 |
INTRODUCTION |
The tight junction (TJ)1
is localized at cell-cell contact sites in epithelial and endothelial
cells. It serves as a paracellular barrier to restrict the movement of
ions and proteins across tissue boundaries (1-4). This barrier
function is essential for the maintenance of tissue environments.
Dysfunction of the TJ occurs in response to a variety of inflammatory
stimuli and also during ischemia, leading to tissue edema and damage.
Therefore, analysis of TJ regulation could lead to an understanding of
normal physiology as well as pathology and to the identification of
novel therapeutic targets.
The molecular components of the TJ are being discovered and so far
include ZO-1 (5), ZO-2 (6), ZO-3 (7), cingulin (8), 7H6 antigen (9),
Rab3b (10), and symplekin (11). In addition to these peripheral
membrane proteins, occludin was discovered as an integral membrane
protein of the TJ having four transmembrane domains (12). The carboxy
tail of occludin is linked to the actin cytoskeleton via ZO-1, ZO-2 and
ZO-3 (13-15). Recent work has also identified members of the claudin
family as TJ components that have four transmembrane domains but no
sequence similarity to occludin (16-18). It has not yet been defined
how these novel proteins interact with occludin or other TJ components. However, as the protein architecture of the TJ is revealed, analysis of
function of the TJ on a molecular basis becomes possible.
The formation and maintenance of the TJ has been considered to be
regulated not only by the specific proteins of cell-cell junctions but
also by the perijunctional actin cytoskeleton (19). Botulinum C3 toxin,
which ADP-ribosylates and inactivates Rho, has been shown to disrupt
perijunctional actin, resulting in TJ dysfunction in epithelial cells
(20). Also, mutants of RhoA and Rac1 disrupted TJ functions (21). Thus,
signaling pathways transduced by the Ras-related small GTPase Rho
family members like Rho and Rac1, which control the actin cytoskeleton,
have been implicated in the regulation of the TJ (see Refs. 19 and 22),
whereas involvement of downstream signaling events of Rho in TJ
function is still lacking. Recently, several downstream targets of Rho
have been identified (see Refs. 22 and 23). Among these, p160ROCK/Rho
kinase (24, 25), one of the key effectors of Rho, has been shown to be
a serine-threonine kinase that is involved in the regulation of actin
organization, cellular morphology, and cellular transformation (26,
27).
In this study, the basis of effects of inflammatory stimuli on TJ
permeability was investigated. The role of the cytoskeleton and the
possibility of direct effects on occludin were explored. Using ECV304
cells as a model system, lysophosphatidic acid (LPA) and histamine were
found to increase the paracellular permeability of the tracer
horseradish peroxidase (HRP). LPA is a glycerophospholipid that is
secreted from activated platelets, mediating tissue regeneration and
wound healing (28), and can induce an increase in TJ permeability in
brain endothelial cells (29). Also, histamine causes vascular leakiness
in vivo, but mechanisms are not completely clear yet (see
Ref. 30). Because LPA activates Rho and its targets (22, 28), the
involvement of these was studied using a dominant-negative mutant of
RhoA (RhoA T19N) considered to be the inactive GDP-bound form of RhoA
(31, 32) and a specific p160ROCK inhibitor Y-27632 (25). Evidence is
provided that TJ permeability is regulated by
RhoA-p160ROCK-dependent and -independent mechanisms and
that occludin is a target for receptor-initiated signaling events
regulating its phosphorylation.
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EXPERIMENTAL PROCEDURES |
Reagents--
All tissue culture materials were from Life
Technologies, Inc. Gel electrophoresis reagents were from Bio-Rad. HRP
was from Sigma. Cytochalasin D and
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) were from Calbiochem. Other reagents used were of the
highest grade commercially available. Y-27632 (25), a specific inhibitor of p160ROCK, was a generous gift from WelFide (Osaka, Japan).
Pervanadate was prepared as described previously (33).
Antibodies--
The rat monoclonal antibody (MOC37) and rabbit
polyclonal antibody were both raised against a fragment of occludin
fused to glutathione S-transferase (34). The anti-ZO-1
monoclonal antibody and the anti-phosphotyrosine antibody PY20 were
from Transduction Laboratories (Lexington, KY). The
anti-phosphotyrosine antibody 4G10 was purchased from Upstate
Biotechnology (Lake Placid, NY). The monoclonal antibody against RhoA
was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A purified
polyclonal antibody recognizing Thr18- and
Ser19-phosphorylated myosin light chain (MLC) was raised
against the synthetic peptide RPQRApTpSNVFAMK (where p indicates
phosphorylation), as described previously (35).
Cell Culture--
ECV304 cells were obtained from the European
Collection of Animal Cell Cultures (Salisbury, UK) and cultured at
37 °C under an atmosphere of 5% CO2 in DME containing
10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Replication-defective Recombinant Adenovirus--
RhoA T19N,
from Professor Yoshimi Takai (Osaka University, Osaka, Japan), or LacZ
was placed into pAdex1CAwt under a CA promoter comprising a
cytomegalovirus enhancer and a chicken
-actin promoter (36) to give
pAdex RhoA T19N and pAdex LacZ, respectively. A recombinant adenovirus
was constructed by in vitro homologous recombination in 293 cells using pAdex RhoA T19N or pAdex LacZ and the adenovirus
DNA-terminal protein complex by a method previously described (37).
Recombinant Adenoviral Gene Transfer--
After ECV304 cells had
attained confluence, they were infected with recombinant adenovirus
expressing either LacZ or RhoA T19N. The viruses were diluted in
serum-depleted medium at a multiplicity of infection of 30 particles/cell and incubated for 60 min. The viral suspension was
removed by washing twice with serum-depleted DME, and the cells were
cultured with serum-depleted DME for 48 h.
