1 Department of Experimental Immunohematology, CLB and Laboratory for Clinical
and Experimental Immunology, Academic Medical Center, Plesmanlaan 125 1066 CX,
Amsterdam
2 Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066
CX Amsterdam, The Netherlands
* Author for correspondence (e-mail: p_hordijk{at}clb.nl )
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The loss of cell-cell adhesion, which is induced by Tat-RacV12, occurred in
parallel to and was dependent upon the intracellular production of reactive
oxygen species (ROS). Moreover, Tat-RacV12 induced an increase in tyrosine
phosphorylation of a component the VE-cadherin-catenin complex, which was
identified as -catenin. The functional relevance of this signaling
pathway was further underscored by the observation that endothelial cell
migration, which requires a transient reduction of cell-cell adhesion, was
blocked when signaling through ROS was inhibited.
In conclusion, Rac-mediated production of ROS represents a previously unrecognized means of regulating VE-cadherin function and may play an important role in the (patho)physiology associated with inflammation and endothelial damage as well as with endothelial cell migration and angiogenesis.
Key words: pHUVEC, Rac, Rho, Reactive oxygen species, VE-cadherin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Like the other cadherins, VE-cadherin mediates calcium-dependent,
homophylic intercellular adhesion and is an important regulator of endothelial
permeability (Corada et al.,
1999; Hordijk et al.,
1999
). Inhibition of VE-cadherin-mediated cell-cell adhesion has
pronounced effects on the organisation of the endothelial actin cytoskeleton
and vice versa (Hordijk et al.,
1999
; Wojciak-Stothard et al.,
1998
). Transfection studies have shown that VE-cadherin is
involved in endothelial cell migration
(Breviario et al., 1995
) and
survival (Carmeliet et al.,
1999
) and appears to be required for the organisation of vascular
structures in embryoid bodies, angiogenesis and tumor growth
(Liao et al., 2000
). The
mechanisms that control the function of VE-cadherin are not well understood,
but they have been suggested to involve indirect signaling through changes in
the actin cytoskeleton and more direct signaling through tyrosine
phosphorylation of VE-cadherin or its associated proteins
(Esser et al., 1998
;
Lampugnani et al., 1997
).
Recent studies have underscored the important role for the actin
cytoskeleton in regulating cadherin function and cell-cell adhesion in
epithelial and endothelial cells. Consequently, Rho family GTPases have been
identified as regulators of cadherin-based cell-cell adhesion. In epithelial
cells, Rac, Rho and CDC42 have all been implicated in the formation and
control of E-cadherin-mediated cell-cell adhesion
(Braga et al., 1997;
Braga et al., 1999
;
Hordijk et al., 1997
;
Takaishi et al., 1997
). In
endothelial cells, the functional link between Rho-like GTPases and
VE-cadherin function is less clear. Inhibition of Rho-dependent contractility
has been shown to prevent receptor-mediated increases in endothelial
permeability (Nieuw Amerongen et al., 1998), whereas others have shown that
activation of Rho using bacterial toxins was not sufficient to perturb
endothelial integrity (Vouret-Craviari et al., 1999). Dominant-negative Rac
(RacN17) has been described as blocking as well as promoting thrombin-induced
permeability in transfected HUVEC
(Vouret-Craviari et al., 1998
;
Wojciak-Stothard et al.,
2001
). In both of these studies, active Rac (Rac V12) was found to
mimic the effects of thrombin in stimulating permeability. In contrast to this
studies, Braga et al. (Braga et al.,
1999
) have described endothelial cell-cell adhesion as independent
from either Rho or Rac activity. However, this study was performed using
unstimulated cells, which may explain the different effects of the Rho and Rac
GTPases.
In previous work, we showed that activation of Rac promotes E-cadherin
function in normal and Ras-transformed epithelial cells
(Hordijk et al., 1997). We
therefore investigated whether introduction of constitutively active Rac would
also affect the localisation and function of VE-cadherin in primary human
endothelial cells. Since these cells are notoriously difficult to manipulate
using classical transfection or retroviral transduction techniques, we
developed cell-penetrating variants of Rho-like GTPases by fusion with the
cell-penetrating sequence of HIV-Tat
(Nagahara et al., 1998
). The
main advantages of using such cell-penetrating GTPases are that one can
analyse cellular responses in primary human cells directly after addition of
the proteins, which is comparable to adding receptor agonists, and that all
cells in the culture are transduced, which allows the combination of
functional and biochemical assays with morphological analysis.
