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INTRODUCTION |
The tight junction (TJ)1
forms a barrier to the diffusion of toxins, allergens, and pathogens
across the epithelial tissue. Three types of transmembrane proteins,
occludin, claudins, and junction adhesion molecule, have been
identified at the TJ (1-3). The intracellular domains of these
transmembrane proteins interact with a number of plaque proteins, which
in turn anchors TJ protein complex to the actin cytoskeleton (4, 5).
Although the specific interactions among TJ proteins are yet to be
delineated, there is evidence to support occludin interaction with
zonula occludens (ZO)-1, ZO-2, and ZO-3 (5-7). These protein-protein
interactions are crucial for the assembly of TJ and the maintenance of
epithelial barrier functions (6). A significant body of evidence
indicates that TJ is under dynamic regulation by intracellular
signaling molecules. Although only little is known about the specific
interactions of signaling molecules with the TJ proteins, a number of
signaling molecules, including c-Src, c-Yes, protein kinase C, and
G-proteins, appear to be localized at the vicinity of TJ (1). A recent study indicates that signaling molecules such as phosphatidylinositol 3-kinase, c-Yes, and protein kinase C
may be associated with the
C-terminal tail of occludin (8). Additionally, pharmacologic modulations of the activity of a number of signaling molecules affect
the TJ permeability in a variety of epithelia (9-19).
Tyrosine kinase activity is required for both the disassembly (15-19)
and the assembly (20-23) of TJ in different epithelial monolayers.
Although protein tyrosine phosphorylation is associated with the
disruption of TJ in MDCK and Caco-2 cell monolayers (15, 24, 25),
tyrosine kinase inhibitors prevent oxidative stress (16-17, 19)- and
acetaldehyde-induced (18) disruption of the TJ in Caco-2 cell
monolayers. Hepatocyte growth factor-mediated disruption of TJ in
gastric epithelial and MDCK cell monolayers was associated with
tyrosine phosphorylation of ZO-1 (26, 27). Our previous study showed
that oxidative stress-induced disassembly of TJ in Caco-2 cell
monolayer was associated with the tyrosine phosphorylation of occludin,
ZO-1, E-cadherin, and
-catenin and dissociation of occludin from
ZO-1 and the actin cytoskeleton (19).
Oxidative stress activates Src family kinases in embryonic fibroblasts
and Xenopus eggs (30-32), whereas c-Src activity was reduced by oxidative stress in mesangial cells (33). The role of Src
family kinases in oxidative stress-mediated alteration of cell
functions was shown in embryonic cells and endothelial cells by using
selective inhibitors of Src family kinases (31, 32, 34, 35). Although
inhibitor of Src family kinases delay H2O2-induced endothelial permeability (34) and
prevent the assembly of TJ in canine kidney epithelial cells (23),
there is no direct evidence for the oxidative stress-induced activation
of c-Src in epithelial tissue and its role in the regulation of TJ. The expression of constitutively active v-Src in MDCK (28) and
Caco-2 (29) cells resulted in the disruption of adherens junction with no significant effect on TJ. A recent study suggested that c-Yes may be
associated with TJ and play a role in the assembly of TJ in kidney
epithelium (22).
Data in the present study demonstrate that c-Src is involved in the
de-stabilization of TJ. We transfected Caco-2 cells with empty
expression vector, pUSEc-Src (wild type), and
pUSEc-SrcK297R (kinase-inactive mutant) to determine the
role of c-Src in regulation of the epithelial TJ. We show that 1)
oxidative stress induces activation and membrane translocation of c-Src
in Caco-2 cells, 2) oxidative stress-induced increase in paracellular
permeability is prevented by PP2, the inhibitor of Src family kinases,
3) expression of kinase-inactive c-SrcK297R mutant delays
oxidative stress-induced increase in TJ permeability, tyrosine
phosphorylation of ZO-1 and
-catenin, and redistribution of occludin
and ZO-1, whereas the expression of wild type c-Src induces slight
potentiation of the effect of oxidative stress, and 4) expression of
c-SrcK297R accelerates the assembly of TJ during the
recovery after calcium switch.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Cell culture reagents including LipofectAMINE and
Geneticin were purchased from Invitrogen. EGTA,
xanthine oxidase, xanthine, streptavidin-agarose, fluorescein
isothiocyanate-conjugated inulin, protease inhibitors, and
protein-A-Sepharose were purchased from Sigma. PP2 (AG1879,
4-amino-5[chlorophenyl]-7-[t-butyl]pyrazolo[3-4-d]pyramidine) was purchased from Calbiochem. All other chemicals were of analytical grade purchased either from Sigma or Fisher. Pre-cast
SDS-polyacrylamide gels were purchased from Invitrogen.
Antibodies--
Mouse monoclonal anti-occludin and rabbit
polyclonal anti-occludin and anti-ZO-1 antibodies were purchased from
Zymed Laboratories Inc. (South San Francisco, CA).
Biotin-conjugated anti-phosphotyrosine, mouse monoclonal
anti-
-catenin, and horseradish peroxidase-conjugated anti-phosphotyrosine antibodies were from Transduction Laboratories (Lexington, KY). Fluorescein isothiocyanate-conjugated
anti-phosphotyrosine and Cy3-conjugated goat anti-rabbit IgG were
purchased from Sigma. Alexa-fluor® 568-conjugated
anti-mouse IgGs were from Molecular Probes (Eugene, OR). Mouse
monoclonal anti-c-Src antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and rabbit polyclonal
anti-c- Src[pY418] antibody was from BioSource (Camarillo, CA).