HRP Flux Measurement--
ECV304 cells were seeded onto 0.4-µm
polycarbonate Transwell filters (Costar Corp., Cambridge, MA). After
attaining confluence, the cells were incubated with serum-depleted DME
with or without recombinant adenoviral gene transfer for 48 h. For
pretreatment with compound, vehicle or Y-27632 at a final concentration
of 10 µM was added, and the incubation was continued for
60 min. Medium was then replaced with fresh serum-free DME in the
presence or absence of 10 µM Y-27632 and agonists at the
indicated concentrations. To the upper chambers, HRP dissolved in
serum-free DME was added to give a final concentration of 0.5 µM. The upper chambers contained 200 µl of medium, and
the lower chambers contained 800 µl of medium. One hour after the
start of the experiment, 50 µl of medium was collected from the lower
chambers. The HRP content of the samples was evaluated
spectrophotometrically by assaying peroxidase activity in buffer
containing 0.5 mM guaiacol, 50 mM
Na2HPO4, and 0.6 mM H2O2 and measuring absorbance at 470 nm. Data
from five independent experiments, each in triplicate, are shown as the
means ± S.E. Statistical significance, calculated using
Student's t test, was taken as p < 0.001.
Immunofluorescence--
All procedures were performed at room
temperature. Cells were fixed in 3% paraformaldehyde in PBS for 15 min. After fixation, the cells were rinsed and permeabilized by
incubation with 0.2% Triton X-100 in PBS for 15 min. After rinsing,
the cells were blocked in 1% BSA in PBS for 15 min and incubated with
100 ng/ml fluorescein isothiocyanate-conjugated phalloidin (Molecular
Probes, Inc., Eugene, OR) in blocking solution for 60 min. After the
final rinse, the cells were mounted with fluorescent mounting medium (DAKO, Carpinteria, CA) and examined using a AxioskopTM
fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY) fitted with
100× objectives. Photographs were taken using 400 ASA T-MAX film
(Eastman Kodak Co.).
Gel Electrophoresis and Immunoblotting--
The protein content
of samples was determined using the Bio-Rad protein assay. Samples were
resolved by one-dimensional SDS-PAGE and then electrophoretically
transferred to polyvinylidene difluoride membranes (0.2 µm pore size;
ATTO, Tokyo, Japan). The membrane was subjected to immunoblotting as
described previously (38).
Immunoprecipitation--
Cells were rinsed twice with ice-cold
PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2 and then lysed with boiling-hot SDS-IP buffer (25 mM Hepes/NaOH, pH 7.4, 4 mM EDTA, 25 mM NaF, 1% SDS, 1 mM
Na3VO4). After the lysates had been heated at
100 °C for 3 min and cooled, a 9-fold volume of ice-cold Nonidet
P-40-IP buffer (25 mM Hepes/NaOH, pH 7.4, 150 mM NaCl, 4 mM EDTA, 25 mM NaF, 1%
Nonidet P-40, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin) was added. Lysates were passed 10 times through a
27-gauge needle and then gently mixed for 30 min at 4 °C. After
centrifugation (10,000 × g for 30 min), the
supernatant was collected. For immunoprecipitation, 4 µl of anti-occludin polyclonal antibody and a 15-µl bed volume of GammaBind Plus Sepharose (Amersham Pharmacia Biotech) were added to each sample
and mixed for 3 h at 4 °C. Beads were washed five times with 1 ml of Nonidet P-40-IP buffer, from which immunoprecipitates were eluted
by boiling in Laemmli sample buffer (39) for 5 min. Samples were then
separated by gel electrophoresis followed by immunoblotting.
In Vitro Phosphatase Treatment--
After immunoprecipitation,
the beads were washed three times with 1 ml of Nonidet P-40-IP buffer
and three times with 1 ml of either AP buffer (50 mM
Tris-HCl, pH 9.0, 1 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) for
alkaline phosphatase treatment or LP buffer (50 mM
Tris-HCl, pH 7.5, 1 mM MnCl2, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) for
protein phosphatase treatment. They were then resuspended in 100 µl
of AP buffer or LP buffer and incubated with or without calf intestine
alkaline phosphatase (Takara Shuzo Co., Ltd., Ohtsu, Japan) or with or
without
protein phosphatase (New England Biolabs, Inc., Beverly,
MA), respectively. To block phosphatase activity, a phosphatase
inhibitor mixture (100 mM
-glycerophosphate, 25 mM NaF, 4 mM EDTA, 1 mM
Na3VO4) was used. After a 1-h incubation at
30 °C with occasional mixing, beads were washed three times with 1 ml of Nonidet P-40-IP buffer and boiled with Laemmli sample buffer to
elute the immunoprecipitates.
[32P]Phosphorylation Analysis--
Confluent
cultures of ECV304 cells on 9-cm diameter dishes were rinsed twice with
phosphate-free M199 containing 0.5% fetal calf serum and 2 mM glutamine. The cells were then incubated in 10 ml of
this medium containing 1 mCi [32P]orthophosphate (ICN
Biomedicals, Costa Mesa, CA) for 4 h under an atmosphere of 5%
CO2. Vehicle or factors were then added as required, and
the incubation was continued for an additional 10 min. At 4 °C, the
cultures were then rapidly rinsed twice in PBS (magnesium- and
calcium-free) and lysed in 1 ml of extraction buffer containing 1%
(v/v) Triton X-100, 0.1% (w/v) SDS, 25 mM Hepes, 2 mM EDTA, 0.1 M NaCl, 25 mM NaF, 1 mM Na3VO4, pH 7.6 (adjusted with
NaOH), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
soybean trypsin inhibitor, 0.1 units/ml
2-macroglobulin,
and 10 µg/ml leupeptin. The extracts were collected by scraping the
dish and then centrifuged at 10,000 × g for 10 min.
