From the Renal Division, Department of Medicine,
Brigham and Women's Hospital and Harvard Medical School, Boston,
Massachusetts 02115, the
Department of Surgery, Children's
Hospital, Boston, Massachusetts 02115, and the § Department
of Pediatrics and Medicine, Division of Nephrology and Hypertension,
University of California in San Diego,
La Jolla, California 92093
Received for publication, December 20, 2000, and in revised form, March 26, 2001
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ABSTRACT |
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Oxidative stress compromises the tight
junction, but the mechanisms underlying its recovery remain unclear. We
developed a model in which oxidative stress reversibly disrupts
the tight junction. Exposure of Madin-Darby canine
kidney cells to hydrogen peroxide markedly reduced
transepithelial resistance and disrupted the staining patterns of the
tight junction proteins ZO-1 and occludin. These changes were reversed
by catalase. The short-term reassembly of tight junctions was not
dependent on new protein synthesis, suggesting that recovery occurs
through re-utilization of existing proteins. Although ATP levels were
reduced, the reduction was insufficient to explain the observed
changes, since a comparable reduction of ATP levels (with
2-deoxy-D-glucose) did not induce these changes. The
intracellular hydrogen peroxide scavenger pyruvate protected
Madin-Darby canine kidney cells from loss of transepithelial resistance
as did the heavy metal scavenger
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. Of a wide variety of agents examined, only tyrosine kinase inhibitors and protein kinase C inhibitors markedly inhibited tight junction reassembly. During reassembly, tyrosine phosphorylation in or near the
lateral membrane, was detected by immunofluorescence. The tyrosine
kinase inhibitors genistein and PP-2 inhibited the recovery of
transepithelial resistance and perturbed the relocalization of ZO-1 and
occludin to the tight junction, indicating that tyrosine kinases,
possibly members of the Src family, are critical for reassembly after
oxidative stress.
Many disease states of the kidney such as ischemia/reperfusion,
inflammation, or toxic injury to the kidney and gut lead to loss of the
epithelial barrier. Reactive oxygen species are directly involved in
the pathophysiology of some of these diseases. Targets for hydrogen
peroxide
(H2O2)1
include DNA, proteins, and lipids. Reported effects of hydrogen peroxide on renal epithelial cell lines include DNA damage with induction of apoptosis or necrosis (1-3), decreased activity of
membrane transporters (4), and membrane lipid peroxidation (5).
Hydrogen peroxide decreases transepithelial resistance (TER) and
increases transcellular permeability of MDCK cell monolayers (6, 7),
which is also well documented in non-kidney epithelial cell lines
(8-11) and endothelial cell lines (12-15). However, the biochemical
and subcellular effects of hydrogen peroxide on tight junction (TJ)
proteins have not been studied, nor have they been distinguished from
effects due to partial ATP depletion. Short-term depletion and
repletion of intracellular ATP in cultured cells has been used to study
the disassembly and/or reassembly of junctions as a model of organ
ischemia and reperfusion (16-20). However, little is known about the
reassembly of the tight junction after exposure to reactive oxygen
species due to the lack of a reproducible model system.
We have now developed and analyzed a model of reversible hydrogen
peroxide-induced disassembly and reassembly of the TJ in vitro and show that the reassembly pathway, as monitored by
physiological, biochemical, and immunocytochemical parameters, depends
upon tyrosine kinase activity. Furthermore, the cellular and
biochemical consequences of H2O2 exposure are
distinct from those caused by ATP depletion-repletion.
Cell Culture and Materials--
Madin-Darby canine kidney cells
(MDCK, type II) were obtained from American Type Tissue Culture
Collection (Rockville, MD) and maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 5% fetal calf serum, 50 IU/ml
penicillin, and 50 µg/ml streptomycin. Cells were incubated at
37 °C in 5% CO2 and were passaged weekly. DMEM was from
Cellgro (Herndon, VA), streptomycin, penicillin, and fetal calf serum
from Sigma. Plasticware was from Falcon (Lincoln Park, NJ). Transwells
were obtained from Costar (Cambridge, MA), the ohm meter (Millicell
ERS) from Millipore (Bedford, MA). Anti-ZO-1 rat monoclonal antibody
(R40.76) was kindly supplied by Dr. D. Goodenough (Harvard Medical
School, Boston). Anti-E-cadherin antibodies were isolated from
hybridoma cells (rr-1) kindly provided by Barry Gumbiner (University of
California, San Francisco). Anti-ZO-2 and anti-occludin polyclonal
rabbit antibodies were from Zymed Laboratories
Inc. (San Francisco, CA), anti-phosphotyrosine antibody was
from Upstate (4G10), Upstate Biotechnology, Inc., Lake Placid, NY. All other reagents used in these experiments were of analytical grade.
Hydrogen Peroxide Experiments--
Confluent monolayers were
cultured in DMEM without fetal calf serum 24 h prior to the
exposure with specific concentrations of hydrogen peroxide and
throughout the experiment. To terminate the exposure to hydrogen
peroxide, catalase was added into the media at a final concentration of
5000 units/ml. Control experiments were performed with monolayers in
serum free DMEM with and without catalase or Me2SO.
