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VE-cadherin regulates endothelial actin activating Rac and increasing membrane association of Tiam - PubMed

VE-cadherin regulates endothelial actin activating Rac and increasing membrane association of Tiam

Maria Grazia Lampugnani et al. Mol Biol Cell. 2002 Apr.

Abstract

Previously published reports support the concept that, besides promoting homotypic intercellular adhesion, cadherins may transfer intracellular signals. However, the signaling pathways triggered by cadherin clustering and their biological significance are still poorly understood. We report herein that transfection of VE-cadherin (VEC) cDNA in VEC null endothelial cells induces actin rearrangement and increases the number of vinculin positive adhesion plaques. VEC expression augments the level of active Rac but decreases active Rho. Microinjection of a dominant negative Rac mutant altered stress fiber organization, whereas inhibition of Rho was ineffective. VEC expression increased protein and mRNA levels of the Rac-specific guanosine exchange factor Tiam-1 and induced its localization at intercellular junctions. In addition, in the presence of VEC, the amounts of Tiam, Rac, and the Rac effector PAK as well as the level of PAK phosphorylation were found increased in the membrane/cytoskeletal fraction. These observations are consistent with a role of VEC in localizing Rac and its signaling partners in the same membrane compartment, facilitating their reciprocal interaction. Through this mechanism VEC may influence the constitutive organization of the actin cytoskeleton.

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Figures

Figure 1
Figure 1

Endothelial markers and junctional molecules in VEC null and positive EC. (A) When examined by immunofluorescence microscopy the neo-expressed VEC was concentrated to intercellular contacts in VEC positive cells (b). In both VEC null and positive cells the endothelial junctional markers PECAM (c and d) and JAM (e and f) were concentrated at cell-cell contacts. Bar, 20 μm. (B) VEC null cells were found positive in Western blot for the endothelial-selective VEGF and angiopoietin 1 receptors VEGFR-2 and Tie 2, respectively, as well as for the transforming growth factor-β coreceptor endoglin. The level of endothelial markers was comparable in VEC null and positive EC.

Figure 2
Figure 2

VEC expression modifies actin and vinculin organization in EC. VEC null and VEC positive cells formed morphologically different monolayers as observed at phase contrast (a and b). The organization of actin stress fibers was deeply modified by the expression of VEC (c and d). Actin stress fibers were thick and short in VEC positive cells (d) in comparison with the thin and elongated fibers in VEC null cells (c). Distribution of vinculin positive adhesion plaques (e and f) at the end of the actin stress fibers reflected the different setting of actin microfilaments. Vinculin positive plaques were numerous and marked the cell margins in VEC positive cells (f). They were few, thin, and mostly polar in VEC null cells (e). EC isolated from 9.0-d-postcoitum embyros with wild-type (h and l) or homozygous null mutation of VEC gene (g and i) showed differences in thickness and organization of actin stress fibers and vinculin positive adhesion plaques comparable with EC differentiated from embryonic stem (ES) cells. Cells in c–l were double stained for vinculin and actin. Black bar, 100 μm; white bar, 20 μm.

Figure 3
Figure 3

VEC antibodies disorganize actin and vinculin in VEC positive murine EC and in HUVEC. VEC positive murine EC (A) responded to monoclonal antibodies in a way comparable to HUVEC (B). As reported previously in detail (Corada et al., 1999) VEC antibodies induced release of VEC from cell-cell junctions (f in A and B in comparison with control antibodies in e in A and B). This effect paralleled a strong reduction of actin stress fibers (compare b with control a both in A and B) and loss of the vinculin positive adhesion plaques (compare d with control c both in A and B). The cells shown in the figure were treated with the antibodies for 7 h. For analysis in immunofluorescence microscopy they were double labeled for vinculin (c and d) and actin (a and b) or labeled for VEC (e and f). Bar, 20 μm.