Using this approach we show here that, in contrast to epithelial cells, introduction of active Rac in primary human endothelial cells disturbs VE-cadherin localisation and reduces cell-cell adhesion. This was paralleled by and dependent on the synthesis of reactive oxygen species (ROS) and was also accompanied by tyrosine phosphorylation of the VE-cadherin complex. This mode of regulation is likely to be relevant during a variety of (patho)physiological processes, such as inflammation, endothelial damage following ischemia, endothelial cell migration and angiogenesis.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture
Human endothelial cells were harvested from umbilical veins (HUVECs) as
described previously (Brinkman et al.,
1994) and maintained in RPMI 1640 (Gibco, Grand Island, NY)
supplemented with 10% (v/v) heat-inactivated human serum (HSA, CLB), 2 mM
glutamine (Gibco), 100 units/ml penicillin and 100 µg/ml streptomycin
(Gibco) in fibronectin-coated cultures flasks. The cells were used from
passages two to four.
NIH3T3 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) containing 10% heat-inactivated foetal calf serum (FCS, Gibco), 2 mM glutamine and 100 units/ml penicillin and 100 µg/ml streptomycin. Ras-transformed epithelial cells, Madin Darby Canine Kidney-f3 (MDCK-f3), were cultured in Dulbecco's Modified Epithelial Medium (DMEM, Gibco) containing 10% heat-inactivated fetal calf serum (FCS, Gibco), 100 units/ml penicillin and 100 µg/ml streptomycin.
Retroviral tranduction and transfection assays
Immortalized HUVEC were transduced as described
(Michiels et al., 2000) with a
MMLV-based amphotrophic retrovirus that contained the GFP-human ß-actin
fusion protein. The retroviral construct was generated by cloning the GFP
actin from the pEGFP-actin vector (Clontech, Palo Alto, USA) using
NheI-BamHI restiction digestion into pBluescript and
subsequently NotI (blunted)-EcoRI into the
SwaI-EcoRI sites of the retroviral vector.
Protein purification and transduction
To produce Tat-fusion proteins, PCR products encoding human Rac1 (V12 and
N17 mutants) and RhoA (V14 and N19 mutants) were cloned as
KpnI/EcoRI fragments into the pTat-HA factor
(Nagahara et al., 1998),
sequenced and transformed into the BL21(DE3) strain. Rac primers: forward,
GATCGGTACCCAGGCCATCAAGTGTG-TGGT; reverse, GATCGAATTCTTACAAACAGCAGGCATTTTC-TC;
Rho primers: forward, 5'-GATCGGTACCGCTGCCATCCGG-AAGAAACT-3';
reverse, 5'-GATCGAATTCTCACAAGACAAGG-CAACCAG-3'. Transformed
bacteria were obtained from an overnight culture, resuspended and sonicated in
Z-buffer (8 M Urea, 100 mM NaCl and 20 mM Hepes, pH 8.0). Cleared lysates,
produced by the addition of 20 mM imidazole, were loaded onto a Ni-NTA column
(Qiagen) as described (Nagahara et al.,
1998
). Tat-fusion proteins were eluted with 1 M imidazole in Z
buffer, diluted five times with 20 mM Hepes buffer pH 8.0 and applied to a
Source 30Q column (Pharmacia Biotech, Uppsala, Sweden). After washing, bound
proteins were eluted with 1 M NaCl, desalted on PD-10 columns with PBS with 1
mM Ca2+, snap frozen in 10% (v/v) glycerol and stored at
-80°C.
For transduction, proteins were added directly to the cells in normal culture medium at a final concentration of 50 nM. For controls, we used the Tat-PTD peptide (YGRKKRRQRRR), which was dissolved in PBS/1 mM Ca2+.
Permeability assays
Permeability of pHUVEC monolayers, cultured for 4-5 days on Transwell
filters (0.4 µm pore size, 12 mm diameter; Costar, Cambridge, MA, USA), was
assessed with FITC-labeled dextran
(Hordijk et al., 1999).
Phalloidin staining of cells on a filter, cultured in parallel, was used to
confirm confluency of the monolayers used in the permeability assays. The
endothelial cell monolayers were preincubated for 30 minutes with 50 nM of
Tat-Rac V12, Tat-Rho V14 or an antibody to VE-cadherin as a positive control
(10 µg/ml; c175). FITC-dextran 3000 (10 µg/ml; Molecular Probes, Leiden,
The Netherlands) was then added to the upper compartment, and fluorescence in
the lower compartment was measured after 2 hours with a spectrofluorimeter
(
ex 485 nm;
em 525 nm). Permeability of
untreated monolayers was set at 100% (absolute permeability was in the range
of 5-10% and depended on donor variability).
Immunocytochemistry
Cells that were cultured to subconfluency were incubated with TAT-proteins
or the TAT-peptide in PBS with 1 mM Ca2+ for various time periods,
fixed and permeabilized with 2% paraformaldehyde and 0.5% (v/v) Triton-X100 in
wash buffer (PBS containing 0.5% (v/v) HSA, 1 mM Ca2+) for 20
mintes at room temperature (RT). Cells were stained with the indicated mouse
monoclonal antibodies, washed and incubated with Alexa-488-conjugated
goat-anti-mouse Ig antibodies in combination with Texas Red phalloidin (1
U/ml) to visualize F-actin. Images were recorded with a ZEISS LSM 510 confocal
laser scanning microscope. Immunofluorescent staining for the HA-epitope
confirmed protein transduction of all cells in the culture (not shown). For
time-lapse confocal microscopy, cells were mounted in culture medium in a
temperature-controlled incubation chamber kept at 37°C.