After preclearing with protein A-Sepharose (Amersham Pharmacia
Biotech), occludin was immunoprecipitated using 2.5 µg of
anti-occludin antibody (Zymed Laboratories Inc., South
San Francisco, CA) in the presence of protein A-Sepharose. The beads
were washed five times with extraction buffer, and protein was eluted
into Laemmli sample buffer, resolved by SDS-PAGE (8% acrylamide), and
transferred to Immobilon P membrane (Millipore, Bedford, MA).
[32P]Phosphate-labeled protein was detected by
autoradiography, and occludin protein was then detected by probing the
filters with the anti-occludin antibody. In this manner, after
quantitation of band intensity by densitometry, the relative amount of
radiolabeled phosphate per occludin protein could be estimated.
Phosphoamino acid analysis was performed on the labeled bands
according to the procedures described previously (40).
For two-dimensional gel electrophoresis, immunoprecipitated occludin
from [32P]orthophosphate-labeled cells was solubilized in
50 µl of buffer containing 0.5% SDS, 0.1 M
dithiothreitol, 1 mM EDTA, 25 mM Tris/HCl, pH
8.0, and heated at 100 °C for 5 min. The eluted occludin was then
freeze-dried and redissolved in 50 µl of a solution containing 9.5 M urea, 4% Triton X-100, 0.1 M dithiothreitol,
2% Pharmalyte® 3.5-10 (Amersham Pharmacia Biotech), 0.05%
bromphenol blue. Isoelectric focusing (400 V for 16 h) was
performed in tube gels containing 9.5 M urea, 4% Triton
X-100, 1% Pharmalyte 4-6.5, 1% Pharmalyte 5-8 in polymerized 3%
acrylamide, 0.15% bisacrylamide. The proteins in the tube gels were
then equilibrated in Laemmli sample buffer, transferred to slab gels,
and resolved by SDS-PAGE (8% polyacrylamide). After transfer to
nitrocellulose, [32P]phosphate incorporated into protein
was detected by autoradiography, and signal corresponding to occludin
protein was then revealed by subsequent immunoblotting of the filter
with anti-occludin antibody.
Phosphatase treatment of radiolabeled occludin was performed
essentially as described by Meisenhelder and Hunter (41). Thus, an
immune complex containing occludin from
[32P]orthophosphate-labeled cells was prepared as
described above. To remove interfering salts and detergents, the beads
(50-µl packed volume) were washed twice with 1 ml of wash buffer
containing 1% Triton X-100, 0.1 M NaCl, 25 mM
Hepes-NaOH, pH 7.4. They were then switched to phosphatase buffer by
washing twice with a solution containing 20 mM Mes-NaOH, 1 mM MgCl2, 0.8 mM dithiothreitol, 4 µg/ml leupeptin, 4 µg/ml soybean trypsin inhibitor, pH 5.5. The
beads were then incubated with 50 µl of the phosphatase buffer in the
absence or presence of 0.2 units of potato acid phosphatase (Calbiochem). Phosphatase activity was blocked as described (41). After
1 h at 37 °C, the reaction was quenched by the addition of 1 ml
of ice-cold wash buffer. The suspension was briefly (<10 s)
centrifuged to pellet the beads. Protein was eluted into Laemmli sample
buffer, resolved by SDS-PAGE, and transferred to nitrocellulose as
described above. [32P]Phosphate incorporated into protein
was detected by autoradiography, and occludin protein was revealed by immunoblotting.
 |
RESULTS |
LPA and Histamine Increase Paracellular Flux of HRP in ECV304
Cells--
The effects of LPA on the TJ permeability of ECV304 cell
monolayers were investigated by measuring paracellular flux of HRP. Using cultures on Transwell filters, HRP was added to the apical chambers in the presence or absence of LPA. HRP that passed via the
paracellular pathway to enter the basolateral chamber was quantified by
assaying peroxidase activity spectrophotometrically. The HRP activity
detected was compared with that of control cells. As shown in Fig.
1a, LPA induced a greater flux
of HRP in a dose-dependent manner. The involvement of RhoA
was examined by using adenovirus-mediated overexpression of RhoA T19N,
a dominant-negative mutant of RhoA. LacZ overexpression, confirmed by
X-gal (5-bromo-4-chloro-3-indolyl
-D-galactopyranoside)
staining (data not shown), was used as a control. In these experiments,
the increase in HRP flux in response to LPA was similar in noninfected
cells and LacZ-overexpressing cells. In contrast, LPA failed to
stimulate an increase in HRP flux in RhoA T19N-overexpressing cells
(Fig. 1b). Next, the role of p160ROCK was investigated using
the specific p160ROCK inhibitor Y-27632 (25). Pretreatment with 10 µM Y-27632 blocked the increase in HRP flux induced by
LPA (Fig. 1b). These data indicate that RhoA and its target
p160ROCK transduce the action of LPA to increase TJ permeability in
ECV304 cells.

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Fig. 1.
LPA-stimulated HRP paracellular flux in
ECV304 cells via a RhoA-p160ROCK-dependent pathway.
a, ECV304 cells were treated with or without LPA at the
indicated concentrations. HRP flux increased in a
dose-dependent manner. Data shown are the mean ± S.E.
of five independent experiments expressed as fold increase compared
with the value for control cells. b, noninfected cells,
LacZ-overexpressing cells (LacZ), RhoA T19N-overexpressing
cells (RhoA T19N), or cells pretreated with 10 µM Y-27632 for 60 min (Y-27632) were subjected
to HRP flux measurement in the presence or absence of 1 µM LPA as described under "Experimental Procedures."
Data are shown as fold increase of the mean ± S.E. of five
independent experiments, compared with the value for noninfected cells
in the absence of LPA. The asterisk indicates significant
differences from the HRP flux of noninfected cells in the absence of
LPA (p < 0.001).