Cell Culture--
Caco-2 and MDCK cells, purchased from American
Type Culture Collection, Manassas, VA, were maintained under standard
cell culture conditions at 37 °C in DMEM containing 10% (v/v) fetal bovine serum. Cells were grown on 60-mm plates to 80% confluence were
used for transfection with different expression constructs.
Expression Constructs--
The empty expression vector,
pUSE(
), pUSEc-Src (wild type c-Src
gene), and pUSEc-SrcK297R (kinase-inactive
c-Src mutant gene) were purchased from Upstate
Biotechnology, Inc. cDNA was inserted as Klenow-treated
NotI-ClaI fragment into the EcoRV site
of the pUSE(
) multiple cloning site. The substitution of
lysine 297 with arginine in kinase domain abolishes the
phosphotransferase activity. Kinase-inactive c-Src serves as a dominant
negative and causes cell cycle arrest (36).
Transfection and Cloning--
Caco-2 cells at 80% confluence
(in 60-mm dishes) were washed with Opti-MEM and LipofectAMINE according
to vendor instruction. Five µg of pUSE(
),
pUSEc-Src, or pUSEc-SrcK297R were
preincubated with LipofectAMINE at 25 °C for 20 min and placed onto
the cells in a volume of 500 µl. After incubation at 37 °C for
3 h (with intermittent rocking every half-hour) regular medium was
added. After 96 h of incubation cells were harvested, split into
different plates, and grown in the presence of Geneticin (800 µg/ml).
After 2 weeks, cell colonies were cloned using cloning cylinders. Eight different clones each of Caco-2(EV), Caco-2(WT), and Caco-2(KI) were
isolated and subcultured. These subclones were examined under phase
contrast microscopy for gross morphology and analyzed for overexpression of c-Src protein by immunoblot analysis.
Treatment with Oxidative Stress--
Confluent monolayers of
different subclones of Caco-2 cells or MDCK cells were bathed in PBS
(Dulbecco's saline containing 1.2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 0.6%
bovine serum albumin). Oxidative stress was induced as described
previously (19) by administering a mixture of xanthine oxidase (20 milliunits/ml) and xanthine (0.5 mM) (XO + X) in the
absence or presence of PP2 (10 µM). Control cell
monolayers received PBS without XO + X.
Calcium-switch Experiment--
Confluent monolayers of
Caco-2(EV), Caco-2(WT), or Caco-2(KI) cells were washed in DMEM and
treated with 4 mM EGTA in DMEM for 20 min. EGTA solution
was removed, and cell monolayers were washed three times with DMEM.
EGTA-treated cell monolayers were then incubated in regular DMEM
without EGTA for 6-8 h under standard cell culture conditions.
Reassembly of TJ and restoration of barrier function were assessed at
varying times by measuring the transepithelial electrical resistance
(TER) and inulin flux.
Measurement of TER--
TER was measured as described previously
(15) using a Millicell-ERS Electrical Resistance System (Millipore,
Bedford, MA). TER was calculated as ohms/cm2 by
multiplying it with the surface area of the monolayer (0.33 cm2). The resistance of the supporting membrane in
Transwells (which is usually around 30 ohms/cm2) was
subtracted from all readings before calculations.
Unidirectional Flux of Inulin--
Cell monolayers in Transwells
were incubated under different experimental conditions in the presence
of 5 µg/ml fluorescein isothiocyanate-conjugated inulin in the basal
well. At varying times after experimental treatments, 150 µl of
apical medium and 50 µl of basal medium were withdrawn. Fluorescence
was measured in a FLX 800 microplate fluorescence reader (BioTEK
Instruments Inc, Winooski, VT). The flux into the apical well was
calculated as the percent of total inulin administered into the basal
well/h/per cm2 of surface area.
Immunofluorescence Microscopy--
Under different experimental
conditions, Caco-2(EV), Caco-2(WT), and Caco-2(KI) cell monolayers were
washed in PBS, fixed in acetone:methanol (1:1), and blocked in 20 mM Tris, pH 7.2, 150 mM NaCl, and 1% bovine
serum albumin as described before (19). Cell monolayers were incubated
with primary antibodies (anti-occludin and anti-ZO-1 antibodies) for
1 h followed by incubation for 1 h with secondary antibodies
(Alexa-fluor® 568-conjugated anti-mouse IgG and
Cy3-conjugated anti-rabbit IgG). The fluorescence was analyzed as
described before (19) by confocal laser-scanning microscopy (Bio-Rad
MRC1024). Images were processed using the software Confocal Assistant
4.02 and Adobe Photoshop (Adobe Systems Inc., San Jose, CA).
Preparation of Plasma Membrane Fraction--
Caco-2(EV),
Caco-2(WT), and Caco-2(KI) cell monolayers (in 24-mm Transwells) were
washed twice with ice-cold PBS and once with lysis buffer-F (PBS
containing 10 mM
-glycerophosphate, 2 µg/ml leupeptin,
10 µg/ml aprotinin, 10 µg/ml bestatin, 10 µg/ml pepstatin-A, and
1 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride). Cells were scraped and homogenized as described before (19).
