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
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ABSTRACT |
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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 -catenin and plakoglobin. Growing evidence
indicates that
-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
-catenin stimulated HMEC-1 cell growth, which suggests
that the
-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
-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
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INTRODUCTION |
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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 -catenin and plakoglobin, which are members of the
armadillo gene family (44). Both
-catenin and
plakoglobin associate with
-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 -catenin and
plakoglobin, and previous studies indicate that plakoglobin is
recruited to sites of endothelial cell-cell contact after
-catenin.
Likewise, plakoglobin is displaced from intercellular junctions before
-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,
-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
-catenin and leads to the translocation of
-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
-catenin in the cytosol is controlled by
glycogen synthase kinase-3
(GSK-3
), which is thought to
phosphorylate the
-catenin amino terminal domain (62).
Phosphorylation of
-catenin by GSK-3
is inhibited by Wnt
signaling. In the absence of a Wnt signal,
-catenin associates with
the tumor-suppressor protein adenomatous polyposis coli (APC) as well
as several other components of a destruction complex that targets
-catenin for degradation (21). Inappropriate
accumulation of
-catenin in intestinal epithelial cells is now
thought to be a key event in the progression of colon carcinoma, and
-catenin mutations have also been found in a variety of other human
tumors. The finding that mutations in
-catenin or APC precede
colon-tumor formation has led to the current view that
-catenin is
an oncogene (45).
A number of recent studies suggest that the Wnt/-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
-catenin and
increased endothelial proliferation (59). Recently,
-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
-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 -catenin increased HMEC-1 cell
growth rates, whereas a mutant TCF-4 polypeptide that inhibits
-catenin signaling decreased endothelial growth. Together, these
observations provide direct evidence that the
-catenin signaling
pathway regulates endothelial growth and raise the possibility that
this pathway is important during vascular development and tumor angiogenesis.
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MATERIAL AND METHODS |
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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-31. pcDNA-3
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
-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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Construction of VE-cadherin, plakoglobin, and -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
Xenopus
N-
-catenin and
Xenopus
GSK-
-catenin were kindly provided by Drs. D. Kimelman and P. McCrea (62). The
-catenin constructs
were subcloned into pDIVA using EcoRI/HindIII sites. A human TCF-4-
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-
-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), -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.
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RESULTS |
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A mutant VE-cadherin disrupts HMEC-1 intercellular junctions.
VE-cadherin and the cadherin-associated proteins -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
-catenin accumulation at cell-cell
junctions (Fig. 2B). In contrast,
-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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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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-Catenin-TCF signaling pathway regulates HMEC-1 cell
growth.
In a number of other model systems, the displacement of
-catenin
from intercellular junctions by plakoglobin correlates with increased
-catenin signaling and increased cell proliferation (14,
46). To directly test the role of
-catenin in the modulation of vascular endothelial cell growth, two mutant
-catenin constructs that lack the GSK-3
phosphorylation sites were expressed in HMEC-1 cells. One mutant lacks the first 46 amino terminal residues of
-catenin (
N-
-catenin), whereas the other mutant comprises
point mutations in the residues thought to be phosphorylated by
GSK-3
(
GSK-
-catenin) (62). Both the
N-
-catenin mutant (not shown) and the
GSK-
-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
GSK-
-catenin or
N-
-catenin exhibited increased growth rates compared with control cells expressing empty DIVA vector (Fig. 7B). These
data suggested that
-catenin may regulate endothelial growth rates by activating the
-catenin-TCF pathway.
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DISCUSSION |
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In the current study, a replication-deficient retroviral system
was used to determine the role of VE-cadherin, plakoglobin, and
-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
-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,
-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
-catenin binding domain of the E-cadherin cytoplasmic tail, which
suggests that inhibition of
-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
-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 -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
-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 -catenin signaling (not shown), plakoglobin and
-catenin are known to regulate both proliferation and
apoptosis (64). The observation that
plakoglobin.myc displaced endogenous
-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
-catenin on cell proliferation. Indeed, we
observed that expression of
-catenin mutants that lack the GSK-3
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
-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
-catenin
signaling (12, 22, 30, 58), and we cannot rule out the
possibility that plakoglobin regulates endothelial growth independently
from
-catenin.
Numerous studies have demonstrated that mutant forms of -catenin
that lack the amino terminal GSK-3
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
-catenin is
upregulated (data not shown) in a mouse model of angiosarcomas
(5). However, HMEC-1 cell lines expressing
-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
-catenin mutations play a role in endothelial tumorigenesis.
Growing evidence indicates that Wnt family growth factors and
-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
-catenin in angiogenesis are not yet clear, the
cytoplasmic pool of
-catenin was found to be upregulated in
endothelial cells in an experimental model of cardiac infarction, which
suggests an important role for
-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-
-catenin
signaling pathway may play an important role in vasculogenesis during
development and angiogenesis associated with wound healing or tumor growth.
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ACKNOWLEDGEMENTS |
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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 -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.
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FOOTNOTES |
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* 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.
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