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The effects of histamine on HRP flux in ECV304 cells were then
examined. Like LPA, histamine increased HRP flux in a
dose-dependent manner in ECV304 cells (Fig.
2a). The roles of RhoA and
p160ROCK were again studied. In noninfected cells and
LacZ-overexpressing cells, histamine increased HRP flux to similar
levels (Fig. 2b). However, overexpression of RhoA T19N had
no significant effect on the increase in HRP flux induced by histamine
(Fig. 2b). Also, inhibition of p160ROCK using Y-27632 failed
to inhibit the increase in HRP flux in response to histamine (Fig.
2b). Thus, in contrast to the response to LPA, histamine
appears to stimulate an increase in TJ permeability in ECV304 cells
independently of RhoA and p160ROCK.

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Fig. 2.
Histamine caused greater HRP paracellular
flux in ECV304 cells via a RhoA-p160ROCK-independent pathway.
a, in ECV304 cells, HRP flux was measured in the presence or
absence of histamine at the concentrations indicated. Histamine caused
a dose-dependent increase in HRP flux. Data shown are the
mean ± S.E. of five independent experiments expressed as fold
increase compared with control cells. b, HRP flux of
noninfected cells, LacZ-overexpressing cells (LacZ), RhoA
T19N-overexpressing cells (RhoA T19N), or cells pretreated
with 10 µM Y-27632 for 60 min (Y-27632) was determined in
the presence or absence of 1 µM histamine. Data are shown
as fold increase of the mean ± S.E. of five independent
experiments, compared with the value for noninfected cells in the
absence of histamine. The asterisk indicates significant
differences from the HRP flux of noninfected cells in the absence of
histamine (p < 0.001).
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Changes in the Actin Cytoskeleton in Response to LPA and
Histamine--
To determine whether LPA and histamine caused changes
in the actin cytoskeleton in ECV304 cells, F-actin was visualized by staining with fluorescein isothiocyanate-conjugated phalloidin (Fig.
3). In noninfected cells and
LacZ-overexpressing cells as controls, pericellular actin bundles were
seen, and stress fibers were hardly detectable in the cell bodies (Fig.
3, a and b), whereas subtle reorganization of
pericellular actin bundles was observed in RhoA T19N-overexpressing
cells and cells pretreated with 10 µM Y-27632 (Fig. 3,
c and d). Noninfected cells and
LacZ-overexpressing cells that were treated with 1 µM LPA
showed F-actin bundles in stress fibers and some gaps between cells
(Fig. 3, e and f). In contrast, in the cells
overexpressing RhoA T19N, LPA failed to induce F-actin bundles (Fig.
3g). Pretreatment with 10 µM Y-27632 also
prevented stress fiber formation in response to LPA (Fig. 3h).

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Fig. 3.
Changes in the actin cytoskeleton caused by
LPA and histamine. F-actin was visualized by staining cells with
fluorescein isothiocyanate-labeled phalloidin. In noninfected cells
(a) and LacZ-overexpressing cells (b),
pericellular actin bundles were seen (c and d),
and stress fibers were hardly detectable in cell bodies. In noninfected
cells (e) and LacZ-overexpressing cells (f)
stimulated with 1 µM LPA for 10 min, F-actin bundles in
stress fibers were seen. In contrast, in the cells overexpressing RhoA
T19N (g) or pretreated with 10 µM Y-27632 for
60 min (h), LPA failed to form F-actin bundles. Also,
noninfected cells (i) and LacZ-overexpressing cells
(j) stimulated with 1 µM histamine
(Hist.) for 10 min showed F-actin bundles in stress fibers.
In the cells overexpressing RhoA T19N (k) or pretreated with
10 µM Y-27632 for 60 min (l), histamine did
not induce formation of F-actin bundles. Bar, 5 µm.
Cont., control.
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Similar to the effect of LPA, 1 µM histamine induced
F-actin bundles in stress fibers in noninfected cells and
LacZ-overexpressing cells (Fig. 3, i and j). Both
overexpression of RhoA T19N and pretreatment with Y-27632 also blocked
formation of stress fibers in response to histamine (Fig. 3,
k and l). These data show that LPA and histamine
induce reorganization of pericellular actin bundles and stimulation of
stress fiber formation in ECV304 cells and that both events are
mediated by RhoA and p160ROCK.
Changes in Occludin Electrophoretic Mobility in Response to LPA and
Histamine--
Although LPA and histamine clearly had effects on the
actin-based cytoskeleton, the possibility of direct effects on TJ
proteins was also explored. The main reason for this was because the
cytoskeletal effects of histamine were blocked by inhibition of
RhoA-p160ROCK signaling, whereas effects on TJ permeability were
unaffected. By immunocytochemistry, it was shown that the localization
of occludin or ZO-1 was not altered in response to either LPA or histamine (data not shown). The possibility of effects of LPA and
histamine on biochemical changes in the TJ protein occludin was then
examined. Occludin immunoprecipitates from cells stimulated with either
LPA or histamine were resolved by SDS-PAGE and then analyzed by
immunoblotting with an anti-occludin monoclonal antibody. This revealed
that the electrophoretic mobility of occludin in gels was altered
within minutes in response to both LPA and histamine in a
dose-dependent manner. This alteration was visualized as a
retarded mobility during SDS-PAGE (Fig.
4, a and b). The
dose dependences were similar to those that caused an increase in HRP flux (see Figs. 1 and 2). As a control, expression levels and electrophoretic mobility of ZO-1 were not changed in response to LPA or
histamine (Fig. 4, a and b).

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Fig. 4.
