1 Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208; and 2 Department of Surgery, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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ABSTRACT |
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The signal transduction pathways that lead to
disruption of pulmonary endothelial monolayer integrity by transforming
growth factor-1 (TGF-
1) have not been elucidated.
The purpose of this investigation was to determine whether disassembly
of the adherens junction is temporally associated with the
TGF-
1-induced decrease in pulmonary endothelial monolayer integrity.
Measurement of albumin clearance and electrical resistance showed that
monolayer integrity started to decrease between 1 and 2 h post-TGF-
1
treatment and continued to slowly decrease over the next 6 h.
Immunofluorescence microscopy of monolayers between 2 and 3 h
post-TGF-
1 showed that
-catenin, plakoglobin,
-catenin, and
cadherin-5 were colocalized both at the cell periphery and in newly
formed bands that are perpendicular to the cell-cell border. At 4 h
post-TGF-
1, cells began separating; however,
- and
-catenin,
plakoglobin, and cadherin-5 could still be found at the cell periphery
at areas of cell separation and in strands between separated cells. By 8 h, these junctional proteins were no longer present at the cell periphery at areas of cell separation. The myosin light chain kinase
inhibitor KT-5926 prevented the TGF-
1-induced change in integrity
but did not inhibit the formation of actin stress fibers or the
formation of bands containing adherens junction proteins that were
perpendicular to the cell-cell junction. Overall, these results suggest
that adherens junction disassembly occurs after cell separation during
TGF-
1-induced decreases in pulmonary endothelial monolayer integrity
and that the loss of integrity may be due to the activation of a myosin
light chain kinase-dependent signaling cascade.
cadherin; catenin; myosin; myosin light chain; myosin light chain kinase; actin; vascular endothelial cells; immunofluorescence microscopy; KT-5926
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INTRODUCTION |
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INCREASES IN THE PARACELLULAR FLUX of macromolecules can result from changes in endothelial cell shape and the formation of intercellular gaps. Endothelial cell shape and gap formation are believed to be controlled by signal transduction pathways that alter the balance of competing adhesive and contractile forces (reviewed in Refs. 16, 27). Endothelial cell-cell and cell-matrix contacts tether endothelial cells to each other and to the extracellular matrix, respectively, and act against centripetal tension generated by actomyosin motors. Thus the loss of cell-cell or cell-matrix attachment or the activation of contractility will alter the balance of forces within the cell, resulting in cell retraction and a decrease in endothelial monolayer integrity.
A number of investigators (15, 20, 29, 30, 39) have demonstrated that thrombin and histamine stimulate an actin-myosin motor system within endothelial cells that causes cells to contract. The signaling cascade is similar to what has been observed in smooth muscle cells where activation of myosin light chain (MLC) kinase (MLCK) leads to the phosphorylation of MLC and, subsequently, cell contraction. Treatment of endothelial monolayers with thrombin has been shown to activate MLCK, causing decreases in barrier integrity as demonstrated by observed increases in paracellular protein permeability (15, 20). Taken together, these studies suggested that physiological agents can alter the endothelial monolayer integrity through the activation of MLCK, which subsequently results in cell contraction.
The adherens junction, which is composed of cadherins and catenins, is
one type of cell junction that maintains endothelial cell-cell
adhesion. The cadherins are a family of structurally related proteins
that bind in a Ca2+-dependent,
homophilic fashion and have been implicated in modulating the integrity
of endothelial cell monolayers (2, 26). Cadherin-5, also called
VE-cadherin, is the cadherin found specifically on endothelial cells
(25, 26). The cytoplasmic tail of cadherins is very well conserved
between the different types of cadherins as well as across species
(17). This cytoplasmic tail has been shown to bind to the actin
cytoskeleton via cytoplasmic proteins called catenins. The catenins
have been classified on the basis of molecular mass as
-catenin (102 kDa),
-catenin (92 kDa), and
-catenin (82 kDa),
with
-catenin being homologous to plakoglobin (reviewed in Refs. 21,
44). The cytoplasmic tail of cadherin binds to either
-catenin or
plakoglobin but not both as demonstrated by the inability to
coprecipitate
-catenin and plakoglobin in the same cadherin-catenin
complex (1, 23).
