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Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments

Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular... JCB: ARTICLE Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments 1,2 1 4 4 Maria Grazia Lampugnani, Fabrizio Orsenigo, Maria Cristina Gagliani, Carlo Tacchetti, 1,2,3 and Elisabetta Dejana 1 2 IFOM, Fondazione Italiana per la Ricerca sul Cancro Institute of Molecular Oncology, Mario Negri Institute for Pharmacological Research, and Department of Biomolecular and Biotechnological Sciences, Faculty of Sciences, University of Milan, 20139 Milan, Italy Department of Experimental Medicine, University of Genova, 16146 Genova, Italy eceptor endocytosis is a fundamental step in con- VEC is absent or not engaged at junctions, VEGFR-2 is trolling the magnitude, duration, and nature of cell internalized more rapidly and remains in endosomal R signaling events. Confl uent endothelial cells are con- compartments for a longer time. Internalization does not tact inhibited in their growth and respond poorly to the terminate its signaling; instead, the internalized receptor proliferative signals of vascular endothelial growth factor is phosphorylated, codistributes with active phospholipase (VEGF). In a previous study, we found that the association C–γ, and activates p44/42 mitogen- activated protein of vascular endothelial cadherin (VEC) with VEGF receptor kinase phosphorylation and cell proliferation. Inhibition of (VEGFR) type 2 contributes to density- dependent growth VEGFR-2 internalization reestablishes the contact inhibition inhibition (Lampugnani, G.M., A. Zanetti, M. Corada, of cell growth, whereas silencing the junction-associated T. Takahashi, G. Balconi, F. Breviario, F. Orsenigo, density-enhanced phosphatase-1/CD148 phosphatase A. Cattelino, R. Kemler, T.O. Daniel, and E. Dejana. restores VEGFR-2 internalization and signaling. Thus, 2003. J. Cell Biol. 161:793–804). In the present study, VEC limits cell proliferation by retaining VEGFR-2 at we describe the mechanism through which VEC reduces the membrane and preventing its internalization into VEGFR-2 signaling. We found that VEGF induces the signaling compartments. clathrin- dependent internalization of VEGFR-2. When Introduction Endothelial cells are contact inhibited in their growth and lose nucleus and modulate cell transcription. In tumor cells, the neg- the capacity to respond to growth factors when they reach ative effect of epithelial cadherin (E-cadherin) on cell growth is confl uence. This phenomenon is mediated by different concurrent a result of its capacity to bind β-catenin and inhibit its translo- mechanisms. Molecules at cell to cell junctions, such as cad- ca tion to the nucleus. This effect is detected in tumor cell lines herins, may transfer signals that reduce the capacity of the cells to in which cytosolic β-catenin ubiquitination and destruction is respond to proliferative stimuli (Dejana, 2004; Gumbiner, 2005). impaired (St Croix et al., 1998; Mueller et al., 2000; Gottardi Cadherins are located at intercellular adherens junctions and are et al., 2001; Stockinger et al., 2001; Bryant and Stow, 2005). linked to different intracellular partners that include β-catenin, Endothelial cells express a cell-specifi c cadherin called plakoglobin, p120, Src (Gumbiner, 2005), csk (Baumeister et al., vascular endothelial cadherin (VEC). This protein exerts a neg- 2005), and density-enhanced phosphatase-1 (DEP-1)/CD148. ative effect on cell growth by binding VEGF receptor (VEGFR) β-catenin, plakoglobin, and p120 can also translocate to the type 2 and inhibiting its signaling activity (Carmeliet et al., 1999; Shay-Salit et al., 2002; Lampugnani et al., 2003; Correspondence to Elisabetta Dejana: [email protected] Dejana, 2004). VEGF is a major growth factor for endothelial Abbreviations used in this paper: EEA-1, early endosomal antigen-1; DEP-1, cells and plays an important role in the formation of new ves- density-enhanced phosphatase-1; GSH, glutathione; HUVEC, human umbilical sels during embryogenesis and in proliferative diseases (Alitalo vein endothelial cell; PY, phosphotyrosine; VEC, vascular endothelial cadherin; VEGFR, VEGF receptor. et al., 2005; Carmeliet, 2005; Ferrara and Kerbel, 2005). In The online version of this article contains supplemental material. blood endothelium, the activities of VEGF are mediated by its © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 174, No. 4, August 14, 2006 593–604 http://www.jcb.org/cgi/doi/10.1083/jcb.200602080 JCB 593 THE JOURNAL OF CELL BIOLOGY interaction with two tyrosine kinase receptors, VEGFR-1 (fl t-1) Little is known about the internalization pathways fol- and -2 (fl k/KDR), as well as neuropilins. The growth signals are lowed by VEGFR-2 or their functional signifi cance (Labrecque transferred, to a large extent, through the activation of PLC-γ, et al., 2003; Bhattacharya et al., 2005; Mitola et al., 2006). It has PKC, and subsequently p44/42 MAPK (Takahashi et al., 1999, been reported that cadherins may infl uence growth factor re- 2001; Matsumoto et al., 2005; Singh et al., 2005). ceptor internalization, but the extent to which they do depends We found that in contact-inhibited endothelial cells, on the cadherin or growth factor receptor. In tumor cell lines, VEGFR-2 forms a complex with VEC that results in the inhi- N-cadherin forms a complex with FGF receptor 1 that inhibits bition of its tyrosine phosphorylation and, consequently, in the its internalization and degradation. This causes a sustained FGF attenuation of MAPK activation. This effect was attributed to the signaling and abnormal cell growth (Suyama et al., 2002). In phosphatase DEP-1/CD148 that, by binding β-catenin and p120, contrast, E-cadherin cointernalizes with FGF receptor 1, which may associate with the cadherin–receptor complex and dephos- facilitates its nuclear translocation and signaling activity (Bryant phorylate the receptor (Lampugnani et al., 2003). In this study, and Stow, 2005; Bryant et al., 2005). we go further by describing another aspect of this phenomenon. In this study, we analyzed the role of VEC on VEGFR-2 Upon activation with specifi c ligands, growth factor recep- internalization and signaling in endothelial cells. We found tors are internalized via clathrin-dependent and - independent path- that the receptor is internalized more rapidly and effi ciently ways. In many cases, this process leads to signaling termination via when VEC is absent or not clustered at intercellular contacts. degradation of the activated receptor complex. Therefore, internal- Strikingly , internalization does not terminate receptor signaling, ization is considered an important mechanism through which cells which instead continues in endosomes. This may explain why may control the intensity and duration of signal transduction. VEC-null cells present increased and uncontrolled growth. However, more recent fi ndings indicate that internaliza- tion is not just a sink through which receptors are degraded Results (Di Fiore and De Camilli, 2001; Miaczynska et al., 2004). On the contrary, some receptors, such as TGF-β, EGF, or NGF recep- VEC expression inhibits VEGFR-2 tors, can maintain their signaling activity from within intra- internalization cellular compartments (Suyama et al., 2002; Di Guglielmo We fi rst investigated whether the establishment of cell to et al., 2003; Bryant et al., 2005; Sigismund et al., 2005). cell contact modulates VEGFR-2 internalization. Using freshly Figure 1. VEC clustering at cell–cell con- tacts inhibits VEGFR-2 endocytosis. (A) The internalization of VEGFR-2 from the plasma membrane was analyzed in sparse and con- fl uent HUVECs treated with VEGF for 5 min. To detect the internalized receptor, cells were treated with a recombinant single chain anti- body to human VEGFR-2, scFvA7, and acid washed before fi xation and processing for immunofl uorescence microscopy. Internalized VEGFR-2 appears in a vesicular pattern that is more abundant in sparse than in confl uent cultures. (bottom) The granular staining after different incubation lengths with VEGF was quantifi ed using the ImageJ program (see Materials and methods). The results (referred to as events per cell) reported in the graph are means ± SD ( error bars) of three independent experiments. At least seven random fi elds were analyzed for each time point in each experiment. (B) VEC-null and -positive confl u- ent cultures were treated as described in A, but the anti–mouse VEGFR-2 clone Avas12α1 was used. The micrographs show a typical vesicular labeling pattern after a 10-min treat- ment with VEGF. The staining appears more abundant in VEC-null than in VEC-positive cells. (bottom) The time course of vesicular labeling in response to VEGF was analyzed as described in A. The binding of the antibody does not activate VEGFR-2 nor does it induce its internalization (for details see Fig. S5, avail- able at http://www.jcb.org/cgi/ content/full/ jcb.200602080/DC1). In A and B, nuclei stained with DAPI appear blue. *, P ≤ 0.05; **, P ≤ 0.01 by comparing sparse versus con- fl uent (A, bottom) and VEC-null versus -positive (B, bottom) cells by analysis of variance and the Duncan test. Bars (A), 15 μm; (B) 20 μm. 594 JCB • VOLUME 174 • NUMBER 4 • 2006 isolated human umbilical vein endothelial cells (HUVECs) than -positive cells. Receptor degradation exceeds recycling by stimu lated with VEGF, we observed that VEGFR-2 endo- about fi vefold in both cell types, but both parameters are signifi - cytosis, which was evaluated by immunofl uorescence labeling cantly increased in the absence of VEC (Fig. 2, C and D). These of intracellular vesicular compartments, was signifi cantly re- data indicate that a higher amount of VEGFR-2 is internalized, duced by cell density (Fig. 1 A). Time course analysis revealed degraded, and recycled in the absence of VEC. that in sparse cells, the number of receptor-positive vesicles in- Internalization of growth factor receptors may follow creased more rapidly and to a larger extent than in confl uent clathrin-dependent or -independent pathways. Among the lat- cells (Fig. 1 A, bottom). ter, caveolae have been shown to regulate receptor internali- This fi rst observation suggested that the establishment of zation directed toward degradation (Di Guglielmo et al., cell to cell contact reduced VEGFR-2 internalization. Because 2003; Sigismund et al., 2005). Our codistribution experi- VEC plays a role in VEGFR-2 signaling, we investigated ments of VEGFR-2 with early endosomal antigen-1 (EEA-1) whether VEC could be involved inVEGFR-2 internalization. and caveolin-1 show that VEGFR-2 internalizes mostly in We compared syngenic endothelial cell lines differing for the ex- EEA-1–positive early endosomes (Fig. 3) and to a very low pression of VEC. These cells had been characterized previously extent in caveolae. Colocalization of VEGFR-2 and the caveolar in detail and presented superimposable levels of VEGFR-2 (see component PV-1 (Stan et al., 2004; Stan, 2005) was also neg- Fig. 8; Lampugnani et al., 2002, 2003). As shown in Fig. 1 B, ligible (unpublished data). To control whether caveolae were after the addition of VEGF, the number of VEGFR-2– containing ex pressed correctly and to a comparable extent in both VEC- vesicular compartments is markedly higher in the absence of null and -positive cells, we costained these structures with VEC. Quantifi cation of the amount of biotinylated receptor PV-1 and caveolin antibodies. As shown in Fig. S1 (available that was internalized, degraded, or recycled back to the plasma at http://www.jcb.org/cgi/ content/full/jcb.200602080/DC1), membrane is reported in Fig. 2. In VEC-null endothelium, the the extensive and comparable colocalization of caveolin and receptor is internalized more quickly and to a higher extent than PV-1 could be observed in both cell types, suggesting structural in VEC-positive cells (Fig. 2, A and B). integrity of the caveolar compartment. In VEC-null cells, receptor internalization kinetics appear The preferential distribution of VEGFR-2 in EEA-1– faster in biotinylation than in immunofl uorescence experiments. positive endosomes was further confi rmed using immuno-EM This apparent discrepancy may be caused by internalization (Fig. 3 C). In addition, silencing clathrin heavy chain expres- compartments, which can be measured in biotinylation experi- sion either by siRNA (see Fig. 8 and Fig. S3, available at ments because they are protected from glutathione (GSH) re- http://www.jcb.org/cgi/content/full/jcb.200602080/DC1) or dis- duction but are not yet clustered in structures resolvable in rupting clathrin-coated pits by hypertonic medium blocked immunofl uorescence microscopy. receptor internalization both in VEC-positive and -null cells (Fig. 4). The overall amount of internalized receptor for the dura- In contrast, after incubation with fi lipin at a concentration able tion of the experiment is about fourfold more in VEC-null to fully disrupt lipid rafts and caveolae (Schnitzer et al., 1994; Figure 2. Internalization, degradation, and recycling of biotinylated VEGFR-2 are inhibited in VEC-positive cells. (A) Internalization and re- cycling of VEGFR-2 was measured after cell surface biotinylation with thiol-cleavable Sulfo- NHS-SS-Biotin. At selected time points, biotin was cleaved by GSH followed by immuno- precipitation of VEGFR-2 and probing with HRP-streptavidin as described in Materials and methods. Recycling was measured as the amount of biotinylated receptor reappearing on