Endothelial cell migration assay
To monitor endothelial cell migration, pHUVECs were serum-starved
overnight, detached with 5 mM EDTA and plated on fibronectin-coated Transwell
filters (8 µm pore size, 6.5 mm diameter; Costar) in serum-free RPMI 1640
medium. Medium containing 0-10% FCS was added to the lower chamber and the
cells were allowed to migrate for 3-5 hours. Next, the cells were fixed in 2%
paraformaldehyde containing 0.5% (v/v) human serum albumin (HSA) and 1 mM
Ca2+. Cells from the upper compartment were removed with a cotton
swab. Nuclei of migrated cells were stained with Hoechst 33258 and counted by
fluorescence microscopy.
Measurement of reactive oxygen species
To measure generation of reactive oxygen species (ROS) in endothelial
cells, pHUVECs cultured on fibronectin-coated glass coverslips were loaded
with dihydrorodamine 123 (DHR, 30 µM; Molecular Probes) for 30 minutes,
washed and subsequently transduced with the Tat-peptide or Tat-fusion
proteins. Fluorescence of DHR was quantified by time-lapse confocal
microscopy. Intensity values are shown as the percentage increase relative to
the values at the start of the experiment.
Immunoprecipitaton and western blotting
Cells were grown to confluency in 6-well plates, washed and lysed for 10
minutes in 0.5 ml lysis buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1%
NP-40, 0.5 mM orthovanadate with the addition of protease inhibitor cocktail
tablets (Boehringer Mannheim, Mannheim, Germany) and, where indicated, in the
presence of 0.1% SDS. The lysates were precleared with 25 µl Protein
A-sepharose beads (Pharmacia Biotech). Next, the lysates were incubated with
25 µl of Protein A beads, coated with 10 µg/ml VE-cadherin (7H1) or
anti--catenin antibodies for 1 hour at 4°C under continuous mixing.
The beads were extensively washed in lysis buffer, and proteins were eluted by
boiling in sample buffer. Samples were run on 10% SDS-PAGE under reducing
conditions, transferred onto 0.2 µm nitrocellulose filters (Schleicher
& Schuell, Dassel, Germany), which were blocked with 5% dried milk protein
in TBST buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.5% (v/v) Tween-20)
and incubated with the appropriate antibodies (to VE-cadherin (7H1),
-catenin or PY-20, all diluted 1:1000 in blocking buffer). This was
followed by incubation with rabbit anti-mouse (R
M) IgG-HRP (1:1000,
DAKO) at room temperature. The immunoreactive bands were visualised with the
ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, England).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The effects of Tat-RacV12 were also tested in Ras-transformed MDCKf3 cells,
which are known to revert from a fibroblastoid to a more epithelial phenotype
upon expression of constitutively active Rac
(Hordijk et al., 1997).
Time-lapse analysis showed that these MDCK cells, in contrast to NIH3T3 cells,
did not show either contraction or lamellipodia formation following
transduction with Tat-RacV12. However, incubation with Tat-RacV12 induced a
clear increase in cortical F-actin and recruitment of ß-catenin to
cell-cell contact sites (Fig.
1c,d). These effects were already seen after 3 hours, but were
most obvious following overnight incubation, indicating that the
cell-penetrating Rac protein, similar to its retrovirally expressed
equivalent, promotes cell-cell adhesion in these Ras-transformed epithelial
cells. These data demonstrate that protein transduction of RacV12 induces
similar phenotypic changes to RacV12 proteins expressed through microinjection
(Ridley et al., 1992
) or
retroviral transduction (Hordijk et al.,
1997
).
Transduction of Rac proteins in primary HUVECs
Given the stimulatory effect of Rac on E-cadherin function in epithelial
cells (Hordijk et al., 1997),
we analysed its effect on cadherin-based adhesion and barrier function of
primary HUVECs. The Tat-RacV12 protein induced pronounced but transient
cellular contraction, followed by extensive cell spreading and lamellipodia
formation. This was clearly visualized in immortalized HUVEC
(Fontijn et al., 1995
) that
expressed a GFP-actin fusion protein (Fig.
2A). Immunocytochemical analysis of subconfluent pHUVEC showed
that Tat-RacV12 induced endothelial contractility and intercellular gap
formation after only 15 minutes of transduction
(Fig. 2Bb), and prominent loss
of cell-cell adhesion and formation of lammelipodia was observed after 30
minutes. Furthermore, this Tat-RacV12-induced contractility and loss of
cell-cell adhesion was accompanied by a rapid increase in the levels of
F-actin stress fibers and in altered VE-cadherin distribution
(Fig. 2Bc). Complementary FACS
analysis showed that under these circumstances the surface expression of
VE-cadherin remained unaltered (not shown). In contrast, transduction of
Tat-RacN17 protein did not induce significant changes in the cytoskeleton or
in VE-cadherin distribution (Fig.
