Regulation of endothelial barrier function and growth by VE-cadherin, plakoglobin, and beta -catenin

Kala Venkiteswaran1,*, Kanyan Xiao1,*, Susan Summers1, Cathárine C. Calkins1, Peter A. Vincent2, Kevin Pumiglia3, and Andrew P. Kowalczyk1

1 Departments of Dermatology, Cell Biology, and Emory Skin Diseases Research Center, Emory University School of Medicine, Atlanta, Georgia 30322; and 2 Center for Cardiovascular Sciences and 3 Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

VE-cadherin is an endothelial-specific cadherin that plays a central role in vascular barrier function and angiogenesis. The cytoplasmic domain of VE-cadherin is linked to the cytoskeleton through interactions with the armadillo family proteins beta -catenin and plakoglobin. Growing evidence indicates that beta -catenin and plakoglobin play important roles in epithelial growth and morphogenesis. To test the role of these proteins in vascular cells, a replication-deficient retroviral system was used to express intercellular junction proteins and mutants in the human dermal microvascular endothelial cell line (HMEC-1). A mutant VE-cadherin lacking an adhesive extracellular domain disrupted endothelial barrier function and inhibited endothelial growth. In contrast, expression of exogenous plakoglobin or metabolically stable mutants of beta -catenin stimulated HMEC-1 cell growth, which suggests that the beta -catenin signaling pathway was active in HMEC-1 cells. This possibility was supported by the finding that a dominant-negative mutant of the transcription factor TCF-4, designed to inhibit beta -catenin signaling, also inhibited HMEC-1 cell growth. These observations suggest that intercellular junction proteins function as components of an adhesion and signaling system that regulates vascular barrier function and growth.

adherens junction; angiogenesis; Wnt signaling


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADHESIVE INTERCELLULAR JUNCTIONS mediate contact between adjacent cells and provide positional cues to cells during development, wound healing, and other processes that require dynamic cell-cell contact (37, 52, 53, 61). Cadherins are a family of calcium-dependent, cell-cell adhesion molecules that play important roles in intercellular junction assembly, cell adhesion, and tissue morphogenesis (reviewed in Ref. 3). The adhesive interactions mediated by cadherin extracellular domains are coupled to the interior of cells by a series of cytoskeletal linking proteins that associate with the cadherin cytoplasmic domain. The cytoplasmic domains of the classical cadherins interact with beta -catenin and plakoglobin, which are members of the armadillo gene family (44). Both beta -catenin and plakoglobin associate with alpha -catenin, a vinculin homologue that plays a key role in linking the cadherin complex to the actin cytoskeleton (42). The interaction of the cadherin-catenin complex with the cytoskeleton is thought to increase cadherin-mediated adhesion, whereas the disruption of the cadherin-catenin complex leads to the downregulation of cell adhesion, increased cell motility, and in some cases, tumorigenesis (7).

Endothelial cells form a continuous cell layer along the wall of blood vessels and control the movement of solutes and fluid from the vascular space to the tissues (50). Vascular endothelial cells express a unique member of the cadherin family, termed VE-cadherin (cadherin-5), which has been shown to play an important role in the establishment and maintenance of endothelial monolayer integrity (10, 15). VE-cadherin associates with both beta -catenin and plakoglobin, and previous studies indicate that plakoglobin is recruited to sites of endothelial cell-cell contact after beta -catenin. Likewise, plakoglobin is displaced from intercellular junctions before beta -catenin when endothelial junctions are disrupted, as is observed before cell migration (35). Using antisense oligonucleotides, Schnittler et al. (49) found that inhibition of plakoglobin expression compromised cell-cell adhesion in endothelial cells exposed to fluid shear stress. These findings led to the conclusion that plakoglobin is required for endothelial intercellular junctions to resist mechanical stress and suggest that plakoglobin plays a particularly important role in endothelial cell-cell adhesion and barrier function.

In addition to a structural role in intercellular junction assembly, beta -catenin is recognized as a key component of the Wnt signal transduction pathway (8, 57). Members of the Wnt family play important roles in normal tissue morphogenesis and are also recognized as oncogenic growth factors in certain human tumors (51). The elucidation of the Wnt signal transduction pathway has led to the current model in which Wnt signaling stabilizes a cytoplasmic pool of beta -catenin and leads to the translocation of beta -catenin to the nucleus; there it interacts with members of the TCF/LEF family of transcription factors to control the expression of genes such as cyclin D and c-myc (24, 25, 54). The accumulation of beta -catenin in the cytosol is controlled by glycogen synthase kinase-3beta (GSK-3beta ), which is thought to phosphorylate the beta -catenin amino terminal domain (62). Phosphorylation of beta -catenin by GSK-3beta is inhibited by Wnt signaling. In the absence of a Wnt signal, beta -catenin associates with the tumor-suppressor protein adenomatous polyposis coli (APC) as well as several other components of a destruction complex that targets beta -catenin for degradation (21). Inappropriate accumulation of beta -catenin in intestinal epithelial cells is now thought to be a key event in the progression of colon carcinoma, and beta -catenin mutations have also been found in a variety of other human tumors. The finding that mutations in beta -catenin or APC precede colon-tumor formation has led to the current view that beta -catenin is an oncogene (45).