the cell surface (and therefore not protected by GSH cleavage) at the indicated time points after 10 min of internalization in the presence of VEGF (indicated as 0 min in recycling panel). Total, total amount of labeled receptor after Sulfo-NHS-SS-Biotin labeling at 4°C (with- out GSH treatment). GSH, treatment with re- ducing GSH to remove any labeling on the residual surface-exposed receptor. (B) Densito- metric analysis of internalization expressed as a percentage of total labeling. (C and D) Quantitation of receptor degradation and re- cycling as a percentage of the amount of receptor internalized after 10 min with VEGF. Although A presents a representative experi- ment, the values in B–D are the means of four independent experiments ± SD (error bars). **, P ≤ 0.01; *, P ≤ 0.05 comparing VEC-null with -positive cells, respectively, by analysis of variance and the Duncan test. VE-CADHERIN AND VEGFR-2 INTERNALIZATION • LAMPUGNANI ET AL. 595 Figure 3. Internalized VEGFR-2 colocalizes with EEA-1– positive compartments by immunofl uorescence analysis. HUVECs, VEC-null, and VEC-positive cells were double la- beled for VEGFR-2 and either EEA-1 or caveolin-1. Cells were activated with VEGF for 10 min. (A) Representative exam- ples of confocal images for each antigen and their respective merges (boxed areas; 3.5-fold magnifi cation) are shown for confl uent HUVECs after treatment with VEGF. VEGFR-2 was revealed with an AlexaFluor488-conjugated secondary anti- body and is shown in green. EEA-1 and caveolin revealed with AlexaFluor647-conjugated secondary antibodies are shown in red. Nuclei appear blue after DAPI staining. Bars, 10 μm. (B) To quantify colocalization events, images were an- alyzed using the ImageJ colocalization plugin (as described in Materials and methods). The graphs present the number of colocalization events normalized for the number of VEGFR-2– positive compartments. After VEGF treatment, 45–55% of VEGFR-2–positive compartments showed colocalization with EEA-1 in all of the situations examined. Colocalization of internalized VEGFR-2 with caveolin-1 was negligible. Values are the mean of at least three experiments ± SD (error bars). In each experiment, at least fi ve random fi elds were analyzed for each point. *, P ≤ 0.01 by t test. (C, a and b) Immuno- gold labeling of EEA-1 (10 nm gold; arrows) and VEGFR-2 (15 nm gold; arrowheads) on an ultrathin cryosection of VEC-null and -positive cells treated with VEGF for 10 min. The panels display VEGFR-2 labeling of EEA-1–positive endo- somes in VEC-null rather than in VEC-positive cells. (c and d) Under the same conditions, morphologically identifi ed caveo- lae are devoid of VEGFR-2. Bar (a), 227 nm; (b) 222 nm; (c) 350 nm; (d) 370 nm. Andriopoulou et al., 1999), receptor internalization did not sig- We fi rst tested whether the internalized receptor retained tyro- nifi cantly change (Fig. 4). sine phosphorylation by cell fractionation on an iodixanol gradient We also performed experiments at different time points (Yeaman et al., 2001). Antibodies recognizing phosphotyrosine (5, 7, 10, 20, and 30 min) after the addition of VEGF in VEC- (PY) 1214– and PY1054/59–VEGFR-2 were used. As shown in null and -positive cells using caveolin and PV-1 as markers of Fig. 5, in the absence of VEC, a higher amount of phosphorylated caveolae and EEA-1 and Rab-5 as markers of early endosomes. VEGFR-2 is detected in intracellular fractions, whereas in the At all time points considered, we could not see internalized presence of VEC, the phosphorylated receptor remains preferen- VEGFR-2 in vesicles positive to either caveolin or PV-1 but tially in fractions corresponding to peripheral plasma membranes. only in bona fi de early endosomes (unpublished data). These Phosphorylation of VEGFR-2 tyrosine 1175 is required data strongly suggest that, at least within the experimental con- for binding and activation of PLC-γ, which is the major effector ditions used, VEGFR-2 endocytosis in endothelial cells is mostly of VEGF-mediated cell proliferation (Takahashi et al., 2001). By clathrin dependent. using antibodies specifi c for PY1175–VEGFR-2, we observed that in the absence of VEC, a higher amount of internalized Internalized VEGFR-2 retains receptor was phosphorylated at this specifi c tyrosine (Fig. 6, signaling activity A and B). Consistently, more internalized PY1175–VEGFR-2 In the absence of VEC, endothelial cells respond more effec- was found in sparse than in confl uent HUVECs (Fig. S2, available tively to growth signals transferred by VEGFR-2. From the at http://www.jcb.org/cgi/content/full/jcb.200602080/DC1). aforementioned observations, we concluded that VEGFR-2 is To further prove that PLC-γ could be activated by the inter- internalized more quickly and to a larger extent in VEC-null nalized receptor, we stained the cells with antibodies directed cells than in VEC-positive cells. Therefore, we asked whether to the active PY783–PLC-γ. As shown in Fig. 7, active PLC-γ the receptor retains its signaling activity when sequestered in codistributes with internalized VEGFR-2 more effectively in intracellular compartments. the absence than in the presence of VEC. 596 JCB • VOLUME 174 • NUMBER 4 • 2006 Figure 4. VEGFR-2 is internalized through a clathrin-dependent pathway. (A) VEC-null and -positive cells were transfected with either Stealth siRNA targeting the mouse clathrin heavy chain or negative Stealth siRNA control duplex (and used 72 h later), or the cells were treated with hypertonic medium (0.45 M sucrose for 30 min) or 1 μg/ml fi lipin for 1 h. The micrographs show VEGFR-2 immuno- fl uorescence staining after VEGF treatment for 10 min. Both clathrin heavy chain siRNA and hypertonic medium strongly reduced VEGFR-2 vesicular patterning both in VEC-null and -positive cells. Using fi lipin to interfere with the caveolar compartment had no effect on either cell type. The negative Stealth siRNA control duplex produced results that were indistinguishable from untreated cells (not depicted). Nuclei appear blue after DAPI staining. Bar, 20 μm. (B) Column graphs represent VEGFR-2 vesicu- lar labeling in VEGF-treated cells quantifi ed by ImageJ. Ctr, control; HM, hypertonic medium. Values normalized per cell are the mean of three independent experiments ± SD (error bars). In each experiment, at least fi ve inde- pendent fi elds were analyzed for each point. *, P ≤ 0.01 versus control values by analysis of variance and the Dunnet test. Inhibition of VEGFR-2 internalization receptor is internalized more effi ciently and retains its active state affects its proliferative signaling for a longer time, leading to continuous proliferative signaling. Overall, the aforementioned data indicate that VEC limits receptor To further test this hypothesis, we prevented receptor activation and internalization. In the absence of this protein, the internalization by silencing clathrin with two specifi c siRNAs Figure 5. VEC expression reduces the amount of PY–VEGFR-2 in internal compartments. Extracts of VEGF-treated VEC-null and -positive cells were fraction- ated on an iodixanol gradient as described in Materials and methods. Samples representative of the total protein content of each fraction were analyzed by SDS-PAGE and Western blotting. As expected, VEC concentrates in lower density fractions corresponding to plasmatic membranes, whereas clathrin and EEA-1 are enriched in higher density fractions corresponding to internal membranes (Yeaman et al., 2001). Upon stimulation with VEGF, PY1214–VEGFR-2 is enriched in fractions corresponding to the internal membranes in VEC-null cells. The graph shows the ratio between the phosphorylated receptor and the total receptor present in each fraction. For quantifi cation, in each fraction, we considered the band with the molecular mass of the mature form of the receptor at the plasma membrane ( 210 kD; see fraction 1 and arrowheads; Takahashi and Shibuya, 1997). We chose this band by making the assumption that full-length VEGFR-2 represents the signaling form of the receptor. At increasing density, a lighter band appears both in VEC-null and -positive cells (asterisks). It is likely that this band derives from VEGFR-2 processing in internal compartments (for instance, proteolytic or intermediate synthesis products) and is indeed not present in the peripheral membrane fractions. Comparable results were obtained using an antibody to PY1054/59–VEGFR-2 (not depicted). VE-CADHERIN AND VEGFR-2 INTERNALIZATION • LAMPUGNANI ET AL. 597 VEC mutants lacking either the β-catenin– or p120-binding domains were unable or less effi cient, respectively, to coimmuno- precipitate VEGFR-2. We found that these mutants were also unable to signifi cantly prevent VEGFR-2 internalization (Fig. S4, available at http://www.jcb.org/cgi/content/full/ jcb.200602080/DC1), suggesting that receptor internalization is reduced as a consequence of binding to VEC. Similar to E-cadherin (for review see Bryant and Stow, 2004), VEC can be internalized through clathrin-coated pits (Xiao et al., 2005). We tested whether VEC codistributes with VEGFR-2 in intracellular compartments. As reported in Fig. 9 upon VEGF activation, no signifi cant codistribution of VEC with VEGFR-2–positive vesicles is detected in VEC-positive cells and HUVECs (Fig. S4). Only junctional colocalization can be observed (Fig. 9). The lack of codistribution in internal compartments was confi rmed by immuno-EM (unpublished data). Collectively, these data suggest that the receptor internal- izes upon dissociation from VEC. VEGFR-2 phosphorylation is required for internalization and signaling In previous studies, we showed that the phosphatase DEP-1/ CD148 can reduce VEGFR-2 signaling. DEP-1/CD148 can Figure 6. Internalized VEGFR-2 is phosphorylated at tyrosine 1175. (A) VEC-null and -positive cells were stimulated with VEGF as in Fig. 1, fi xed, associate with β-catenin and p120 (Holsinger et al., 2002; and processed for immunofl uorescence microscopy. The vesicular labeling Palka et al., 2003) and with the VEC–VEGFR-2 complex, re- pattern observed with PY1175–VEGFR-2 antibody after stimulation with ducing VEGFR-2 phosphorylation (Lampugnani et al., 2003). VEGF was signifi cantly more abundant in VEC-null than in VEC-positive cells. Nuclei appear blue after DAPI staining. Bar, 20 μm. (B) Images were Therefore, we tested whether DEP-1/CD148 could also reduce analyzed by ImageJ to quantify PY115–VEGFR-2–positive compartments receptor internalization. (see Materials and methods). The graph presents the mean ± SD (error As reported in Fig. 10 A, in endothelial cells transfected bars) calculated from 18 random fi elds that were analyzed through ImageJ in fi ve independent experiments and is normalized to the number of cells with DEP-1/CD148 siRNA, VEGFR-2 internalization is signi- per fi eld. In each experiment, at least three random fi elds were analyzed. fi cantly higher. This effect is accompanied by an increase in *, P ≤ 0.01 comparing VEC-null with -positive cells after VEGF by analysis VEGFR-2 phosphorylation and MAPK activation (Lampugnani of variance and the Duncan test. et al., 2003). These data suggest that retention of VEGFR-2 at the membrane by VEC allows its dephosphorylation by DEP-1/ (Figs. 4, 8, and S3) that target independent sequences of clathrin CD148 and limits its internalization and signaling. heavy chain messenger. As expected, VEGF-induced phosphor- ylation of VEGFR-2 and activation of p44/42 MAPK were Discussion higher in the absence than in the presence of VEC (Fig. 8). When siRNA clathrin was applied to VEC-null cells, both re- In this study, we report a novel aspect of the mechanism through ceptor and MAPK phosphorylation dropped to values compara- which VEC expression and clustering inhibits VEGFR-2 prolif- ble with those of VEC-positive cells. In contrast, in the presence erative signaling. We found that in the absence of VEC or in of VEC, the effect of siRNA clathrin was either weak or conditions in which VEC is not clustered at adherens junctions undetectable. Consistently, the inhibition of MAPK activation as in sparse cells, VEGFR-2 is endocytosed to a higher extent in was also observed by treating the cells with hypertonic medium, intracellular compartments, from where it maintains its signaling whereas treatment with fi lipin did not modify MAPK activa- activity. VEC could therefore reduce receptor activity by in- tion in response to VEGF either in VEC-positive or -null cells hibiting VEGFR-2 internalization and promoting its inactivation (unpublished data). Overall, these results strongly suggest that at the cell surface. internalization protects the receptor from dephosphorylation In our experimental conditions, VEGFR-2 is internal- and, therefore, increases and prolongs its proliferative signaling. ized in early endosomes mostly through a clathrin-dependent pathway. We were unable to detect caveolin-1–positive vesi- VEC association with VEGFR-2 cles containing VEGFR-2, and we could not inhibit receptor is required for receptor retention internalization using a caveolae-perturbing drug such as fi lipin at the plasma membrane (Schnitzer et al., 1994; Andriopoulou et al., 1999). Other We then investigated the mechanism through which VEC in- studies found the codistribution of VEGFR-2 with caveolin-1 hibits VEGFR-2 internalization. In a previous study, we found (Labrecque et al., 2003; Bhattacharya et al., 2005; Ikeda et al., that VEC forms a complex with VEGFR-2, and we analyzed the 2005), and we cannot exclude that under different experimental domains of VEC involved in this process (Lampugnani et al., 2003). conditions, VEGFR-2 may be internalized through caveolae. 