Occludin electrophoretic mobility was altered
in response to LPA and histamine. Occludin was immunoprecipitated
with an anti-occludin polyclonal antibody from cells stimulated with
LPA (a) and histamine (b) at various doses (for
10 min) and times (at 1 µM) as indicated.
Immunoprecipitates were resolved by SDS-PAGE and then analyzed by
immunoblotting with an anti-occludin monoclonal antibody (MOC37). As a
control, 40 µg of protein of whole cell lysates from cells in the
same condition were resolved by SDS-PAGE and then analyzed by
immunoblotting with an anti-ZO-1 antibody (a and
b).
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Phosphorylation Analysis of Occludin--
The biochemical basis of
the change in electrophoretic mobility of occludin was investigated.
One possibility was that this was due to changes in phosphorylation of
the protein. In initial experiments, cells were metabolically labeled
with [32P]orthophosphate, and phosphorylation of occludin
was examined after immunoprecipitation and resolution by SDS-PAGE. In
control cells, phosphate incorporation into occludin was detected.
However, after stimulation with histamine or LPA, even though a band
shift in occludin was observed, an increase in phosphate labeling of the protein was not detected (Fig.
5a). The intensity of labeled bands was quantitated by densitometry, and similar results were obtained in other experiments.

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Fig. 5.
Phosphorylation analysis of occludin.
Panel a, cells were metabolically labeled with
[32P]orthophosphate and then treated for 10 min with
vehicle (Cont.), 1 µM histamine
(Hist.) or 1 µM LPA. Occludin was extracted,
immunoprecipitated, resolved by SDS-PAGE, and detected by
immunoblotting (Protein) or autoradiography
(32P). Occludin from control cells migrates as a
single band but becomes a doublet after treatment of cells with LPA or
histamine (arrowheads). However, as confirmed by
densitometry, changes in total phosphorylation of occludin were not
detectable. Panel b, occludin immunoprecipitates
(IP) from cells stimulated for 10 min with either vehicle
(Cont.), 1 µM LPA, 1 µM
histamine (Hist.), or 100 µM pervanadate
(PV) were resolved by SDS-PAGE and immunoblotted with either
anti-occludin antibody or the anti-phosphotyrosine antibodies PY20 and
4G10. The brackets indicate more slowly migrating bands of
occludin from cells treated with pervanadate. Panel c, the
bands corresponding to occludin in the autoradiogram of panel
a were excised and subject to partial acid hydrolysis, and the
released phosphoamino acids were analyzed by high voltage
electrophoresis in two dimensions. The migration of phosphoserine
(S), phosphothreonine (T) and phosphotyrosine
(Y), as detected by ninhydrin staining, is indicated in the
Standards panel.
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Phosphoamino Acid Analysis--
Amino acid residues phosphorylated
in occludin were then characterized and investigated to see if these
changed in response to cell stimulation. Both immunological and
labeling procedures were used. Occludin immunoprecipitates from cells
treated with either 1 µM LPA or 1 µM
histamine were resolved by SDS-PAGE and immunoblotted with
anti-occludin antibody, revealing the mobility shift, and then with the
anti-phosphotyrosine antibodies PY20 or 4G10 (Fig. 5b). As a
control, cells were treated with pervanadate, a membrane-permeable
peroxide derivative of vanadate and a potent inhibitor of tyrosine
phosphatases (see Ref. 33). Occludin from pervanadate-treated cells
showed a change in electrophoretic mobility and clear immunoreactivity
with PY20 and 4G10 (Fig. 5b). In contrast, tyrosine
phosphorylation of occludin was not detected with PY20 or 4G10 in
immunoprecipitates from cells stimulated with LPA or histamine (Fig.
5b).
Phosphorylation was then analyzed by phosphoamino acid analysis of
metabolically radiolabeled occludin using high voltage electrophoresis
in two dimensions. Occludin from control cells was phosphorylated
mainly on serine residues, phosphothreonine was barely detectable, and
phosphotyrosine was not detected (Fig. 5c). The phosphoamino
acid composition of occludin from LPA and histamine-treated cells was
very similar to that of control cells (Fig. 5c). Thus,
phosphorylation of occludin may involve subtle changes in
phosphorylation of serine or threonine residues. Tyrosine phosphorylation does not seem to play a role.
Two-dimensional Gel Analysis--
Occludin phosphorylation in
[32P]orthophosphate-labeled cells was also analyzed by
two-dimensional gel electrophoresis. Occludin from control cells
migrated as a series of at least six discrete spots (Fig.
6A, panel a,
arrows), suggesting differentially, post-translationally modified protein. The five most acidic spots appeared as doublets consisting of a more abundant lower spot and a less abundant, slightly
retarded, more acidic spot. Out of the six spots, the most basic was
not detectable as phosphorylated (Fig. 6A,
cf. panels a and b,
arrows). As the pI of occludin decreased, phosphate was detected mainly in the lower, more abundant component of the pairs.
After stimulation with histamine (Fig. 6A, panels
c and d) or LPA (Fig. 6A, panels
e and f), occludin still migrated as a series of spots.
However, the migration of these was different from that of occludin
from vehicle-treated cells. In particular, the distribution of occludin
between the two spots in the more acidic forms of the protein was
shifted such that the upper member of the pair was predominant or more
equal to that of the lower member of the pair. This is seen fairly well
with the second least basic form of occludin (Fig.
6A, panels c-f, arrows). Enlarged views of these regions of the blots are shown in Fig. 6B.
Although a detectable increase in phosphorylation of occludin was not
observed, the decrease in pI of the protein is consistent with
increased phosphorylation. The lack of increase in detectable
phosphorylation may again be due to the fact that occludin in resting
cells is already substantially phosphorylated, and the phosphorylation responsible for the pI and band shift is difficult to detect in this
background. Nevertheless, the two-dimensional gel analysis is
consistent with the possibility that occludin phosphorylation is
altered in response to histamine and LPA treatment.

