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Cardiovascular Research Advance Access originally published online on May 19, 2008
Cardiovascular Research 2008 79(4):679-688; doi:10.1093/cvr/cvn127
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Nuclear targeting of β-catenin and p120ctn during thrombin-induced endothelial barrier dysfunction

Cora M.L. Beckers1, Juan J. García-Vallejo2, Victor W.M. van Hinsbergh1 and Geerten P. van Nieuw Amerongen1,*

1 Department for Physiology, VU University Medical Center, Institute for Cardiovascular Research, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
2 Department of Molecular Cell Biology and Immunology, van der Boechorststraat 7, 1108 BH Amsterdam, The Netherlands

* Corresponding author. Tel: +31 20 4441748; fax: +31 20 4448255. E-mail address: nieuwamerongen{at}vumc.nl

Received 29 November 2007; revised 8 May 2008; accepted 13 May 2008

Time for primary review: 33 days


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Cytosolic and nuclear localization of β-catenin was observed in leaky vessels and in tumours. Several lines of evidence indicate that nuclear β-catenin facilitates angiogenesis. We hypothesized that nuclear β-catenin liberated from endothelial junctional complexes marks the transition from hyperpermeability to angiogenesis. The aim of this study was, therefore, to investigate the fate of β-catenin and the related catenin p120catenin (p120ctn), during disruption of the endothelial barrier function in human umbilical vein endothelial cells (ECs).

Methods and results: The hyperpermeability-inducer thrombin caused a Rho kinase-dependent redistribution of β-catenin from the membrane to the cytosol as evidenced by the western blot analysis of membrane and cytosol fractions and by immunohistochemistry. Glycogen synthase kinase 3β, which phosphorylates cytosolic β-catenin and thereby facilitates its proteasomal degradation, was inhibited by thrombin. The analysis of nuclear extracts demonstrated a thrombin-induced nuclear accumulation of β-catenin as well as p120ctn. Thrombin stimulation activated β-catenin-mediated transcriptional activity as evidenced by reporter assays. Finally, real-time-PCR revealed increased mRNA levels of several β-catenin target genes.

Conclusion: Thrombin induced a cytosolic stabilization of membrane-liberated β-catenin, which, together with p120ctn, subsequently translocated to the nucleus where it induces several β-catenin target genes. This supports the suggestion that membrane-liberated β-catenin and p120ctn contribute to angiogenic responses of ECs following episodes of vascular leakage.

KEYWORDS Angiogenesis; Capillary permeability; Cell communication; Signal transduction; Vasoactive agents


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Endothelial cells (ECs) form a barrier between the blood and the surrounding tissue and actively control the exchange of nutrients and proteins between these compartments.1,2 During inflammation or exposure to ischaemia and other angiogenic stimuli, the EC junctions become loosened, which might result in the generation of focal intercellular gaps that facilitate paracellular passage of fluids, solutes, and proteins. Subsequent to loosening of the cell junctions, the ECs may form new vessels. In most types of ECs, the adherens junctions (AJs) play a dominant role in the endothelial barrier function.2 The disassembly of junctional complexes of the AJs is accompanied by liberation of β-catenin.3 Interestingly, β-catenin can also induce transcriptional activation in the nucleus of various types of cells.4,5 In Caenorhabditis elegans, the adhesion and the signalling function of β-catenin are performed by two different β-catenin homologues, leaving the possibility that in the mammalian system separate pools of β-catenin exist.6 To date, it is unknown whether disintegration of AJs in ECs is accompanied by nuclear translocation of β-catenin to the nucleus.7

The intercellular bridges in the AJs of neighbouring ECs are formed by homotypic interactions of the transmembrane protein vascular endothelial (VE)-cadherin.8,9 β-Catenin participates in the adhesion complex by connecting the F-actin cytoskeleton to VE-cadherin, an interaction that also involves {alpha}-catenin.10,11 A third catenin, p120catenin (p120ctn), of which four isoforms have been identified, is also present in AJs of ECs and stabilizes the AJ complex.12