598 JCB • VOLUME 174 • NUMBER 4 • 2006 Figure 7. Colocalization of internalized VEGFR-2 and acti- vated PLC-𝛄 . (A) VEC-null and -positive cells were labeled in vivo with antibodies to VEGFR-2, stimulated with VEGF as in Fig. 6, acid washed, fi xed, and processed for immunofl uo- rescence microscopy. Cells were double labeled with an anti- body that recognizes PLC-γ only when phosphorylated at tyrosine 783. VEGFR-2 was revealed with an AlexaFluor488- conjugated secondary antibody and is shown in green. PY783–PLC-γ, which was revealed with an AlexaFluor647- conjugated secondary antibody, is shown in red. For each cell type, the bottom panel on the right (threefold magnifi ca- tion of the boxed areas) shows the colocalization of VEGFR-2 and PY783–PLC-γ (pink) set upon the VEGFR-2 background (gray). This was obtained through the ImageJ colocalization plugin (see Materials and methods for details). Arrows point to the colocalization of PY783–PLC-γ and VEGFR-2. Bars, 10 μm. (B) The confocal images were analyzed through ImageJ to calculate the number of colocalization events. These values, which were normalized over the number of VEGFR-2– positive compartments per cell, are presented in the graph. After VEGF treatment, colocalization was about fi vefold higher in VEC-null than in VEC-positive cells. Values in the graph are the mean from three independent experiments ± SD (error bars). In each experiment, each point was calcu- lated from at least fi ve random fi elds. *, P ≤ 0.01 comparing VEC-null with -positive cells after VEGF by analysis of vari- ance and the Duncan test. However, the observed association with caveolin-1 may also in transgenic mice reduces permeability and angiogenic response represent a mechanism of receptor compartmentalization at the to VEGF (Bauer et al., 2005). plasma membrane. It was found that caveolin-1 would form It has been reported that VEGFR-2 internalization and degra- a molecular complex with VEGFR-2 that inhibits receptor acti- dation are regulated by ubiquitination through a Cbl-dependent vation in resting cells. Upon activation of the cells with VEGF, mechanism (Duval et al., 2003) or C-tail serine phosphorylation caveolin-1 is phosphorylated, and the complex rapidly disso- by activated PKC (Singh et al., 2005). These mechanisms may ciates (Labrecque et al., 2003). Thus, it is tempting to specu- coexist and may be responsible for the amount of receptor degra- late that similar to TGF-β receptor (Di Guglielmo et al., 2003), dation reported here. once VEGFR-2 is released from the caveolin-1 complex, it Our observations support the hypothesis that internal- becomes available for internalization in clathrin-coated pits. ized VEGFR-2 maintains its activity. These data are in agree- In agreement with this model, the overexpression of caveolin-1 ment with recent publications indicating that signaling through Figure 8. Silencing clathrin expression in- hibits VEGFR-2 phosphorylation and signaling in VEC-null cells. VEC-null and -positive cells were transfected with Stealth siRNA-targeting mouse clathrin heavy chain. Two oligonucleo- tides (a and b; respective sequences are re- ported in Materials and methods) were used that target two independent sequences of the clathrin heavy chain mRNA. Negative Stealth siRNA duplex was used as a control. After 72 h, cells were treated with VEGF or control me- dium for 10 min. They were then extracted and processed for Western blotting. Clathrin siRNA reduced clathrin heavy chain levels by 75 (oligonucleotide a) and 90% (oligonucleotide b) in both cell types. VEGFR-2 phosphorylation at tyrosine 1214 and p44/42 MAPK phos- phorylation in response to VEGF were strongly inhibited in VEC-null cells, whereas these pa- rameters were only barely affected in VEC- positive cells. Comparable results were obtained for PY1054/59–VEGFR-2 (not depicted). The graphs show the quantifi cation of these effects as means ± SD (error bars) of fi ve independent experiments. Fold increase after VEGF is shown for PY1214–VEGFR-2 and phospho-p44/42 MAPK. *, P ≤ 0.01 versus negative siRNA by variant analysis and the Dunnet test. VE-CADHERIN AND VEGFR-2 INTERNALIZATION • LAMPUGNANI ET AL. 599 A novel aspect of our work is that VEC inhibits VEGFR-2 internalization, thereby reducing its cell growth signaling activity. How can VEC inhibit receptor endocytosis? A likely hypothesis is that VEC retains VEGFR-2 at the membrane by binding to it. In addition to our studies, others have reported (Shay-Salit et al., 2002; Lampugnani et al., 2003; Weis et al., 2004; Lambeng et al., 2005) that VEGFR-2 couples with VEC. This process requires the binding of VEC to β-catenin and, to a lesser extent, to p120. In the present study, we found that mutants of VEC lacking the cytoplasmic domain responsible for binding either β-catenin or p120 and unable to associate with VEGFR-2 (Lampugnani et al., 2003) do not prevent VEGFR-2 internaliza- tion. This supports the idea that VEC–VEGFR-2 coupling is re- quired to inhibit receptor endocytosis. Cadherins themselves are endocytosed via several dif- ferent routes, including clathrin-dependent (Le et al., 1999; Palacios et al., 2002; Ivanov et al., 2004; Izumi et al., 2004) Figure 9. VEC does not codistribute with VEGFR-2 in internal compartments. and -independent pathways (Akhtar and Hotchin, 2001; Paterson VEC-positive cells were treated with VEGF for 10 min, double stained for et al., 2003). Therefore, cotraffi cking of receptor and cadherin VEGFR-2 and VEC, and analyzed by confocal microscopy. Besides junc- tional staining, VEC did not show any obvious vesicular pattern. VEGFR-2 complexes is possible (for reviews see Bryant and Stow, 2004, and VEC appeared to codistribute only at cell–cell contacts and not in intra- 2005; Cavallaro and Christofori, 2004). However, under our cellular compartments. The bottom panel on the right (2.6-fold magnifi cation experimental conditions, we could detect VEC in intracellular of the boxed areas) shows the colocalization of VEGFR-2 and VEC (yellow) set upon the VEC background (gray). This was obtained through the colo- compartments (Xiao et al., 2005), but we could not see codistri- calization plugin of ImageJ (see Materials and methods for details). Arrows bution with the receptor. Thus, it is likely that the receptor dis- point to the junctional colocalization of VEGFR-2 and VEC. Bars, 10 μm. sociates from VEC before internalization (Fig. 9). In a previous study, the phosphatase DEP-1/CD148 was growth factor receptors does not occur only at the cell mem- found to play a role in the inhibitory effect of VEC on VEGR-2 brane but may continue even more effectively from intracellular signaling (Lampugnani et al., 2003). This phosphatase associ- compartments (Di Fiore and De Camilli, 2001; Sorkin and Von ates with VEC through its binding to β-catenin and p120 and, Zastrow, 2002; Miaczynska et al., 2004; Le Roy and Wrana, in this way, reduces VEGFR-2 phosphorylation ( Lampugnani 2005). Clathrin-dependent internalization of TGF-β in early en- et al., 2003). We report that DEP-1/CD148 could prevent dosomes, where the Smad2 anchor SARA is enriched, promotes VEGFR-2 internalization along with the reduction of recep- TGF-β signaling (Di Guglielmo et al., 2003). EGF receptor is tor phosphorylation and signaling. Therefore, it is possible that internalized shortly after ligand addition in intracellular com- VEC, by retaining VEGFR-2 at the cell surface, allows its de- partments together with its downstream signaling factors shc, phosphorylation by DEP-1/CD148, which, in turn, inhibits its Grb2, and mSOS (Di Guglielmo et al., 1994) and maintains internalization and signaling. its signaling activity (Pennock and Wang, 2003). Similarly, Besides VEC, VEGFR-2 was found to bind to integrins (Soldi the specifi c activation of endosome-associated PDGF receptor et al., 1999). Another study reported that when cells are plated leads to the activation of its major signaling pathways (Wang on collagen I, the phosphatase SHP2 can associate with VEGFR-2 et al., 2004). Thus, as for VEGFR-2, endocytic transport is im- and stimulate its internalization. SHP2 activates Src, which in portant not only for receptor turnover but also for regulating turn activates dynamin II–dependent receptor internalization signal transduction and for mediating the formation of special- (Mitola et al., 2006). Interestingly, this phenomenon does not ized signaling complexes. occur when cells are plated on vitronectin, and SHP2 does not Figure 10. DEP-1/CD148 silencing in VEC-positive cells in- creases internalization, tyrosine phosphorylation, and activity of VEGFR-2. VEC-positive cells were transfected with either Stealth siRNA-targeting mouse DEP-1/CD148 or a negative Stealth siRNA control duplex. After 72 h, cells were treated with VEGF for 10 min and either fi xed and processed for immuno- fl uorescence microscopy or extracted and processed for Western blotting. DEP-1/CD148 siRNA reduced DEP-1/ CD148 protein by 45% (B). (A) VEGFR-2–positive vesicular compartments were found to be signifi cantly higher (>80%) in VEGF-treated cells after transfection with DEP-1/CD148 siRNA. Means of three independent experiments ± SD (error bars) are shown. In each experiment, at least fi ve random fi elds were analyzed. *, P ≤ 0.01 comparing negative with DEP-1 siRNA interference by analysis of variance and the Duncan test. (B) Phosphorylation of VEGFR-2 at tyrosine 1214 and of p44/42 MAPK was increased by 80 and 60%, respectively, after DEP-1/CD148 siRNA transfection in VEGF-treated cells. 600 JCB • VOLUME 174 • NUMBER 4 • 2006 bind to VEGFR-2 (Mitola et al., 2006). These observations sug- RNA interference Cells were seeded as described in Cell types and culture conditions. 20 h gest that the capacity of different adhesive proteins to complex later, cells were washed once with OptiMEM (Life Technologies) and trans- with growth factor receptors and modulate their internalization fected with 40 nM of Stealth oligonucleotides (Invitrogen) using 2 μl/ml and signaling may be a general paradigm. In this way, cells may LipofectAMINE 2000 (Invitrogen) in OptiMEM according to the manufac- turer’s instructions. After 5 h, the transfection medium was removed, and modulate their growth and survival as a function of density and complete culture medium was added. Cells were cultured for a further 72 h interaction with specifi c matrix proteins. before stimulation and processing for immunofl uorescence and extraction. In conclusion, the results reported in this study are con- Stealth oligonucleotides were used as follows: a, 5′-G C A G U U G U U- C A U A C C C A U C U U C U U A -3′ (start nucleotide was 2,118 bases down- sistent with the idea that VEGFR-2 proliferative signaling is in- stream of the start codon); b, 5′-G A A G A A C U C U U U G C C C G G A A A U U U A -3′ creased by endocytosis. The inhibitory role of VEC is likely that (start nucleotide was 1,284 bases downstream of the start codon) to target of binding and retaining the receptor at the cell surface, prevent- mouse clathrin heavy chain; and 5′-U C G A G C C A G U G A G C A U G U U U G G- A A A -3′ (start nucleotide was 4,023 bases downstream of the start codon) ing its endocytosis, and favoring inactivation by DEP-1/CD148. to target mouse DEP-1/CD148. As a control, a Stealth siRNA-negative This suggests that the modulation of VEC–VEGFR-2 complex control duplex oligonucleotide (Invitrogen) with a C/G content equivalent formation may be a novel strategy to regulate VEGF proliferative to the positive oligonucleotide was used. signaling and, therefore, to inhibit or stimulate angiogenesis. Internalization assays Microscopy. Cells were treated in vivo with anti–VEGFR-2 antibody and acid washed before fi xation (Ehrlich et al., 2001; Di Guglielmo et al., Materials and methods 2003) as follows. We used monoclonal antibodies to the extracellular domain of human and mouse VEGFR-2 (see Primary antibodies) that are Primary antibodies described by the manufacturers to be devoid of biological activity. As this For the detection of VEGFR-2, anti–human VEGFR-2 (single chain recom- aspect was crucial to the assay, we tested the effect of these antibodies on binant; clone scFvA7 with E tag; RDI and Fitzgerald) and anti–mouse basal and VEGF-stimulated phosphorylation and internalization of VEGFR-2. VEGFR-2 (rat clone Avas12α1; RDI and Fitzgerald) were used for immuno- We found that these antibodies do not stimulate the phosphorylation fl uorescence; rabbit polyclonal C-1158 (sc504; Santa Cruz Biotechnology, of VEGFR-2 in either condition. These results are reported in Fig. S5 Inc.) was used for Western blotting. Antibodies to tyrosine-phosphorylated (available at http://www.jcb.org/cgi/content/full/jcb.200602080/DC1). VEGFR-2 were rabbit polyclonal PY1214 and PY1054/59 (Biosource Anti–mouse VEGFR-2 was dialyzed for 3 h against PBS (with one change) International) and rabbit polyclonal PY1175, which was provided by to remove azide. Cells were precooled for 30 min on ice and treated with M. Shibuya (University of Tokyo, Tokyo, Japan). Antibodies to clathrin 10 μg/ml of antibody for 1 h on ice with gentle agitation. Before stimula- heavy chain were mouse monoclonal cloneX22 (Affi nity BioReagents, Inc.) tion, cells were washed with ice-cold 1% BSA in MCDB 131 to remove for immunofl uorescence and mouse monoclonal clone 23 (R&D Systems) unbound antibody, and fresh medium was added. Cells were stimulated for Western blotting. Antibody