Figure 4
Figure 4

Activity of Rac-1 and Rho-A in VEC null and VEC positive EC. (A) GTP-bound Rac recovered in pulldown experiments with recombinant PBD-GST was twice as much in VEC positive in comparison with VEC null EC. The mean fold increase over control VEC null ± SD of five experiments is reported in the right panel. This difference was slightly reduced (to 1.6 increase), but still significant, after stimulation with FCS for 9 min. (B) GTP-bound Rho could not be recovered in pulldown experiments with recombinant RBD-GST from unstimulated VEC null or VEC positive EC. After stimulation with FCS for 3 min GTP-Rho became measurable and it was 1.6-fold more in VEC null than in VEC positive cells. The mean fold increase over FCS-stimulated VEC positive ± SD of five experiments is shown in the right panel.

Figure 5
Figure 5

Actin reorganization in response to N17Rac microinjection in VEC null and VEC positive EC. VEC positive EC responded to microinjected N17Rac with a strong reduction of actin stress fibers (a). After the same treatment VEC null did not show appreciable modification of actin microfilaments (c). Microinjected cells were TRITC-dextran labeled as shown in b and d. They are indicated by arrowheads in a and c. The effects presented in the figure were observed 1 h after microinjection, but similar responses were already present 30 min after injection (our unpublished data). Bar, 20 μm.

Figure 6
Figure 6

Actin reorganization in response to L63Rho and C3 microinjection in VEC null and VEC positive EC. (A) Microinjection of the constitutively active L63Rho induced formation of thick cables of actin stress fibers both in VEC positive and null EC (a and e). In null cells this effect was followed by contraction (e, arrowheads, and g). In contrast, microinjected VEC positive cells remained mostly flat (a, arrowheads, and c). Microinjection of C3 transferase did not modify actin stress fibers either in VEC null (f) or VEC positive EC (b). Microinjected cells were TRITC-dextran labeled as shown in c, d, g, and h. The figure reports the effects observed 1 h after microinjection. Bar, 30 μm. (B) In keratinocytes, the same preparation of C3 transferase as in A (0.1 mg/ml for 1 h) microjniected on the same day as for the experiments in A induced dissolution (arrows, in microinjected cells, l) of the peripheral ring of actin stress fibers (arrowhead, in control cells, l) and diffusion of E-cadherin from cell-cell contacts (asterisks, m). i shows microjniected cells labeled with TRITC-dextran.

Figure 7
Figure 7

(A) Junctional localization of Tiam requires VEC expression and clustering. VEC positive (b) but not negative EC (a) localized Tiam to intercellular junctions. Antibodies to VEC that disrupted VEC clusters at junctions (Figure 3) also induced disappearance of Tiam from cell-cell contacts (c). Bar, 20 μm. (B) Distribution of Rac activators and effectors between soluble and particulate fractions in VEC null and positive EC. When tested by Western blot, the Rac GEF Tiam was twofold concentrated in the particulate fraction of VEC positive (+/+) than in VEC null (−/−) EC. VEC was also selectively distributed in this fraction. The position of mw markers is reported on the right. (C) Northern blot analysis at Tiam mRNA. Expression of VEC (+/+) was accompanied by a twofold increase of Tiam mRNA in comparison to null (−/−) cells. The position of molecular weight markers is indicated on the right.

Figure 8
Figure 8

Effect of VEC antibodies on Rac-1 activity and distribution between soluble and particulate fraction. (A) Incubation of EC with VEC antibodies for 7 h did not modify the amount of GTP-Rac recovered in pulldown experiments with PBD-GST in both VEC positive and negative EC. (B) Rac is 2 ± 0.4-fold (mean ± SD from five experiments) more concentrated in the pellet of VEC positive than VEC null EC. Incubation of the cells with a VEC mAb reduced the particulate Rac by ∼50% in VEC positive EC, leading to values similar to VEC null cells. Graphics on the right report Rac fold increase in the respective fractions. For soluble and particulate fractions the reference level is soluble Rac in control VEC null. For the total, the reference value is Rac in control VEC null. The mean ± SD of three experiments is reported.