β-Catenin is also a key player in the Wnt signalling pathway. Here it drives gene transcription upon nuclear translocation.4,13 Cytosolic β-catenin is constitutively bound to a destruction complex containing axin and adenomatosis poliposis coli (APC) as well as casein kinase-1 (CK-1) and glycogen synthase kinase (GSK)-3β. The latter two kinases phosphorylate β-catenin, thereby inactivating it and targeting it for ubiquitination and subsequent degradation by proteasomes.14 Due to this rapid degradation, there are very low levels of non-phosphorylated (active) β-catenin in the cytosol under normal non-stimulated conditions.15,16 In the presence of Wnt signalling and as a response to growth factor signalling, cytosolic β-catenin phosphorylation and degradation are prevented via inhibition of GSK3β.17 GSK3β phosphorylation can be stimulated in human umbilical vein endothelial cells (HUVECs).18 In other cells, it has been shown that stabilization of β-catenin enables β-catenin to translocate to the nucleus and to induce transcription.19 It is still unknown how β-catenin shuttles between the cytosol and nucleus,7 but once β-catenin accumulates in the nucleus it serves as a co-transcription factor to enhance T-cell factor/leukocyte enhancing factor (TCF/LEF) inducible gene transcription.4,20 Among β-catenin target genes that have been linked to cancer and angiogenesis are c-myc,21 cyclinD1,22,23 cox-2,24,25 IL-8,26 and endothelin-1.27 Evidence is accumulating that β-catenin-mediated gene transcription only occurs if p120ctn binds, and thereby inhibits the β-catenin target gene repressor Kaiso.28,29

β-Catenin is an important factor in the development of organs, and in angiogenesis.30 In the adult, angiogenesis is mainly associated with pathological conditions, such as inflammation and ischaemia.31 Blankesteijn et al.16 reported that β-catenin accumulated in the cytosol, and partially in the nucleus of vascular cells, after myocardial ischaemia. In contrast, cells of normal adult vasculature rarely accumulate β-catenin in the cytoplasm or nucleus.15,16 Vascular leakage and angiogenesis are closely related processes.32 Disassembly of the AJs is a requirement for angiogenesis and remodelling of the vascular bed.33 The liberated β-catenin might provide a pool for nuclear translocation and subsequent gene transcription,4,7,30 and thus contribute to angiogenesis.

To clarify whether in HUVECs junction disassembly and nuclear accumulation of β-catenin reflect one pool of β-catenin, we studied whether upon stimulation by thrombin, which induces junction disintegration,34 membrane-localized β-catenin and p120ctn can translocate and stabilize in the cytosol. Subsequently, we investigated whether stabilization of β-catenin was accompanied by translocation to the nucleus and whether this relocalization activated the β-catenin reporter Topflash and enhanced β-catenin target gene expression.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Sources of reagents can be found in the expanded Materials and Methods section in the Supplementary material online. HUVECs were cultured as previously described35 and transfected with the TCF reporter constructs Topflash and Fopflash using the Amaxa technology. Cytosol and membrane fractions were obtained as described by Kouklis et al.,36 whereas nuclear fractions were obtained as described by Nilsson et al.37 For quantification of western blots AIDA Image Analyzer software was used. 3D digital fluorescence imaging microscopy was performed with a Zeiss Axiovert 200 MarianasTM inverted microscope under the control of Slidebook software as described previously.38 mRNA isolation, cDNA synthesis, and real-time-PCR were performed as described previously.39 Primers are described in Table 1 in the Materials and Methods section of the Supplementary material online.

Data were compared by paired sample t-test. Probability values of less than 0.05 were considered to be significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Subcellular localization of β-catenin in confluent human umbilical vein endothelial cells and endothelial cells in situ
To investigate the fate of β-catenin before and after stimulation of endothelial permeability, we determined the subcellular distribution of β-catenin in confluent HUVECs, which were counter-stained for F-actin. Using a general β-catenin antibody (Sigma C2206), β-catenin was encountered at the cell margins, where it formed a continuous lining (Figure 1A, green). A complex staining pattern of β-catenin was locally observed (Figure 1A, arrow). Detailed analysis of such regions revealed a network-like structure (Figure 1B) that co-localized with VE-cadherin (data not shown). Furthermore, at specific sites (box in Figure 1A, enlarged in Figure 1C), β-catenin was curled around actin strands of the peripheral belt of F-actin filaments. Under basal conditions, β-catenin was not detectable in the nucleus, and only tiny amounts of immunostaining were present in the cytosol.