Trypan Blue Exclusion Assay--
After treatment of MDCK
monolayers with 5 mM H2O2 or
2-deoxy-D-glucose (dGlc) for various times, cells
were washed with PBS twice, and exposed to 0.4% trypan blue in PBS
supplemented with 1.5 mM CaCl2 and 2 mM MgCl2 for 5 min. The cells were examined using a Nikon Diaphot inverted microscope, and the number of viable and
nonviable cells was determined.
Apoptosis Assay--
MDCK cells were grown to confluency on
glass coverslips and exposed to H2O2 with or
without treatment with catalase, genistein, or PP-2. After fixation of
the monolayers with 4% paraformaldehyde in PBS for 30 min, apoptosis
was detected with the ApopTagTM Peroxidase kit (Intergen,
Purchase, NY), labeling free 3'-OH termini of DNA strand breaks,
according to the manufacturers instructions. Apoptotic cells were
counted under an inverted microscope (Nikon).
ATP Depletion and Repletion--
Depletion of intracellular ATP
was achieved by using the glycolytic inhibitor
2-deoxy-D-glucose. In brief, confluent MDCK monolayers were
serum starved in serum-free DMEM for 24 h, washed with PBS three
times, and exposed to PBS containing 1.5 mM
CaCl2, 2 mM MgCl2, and 12 mM 2-deoxy-D-glucose for different time
periods. Repletion of intracellular ATP levels was achieved by changing the media to serum-free DMEM. Control experiments were performed in
serum-free DMEM alone.
TER Measurements--
MDCK cells were plated on polycarbonate
filters (Transwell, Costar) at confluent density (roughly 2 × 105 cells/cm2) and maintained in serum
containing media for 48 h to establish tight monolayers. 24 h
before TER measurement cells were exposed to serum-free DMEM. In some
experiments, known scavengers of hydrogen peroxide or modulators of
intracellular signaling pathways were preincubated in serum-free DMEM
before exposure to H2O2 on both sides of the
transwell. TER was measured at different time points after treatment
with these reagents and/or hydrogen peroxide with a Millipore
electrical resistance system, as described previously (21).
Measurements were expressed in ohm × cm2, as a mean
of the original readings after subtraction of background values. Basal
TER measurement of confluent monolayers were recorded between 150 and
300 ohm × cm2.
ATP Measurements--
Measurements of total cellular ATP content
were performed with a luciferase-based ATP determination kit (Sigma).
After rinsing the monolayer with PBS three times, 300 µl of somatic
cell ATP-releasing agent solubilized the cells. Cells were immediately
scraped, aspirated, and cleared of insoluble material by spinning for 5 min at 10,000 rpm. The ATP content of 100 µl of supernatant was
measured with 100 µl of Sigmas diluted luciferase-containing ATP
assay mixture in an automated chemiluminescence counter (Berthold, Bad
Wildholz, Germany). ATP levels are expressed as percent of the initial
value after subtraction of background readings.
Immunocytochemistry--
Confluent monolayers of MDCK cells were
grown on clear coverslips. At specific time points during the
experiment, the monolayers were rinsed twice with PBS, fixed with 100%
methanol ( Statistics--
The data are given as mean values ± S.E.
(n), where n refers to the number of measurements
performed. The paired Student's t test was used to compare
mean values within one experimental series. Data from two groups were
compared by unpaired t test. A p value of <0.05
was accepted to indicate statistical significance.
H2O2 Reversibly Decreases Transepithelial
Resistance in MDCK Cell Monolayers in a Concentration and Time
Dependent Warmer--
Low concentrations of
H2O2 (0.5 and 0.75 mM) had no
detectable effect on TER even after treatment for 8 h and 1 mM H2O2 only lowered TER by 9 ± 2%. Intermediate concentrations of H2O2
(2.5 mM) caused a gradual decrease in TER to values near
zero over 8 h. Higher concentrations (5 and 10 mM)
caused a more rapid loss of TER within the first 3 h (Fig.
1A). Treatment of MDCK
monolayers with 5000 units/ml catalase reversed the decrease of TER for
all H2O2 concentrations used. Recovery of TER
to control values took about 3 h for cells treated with 2.5 mM H2O2, 4.5 h for cells treated with 5 mM H2O2, and did not
occur within 8 h after exposure to 10 mM
H2O2 (Fig. 1B). However, 20 h
after catalase treatment, TER values of the cells treated with 10 mM H2O2 reached baseline values
(data not shown). Changing media or exposure to catalase alone had no
effect on TER. Based on these results, a H2O2
concentration of 5 mM was chosen for further experiments,
as TER reproducibly decreased to 26 ± 3% (n = 35) of baseline within 30 min and complete recovery occurred within
4-6 h in the presence of catalase (Fig. 1B). This
represents a new and reproducible model for reversible H2O2-dependent disassembly and
reassembly. In addition, the rate of recovery of TER after treatment
with 5 mM H2O2 depended upon incubation time. Incubation of MDCK monolayers with 5 mM
H2O2 for 45 or 60 min before treatment with
5000 units/ml catalase resulted in a slower recovery compared with
incubation for 30 min (Fig. 1C). Recovery to control values
of TER was complete after ~12 h for a 45-min incubation and 20 h
for a 60-min incubation (data not shown).