2Bd). When Tat-RacV12-treated cells were left overnight,
VE-cadherin distribution and cell-cell adhesion were restored (not shown),
indicating that the protein was not toxic to the cells and that its effects
were reversible. Together, these results show that the Tat-RacV12 protein
induces a rapid loss of VE-cadherin-mediated cell-cell adhesion followed by
the induction of a `Rac-phenotype', as deduced from the induction of
lamellipodia.
|
It has been reported that control of endothelial cell-cell adhesion by
Rho-like GTPases is dependent on the confluency of the cells
(Braga et al., 1999). The
effects of Tat-RacV12 were therefore analysed in cultures of different cell
density. The data in Fig. 2C
show that in confluent monolayers, Tat-RacV12 induces mainly stress fibers and
small gaps in between the cells. Occasionally, formation of ruffles can also
be observed. At subconfluent monolayers, the Tat-RacV12-induced cellular
contraction results in a more pronounced loss of cell-cell adhesion,
accompanied by formation of lamellae and ruffles. Thus, the effects of
Tat-RacV12, that is, induction of contractility and gap formation, seem not to
be qualitatively different, but a more general loss of cell-cell adhesion and
appearance of lamellae is most clear at lower densities.
Role of Rho GTPase in Rac-mediated responses
As the effects of Tat-RacV12 were accompanied by transient cellular
contraction, we tested Rho-mediated contractility for a role in this response.
Pretreament of pHUVECs with the C3 exo-enzyme from Clostridium
botulinum prevented Tat-RacV12-induced stress fiber formation and loss of
cell-cell adhesion (Fig. 3A),
suggesting that in HUVECs, as in fibroblasts
(Ridley and Hall, 1992), the
Rac phenotype requires Rho activity. To assess whether the effects of Rac
could be mimicked by constitutively active Rho, we transduced pHUVECs with
Tat-RhoV14. The active Rho protein rapidly induced pronounced stress fiber
formation and prominent focal adhesions, as revealed by vinculin staining
(Fig. 3B), but did not induce
significant changes in junctional VE-cadherin staining or loss of cell-cell
adhesion (Fig. 3B). Small gaps
in between the cells could be observed, but this effect was not comparable
with the more general loss of cell-cell adhesion induced by transduction of
Tat-RacV12 (compare Fig. 3B)
with Fig. 2A). Tat-RhoN19, the
inactive mutant of Rho, had little effect on the cellular F-actin distribution
and did not significantly alter VE-cadherin distribution
(Fig. 3B).
|
To analyse whether the Tat-RacV12-induced loss of cell-cell adhesion and
gap formation would result in reduced monolayer integrity we performed
permeability assays. As shown in Fig.
4A, Tat-RacV12 increased endothelial monolayer permeability by
25-30% compared with basal values. Tat-RhoV14 only partially mimicked the
response of Tat-RacV12 and increased permeability by approximately 15%. An
antibody against VE-cadherin was used as a positive control. As shown in
Fig. 4B, Tat-RacV12 also
induced gap formation and loss of cell-cell adhesion in highly confluent cells
that were grown on the filters. The results obtained with Tat-RacV12 and
Tat-RhoV14 are in line with the immunofluorescence analysis shown in Figs
2A and
3B and indicate that, although
Rho activity is required for Rac-mediated loss of cell-cell adhesion, the
Tat-RhoV14 protein by itself is not sufficient to fully mimic this effect. The
combination of the constitutively active Rac and Rho proteins did not result
in an additional increase in the Rac-induced increase in permeability (not
shown). The Tat-RacV12-mediated increase in endothelial permeability in HUVEC
is in agreement with recent data from Wojciak-Stothard et al.
(Wojciak-Stothard et al.,
2001) who used adenovirus to express active Rac proteins in
primary HUVEC.
|
Induction of ROS by Tat-RacV12 in primary HUVECs
In phagocytes, the Rac2 protein is essential in the synthesis of ROS
through its activation of the NADPH oxidase complex
(Diebold and Bokoch, 2001).
Exposure to ROS, in particular H2O2, has been show to
reduce cell-cell adhesion in epithelial cells
(Volberg et al., 1992
) and to
affect endothelial cell function (Lum and
Roebuck, 2001
). Since Rac activation has also been implicated in
the production of ROS in endothelial cells
(Deshpande et al., 2000
), we
analyzed whether production of ROS was induced by Tat-RacV12. As shown in
Fig. 5A, Tat-RacV12 induced a
rapid (within 5 minutes) increase in DHR (dihydrorodamine 123) fluorescence,
indicating that ROS were formed. This effect lasted for approximately 60
minutes, after which time a decline in DHR fluorescence was observed. These
kinetics paralled the effects on cell-cell adhesion. In contrast, neither the
Tat-peptide nor Tat-RhoV14 (not shown) induced ROS production.