-Catenin, which has been shown to associate with
the actin cytoskeleton, binds with
-catenin and plakoglobin, thereby
linking the cadherin-catenin complex to the actin cytoskeleton (1, 32,
34).
Recently, Lampugnani et al. (25) demonstrated that the association of
cadherin-5 with -catenin or plakoglobin in endothelial cells was
dependent on the duration of cell-cell interaction. These investigators
showed that complexes consisting of cadherin-
-catenin-
-catenin were localized to the cell junction before those complexes
consisting of cadherin-plakoglobin-
-catenin, with the latter
adherens junction complex being formed after the cell monolayers
were confluent for 72 h. This order was reversed because cell-cell
adhesion decreases when endothelial cells prepare to migrate; that is,
plakoglobin was found to leave the junction before the disappearance of
-catenin. These findings led the investigators to postulate that the
localization of
-catenin and plakoglobin to the junction may be
related to the strength and/or integrity of the endothelial cell
junction. Similar findings have also been reported by Schnittler et al. (38).
Transforming growth factor (TGF)-1 is a 25-kDa disulfide-linked
homodimer that has been implicated as a mediator in altering the
structure and function of the vascular wall. TGF-
1 has been shown to
alter endothelial cell phenotype as demonstrated by a decrease in
cell-cell contact, inducing a reorganization of the actin cytoskeleton
of the cell, and by a decrease in the localization of the tight
junction protein ZO-1 at the cell periphery (11, 28). The ability of
TGF-
1 to alter endothelial cell phenotype suggests that this
cytokine-like growth factor may play a role in the regulation of
vascular endothelial integrity. This may occur with pulmonary
hypertension where there is an elevated level of TGF-
1 in the vessel
wall (8) and an increased transvascular flux of water and
macromolecules across the endothelial cell monolayer (36, 40). Botney
et al. (8) found that TGF-
1-expression in vessels of hypoxia-induced
pulmonary hypertension was decreased in the medial smooth muscle layer.
However, immunohistochemical staining of TGF-
1 was more intense in
endothelial cells of hypertensive vessels compared with that in
endothelial cells in normotensive vessels. As reviewed by Stenmark et
al. (41), a loss of endothelial integrity and the increased edema and
transudation of mitogenic plasma factors into the subendothelial space
have been implicated in initiating proliferation and protein synthesis
in both smooth muscle cells and fibroblasts, both important factors in
vascular remodeling found in pulmonary hypertension. Thus
TGF-
1-induced changes in endothelial morphology may play a role in
mediating decreased vascular integrity that leads to vascular remodeling.
The signal transduction pathways that lead to the TGF-1-induced
decrease in endothelial cell monolayer integrity are not known. We
hypothesized that disassembly of the cell-cell adherens junction was,
in part, responsible for the TGF-
1-induced decrease in endothelial
monolayer integrity. Studies were undertaken to determine the time
course and dose response of the TGF-
1-induced increase in
permeability immediately after its addition to endothelial monolayers.
Endothelial monolayer permeability was assessed by measuring changes in
albumin clearance and electrical resistance. Immunofluorescence
microscopy was then used to determine the kinetics of disassembly of
cell-cell adherens junction after treatment of endothelial cell
monolayers with TGF-
1. Colocalization of
-catenin and plakoglobin
was performed to determine whether cadherin-catenin complexes
consisting of plakoglobin would disassemble before those complexes
consisting of
-catenin as described by Lampugnani et al. (25).
Colocalization of
-catenin and actin was also performed to determine
whether the changes in these two structures are temporally associated.
We also used the MLCK inhibitor KT-5926 to determine whether
TGF-
1-induced changes in endothelial integrity and rearrangement of
the adherens junction were dependent on MLC phosphorylation, which
would suggest a role for cell contraction.
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METHODS |
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Endothelial cell culture. Calf pulmonary arterial endothelial cells (CPAECs; American Type Culture Collection) were grown in modified Eagle's medium (MEM; GIBCO BRL) supplemented with 20% fetal bovine serum (Sterile Systems), nonessential amino acids (10 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml; all from GIBCO BRL). The cells were split 1:4 every fifth day. All experiments were performed between passages 18 and 24.