The plasma membrane pellet was suspended in 500 µl of lysis buffer F. Protein was measured by the BCA method (Pierce). Membrane fraction was
either mixed with an equal volume of Laemmli sample buffer (2×
concentrated) and heated at 100 °C for 5 min or dissolved in lysis
buffer N (20 mM Tris, pH 7.4, containing 0.2% Nonidet
P-40, 0.1% sodium deoxycholate, and a mixture of protease inhibitors
as described above for lysis buffer-F) for immunoprecipitation of
c-Src.
Immunoprecipitation--
After XO + X treatment, Caco-2(EV),
Caco-2(WT), and Caco-2(KI) cell monolayers (24 mm) were washed with
ice-cold 20 mM Tris, pH 7.4, and the proteins were
extracted in lysis buffer D (0.3% SDS in 10 mM Tris
buffer, pH 7.4, containing 1 mM vanadate and 0.33 mM phenylmethylsulfonyl fluoride) under denaturing
conditions (heating at 100 °C for 5 min). Phosphotyrosine was
immunoprecipitated as described before (19) using biotin-conjugated
anti-phosphotyrosine antibody. Immune complexes were isolated by
precipitation using streptavidin-agarose. Immunoprecipitates were
immunoblotted for ZO-1 and
-catenin using rabbit polyclonal
anti-ZO-1 and mouse monoclonal anti-
-catenin antibodies,
respectively. For immune complex c-Src kinase assay, proteins from
plasma membrane fraction were extracted under native conditions with
lysis buffer N. c-Src was immunoprecipitated by using mouse monoclonal
anti c-Src antibodies.
Immunoblot Analysis--
Proteins in different samples were
separated by SDS-polyacrylamide gel (4-12% gradient) electrophoresis
and electroblotted into polyvinylidene difluoride membranes. Membranes
were probed for ZO-1,
-catenin, c-Src, or c-Src[pY418] using
rabbit polyclonal anti-ZO-1 and anti-c-Src[pY418] and mouse
monoclonal anti-c-Src and anti-
-catenin antibodies in combination
with horseradish peroxidase-conjugated anti-mouse IgG or horseradish
peroxidase-conjugated anti-rabbit IgG antibodies. Blots were developed
using ECL chemiluminescence Kit (Amersham Biosciences.
Tyrosine Kinase Assay--
Plasma membrane and soluble fractions
or anti-c-Src immune complexes were incubated in an assay mixture
containing 50 mM imidazole, pH 7.4, 150 mM
NaCl, 2 mM MnCl2, 12 mM
MgCl2, 0.17 mM ATP, 0.1 mM sodium
orthovanadate, 20 mM p-nitrophenyl phosphate,
0.1% Triton X-100, 5 µg of poly-(Glu-Tyr) peptide substrate, and 1 µCi of [
-32P]ATP at 25 °C for 30 min. After
incubation proteins were precipitated with an equal volume of 5%
trichloroacetic acid, and a 50-µl aliquot of trichloroacetic
acid-soluble fraction was blotted on to P81 Whatman filter discs. Discs
were washed in 0.5% phosphoric acid. Radioactivity in air-dried discs
was counted in a scintillation counter, Beckman LS500TA (Beckman
Coulter, Inc., Fullerton, CA). Activity was calculated as units of pmol
of phosphate incorporated to substrate/h and presented as units/mg of
protein in plasma membrane or soluble fractions or protein used for
immunoprecipitation of c-Src. Activity present in corresponding immune
complexes prepared using pre-immune mouse IgG was subtracted from the
activity in anti-c-Src immune complexes.
Statistics--
Comparison between two groups was made by the
Student's t tests for grouped data. The significance in all
tests was derived at the 95% or greater confidence level.
 |
RESULTS |
Oxidative Stress Induces Activation and Membrane Translocation of
c-Src--
The activation of c-Src by oxidative stress in Caco-2 cells
was determined by measuring the tyrosine kinase activity, assessing the
phosphorylation of c-Src on tyrosine 418, and the translocation of
c-Src into plasma membranes. Treatment with XO + X resulted in a rapid
increase in overall tyrosine kinase activity in the plasma membrane
fraction, which was associated with a decrease in tyrosine kinase
activity in the soluble fraction (Fig.
1A). Immune complex tyrosine
kinase assay showed that the activity of c-Src in the plasma membrane
was also rapidly increased by XO + X (Fig. 1B).
Immunoprecipitation of phosphotyrosine followed by immunoblot analysis
for c-Src showed that XO + X treatment resulted in a rapid increase in
tyrosine phosphorylation of c-Src in Caco-2 cells (Fig. 1C).
Immunoblot analysis also shows that XO + X induced a rapid increase
in the phosphorylation of c-Src on tyrosine 418 (Fig. 1D),
whereas the level of total c-Src was unaffected. The level of c-Src in
the plasma membrane was rapidly increased by XO + X treatment (Fig.
2A), which was associated with
a concomitant decrease in c-Src in the soluble fraction. The level of
phosphorylation of c-Src on tyrosine 418 was increased in both plasma
membrane and soluble fractions (Fig. 2B).

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Fig. 1.
Tyrosine phosphorylation and activation of
c-Src by oxidative stress. A, tyrosine kinase activity
was measured in plasma membrane ( ) and soluble ( ) fractions of
Caco-2 cell monolayers treated with XO + X for varying lengths of time.