|
To determine whether ROS were involved in Tat-RacV12-mediated loss of cell-cell adhesion, pHUVEC were pretreated with the oxygen scavenger N-acetyl-cysteine (N-AC) and then incubated with Tat-RacV12. N-AC, which by itself left the cytoskeleton and VE-cadherin distribution unaltered, was found to prevent the Tat-RacV12-induced production of ROS (not shown) and blocked the concomitant loss of cell-cell contacts (Fig. 5Bc), indicating that ROS are essentially involved in Rac-mediated loss of cell-cell adhesion and VE-cadherin redistribution. In contrast, the induction of stress fibers was not inhibited in N-AC-pretreated cells, demonstrating that N-AC is not toxic for the cells and did not interfere with (Rho-dependent) cell signaling events (Fig. 5Bc). The addition of 1 mM H2O2 mimicked the Tat-RacV12-induced loss of cell-cell adhesion (not shown).
Induction of tyrosine phosphorylation
The pathways that regulate VE-cadherin-mediated cell-cell adhesion from
within the endothelial cells are poorly defined. Cellular contractility has
been proposed to be important, but our present data indicate that the
induction of Rho-mediated contractility is not sufficient to disrupt
VE-cadherin function (Fig. 3B).
Others have reported an important role for tyrosine phosphorylation of the
VE-cadherin complex as a means to regulate cell-cell adhesion in epithelial
and endothelial cells (Braga et al.,
1999; Esser et al.,
1998
; Volberg et al.,
1992
). In this regard, tyrosine phosphorylation of VE-cadherin,
ß-catenin and
-catenin have been reported, although the kinase
that is involved in phosphorylation of the cadherin-catenin complex in
endothelial cells has not been identified.
To investigate the induction of tyrosine phosphorylation, the cellular
distribution of phosphotyrosine was analysed in pHUVEC that had been
transduced with Tat-RacV12. The data in
Fig. 6A show that Tat-RacV12
induced an increase in phosphotyrosine content, in particular at cell borders.
To detect tyrosine phosphorylation at adherens junctions, phosphotyrosine was
detected using a monoclonal antibody, and, owing to the lack of a suitable
polyclonal VE-cadherin antibody, adherens junctions were marked using a
polyclonal anti-ß-catenin antibody. Colocalisation of phosphotyrosine and
ß-catenin could be observed following transduction with Tat-RacV12,
indicating that junctional proteins become phosphorylated
(Fig. 6B). To test for specific
changes in tyrosine phosphorylation of the VE-cadherin complex, the fusion
protein was immunoprecipitated under non-denaturing conditions following
transduction of the cells with Tat-RacV12. Within 10-20 minutes, increased
tyrosine phosphorylation was detected in the immunoprecipitate, albeit not of
VE-cadherin itself (migrating at around 130 kDa), but of an associated protein
with a molecular weight of approximately 100-110 kDa
(Fig. 6C). This apparent
molecular weight corresponds to that of -catenin. Subsequent
immunoprecipitation under denaturing conditions indicated tyrosine
phosphorylation of
-catenin (molecular weight, 105 kDa) upon
transduction of pHUVEC with Tat-RacV12
(Fig. 6D). This response was
inhibited when the cells were preincubated with N-AC, indicating that the
phosphorylation of
-catenin is dependent on Rac-mediated production of
ROS.
|
To further establish the significance of Rac-mediated ROS production for endothelial cell function, we analysed migration of pHUVEC across fibronectin-coated Transwell filters. Serum increased endothelial migration five-fold after 3 hours and 15-fold after 5 hours of migration (Fig. 7). No differences were observed in the migration induced by either 5 or 10% serum. Transduction of pHUVEC with Tat-RacV12 did not promote either the spontaneous or serum-induced migration (not shown). However, pretreatment of the pHUVEC with N-AC almost completely abrogated serum-induced migration of pHUVEC, underscoring the importance of cellular ROS production for endothelial cell motility.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The analysis of the role of Rac and Rho proteins in primary human
endothelial cells was simplified significantly by using protein transduction,
which has previously proven successful for a variety of proteins, including
the small GTPase Rho in chicken osteclasts
(Chellaiah et al., 2000) as
well as in human eosinophils (Alblas et
al., 2001
).
The major finding of this study is that RacV12 induces loss of endothelial
cell-cell adhesion through a pathway that involves the production of ROS and
tyrosine phosphorylation of the VE-cadherin complex. This negative effect of
RacV12 on VE-cadherin function is in sharp contrast to its effect in
epithelial cells where Rac and its exchange factor Tiam-1 promote E-cadherin
function (Hordijk et al.,
1997). Considering the fact that the basic principles of
cadherinmediated homotypic adhesion (composition of the cadherincatenin
complexes, regulation through the actin cytoskeleton) are very similar in
epithelial cells and in endothelial cells, this result represents an
intriguing discrepancy.