A number of recent studies suggest that the Wnt/beta -catenin signaling pathway may play a role in vascular endothelial growth control. For example, transfection of endothelial cells with one member of the Wnt family, Wnt-1, results in increased accumulation of beta -catenin and increased endothelial proliferation (59). Recently, beta -catenin was found to accumulate in the cytosol of vascular endothelial cells that surround injured myocardium in a mouse model of myocardial infarction, which suggests that a beta -catenin signaling pathway may be activated in endothelial cells during inflammation and angiogenesis (9). Endothelial cells also have been found to express members of the frizzled family of Wnt receptors, which suggests that the Wnt family of growth factors plays an important role in cardiovascular growth control and morphogenesis (16, 27).

To test the possibility that intercellular junction proteins regulate both endothelial growth and barrier function, we used a replication-deficient retroviral system to introduce cDNA constructs that encode various intercellular junction components into a well-characterized human dermal microvascular endothelial cell line, HMEC-1 (1). The results of this study indicate that VE-cadherin and plakoglobin play key roles not only in the establishment of endothelial barrier function but also in the regulation of vascular endothelial growth. Interestingly, expression of metabolically stable mutants of beta -catenin increased HMEC-1 cell growth rates, whereas a mutant TCF-4 polypeptide that inhibits beta -catenin signaling decreased endothelial growth. Together, these observations provide direct evidence that the beta -catenin signaling pathway regulates endothelial growth and raise the possibility that this pathway is important during vascular development and tumor angiogenesis.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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Cell culture and retrovirus infection. HMEC-1 cells were cultured in MCDB131 medium (Invitrogen, Carlsbad, CA) containing fetal bovine serum (FBS, HyClone, Logan, UT) supplemented with L-glutamine, cAMP (Sigma, St. Louis, MO), hydrocortisone (Sigma), epidermal growth factor (Intergen, Purchase, NY), and an antibiotic/antimycotic (Invitrogen). For retroviral production, replication-deficient retrovirus was produced using 293-10A1 cells (Imgenex, San Diego, CA). For virus preparation, 2 × 106 293-10A1 cells were seeded into 100-mm dishes for 12 h and then transfected with 30 µg of retroviral plasmid DNA using calcium phosphate precipitation. After 8 h, the growth medium was replaced with fresh medium, and 12 h later, the cells were fed again and incubated at 32°C. Supernatant that contained amphotropic virus was harvested and filtered through a 0.45-µm filter. To establish stable cell lines, HMEC-1 cells were incubated in fresh medium and infected in six-well dishes at 70% confluence with 4 ml of virus supernatant in the presence of 8 µg/ml Polybrene (Sigma). Substrate-attached cells were incubated at 32°C for 15 min with virus and then centrifuged at 900 ×g for 30 min at 32°C. The medium was discarded, and the cells were supplemented with fresh medium and returned to 37°C. After two or three rounds of infection, the cells were transferred into selection medium that contained 0.1 µg/ml puromycin. Resistant cells were pooled and then screened for expression of the protein of interest by Western blot and immunofluorescence microscopy. In each case, at least two independently derived populations were isolated and analyzed. For branching morphogenesis assays, HMEC-1 cells expressing various junctional proteins were seeded into six-well dishes coated with Matrigel (Becton Dickinson, Bedford, MA) and cultured for 24 h. For growth-curve studies, HMEC-1 cells were seeded at a density of 50,000 cells per 35-mm plate and allowed to attach overnight in the presence of 10% serum. To measure growth in low serum, cells were seeded in normal growth medium and allowed to attach for 18 h, washed once with PBS, and then cultured in medium with 1% FBS. After various amounts of time, the cells were harvested by trypsinization and counted using a Coulter particle counter (9914591-C, Beckman Coulter City, FL).