B, tyrosine kinase activity was measured in the immune
complexes of c-Src prepared from the plasma membranes of Caco-2 cell
monolayers treated with XO + X for varying lengths of time.
C, at varying times after treatment with XO + X proteins
were extracted under denaturing conditions. Phosphotyrosine
(PY) and c-Src were immunoprecipitated (IP)
followed by immunoblot (IB) analysis for c-Src and
phosphotyrosine, respectively. D, whole protein extracts
prepared from cell monolayers treated with XO + X for varying times
were immunoblotted for total c-Src and active c-Src[pY418] using
specific antibodies.
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Fig. 2.
Activation and membrane translocation of
c-Src by oxidative stress. At varying times after treatment with
XO + X, plasma membranes (PM) and soluble fractions
(SF) were isolated. These fractions were immunoblotted for
total c-Src (A) and active c-Src[pY418]
(B).
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The Activity of Src Family Kinases Is Involved in Oxidative
Stress-induced Disruption of TJ--
Our previous studies demonstrated
that tyrosine kinase activity plays a role in the oxidative
stress-induced increase in TJ permeability (15-19). The role of Src
family kinase activity in oxidative stress-induced TJ permeability was
determined by evaluating the effect of PP2, a selective inhibitor of
Src family kinases. Administration of PP2 significantly blocked the XO + X-induced decrease in TER (Fig.
3A) and increase in inulin
permeability (Fig. 3B). PP2 also prevented XO + X-induced
redistribution of occludin and ZO-1 from the intercellular junctions
(Fig. 3C). Cell viability was evaluated and compared between
groups by several methods, such as lactate dehydrogenase release, DNA
fragmentation, trypan blue exclusion, and nuclear staining with
propidium iodide. All methods showed that there was less than 0.03%
cell death, and these values were similar for non-treated and XO + X-treated cell monolayers.

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Fig. 3.
Activity of Src family kinases is involved in
oxidative stress-induced disruption of tight junction. Caco-2 cell
monolayers were incubated with PBS ( ) and PBS containing XO + X in
the absence ( ) or in the presence ( ) of PP2 (10 µM)
for varying lengths of time. TER (A) was measured at varying
times, whereas inulin flux (B) was measured after 3 h
of incubations. Values are the mean ± S.E. (n = 6). Cell monolayers incubated for 3 h with XO + X without or with
PP2 were fixed and labeled for occludin and ZO-1 by immunofluoresence
staining (C).
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Expression of Wild Type c-Src and Kinase-inactive c-Src Mutant in
Caco-2 Cells--
Caco-2 subclones, Caco-2(EV), Caco-2(WT), and
Caco-2(KI), expressing the empty vector, wild type c-Src, and
kinase-inactive c-SrcK297R mutant, respectively, were isolated.
Immunoblot analysis shows an overexpression of c-Src in Caco-2(WT) and
Caco-2(KI) cells compared with c-Src expression in Caco-2(EV) cells
(Fig. 4A). The gross
morphology of these clones in culture was not very different from one
another, except that Caco-2(KI) cells grew with slightly greater
density (8-12% more cells per unit area) than the others (data not
shown). TER tends to be slightly low in Caco-2(WT) cell monolayer and
high in Caco-2(KI) cell monolayer compared with the TER in Caco-2(EV)
cell monolayer (Fig. 4B). Similarly, inulin permeability
tends to be slightly high in Caco-2(WT) cell monolayer and low in
Caco-2(KI) cell monolayer compared with that in Caco-2(EV) cell
monolayer (Fig. 4C). The differences between Caco-2(WT) and
Caco-2(KI) were statistically significant.

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Fig. 4.
Expression of wild type c-Src and
kinase-inactive c-Src(K297R) mutant in Caco-2 cells.
Extracts from four subclones each of empty vector
(EV)-transfected Caco-2(EV), wild type c-Src-transfected
Caco-2(WT) and kinase-inactive c-Src(K297R)-transfected
Caco-2(KI) cells were immunoblotted for total cellular c-Src
using the mouse monoclonal anti-c-Src antibody (A). TER
(B) and inulin permeability (C) were measured in
Caco-2(EV) (white bars), Caco-2(WT) (hatched
bars), Caco-2(KI) (black bars) cells grown on
Transwells after incubation with PBS for 1 h. Values are the
mean ± S.E. (n = 4). Values for Caco-2(KI) are
not significantly different from the values for Caco-2(EV).
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Expression of Kinase Inactive c-Src(K297R) Mutant Delays
Oxidative Stress-induced Increase in Paracellular
Permeability--
Four distinct clones each from Caco-2(EV),
Caco-2(WT), and Caco-2(KI) were analyzed. Treatment with XO + X
resulted in a time-dependent decrease in TER in Caco-2(EV)
cell monolayers (Fig. 5A). The
rates of decrease in TER by XO + X were significantly low in Caco-2(KI) cell monolayers, whereas those in Caco-2(WT) cell monolayers were greater as compared with Caco-2(EV) cell monolayers. At 1 h of XO + X treatment, the TERs for Caco-2 (EV), Caco-2(WT), and Caco-2(KI) monolayers were 73 ± 5, 38 ± 3, and 108 ± 8% of
corresponding basal values, respectively. Similarly, inulin
permeability measured at 2 h of oxidative stress was significantly
low in Caco-2(KI) cell monolayers, whereas it was greater in Caco-2(WT)
cell monolayers as compared with inulin permeability in Caco-2(EV) cell
monolayers (Fig. 5B).