Although Rho-mediated cell contractility was required for RacV12-induced
loss of cell-cell adhesion, Tat-RhoV14 showed limited effects on cell-cell
junctions and monolayer permeability. These results may be explained by the
fact that experiments were performed in the presence of 10% serum, a known
activator of Rho, so that transduction with exogenous active Rho would have
limited additional effects. Another explanation might be that Rho activity per
se is not sufficient to open intercellular contacts and that RacV12, in
addition to inducing Rho-dependent contractility, activates a complementary
Rho-independent pathway that negatively controls endothelial cell-cell
adhesion. This explanation is supported by our observation that scavenging ROS
reduces Rac-mediated loss of cell-cell adhesion but does not affect the
Rho-dependent formation of stress fibers. Recently, RacV12-mediated loss of
cadherin-dependent adhesion was also described in keratinocytes
(Braga et al., 2000). However,
since the intracellular signaling underlying this effect was not studied, it
is not clear whether similar pathways control cadherin function in
keratinocytes and in endothelial cells. We have not been able to detect
increased production of ROS by Tat-RacV12 in epithelial cell lines, and it
needs to be investigated whether this pathway may be prevalent in primary
cells. Finally, transduction of primary HUVEC with Tat-N17-Rac or Tat-N19-Rho
proteins did not disturb VE-cadherin localization at cell-cell borders,
indicating that, at least within the time frame of these experiments, Rac or
Rho activity is not required to keep VE-cadherin at cellular junctions, which
is in agreement with earlier studies (Braga
et al., 1999
).
The modulatory role of ROS in cell-cell adhesion is well known and is
largely based on the addition of (high concentrations of)
H2O2 to endothelial and epithelial cells, resulting in
increased permeability, cellular injury and cell death
(Lum and Roebuck, 2001).
However, increasing evidence indicates that at low levels, ROS can also
function as signaling molecules participating in the regulation of fundamental
cellular processes such as cell growth, cell division and apoptosis
(Finkel, 1999
). A number of
previous studies have suggested that Rac is involved in ROS production in
endothelial cells, as it is in neutrophils, although it is not certain which
type of ROS-generating enzyme is expressed in these cells. Rac-mediated ROS
production has been implicated in TNF-
-induced endothelial apoptosis
(Deshpande et al., 2000
) and
shear-stress induced tyrosine phosphorylation
(Yeh et al., 1999
) but to our
knowledge, production of intracellular peroxide has so far not been implicated
in the regulation of VE-cadherin function.
The mechanism of action of ROS-mediated loss of cadherin function is not
established, but peroxide has been proposed to increase oxydation of crucial
cysteine residues in tyrosine phosphatases
(Rhee et al., 2000), leading
to inactivation. This results in a net increase in tyrosine phosphorylation,
which could lead to increased phosphorylation of the cadherincatenin complex,
an event that is generally associated with reduced cell-cell adhesion. Our
data are in agreement with this hypothesis in that Tat-RacV12-induced ROS
production is accompanied by ROS-dependent phosphorylation of
-catenin,
which links the VE-cadherin complex to the cortical actin cytoskeleton. It is
not known which phosphatase or kinase may be involved in this signaling,
although it is intriguing that association of the SHP2 tyrosine phosphatase
with VE-cadherin has recently been reported to be modulated by thrombin
(Ukropec et al., 2000
).
Previous studies demonstrated a lack of
-catenin phosphorylation
(Andriopoulou et al., 1999
;
Lampugnani et al., 1997
).
However, these studies were performed under different conditions and therefore
difficult to compare. Although Tat-RacV12 induces delocalization of the
VE-cadherin-catenin complex, we have not been able to detect
Tat-RacV12-mediated dissociation of the complex, suggesting that the complex
as it exists at the cell-cell junctions becomes disconnected from the cortical
actin cytoskeleton, resulting in loss of adhesion.
The role of Rac-mediated ROS production in reducing VE-cadherin function
may explain the endothelial damage that is associated with, for instance,
ischemia-reperfusion injury, an effect that is known to be accompanied by the
formation of ROS. Moreover, the modulation of VE-cadherin function through the
intracellular levels of ROS appears to also be relevant for migration
(Fig. 7), indicating that
inhibition of ROS production may interfere with angiogenesis. This hypothesis
is based on the fact that transient reduction of cadherin function is required
for efficient endothelial cell migration
(Cai et al., 1999;
Liao et al., 2000
). The
general roles that intracellular ROS levels may play in endothelial integrity
are further underscored by our finding that thrombin-mediated loss of
cell-cell adhesion was also blocked following ROS scavenging (not shown),
although we have not yet been able to detect thrombin-induced formation of
ROS.
In conclusion, the Rac-ROS signaling pathway seems to be an important regulator of VE-cadherin function and cell-cell adhesion in primary human endothelial cells. Future research will focus on the type of oxidase that is relevant for endothelial cells and the pathway that leads to Rac-mediated phosphorylation of the cadherin-complex and the concomitant loss of cadherin function.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alblas, J., Ulfman, L., Hordijk, P. and Koenderman, L.