Experimental protocol. The endothelial
cells were seeded (8 × 104
cells/cm2) and grown to
confluence (3-5 days) on tissue culture-treated polycarbonate
micropore membranes (13-mm diameter, 0.4-µm pore size; Transwell,
Costar) for albumin clearance, on gelatinized wells for electrical
resistance measurement, on glass coverslips for immunofluorescence
microscopy, and on tissue culture-treated 35- and 60-mm-diameter plates
(Sarstedt) for assessment of the soluble and insoluble protein pools
and MLC phosphorylation, respectively. The day before the experiment,
the medium was changed to MEM containing penicillin-streptomycin and
5% serum. TGF-1 (porcine platelet) was purchased from R&D Systems.
Antibodies to TGF-
1 and preimmune serum were also purchased from R&D
Systems. KT-5926, an inhibitor of MLCK, was from Kamiya Biomedical. All
incubations with TGF-
1 and KT-5926 were performed in MEM containing
penicillin-streptomycin and 5% serum.
Assessment of 125I-labeled albumin clearance. Measurement of 125I-labeled albumin clearance (in µl/min) as described by Cooper et al. (12) was used to assess the changes in the diffusive permeability of albumin across the endothelial monolayers. This technique allowed the measurement of transendothelial albumin transport in the absence of an oncotic pressure gradient and a changing hydrostatic pressure gradient (12). The dual-chamber monolayer system consisted of a 0.2-ml luminal chamber (containing a filter lined with a confluent endothelial monolayer) that floated in a larger 25-ml abluminal chamber. To ensure complete mixing, the abluminal chamber was stirred constantly, and both chambers were kept at a constant 37°C in a thermostatically controlled water bath. A concentration gradient of purified 125I-labeled albumin was established across the endothelial monolayer. Present in the abluminal chamber was 25 ml of MEM containing 0.5% bovine serum albumin (BSA; Sigma). To the luminal chamber, we added 200 µl of the same BSA-containing MEM solution as well as the 125I-labeled albumin. The protocol for the measurement of albumin clearance consisted of obtaining 400-µl samples from the abluminal chamber every 5 min for 60 min.
As discussed by Cooper et al. (12), the volume of the luminal chamber, which is cleared of the albumin tracer into the abluminal chamber, represents the total activity of the abluminal chamber. The change in clearance volume during the interval between sampling points was calculated by dividing the amount of albumin flux during the interval by the luminal tracer concentration. The clearance volume of albumin at each time point (Valb,t) was calculated by summing the incremental clearance volumes up to that time point
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RESULTS |
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TGF-1-induced increase in endothelial monolayer
permeability. Experiments were first performed to
characterize the change in permeability after the addition of TGF-
1
to CPAECs. Figure 1 shows the dose response
of the TGF-
1-induced increase in albumin clearance. Active TGF-
1
was added at concentrations of 0.001, 0.01, 0.1, 1.0, and 10.0 ng/ml to
the luminal chamber of the monolayer system and incubated for 18 h. At
the end of 18 h, albumin clearance was determined as outlined in
METHODS. The addition of 1.0 and 10.0 ng active TGF-
1/ml to the endothelial monolayer increased permeability threefold. Doses of 0.1 ng active TGF-
1/ml and lower showed no change in monolayer permeability at 18 h. Because 1.0 ng/ml
of TGF-
1 produced a maximal response, this dose was used to
determine the time course of the change in albumin clearance (Fig.
2). Time-course experiments showed that
TGF-
1 produces a significant increase in permeability within 3 h
after its addition to endothelial monolayers
(P < 0.05). The maximal threefold
increase in albumin clearance was achieved at 6 h because there was no significant difference in albumin clearance determined at 6 or 18 h
after the addition of TGF-
1. To demonstrate the specificity of this
response, we coincubated monolayers with TGF-
1 and antisera to
TGF-
1. As seen in Fig. 3, the addition
of TGF-
1 antisera or preimmune chicken serum alone did not alter
endothelial monolayer permeability. In contrast, the addition of
TGF-
1 antisera in conjunction with TGF-
1 inhibited the typical
threefold increase in albumin clearance.
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ECIS was used next to determine the changes in endothelial cell
permeability by measuring changes in electrical resistance. This
technique has the benefit of assessing changes in endothelial shape at
1-min intervals, giving near to real time assessment for the changes in
permeability. Figure
4B shows
tracings of the first 6 h from two typical experiments where monolayers
were treated with TGF-1 (1.0 ng/ml). The dotted and solid tracings
show that electrical resistance started to drop 1 and 2 h,
respectively, after the addition of TGF-
1, demonstrating the initial
variability in endothelial monolayer integrity. Dose-response
experiments were performed, and the data were plotted over 20 h at
30-min intervals (Fig. 4A).