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Fig. 5.
Effect of XO + X on TER (A)
and inulin permeability (B). Four subclones each
of Caco-2(EV) ( or white bar), Caco-2(WT) ( or
hatched bar), and Caco-2(KI) ( or black bar)
were treated with XO + X for varying times. TER (A) was
measured at varying times and calculated as percent of basal values.
The basal TER of monolayers varied from 400 to 500 ohms/cm2. Values are the average of triplicates for each
clone. Inulin permeability (B) was measured during 3 h
of incubation and calculated as percent flux/h/cm2. Values
are the mean ± S.E. (n = 4). Asterisks
indicate the values that are significantly (p < 0.05)
different from the values for Caco-2(EV) cell monolayers.
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Expression of Kinase Inactive c-SrcK297R Mutant Delays Oxidative
Stress-induced Redistribution of Occludin and
ZO-1--
Immunofluorescence staining and confocal microscopy
showed a co-localization of occludin (Fig.
6A) and ZO-1 (Fig.
6B) at the intercellular junctions of Caco-2(EV),
Caco-2(WT), and Caco-2(KI) cell monolayers. In Caco-2(EV) and
Caco-2(WT) cell monolayers treated with XO + X for 3 h, the
fluorescence for occludin (Fig. 6A) and ZO-1 (Fig.
6B) was found to be low at the intercellular junctions,
whereas it was increased in the intracellular compartment. On the other
hand, fluorescence for both occludin and ZO-1 remained co-localized at
the intercellular junctions of Caco-2(KI) cell monolayers after 3 h of treatment with XO + X.

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Fig. 6.
Effect of XO + X on immunofluorescence
localization of occludin (A) and ZO-1
(B). Caco-2(EV), Caco-2(WT), and Caco-2(KI) cell
monolayers were incubated for 3 h with PBS alone or with PBS
containing XO + X. After incubation, cell monolayers were fixed and
labeled for occludin (A) and ZO-1 (B) by
immunofluorescence staining as described under "Experimental
Procedures" using mouse monoclonal anti-occludin antibodies and
rabbit polyclonal anti-ZO-1 antibodies. Images were collected by
confocal microscopy.
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Expression of Kinase-inactive c-Src Mutant Reduces Oxidative
Stress-induced Activation of c-Src--
Studies described above show
that oxidative stress induces activation and membrane translocation of
c-Src in Caco-2 cell monolayers. To determine a similar activation of
c-Src in different subclones, plasma membrane and soluble fractions
were isolated from the control and the XO + X-treated Caco-2(EV),
Caco-2(WT), and Caco-2(KI) cell monolayers. Distribution of total and
active c-Src[pY418] in the plasma membrane fraction was determined by
immunoblot analysis using anti-Src and anti-Src[pY418] antibodies,
respectively. Total amount of c-Src present in plasma membranes was
increased by XO + X treatment in all subclones (Fig.
7A). Active c-Src was not detectable in the plasma membrane of control cell monolayers, but it
was present in XO + X-treated cell monolayers (Fig. 7B). However, the amount of c-Src[pY418] present in the plasma membranes of Caco-2(KI) cells was much less than the amounts of c-Src[pY418] present in the membranes from Caco-2(EV) and Caco-2(WT) cells. We also
measured the c-Src kinase activity in the plasma membranes by immune
complex tyrosine kinase activity. Very low levels of c-Src kinase
activity were detected in the plasma membranes of untreated Caco-2(EV),
Caco-2(WT), and Caco-2(KI) cell monolayers, whereas the activity was
increased by XO + X treatment (Fig. 7C). However, c-Src
kinase activity in XO + X-treated Caco-2 (KI) cells was significantly
less than that in XO + X-treated Caco-2(EV) and Caco-2(WT) cells. The
activity in XO + X-treated Caco-2 (WT) cells was slightly greater than
that in Caco-2(EV) cells.

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Fig. 7.
XO + X-induced membrane translocation and
activation of c-Src. Caco-2(EV), Caco-2(WT), and Caco-2(KI) cell
monolayers were incubated with PBS or PBS containing XO + X for 3 h. After treatment, plasma membranes were isolated and analyzed by
immunoblotting for total c-Src (A) and active c-Src[pY418]
(B) as described under "Experimental Procedures" using
mouse monoclonal anti-c-Src and rabbit polyclonal anti-c-Src[pY418]
antibodies. c-Src kinase activity (C) was measured by immune
complex c-Src kinase assay; values are the mean ± S.E.
(n = 3), and asterisks indicate values that
are statistically different from the values for Caco-2(EV) cells.
U, units.
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Oxidative Stress Induces Tyrosine Phosphorylation of ZO-1 and
-Catenin--
Our previous study demonstrated that oxidative stress
increases tyrosine phosphorylation of junctional proteins, such as ZO-1 and
-catenin, which appear to be important in the disassembly of TJ
(19). Immunoprecipitation of phosphotyrosine from denatured extracts of
cell monolayers followed by immunoblot analysis for ZO-1 and
-catenin detected no tyrosine-phosphorylated ZO-1 and
-catenin in
nontreated Caco-2(EV), Caco-2(WT), and Caco-2(KI) cell monolayers (Fig.
