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17 ß-Estradiol transiently disrupts adherens junctions in endothelial cells

Tanja Groten^*,1, Amy A. Pierce^*, Arthur C. Huen and H. William Schnaper^*

^* Department of Pediatrics, Northwestern University and Children’s Memorial Research Center; and
Department of Pathology, Northwestern University, Chicago, Illinois, USA

Correspondence: University of Ulm, Department of Obstetrics and Gynecology, Prittwitzstr. 43, Ulm 89075, Germany. E-mail: tanja.groten{at}web.de

SPECIFIC AIMS

We hypothesized that 17 ß-estradiol (E2) targets the adherens junction (AJ) complex in endothelial cells via a membrane-associated, non-nuclear signaling pathway. E2 thereby may release the endothelial cells from their quiescent stage in the confluent monolayer and contribute to increased endothelial monolayer permeability and cell activation. Consequently, we chose to investigate whether E2: 1) stimulates disruption of the VE-cadherin-mediated endothelial cell-cell junctions; and thereby 2) may enhance the responsiveness of endothelial cells to stimuli such as VEGF.

PRINCIPAL FINDINGS

1. E2 specifically enhances utHMVEC monolayer permeability
The effect of E2 on endothelial monolayer permeability was tested on uterine microvascular endothelial cells (utHMVEC) in a transwell filter system using FITC-labeled dextran as a marker molecule. At 30 and 60 min, the concentration of dextran was twice as high in the wells below the E2-treated monolayers as in wells below nontreated cells. This effect was not caused by progesterone, and was inhibited by the ER antagonist ICI 182,780.

2. E2 treatment transiently redistributes adherens junction proteins
Western blot analysis of TX-100-soluble (cytoskeleton-free) and TX-100-insoluble (cytoskeleton-bound) as well as membrane and cytosolic fractions of E2-treated endothelial cells showed a significant redistribution of the AJ protein -catenin into the cytoskeleton-free pool and from the membrane fraction. This effect occurred transiently, 20–40 min after treatment with E2.

3. E2 transiently disconnects -catenin from the adherens junction complex
To further investigate whether this redistribution of -catenin represents not only a disconnection of the AJ complex from the cytoskeleton but also a disruption of the complex, we performed coimmunostaining and coimmunoprecipitation experiments of E2-treated endothelial cells. The results showed a transient decrease of the association of -catenin with VE-cadherin, the main component of endothelial cell AJs (Fig. 1 ).

View larger version (67K): [in this window] [in a new window]   Figure 1. E2 transiently dissociates -catenin from VE-cadherin in endothelial cells. A) Confluent HUVEC monolayers were treated with 10 nM E2 for the indicated duration. Cells were stained for VE-cadherin (green) and -catenin (red). Pictures were merged to demonstrate colocalization of the two proteins. Ratio images were constructed by displaying the ratio in 32 color hues, as shown in the ratio bar, with the lowest ratio (no -catenin staining) displayed at the blue end and the highest ratio (-catenin/VE-cadherin=0.75) at the red end of the ratio spectrum. Scale bar = 10 µm. B) Western blot analysis of VE-cadherin immunoprecipitates from TX-100-soluble fractions of E2-treated HUVEC. Shown is one representative experiment. C) Bar graphs represent means ± SE of the fold change of the ratio -catenin/VE-cadherin in the immunoprecipitates (*P<0.05, n=8).

4. E2 treatment induces redistribution of phosphotyrosine signal to the membrane and to the adherens junctions in endothelial cells
Western blot analysis of immunoprecipitated AJ proteins demonstrated that E2 enhances the association of phosphotyrosine with the AJ complex after 20 min treatment. Consistently, phosphotyrosine immunoprecipitated from membrane fractions of E2-treated endothelial cells showed increased association with VE-cadherin within the same time course.

5. The E2 effects on adherens junction protein redistribution were estrogen receptor (ER)-dependent, specific for 17 ß-estradiol, and mediated through membrane-associated signaling
Endothelial cell monolayers were treated with E2 or its derivatives and coimmunostaining for VE-cadherin and -catenin was performed. E2 or its non-cell membrane-permeable derivative, E2-BSA, decreased VE-cadherin/-catenin colocalization. This effect was blocked by the ER antagonist ICI 182,780 and was not induced by the biologically inactive 17 -estradiol or progesterone. Thus, AJ dissolution was specific for 17 ß-estradiol, was ER-dependent, and could be provoked by membrane-impermeable E2.

6. The Src inhibitor PP2 blocks E2-induced disruption and phosphorylation of adherens junction complexes and E2-induced Src membrane translocation in endothelial cells
Western blot and immunostaining analyses demonstrated that the effects of E2 on AJs in endothelial cells could be blocked in the presence of PP2 (Fig. 2 ). In support of these data, Western blot analysis of membrane fractions of E2-treated endothelial cells demonstrated a transient increase of Src family kinases in the membrane fraction. This increase could also be blocked in the presence of PP2. LY294002 and PD98059 did not block the E2-induced phosphorylation of AJ complex components, suggesting that the response is Src-dependent but PI-3-kinase- and ERK-independent (Fig. 2) .