Recovery from Hydrogen Peroxide Is Not Dependent on New Protein
Synthesis--
To test whether the recovery of TER after treatment
with hydrogen peroxide and catalase is dependent on synthesis of new TJ or other proteins, MDCK monolayers were coincubated with the protein synthesis inhibitor cycloheximide (50 µg/ml) and 5 mM
H2O2 before scavenging with catalase. Recovery
of TER was nearly identical in cycloheximide-treated cells over the
initial 6 h (Fig. 1D). At later time points, TER began
to drop in the cycloheximide-treated monolayers, most likely reflecting
the general effects of inhibiting protein synthesis. This result
indicates that, for the early recovery of barrier function, new protein
synthesis is not necessary.
Cell Viability of MDCK Cells after Exposure to
H2O2 Depended Upon Exposure Time--
Trypan
blue uptake was observed in 0.1 ± 0.1% of the cells under
baseline conditions. The number of trypan blue positive cells increased
to 0.7 ± 0.1% after 30 min exposure to 5 mM
H2O2 and 2.5 ± 0.6% after 6 h (Fig.
2A). After scavenging of
H2O2 with catalase for 5.5 h, the number
of trypan blue positive cells remained at the same value for 30 min of
H2O2 exposure (0.7 ± 0.3%,
n = 10). PP-2 and genistein did not increase the number
of trypan blue positive cells (Fig. 2B).
The number of apoptotic cells was 0.18 ± 0.1% under control
conditions and did not increase significantly after 30 min of exposure
to 5 mM H2O2 or after exposure to
PP-2 or genistein for 6 h with or without catalase (0.2% ± 0.1%, Fig. 2C). After 6 h exposure to hydrogen
peroxide 1.1 ± 0.06% of the cells stained positive for apoptosis.
The Initial Fall in TER after Exposure to Hydrogen Peroxide for 30 min Was Reduced by Intra- and Extracellular Scavengers of Hydrogen
Peroxide--
After establishing the conditions under which a constant
fall and recovery of TER occurred reproducibly (5 mM
H2O2 for 30 min), different scavengers or
inhibitors of intracellular signaling pathways were used to determine
whether the TER reduction was due to oxidative stress, and to
investigate specific targets for hydrogen peroxide.
Pyruvate scavenges hydrogen peroxide by direct nonenzymatic reduction
of H2O2 to water while undergoing
decarboxylation at the 1-carbon position (22). Pyruvate is also readily
taken up by cells and can thus serve as an intracellular scavenger of
H2O2. Co-incubation of pyruvate (5 mM) with hydrogen peroxide completely abolished the loss of
TER, indicating that pyruvate is effectively scavenging intracellular
H2O2 (Fig.
3A, n = 4). In
another set of experiments MDCK cells were preincubated with 5 mM pyruvate in serum-free DMEM for 16 h. Before
exposure to H2O2, the cells were washed with
PBS to eliminate extracellular pyruvate. This preincubation with
pyruvate significantly reduced the drop in TER to 62 ± 5% of
control. Recovery to baseline after addition of catalase was
accelerated to ~2 versus 5 h under control conditions (Fig. 3A, n = 4). Pyruvate alone did not
influence TER. Other scavengers were preincubated for 1 h and
coincubated with hydrogen peroxide for 30 min and the fall in TER was
recorded (Fig. 3B). The reducing agent dithiothreitol
reduced the initial fall of TER to 57 ± 7% (dithiothreitol, 0.5 mM, n = 7). Superoxide dismutase (30 units/ml, n = 7) had a similar effect in reducing the
fall to 50 ± 4%. In marked contrast, the membrane permeable
hydrogen peroxide and hydroxyl radical scavenger dimethylthiourea (10 mM, n = 7) and the hydroxyl radical
scavenger 5,5-dimethylpyrroline N-oxide (10 mM,
n = 7) had no effect on the initial fall in TER.
Two Heavy Metal Chelators Reduce the Fall in TER after Exposure to
H2O2--
Preincubation for 1 h before
exposure to H2O2, with the cell permeable heavy
metal chelator TPEN
(N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, 1 µM), which acts as an intracellular scavenger, reduced
the fall in TER at 30 min to 58 ± 3% (Fig. 3C).
Furthermore, the subsequent decline of TER was significantly reduced
and recovery from the exposure to H2O2 was
faster and complete after ~2 h (Fig. 3B, n = 6). The
cell permeable iron chelator deferoxamine (0.1 mM) was
preincubated for 16 h and subsequently reduced the initial fall in
TER to 56 ± 7% (Fig. 3C, n = 6).
Exposure to TPEN or deferoxamine alone had no effect on TER.