8). XO + X treatment resulted in the
appearance of tyrosine-phosphorylated ZO-1 and
-catenin. The amount
of tyrosine-phosphorylated ZO-1 and
-catenin was found to be less in
XO + X-treated Caco-2(KI) cells, whereas it was greater in Caco-2(WT)
cells monolayers compared with that in XO + X-treated Caco-2(EV) cell
monolayers.

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Fig. 8.
XO + X-induced tyrosine phosphorylation ZO-1
and -catenin. Caco-2(EV), Caco-2(WT), and
Caco-2(KI) cell monolayers were incubated in the absence or presence XO + X for 3 h. Phosphotyrosine (PY) from proteins
extracted under denaturing conditions were immunoprecipitated using
biotin-conjugated anti-phosphotyrosine antibody. Immunoprecipitates
(IP) were then analyzed for ZO-1and -catenin by
immunoblot (IB) analysis using rabbit polyclonal anti-ZO-1
and mouse monoclonal anti- -catenin antibodies.
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Expression of Kinase-inactive c-Src(K297R) Mutant Accelerates
Calcium-mediated Assembly of TJ--
To determine the role of c-Src in
the assembly of TJ we evaluated the rate of recovery of barrier
function after the disruption of TJ by calcium depletion. Treatment
with EGTA for 20 min rapidly reduced the TER of Caco-2 (EV), Caco-2
(WT), and Caco-2 (KI) cell monolayers to around 70 ohms/cm2. Replacement of calcium resulted in a
time-dependent increase in TER in all cell monolayers (Fig.
9A). However, the rates of increase in TER in Caco-2 (KI) cell monolayers were dramatically high
compared with those in Caco-2 (EV) cell monolayers, whereas those in
Caco-2(WT) cell monolayers were significantly lower than those in
Caco-2(EV) cell monolayers. After 4 h of recovery the TER was
found to be 192 ± 18, 125 ± 3, and 421 ± 33 ohms/cm2 in Caco-2(EV), Caco-2(WT), and Caco-2(KI) cell
monolayers, respectively. Similarly, inulin permeability measured after
3 h of recovery was significantly low in Caco-2(KI) cell
monolayers, whereas it was high in Caco-2(WT) cell monolayers as
compared with that in Caco-2(EV) cell monolayers (Fig. 9B).
Immunofluorescence staining and confocal microscopy showed a
redistribution of occludin (Fig. 10A) and ZO-1 (Fig.
10B) from the intercellular junctions of Caco-2(EV), Caco-2(WT), and Caco-2(KI) cell monolayers into the intracellular compartments by EGTA treatment. After 3 h of recovery only minimal amounts of occludin and ZO-1 were reorganized at the intercellular junctions in Caco-2(EV) and Caco-2(WT) cell monolayers, whereas major
portions of occludin and ZO-1 were reorganized at the intercellular junctions of Caco-2(KI) cell monolayers.

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Fig. 9.
TER (A) and inulin
permeability (B) during reassembly of TJ after calcium
switch. Four subclones each of Caco-2(EV) ( or white
bar), Caco-2(WT) ( or hatched bar), and Caco-2(KI)
( or black bar) were incubated for 20 min with EGTA
followed by washing and replacement of calcium. TER (A) was
measured at varying times and calculated as ohms/cm2.
Values are the average of triplicates for each clone. Inulin
permeability (B) was measured after 4 h of recovery and
calculated as percent flux/h/cm2. Values are the mean ± S.E. (n = 4). Asterisks indicate that the
values that are significantly (p < 0.05) different
from the values for Caco-2(EV) cell monolayers.
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Fig. 10.
Immunofluorescence localization of occludin
during reassembly of TJ. Caco-2(EV), Caco-2(WT), and Caco-2(KI)
cell monolayers were incubated for 20 min with EGTA followed by washing
and replacement of calcium. At 0 and 3 h of recovery, cell
monolayers were fixed and labeled for occludin (A) and ZO-1
(B) by immunofluorescence staining as described under
"Experimental Procedures" using mouse monoclonal anti-occludin and
rabbit polyclonal anti-ZO-1 antibodies. Images were collected by
confocal microscopy.
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The Activity of Src Family Kinases Is Involved in Oxidative
Stress-induced Disruption of TJ in MDCK Cell Monolayer--
The above
studies show that c-Src is required for the oxidative stress-induced
disruption of TJ in the Caco-2 cell monolayer. Interestingly, this
observation contrasts the previous observation by Meyer et
al. (23) that Src kinase activity is required for the assembly of
TJ in MDCK cell monolayers. To determine whether there is a difference
in the mechanism of oxidative stress-induced disruption of TJ between
Caco-2 and MDCK cell monolayers, the effect of XO + X in the absence or
presence of PP2 on TJ permeability was evaluated. Administration of XO + X induced a decrease in TER (Fig.
11A), an increase in inulin
permeability (Fig. 11B), and redistribution of occludin
(Fig. 11C). PP2 significantly blocked the XO + X-induced
changes in TER, inulin flux, and redistribution of occludin.

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Fig. 11.