Construction of pDIVA retroviral expression vector. To construct the pDIVA retroviral expression vector, an encephalomyocarditis virus-internal ribosome entry site (EMCV-IRES) element was generated by PCR amplification using pCite4a as a template and custom primers that inserted XbaI and BglII ends onto the resulting PCR product. The PCR product was gel purified, extracted with a GeneClean Spin kit, and cloned into pCR-script (IRES-script). Similarly, a cloned puromycin resistance gene (pur) in pCR-script (pur-script) was generated using pBabe-puro (39) as a template. The custom primers were designed to contain BamHI/SstI ends with pur in frame with the 11th codon of the IRES to promote optimal bicistronic expression. To assemble the bicistronic cassette, the BamHI/SstI fragment that contained pur was ligated into BglII/SstI-digested IRES-script, thereby generating IRES/pur-script. A test plasmid, pDICE, was generated to verify bicistronic expression. This plasmid was created by inserting an Xba/Ecl136II fragment from IRES-script into XbaI/Eco47II digested pcDNA-3Delta 1. pcDNA-3Delta 1 is identical to pcDNA-3 (Invitrogen), except the nucleotide sequence from 1,400 to 3,400 bp was deleted, thus removing the neomycin-resistance cassette and the SV-40 origin of replication. The LacZ marker gene was cloned into the HindIII/NotI sites of pDICE, thereby generating pDICE-Lac-Z. This plasmid was transfected into 293 or CHO cells using Lipofectamine (Invitrogen). Cells were split into media containing puromycin 48 h after transfection (1 µg/ml 293-T cells, 7 µg/ml CHO cells). In both cases, the pooled antibiotic resistant cells were >95% positive for beta -galactosidase, which demonstrates that the bicistronic IRES-puromycin cassette was functioning as expected. To generate a bicistronic retroviral expression vector, the SV-40 promoter and puromycin resistance elements of pBabe-puro were replaced with the IRES-puromycin cassette. A Bam/Sal (filled-in) fragment of pDICE that contained the polylinker and IRES-puro cassette were cloned into BamHI/ClaI (filled-in) digested pBabe-puro to create a functional bicistronic retroviral expression vector termed pDIVA (Fig. 1A).


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Fig. 1.   Retroviral vector and cDNA constructs. A: schematic diagram of the pDIVA retroviral vector. B: schematic of VE-cadherin dominant-negative mutant (IL-2R-VE-cadcyto). Extracellular domain of VE-cadherin was replaced by the nonadhesive extracellular domain of the interleukin-2 (IL-2) receptor. Carboxyl terminal domain of the cadherin mutant is followed by a myc epitope tag. C: schematic of full-length plakoglobin with a carboxyl terminal myc tag. Refer to MATERIALS AND METHODS for additional information. IRES, internal ribosome entry site; LTR, long terminal repeat; gag, group antigens; ampr, ampicillin resistance element; puror, puromycin resistance element; pBS Ori, origin of replication.

Construction of VE-cadherin, plakoglobin, and beta -catenin cDNA reagents. A fragment of the VE-cadherin cytoplasmic domain that encodes amino acids 621-784 (10) was constructed by PCR using Vent polymerase and the following primers: 5' primer, 5'-ATGGAAGCTTCGGCGGCGGCTCCGGAAGCAGGC; and the 3' primer, 5'-ACGTCTCGAGCTACAAGTCCTCTTCAGAAATGAGCTTTTGCTCCACATACAGCAGCTCCTCCCGGGG. This 3' primer includes an in-frame c-myc epitope tag followed by a stop codon. The resulting PCR product was ligated into pBluescript, sequenced, and subcloned into a cytomegalovirus (CMV) expression vector in frame with the interleukin-2 (IL-2) receptor (34). The resulting construct, IL-2R-VE-cadcyto, was then subcloned into the pDIVA expression vector (Fig. 1B). A cDNA encoding full-length human plakoglobin with a carboxyl terminal myc epitope tag in pBluescript was constructed as described previously (33). Plakoglobin.myc was subcloned into pDIVA using Sal/HindIII restriction sites (Fig. 1C). Myc-tagged XenopusDelta N-beta -catenin and XenopusDelta GSK-beta -catenin were kindly provided by Drs. D. Kimelman and P. McCrea (62). The beta -catenin constructs were subcloned into pDIVA using EcoRI/HindIII sites. A human TCF-4-Delta N31 construct in a retroviral expression vector was kindly provided by E. R. Fearon (29).

TCF-4 RNA expression. HMEC-1 cells were cultured for 24-48 h, and total cellular RNA was isolated using Tri-Reagent (Sigma) according to the manufacturer's instructions. To remove possible DNA contamination, 1 µg of total RNA sample was treated with 1 µl of amplification-grade DNase I (Invitrogen) in a 10-µl volume for 15 min at room temperature. After adding 1 µl of 25 mM EDTA, DNase I was heat-inactivated for 10 min at 65°C. For reverse transcription, 5 µg of total RNA was used with the SuperScript First-Strand Synthesis system and oligo(dT)12-18 (Invitrogen), according to the manufacturer's instructions. PCR was performed on a Perkin-Elmer cycler 2400 with 2 µl of reverse-transcribed RNA in a 50-µl reaction with the final MgCl2 concentration being 4 mM. The following primers for human TCF-4 were used: 5' primer, 5'-GAAACCCACCTCCACACTTACCA; and 3' primer, 5'-GGGGCTTCTTCTTTTCTTCTTCCT. The PCR products were analyzed by agarose gel electrophoresis using 1.5% agarose (EM Science).