Dose-response experiments (Fig. 4A)
revealed that concentrations of TGF-
1 ranging from 0.05 to 1.0 ng/ml
produce similar decreases in electrical resistance over the first
4-5 h, beginning between 1 and 2 h after the addition of TGF-
1.
Higher doses (0.5 and 1.0 ng/ml) continued to decrease electrical
resistance, with maximum levels being reached at 8-9 h, whereas
the electrical resistance of the lower doses (0.05 and 0.01 ng/ml)
began to return to basal levels during the next 10 h of the
experimental period. Comparison of ECIS and albumin clearance data
revealed that the time course of changes in protein permeability and
electrical resistance is consistent. The addition of TGF-
1 did not
affect cell viability because the cells were able to exclude trypan
blue and tested negative for apoptosis after 24 h of treatment with 1.0 ng TGF-
1/ml (data not shown).
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TGF-1-induced rearrangement of
adherens junctions. The changes in both the actin
cytoskeleton and
-catenin were examined by immunofluorescence
microscopy during treatment with 1 ng/ml of TGF-
1. Shown in Fig.
5 is staining for F-actin
(A,
C, E,
and G) and
-catenin
(B,
D, F,
and H) for control cells
(A and
B) and after 2 (C and
D), 4 (E and
F), and 8 (G and
H) h of incubation with TGF-
1.
Figure 5C shows that there was a loss
of the actin peripheral band and an increase in actin stress fibers 2 h
after the addition of TGF-
1. Although some cell-cell separation
started at 2 h, the majority of cell-cell contacts was still intact as shown by the
-catenin staining at this time (Fig.
5D). At 4 h, the cells were
separated, and strands were formed that linked the endothelial cells to
one another. Interestingly,
-catenin was still located at the cell
periphery between strands when the cells were separated at 4 h (Fig.
5F, arrow); however, by 8 h post-TGF-
1,
-catenin was no longer present at the cell periphery (Fig. 5H). These results suggest
that the actin cytoskeleton reorganizes before disruption of the
cell-cell junction and that disassembly of the adherens junction as
demonstrated by the loss of
-catenin occurs after the cells have
separated.
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Previous studies (25, 38) have suggested that the loss of cell-cell
junctions containing plakoglobin results in a decrease in junction
strength. We used dual-label immunofluorescence microscopy to determine
whether plakoglobin was lost from the cell-cell junction before or
during TGF-1-induced cell separation. We also colocalized cadherin-5
and
-catenin with
-catenin because these are also important in
the formation of adherens junctions. CPAECs were stained for
-catenin in conjunction with staining for actin, plakoglobin,
-catenin, and cadherin-5 after treatment with TGF-
1 (1.0 ng/ml)
for 2-3 h ("initial" stage of cell separation). Figure 6, A,
C, E,
and G, shows that
-catenin remained
at the cell border before cell separation, but
-catenin was also
found in projections that were perpendicular to the cell periphery.
Whether these structures are filipodia, as suggested by Esser et al.
(14) for vascular endothelial growth factor (VEGF)-treated endothelial
cells, or are fingerlike projections, as shown by Baluk et al. (6),
could not be determined from these micrographs. The colocalization of
-catenin and actin during the initial stage of cell separation (Fig.
6, A and
B) suggests that these
fingerlike projections may be precursors to the perpendicular bands
containing
-catenin at the cell border (Fig.
7). Plakoglobin,
-catenin, and
cadherin-5 are also present at the cell border before cell separation
and are colocalized with
-catenin (Fig. 6,
D, F,
and H, respectively). Plakoglobin,
-catenin,
-catenin, and cadherin-5 were all found to colocalize
at the cell-cell junction in control cells (data not shown). Single
staining of each of these proteins also produced staining patterns
similar to what is shown in Figs. 6 and 7, indicating that
colocalization is not due to background fluorescence from the different
probes (data not shown). Together, the data indicate that
-catenin,
-catenin, plakoglobin, and cadherin-5 appear to realign with
reorganizing actin polymers into fingerlike projections that are
perpendicular to the cell border.