Activity of Src family kinases is involved
in oxidative stress-induced disruption of tight junction in MDCK cell
monolayers. MDCK cell monolayers were incubated with PBS ( ) and
PBS containing XO + X in the absence ( ) or presence ( ) of PP2 (10 µM) for varying lengths of time. TER (A) was
measured at varying times, whereas inulin flux (B) was
measured after 3 h of incubations. Values are mean ± S.E.
(n = 6). Cell monolayers incubated for 3 h with XO + X without or with PP2 were fixed and labeled for occludin by
immunofluoresence staining (C).
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DISCUSSION |
The proto-oncogene, c-Src, is a non-receptor tyrosine kinase that
plays a crucial role in a number of physiological functions of normal
cells, such as cell proliferation, differentiation, and cell migration
(37). c-Src is transiently activated during growth factor receptor
activation and mitosis (38), whereas prolonged activation of c-Src due
to molecular modifications or mutation leads to cell transformation.
Elevations of c-Src kinase activity have been altered in a variety of
human cancers, including colon cancer (39, 40). c-Src tyrosine
phosphorylates a wide variety of cellular proteins, including
platelet-derived growth factor, epidermal growth factor, and
CSF-1 receptors, focal adhesion kinase, proline-rich tyrosine
kinase-2, Cdk-activating kinase, vinculin, and lactate dehydrogenase
(41). Localization of c-Src at the TJ of epithelial tissue
suggests that Src kinase activity may play an important role in the
regulation of TJ structure and function (1). Our recent studies
indicated that tyrosine kinase activity and protein tyrosine
phosphorylation play an important role in the regulation of TJ in the
Caco-2 cell monolayer (15-19). These studies raised the issue of
whether c-Src plays a role in the regulation of TJ under basal
condition or under oxidative stress. Here we show that overexpression
of kinase-inactive c-Src mutant delays oxidative stress-induced
increase in paracellular permeability and accelerates calcium-mediated
assembly of TJ.
Inhibition of XO + X-induced decrease in TER, increase in inulin
permeability, and redistribution of occludin and ZO-1 from the
intercellular junctions by PP2, a Src family kinase inhibitor, indicate
that the activity of Src family kinases is involved in the oxidative
stress-induced disruption of TJ and increase in paracellular
permeability. A previous study showed a similar inhibition of hydrogen
peroxide-induced paracellular permeability in an endothelial cell
monolayers (34); however, redistribution of TJ proteins or activation
of Src family kinases were not analyzed. Our present data show that the
tyrosine kinase activity of c-Src in the plasma membrane was strongly
increased by XO + X treatment. Additionally, treatment with XO + X
induced tyrosine phosphorylation of c-Src, particularly phosphorylation
on tyrosine 418, and translocation of c-Src protein into
the plasma membrane. Tyrosine 418 is an autophosphorylation site, and
therefore, the increase in phosphorylation at this site is an indicator
of the activity of c-Src. These results clearly demonstrate that
oxidative stress rapidly activates c-Src in the Caco-2 cell monolayer
and raises the issue of whether c-Src activity plays a role in the
disruption of TJ. However, there is a significant interval between the
activation of c-Src and the decrease in TER. It is quite likely that a
rapid activation of c-Src sets the stage for subsequent events that
lead to the opening of TJ. Evidence indicates that disruption of TJ by
oxidative stress involves protein kinase C activity, dephosphorylation
of occludin on serine and threonine residues, and reorganization of
actin cytoskeleton. These may well be some of the intermediate steps
involved in TJ disruption by oxidative stress.
To determine the role of c-Src in regulation of TJ, we transfected
Caco-2 cells with the wild type c-Src or kinase-inactive c-SrcK297R
mutant. The overexpression of wild type c-Src and kinase-inactive c-Src(K297R) mutant was determined by the increased levels of total
c-Src present in the extracts of Caco-2(EV), Caco-2(WT), and Caco-2(KI)
subclones. c-Src levels in Caco-2(WT) and Caco-2(KI) cells were greater
than that in Caco-2(EV) cells. Although the gross morphology of
different clones was similar, there was a tendency for Caco-2(KI) cells
to grow more densely than Caco-2(EV) and Caco-2(WT) cells. The
paracellular permeability tends to be slightly low in Caco-2 (KI-Src)
cell monolayers and greater in the Caco-2 (WT-Src) cell monolayer
compared with that in Caco-2 (EV) cell monolayers, although these
differences were not statistically significant.
To determine the role of c-Src in the oxidative stress-induced
disruption of TJ, we evaluated the effect of XO + X on paracellular permeability and redistribution of TJ proteins in Caco-2(EV), Caco-2(WT), and Caco-2(KI) cell monolayers. The rate of oxidative stress-induced increase in permeability was slightly greater in Caco-2(WT) cell monolayers, whereas it was dramatically delayed in
Caco-2(KI) cell monolayers when compared with that in Caco-2(EV) cell
monolayers. This observation supports the view that activation of c-Src
plays an important role in oxidative stress-induced disassembly of TJ.
It was further supported by the confocal immunofluorescence microscopic
observation that oxidative stress causes a reorganization of TJ
proteins occludin and ZO-1 in Caco-2(EV) and Caco-2(WT) cell
monolayers, whereas it is relatively unaffected in Caco-2(KI) cell monolayers.