Figure 1
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  		Figure 1 Subcellular localization of β-catenin in confluent human umbilical vein endothelial cells and ECs in situ. (A) Confluent ECs grown on glass cover slips, fixed and stained for β-catenin (green), F-actin (red), and the nuclei (blue). The arrow indicates the complex β-catenin structures at the cell margins. (B) Enlargement of more complex localization of β-catenin as indicated by the arrow in (A). Network-like organization of β-catenin (green). (C) Higher magnification of the peripheral belt of resting ECs as indicated by the white box in (A). (D) Human umbilical cord veins were cannulated and incubated in serum-free M199+1% HSA for 1 h (see Materials and Methods). Veins were fixed and stained for active β-catenin (green).

 
We verified, in freshly isolated umbilical cord veins in situ, the presence of β-catenin by using the general β-catenin antibody and the antibody {alpha}ABC that recognizes active β-catenin, as shown for the latter in Figure 1D. In both cases, β-catenin was mainly located at the cell-margins of the ECs. 3D-deconvolution microscopy combined with 3D-reconstruction confirmed the network-like structure of β-catenin at the cell-margins in situ (data not shown).

3.2 Cytosolic accumulation of β-catenin in thrombin-stimulated human umbilical vein endothelial cells
To investigate whether β-catenin redistributes during hyperpermeability responses, confluent HUVECs were stimulated by thrombin. Thirty minutes after stimulation by thrombin (1 U/mL), β-catenin (green) was present at the end of the stress fibres (red), but had disappeared at sites where gaps between the ECs had been formed (Figure 2A; arrows mark interendothelial gaps). In the cytosol, β-catenin did no longer co-localize with VE-cadherin (data not shown). Subsequently, we determined whether β-catenin redistributed from the membrane to the cytosol during thrombin stimulation of confluent HUVECs by quantifying the amounts of β-catenin in isolated membrane and cytosol fractions (Figure 2B). The western blot analysis demonstrated that in untreated cells 93 ± 9% (n = 3) of the β-catenin is present in the membrane and 7 ± 1% (n = 3) in the cytosol (Figure 2B, first lane). During a 1 h stimulation by thrombin, the amount of β-catenin in the membrane fraction slowly decreased, while it increased simultaneously in the cytosol fraction. The cytosolic β-catenin was significantly increased after stimulation with thrombin for 30 and 60 min (from 7 ± 1 to 22 ± 4% and 22 ± 5%, respectively, n = 3 P < 0.05) when compared with the untreated cells (Figure 2B). Similarly, p120ctn increased in the cytosol after thrombin stimulation (Figure 2C).


Figure 2
Figure 2
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  		Figure 2 Cytosolic accumulation of β-catenin in thrombin stimulated human umbilical vein endothelial cells. (A) The left panels show resting ECs and the right panels shows thrombin stimulated (1 U/mL) ECs. Cells were stained for β-catenin (green, upper panels) and F-actin (red, middle panels). The lower panels show the merged channels. The arrows indicate gaps between the ECs. (B) Western blot analysis for β-catenin of membrane (upper panel) and cytosol (middle panel) fractions after exposure to thrombin for the indicated times. In both cases, β-actin was used as a loading control. The lower panel shows the quantitative analysis of the western blot data. The relative content of β-catenin in the cytosol and membrane fractions was calculated as described in the Supplementary material online, Material and Methods. The white bars represent the cytosol fraction. The black bars represent the membrane fraction. Values are the mean ± SD of three separate experiments, *P < 0.05 when compared with the control. (C) Western blots of membrane (upper panel) and cytosol (lower panel) fractions were stained for p120ctn. β-Actin was used as a loading control.

 
Concurrent with our previous observation that thrombin-induced hyperpermeability was inhibited by the Rho kinase inhibitor Y-27632, pre-treatment of the ECs with Y-27632 inhibited thrombin-induced stress fibre and gap formation (see Supplementary material online, Figure S1A), as well as the redistribution of β-catenin towards the cytosol of thrombin-stimulated ECs as shown by the western blot analysis (see Supplementary material online, Figure S1B). While thrombin increased cytosolic β-catenin to 120% of control values, the β-catenin content of the cytosol dropped to 74% of control values in thrombin-stimulated cells that had been pre-incubated with Y27632. Thus, thrombin-induced β-catenin redistribution in confluent ECs is dependent on the activation of Rho kinase.