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We next assessed the colocalization of -catenin,
-catenin,
plakoglobin, cadherin-5, and actin immediately after the cells began to
separate. CPAECs were stained for
-catenin in conjunction with
staining for actin, plakoglobin,
-catenin, and cadherin-5 after
treatment with TGF-
1 (1.0 ng/ml) for 3-4 h
("transitional" stage of cell separation). Figure 7 shows that
all four adherens junction proteins, as well as actin, were colocalized
in the strands that connected the cells to one another. Interestingly,
-catenin remained colocalized with plakoglobin,
-catenin, and
cadherin-5 at the cell periphery between strands in cells that were
separated (Fig. 7,
C-H),
suggesting that the adherens junction had yet to disassemble.
Colocalization of actin with
-catenin was not consistent because
areas showing
-catenin staining did not always show actin stress
fibers. The realignment of adherens junction proteins into perpendicular bands at the cell border may be a precursor to the strand
formation observed later in the TGF-
1 time course (Fig. 7).
TGF-1-induced increase in MLC
phosphorylation. Previous studies (15, 20) have
demonstrated that activation of an MLCK-dependent signaling pathway
contributes to thrombin-induced endothelial cell separation, suggesting
that cell separation is caused by an increase in cell contraction. In
addition, Goeckeler and Wysolmerski (20) have shown that
after MLC phosphorylation, myosin interacts with reorganizing actin as
demonstrated by myosin shifting from a detergent-soluble protein pool
to an insoluble (actin-associated) protein pool during cell separation.
We were interested in whether this observed MLC phosphorylation and
shift in myosin to the actin-associated pool occurs in endothelial
cells after treatment with TGF-
1. Figure
8 shows that TGF-
1 induces MLC
phosphorylation beginning at 2 h (23.0 ± 1.5% monophosphate and
8.2 ± 3.6% diphosphate) compared with that in control treated
cells (15.0 ± 3.0% monophosphate and 0.0 ± 0% diphosphate).
Maximum levels of MLC phosphorylation were observed at 6 h
posttreatment with TGF-
1 (21.9 ± 2.7% monophosphate and 32.8 ± 9.0% diphosphate). The elevation in MLC phosphorylation led to
an increased interaction of myosin with actin as demonstrated by the
significant shift of myosin to the actin-associated pool (vehicle: 40 ± 5% soluble and 60 ± 5% insoluble; TGF-
1: 14 ± 3%
soluble and 86 ± 3% insoluble; P < 0.05; Fig. 9). Together, these results indicate that TGF-
1 increases the phosphorylation of
MLC and causes an increased interaction between myosin and actin. Thus TGF-
1-induced endothelial cell separation may be caused
by an increase in cell contraction.
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To determine whether activation of the MLCK-dependent pathway plays a
role in TGF-1-induced decreases in endothelial integrity, we added
the MLCK inhibitor KT-5926 to endothelial monolayers and assessed the
changes in integrity with ECIS (30, 33). Figure
10 shows that coincubation of KT-5926
(1.0 µM) with TGF-
1 (1.0 ng/ml) prevents the decrease in barrier
function caused by TGF-
1 alone. Dual-label immunofluorescence
analysis revealed that endothelial cell monolayers treated with both
KT-5926 and TGF-
1 (for either 3 or 8 h) do not form the
intracellular gaps that are observed in monolayers treated with
TGF-
1 alone (compare Fig. 11 with Fig.
5). Interestingly, the actin stress-fiber formation and the fingerlike
projections that are observed in TGF-
1-treated cells were also seen
in cells that were cotreated with the MLCK inhibitor KT-5926 (Fig.
11J, arrows). Together, the results
indicate that KT-5926 inhibits the formation of intracellular gaps by
TGF-
1, thus preventing the loss of endothelial barrier integrity
that is induced by TGF-
1 alone.
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DISCUSSION |
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Previous studies (11, 28, 42) have demonstrated that
TGF-1 causes changes in the endothelial cell phenotype that would result in a decrease in endothelial monolayer integrity. We have extended these findings by showing that TGF-
1 causes a
dose-dependent decrease in monolayer integrity that begins 1-2 h
after treatment. We have further demonstrated that the loss of
endothelial integrity induced by TGF-
1 is not due to a disassembly
of the adherens junction but is dependent on MLC phosphorylation,
suggesting that contraction plays an important role in this response.