Oxidative stress induced a translocation of c-Src to the plasma
membranes and increased the level of active c-Src[pY418] in the
plasma membranes on Caco-2(EV) cells. The membrane translocation of
total c-Src was found to be similar in Caco-2(EV), Caco-2(WT), and
Caco-2(KI) cells. However, the level of active c-Src[pY418] present
in the plasma membrane was different from each other. The amount
of active c-Src[pY418] present in plasma membrane of XO + X-treated
Caco-2(WT) cells was slightly greater than that in membranes of XO + X-treated Caco-2(EV) cells, whereas the amount of active c-Src[pY418]
in plasma membranes of XO + X-treated Caco-2(KI) cells was much lower
compared with those in XO + X-treated Caco-2(EV) and Caco-2(WT) cells.
These results indicate that translocation of total amount of c-Src to
plasma membrane was unaffected by the overexpression of wild type c-Src
or kinase-inactive c-Src(K297R). However, translocation of inactive
c-Src(K297R) may have resulted in the presence of low levels of
active c-Src[pY418] in the plasma membrane of XO + X-treated
Caco-2(KI) cell monolayers. This observation was further supported by
similar differences in the c-Src tyrosine kinase activity in the plasma
membranes of Caco-2(EV) Caco-2(WT), and Caco-2(KI)cells. Furthermore,
the extent of tyrosine phosphorylation of ZO-1 and
-catenin in XO + X-treated Caco-2(KI) cell monolayer was low compared with that in XO + X-treated Caco-2(EV) and Caco-2(WT) cell monolayers. ZO-1 and
-catenin tyrosine phosphorylation was greater in XO + X-treated
Caco-2(WT) cells. Our recent study demonstrated that oxidative stress
induces tyrosine phosphorylation of proteins of TJ and adherens
junction, in particular tyrosine phosphorylation of ZO-1 and
-catenin associated with the actin cytoskeleton (19). These results
demonstrate that the oxidative stress-induced c-Src activation in the
plasma membrane and tyrosine phosphorylation of ZO-1 and
-catenin
were attenuated by the dominant negative expression of kinase-inactive
c-Src in Caco-2 cells.
The outcome of this study that the tyrosine kinase activity of c-Src is
involved in the disruption of TJ in human intestinal epithelium is in
contrast to the previous reports that Src family kinase activity is
required for the assembly of TJ in the canine kidney epithelium (23).
There are two likely explanations for such a discrepancy in the role of
Src kinase activity in the regulation of TJ. First, the possibility
exists that there is a cell type-dependent difference in
signaling pathways and their role in regulation of TJ. Second, it is
quite likely that Src kinase activity is required for both the assembly
and the disassembly of TJ; different members of Src family kinase may
be involved in regulation of TJ. To determine whether there is a
cell type-dependent difference in the mechanism of
XO + X-induced increase in TJ permeability, we evaluated the effect of
XO + X in the absence and presence of PP2. The results showed that XO + X induces a decrease in TER, an increase in inulin permeability, and
redistribution of occludin in MDCK cell monolayers. These XO + X-induced changes in TER, inulin flux, and redistribution of occludin
were prevented by PP2. This observation in the MDCK cell monolayer is
similar to the observation made in Caco-2 cells, suggesting that the
mechanism of TJ regulation is similar in both Caco-2 and MDCK cell
monolayers. However, the new observation in combination with a previous
study by Meyer et al. (23) demonstrates that Src activity is
required for both the disruption and the assembly of TJ, suggesting
that distinct members of Src family kinases may be involved in the assembly and the disruption of TJ. As shown by our present data c-Src
activity may regulate the disassembly of TJ, whereas other Src kinases
such as c-Yes may be involved in the assembly of TJ. C-Yes appears to
bind occludin C-terminal peptide in vitro (8) and play a
role in the assembly of TJ in MDCK cell monolayers (21).
The present study also shows that the expression of wild type and
kinase-inactive c-Src alters the ability of Caco-2 cells to reassemble
TJ after the disruption by calcium depletion. As shown previously in
MDCK cell monolayers (42), calcium depletion resulted in a rapid
disruption of the TJ and increase in paracellular permeability in
Caco-2(EV) cell monolayer. Replacement of calcium resulted in a gradual
reassembly of TJ and restoration of paracellular barrier function.
Interestingly enough, restoration of barrier function was delayed in
Caco-2(WT) cells, whereas it was dramatically accelerated in Caco-2(KI)
cells. This difference in the rate of restoration of barrier function
in Caco-2(EV), Caco-2(WT), and Caco-2(KI) cells was supported by
immunofluorescence localization of the TJ proteins occludin and ZO-1.
After 3 h of recovery, occludin and ZO-1 remained disorganized in
the intracellular compartments of Caco-2(EV) and Caco-2(WT), whereas
occludin and ZO-1 were reorganized at the intercellular junctions of
Caco-2(KI) cell monolayer. These data indicate that the expression of
wild type c-Src delays the assembly of TJ, whereas the expression of
kinase-inactive c-Src(K297R) accelerates the assembly of TJ and
restoration of the barrier function.
In summary, this study shows that c-Src tyrosine kinase activity delays
the calcium-mediated assembly and accelerates the oxidative
stress-induced disassembly of TJ in Caco-2 cell monolayers. Results
also indicate that activation of c-Src and its activity play a crucial
role in the oxidative stress-induced disruption of TJ and increase in
paracellular permeability.