Immunofluorescence. HMEC-1 cells cultured on glass coverslips were washed in PBS, fixed in ice-cold methanol for 3 min at -20°C, and processed for immunofluorescence microscopy. The following antibodies were used for immunolocalization: rabbit anti-myc antibody 2030 was kindly provided by Dr. J. Stanley and used at a 1:500 dilution, a custom goat anti-FLAG antibody generated by Bethyl Laboratories was used at a 1:500 dilution, the mouse monoclonal antibody 11E4 was kindly provided by Dr. M. Wheelock and used at a 1:50 dilution, a mouse anti-beta -catenin antibody (Transduction Laboratories, Levington, KY) was used at a 1:50 dilution, mouse anti-VE-cadherin (Cad-5 Transduction Lab) was used at a 1:100 dilution, and a mouse anti-IL-2 receptor antibody (Biosource, Camarillo, CA) was used at a 1:100 dilution. Species-appropriate secondary antibodies labeled with rhodamine or fluorescein (Kirkegaard and Perry Laboratories, Gaithersburg, MD) were used at a 1:100 dilution. Coverslips were mounted and examined using a Leica DMR-E fluorescence microscope equipped with a Hamamatsu Orca camera. Images were captured and processed using Open Lab software (Improvision, Lexington, MA).

Western blot analysis. HMEC-1 cells were harvested in Laemmli gel sample buffer (Bio-Rad Laboratories, Hercules, CA) and analyzed by SDS-PAGE and immunoblot using antibodies directed against the myc epitope tag (2030), plakoglobin (11E4), beta -catenin antibody, or VE-cadherin antibody (Cad-5, Transduction Laboratories). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit immunoglobulin (Bio-Rad) were used at a 1:3,000 dilution and detected using ECL (Parmacia, Piscataway, NJ).

Measurement of endothelial barrier function. Endothelial barrier function was monitored using electric cell substrate-impedance sensing (ECIS, Applied Biosystems) as previously described (26). Barrier function was also monitored by culturing cells on Transwell polycarbonate filter membranes with 24-mm diameter inserts and 0.4-µm pore size (Corning Costar, Cambridge, MA) as previously described (31, 32). Briefly, HMEC-1 cells were grown to confluence and the medium in the apical chamber was replaced with 1.5 ml of growth medium that contained 125I-albumin (ICN Biomedicals, Costa Mesa, CA). At various times, 200-µl samples were withdrawn from the basal chamber and the amount of radioactivity present in the basal chamber was measured by using an LKB 1274 gamma counter.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

A mutant VE-cadherin disrupts HMEC-1 intercellular junctions. VE-cadherin and the cadherin-associated proteins beta -catenin and plakoglobin have been implicated as critical regulators of endothelial junction assembly. However, due to the difficulty associated with expressing exogenous cDNAs in normal endothelial cells, most studies of endothelial intercellular junction proteins have used model systems for endothelial cells such as ECV304 (23), CHO (40), or L-cells (32) rather than normal human endothelial cells. To circumvent this problem, we established a replication-deficient retroviral system (Fig. 1A) to express various intercellular junction proteins and mutants in the well-characterized dermal microvascular endothelial cell line HMEC-1 (1). To test this system and to explore the relationship between endothelial cell-cell adhesion and growth control, we constructed a mutant VE-cadherin cDNA in which the extracellular domain of VE-cadherin was replaced by the nonadhesive extracellular domain of the IL-2 receptor (Fig. 1B). Similar cadherin mutants have been used to disrupt intercellular junctions in epithelial model systems (2, 28, 41, 43, 63). As shown in Fig. 2, expression of the IL-2R-VE-cadcyto mutant in HMEC-1 cells disrupted intercellular junction assembly as determined by immunofluorescence analysis. Control HMEC-1 cell lines expressing empty DIVA retroviral vector exhibited extensive beta -catenin accumulation at cell-cell junctions (Fig. 2B). In contrast, beta -catenin was distributed in a diffuse pattern in HMEC-1 cells expressing the IL-2R-VE-cadcyto mutant (Fig. 2E). Similar results were obtained for other intercellular junction components such as plakoglobin and VE-cadherin (not shown). To determine whether the disruption of intercellular junctions was associated with changes in HMEC-1 function, control HMEC-1 and cells expressing the IL-2R-VE-cadcyto mutant were seeded onto Matrigel and the formation of branching networks was monitored. In contrast with control cells that formed extensive networks (Fig. 2C), HMEC-1 cells expressing the IL-2R-VE-cadcyto mutant failed to organize into continuous networks when seeded onto Matrigel (Fig. 2F). These data confirm previous studies that indicate that VE-cadherin plays an important role in endothelial branching morphogenesis during neovascularization (11, 36, 38).