Tyrosine Kinase or PKC Inhibition Prevents Recovery of TER--
TJ
assembly is known to be modulated by a variety of signaling
mechanisms (9, 16, 17, 23-28). In a broad screen of many inhibitors of
signaling events known to modulate the TJ, the most consistent results
were obtained with the two tyrosine kinase inhibitors PP-2
(4-amino-5-(4-chlorophenyl)-7-(t-butyl)-pyrazolo[3,4-d]pyrimidine, 1 or 10 µM) and genistein (4',5,7-trihydroxyisoflavone,
50 or 100 µM), both of which inhibit Src family tyrosine
kinases, although genistein is also a broad spectrum tyrosine kinase
inhibitor. MDCK cells were preincubated with these agents for 1 h
before being exposed to 5 mM H2O2.
The drop of TER was not different from a control experiment, but the
recovery after 5000 units/ml catalase was virtually abolished (Fig.
4, A and B,
n = 6), indicating a role for tyrosine kinase in the
reassembly of the TJ but not the initial disassembly. Control
experiments with PP-2, genistein, or Me2SO alone showed no
changes in baseline TER (n = 4, data not shown). Thus,
tyrosine kinase activity, most likely due to a Src family member,
appears to be important for the recovery of the TJ after exposure to
hydrogen peroxide. PKC has also been shown to regulate TJ assembly in
other models (24, 29, 30). Three PKC inhibitors were tested for their
ability to inhibit the recovery of TER after exposure to hydrogen
peroxide. Calphostin C (500 nM, n = 7),
bisindolylmaleimide I (500 nM, n = 7), and chelerythrine (1 µM, n = 7) reduced TER
at 6 h by 73 ± 5, 55 ± 2, and 42 ± 5%,
respectively, when compared with control (Fig. 4C). The
agents did not decrease TER in control experiments (n = 3).
ATP Levels Are Similar in dGlc and
H2O2-treated Cells but Partial ATP Depletion
with dGlc Does Not Decrease TER--
Intracellular ATP levels decrease
in various cell lines after exposure to H2O2
(31, 32). In MDCK cells, ATP levels have been shown to decrease with 1 mM H2O2 (7). In our experiments, exposure of MDCK monolayers to 5 mM
H2O2 or 12 mM
2-deoxy-D-glucose for 30 min resulted in a partial ATP
depletion to 21.4 ± 4 and 20.7 ± 2% of control,
respectively (Fig. 5, A and
B, n = 4-8). Furthermore, the time course
for the recovery of cellular ATP content after treatment with either
5000 units/ml catalase (H2O2 group) or
repletion with control media (dGlc group) after 30 min was similar.
Media changes or catalase treatment had no effect on the intracellular
ATP concentration (98.7 ± 3 and 96.3 ± 4% after after
6 h, respectively). Therefore, 12 mM dGlc was used in
the following experiments to examine the effect of partial ATP
depletion on TER, extractability and localization of TJ proteins.
In marked contrast to the effects of 5 mM
H2O2 on TER (see Fig. 1), treatment with 12 mM dGlc was not associated with a detectable drop in
transepithelial resistance. On the contrary, at later time points, a
rise in TER of partially ATP depleted monolayers to 25.5 ± 5%
above the TER of repleted monolayers was observed (Fig. 5C,
n = 4). Consistent with this finding, in dGlc-treated cells, no increase in trypan blue uptake was recorded
(n = 10). The finding that TER is unchanged in cells
ATP depleted to 20% of control levels is consistent with previous work
that indicates that the threshold for ATP depletion-induced declines in
TER occurs at around 5-10% of normal ATP levels (4, 5, 7, 17, 33).
After Exposure to H2O2 but Not dGlc,
Staining of Junctional Proteins Shows a Partial Breakdown of the Tight
Junction and Adherens Junction--
Confocal microscopy revealed that
H2O2 induces a reversible, partial breakdown of
ZO-1 in the TJ after 30 min (Fig.
6A, b, f, and j).
Prolonged exposure of MDCK cells to 5 mM
H2O2 resulted in a discontinuous ZO-1 staining
pattern throughout the monolayer (Fig. 6A, c, g,
and k, n = 4 for each observation). Occludin
and E-cadherin showed a similar pattern of redistribution and
broadening of the staining. Treatment with 5000 units/ml catalase
prevented the disruption of the TJ and after 6 h a normal
distribution of all three junctional proteins was demonstrated (Fig.
6A, d, h, and l). The exposure of MDCK
cells to 12 mM dGlc had no effect on the distribution of
ZO-1, occludin, or E-cadherin, indicating that these changes, like the
drop in TER, could not be explained by lower ATP levels (Fig. 6B,
a and b, data shown for ZO-1 only, n = 3 for each
observation). The ZO-1 staining remained continuous but appeared
slightly more jagged during later time points.