(2001). Activation of rhoa and rock are essential for detachment
of migrating leukocytes. Mol. Biol. Cell
12,2137
-2145.
Andriopoulou, P., Navarro, P., Zanetti, A., Lampugnani, M. G.
and Dejana, E. (1999). Histamine induces tyrosine
phosphorylation of endothelial cell-to-cell adherens junctions.
Arterioscler. Thromb. Vasc. Biol.
19,2286
-2297.
Braga, V. M., Machesky, L. M., Hall, A. and Hotchin, N. A.
(1997). The small GTPases Rho and Rac are required for the
establishment of cadherin-dependent cell-cell contacts. J. Cell
Biol. 137,1421
-1431.
Braga, V. M., Del Maschio, A., Machesky, L. and Dejana, E.
(1999). Regulation of cadherin function by Rho and Rac:
modulation by junction maturation and cellular context. Mol. Biol.
Cell 10,9
-22.
Braga, V. M., Betson, M., Li, X. and Lamarche-Vane, N.
(2000). Activation of the small GTPase Rac is sufficient to
disrupt cadherin-dependent cell-cell adhesion in normal human keratinocytes.
Mol. Biol. Cell 11,3703
-3721.
Breviario, F., Caveda, L., Corada, M., Martin-Padura, I.,
Navarro, P., Golay, J., Introna, M., Gulino, D., Lampugnani, M. G. and Dejana,
E. (1995). Functional properties of human vascular
endothelial cadherin (7B4/cadherin-5), an endothelium-specific cadherin.
Arterioscler. Thromb. Vasc. Biol.
15,1229
-1239.
Brinkman, H. J., Mertens, K., Holthuis, J., Zwart-Huinink, L. A., Grijm, K. and van Mourik, J. A. (1994). The activation of human blood coagulation factor X on the surface of endothelial cells: a comparison with various vascular cells, platelets and monocytes. Br. J. Haematol. 87,332 -342.[Medline]
Cai, T., Fassina, G., Morini, M., Aluigi, M. G., Masiello, L., Fontanini, G., D'Agostini, F., De Flora, S., Noonan, D. M. and Albini, A. (1999). N-acetylcysteine inhibits endothelial cell invasion and angiogenesis. Lab. Invest. 79,1151 -1159.[Medline]
Carmeliet, P. (2000). Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6, 389-395.[Medline]
Carmeliet, P., Lampugnani, M. G., Moons, L., Breviario, F., Compernolle, V., Bono, F., Balconi, G., Spagnuolo, R., Oostuyse, B., Dewerchin, M. et al. (1999). Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 98,147 -157.[Medline]
Chellaiah, M. A., Soga, N., Swanson, S., McAllister, S.,
Alvarez, U., Wang, D., Dowdy, S. F. and Hruska, K. A. (2000).
Rho-A is critical for osteoclast podosome organization, motility, and bone
resorption. J. Biol. Chem.
275,11993
-12002.
Corada, M., Liao, F., Lindgren, M., Lampugnani, M. G., Brevario,
F., Frank, R., Muller, W. A., Hicklin, D. J., Bohlen, P. and Dejana, E.
(2001). Monoclonal antibodies directed to different regions of
vasculair endothelial cadherin extracellulair domain affect adhesion and
clustering of the protein and modulate endothelial permeability.
Blood 97,1679
-1684.
Corada, M., Mariotti, M., Thurston, G., Smith, K., Kunkel, R.,
Brockhaus, M., Lampugnani, M. G., Martin-Padura, I., Stoppacciaro, A., Ruco,
L. et al. (1999). Vascular endothelial-cadherin is an
important determinant of microvascular integrity in vivo. Proc.
Natl. Acad. Sci. USA 96,9815
-9820.
Dejana, E., Bazzoni, G. and Lampugnani, M. G. (1999). Vascular endothelial (VE)-cadherin: only an intercellular glue? Exp. Cell Res. 252, 13-19.[Medline]
Deshpande, S. S., Angkeow, P., Huang, J., Ozaki, M. and Irani,
K. (2000). Rac1 inhibits TNF-alpha-induced endothelial cell
apoptosis: dual regulation by reactive oxygen species. FASEB
J. 14,1705
-1714.
Diebold, B. A. and Bokoch, G. M. (2001). Molecular basis for Rac2 regulation of phagocyte NADPH oxidase. Nat. Immunol. 2,211 -215.[Medline]
Esser, S., Lampugnani, M. G., Corada, M., Dejana, E. and Risau,
W. (1998). Vascular endothelial growth factor induces
VE-cadherin tyrosine phosphorylation in endothelial cells. J. Cell
Sci. 111,1853
-1865.