to EEA-1 was goat polyclonal N-19 with 80 ng/ml VEGF and transferred to 37°C. After the indicated time (sc-6415; Santa Cruz Biotechnology, Inc.); antibody to caveolin-1 was rabbit intervals, cells were placed on ice and acid washed (three washes with 2+ 2+ polyclonal N-20 (sc-894; Santa Cruz Biotechnology, Inc.); and antibody ice-cold 50 mM glycine in Ca /Mg HBSS, pH 2.5, and two washes with 2+ 2+ to VEC was goat polyclonal C-19 (sc-6458; Santa Cruz Biotechnology, Ca /Mg HBSS, pH 7.5) to remove the antibody from the cell surface. Inc.) and mouse monoclonal BV6 and BV9 (produced in our laboratory; Cells were then fi xed as described in Immunofl uorescence microscopy. To Corada et al., 2001). Antibody to PY783-PLCγ, total p42/44 MAPK, and reveal the distribution of the primary antibody, AlexaFluor488-conjugated phospho-p42/44 MAPK was rabbit polyclonal (Cell Signaling). Antibody donkey anti–rat (Invitrogen) was used for the rat anti–mouse VEGFR-2. For to DEP-1/CD148/CD148 was goat polyclonal (R&D Systems), and anti- the recombinant E-tagged anti–human VEGFR-2, rabbit anti–E-tag (Abcam) body to PV-1 was rat monoclonal (provided by R. Stan, Dartmouth Medical followed by AlexaFluor488-conjugated donkey anti–rabbit (Invitrogen) School, Lebanon, NH). were used. Cell surface biotinylation and immunoprecipitation. Internalization, Cell types and culture conditions recycling, and degradation were measured as described previously by Endothelial cells with a homozygous null mutation of the VEC gene (VEC Fabbri et al. (1999) with the following modifi cations. Cells were put on ice 2+ 2+ null) and the cell lines derived from them through retroviral gene transfer and washed three times with ice-cold PBS containing Ca and Mg 2+ 2+ 2+ 2+ and expressing wild-type (VEC positive) or various VEC mutant constructs (Ca /Mg PBS). For surface biotinylation, cells in Ca /Mg PBS were were generated and characterized as described previously in detail treated with 0.5 mg/ml of thiol-cleavable Sulfo-NHS-S-S-Biotin (Pierce (Lampugnani et al., 2003). For all of the experiments, 50,000 cells/cm Chemical Co.) for 1 h on ice. They were then washed on ice twice with 2+ 2+ (to reach confl uence within 24 h) were seeded in complete culture medium Ca /Mg PBS, once with MCDB 131, and once with 1% BSA MCDB and cultured without medium change for 72 h. Cells were then washed 131. 80 ng/ml VEGF in fresh 1% BSA MCDB 131 was added, and cells once with MCDB 131 (Life Technologies) and starved in 1% BSA in MCDB were incubated at 37°C for the time indicated to allow internalization. The 131 (starving medium) for 18–20 h. 2 h before activation, cells were cultures were then put back on ice and washed three times with ice-cold 2+ 2+ washed once with MCDB 131 and further incubated in fresh starving Ca /Mg PBS. Samples were incubated twice for 20 min with 45 mM of medium. Cells were treated with 80 ng/ml VEGF (human recombinant the membrane-nonpermeable reducing agent GSH in 75 mM NaCl, with VEGF 165; PeproTech) in fresh starving medium (fresh starving medium 75 mM NaOH and 1% BSA added just before use (stripping buffer). Cells 2+ 2+ alone was used for controls) for the indicated intervals at 37°C. If not were further washed twice on ice with Ca /Mg PBS and incubated for 2+ 2+ otherwise indicated, VEGF treatment was for 10 min. 15 min with iodoacetamide (in Ca /Mg PBS with 1% BSA; quenching HUVECs were cultured in MCDB 131 with endothelial cell supple- buffer) to quench free sulfo-reactive groups. To evaluate total labeling, ments as described previously (Lampugnani et al., 2003). For the experi- a sample for each cell type was not reduced with GSH. To control back- ments 1,800 and 42,000 cells/cm were seeded to obtain sparse and ground, a sample was labeled and reduced without incubation at 37°C. 2+ 2+ confl uent cultures, respectively. HUVECs were then treated as described in For immunoprecipitation, cells were washed with Ca /Mg PBS this section for mouse endothelial cells except that starving was reduced to and extracted on ice in 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Triton 6 h before stimulation with VEGF. X-100, 1% NP-40, and a cocktail of protease inhibitors (Set III; Calbiochem). Extracts were precleared for 90 min with protein A–agarose beads and Filipin and hypertonic treatments incubated overnight with 5 μg anti–VEGFR-2 (rabbit sc-504), and the Caveolae organization was altered by treatment with 1 μg/ml fi lipin immunocomplexes were collected on protein A–agarose beads for 90 min. (fi lipin III from Streptomyces fi lipinensis; Sigma-Aldrich) for 1 h (Schnitzer After fi ve washes in extraction buffer (the last one containing 0.1% Triton et al., 1994; Andriopoulou et al., 1999). Clathrin pit–mediated endocyto- X-100), proteins were eluted by boiling for 10 min in nonreducing laemmli sis was inhibited using hypertonic medium (0.45 M sucrose in MCDB 131 sample buffer. Samples were analyzed by SDS-PAGE followed by Western with 1% BSA) for 30 min to affect as described previously (Heuser and blotting on nitrocellulose membrane and revealed by ECL chemilumines- Anderson, 1989; Ehrlich et al., 2001). Cells were then stimulated and cence. Band intensity was quantifi ed by ImageJ analysis (National Insti- assayed as indicated in the specifi c sections. tutes of Health; freely available at http://rsb.info.nih.gov/ij/). VE-CADHERIN AND VEGFR-2 INTERNALIZATION • LAMPUGNANI ET AL. 601 To quantify VEGFR-2 recycling and degradation, cells were labeled fl asks as described in Cell types and culture conditions. Three fl asks per as described in the fi rst paragraph of this section, and endocytosis was al- gradient were used. After the indicated treatments, cultures were put on ice lowed for 10 min in the presence of 80 ng/ml VEGF (peak time for VEGF- and washed twice with ice-cold PBS. Cells were scraped in 1.2 ml of ice- induced VEGFR-2 internalization both in VEC-null and -positive cells as cold isotonic buffer/fl ask (20 mM Hepes-KOH, pH 7.5, 0.25 M sucrose, determined in internalization experiments). Samples were then reduced as 90 mM KO-acetate, 2 mM Mg-acetate, 0.5 mM Na-vanadate, 1 mM NaF, described in the fi rst paragraph of this section to remove the label from the 10 mM pyrophosphate, 3 mM β-glycerophasphate, 1 mM pefabloc, residual cell surface receptor. The internalized fraction was chased by re- 40 U/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin) and ho- incubation at 37°C for 10 and 20 min in duplicate samples. One sample mogenized with a Dounce homogenizer. Nuclei and residual intact cells was reduced to evaluate the amount of VEGFR-2 that recycled back to the were pelleted by centrifugation at 1,400 g for 5 min at 4°C. The superna- plasma membrane, and the other sample was left unreduced to measure tants were separated in three equal aliquots and mixed with iodixanol degradation. The samples were then processed as described in the fi rst (OptiPrep; Axis-Shield) and homogenization buffer to generate 30, 20, paragraph of this section. VEGFR-2 degradation was calculated by sub- and 10% iodixanol solutions. They were then loaded into 11.2-ml tracting the value of residual biotinylated receptor after incubation at 37°C OptiSeal tubes (Beckman Coulter) and ultracentrifuged at 353,000 g for without reduction (i.e., internalized + recycled − degraded) from the total 3 h in a rotor (VT 65.1; Beckman Coulter). 600-μl fractions were collected pool of internalized receptor. VEGFR-2 recycling was calculated by sub- from the top of the gradient, and protein concentration (bicinchoninic acid tracting both the degradation value and the value of residual biotinylated reagent; Pierce Chemical Co.) and density (OD at 244 nm as indicated by receptor after incubation at 37°C and reduction (i.e., internalized − recycled the OptiPrep manufacturer) were determined. The fractions were then − degraded) from the total pool of internalized receptor. boiled in the presence of reducing laemmli sample buffer. Samples of each fraction containing the same amount of protein for the different cell types to be compared and representative of the total protein content of each Immunofl uorescence microscopy fraction were analyzed by SDS-PAGE followed by Western blotting. Cells were cultured in 35-mm diameter petri dishes as described in Cell For total cell extracts, cells were washed twice in PBS, extracted in types and culture conditions. After the treatments indicated in the specifi c 2× boiling laemmli sample buffer (200 μl for a 35-mm petri dish) containing sections, culture medium was removed, and cells were fi xed in 1% PFA in 100 mM DTT, scraped, and boiled for a further 10 min. Parallel samples 2.5 mM triethanolamine, pH 7.5, containing 0.1% Triton X-100 and 0.1% were extracted without DTT, and protein concentration was determined NP-40 for 25 min at RT (Lallemand et al., 2003). Before staining, 0.5% by bicinchoninic acid analysis. A total of 15 μg of protein was loaded in Triton X-100 in PBS was added for 10 min at RT. In some experiments, each lane and separated by SDS-PAGE, transferred onto nitrocellulose, immunofl uorescence microscopy for VEGFR-2 was performed using the and immunoblotted with the indicated antibodies. Avas12α1 antibody after cell fi xation (in vitro staining). The fi xation/ For detection, HRP-conjugated horse anti–mouse, goat anti–rabbit permeabilization method applied (Lallemand et al., 2003) allows an optimal (Cell Signaling), and rabbit anti–goat (DakoCytomation) secondary anti- observation of VEGFR-2 in internal compartments with in vitro staining bodies and ECL chemiluminescence reagent (GE Healthcare) were used. (primary antibody after cell fi xation). Data obtained with in vivo (see Internal- Films were scanned and bands were quantifi ed using ImageJ set on the un- ization assays) and in vitro staining were superimposable for both VEC’s calibrated OD function. Adobe Photoshop 7.0, Excel X for MacIntosh, and effect on VEGFR-2 vesicular labeling and the codistribution of VEGFR-2 Adobe Illustrator 11 for the PC were used to produce the fi gures presented. with markers of specifi c compartments such as EEA-1 and caveolin-1. A com- parison of VEGFR-2 vesicular distribution after in vivo staining (with and Online supplemental material without acid wash before fi xation) is shown in Fig. S5. Fig. S1 shows that PV-1 colocalizes with caveolin in VEC-null and -positive In some experiments (in particular for clathrin detection), fi xation was cells. Fig. S2 shows that cell confl uence modulates VEGF-induced PY1175– performed in 1% PFA, and permeabilization was performed with 0.02% VEGFR-2–positive compartments in HUVECs. Fig. S3 shows that clathrin saponin that was maintained for all of the staining procedure. Fluorophore- siRNA inhibits the formation of clathrin-positive vesicular compartments. labeled secondary antibodies produced in donkey had minimal cross- Fig. S4 shows that the cytoplasmic domain of VEC is required to modulate reactivity to other species except for the targeted species (Invitrogen). VEGFR-2 internalization from the plasma membrane. Fig. S5 shows that Anti–VEGFR-2 was revealed with AlexaFluor488-conjugated secondary the antibody Avas12α1 does not modify either the basal or VEGF- stimulated antibodies (anti–rat for anti–mouse VEGFR-2 and anti–rabbit for anti– human tyrosine phosphorylation of VEGFR-2 and does not induce VEGFR-2 inter- VEGFR-2). In double labeling experiments, AlexaFluo488- and -647 fl uoro- nalization. Online supplemental material is available at http://www.jcb. chromes were used to stain each of the two antigens, respectively. org/cgi/content/full/jcb.200602080/DC1. Samples were observed under a fl uorescence microscope (DMR; Leica) using 63× and 100× lenses. Images were captured using a charge- We are most grateful to Kendra Swirsding for editing the manuscript. coupled camera (model 3; Hamamatsu) before processing through Adobe This work was supported by the Associazione Italiana per la Ricerca sul Photoshop for MacIntosh. Quantifi cation of vesicular labeling was per- Cancro, the European Community (grants QLRT-2001-02059, Integrated Proj- formed using the ImageJ program (version 10.2). For comparison pur- ect Contract LSHG-CT-2004-503573, NoE MAIN 502935, and NoE EVGN poses, different sample images of the same antigen were acquired under 503254), Italian Ministry of Health, Ministero dell’Università e della Ricerca/ constant acquisition settings. The best-fi t lower threshold to eliminate most Fondo degli Investimenti della Ricerca di Base (grants RBNE01MWA_009 of the signal background was determined using the threshold tool and con- and RBNE01F8LT_007), and the Cariplo Foundation. C. Tacchetti received fi rmed by visual inspection and count of one-pixel dimension particles. a grant from the Telethon Foundation (GTF03001). In some cases, the same sample was analyzed at different lower thresholds to determine the best fi t. Upper threshold was always set at 255. Particles Submitted: 14 February 2006 with a minimum size of fi ve pixels were counted. For colocalization analy- Accepted: 13 July 2006 sis, images were acquired using a confocal microscope (TCS SP2 AOBS; Leica) with a 63× objective and a 3× zoom. Colocalization was quanti- fi ed using the colocalization plugin of ImageJ. The channel ratio was al- References ways set at 90%. 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Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments

Lampugnani, Maria Grazia; Orsenigo, Fabrizio; Gagliani, Maria Cristina; Tacchetti, Carlo; Dejana, Elisabetta