Figure 9
Figure 9

PAK phosphorylation and distribution between soluble and particulate compartment. PAK was 1.6 ± 0.1-fold (mean ± SD of five experiments) concentrated in the particulate fraction of VEC positive cells (lower lane in middle gel, PAK). The lanes marked Ser 199–204 and Thr 423 are Western blot performed using two antibodies that recognize PAK only when phosphorylated in these respective positions. Particulate PAK was 2.5- (Ser 199–204) and 2-fold (Thr 423) more phosphorylated in VEC positive than in VEC null cells. Treatment with VEC antibodies for 7 h reduced phosphorylation by 50–60% in both positions. The graphics on the right report the ratio between the respective phospho-PAK and PAK in the same fraction. For soluble and particulate fractions the reference value is the ratio phospho-PAK/PAK of soluble control VEC null EC for the respective amino acid. For the total the reference value is the ratio phospho-PAK/PAK of control VEC null. They report the mean ± SD of three experiments. The dash on the right of each gel panel indicates the position of the 66-kDa marker.

Figure 10
Figure 10

Association of catenins to VEC mutants. Mutant cDNAs with deletion of the cytoplasmic domain required for p120 (Δp120), or β-catenin (Δβcat) binding, or with substitution of the extracellular domain with the extracellular domain of the IL-2 receptor (IL2-VE) (bottom) were transduced into VEC null cells. By immunoprecipitation and Western blot analysis VEC mutants were expressed at the expected molecular weight of ∼110 kDa for Δp120 and Δβcat and 50 kDa for IL2-VE, in comparison to 120 kDa of the full-length VEC (top). Δp120 mutant coimmunoprecipitated only with β-catenin, Δβcat mutant only with p120, and IL2-VE chimera with both β-catenin and p120. The total amount of both β-catenin and p120 in the different cell types was not significantly modified (our unpublished data). The position of molecular weight markers is shown on the right.

Figure 11
Figure 11

Junctional distribution of VEC mutants and associated catenins. Δβcat and Δp120 distributed to intercellular junctions (d and g, respectively) as wild-type VEC (a). Δβcat codistributed with p120 (f), but not βcat (e), whereas Δp120 codistributed with β-catenin (h), but not p120 (i). IL2-VE and associated β-catenin and p120 did not show preferential junctional distribution (l–n). Bar, 20 μm.

Figure 12
Figure 12

Actin organization, Rac activity, and Tiam distribution in soluble and particulate fractions in VEC mutants. (A) Compared with wild-type VEC none of the mutants induced actin reorganization. (B) In parallel, VEC mutants did not increase the level of activated Rac measured in pulldown experiments with PBD-GST (GTP-Rac was 2–3-fold less than in wild-type VEC). (C) Tiam was not concentrated in the particulate compartment of any VEC mutants in contrast to wild-type VEC. Tiam associated to particulate fraction was 1.7–2.3-fold more in wild-type than in mutant cells, whereas the reactivity in the total extract of VEC positive cells was 1.8–2-fold more than in the other transfectants. Bar, 20 μm (A).

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References

    1. Anastasiadis PZ, Reynolds AB. The p120 catenin family. complex roles in adhesion, signaling and cancer. J Cell Sci. 2000;113:1319–1334. - PubMed
    1. Anastasiadis PZ, Moon SY, Thoreson MA, Mariner DJ, Crawford HC, Zheng Y, Reynolds AB. Inhibition of RhoA by p120 catenin. Nat Cell Biol. 2000;2:637–644. - PubMed
    1. Aspenstrom P. Effectors for the Rho GTPases. Curr Opin Cell Biol. 1999;11:95–102. - PubMed
    1. Balconi G, Spagnuolo R, Dejana E. Development of endothelial cell lines from embryonic stem cells. A tool for studying genetically manipulated endothelial cells in vitro. Arterioscler Thromb Vasc Biol. 2000;20:1443–1451. - PubMed
    1. Ben-Ze'ev A, Geiger B. Differential molecular interactions of beta-catenin and plakoglobin in adhesion, signaling and cancer. Curr Opin Cell Biol. 1998;10:629–639. - PubMed

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