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Fig. 2.   Expression of a dominant-negative cadherin mutant disrupts intercellular junction assembly in human dermal microvascular endothelial-1 cells (HMEC-1). Control HMEC-1 and cells expressing the IL-2R-VE-cadcyto mutant were fixed in methanol and processed for indirect immunofluorescence using antibodies directed against IL-2R (A, D) or beta -catenin (B, E). Control HMEC-1 (C) and cells expressing the IL-2R-VE-cadcyto mutant (F) were seeded onto Matrigel and the formation of branching networks was visualized. Full-length plakoglobin with a carboxyl terminal myc epitope tag was also expressed. Control HMEC-1 (G) or HMEC-1 cells expressing plakoglobin.myc (H) were fixed in methanol and processed for immunofluorescence using antibodies directed against the myc epitope tag. Plakoglobin.myc polypeptide exhibited extensive colocalization with VE-cadherin (not shown). Bar = 50 µm.

To investigate the role of other endothelial proteins in intercellular junction assembly, a cDNA encoding full-length human plakoglobin with a carboxyl terminal myc epitope tag (Fig. 1C) was also expressed in HMEC-1 cells. As shown in Fig. 2H, the plakoglobin. myc polypeptide was expressed uniformly in HMEC-1 cells and assembled into intercellular junctions where it colocalized with endogenous VE-cadherin (not shown). To further characterize these cell lines, Western blot analysis was carried out as shown in Fig. 3. HMEC-1 cell lines expressing plakoglobin.myc exhibited an increase in total plakoglobin levels and a slight decrease in total beta -catenin levels. HMEC-1 cells expressing the IL-2R-VE-cadcyto mutant cadherin exhibited little or no change in plakoglobin or beta -catenin levels. However, endogenous VE-cadherin levels were dramatically decreased in HMEC-1 cells that expressed the IL-2R-VE-cadcyto mutant. These findings are similar to previous results indicating that dominant-negative mutants of cadherins cause disrupted junctions and a downregulation of endogenous cadherins (41, 43, 55).


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Fig. 3.   Western blot analysis of HMEC-1 cell lines. Control cells and HMEC-1 cells expressing plakoglobin. myc or the IL-2R-VE-cadcyto mutant were grown for 24 h, and Western blot analysis was carried out using antibodies directed against myc epitope tag, plakoglobin, beta -catenin, or VE-cadherin. Note that the VE-cadherin antibody is directed against the VE-cadherin cytoplasmic domain and thus recognizes both endogenous VE-cadherin as well as the IL-2R-VE-cadcyto mutant. Loading of the SDS-PAGE gels was normalized for equal protein. Cpm, counts/min.

VE-cadherin and plakoglobin regulate HMEC-1 cell barrier function and growth. The establishment and maintenance of the endothelial barrier is an important function of intercellular junctions in vascular endothelial cells. To determine whether expression of the IL-2R-VE-cadcyto mutant or plakoglobin.myc altered endothelial barrier function, HMEC-1 cell lines were analyzed using ECIS (26). HMEC-1 cell lines were seeded at confluent densities and electrical resistance measurements were taken in real time over the course of 3 days. As shown in Fig. 4A, HMEC-1 cells that expressed the mutant VE-cadherin exhibited a dramatic delay in the establishment of electrical resistance compared with control cells that expressed the empty DIVA vector. Interestingly, HMEC-1 cells expressing plakoglobin.myc consistently exhibited increased barrier function. These results were confirmed using 125I-albumin flux as a measure of HMEC-1 barrier function in cell lines seeded onto polycarbonate filter membranes. HMEC-1 cells expressing plakoglobin exhibited decreased 125I-albumin flux, whereas HMEC-1 cells expressing the IL-2R-VE-cadcyto mutant exhibited increased 125I-albumin flux (Fig. 4B). These data indicate that VE-cadherin and plakoglobin play central roles in endothelial junction assembly and in the establishment of endothelial barrier function.


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Fig. 4.   Expression of plakoglobin.myc increases HMEC-1 barrier function. A: control cells and HMEC-1 cells expressing plakoglobin.myc or the IL-2R-VE-cadcyto mutant were analyzed using electric cell substrate impedance sensing (ECIS). HMEC-1 cell lines were seeded at confluent densities, and electrical resistance measurements were taken in real time over the course of 3 days. B: as a second assay for barrier function, HMEC-1 cell lines were seeded onto polycarbonate membranes and barrier function was measured by using 125I-albumin flux from the apical to basal chamber of Transwell inserts at 48 h postseed. Both assays indicate that plakoglobin.myc expression increases barrier function, whereas the IL-2R-VE-cadcyto mutant decreases barrier function.