The Journal of Cell Biology , Volume 174 (4) – Aug 14, 2006
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Copyright © 2006, The Rockefeller University Press
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0021-9525
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Abstract

JCB: ARTICLE Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments 1,2 1 4 4 Maria Grazia Lampugnani, Fabrizio Orsenigo, Maria Cristina Gagliani, Carlo Tacchetti, 1,2,3 and Elisabetta Dejana 1 2 IFOM, Fondazione Italiana per la Ricerca sul Cancro Institute of Molecular Oncology, Mario Negri Institute for Pharmacological Research, and Department of Biomolecular and Biotechnological Sciences, Faculty of Sciences, University of Milan, 20139 Milan, Italy Department of Experimental Medicine, University of Genova, 16146 Genova, Italy eceptor endocytosis is a fundamental step in con- VEC is absent or not engaged at junctions, VEGFR-2 is trolling the magnitude, duration, and nature of cell internalized more rapidly and remains in endosomal R signaling events. Confl uent endothelial cells are con- compartments for a longer time. Internalization does not tact inhibited in their growth and respond poorly to the terminate its signaling; instead, the internalized receptor proliferative signals of vascular endothelial growth factor is phosphorylated, codistributes with active phospholipase (VEGF). In a previous study, we found that the association C–γ, and activates p44/42 mitogen- activated protein of vascular endothelial cadherin (VEC) with VEGF receptor kinase phosphorylation and cell proliferation. Inhibition of (VEGFR) type 2 contributes to density- dependent growth VEGFR-2 internalization reestablishes the contact inhibition inhibition (Lampugnani, G.M., A. Zanetti, M. Corada, of cell growth, whereas silencing the junction-associated T. Takahashi, G. Balconi, F. Breviario, F. Orsenigo, density-enhanced phosphatase-1/CD148 phosphatase A. Cattelino, R. Kemler, T.O. Daniel, and E. Dejana. restores VEGFR-2 internalization and signaling. Thus, 2003. J. Cell Biol. 161:793–804). In the present study, VEC limits cell proliferation by retaining VEGFR-2 at we describe the mechanism through which VEC reduces the membrane and preventing its internalization into VEGFR-2 signaling. We found that VEGF induces the signaling compartments. clathrin- dependent internalization of VEGFR-2. When Introduction Endothelial cells are contact inhibited in their growth and lose nucleus and modulate cell transcription. In tumor cells, the neg- the capacity to respond to growth factors when they reach ative effect of epithelial cadherin (E-cadherin) on cell growth is confl uence. This phenomenon is mediated by different concurrent a result of its capacity to bind β-catenin and inhibit its translo- mechanisms. Molecules at cell to cell junctions, such as cad- ca tion to the nucleus. This effect is detected in tumor cell lines herins, may transfer signals that reduce the capacity of the cells to in which cytosolic β-catenin ubiquitination and destruction is respond to proliferative stimuli (Dejana, 2004; Gumbiner, 2005). impaired (St Croix et al., 1998; Mueller et al., 2000; Gottardi Cadherins are located at intercellular adherens junctions and are et al., 2001; Stockinger et al., 2001; Bryant and Stow, 2005). linked to different intracellular partners that include β-catenin, Endothelial cells express a cell-specifi c cadherin called plakoglobin, p120, Src (Gumbiner, 2005), csk (Baumeister et al., vascular endothelial cadherin (VEC). This protein exerts a neg- 2005), and density-enhanced phosphatase-1 (DEP-1)/CD148. ative effect on cell growth by binding VEGF receptor (VEGFR) β-catenin, plakoglobin, and p120 can also translocate to the type 2 and inhibiting its signaling activity (Carmeliet et al., 1999; Shay-Salit et al., 2002; Lampugnani et al., 2003; Correspondence to Elisabetta Dejana: [email protected] Dejana, 2004). VEGF is a major growth factor for endothelial Abbreviations used in this paper: EEA-1, early endosomal antigen-1; DEP-1, cells and plays an important role in the formation of new ves- density-enhanced phosphatase-1; GSH, glutathione; HUVEC, human umbilical sels during embryogenesis and in proliferative diseases (Alitalo vein endothelial cell; PY, phosphotyrosine; VEC, vascular endothelial cadherin; VEGFR, VEGF receptor. et al., 2005; Carmeliet, 2005; Ferrara and Kerbel, 2005). In The online version of this article contains supplemental material. blood endothelium, the activities of VEGF are mediated by its © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 174, No. 4, August 14, 2006 593–604 http://www.jcb.org/cgi/doi/10.1083/jcb.200602080 JCB 593 THE JOURNAL OF CELL BIOLOGY interaction with two tyrosine kinase receptors, VEGFR-1 (fl t-1) Little is known about the internalization pathways fol- and -2 (fl k/KDR), as well as neuropilins. The growth signals are lowed by VEGFR-2 or their functional signifi cance (Labrecque transferred, to a large extent, through the activation of PLC-γ, et al., 2003; Bhattacharya et al., 2005; Mitola et al., 2006). It has PKC, and subsequently p44/42 MAPK (Takahashi et al., 1999, been reported that cadherins may infl uence growth factor re- 2001; Matsumoto et al., 2005; Singh et al., 2005). ceptor internalization, but the extent to which they do depends We found that in contact-inhibited endothelial cells, on the cadherin or growth factor receptor. In tumor cell lines, VEGFR-2 forms a complex with VEC that results in the inhi- N-cadherin forms a complex with FGF receptor 1 that inhibits bition of its tyrosine phosphorylation and, consequently, in the its internalization and degradation. This causes a sustained FGF attenuation of MAPK activation. This effect was attributed to the signaling and abnormal cell growth (Suyama et al., 2002). In phosphatase DEP-1/CD148 that, by binding β-catenin and p120, contrast, E-cadherin cointernalizes with FGF receptor 1, which may associate with the cadherin–receptor complex and dephos- facilitates its nuclear translocation and signaling activity (Bryant phorylate the receptor (Lampugnani et al., 2003). In this study, and Stow, 2005; Bryant et al., 2005). we go further by describing another aspect of this phenomenon. In this study, we analyzed the role of VEC on VEGFR-2 Upon activation with specifi c ligands, growth factor recep- internalization and signaling in endothelial cells. We found tors are internalized via clathrin-dependent and - independent path- that the receptor is internalized more rapidly and effi ciently ways. In many cases, this process leads to signaling termination via when VEC is absent or not clustered at intercellular contacts. degradation of the activated receptor complex. Therefore, internal- Strikingly , internalization does not terminate receptor signaling, ization is considered an important mechanism through which cells which instead continues in endosomes. This may explain why may control the intensity and duration of signal transduction. VEC-null cells present increased and uncontrolled growth. However, more recent fi ndings indicate that internaliza- tion is not just a sink through which receptors are degraded Results (Di Fiore and De Camilli, 2001; Miaczynska et al., 2004). On the contrary, some receptors, such as TGF-β, EGF, or NGF recep- VEC expression inhibits VEGFR-2 tors, can maintain their signaling activity from within intra- internalization cellular compartments (Suyama et al., 2002; Di Guglielmo We fi rst investigated whether the establishment of cell to et al., 2003; Bryant et al., 2005; Sigismund et al., 2005). cell contact modulates VEGFR-2 internalization. Using freshly Figure 1. VEC clustering at cell–cell con- tacts inhibits VEGFR-2 endocytosis. (A) The internalization of VEGFR-2 from the plasma membrane was analyzed in sparse and con- fl uent HUVECs treated with VEGF for 5 min. To detect the internalized receptor, cells were treated with a recombinant single chain anti- body to human VEGFR-2, scFvA7, and acid washed before fi xation and processing for immunofl uorescence microscopy. Internalized VEGFR-2 appears in a vesicular pattern that is more abundant in sparse than in confl uent cultures. (bottom) The granular staining after different incubation lengths with VEGF was quantifi ed using the ImageJ program (see Materials and methods). The results (referred to as events per cell) reported in the graph are means ± SD ( error bars) of three independent experiments. At least seven random fi elds were analyzed for each time point in each experiment. (B) VEC-null and -positive confl u- ent cultures were treated as described in A, but the anti–mouse VEGFR-2 clone Avas12α1 was used. The micrographs show a typical vesicular labeling pattern after a 10-min treat- ment with VEGF. The staining appears more abundant in VEC-null than in VEC-positive cells. (bottom) The time course of vesicular labeling in response to VEGF was analyzed as described in A. The binding of the antibody does not activate VEGFR-2 nor does it induce its internalization (for details see Fig. S5, avail- able at http://www.jcb.org/cgi/ content/full/ jcb.200602080/DC1). In A and B, nuclei stained with DAPI appear blue. *, P ≤ 0.05; **, P ≤ 0.01 by comparing sparse versus con- fl uent (A, bottom) and VEC-null versus -positive (B, bottom) cells by analysis of variance and the Duncan test. Bars (A), 15 μm; (B) 20 μm. 594 JCB • VOLUME 174 • NUMBER 4 • 2006 isolated human umbilical vein endothelial cells (HUVECs) than -positive cells. Receptor degradation exceeds recycling by stimu lated with VEGF, we observed that VEGFR-2 endo- about fi vefold in both cell types, but both parameters are signifi - cytosis, which was evaluated by immunofl uorescence labeling cantly increased in the absence of VEC (Fig. 2, C and D). These of intracellular vesicular compartments, was signifi cantly re- data indicate that a higher amount of VEGFR-2 is internalized, duced by cell density (Fig. 1 A). Time course analysis revealed degraded, and recycled in the absence of VEC. that in sparse cells, the number of receptor-positive vesicles in- Internalization of growth factor receptors may follow creased more rapidly and to a larger extent than in confl uent clathrin-dependent or -independent pathways. Among the lat- cells (Fig. 1 A, bottom). ter, caveolae have been shown to regulate receptor internali- This fi rst observation suggested that the establishment of zation directed toward degradation (Di Guglielmo et al., cell to cell contact reduced VEGFR-2 internalization. Because 2003; Sigismund et al., 2005). Our codistribution experi- VEC plays a role in VEGFR-2 signaling, we investigated ments of VEGFR-2 with early endosomal antigen-1 (EEA-1) whether VEC could be involved inVEGFR-2 internalization. and caveolin-1 show that VEGFR-2 internalizes mostly in We compared syngenic endothelial cell lines differing for the ex- EEA-1–positive early endosomes (Fig. 3) and to a very low pression of VEC. These cells had been characterized previously extent in caveolae. Colocalization of VEGFR-2 and the caveolar in detail and presented superimposable levels of VEGFR-2 (see component PV-1 (Stan et al., 2004; Stan, 2005) was also neg- Fig. 8; Lampugnani et al., 2002, 2003). As shown in Fig. 1 B, ligible (unpublished data). To control whether caveolae were after the addition of VEGF, the number of VEGFR-2– containing ex pressed correctly and to a comparable extent in both VEC- vesicular compartments is markedly higher in the absence of null and -positive cells, we costained these structures with VEC. Quantifi cation of the amount of biotinylated receptor PV-1 and caveolin antibodies. As shown in Fig. S1 (available that was internalized, degraded, or recycled back to the plasma at http://www.jcb.org/cgi/ content/full/jcb.200602080/DC1), membrane is reported in Fig. 2. In VEC-null endothelium, the the extensive and comparable colocalization of caveolin and receptor is internalized more quickly and to a higher extent than PV-1 could be observed in both cell types, suggesting structural in VEC-positive cells (Fig. 2, A and B). integrity of the caveolar compartment. In VEC-null cells, receptor internalization kinetics appear The preferential distribution of VEGFR-2 in EEA-1– faster in biotinylation than in immunofl uorescence experiments. positive endosomes was further confi rmed using immuno-EM This apparent discrepancy may be caused by internalization (Fig. 3 C). In addition, silencing clathrin heavy chain expres- compartments, which can be measured in biotinylation experi- sion either by siRNA (see Fig. 8 and Fig. S3, available at ments because they are protected from glutathione (GSH) re- http://www.jcb.org/cgi/content/full/jcb.200602080/DC1) or dis- duction but are not yet clustered in structures resolvable in rupting clathrin-coated pits by hypertonic medium blocked immunofl uorescence microscopy. receptor internalization both in VEC-positive and -null cells (Fig. 4). The overall amount of internalized receptor for the dura- In contrast, after incubation with fi lipin at a concentration able tion of the experiment is about fourfold more in VEC-null to fully disrupt lipid rafts and caveolae (Schnitzer et al., 1994; Figure 2. Internalization, degradation, and recycling of biotinylated VEGFR-2 are inhibited in VEC-positive cells. (A) Internalization and re- cycling of VEGFR-2 was measured after cell surface biotinylation with thiol-cleavable Sulfo- NHS-SS-Biotin. At selected time points, biotin was cleaved by GSH followed by immuno- precipitation of VEGFR-2 and probing with HRP-streptavidin as described in