Finkel, T. (1999). Signal transduction by reactive oxygen species in non-phagocytic cells. J. Leukoc. Biol. 65,337 -340.[Abstract]
Fontijn, R., Hop, C., Brinkman, H. J., Slater, R., Westerveld, A., van Mourik, J. A. and Pannekoek, H. (1995). Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. Exp. Cell Res. 216,199 -207.[Medline]
Hordijk, P. L., ten Klooster, J. P., van der Kammen, R. A.,
Michiels, F., Oomen, L. C. and Collard, J. G. (1997).
Inhibition of invasion of epithelial cells by Tiam1-Rac signaling.
Science 278,1464
-1466.
Hordijk, P. L., Anthony, E., Mul, F. P., Rientsma, R., Oomen, L.
C. and Roos, D. (1999). Vascular-endothelial-cadherin
modulates endothelial monolayer permeability. J. Cell
Sci. 112,1915
-1923.
Lampugnani, M. G., Corada, M., Caveda, L., Breviario, F., Ayalon, O., Geiger, B. and Dejana, E. (1995). The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J. Cell Biol. 129,203 -217.[Abstract]
Lampugnani, M. G., Corada, M., Andriopoulou, P., Esser, S.,
Risau, W. and Dejana, E. (1997). Cell confluence regulates
tyrosine phosphorylation of adherens junction components in endothelial cells.
J. Cell Sci. 110,2065
-2077.
Liao, F., Li, Y., O'Connor, W., Zanetta, L., Bassi, R.,
Santiago, A., Overholser, J., Hooper, A., Mignatti, P., Dejana, E., Hicklin,
D. J. and Bohlen, P. (2000). Monoclonal antibody to vascular
endothelial-cadherin is a potent inhibitor of angiogenesis, tumor growth, and
metastasis. Cancer Res.
60,6805
-6810.
Lum, H. and Roebuck, K. A. (2001). Oxidant
stress and endothelial cell dysfunction. Am. J. Physiol. Cell
Physiol. 280,C719
-C741.
Michiels, F., van der Kammen, R. A., Janssen, L., Nolan, G. and Collard, J. G. (2000). Expression of Rho GTPases using retroviral vectors. Methods Enzymol. 325,295 -302.[Medline]
Nagahara, H., Vocero-Akbani, A. M., Snyder, E. L., Ho, A., Latham, D. G., Lissy, N. A., Becker-Hapak, M., Ezhevsky, S. A. and Dowdy, S. F. (1998). Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat. Med. 4,1449 -1452.[Medline]
Noren, N. K., Liu, B. P., Burridge, K. and Kreft, B.
(2000). p120 catenin regulates the actin cytoskeleton via Rho
family GTPases. J. Cell Biol.
150,567
-580.
Rhee, S. G., Bae, Y. S., Lee, S. R. and Kwon, J. (2000). Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Science STKE 53,1 -6.
Ridley, A. J. and Hall, A. (1992). The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70,389 -399.[Medline]
Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. and Hall, A. (1992). The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70,401 -410.[Medline]
Takaishi, K., Sasaki, T., Kotani, H., Nishioka, H. and Takai,
Y. (1997). Regulation of cell-cell adhesion by rac and rho
small G proteins in MDCK cells. J. Cell Biol.
139,1047
-1059.
Ukropec, J. A., Hollinger, M. K., Salva, S. M. and Woolkalis, M.
J. (2000). SHP2 association with VE-cadherin complexes in
human endothelial cells is regulated by thrombin. J. Biol.
Chem. 275,5983
-5986.
van Nieuw Amerongen, G. P., Draijer, R., Vermeer, M. A. and van
Hinsbergh, V. W. (1998). Transient and prolonged increase in
endothelial permeability induced by histamine and thrombin: role of protein
kinases, calcium, and RhoA. Circ. Res.
83,1115
-1123.
Volberg, T., Zick, Y., Dror, R., Sabanay, I., Gilon, C., Levitzki, A. and Geiger, B. (1992). The effect of tyrosine-specific protein phosphorylation on the assembly of adherens-type junctions. EMBO J. 11,1733 -1742.[Abstract]
Vouret-Craviari, V., Boquet, P., Pouyssegur, J. and
Obberghen-Schilling, E. 1998. Regulation of the actin
cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in
endothelial barrier function. Mol. Biol. Cell
9,2639
-2653.
Wojciak-Stothard, B., Entwistle, A., Garg, R. and Ridley, A. J. (1998). Regulation of TNF-alpha-induced reorganization of the actin cytoskeleton and cell-cell junctions by Rho, Rac, and Cdc42 in human endothelial cells. J. Cell Physiol. 176,150 -165.[Medline]
Wojciak-Stothard, B., Potempa, S., Eichholtz, T. and Ridley, A.
J. (2001). Rho and Rac but not Cdc42 regulate endothelial
cell permeability. J. Cell Sci.
114,1343
-1355.
Yeh, L. H., Park, Y. J., Hansalia, R. J., Ahmed, I. S., Deshpande, S. S., Goldschmidt-Clermont, P. J., Irani, K. and Alevriadou, B. R. (1999). Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS. Am. J. Physiol 276,C838 -C847.[Medline]