Interestingly, we also found that inhibition of MLC
phosphorylation did not prevent the morphological changes in
adherens junction structure or actin stress-fiber formation induced by
TGF-
1 alone, even though gap formation and increased permeability
were inhibited.
Our results agree with those of Coomber (11), who, using
monolayers of CPAECs, found that endothelial cells lose their
cobblestone morphology and assume a pleiomorphic shape after an 18-h
incubation with TGF-1. The data presented here are also similar to
those of Sutton et al. (42), who showed that when TGF-
1 was added to
newly confluent cultures of bovine aortic endothelial cells for 5 days,
a number of cells in the monolayer detached, whereas the remaining
adherent cells pulled away from one another and became "enlarged and
ragged." These investigators found that the response of these cells
to TGF-
1 was dependent on the proliferative state and the degree of
confluence, with quiescent endothelial cells not showing any response
to TGF-
1.
Our data extend these findings by measuring the changes in albumin
clearance and electrical resistance to assess decreases and/or
increases in monolayer integrity. The use of ECIS allowed the changes
in electrical resistance to be measured in small intervals (1 min) over
a 20-h period. This method allows permeability to be assessed for long
experimental periods and in real time; thus the start of a response and
the transient nature of the response can be accurately determined. As
shown in Fig. 4, electrical resistance began to change between 1 and 2 h after the addition of TGF-1. Resistance continued to decrease over
the next 6 h, reaching a maximum decrease at 8-9 h post-TGF-
1
treatment. The duration of the decrease was dependent on the dose of
TGF-
1 applied to the monolayer, with doses > 0.5 ng/ml maintaining
the decrease in electrical resistance, whereas resistance returned
toward baseline in monolayers treated with doses < 0.5 ng/ml. The
reversibility of the TGF-
1-induced increase in permeability at the
lower doses, as demonstrated by ECIS, may be explained by the
internalization of the TGF-
1 receptor. Anders and colleagues (3, 4)
have demonstrated that on activation of the TGF-
1 receptor, the
receptor-ligand complex is internalized, with downregulation of
heteromeric TGF-
1-receptor activity. These investigators also found
that surface binding recovered from downregulation in 6-8 h. This
would suggest that the lower doses (0.1 ng/ml and below) of TGF-
1
are metabolized during the experimental period shown in Fig. 4,
allowing resistance to return to control levels. However, the higher
doses are not depleted during this time frame; thus the increase in
permeability is maintained for the entire experiment.
Previous investigators (24, 25, 38) have suggested that disassembly of
the adherens junction, i.e., a loss of binding between cadherins and
catenins, with redistribution of the catenins into the cytoplasmic
pool, contributes to the formation of gaps between cells after the
addition of cytokines or other inflammatory mediators. This can be
demonstrated morphologically by a disappearance or loss of
immunofluorescence localization of junctional components at the cell
periphery. An example of this would be the recent work by Kevil et al.
(24) that showed that cadherin-5 and occludin were lost from the cell
border at areas of gap formation after the addition of VEGF to
endothelial monolayers. Although our data show that adherens junction
proteins are lost from the cell periphery after the cells have
completely separated (Fig. 5H),
these junctional proteins were still present at the cell periphery when
the cells initially separated. This is demonstrated in Fig. 7 where
-catenin, plakoglobin,
-catenin, and cadherin-5 are all found at
the cell periphery (arrows) as well as in strands connecting separated cells. Indeed, the presence of adherens junction proteins at the cell
periphery immediately after cell separation suggests that the loss of
cell-cell contact is not the result of a disappearance of junctional
proteins from the cell-cell contact area. This does not rule out the
possibility that homotypic binding of the cadherins was decreased even
though the junctional components are still located at the cell
periphery. Recent studies have suggested that the adhesion strength of
cadherin-mediated junctions may be altered by changes in the
phosphorylation state of the junctional proteins (14), a change in the
lateral clustering of cadherins (45), or a loss of certain adherens
junction proteins from the cadherin-catenin complex (25, 38), all of
which may occur independently of a disappearance of all junctional
components from the cell-cell contact area. Esser et al. (14) recently
showed that VEGF causes an increase in the tyrosine phosphorylation of
cadherin,
-catenin, plakoglobin, and p120 that is associated with a
decrease in endothelial barrier function. Similar to our findings,
Esser et al. also found that the cadherin-catenin complex remained
intact after VEGF treatment and that the adherens junction components
were still localized at the cell periphery when monolayer integrity was
decreased. These findings suggest that phosphorylation of adherens
junction proteins plays a role in governing the integrity of
endothelial monolayers without causing disassembly of the adherens
junction. Whether TGF-
1 alters phosphorylation levels of adherens
junction proteins, thereby decreasing cadherin affinity and strength of the cell-cell junction, remains to be determined.