3.3 Thrombin-induced GSK3β inactivation prevents β-catenin degradation
The cytoplasmic β-catenin accumulation may be solely due to liberation of β-catenin from the adhesion complexes or additionally by protection against proteolytic degradation in the cytosol, which is facilitated by GSK3β phosphorylation of β-catenin. Thrombin may reduce this degradation by inhibitory phosphorylation of GSK3β on Ser9.18 The western blot analysis of whole cell lysates revealed that thrombin increased the phosphorylation of GSK3β on Ser9 in confluent ECs when compared with their control counterparts (Figure 3A, middle panel). Simultaneously, β-catenin levels increased in the cytosol fraction (Figure 3B, upper panel). The GSK3β inhibitor LiCl (10 mM) was used as a positive control for GSK3β phosphorylation and its effect on the stabilization of cytosolic β-catenin (Figure 3). Thrombin-induced GSK3β phosphorylation was not inhibited by Wortmannin (50 nM) or by Akt inhibitor VII (50 µM) (data not shown). Thrombin induced a PI3K/Akt-independent GSK3β phosphorylation, which prevents GSK3β-mediated β-catenin degradation and contributes to enhance cytosolic β-catenin levels.


Figure 3
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  		Figure 3 Thrombin-induced GSK3β inactivation prevents β-catenin degradation. (A) Whole cell lysates of control, thrombin (1 U/mL), and LiCl (10 mM) treated cells were analysed by the western blot analysis. Blots were stained for β-catenin (upper lanes), GSK3β phosphorylated at ser9 (middle lanes) and total GSK3β (bottom lanes). (B) The Western blots of the cytosol fractions were stained for β-catenin (upper lanes) and β-actin (lower lanes). Results are representative of three experiments, P > 0.05.

 
3.4 Thrombin induces nuclear localization of active β-catenin and p120ctn
Next we investigated whether thrombin (1 U/mL) induced translocation of β-catenin and p120ctn to the nucleus of confluent HUVECs. To this end, we used the antibody {alpha}ABC that specifically recognizes the active, non-phosphorylated form of β-catenin. Figure 4A shows that this antibody recognized some activity in the nucleus of non-stimulated confluent ECs. Thrombin stimulation for 30 min increased the degree of immunoreactivity for nuclear β-catenin. However, the western blot analysis of nuclear fractions (Figure 4B, lanes 1 and 2) showed that in addition to the 92 kDa active β-catenin, the antibody also recognized an unidentified protein of 130 kDa. This protein was similarly enhanced by thrombin as the β-catenin. Although the immunostaining may be flawed by the 130 kDa protein, the western blots clearly demonstrate that nuclear β-catenin is enhanced after stimulation of the cells with thrombin (2.8 ± 0.8 fold-increase when compared with control, n = 4, P < 0.05). These results were verified using a NE-PER kit (# 78833) from Pierce to obtain cytosol and nuclear fractions (2.3-fold increase, data not shown).


Figure 4
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  		Figure 4 Thrombin induces nuclear localization of active β-catenin and p120ctn. (A) ECs were stained for active β-catenin (white). Control and thrombin (1 U/mL) treated cells in the upper panel. MG132 (10 µM) treated ECs in the presence or absence of thrombin in the lower panel. (B) ECs were stimulated with thrombin for the indicated times in the presence or absence of MG132. Western blot analysis of nuclear fractions was performed. An unknown 130 kDa nuclear protein and active β-catenin (upper panel) and PARP (lower panel) were stained. Results are representative of four experiments. (C) The western blots of nuclear fractions were stained for p120ctn (upper panel) and PARP (lower panel).

 
Pre-incubation of the proteasome inhibitor MG132 did not affect or only slightly enhance basal β-catenin levels (Figure 4A). Subsequent thrombin stimulation increased the nuclear localization of β-catenin (Figure 4A) as was conformed by the western blot analysis (Figure 4B, lanes 3 and 4). In addition to β-catenin, the nuclear amount of p120ctn also increased (Figure 4C). Proteasome inhibition by MG132 also did not affect the amount of nuclear p120ctn.

These data show that thrombin stimulation of ECs induces membrane liberated β-catenin and p120ctn translocation to the nucleus, but that inhibition of β-catenin degradation alone is not sufficient to increase the nuclear β-catenin levels.