Previous studies have demonstrated that both cadherins and plakoglobin regulate epithelial growth rates (12, 22, 63). Therefore, growth-curve experiments were carried out to determine whether the IL-2R-VE-cadherin mutant or plakoglobin.myc altered vascular endothelial growth rates (Fig. 5). Growth curves demonstrated that HMEC-1 cells expressing the IL-2R-VEcadcyto mutant grew more slowly under normal growth conditions than control cells (Fig. 5A). In contrast, HMEC-1 cells expressing plakoglobin.myc grew slightly faster than controls. These differences in HMEC-1 cell growth rates were magnified when cells were grown in low-serum conditions (Fig. 5B). HMEC-1 cell lines expressing plakoglobin.myc grew approximately twofold more quickly than control HMEC-1 or HMEC-1 cells expressing the IL-2R-VE-cadcyto mutant cadherin. To determine whether expression of plakoglobin.myc was altering the localization of endogenous beta -catenin, the localization of beta -catenin was examined in control HMEC-1 cells and in HMEC-1 cell lines expressing plakoglobin.myc (Fig. 6). In control cells, extensive beta -catenin accumulation was observed at cell-cell junctions (Fig. 6B). In contrast, in HMEC-1 cells expressing plakoglobin, beta -catenin was largely absent from intercellular junctions (Fig. 6D) presumably due to competitive displacement of beta -catenin from cadherin binding sites by plakoglobin.myc.


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Fig. 5.   Plakoglobin increases HMEC-1 cell growth rates. A: control cells expressing empty pDIVA and HMEC-1 cells expressing plakoglobin.myc or the IL-2R-VE-cadcyto mutant were plated at a density of 50,000 cells/well in medium containing 10% serum and were counted at various days after plating. B: HMEC-1 cell lines were plated at a density of 50,000 cells/well, allowed to adhere for 18 h, and counted to determine approximate seeding densities. Cells were then switched to 1% serum medium and counted again on day 4. Fold increase in cell number from day 1 to day 4 was calculated.



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Fig. 6.   Expression of plakoglobin.myc displaces beta -catenin from intercellular junctions in HMEC-1 cells. Control HMEC-1 cells transduced with empty DIVA vector and HMEC-1 cells expressing plakoglobin.myc were cultured for 24 h. Cells were then fixed and processed for dual-label immunofluorescence microscopy using antibodies directed against the myc epitope tag (A, C) to detect plakoglobin.myc and a monoclonal antibody against beta -catenin (B, D). Bar = 50 µm.

beta -Catenin-TCF signaling pathway regulates HMEC-1 cell growth. In a number of other model systems, the displacement of beta -catenin from intercellular junctions by plakoglobin correlates with increased beta -catenin signaling and increased cell proliferation (14, 46). To directly test the role of beta -catenin in the modulation of vascular endothelial cell growth, two mutant beta -catenin constructs that lack the GSK-3beta phosphorylation sites were expressed in HMEC-1 cells. One mutant lacks the first 46 amino terminal residues of beta -catenin (Delta N-beta -catenin), whereas the other mutant comprises point mutations in the residues thought to be phosphorylated by GSK-3beta (Delta GSK-beta -catenin) (62). Both the Delta N-beta -catenin mutant (not shown) and the Delta GSK-beta -catenin mutant were expressed and assembled into endothelial intercellular junctions (Fig. 7A), and both mutants colocalized with VE-cadherin (not shown). Similar to HMEC-1 cells expressing plakoglobin.myc, HMEC-1 cells expressing Delta GSK-beta -catenin or Delta N-beta -catenin exhibited increased growth rates compared with control cells expressing empty DIVA vector (Fig. 7B). These data suggested that beta -catenin may regulate endothelial growth rates by activating the beta -catenin-TCF pathway.


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Fig. 7.   Expression of Delta glycogen synthase kinase-3beta (GSK-beta )-catenin mutant increases HMEC-1 cell growth. A: control HMEC-1 and HMEC-1 cells expressing the Delta GSK-beta -catenin mutant were processed for immunofluorescence using antibodies directed against the myc epitope tag. B: HMEC-1 cells expressing either the Delta GSK-beta -catenin mutant or the Delta N-beta -catenin mutant were plated at a density of 50,000 cells/well. Cells were cultured in medium containing 1% serum and counted at various times after plating. Bar = 50 µm.