-Catenin and plakoglobin have been shown to form distinct complexes
with the cytoplasmic domain of E-cadherin in epithelial cells (1, 10,
23). Distinct cadherin-catenin complexes are also believed to occur in
endothelial cells. A recent study by Schnittler et al. (38) as well as
the study by Lampugnani et al. (25) suggested that a loss of
plakoglobin-containing adherens junctions will weaken endothelial
cell-cell contacts. We used immunofluorescence microscopy to determine
whether plakoglobin was lost from cell-cell junctions before other
adherens junction proteins after TGF-
1 treatment. As seen in Fig. 6,
both plakoglobin and
-catenin remain colocalized at the cell border
before cell separation in TGF-
1-treated monolayers. Immediately
after cell-cell separation (Fig. 7), plakoglobin and
-catenin were
also found at the cell periphery between strands as well as in strands
connecting separating cells. This would suggest that plakoglobin does
not preferentially leave the cell junction before
-catenin during TGF-
1-induced endothelial cell separation.
The colocalization of -catenin, plakoglobin,
-catenin, and
cadherin-5 at the cell periphery immediately after cell-cell separation
indicates that disassembly of the adherens junction does not occur
before cell separation. Alternatively, TGF-
1-induced cell separation
could be caused by an increase in tension due to cell contraction. The
appearance of strands containing actin and adherens junction proteins
between separated cells (Fig. 7) has also been observed during
thrombin-induced decreases in endothelial monolayer integrity (37).
Thrombin has been shown to decrease monolayer integrity by increasing
endothelial cell contraction via a signaling cascade similar to that
observed in smooth muscle cells (15, 20). Thrombin stimulation
increases the activity of MLCK, which subsequently phosphorylates MLC,
thus allowing myosin to interact with actin stress fibers within the
cell. Using a native gel system to separate MLC based on the level of
phosphorylation, we found that TGF-
1 treatment increased the
phosphorylation of MLC. As seen in Fig. 8, this increase was temporally
associated with the decrease in monolayer integrity shown in Figs. 2
and 4. In association with the increase in MLC phosphorylation, there was also an increase in the amount of myosin associated with actin (Fig. 9), suggesting that TGF-
1 causes endothelial cell separation by increasing MLC phosphorylation, which leads to cell contraction.
To verify that MLCK plays a role in TGF-1-induced endothelial cell
separation, we applied KT-5926, a specific inhibitor of MLCK (33), to
both control and TGF-
1-treated endothelial monolayers. Dual-label
immunofluorescence microscopy revealed that KT-5926, when coincubated
with TGF-
1, prevented the formation of intercellular gaps (Fig. 11).
Assessment of the changes in monolayer integrity with ECIS showed that
KT-5926 also prevented the decrease in electrical resistance (Fig. 10)
typically observed in monolayers treated with TGF-
1 alone. The dose
of KT-5926 used in these studies (1 µM) is lower than that used in a
previous study (15) that used pretreatment with KT-5926 to prevent the
change in barrier integrity and the increase in MLC phosphorylation
observed after treating bovine pulmonary arterial endothelial
cells with thrombin. Although KT-5926 will also inhibit
protein kinase (PK) C, the inhibition constant value of KT-5926 for PKC
is 40-fold higher than that for MLCK (33). Also, PKC stimulation via
the addition of phorbol esters does not result in the phosphorylation
of MLC in bovine pulmonary arterial endothelial cells, suggesting that
PKC is not involved in this response (15). Thus the ability of KT-5926
to inhibit these changes suggests that TGF-
1 alters barrier
integrity through an MLCK-dependent pathway that decreases the
contractile activity of the endothelial cell. Alternatively, KT-5926
may preserve monolayer integrity by preventing a loss of homotypic
cadherin-mediated adhesion induced by TGF-
1. As previously stated,
there is a growing body of evidence indicating that phosphorylation of
components of the cadherin-catenin complex may lead to a loss of
adhesive function and the destabilization of the adherens junction
(reviewed in Ref. 13). Although MLCK or a serine/threonine kinase with similar characteristics has not been identified as altering cell-cell adhesion or phosphorylating components of the cadherin-catenin complex,
the data shown in Fig. 11 would allow one to hypothesize that such a
mechanism may exist.