View larger version (26K): [in this window] [in a new window]   Figure 2. Effect of PP2 on E2-induced changes in the adherens junction. A) Western blot for -catenin of membrane fractions of E2-treated EA.hy 926 in the presence and absence of 10 µM PP2. B) Western blot for VE-cadherin and -catenin of immunoprecipitates of VE-cadherin from TX-soluble fractions of E2-treated HUVEC in the presence and absence of 10 µM PP2. C) Western blot of immunoprecipitates of ß-catenin from soluble fractions of EA.hy 926 treated with 10 nM E2 in the presence and absence of 10 µm PP2. D) Western blot of immunoprecipitates of ß-catenin from soluble fractions of EA.hy 926 treated with 10 nM E2 in the presence and absence of 20 µM LY 294002, 10 µM PD 98059, or 10 µM PP2. Shown is one representative experiment (n=4).

7. E2 treatment increases the responsiveness of endothelial cells to stimulation by VEGF
Since VEGF is known to signal differentially (dependent on the engagement of endothelial cells in monolayer) and since this has been related to the engagement of the VEGF receptor in the adherens junction, we tested whether the E2-induced dissociation of the AJ complex might lead to increased responsiveness of the endothelial cells to VEGF. In an in vitro sprouting assay we show that, in the presence of E2, treatment with minimally effective amounts of VEGF distinctly enhances the sprouting response of endothelial cell spheroids.

CONCLUSIONS AND SIGNIFICANCE

Endothelial cells lining the vessels in the adult are physiologically in a resting state. During the female reproductive cycle, angiogenesis emerging from quiescent vessels occurs in the adult organism under the effect of E2 in the absence of initially increased growth factors. This response requires a change from quiescence to an activated state. Such phenotypic change is part of growth factor-mediated effects. However, on the establishment of intercellular junctions and integration into a monolayer, endothelial cells become refractory to growth factor activation. Accordingly, migration and proliferation of endothelial cells emerging from a monolayer, as must occur during angiogenesis, is regulated by interendothelial cell-cell adhesion. Since E2 is known to enhance vessel permeability and increase endothelial cell migration and proliferation, it could activate endothelial cells by disrupting the quiescent monolayer.

In endothelial cells, intercellular adhesions are predominantly formed by the AJ complexes. VE-cadherin is the endothelial cell-specific cadherin mediating homotypic intercellular cell-cell contact. It binds with its intracytoplasmatic C terminus to ß-catenin, which binds to -catenin, which then connects the AJ complex to the cytoskeleton. These linkages, as well as the phosphorylation state of the AJ proteins, regulate the strength of intercellular adhesion, which in turn has been related to the regulation of endothelial cell monolayer integrity. Therefore, we hypothesized that E2 targets the AJ in endothelial cells. Our data showing that -catenin, the linker of the AJ complex to the cytoskeleton, is disconnected from the AJ complex clearly support this hypothesis. These E2 effects were 17 ß-estradiol-specific, ER-dependent, and mediated through a membrane-associated pathway. Evidence for the latter is provided by results indicating that the effect could be provoked by the non-membrane-permeable E2 derivative, E2-BSA, and in the immortalized endothelial cell line EA.hy 926, which has been shown to be unable to perform E2-induced nuclear signaling.

The Src family of tyrosine kinases has been shown to be involved in tyrosine phosphorylation of AJ components and consequently in regulating AJ function. Recent data have indicated that E2 activates Src family kinases in endothelial cells in a membrane receptor-mediated manner. E2 treatment leads to translocation of Src to the membrane, an effect that could be blocked in the presence of the Src family kinase inhibitor PP2. PP2 also blocks the effect of E2 on endothelial cell AJ. Together, these data strongly suggest that E2-induced activation of Src family kinases leads to phosphorylation and disconnection of the AJ complex in endothelial cells.

The action of E2 on AJs in endothelial cells may account for the effect of E2 on endothelial cell monolayer permeability in vivo and vitro and may also explain the angiogenic effects of E2. A hypothetical model for such an action is pictured in Fig. 3 . Under resting conditions in contact-inhibited confluent monolayers, VEGF promotes endothelial survival and homeostasis, whereas in sparse endothelial cells, VEGF promotes proliferation and migration. It has been shown that this differential signaling of VEGF is mediated by engagement of the VEGFR-2 in a complex with VE-cadherin, ß-catenin, and PI-3 kinase in endothelial cells with mature cell-cell junctions. In contrast, in sparse endothelial cells the VEGFR-2 is not engaged in the AJ complex and VEGF stimulates migration and proliferation. Our data showing that E2 enhances the angiogenic response of endothelial cell spheroids to suboptimal concentrations of VEGF are consistent with the hypothesis that E2-mediated, transient changes in AJ integrity may provide a mechanism by which endothelial cells regain responsiveness to growth factor signaling.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic representation of the proposed mechanism by which estrogen-mediated dissolution of the adherens junction complex could increase endothelial cell activation and responsiveness to angiogenic stimuli such as VEGF. E2activation of the estrogen receptor located at the endothelial cell membrane leads to translocation (and activation) of Src (dotted circle). Consequently, the adherens junction complex is phosphorylated (P=phosphorylation), disconnected from the cytoskeleton, and disrupted (VE-cadherin: rectangle with lines, catenin: circle with lines). This dissolution of the adherens junction complex possibly frees the VEGFR (light gray rectangle) from the complex and allows VEGF to stimulate proliferation, migration, and adhesion to the extracellular matrix, consequently activating angiogenesis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2558fje;

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