Genistein and PP-2 Prevent the Reformation of the Tight Junction
after Oxidative Stress--
Immunofluorescence of MDCK monolayers
after double staining with anti-phosphotyrosine and anti-ZO-1
antibodies showed intact staining of the tight junction junction 6 h after exposure to H2O2 and catalase or
catalase alone (Fig. 7, a and
b). In contrast, MDCK cell monolayers that were pretreated
with 10 µM PP-2 (Fig. 7c) or 100 µM genistein (Fig. 7e) for 1 h and
exposed to the same oxidative stress and catalase in the presence of
the inhibitors showed a marked disruption in ZO-1 staining after 6 h that resembled the disruption without catalase scavenging (see Fig.
6c), consistent with the data indicating that these agents
inhibit TER recovery (Fig. 4). Both inhibitors had no effect on the
ZO-1 staining in the presence of catalase without oxidative stress
(Fig. 7, d and f).
Hydrogen Peroxide Induces Tyrosine Phosphorylation along the
Lateral Membrane during Disassembly and Reassembly of the Tight
Junction--
Tyrosine phosphorylation occurred in the lateral
membrane after short-term exposure to hydrogen peroxide (Fig.
8A, b) when compared with
untreated monolayers (Fig. 8A, a), as described before (34,
35). Furthermore, strong tyrosine phosphorylation occurred in the
lateral cell border 6 h after exposure to
H2O2 and scavenging with catalase (Fig.
8A, f, same sample as Fig. 7a). Analysis of
z-sections revealed that the staining for tyrosine phosphorylation and ZO-1 overlapped in the tight junction although additional phosphotyrosine staining was observed in the lateral membrane and the cytosol of the cells (Fig. 8B). Both PP-2
and genistein prevented this tyrosine phosphorylation (Fig. 8A,
d and e), giving rise to a very weak and diffuse
staining pattern which resembled the tyrosine phosphorylation of
monolayers treated with H2O2 alone (Fig.
8A, c). Catalase treatment alone did not induce tyrosine
phosphorylation in the cell junction (Fig. 8A, g). These
experiments imply that tyrosine phosphorylation, possibly in the
lateral membrane near or in the TJ, occurs during TJ recovery, and that
the ability of catalase to induce the recovery of the tight junction
possibly requires these phosphorylation events.
Oxidative stress is known to disturb the permeability barrier in
renal epithelial cells in culture. Transepithelial resistance and
intracellular ATP levels fall after exposure to hydrogen peroxide, whereas the permeability for small solutes across the monolayer increases (6, 7). Initially, a reduced intracellular ATP level was
thought to cause the disruption of the junction (36), but later studies
identified other potential regulators such as intracellular calcium
activity (37), pH (38), PKC (39), and calcium-independent phospholipase
A2 (40) during oxidative stress. Until now, the mechanisms
of tight junction reassembly after oxidative stress that lead to a
reconstituted and functional epithelial cell barrier have not been
investigated. Here, we present a new model in which the disruptive
effect of hydrogen peroxide on physiological, biochemical, and
immunocytochemical parameters of TJ integrity is reversible.
H2O2 (5 mM) consistently dropped transepithelial resistance to 23% of control. Thereafter, the epithelial cell monolayers completely recovered to baseline within 6 h. After recovery, the MDCK monolayers showed a stable TER for several days. The exposure time of 30 min used in our experiments was
associated with only a small decrease in cell viability. This finding
is in good agreement with previous studies in epithelial cells that
show a high resistance against oxidative stress in MDCK cells (5, 32).
The small decrease in viability did not correlate with the observed
changes in transepithelial resistance. After full recovery of TER about
the same number of non-viable or apoptotic cells were observed as
after 30 min of hydrogen peroxide exposure. Importantly, the initial
recovery did not depend on new protein synthesis, which implies that
the recovery does not depend on development of de novo tight
junctions. Increasing the intracellular concentration of the
H2O2 scavenger pyruvate reduced the drop in TER
by 46%, indicating an intracellular effect of H2O2. Several extracellular hydrogen peroxide
scavengers were also effective in reducing the initial fall in TER.
Interestingly, superoxide dismutase had a significant effect on the
initial fall in TER. Superoxide dismutase catalyzes the dismutation of
superoxide anion into hydrogen peroxide. Superoxide anion is generated
proportionally to the oxygen tension in mitochondria or after
activation of NADPH oxidase. Both pathways could be activated under
oxidative stress. Mitochondrial function is severely impaired by
hydrogen peroxide which results in additional generation of reactive
oxidative species. Also, activation of NADPH oxidase by arachidonic
acid has been described in kidney epithelial cells (41) and arachidonic
acid is known to be increased under oxidative stress (40, 42). The
induction of superoxide anion production by hydrogen peroxide could
therefore increase the intracellular hydrogen peroxide even further.