Baluk et al. (6) demonstrated that substance P caused the formation of
fingerlike projections before gap formation in tracheal microvascular
endothelial cells and that after gap formation strands formed between
cells. We found similar results after TGF-1 treatment. Before the
formation of large gaps, actin,
-catenin, plakoglobin,
-catenin,
and cadherin-5 are all colocalized in fingerlike projections that are
perpendicular to the cell border (Fig. 6). These structures are also
similar to the filipodia found in endothelial cells treated with VEGF
(14). Whether these are precursors to the strands seen during cell
separation (Fig. 7) remains to be determined. Interestingly, the
formation of these projections as well as increased actin stress-fiber
formation occurs even in the presence of the MLCK inhibitor KT-5926
(Fig. 11, I and
J). Two interpretations may explain
this finding. The first is that the dose of KT-5926 did not completely
inhibit all of the TGF-
1-induced MLC phosphorylation. Thus, in these
experiments, the amount of MLC phosphorylation inhibited is enough to
prevent the severe changes in morphology that result in the disruption
of endothelial monolayer integrity, but the amount of MLC that is
phosphorylated still permits the formation of stress fibers.
A second interpretation is that a signal transduction pathway
independent of both MLC phosphorylation and increased cell tension is
responsible for the generation of actin stress fibers and the formation
of perpendicular strands containing proteins of the adherens junction.
Rho and Rac, members of the Rho family of small GTPases, have been
shown to be important in the formation of actin stress fibers (35).
Recently, Rho and Rac have been implicated in the formation of
cadherin-mediated junctions as well as in the stabilization of these
junctions in epithelial cells (9), suggesting that these small GTPases
may also be involved in the reorganization of adherens junction
proteins. Although Rac and Rho have been shown to be activated after
TGF-1 exposure in other cell types (5, 31), activation of these
small GTPases by TGF-
1 in endothelial cells has yet to be reported.
Further experiments need to be performed to determine the mechanism of
stress-fiber formation in the presence of KT-5926.
In summary, the addition of TGF-1 to confluent endothelial
monolayers induces a significant decrease in the integrity of these
monolayers. The decrease in endothelial monolayer integrity occurs
after the reorganization of the actin cytoskeleton but before the
disassembly of the adherens junction complex as demonstrated by the
presence of
-catenin,
-catenin, plakoglobin, and cadherin-5 at
the cell periphery after cell separation. It is also apparent that
complexes consisting of plakoglobin and cadherin-5 do not leave the
cell-cell junction before those complexes consisting of
-catenin and
cadherin-5 during TGF-
1-induced cell separation. TGF-
1 elevates
the level of MLC phosphorylation, causing an increased interaction
between myosin and reorganizing actin, both of which are associated
with an increase in cell contraction. Taken together, the data indicate
that TGF-
1 induces endothelial cell separation by initiating cell
contraction and not through the disassembly of adherens junctions.
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ACKNOWLEDGEMENTS |
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We thank Wendy Ward, Debbie Moran, and Maureen Davis for assistance in manuscript preparation.
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FOOTNOTES |
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This work was supported by American Heart Association (New York Affiliate) Grant RG-148-N and National Heart, Lung, and Blood Institute Grant HL-54206.
V. Hurst IV is a predoctoral trainee supported by National Heart, Lung, and Blood Institute Grant T32-HL-07194.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. A. Vincent, Dept. of Physiology and Cell Biology (MC-134), Albany Medical College, 47 New Scotland Ave., Albany, NY 12208-3479 (E-mail: peter_vincent{at}ccgateway.amc.edu).
Received 11 September 1998; accepted in final form 23 December 1998.
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