TCF-4 is a transcription factor known to bind directly to beta -catenin, and these proteins form a transcriptional unit that activates the expression of genes that regulate proliferation (13). To determine whether HMEC-1 endothelial cells express TCF-4, RT-PCR was used to detect the presence of endogenous TCF-4 RNA in HMEC-1 cell lines. Using 293 cellular RNA as a positive control, a single RT-PCR product of the correct molecular mass was detected when using HMEC-1 cellular RNA as a template for the reaction (Fig. 8A). To determine whether the beta -catenin/TCF pathway contributes to HMEC-1 cell growth, HMEC-1 cell lines expressing a dominant-negative TCF-4 mutant were established. This mutant lacks the first 31 amino acids that bind to beta -catenin but retains the conserved TCF DNA binding domain (29). In transient transfections using 293 cells, this TCF mutant inhibited beta -catenin activation of a TCF reporter (not shown) as previously reported (29). When expressed in HMEC-1 cells, the FLAG epitope-tagged TCF-4 dominant-negative mutant was expressed and localized to the nucleus in HMEC-1 cell lines (Fig. 8B). Growth analysis indicated that HMEC-1 cell lines expressing the TCF-4 mutant consistently grew more slowly than control cell lines (Fig. 8C). Together with the data shown in Fig. 7, these data demonstrate that the beta -catenin-TCF pathway is active in vascular endothelial cells and plays a role in the regulation of vascular endothelial growth.


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Fig. 8.   TCF-4 is expressed in HMEC-1 cells, and the expression of a TCF-4 dominant-negative mutant inhibits HMEC-1 cell growth. A: RT-PCR was performed using primers for human TCF-4 to detect endogenous TCF-4 in parental HMEC-1 cells and in HMEC-1 cell lines. B: to test the role of TCF-4 in HMEC-1 cell growth, a FLAG-tagged dominant-negative mutant TCF lacking the amino terminal beta -catenin binding domain was expressed in HMEC-1 (DN-TCF-4) cells. Mutant DN-TCF-4 localized predominantly in the nucleus. C: HMEC-1 cells expressing empty retroviral vector or the DN-TCF-4 mutant were cultured for various amounts of time under low-serum conditions and counted at various times after plating. Bar = 50 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the current study, a replication-deficient retroviral system was used to determine the role of VE-cadherin, plakoglobin, and beta -catenin in the assembly of endothelial junctions and the regulation of endothelial growth. The results of these studies indicate that the armadillo family proteins plakoglobin and beta -catenin regulate vascular endothelial growth. In contrast, endothelial growth was inhibited by dominant-negative mutants of either VE-cadherin or TCF-4. These results provide direct evidence that vascular endothelial cell growth is regulated by plakoglobin, beta -catenin, and the TCF family of transcription factors.

A number of previous studies have demonstrated that VE-cadherin plays an important role in vasculogenesis and angiogenesis (6, 11, 19, 36, 38, 60). The results of our studies using a dominant-negative VE-cadherin mutant confirm the view that VE-cadherin plays an important role in endothelial morphogenesis. Expression of the IL-2R-VE-cadcyto mutant inhibited endothelial junction assembly and compromised endothelial barrier function. In addition, endogenous VE-cadherin levels were downregulated in HMEC-1 cells expressing the IL-2R-VE-cadcyto mutant. Previous studies also reported that epithelial cadherins are downregulated by the expression of cadherin mutants (41, 43, 55), but the precise mechanism by which mutant cadherins cause downregulation of endogenous cadherins is not fully understood. In addition to disrupting endothelial junction assembly, the IL-2R-VE-cadcyto mutant also decreased endothelial growth rates. A similar cadherin mutant comprising the IL-2-receptor extracellular domain and the E-cadherin cytoplasmic domain was found to inhibit keratinocyte growth (63). The growth inhibitory activity required the beta -catenin binding domain of the E-cadherin cytoplasmic tail, which suggests that inhibition of beta -catenin signaling by cadherins functions as a brake on cell proliferation. This notion is supported by recent observations indicating that the cytoplasmic domain of E-cadherin regulates growth of SW480 cells not by modulating adhesion but rather by modulating beta -catenin signaling (20).

In contrast to the IL-2R-VE-cadcyto mutant, expression of full-length plakoglobin enhanced endothelial barrier function and increased endothelial growth rates. Previous time-course studies demonstrated that plakoglobin accumulates at endothelial intercellular junctions after beta -catenin and that the association of plakoglobin correlated with the formation of stable endothelial cell-cell contacts (35). Our results directly demonstrate that increased expression of plakoglobin displaces beta -catenin from vascular endothelial intercellular junctions and increases endothelial barrier function. The mechanism by which plakoglobin increases endothelial barrier function is not known. Plakoglobin. myc expression did not appear to alter the expression or localization of the tight-junction protein ZO-1 (not shown). In previous studies, we demonstrated that plakoglobin couples VE-cadherin to the intermediate filament-binding protein desmoplakin (32). A number of studies have found that desmoplakin is present in vascular (32, 56) and lymphatic (47, 48) endothelial cells as well as blood vessels in vivo (18). Furthermore, recent studies directly demonstrate an important role for desmoplakin in vasculogenesis (17). However, HMEC-1 cells do not express detectable levels of desmoplakin in vitro, and we did not observe increased expression of desmoplakin in HMEC-1 cells expressing plakoglobin.myc (not shown).