H2O2 decreases the intracellular ATP level in
proportion to concentration and duration of exposure (7, 32). A number of groups have shown a decrease of intracellular ATP levels to 16-34%
of control under similar oxidative stress in renal epithelial cell
lines (2, 5, 7). Our observations confirm this reported decrease in
ATP, showing an ATP level of 21% of control after 30 min incubation
with 5 mM H2O2. Interestingly, this
ATP level is higher than ATP levels obtained with the established ATP
depletion model of TJ disassembly in which both glycolysis and
mitochondrial oxidative phosphorylation are inhibited. The two most
widely used reagents for ATP depletion, 2-deoxy-D-glucose
and antimycin A, consistently lower intracellular ATP levels in MDCK
cells to 2-5% of the initial value within minutes (16, 17, 43). This
"maximal" ATP depletion induces the disassembly of the tight
junction, and increases the association of TJ proteins with the
cytoskeleton (17). The reversibility of the effects of ATP depletion on
the TJ of MDCK cells has been established in recent years (16, 17, 33, 44). During the ATP repletion phase, the reassembly of the tight
junction depends on tyrosine phosphorylation of TJ proteins (16). To
confirm that, in our model, the decrease of the intracellular ATP level
is not responsible for the disassembly of the TJ, ATP levels
were reduced to the level observed after exposure to hydrogen peroxide.
Treatment with 2-deoxy-D-glucose for 30 min decreased ATP
to the same level as hydrogen peroxide. This partial ATP depletion to
20% of the initial value failed to cause a decrease in transepithelial resistance. Although these findings indicate that other factors are
more likely to contribute to the disassembly of the tight junction,
they do not rule out an influence of the decreased ATP level on the
regulation of the tight junction under oxidative stress. Reassembly of
the tight junction after partial ATP depletion could not be monitored
since treatment with dGlc did not disturb the tight junction. Hence, it
is conceivable that the reassembly of the TJ after oxidative stress and
total ATP depletion could be regulated in a similar fashion.
It is well recognized that hydrogen peroxide can act as a
protein-tyrosine phosphatase inhibitor (10, 45, 46). In epithelial cells, exposure to hydrogen peroxide or phosphatase inhibitors has been
shown to decrease TER and increase permeability, which could be
prevented by tyrosine kinase inhibition (34, 35). It was suggested that
the general increase in tyrosine phosphorylation could induce the
disassembly of the tight junction, although specific phosphorylation
events in the tight junction were not described. Interestingly, both
genistein and PP-2 did not affect the disassembly of the tight junction
in our study but strikingly prevented the recovery. This suggests an
important role for tyrosine phosphorylation also in the regulation of
TJ reassembly after oxidative stress. Since disassembly and reassembly
of the tight junction are regulated by many, yet most likely distinct,
signaling pathways, our findings led us to investigate the behavior of
tight junction proteins during the reassembly of the junction.
Furthermore, the specific inhibition of PP-2 implies a role for Src
family tyrosine kinases in this process.
Tyrosine phosphorylation that co-localized with ZO-1 as well as the
lateral membrane was observed during disassembly and during the
recovery period but not after 6 h of exposure to
H2O2. This tyrosine phosphorylation and
recovery of continuous staining of ZO-1 after recovery of the TJ were
prevented by PP-2 and genistein. This suggests that tyrosine
phosphorylation of tight junction proteins or their regulatory proteins
is critical during the recovery of the tight junction. However, a
specific target for tyrosine phosphorylation within the tight junction
was not identified.
Tyrosine phosphorylation of occludin, ZO-2, and ZO-3 is known to occur
during the reassembly of the TJ after total ATP depletion followed by
repletion. This tyrosine phosphorylation was inhibited by genistein
(16). In a different model, transfection of Ras into MDCK cells induced
a decrease of TER together with a redistribution of occludin, ZO-1, and
claudin-1 into the cytosol (47). After treatment of these cells with
the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059,
occludin and ZO-1 but not claudin-1 were tyrosine phosphorylated.
Subsequently, all three proteins were recruited to the cell membrane.
ZO-1 was also tyrosine phosphorylated during redistribution into the TJ
together with actin after treatment of human epidermal carcinoma cell
lines with epidermal growth factor (48). In another in vivo
model strong tyrosine phosphorylation of ZO-1 was noted after perfusion
of newborn rat kidneys with protamine sulfate. The tyrosine
phosphorylation correlated with the rapid replacement of the slit
diaphragms between glomerular epithelial foot processes with typical
tight junctions (49). Although these model systems are very different
in nature, these studies, as does ours, suggest that tyrosine
phosphorylation of the tight junction proteins is involved in the
targeting of these proteins into the TJ.
Two groups have reported evidence for tyrosine phosphorylation of
junctional proteins during the disassembly of the junction after
treatment with the tyrosine phosphatase inhibitors vanadate and
phenylarsine (35, 50). Our study indicates a role for tyrosine
phosphorylation during the reassembly process. It thus seems likely
that phosphorylation events are involved in the regulation of both
disassembly and reassembly.
Interestingly, our data also suggests another regulatory mechanism for
TJ reassembly. Although we chose to focus on the role of tyrosine
kinases in TJ assembly, in a broad screen of inhibitors, the only
others that were found to significantly and consistently inhibit TER
recovery after oxidative stress were those active against PKC. This
suggests that serine/threonine phosphorylation of TJ proteins may be
important for their incorporation into the TJ during recovery from
oxidative stress.