3.5 Thrombin induces β-catenin target gene expression in human umbilical vein endothelial cells
Finally, we investigated whether thrombin-activated β-catenin-mediated transcriptional activity. First, HUVECs were transfected with Topflash or Fopflash (a mutant, inactive β-catenin/TCF reporter) and were incubated with 1 U/mL thrombin in 1% HSA for 6 and 18 h. Luciferase activity was found significantly increased when compared with the time-matched control after 18 h of thrombin stimulation in cells transfected with Topflash, but not with Fopflash (Figure 5), indicating that thrombin-mediated β-catenin translocation induces β-catenin/TCF-mediated gene transcription in HUVECs.


Figure 5
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  		Figure 5 Thrombin-mediated activation of β-catenin reporter in human umbilical vein endothelial cells. Cells transfected with 1 µg Topflash or Fopflash were stimulated with 1 U/mL thrombin in 1% HSA for 6 and 18 h. Luciferase activity was determined in three independent experiments. White bars represent the β-catenin reporter Topflash, black bars represent the mutant β-catenin reporter Fopflash. C is control and T is thrombin incubation. Significant P-values as indicated.

 
To test whether β-catenin target genes are indeed differentially expressed, HUVECs were incubated with 1 U/mL thrombin or 10 mM LiCl for 6 and 18 h in 1% HSA. Real-time PCR revealed that LiCl, as expected from our previous data, hardly effected gene expression of any of the genes studied (Figure 6). β-Catenin expression itself was also not significantly regulated by thrombin. The β-catenin target genes c-myc, cyclinD1, cox-2, and IL-8 were significantly enhanced after 6 and/or 18 h of thrombin stimulation as evidenced by the decreased Ct values (Figure 6). The expression of the β-catenin target gene endothelin-1, however, was not altered.


Figure 6
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  		Figure 6 Thrombin mediated β-catenin target gene expression. Human umbilical vein endothelial cells were incubated with 1 U/mL thrombin (T) or 10 mM LiCl (L) for 6 or 18 h in 1% HSA or left untreated (C). Real-time-PCR was performed, as indicated in Materials and Methods (Supplementary material online), for β-catenin and the β-catenin target genes endothelin-1, c-myc, cyclinD1, cox-2, and IL-8. Mean Ct values were determined from four separate experiments and are normalized to the 6 h control ± SD, significant P-values when compared with the time-matched controls as indicated.

 
Taken together, these data indicate that thrombin stimulation of HUVECs induces β-catenin-mediated expression of genes involved in vascular adaptation.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
The present study shows that thrombin, an enhancer of the endothelial barrier dysfunction, induces targeting of membrane-localized β-catenin and p120ctn to the nucleus, resulting in enhanced β-catenin target gene expression. In particular, thrombin induces the release of β-catenin from the membrane, a process that involves Rho kinase-mediated changes of the F-actin cytoskeleton. Thrombin-mediated inactivation of GSK3β stabilizes the membrane-liberated β-catenin in the cytoplasm and subsequently allows its translocation to the nucleus. Based on these data, we propose a model in which both the release of β-catenin from the AJs as well as the stabilization of β-catenin in the cytosol are required for nuclear targeting of β-catenin after thrombin stimulation.

In non-stimulated confluent ECs, β-catenin is recognized as a continuous lining at the membrane, which is well known from the epithelial cell and EC.7,40 The non-phosphorylated β-catenin is here bound to VE-cadherin.41 We observed a network-like organization of β-catenin at areas where the membranes between the ECs overlapped, similar as the structures recently shown by Birukova et al.42 These network-like structures of AJs may enforce the tethering forces between adjacent ECs once they become challenged. In addition, we encountered β-catenin in a new structure, namely a curled network around the peripheral belt of F-actin filaments. In non-stimulated confluent ECs, little β-catenin was observed in the cytosol and no active β-catenin was present in their nuclei. This indicates that either no cytosolic β-catenin pool is formed at all, or that cytosolic β-catenin is efficiently targeted for ubiquitination and degradation. Similarly, little p120ctn was encountered in the cytosol and nucleus of non-stimulated ECs.