Plakoglobin.myc expression increased the growth rates of subconfluent HMEC-1 cells, and this effect was particularly dramatic when HMEC-1 cells were cultured in low serum. Although we did not observe dramatic changes in either apoptosis or in the expression of genes implicated in beta -catenin signaling (not shown), plakoglobin and beta -catenin are known to regulate both proliferation and apoptosis (64). The observation that plakoglobin.myc displaced endogenous beta -catenin from intercellular junctions raises the possibility that the increased growth rates observed in HMEC-1 cell lines expressing plakoglobin.myc might be attributed to effects of beta -catenin on cell proliferation. Indeed, we observed that expression of beta -catenin mutants that lack the GSK-3beta phosphorylation sites increased HMEC-1 cell growth rates, whereas a dominant-negative TCF-4 mutant inhibited HMEC-1 cell growth. Nonetheless, the specific signaling pathways and genes regulated by the beta -catenin-TCF transcriptional complex in endothelial cells are currently unknown. Although the role of plakoglobin in Wnt signaling is still under debate, several studies have suggested that plakoglobin may have direct effects on cell proliferation independent of beta -catenin signaling (12, 22, 30, 58), and we cannot rule out the possibility that plakoglobin regulates endothelial growth independently from beta -catenin.

Numerous studies have demonstrated that mutant forms of beta -catenin that lack the amino terminal GSK-3beta phosphorylation site can lead to a tumorigenic phenotype in some model systems (45, 64). Aberrant vascular growth is observed in endothelial tumors such as hemangiomas and angiosarcomas (4). In contast to hemangiomas, which regress spontaneously, angiosarcomas are rare but aggressive endothelial tumors found primarily in adults. In preliminary studies, we observed that the cytosolic pool of beta -catenin is upregulated (data not shown) in a mouse model of angiosarcomas (5). However, HMEC-1 cell lines expressing beta -catenin mutants did not exhibit growth in soft agar nor did these cells exhibit tumor formation in nude mice (data not shown). Thus it remains to be determined whether beta -catenin mutations play a role in endothelial tumorigenesis.

Growing evidence indicates that Wnt family growth factors and beta -catenin signaling may play important roles in angiogenesis. Transient expression of Wnt-1 stimulates endothelial proliferation in vitro (59), and recent studies also indicate that vascular endothelial cells express members of the Frz'd family of Wnt growth factor receptors (16, 59). Although the roles of Wnt signaling and beta -catenin in angiogenesis are not yet clear, the cytoplasmic pool of beta -catenin was found to be upregulated in endothelial cells in an experimental model of cardiac infarction, which suggests an important role for beta -catenin in endothelial proliferation during angiogenesis (9). Furthermore, knockout mice that lack the Wnt family receptor Fzd-5 exhibit defective yolk sac and placental vasculogenesis (27). These observations, along with the results presented here, suggest that the Wnt-beta -catenin signaling pathway may play an important role in vasculogenesis during development and angiogenesis associated with wound healing or tumor growth.


    ACKNOWLEDGEMENTS

We acknowledge the generosity of investigators who provided cDNA and antibody reagents that made these studies possible. We thank Dr. K. Green for the plakoglobin cDNA and for helpful advice, Dr. E. Dejana for the VE-cadherin cDNA, and Dr. M. Wheelock for the 11E4 antibodies. Thanks also to Drs. Pierre McCrea and D. Kimelman for the beta -catenin mutants, Dr. E. Fearon for the TCF-4 mutant, and Dr. J. Hall for helpful comments and suggestions. Thanks also to Dr. S. LaFlamme for the IL-2 receptor CMV expression cassette.


    FOOTNOTES

* K. Venkiteswaran and K. Xiao contributed equally to this work.

This work was supported by American Cancer Society Grant RPG-00-246-01 (to A. P. Kowalczyk), National Institutes of Health (NIH) Grants K01-AR-02039 (to A. P. Kowalczyk), R01-CA-81419 (to K. Pumiglia), and R29-HL-054206 (to P. Vincent), and Emory Skin Diseases Research Center (NIH Grant P30-AR-042687). C. Calkins was supported by NIH Grant T32-AR-007587.

Address for reprint requests and other correspondence: A. P. Kowalczyk, Dept. of Dermatology, Emory Univ. School of Medicine, 5007 Woodruff Memorial Bldg., 1639 Pierce Dr., Atlanta, GA 30322 (E-mail: akowalc{at}emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00417.2001

Received 27 August 2001; accepted in final form 2 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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