During disassembly of the junctional complex, occludin was found to be
serine/threonine phosphorylated in endothelial cells after exposure to
oxidative stress (15). Other studies in epithelial cells have shown
that activation of PKC and serine/threonine phosphorylation of tight
junction proteins occurs during (re)-assembly of the tight junction.
Activation of PKC stimulates TER development while the inhibition of
PKC induces disassembly of the TJ and inhibits reassembly (24, 29, 30).
Tight junction proteins may be a direct target of PKC. PKC In conclusion, we describe the first reversible model for the study of
oxidative stress on the TJ in epithelial cells. Our results identify
the important role of tyrosine kinases during the reassembly of the
tight junction and establishes differences from other models of tight
junction assembly. Future experiments with the presented model system
will allow the study of the phosphorylation status of specific TJ
proteins after short-term oxidative stress. Also, the specific role of
PKC during the reassembly of the tight junction remains to be
investigated in detail.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C) for 10 min for ZO-1, occludin, and E-cadherin
staining, or 4% paraformaldehyde for 20 min at room temperature (for
anti-phosphotyrosine staining), and stored in PBS at 4 °C. After
blocking (PBS, 5% (v/v) goat serum, 3% (w/v) bovine serum albumin)
for 1 h, cells were incubated for 2 h with primary
antibodies. After washing (4 × 5 min in PBS + 0,05 Triton X-100)
the monolayers were incubated with Texas red or fluorescein
isothiocyanate-conjugated secondary antibodies for 1 h. The cells
were mounted in Fluoromount (Southern Biotechnology Associates Inc.,
Birmingham, AL). The filters were viewed through a ×100 oil immersion
objective with a laser scanning confocal system (model MRC-1024/2p,
Bio-Rad, Cambridge, MA) coupled to a Zeiss Axiovert microscope. Images
were processed using Photoshop software (Adobe, San Jose, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
TER of MDCK monolayers in response to
H2O2. A, effect of different
concentrations of H2O2 on TER after addition of
H2O2 to both sides of the transwell.
B, reversibility of the fall in TER after exposure to
different concentrations of H2O2.
H2O2 was hydrolyzed by the addition of 5000 units/ml catalase after 30 min. C, time response with
exposure to 5 mM H2O2 for different
times before 5000 units/ml catalase was added. D, 50 µM cycloheximide was applied onto the monolayer together
with 5 mM H2O2 to block protein
synthesis. H2O2 was scavenged with catalase
after 30 min. Data are mean ± S.E. of four experiments.
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Fig. 2.
Cell viability after exposure to 5 mM H2O2. A, time
course after exposure of MDCK cells to 5 mM
H2O2 for different times before trypan blue
staining. B, comparison of the percentage of trypan blue
positive cells after exposure to hydrogen peroxide or scavenging with
catalase (cat) in the presence or absence of 100 µM genistein (gen) or 10 µM
PP-2. C The same comparison in different monolayers of the
same passage after staining for apoptotic cells.
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Fig. 3.
Transepithelial resistance of MDCK monolayers
after incubation with pyruvate and TPEN under standard conditions (5 mM H2O2 for 30 min, scavenging with
5000 units/ml catalase). A, preincubation with 5 mM pyruvate for 16 h. The monolayers were washed with
PBS before exposure to H2O2 to remove
extracellular pyruvate (open triangles). Coincubation was
with 5 mM H2O2 and 5 mM
pyruvate (black squares). Control experiment with 5 mM H2O2 and 5000 units/ml catalase
(black circles). B, effect of other scavengers of
reactive oxygen species on the initial fall of TER. Confluent MDCK cell
monolayers were preincubated for 1 h with the scavengers (10 mM 5,5-dimethylpyrroline-N-oxide
(DMPO); 10 mM dimethylthiourea
(DMTU); 0.1 mM deferoxamine (DFO); 1 µM TPEN, 0.5 mM dithiothreitol
(DTT); 30 units/ml superoxide dismutase (SOD); 5 mM pyruvate; 5000 units/ml catalase) in serum-free medium
and co-incubated for 30 min with 5 mM
H2O2. Deferoxamine was preincubated for 16 h. Transepithelial resistance was measured directly before
(co) and after 30 min of coincubation. The change in TER was
calculated as a percentage of the control value (100%).
Asterisks show a significant reduction in the decrease of
TER when compared with the effect of hydrogen peroxide alone.
C, preincubation with 1 µM TPEN for 1 h
and coincubation with H2O2 for 30 min
(black squares) or for 480 min (open squares).
Control experiments with (black circle) and without catalase
(open circles). Data are mean ± S.E.
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Fig. 4.
Effect of tyrosine kinase inhibitors and PKC
inhibitors on the recovery of TER. A, preincubation
with 1 or 10 µmM PP-2 for 1 h and coincubation with
H2O2 for 30 min (black triangles).