Materials and methods. Recycling was measured as the amount of biotinylated receptor reappearing on the cell surface (and therefore not protected by GSH cleavage) at the indicated time points after 10 min of internalization in the presence of VEGF (indicated as 0 min in recycling panel). Total, total amount of labeled receptor after Sulfo-NHS-SS-Biotin labeling at 4°C (with- out GSH treatment). GSH, treatment with re- ducing GSH to remove any labeling on the residual surface-exposed receptor. (B) Densito- metric analysis of internalization expressed as a percentage of total labeling. (C and D) Quantitation of receptor degradation and re- cycling as a percentage of the amount of receptor internalized after 10 min with VEGF. Although A presents a representative experi- ment, the values in B–D are the means of four independent experiments ± SD (error bars). **, P ≤ 0.01; *, P ≤ 0.05 comparing VEC-null with -positive cells, respectively, by analysis of variance and the Duncan test. VE-CADHERIN AND VEGFR-2 INTERNALIZATION • LAMPUGNANI ET AL. 595 Figure 3. Internalized VEGFR-2 colocalizes with EEA-1– positive compartments by immunofl uorescence analysis. HUVECs, VEC-null, and VEC-positive cells were double la- beled for VEGFR-2 and either EEA-1 or caveolin-1. Cells were activated with VEGF for 10 min. (A) Representative exam- ples of confocal images for each antigen and their respective merges (boxed areas; 3.5-fold magnifi cation) are shown for confl uent HUVECs after treatment with VEGF. VEGFR-2 was revealed with an AlexaFluor488-conjugated secondary anti- body and is shown in green. EEA-1 and caveolin revealed with AlexaFluor647-conjugated secondary antibodies are shown in red. Nuclei appear blue after DAPI staining. Bars, 10 μm. (B) To quantify colocalization events, images were an- alyzed using the ImageJ colocalization plugin (as described in Materials and methods). The graphs present the number of colocalization events normalized for the number of VEGFR-2– positive compartments. After VEGF treatment, 45–55% of VEGFR-2–positive compartments showed colocalization with EEA-1 in all of the situations examined. Colocalization of internalized VEGFR-2 with caveolin-1 was negligible. Values are the mean of at least three experiments ± SD (error bars). In each experiment, at least fi ve random fi elds were analyzed for each point. *, P ≤ 0.01 by t test. (C, a and b) Immuno- gold labeling of EEA-1 (10 nm gold; arrows) and VEGFR-2 (15 nm gold; arrowheads) on an ultrathin cryosection of VEC-null and -positive cells treated with VEGF for 10 min. The panels display VEGFR-2 labeling of EEA-1–positive endo- somes in VEC-null rather than in VEC-positive cells. (c and d) Under the same conditions, morphologically identifi ed caveo- lae are devoid of VEGFR-2. Bar (a), 227 nm; (b) 222 nm; (c) 350 nm; (d) 370 nm. Andriopoulou et al., 1999), receptor internalization did not sig- We fi rst tested whether the internalized receptor retained tyro- nifi cantly change (Fig. 4). sine phosphorylation by cell fractionation on an iodixanol gradient We also performed experiments at different time points (Yeaman et al., 2001). Antibodies recognizing phosphotyrosine (5, 7, 10, 20, and 30 min) after the addition of VEGF in VEC- (PY) 1214– and PY1054/59–VEGFR-2 were used. As shown in null and -positive cells using caveolin and PV-1 as markers of Fig. 5, in the absence of VEC, a higher amount of phosphorylated caveolae and EEA-1 and Rab-5 as markers of early endosomes. VEGFR-2 is detected in intracellular fractions, whereas in the At all time points considered, we could not see internalized presence of VEC, the phosphorylated receptor remains preferen- VEGFR-2 in vesicles positive to either caveolin or PV-1 but tially in fractions corresponding to peripheral plasma membranes. only in bona fi de early endosomes (unpublished data). These Phosphorylation of VEGFR-2 tyrosine 1175 is required data strongly suggest that, at least within the experimental con- for binding and activation of PLC-γ, which is the major effector ditions used, VEGFR-2 endocytosis in endothelial cells is mostly of VEGF-mediated cell proliferation (Takahashi et al., 2001). By clathrin dependent. using antibodies specifi c for PY1175–VEGFR-2, we observed that in the absence of VEC, a higher amount of internalized Internalized VEGFR-2 retains receptor was phosphorylated at this specifi c tyrosine (Fig. 6, signaling activity A and B). Consistently, more internalized PY1175–VEGFR-2 In the absence of VEC, endothelial cells respond more effec- was found in sparse than in confl uent HUVECs (Fig. S2, available tively to growth signals transferred by VEGFR-2. From the at http://www.jcb.org/cgi/content/full/jcb.200602080/DC1). aforementioned observations, we concluded that VEGFR-2 is To further prove that PLC-γ could be activated by the inter- internalized more quickly and to a larger extent in VEC-null nalized receptor, we stained the cells with antibodies directed cells than in VEC-positive cells. Therefore, we asked whether to the active PY783–PLC-γ. As shown in Fig. 7, active PLC-γ the receptor retains its signaling activity when sequestered in codistributes with internalized VEGFR-2 more effectively in intracellular compartments. the absence than in the presence of VEC. 596 JCB • VOLUME 174 • NUMBER 4 • 2006 Figure 4. VEGFR-2 is internalized through a clathrin-dependent pathway. (A) VEC-null and -positive cells were transfected with either Stealth siRNA targeting the mouse clathrin heavy chain or negative Stealth siRNA control duplex (and used 72 h later), or the cells were treated with hypertonic medium (0.45 M sucrose for 30 min) or 1 μg/ml fi lipin for 1 h. The micrographs show VEGFR-2 immuno- fl uorescence staining after VEGF treatment for 10 min. Both clathrin heavy chain siRNA and hypertonic medium strongly reduced VEGFR-2 vesicular patterning both in VEC-null and -positive cells. Using fi lipin to interfere with the caveolar compartment had no effect on either cell type. The negative Stealth siRNA control duplex produced results that were indistinguishable from untreated cells (not depicted). Nuclei appear blue after DAPI staining. Bar, 20 μm. (B) Column graphs represent VEGFR-2 vesicu- lar labeling in VEGF-treated cells quantifi ed by ImageJ. Ctr, control; HM, hypertonic medium. Values normalized per cell are the mean of three independent experiments ± SD (error bars). In each experiment, at least fi ve inde- pendent fi elds were analyzed for each point. *, P ≤ 0.01 versus control values by analysis of variance and the Dunnet test. Inhibition of VEGFR-2 internalization receptor is internalized more effi ciently and retains its active state affects its proliferative signaling for a longer time, leading to continuous proliferative signaling. Overall, the aforementioned data indicate that VEC limits receptor To further test this hypothesis, we prevented receptor activation and internalization. In the absence of this protein, the internalization by silencing clathrin with two specifi c siRNAs Figure 5. VEC expression reduces the amount of PY–VEGFR-2 in internal compartments. Extracts of VEGF-treated VEC-null and -positive cells were fraction- ated on an iodixanol gradient as described in Materials and methods. Samples representative of the total protein content of each fraction were analyzed by SDS-PAGE and Western blotting. As expected, VEC concentrates in lower density fractions corresponding to plasmatic membranes, whereas clathrin and EEA-1 are enriched in higher density fractions corresponding to internal membranes (Yeaman et al., 2001). Upon stimulation with VEGF, PY1214–VEGFR-2 is enriched in fractions corresponding to the internal membranes in VEC-null cells. The graph shows the ratio between the phosphorylated receptor and the total receptor present in each fraction. For quantifi cation, in each fraction, we considered the band with the molecular mass of the mature form of the receptor at the plasma membrane ( 210 kD; see fraction 1 and arrowheads; Takahashi and Shibuya, 1997). We chose this band by making the assumption that full-length VEGFR-2 represents the signaling form of the receptor. At increasing density, a lighter band appears both in VEC-null and -positive cells (asterisks). It is likely that this band derives from VEGFR-2 processing in internal compartments (for instance, proteolytic or intermediate synthesis products) and is indeed not present in the peripheral membrane fractions. Comparable results were obtained using an antibody to PY1054/59–VEGFR-2 (not depicted). VE-CADHERIN AND VEGFR-2 INTERNALIZATION • LAMPUGNANI ET AL. 597 VEC mutants lacking either the β-catenin– or p120-binding domains were unable or less effi cient, respectively, to coimmuno- precipitate VEGFR-2. We found that these mutants were also unable to signifi cantly prevent VEGFR-2 internalization (Fig. S4, available at http://www.jcb.org/cgi/content/full/ jcb.200602080/DC1), suggesting that receptor internalization is reduced as a consequence of binding to VEC. Similar to E-cadherin (for review see Bryant and Stow, 2004), VEC can be internalized through clathrin-coated pits (Xiao et al., 2005). We tested whether VEC codistributes with VEGFR-2 in intracellular compartments. As reported in Fig. 9 upon VEGF activation, no signifi cant codistribution of VEC with VEGFR-2–positive vesicles is detected in VEC-positive cells and HUVECs (Fig. S4). Only junctional colocalization can be observed (Fig. 9). The lack of codistribution in internal compartments was confi rmed by immuno-EM (unpublished data). Collectively, these data suggest that the receptor internal- izes upon dissociation from VEC. VEGFR-2 phosphorylation is required for internalization and signaling In previous studies, we showed that the phosphatase DEP-1/ CD148 can reduce VEGFR-2 signaling. DEP-1/CD148 can Figure 6. Internalized VEGFR-2 is phosphorylated at tyrosine 1175. (A) VEC-null and -positive cells were stimulated with VEGF as in Fig. 1, fi xed, associate with β-catenin and p120 (Holsinger et al., 2002; and processed for immunofl uorescence microscopy. The vesicular labeling Palka et al., 2003) and with the VEC–VEGFR-2 complex, re- pattern observed with PY1175–VEGFR-2 antibody after stimulation with ducing VEGFR-2 phosphorylation (Lampugnani et al., 2003). VEGF was signifi cantly more abundant in VEC-null than in VEC-positive cells. Nuclei appear blue after DAPI staining. Bar, 20 μm. (B) Images were Therefore, we tested whether DEP-1/CD148 could also reduce analyzed by ImageJ to quantify PY115–VEGFR-2–positive compartments receptor internalization. (see Materials and methods). The graph presents the mean ± SD (error As reported in Fig. 10 A, in endothelial cells transfected bars) calculated from 18 random fi elds that were analyzed through ImageJ in fi ve independent experiments and is normalized to the number of cells with DEP-1/CD148 siRNA, VEGFR-2 internalization is signi- per fi eld. In each experiment, at least three random fi elds were analyzed. fi cantly higher. This effect is accompanied by an increase in *, P ≤ 0.01 comparing VEC-null with -positive cells after VEGF by analysis VEGFR-2 phosphorylation and MAPK activation (Lampugnani of variance and the Duncan test. et al., 2003). These data suggest that retention of VEGFR-2 at the membrane by VEC allows its dephosphorylation by DEP-1/ (Figs. 4, 8, and S3) that target independent sequences of clathrin CD148 and limits its internalization and signaling. heavy chain messenger. As expected, VEGF-induced phosphor- ylation of VEGFR-2 and activation of p44/42 MAPK were Discussion higher in the absence than in the presence of VEC (Fig. 8). When siRNA clathrin was applied to VEC-null cells, both re- In this study, we report a novel aspect of the mechanism through ceptor and MAPK phosphorylation dropped to values compara- which VEC expression and clustering inhibits VEGFR-2 prolif- ble with those of VEC-positive cells. In contrast, in the presence erative signaling. We found that in the absence of VEC or in of VEC, the effect of siRNA clathrin was either weak or conditions in which VEC is not clustered at adherens junctions undetectable. Consistently, the inhibition of MAPK activation as in sparse cells, VEGFR-2 is endocytosed to a higher extent in was also observed by treating the cells with hypertonic medium, intracellular compartments, from where it maintains its signaling whereas treatment with fi lipin did not modify MAPK activa- activity. VEC could therefore reduce receptor activity by in- tion in response to VEGF either in VEC-positive or -null cells hibiting VEGFR-2 internalization and promoting its inactivation (unpublished data). Overall, these results strongly suggest that at the cell surface. internalization protects the receptor from dephosphorylation In our experimental conditions, VEGFR-2 is internal- and, therefore, increases and prolongs its proliferative signaling. ized in early endosomes mostly through a clathrin-dependent pathway. We were unable to detect caveolin-1–positive vesi- VEC association with VEGFR-2 cles containing VEGFR-2, and we could not inhibit receptor is required for receptor retention internalization using a caveolae-perturbing drug such as fi lipin at the plasma membrane (Schnitzer et al., 1994; Andriopoulou et al., 1999). Other We then investigated the mechanism through which VEC in- studies found the codistribution of VEGFR-2 with caveolin-1 hibits VEGFR-2 internalization. In a previous study, we found (Labrecque et al., 2003; Bhattacharya et al., 2005; Ikeda et al., that VEC forms a complex with VEGFR-2, and we analyzed the 2005), and we cannot exclude that under different experimental domains of VEC involved in this process (Lampugnani et al., 2003). conditions, VEGFR-2 may be internalized through caveolae. 