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Fig. 6.
Two-dimensional gel analysis of occludin
phosphorylation. ECV304 cells were labeled with
[32P]orthophosphate, and occludin was immunoprecipitated
and analyzed by two-dimensional gel electrophoresis as described under
"Experimental Procedures." In A, occludin protein was
detected by immunoblotting (panels a, c, and
e), and corresponding phosphorylation of occludin was
detected by autoradiography (panels b, d, and
f). Occludin was from cells treated for 10 min with either
vehicle (a and b), 1 µM histamine
(c and d), or 1 µM LPA
(e and f). More acid proteins are to the left.
Also shown (B) is an enlargement of the immunoblots
containing protein depicted by the three arrows in
panel a and corresponding regions in panels c and
e.
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Effects of in Vitro Phosphatase Treatment--
Another approach to
investigate if phosphorylation is responsible for electrophoretic
mobility changes in a protein is to study the effects of in
vitro phosphatase treatment (see Ref. 41). Again, cells were
labeled with [32P]orthophosphate and stimulated as
required. In immunoprecipitates not treated with phosphatase, an
increase in total phosphorylation of occludin was not detected, even
though the band shift was observed (Fig.
7a, left-hand
panels). Some of the immunoprecipitates were treated with potato
acid phosphatase, in which case quantitative analysis by densitometry
revealed >95% removal of the radiolabeled phosphate from the protein,
and the occludin band shift was reversed (Fig. 7a,
middle panels). To check that this activity of the
phosphatase preparation was indeed due to phosphatase, it was confirmed
that phosphatase inhibitors had a blocking action (Fig. 7a,
right-hand panels).

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Fig. 7.
Effects of in vitro
phosphatase treatment on occludin electrophoretic mobility.
Panel a, cells were metabolically radiolabeled with
[32P]orthophosphate and treated for 10 min with vehicle
(C), 1 µM histamine (H), or 1 µM LPA (L). After lysis, occludin was
immunoprecipitated, resolved by SDS-PAGE, and analyzed by
immunoblotting (Blot) and autoradiography (32P).
Some of the immunoprecipitates (left panels) were subject to
a sham treatment with potato acid phosphatase (Pase). Others
were treated with phosphatase in the absence (middle panels)
or presence (right panels) of phosphatase inhibitors
(PI). Panel b, occludin immunoprecipitates from
cells stimulated for 10 min with either 1 µM LPA or 1 µM histamine were incubated with or without indicated
doses of alkaline phosphatase (AP) or protein
phosphatase (LP). Phosphatase treatment
dose-dependently reversed the band shift. Panel
c, the activity of phosphatase seen in panel b was not
observed in the presence of a mixture of phosphatase inhibitors
(PI).
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Similarly, alkaline phosphatase (from calf intestine) and
phosphatase (recombinant protein produced in Escherichia
coli), in a dose-dependent manner, could reverse the
effects of both LPA and histamine on the occludin band shift (Fig.
7b). Furthermore, the activities of both alkaline
phosphatase and
phosphatase were blocked by phosphatase inhibitors
(Fig. 7c).
The ability of phosphatases to reverse the effects of LPA and histamine
on the occludin band shift in vitro suggests that the band
shift is due to occludin phosphorylation. Because occludin is already
phosphorylated in control cells, LPA and histamine probably produce
subtle but site-specific changes in occludin phosphorylation. Such
changes would be sufficient to result in altered electrophoretic
mobility, both in the form of an acid shift (see Fig. 6) and apparent
size shift of the protein but not enough to detect changes in total
phosphorylation of occludin.
Involvement of RhoA and p160ROCK in Occludin
Phosphorylation--
The involvement of RhoA and p160ROCK in occludin
phosphorylation induced by LPA and histamine was investigated. In
noninfected cells and cells overexpressing LacZ as a negative control,
LPA and histamine again induced an upward band shift of occludin (Fig. 8). Overexpression of RhoA T19N prevented
the LPA-induced occludin band shift but not that in response to
histamine (Fig. 8, a and b). The expression of
RhoA was determined by the immunoblotting of whole cell lysates from
each condition (Fig. 8, a and b). RhoA expression
was similar in noninfected cells, LacZ-overexpressing cells, and cells
pretreated with Y-27632. The anti-RhoA antibody detected overexpressed
RhoA T19N (Fig. 8, a and b). Also, pretreatment with Y-27632 blocked the ability of LPA to cause an occludin band shift
(Fig. 8a). In contrast, the histamine-induced band shift of
occludin was not blocked by Y-27632 (Fig. 8b). As a control, expression levels and electrophoretic mobility of ZO-1 were unaffected (Fig. 8, a and b). These data suggest that RhoA
and p160ROCK mediate LPA-induced occludin phosphorylation. In contrast,
histamine-stimulated occludin phosphorylation, like its effect on tight
junction permeability (Fig. 2), appears to be mediated by a pathway
that is independent of RhoA and p160ROCK.

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Fig. 8.
Involvement of RhoA and p160ROCK in occludin
phosphorylation and MLC phosphorylation induced by LPA and
histamine. Occludin was immunoprecipitated from noninfected cells,
LacZ-overexpressing cells, RhoA T19N-overexpressing cells, or cells
pretreated with 10 µM Y-27632 for 60 min that were
stimulated for 10 min with either 1 µM LPA (a)
or 1 µM histamine (b). Immunoprecipitates were
resolved by SDS-PAGE and then analyzed by immunoblotting with an
anti-occludin antibody. Whole cell lysates (40 µg of protein) from
each condition were also resolved by SDS-PAGE and then analyzed by
immunoblotting with an anti-ZO-1 antibody, an anti-RhoA antibody, and
an anti-PP-MLC antibody that recognizes MLC phosphorylated on
Thr18 and Ser19 residues.