After stimulation with thrombin, we found relocalization of β-catenin from the membrane to the cytosol. Upon VEGF-mediated disruption of the AJs, VE-cadherin was shown to be internalized via endocytosis43 and subsequently degraded or recycled to the cell membrane.44 Notwithstanding this, Konstantoulaki et al.41 reported that VE-cadherin was relocalized but remained at the plasma membrane after thrombin stimulation of human pulmonary arterial ECs. While β-catenin co-localized with VE-cadherin in the AJs, in agreement with previous reports, this co-localization was largely lost in the cytoplasm.41 Dissociation of β-catenin and VE-cadherin rapidly occurs after phosphorylation of Tyr654 of β-catenin.45 In this respect, it is of interest to note that protein tyrosine phosphorylation is one of the factors that contribute to barrier dysfunction induced by thrombin, together with an increase in cytosolic calcium ion concentration and activation of RhoA/Rho kinase and PKC.41,46 While protein tyrosine phosphorylation may be sufficient to liberate β-catenin from the AJs, our data on the inhibition of Rho kinase indicate that Rho kinase activity was required for the accumulation of β-catenin in the cytosol. This in contrast to the non-canonical Wnt pathway in early development in which RhoA signalling was inhibited without affecting nuclear β-catenin signalling.47 The thrombin-mediated regulation of β-catenin accumulation appears therefore different from the mechanisms observed in early development.

The GSK3β-mediated degradation of β-catenin occurs when the N-terminal part of β-catenin by phosphorylation of its Tyr142 and subsequent structural changes becomes sensitive to serine phosphorylations by CK-1 and GSK-3β.48 The CK-1 phosphorylation facilitates subsequent phosphorylations by GSK3β, which make β-catenin prone to ubiquitination and subsequent degradation by the proteasomes.14 In agreement with earlier data, we demonstrated that thrombin mediates inactivation of GSK3β which depends on PKC activation41 and proceeds independent of the PI3K/Akt pathway.49

The antibody {alpha}ABC recognizes specifically the active, non-phosphorylated form of β-catenin, the form that enters the nucleus and causes gene transcription.20 In addition, it targeted an unidentified 130 kDa protein which was also reported in the original manuscript by van Noort et al.17 Nonetheless, we provide clear evidence that thrombin mediates a transient relocalization of β-catenin as well as p120ctn to the nucleus. Nuclear β-catenin has been associated with EC survival, proliferation, and angiogenesis.5,50 Accumulating evidence suggests that p120ctn is a key mediator in this process.28,51,52 Therefore, β-catenin and p120ctn might form a link between the initial breakdown of the EC barrier and the subsequent angiogenic processes.

Here, we show that thrombin-mediated permeability in HUVECs induces activation of the TCF reporter Topflash as well as the expression of the cancer and angiogenesis-related β-catenin target genes c-myc, cyclinD1, cox-2, and IL-8. We found no effect on endothelin-1 or β-catenin expression itself. Our data are in line with resent data from Ichikawa et al.,53 who showed that prolonged incubation of HUVECs with MMP7 induced disassembly of the AJs, nuclear β-catenin translocation, and increased MMP7 and cox-2, but not cyclinD1 mRNA expression. Of these genes, MMP7 and cyclinD1 contain a Kaiso binding site. In several human cancer cell types, MMP7 is indeed regulated in a Kaiso/p120ctn mediated manner in vivo.52 Our data show nuclear accumulation of p120ctn, which strongly suggests that also in ECs p120ctn plays a role in the β-catenin-mediated gene expression.

In conclusion, in human ECs the adhesive and signalling functions of β-catenin are interconnected. The hyperpermeability inducer thrombin stimulates targeting of membrane-localized β-catenin and p120ctn to the nucleus. Both the release of β-catenin from the AJs as well as thrombin-mediated β-catenin stabilization in the cytosol is required for nuclear targeting. In sharp contrast to the canonical Wnt signalling, where nuclear localization of β-catenin is due to the stabilization of the cytosolic pool of β-catenin, our data strongly suggest that upon thrombin stimulation, it is the pool of active- β-catenin coming from the membrane that relocates to the nucleus. Nuclear accumulation of β-catenin and p120ctn induced transcription of several β-catenin target genes. Future studies have to reveal whether this contributes to an angiogenic response of ECs following episodes of vascular leakage.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary Material is available at Cardiovascular Research Online.


   Funding
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
G.P.v.N.A. was supported by The Netherlands Heart Foundation (The Hague, grant 2003T032). Our laboratory was supported by the EU (EVGN contract LSHM-2003-503254).


   Acknowledgements
 
We thank Martin Kramer and Ing. Clarissa Jungerius for their pleasant company and excellent technical assistance, and Rene JP Musters for sharing his knowledge on 3D wide field microscopy.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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