binding specificity.C, SPR analysis of the sugar-dependent Galectin-1/CD146 interaction. 50 �g/ml of Galectin-1 was incubated with increasing amounts of lactose (upper panel)or maltose (lower panel) and then injected onto CD146 chip. Sensorgrams showing the binding of the Galectin-1 in the different situations are superimposed.Sensorgrams were aligned in the y and x axis at the beginning of injection. The data shown are representative of two separate experiments.

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vival signals such as Akt phosphorylation and down-modula-tion of Bad expression (37). Along the same line, Galectin-1pro-apoptotic effect has already been described for human acti-vated T lymphocytes (36) in which CD45 and CD43 bind toGalectin-1, inducing the activation of AP-1 transcription factorand the down-modulation of bcl-2 proteins (35). However, themechanisms of Galectin-1 induced apoptosis are still debatedsince lower and higher amounts of the protein might act ondifferent transmitting receptors, resulting in diverse apoptoticpathways. For instance, apoptosis induced by low levels ofGalectin-1, but not high, was inhibited with the pan-caspaseinhibitor, zVAD-FMK, which corroborates our results (58).Interestingly, both Galectin-1 and CD146 endothelial cell

expressions are increased in inflammatory conditions (12, 59)and we demonstrated that the absence of CD146 renders endo-thelial cells more susceptible to Galectin-1-induced apoptosisas both down-regulation ofCD146 cell surface expression usingsiRNA and antibody-mediated blockade of CD146 increasedGalectin-1-induced endothelial cell apoptosis.

Thus, it is tempting to speculate that the CD146mucin couldbe a decoy receptor for Galectin-1, avoiding the Galectin-1binding to pro-apoptotic receptors. By scavenging Galectin-1,CD146 could also protect Galectin-1 sensitivity to oxidation orenhances Galectin-1 dimer formation, which could displayadditional or different effects on endothelium. Indeed, dimericGalectin-1 was shown to induce surface exposure of phosphati-dylserine at the plasma membrane of leukemic and promyelo-cytic cells without inducing apoptosis (36, 60). On the otherhand, it is conceivable that CD146 cross-linking by dimericGalectin-1 participates to its role in VEGFR2 signaling involvedin survival pathway (33). Altogether, our results identifiedGalectin-1 as a novel ligand for CD146 and demonstrated thatits interaction with Galectin-1 protects endothelium fromGalectin-1-induced apoptosis.

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FIGURE 4. CD146 protects Galectin-1-induced endothelial cell apoptosis. A, endothelial cell apoptosis was evaluated by 7AAD/Annexin-V staining at 24 husing increasing amounts of Galectin-1 (from 0 to 50 �g/ml) or Galectin-2, used as a negative control. B, endothelial cell apoptosis was evaluated by histone-associated-DNA-fragments released from HUVEC exposed during 6 h to 5 �g/ml and 10 �g/ml of Galectin-1 or 20 nM staurosporin, used as positive control, incomparison with basal conditions, without Galectin-1 (fold/cont). C, ribonucleic acid (RNA)-mediated silencing of CD146 in HUVEC. Western blot analysis (leftpanel) and flow cytometry analysis (right panel) showed down-modulation of CD146 expression. Right panel, CD146 expression on cells was analyzed by MeanFluorescence Intensity (MFI). CD146 expression before transfection is represented on white peak (MFI � 103), CD146-siRNA-transfected cells on gray peak(MFI � 3) and control-siRNA-transfected cells on black peak (MFI � 88). Two independent transfections were shown in Western blot (lanes 1 and 2). D, CD146plays a protective role in the Galectin-1-induced endothelial cell apoptosis. Endothelial cell apoptosis was evaluated by 7AAD/Annexin-V staining at 24 h usingincreasing amounts of Galectin-1 (from 0 to 50 �g/ml). Galectin-1 treatment was performed using either CD146 siRNA-silenced HUVEC or controlsiRNA-transfected HUVEC (left panel). The endothelial apoptosis, induced by Galectin-1 (20 �g/ml) for 24 h, is evaluated by the percentage of Annexin-V/7AAD-positive endothelial cells in comparison with basal conditions, without Galectin-1 (fold/cont) (right panel). E, endothelial cell apoptosis was evaluated by7AAD/Annexin-V staining in HUVEC. Endothelial cells were treated with Galectin-1 (20 �g/ml) alone or after previous exposure to anti-CD146 antibody(S-Endo1 clone, 5 �g/ml). Exposure to anti-CD146 alone serves as a negative control. In A, B, D, and E panels, data are shown compared with control thatcorresponds to basal conditions without Galectin-1 stimulation.

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Galectin-1 Is a Novel Ligand for CD146

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S. LeroyerKarim Fallague, Claudine Schiff, Françoise Dignat-George, Frédéric Vély and Aurélie Nathalie Jouve, Nicolas Despoix, Marion Espeli, Laurent Gauthier, Sophie Cypowyj,

Regulation of Endothelial CellsThe Involvement of CD146 and Its Novel Ligand Galectin-1 in Apoptotic

doi: 10.1074/jbc.M112.418848 originally published online December 7, 20122013, 288:2571-2579.J. Biol. Chem. 

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