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MLC Phosphorylation in Response to LPA and Histamine--
MLC
phosphorylation mediated by Rho and its target Rho kinase/p160ROCK has
been suggested to be a key regulator of cell retraction leading to
increased TJ permeability in endothelial cells (42, 43). Therefore, the
effects of LPA and histamine on MLC phosphorylation in ECV304 cells
were examined. After stimulation with 1 µM LPA or 1 µM histamine, equal protein amounts of cell lysates were resolved by SDS-PAGE and then analyzed by immunoblotting (Fig. 8,
a and b) with an antibody that recognizes
Thr18- and Ser19-phosphorylated MLC (PP-MLC
(35)), a form of the protein produced by stimulation of endothelial
cells with histamine and thrombin (44). In noninfected cells and
LacZ-overexpressing cells, incubation of cells with LPA and histamine
increased PP-MLC immunoreactivity in cell lysates. In contrast, LPA and
histamine failed to increase PP-MLC immunoreactivity in RhoA
T19N-overexpressing cells and cells pretreated with Y-27632. These
results indicate that LPA and histamine induce MLC phosphorylation via
RhoA and p160ROCK in ECV304 cells.
Disrupting TJs Does Not Stimulate Occludin Phosphorylation--
It
is possible that an increase in occludin phosphorylation is responsible
for increased TJ permeability. Alternatively, increased TJ permeability
may trigger occludin phosphorylation. To address this issue, an
actin-depolymerizing agent, cytochalasin D (45), was used to disrupt
TJs, and effects on occludin phosphorylation were examined. The
efficacy of cytochalasin D on TJ structures was confirmed by staining
cells with an anti-occludin antibody (Fig.
9, panel a). As expected,
after cytochalasin D treatment, the cells had detached from their
neighbors, and occludin disappeared from cell-cell adhesion sites (Fig.
9, panel a).

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Fig. 9.
Disruption of TJs does not cause occludin
phosphorylation. Panel a, after treatment of ECV304
cells with either vehicle or 1 µM cytochalasin D for 30 min, cells were fixed, and immunolabeling of occludin was performed.
Occludin was localized at cell-cell contacts in vehicle-treated cells
(left). In contrast, in cytochalasin D-treated cells
(right), occludin localization to the cellular TJs was
disrupted. Bar, 10 µm. Panel b, cells were
stimulated for 10 min with either 1 µM LPA or 1 µM histamine after pretreatment with either vehicle or 1 µM cytochalasin D for 30 min. Occludin immunoprecipitates
and whole cell lysates (40 µg protein) from each condition were
resolved by SDS-PAGE and then analyzed by immunoblotting with an
anti-occludin or, as a control, an anti-ZO-1 antibody,
respectively.
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Cells were then stimulated with either LPA or histamine after
pretreatment with either vehicle or cytochalasin D. The pretreatment with cytochalasin D did not either cause a band shift of occludin or
affect the ability of LPA or histamine to stimulate a mobility shift in
occludin (Fig. 9, panel b). In all cases, equal protein amounts of cell lysates were resolved, as revealed by immunoblotting with anti-ZO-1 antibody (Fig. 9, panel b). Similar results
were obtained by pretreatment of cells with the extracellular calcium chelating agent BAPTA (4 mM, added 30 min before treatment
with LPA or histamine). BAPTA disrupts cell-cell junctions by
preventing cadherin-dependent cell-cell adhesion (33), and
again, the expected disruption of TJ structure was confirmed by
occludin immunocytochemistry (results not shown). These data suggest
that disruption of TJs does not trigger occludin phosphorylation and
that occludin phosphorylation in response to LPA and histamine may be
causal rather than a consequence of increased TJ permeability.
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DISCUSSION |
The present study shows that RhoA and p160ROCK are components of a
signaling pathway coupling LPA receptor stimulation to changes in TJ
permeability. However, other pathways must exist because the
physiologically similar effect of histamine was independent of
RhoA-p160ROCK. Furthermore, the TJ protein occludin is shown to be the
target for G protein-coupled receptor-initiated signaling pathways.
Again, a pathway involving RhoA-p160ROCK is shown to exist, but
RhoA-p160ROCK-independent signaling to occludin can also occur.
Regulation of the function of TJs is considered to be achieved in a
concerted manner by both the cytoskeleton and specific junctional
proteins (1, 2, 19, 46). Regarding the cytoskeleton, inactivation of
the Ras-related GTPase Rho by botulinum C3 toxin was shown to lead to
perijunctional actin reorganization and barrier dysfunction of TJ (20),
suggesting that perijunctional cytoskeleton regulated by Rho can
influence TJ permeability (19). Also, cell contraction mediated by MLC
phosphorylation may play an important role in TJ permeability control
(44). The pathways activating MLC phosphorylation appear to be
transduced by intracellular Ca2+ as well as Rho-associated
kinase/p160ROCK, a downstream target of Rho (44, 47).
Here, using ECV304 cells as a model system, LPA and histamine
were shown to have similar abilities to increase TJ permeability. Both
agents also caused not only actin reorganization, such as the formation
of F-actin bundles and disappearance of perijunctional actin (Fig.
3), but also MLC phosphorylation (Fig. 8). Blocking either RhoA
signaling (by overexpression of the dominant negative RhoA T19N) or
the activity of the RhoA effector p160ROCK (using the pharmacological
inhibitor Y-27632) prevented these cytoskeletal changes in response to
LPA and histamine (Figs. 3 and 8). However, although RhoA T19N or
Y-27632 blocked the LPA-induced increase in TJ permeability, they had
no effect on the response to histamine (Figs. 1 and 2). Therefore, in
the case of LPA, RhoA and p160ROCK are critically involved in the
stimulated increase in TJ permeability. In contrast, histamine has
effects on TJ permeability that are independent of both RhoA-p160ROCK
and effects on the cytoskeleton. Therefore, other mechanistic
possibilities were explored.