598 JCB • VOLUME 174 • NUMBER 4 • 2006 Figure 7. Colocalization of internalized VEGFR-2 and acti- vated PLC-𝛄 . (A) VEC-null and -positive cells were labeled in vivo with antibodies to VEGFR-2, stimulated with VEGF as in Fig. 6, acid washed, fi xed, and processed for immunofl uo- rescence microscopy. Cells were double labeled with an anti- body that recognizes PLC-γ only when phosphorylated at tyrosine 783. VEGFR-2 was revealed with an AlexaFluor488- conjugated secondary antibody and is shown in green. PY783–PLC-γ, which was revealed with an AlexaFluor647- conjugated secondary antibody, is shown in red. For each cell type, the bottom panel on the right (threefold magnifi ca- tion of the boxed areas) shows the colocalization of VEGFR-2 and PY783–PLC-γ (pink) set upon the VEGFR-2 background (gray). This was obtained through the ImageJ colocalization plugin (see Materials and methods for details). Arrows point to the colocalization of PY783–PLC-γ and VEGFR-2. Bars, 10 μm. (B) The confocal images were analyzed through ImageJ to calculate the number of colocalization events. These values, which were normalized over the number of VEGFR-2– positive compartments per cell, are presented in the graph. After VEGF treatment, colocalization was about fi vefold higher in VEC-null than in VEC-positive cells. Values in the graph are the mean from three independent experiments ± SD (error bars). In each experiment, each point was calcu- lated from at least fi ve random fi elds. *, P ≤ 0.01 comparing VEC-null with -positive cells after VEGF by analysis of vari- ance and the Duncan test. However, the observed association with caveolin-1 may also in transgenic mice reduces permeability and angiogenic response represent a mechanism of receptor compartmentalization at the to VEGF (Bauer et al., 2005). plasma membrane. It was found that caveolin-1 would form It has been reported that VEGFR-2 internalization and degra- a molecular complex with VEGFR-2 that inhibits receptor acti- dation are regulated by ubiquitination through a Cbl-dependent vation in resting cells. Upon activation of the cells with VEGF, mechanism (Duval et al., 2003) or C-tail serine phosphorylation caveolin-1 is phosphorylated, and the complex rapidly disso- by activated PKC (Singh et al., 2005). These mechanisms may ciates (Labrecque et al., 2003). Thus, it is tempting to specu- coexist and may be responsible for the amount of receptor degra- late that similar to TGF-β receptor (Di Guglielmo et al., 2003), dation reported here. once VEGFR-2 is released from the caveolin-1 complex, it Our observations support the hypothesis that internal- becomes available for internalization in clathrin-coated pits. ized VEGFR-2 maintains its activity. These data are in agree- In agreement with this model, the overexpression of caveolin-1 ment with recent publications indicating that signaling through Figure 8. Silencing clathrin expression in- hibits VEGFR-2 phosphorylation and signaling in VEC-null cells. VEC-null and -positive cells were transfected with Stealth siRNA-targeting mouse clathrin heavy chain. Two oligonucleo- tides (a and b; respective sequences are re- ported in Materials and methods) were used that target two independent sequences of the clathrin heavy chain mRNA. Negative Stealth siRNA duplex was used as a control. After 72 h, cells were treated with VEGF or control me- dium for 10 min. They were then extracted and processed for Western blotting. Clathrin siRNA reduced clathrin heavy chain levels by 75 (oligonucleotide a) and 90% (oligonucleotide b) in both cell types. VEGFR-2 phosphorylation at tyrosine 1214 and p44/42 MAPK phos- phorylation in response to VEGF were strongly inhibited in VEC-null cells, whereas these pa- rameters were only barely affected in VEC- positive cells. Comparable results were obtained for PY1054/59–VEGFR-2 (not depicted). The graphs show the quantifi cation of these effects as means ± SD (error bars) of fi ve independent experiments. Fold increase after VEGF is shown for PY1214–VEGFR-2 and phospho-p44/42 MAPK. *, P ≤ 0.01 versus negative siRNA by variant analysis and the Dunnet test. VE-CADHERIN AND VEGFR-2 INTERNALIZATION • LAMPUGNANI ET AL. 599 A novel aspect of our work is that VEC inhibits VEGFR-2 internalization, thereby reducing its cell growth signaling activity. How can VEC inhibit receptor endocytosis? A likely hypothesis is that VEC retains VEGFR-2 at the membrane by binding to it. In addition to our studies, others have reported (Shay-Salit et al., 2002; Lampugnani et al., 2003; Weis et al., 2004; Lambeng et al., 2005) that VEGFR-2 couples with VEC. This process requires the binding of VEC to β-catenin and, to a lesser extent, to p120. In the present study, we found that mutants of VEC lacking the cytoplasmic domain responsible for binding either β-catenin or p120 and unable to associate with VEGFR-2 (Lampugnani et al., 2003) do not prevent VEGFR-2 internaliza- tion. This supports the idea that VEC–VEGFR-2 coupling is re- quired to inhibit receptor endocytosis. Cadherins themselves are endocytosed via several dif- ferent routes, including clathrin-dependent (Le et al., 1999; Palacios et al., 2002; Ivanov et al., 2004; Izumi et al., 2004) Figure 9. VEC does not codistribute with VEGFR-2 in internal compartments. and -independent pathways (Akhtar and Hotchin, 2001; Paterson VEC-positive cells were treated with VEGF for 10 min, double stained for et al., 2003). Therefore, cotraffi cking of receptor and cadherin VEGFR-2 and VEC, and analyzed by confocal microscopy. Besides junc- tional staining, VEC did not show any obvious vesicular pattern. VEGFR-2 complexes is possible (for reviews see Bryant and Stow, 2004, and VEC appeared to codistribute only at cell–cell contacts and not in intra- 2005; Cavallaro and Christofori, 2004). However, under our cellular compartments. The bottom panel on the right (2.6-fold magnifi cation experimental conditions, we could detect VEC in intracellular of the boxed areas) shows the colocalization of VEGFR-2 and VEC (yellow) set upon the VEC background (gray). This was obtained through the colo- compartments (Xiao et al., 2005), but we could not see codistri- calization plugin of ImageJ (see Materials and methods for details). Arrows bution with the receptor. Thus, it is likely that the receptor dis- point to the junctional colocalization of VEGFR-2 and VEC. Bars, 10 μm. sociates from VEC before internalization (Fig. 9). In a previous study, the phosphatase DEP-1/CD148 was growth factor receptors does not occur only at the cell mem- found to play a role in the inhibitory effect of VEC on VEGR-2 brane but may continue even more effectively from intracellular signaling (Lampugnani et al., 2003). This phosphatase associ- compartments (Di Fiore and De Camilli, 2001; Sorkin and Von ates with VEC through its binding to β-catenin and p120 and, Zastrow, 2002; Miaczynska et al., 2004; Le Roy and Wrana, in this way, reduces VEGFR-2 phosphorylation ( Lampugnani 2005). Clathrin-dependent internalization of TGF-β in early en- et al., 2003). We report that DEP-1/CD148 could prevent dosomes, where the Smad2 anchor SARA is enriched, promotes VEGFR-2 internalization along with the reduction of recep- TGF-β signaling (Di Guglielmo et al., 2003). EGF receptor is tor phosphorylation and signaling. Therefore, it is possible that internalized shortly after ligand addition in intracellular com- VEC, by retaining VEGFR-2 at the cell surface, allows its de- partments together with its downstream signaling factors shc, phosphorylation by DEP-1/CD148, which, in turn, inhibits its Grb2, and mSOS (Di Guglielmo et al., 1994) and maintains internalization and signaling. its signaling activity (Pennock and Wang, 2003). Similarly, Besides VEC, VEGFR-2 was found to bind to integrins (Soldi the specifi c activation of endosome-associated PDGF receptor et al., 1999). Another study reported that when cells are plated leads to the activation of its major signaling pathways (Wang on collagen I, the phosphatase SHP2 can associate with VEGFR-2 et al., 2004). Thus, as for VEGFR-2, endocytic transport is im- and stimulate its internalization. SHP2 activates Src, which in portant not only for receptor turnover but also for regulating turn activates dynamin II–dependent receptor internalization signal transduction and for mediating the formation of special- (Mitola et al., 2006). Interestingly, this phenomenon does not ized signaling complexes. occur when cells are plated on vitronectin, and SHP2 does not Figure 10. DEP-1/CD148 silencing in VEC-positive cells in- creases internalization, tyrosine phosphorylation, and activity of VEGFR-2. VEC-positive cells were transfected with either Stealth siRNA-targeting mouse DEP-1/CD148 or a negative Stealth siRNA control duplex. After 72 h, cells were treated with VEGF for 10 min and either fi xed and processed for immuno- fl uorescence microscopy or extracted and processed for Western blotting. DEP-1/CD148 siRNA reduced DEP-1/ CD148 protein by 45% (B). (A) VEGFR-2–positive vesicular compartments were found to be signifi cantly higher (>80%) in VEGF-treated cells after transfection with DEP-1/CD148 siRNA. Means of three independent experiments ± SD (error bars) are shown. In each experiment, at least fi ve random fi elds were analyzed. *, P ≤ 0.01 comparing negative with DEP-1 siRNA interference by analysis of variance and the Duncan test. (B) Phosphorylation of VEGFR-2 at tyrosine 1214 and of p44/42 MAPK was increased by 80 and 60%, respectively, after DEP-1/CD148 siRNA transfection in VEGF-treated cells. 600 JCB • VOLUME 174 • NUMBER 4 • 2006 bind to VEGFR-2 (Mitola et al., 2006). These observations sug- RNA interference Cells were seeded as described in Cell types and culture conditions. 20 h gest that the capacity of different adhesive proteins to complex later, cells were washed once with OptiMEM (Life Technologies) and trans- with growth factor receptors and modulate their internalization fected with 40 nM of Stealth oligonucleotides (Invitrogen) using 2 μl/ml and signaling may be a general paradigm. In this way, cells may LipofectAMINE 2000 (Invitrogen) in OptiMEM according to the manufac- turer’s instructions. After 5 h, the transfection medium was removed, and modulate their growth and survival as a function of density and complete culture medium was added. Cells were cultured for a further 72 h interaction with specifi c matrix proteins. before stimulation and processing for immunofl uorescence and extraction. In conclusion, the results reported in this study are con- Stealth oligonucleotides were used as follows: a, 5′-G C A G U U G U U- C A U A C C C A U C U U C U U A -3′ (start nucleotide was 2,118 bases down- sistent with the idea that VEGFR-2 proliferative signaling is in- stream of the start codon); b, 5′-G A A G A A C U C U U U G C C C G G A A A U U U A -3′ creased by endocytosis. The inhibitory role of VEC is likely that (start nucleotide was 1,284 bases downstream of the start codon) to target of binding and retaining the receptor at the cell surface, prevent- mouse clathrin heavy chain; and 5′-U C G A G C C A G U G A G C A U G U U U G G- A A A -3′ (start nucleotide was 4,023 bases downstream of the start codon) ing its endocytosis, and favoring inactivation by DEP-1/CD148. to target mouse DEP-1/CD148. As a control, a Stealth siRNA-negative This suggests that the modulation of VEC–VEGFR-2 complex control duplex oligonucleotide (Invitrogen) with a C/G content equivalent formation may be a novel strategy to regulate VEGF proliferative to the positive oligonucleotide was used. signaling and, therefore, to inhibit or stimulate angiogenesis. Internalization assays Microscopy. Cells were treated in vivo with anti–VEGFR-2 antibody and acid washed before fi xation (Ehrlich et al., 2001; Di Guglielmo et al., Materials and methods 2003) as follows. We used monoclonal antibodies to the extracellular domain of human and mouse VEGFR-2 (see Primary antibodies) that are Primary antibodies described by the manufacturers to be devoid of biological activity. As this For the detection of VEGFR-2, anti–human VEGFR-2 (single chain recom- aspect was crucial to the assay, we tested the effect of these antibodies on binant; clone scFvA7 with E tag; RDI and Fitzgerald) and anti–mouse basal and VEGF-stimulated phosphorylation and internalization of VEGFR-2. VEGFR-2 (rat clone Avas12α1; RDI and Fitzgerald) were used for immuno- We found that these antibodies do not stimulate the phosphorylation fl uorescence; rabbit polyclonal C-1158 (sc504; Santa Cruz Biotechnology, of VEGFR-2 in either condition. These results are reported in Fig. S5 Inc.) was used for Western blotting. Antibodies to tyrosine-phosphorylated (available at http://www.jcb.org/cgi/content/full/jcb.200602080/DC1). VEGFR-2 were rabbit polyclonal PY1214 and PY1054/59 (Biosource Anti–mouse VEGFR-2 was dialyzed for 3 h against PBS (with one change) International) and rabbit polyclonal PY1175, which was provided by to remove azide. Cells were precooled for 30 min on ice and treated with M. Shibuya (University of Tokyo, Tokyo, Japan). Antibodies to clathrin 10 μg/ml of antibody for 1 h on ice with gentle agitation. Before stimula- heavy chain were mouse monoclonal cloneX22 (Affi nity BioReagents, Inc.) tion, cells were washed with ice-cold 1% BSA in MCDB 131 to remove for immunofl uorescence and mouse monoclonal clone 23 (R&D