Control experiment with H2O2 and catalase alone
(black circles, n = 4). B,
preincubation with 50 and 100 µM genistein for 1 h
and coincubation with H2O2 for 30 min
(open triangles). Control experiment (black
circles). C, effect of the PKC inhibitors calphostin C
(cal, 500 nM, open diamond),
bisindolylmaleimide I (bis, 500 nM, open
circles), and chelerythrine (che, 1 µM,
closed diamond) on TER during reassembly of the tight
junction. Data are mean ± S.E.
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Fig. 5.
Intracellular ATP levels after exposure to
H2O2 or 2-deoxy-D-glucose.
A, exposure to 5 mM H2O2
for 30 min (closed circles) or throughout the experiment (8 h, closed squares). B, exposure to 12 mM dGlc for 30 min (open squares) or throughout
the experiment (8 h, closed squares). C,
transepithelial resistance of MDCK monolayers after exposure to dGlc
under the conditions of the model. 12 mM dGlc, repleted
after 30 min with serum-free DMEM (open squares) or not
repleted (8 h, closed squares). Note, that TER of dGlc
exposed monolayers rose above TER of short-term exposed monolayers.
Data are mean ± S.E.
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Fig. 6.
Confocal microscopy of the junctional complex
after exposure to hydrogen peroxide. A, type II MDCK
monolayers were fixed in 100% methanol and double-stained with
anti-ZO-1 (a-d) and anti-occludin (e-h) or the
adherens junction protein E-cadherin (i-l) under control
conditions, after exposure to hydrogen peroxide for 30 min or 6 h
or after scavenging of hydrogen peroxide after 30 min with catalase for
a total of 6 h. Note the disruption of the staining for all three
junctional proteins at 30 min and 6 h. Bar, 30 µmm.
B, confocal microscopy of ZO-1 immunofluorescence in control
(a) or dGlc-treated (b) monolayers. Prolonged
exposure to 12 mM dGlc induced only a slightly more jagged
appearance of the ZO-1 staining. The partial ATP depletion did not
disrupt the ZO-1 staining even after 6 h of treatment.
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Fig. 7.
ZO-1 staining during recovery in the presence
of the tyrosine kinase inhibitors. Confocal microscopy of the
junctional complex after exposure to H2O2 for
30 min and catalase for a total of 6 h in the absence
(a and b) or presence of 10 µM PP-2
(c and d), or 100 µM genistein
(e and f). After fixation with 4%
paraformaldehyde, the monolayers were stained with antibodies for ZO-1
and secondary antibodies coupled to Texas red. Shown here is the ZO-1
staining pattern (n = 3 for each observation). Note the
marked disruption of the staining pattern in the presence of the
tyrosine kinase inhibitors (c and e) but not the
controls (d and f). Bar, 30 µm.
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Fig. 8.
Immunostaining for phosphotyrosine.
A, short-term exposure to H2O2
caused increased tyrosine phosphorylation in the region of the lateral
membrane (b) when compared with control (a).
Strong tyrosine phosphorylation in the same region also occurred during
the recovery period (f) which was diminished in the PP-2 and
genistein-treated monolayers (d and e, same
samples as shown in Fig. 7). Monolayers after exposure to
H2O2 for 6 h (c) or catalase
alone (g) showed a similar background staining.
Bar, 30 µm. B, the tyrosine phosphorylation was
further analyzed in the z axis by confocal microscopy.
Top panel, ZO-1 staining of the TJ after reassembly;
middle panel, tyrosine phosphorylation of the same
z axis; bottom panel, co-localization of both
staining patterns after independent sampling of both emission
wavelengths. Although co-localization occurred, tyrosine
phosphorylation can be observed throughout the lateral membrane and in
the cytosol. The slide surface is indicated with horizontal
bars, the lateral cell borders with vertical arrows.
Bar, 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is a
member of the atypical PKC subgroup and has been localized to the TJ
(29). PKC activation was also detected during TJ reassembly after the
calcium switch. Under these conditions, the TJ protein occludin was
serine/threonine phosphorylated but not tyrosine phosphorylated
(51).
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ACKNOWLEDGEMENT |
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We thank Michelle Lowe for excellent technical assistance with the confocal microscope.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by Deutsche-Forschungs-Gemeinschaft, Bonn, Germany, Grant Me 1760/1-1.
** Supported by National Institutes of Health Grant GM55223 and a Clinical Scientist Award from the National Kidney Foundation.
Supported by National Institutes of Health Grant DK53507. To
whom correspondence should be addressed: University of California in
San Diego, Depts. of Pediatrics and Medicine, Div. of Nephrology and
Hypertension, 9500 Gilman Dr., La Jolla, CA 92093-0693. Tel.: 858-822-3482; Fax: 858-822-3483; E-mail: snigam@ucsd.edu.
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M011477200
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ABBREVIATIONS |
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The abbreviations used are: H2O2, hydrogen peroxide; TJ, tight junction; TER, transepithelial resistance; dGlc, 2-deoxyglucose; MDCK, Madin-Darby canine kidney; ZO, zonula occludens; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.
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