Direct effects of signaling on protein components of the TJ were
examined. By immunoblot analysis, stimulation of cells with either LPA
or histamine obviously had an effect on occludin, causing its
electrophoretic retardation when analyzed by SDS-PAGE. The dose
dependence of this effect was similar to that required for the
increases in TJ permeability. The biochemical basis of this effect on
occludin was analyzed and was highly likely due to an alteration in
phosphorylation. Indeed, the reversal of the LPA and histamine-induced
band shift in occludin by in vitro phosphatase treatment is
consistent with the band shift due to an increased phosphorylation. The
anti-phosphotyrosine antibodies PY20 and 4G10 did not react with
occludin from stimulated cells, suggesting that the decrease in
electrophoretic mobility of occludin was not due to an increase in its
tyrosine phosphorylation. By metabolic labeling with
[32P]phosphate, occludin was shown to be phosphorylated
mainly on serine residues in control cells. However, even after
stimulation with LPA or histamine, it was difficult to detect an
increase in phosphorylation of either serine or threonine residues.
Presumably, changes in phosphorylation of occludin are difficult to
detect because of the high basal level of phosphorylation. The changes are inferred to be on serine or threonine residues because of our
inability to detect tyrosine phosphorylation either by immunoblotting or phosphoamino acid analysis from labeled cells. The complexity of
occludin phosphorylation was revealed by two-dimensional gel analysis.
Occludin from control cells migrated as a series of pI variants, the
more acidic of which were detectable as phosphorylated. LPA or
histamine stimulation again resulted in changes in electrophoretic mobility of occludin, notably to more acidic, electrophoretically retarded forms, consistent with increased phosphorylation. Even by
two-dimensional gel analysis it was difficult to detect gross changes
in occludin phosphorylation, but again, this appears to be due to the
high basal level of phosphorylation.
Phosphorylation of occludin has now been reported in several different
situations. Sakakibara et al. (48) show that occludin is
phosphorylated on serine-threonine residues during the formation of
cell-cell contacts in MDCK cells. During the TJ assembly of Xenopus laevis embryos, occludin dephosphorylation was
observed (49). Also vascular endothelial growth factor stimulated TJ permeability and occludin phosphorylation in retinal endothelial cells
(50). Regarding sites of phosphorylation and function, the relationship
between occludin phosphorylation in these situations is not clear.
It is possible that occludin phosphorylation may be a cause or
consequence of increased TJ permeability. In our study, we would
suggest that it may be causal and, also, capable of being mechanistically independent of changes in the cytoskeleton. In cells
where TJ structure was disrupted by treatment with cytochalasin D or
BAPTA, occludin phosphorylation was not affected. In these same cells,
effects of LPA and histamine could still be observed. Thus, occludin
phosphorylation (the band shift) does not appear to be a consequence of
disruption of junctions, suggesting that it may play a causal role. In
the case of LPA, a RhoA-p160ROCK pathway couples receptor-stimulated
signaling events to changes in the actin cytoskeleton (stress fiber
formation, MLC phosphorylation), changes in TJ permeability, and
occludin phosphorylation. With LPA, because all events were blocked by
interfering with the RhoA-p160ROCK pathway, it is difficult to ascribe
functional importance to any of them in particular. However, with
histamine, a different situation was found. In this case, blocking the
RhoA-p160ROCK pathway had differential effects on the cytoskeleton,
occludin phosphorylation, and increase in TJ permeability. Interfering
with RhoA-p160ROCK signaling blocked the cytoskeletal changes in
response to histamine, whereas both occludin phosphorylation and the
increase in TJ permeability were unaffected. This raises the
possibility that occludin phosphorylation may be a key regulator of TJ
permeability, acting independently of cytoskeletal events.
The precise mechanisms involved in occludin phosphorylation and changes
in TJ permeability have yet to be elucidated. It is not clear whether
LPA and histamine use parallel, independent signaling pathways to
regulate occludin phosphorylation. p160ROCK appears to be a component
of the signaling pathway activated by LPA but not by histamine. Another
possibility is that different pathways are activated with convergence,
perhaps at the point of a common kinase responsible for occludin
phosphorylation. How phosphorylation changes in occludin may regulate
TJ permeability has yet to be understood. Conformational changes of
occludin might affect the association with peripheral membrane proteins
of the TJ linked to the actin cytoskeleton. However, in our present
study, LPA and histamine did not have any apparent effects on the
localization of TJ proteins occludin and ZO-1 (data not shown),
indicating that this aspect of the protein architecture of the TJ is
maintained after stimulation with these agonists. Also, the interaction
of occludin with other TJ membrane proteins such as claudins (16-18) might alter in response to occludin phosphorylation.
In conclusion, our results demonstrate that TJ permeability is
regulated by distinct mechanisms that are
RhoA-p160ROCK-dependent and -independent. RhoA and p160ROCK
appear to be crucial regulators of the cytoskeleton that may partly
determine TJ permeability. Also, evidence is provided that
receptor-initiated signaling events can trigger phosphorylation of the
TJ-specific membrane protein occludin on serine-threonine residues via
RhoA-p160ROCK-dependent and RhoA-p160ROCK-independent
pathways. Recently, LPA was found to be accumulated in human
atherosclerotic lesions that are prone to thrombotic complications,
indicating LPA as an atherothrombogenic molecule (51). The analysis of
regulatory mechanisms controlling TJ permeability could lead to the
understanding of the physiology as well as the pathology of vascular
disorders caused by inflammation, ischemia, and atherosclerosis and,
therefore, direct us to the identification of novel therapeutic targets.