Systems) unbound antibody, and fresh medium was added. Cells were stimulated for Western blotting. Antibody to EEA-1 was goat polyclonal N-19 with 80 ng/ml VEGF and transferred to 37°C. After the indicated time (sc-6415; Santa Cruz Biotechnology, Inc.); antibody to caveolin-1 was rabbit intervals, cells were placed on ice and acid washed (three washes with 2+ 2+ polyclonal N-20 (sc-894; Santa Cruz Biotechnology, Inc.); and antibody ice-cold 50 mM glycine in Ca /Mg HBSS, pH 2.5, and two washes with 2+ 2+ to VEC was goat polyclonal C-19 (sc-6458; Santa Cruz Biotechnology, Ca /Mg HBSS, pH 7.5) to remove the antibody from the cell surface. Inc.) and mouse monoclonal BV6 and BV9 (produced in our laboratory; Cells were then fi xed as described in Immunofl uorescence microscopy. To Corada et al., 2001). Antibody to PY783-PLCγ, total p42/44 MAPK, and reveal the distribution of the primary antibody, AlexaFluor488-conjugated phospho-p42/44 MAPK was rabbit polyclonal (Cell Signaling). Antibody donkey anti–rat (Invitrogen) was used for the rat anti–mouse VEGFR-2. For to DEP-1/CD148/CD148 was goat polyclonal (R&D Systems), and anti- the recombinant E-tagged anti–human VEGFR-2, rabbit anti–E-tag (Abcam) body to PV-1 was rat monoclonal (provided by R. Stan, Dartmouth Medical followed by AlexaFluor488-conjugated donkey anti–rabbit (Invitrogen) School, Lebanon, NH). were used. Cell surface biotinylation and immunoprecipitation. Internalization, Cell types and culture conditions recycling, and degradation were measured as described previously by Endothelial cells with a homozygous null mutation of the VEC gene (VEC Fabbri et al. (1999) with the following modifi cations. Cells were put on ice 2+ 2+ null) and the cell lines derived from them through retroviral gene transfer and washed three times with ice-cold PBS containing Ca and Mg 2+ 2+ 2+ 2+ and expressing wild-type (VEC positive) or various VEC mutant constructs (Ca /Mg PBS). For surface biotinylation, cells in Ca /Mg PBS were were generated and characterized as described previously in detail treated with 0.5 mg/ml of thiol-cleavable Sulfo-NHS-S-S-Biotin (Pierce (Lampugnani et al., 2003). For all of the experiments, 50,000 cells/cm Chemical Co.) for 1 h on ice. They were then washed on ice twice with 2+ 2+ (to reach confl uence within 24 h) were seeded in complete culture medium Ca /Mg PBS, once with MCDB 131, and once with 1% BSA MCDB and cultured without medium change for 72 h. Cells were then washed 131. 80 ng/ml VEGF in fresh 1% BSA MCDB 131 was added, and cells once with MCDB 131 (Life Technologies) and starved in 1% BSA in MCDB were incubated at 37°C for the time indicated to allow internalization. The 131 (starving medium) for 18–20 h. 2 h before activation, cells were cultures were then put back on ice and washed three times with ice-cold 2+ 2+ washed once with MCDB 131 and further incubated in fresh starving Ca /Mg PBS. Samples were incubated twice for 20 min with 45 mM of medium. Cells were treated with 80 ng/ml VEGF (human recombinant the membrane-nonpermeable reducing agent GSH in 75 mM NaCl, with VEGF 165; PeproTech) in fresh starving medium (fresh starving medium 75 mM NaOH and 1% BSA added just before use (stripping buffer). Cells 2+ 2+ alone was used for controls) for the indicated intervals at 37°C. If not were further washed twice on ice with Ca /Mg PBS and incubated for 2+ 2+ otherwise indicated, VEGF treatment was for 10 min. 15 min with iodoacetamide (in Ca /Mg PBS with 1% BSA; quenching HUVECs were cultured in MCDB 131 with endothelial cell supple- buffer) to quench free sulfo-reactive groups. To evaluate total labeling, ments as described previously (Lampugnani et al., 2003). For the experi- a sample for each cell type was not reduced with GSH. To control back- ments 1,800 and 42,000 cells/cm were seeded to obtain sparse and ground, a sample was labeled and reduced without incubation at 37°C. 2+ 2+ confl uent cultures, respectively. HUVECs were then treated as described in For immunoprecipitation, cells were washed with Ca /Mg PBS this section for mouse endothelial cells except that starving was reduced to and extracted on ice in 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Triton 6 h before stimulation with VEGF. X-100, 1% NP-40, and a cocktail of protease inhibitors (Set III; Calbiochem). Extracts were precleared for 90 min with protein A–agarose beads and Filipin and hypertonic treatments incubated overnight with 5 μg anti–VEGFR-2 (rabbit sc-504), and the Caveolae organization was altered by treatment with 1 μg/ml fi lipin immunocomplexes were collected on protein A–agarose beads for 90 min. (fi lipin III from Streptomyces fi lipinensis; Sigma-Aldrich) for 1 h (Schnitzer After fi ve washes in extraction buffer (the last one containing 0.1% Triton et al., 1994; Andriopoulou et al., 1999). Clathrin pit–mediated endocyto- X-100), proteins were eluted by boiling for 10 min in nonreducing laemmli sis was inhibited using hypertonic medium (0.45 M sucrose in MCDB 131 sample buffer. Samples were analyzed by SDS-PAGE followed by Western with 1% BSA) for 30 min to affect as described previously (Heuser and blotting on nitrocellulose membrane and revealed by ECL chemilumines- Anderson, 1989; Ehrlich et al., 2001). Cells were then stimulated and cence. Band intensity was quantifi ed by ImageJ analysis (National Insti- assayed as indicated in the specifi c sections. tutes of Health; freely available at http://rsb.info.nih.gov/ij/). VE-CADHERIN AND VEGFR-2 INTERNALIZATION • LAMPUGNANI ET AL. 601 To quantify VEGFR-2 recycling and degradation, cells were labeled fl asks as described in Cell types and culture conditions. Three fl asks per as described in the fi rst paragraph of this section, and endocytosis was al- gradient were used. After the indicated treatments, cultures were put on ice lowed for 10 min in the presence of 80 ng/ml VEGF (peak time for VEGF- and washed twice with ice-cold PBS. Cells were scraped in 1.2 ml of ice- induced VEGFR-2 internalization both in VEC-null and -positive cells as cold isotonic buffer/fl ask (20 mM Hepes-KOH, pH 7.5, 0.25 M sucrose, determined in internalization experiments). Samples were then reduced as 90 mM KO-acetate, 2 mM Mg-acetate, 0.5 mM Na-vanadate, 1 mM NaF, described in the fi rst paragraph of this section to remove the label from the 10 mM pyrophosphate, 3 mM β-glycerophasphate, 1 mM pefabloc, residual cell surface receptor. The internalized fraction was chased by re- 40 U/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin) and ho- incubation at 37°C for 10 and 20 min in duplicate samples. One sample mogenized with a Dounce homogenizer. Nuclei and residual intact cells was reduced to evaluate the amount of VEGFR-2 that recycled back to the were pelleted by centrifugation at 1,400 g for 5 min at 4°C. The superna- plasma membrane, and the other sample was left unreduced to measure tants were separated in three equal aliquots and mixed with iodixanol degradation. The samples were then processed as described in the fi rst (OptiPrep; Axis-Shield) and homogenization buffer to generate 30, 20, paragraph of this section. VEGFR-2 degradation was calculated by sub- and 10% iodixanol solutions. They were then loaded into 11.2-ml tracting the value of residual biotinylated receptor after incubation at 37°C OptiSeal tubes (Beckman Coulter) and ultracentrifuged at 353,000 g for without reduction (i.e., internalized + recycled − degraded) from the total 3 h in a rotor (VT 65.1; Beckman Coulter). 600-μl fractions were collected pool of internalized receptor. VEGFR-2 recycling was calculated by sub- from the top of the gradient, and protein concentration (bicinchoninic acid tracting both the degradation value and the value of residual biotinylated reagent; Pierce Chemical Co.) and density (OD at 244 nm as indicated by receptor after incubation at 37°C and reduction (i.e., internalized − recycled the OptiPrep manufacturer) were determined. The fractions were then − degraded) from the total pool of internalized receptor. boiled in the presence of reducing laemmli sample buffer. Samples of each fraction containing the same amount of protein for the different cell types to be compared and representative of the total protein content of each Immunofl uorescence microscopy fraction were analyzed by SDS-PAGE followed by Western blotting. Cells were cultured in 35-mm diameter petri dishes as described in Cell For total cell extracts, cells were washed twice in PBS, extracted in types and culture conditions. After the treatments indicated in the specifi c 2× boiling laemmli sample buffer (200 μl for a 35-mm petri dish) containing sections, culture medium was removed, and cells were fi xed in 1% PFA in 100 mM DTT, scraped, and boiled for a further 10 min. Parallel samples 2.5 mM triethanolamine, pH 7.5, containing 0.1% Triton X-100 and 0.1% were extracted without DTT, and protein concentration was determined NP-40 for 25 min at RT (Lallemand et al., 2003). Before staining, 0.5% by bicinchoninic acid analysis. A total of 15 μg of protein was loaded in Triton X-100 in PBS was added for 10 min at RT. In some experiments, each lane and separated by SDS-PAGE, transferred onto nitrocellulose, immunofl uorescence microscopy for VEGFR-2 was performed using the and immunoblotted with the indicated antibodies. Avas12α1 antibody after cell fi xation (in vitro staining). The fi xation/ For detection, HRP-conjugated horse anti–mouse, goat anti–rabbit permeabilization method applied (Lallemand et al., 2003) allows an optimal (Cell Signaling), and rabbit anti–goat (DakoCytomation) secondary anti- observation of VEGFR-2 in internal compartments with in vitro staining bodies and ECL chemiluminescence reagent (GE Healthcare) were used. (primary antibody after cell fi xation). Data obtained with in vivo (see Internal- Films were scanned and bands were quantifi ed using ImageJ set on the un- ization assays) and in vitro staining were superimposable for both VEC’s calibrated OD function. Adobe Photoshop 7.0, Excel X for MacIntosh, and effect on VEGFR-2 vesicular labeling and the codistribution of VEGFR-2 Adobe Illustrator 11 for the PC were used to produce the fi gures presented. with markers of specifi c compartments such as EEA-1 and caveolin-1. A com- parison of VEGFR-2 vesicular distribution after in vivo staining (with and Online supplemental material without acid wash before fi xation) is shown in Fig. S5. Fig. S1 shows that PV-1 colocalizes with caveolin in VEC-null and -positive In some experiments (in particular for clathrin detection), fi xation was cells. Fig. S2 shows that cell confl uence modulates VEGF-induced PY1175– performed in 1% PFA, and permeabilization was performed with 0.02% VEGFR-2–positive compartments in HUVECs. Fig. S3 shows that clathrin saponin that was maintained for all of the staining procedure. Fluorophore- siRNA inhibits the formation of clathrin-positive vesicular compartments. labeled secondary antibodies produced in donkey had minimal cross- Fig. S4 shows that the cytoplasmic domain of VEC is required to modulate reactivity to other species except for the targeted species (Invitrogen). VEGFR-2 internalization from the plasma membrane. Fig. S5 shows that Anti–VEGFR-2 was revealed with AlexaFluor488-conjugated secondary the antibody Avas12α1 does not modify either the basal or VEGF- stimulated antibodies (anti–rat for anti–mouse VEGFR-2 and anti–rabbit for anti– human tyrosine phosphorylation of VEGFR-2 and does not induce VEGFR-2 inter- VEGFR-2). In double labeling experiments, AlexaFluo488- and -647 fl uoro- nalization. Online supplemental material is available at http://www.jcb. chromes were used to stain each of the two antigens, respectively. org/cgi/content/full/jcb.200602080/DC1. Samples were observed under a fl uorescence microscope (DMR; Leica) using 63× and 100× lenses. Images were captured using a charge- We are most grateful to Kendra Swirsding for editing the manuscript. coupled camera (model 3; Hamamatsu) before processing through Adobe This work was supported by the Associazione Italiana per la Ricerca sul Photoshop for MacIntosh. Quantifi cation of vesicular labeling was per- Cancro, the European Community (grants QLRT-2001-02059, Integrated Proj- formed using the ImageJ program (version 10.2). For comparison pur- ect Contract LSHG-CT-2004-503573, NoE MAIN 502935, and NoE EVGN poses, different sample images of the same antigen were acquired under 503254), Italian Ministry of Health, Ministero dell’Università e della Ricerca/ constant acquisition settings. The best-fi t lower threshold to eliminate most Fondo degli Investimenti della Ricerca di Base (grants RBNE01MWA_009 of the signal background was determined using the threshold tool and con- and RBNE01F8LT_007), and the Cariplo Foundation. C. Tacchetti received fi rmed by visual inspection and count of one-pixel dimension particles. a grant from the Telethon Foundation (GTF03001). In some cases, the same sample was analyzed at different lower thresholds to determine the best fi t. Upper threshold was always set at 255. Particles Submitted: 14 February 2006 with a minimum size of fi ve pixels were counted. For colocalization analy- Accepted: 13 July 2006 sis, images were acquired using a confocal microscope (TCS SP2 AOBS; Leica) with a 63× objective and a 3× zoom. Colocalization was quanti- fi ed using the colocalization plugin of ImageJ. The channel ratio was al- References ways set at 90%. 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Journal

The Journal of Cell Biology – Pubmed Central

Published: Aug 14, 2006

References