Cytochalasin B modulation of Caco-2 tight junction barrier:
role of myosin light chain kinase
Thomas Y.
Ma1,2,
Neil T.
Hoa1,2,
Daniel D.
Tran1,2,
Vuong
Bui1,2,
Ali
Pedram1,3,
Susan
Mills1,2, and
Margaret
Merryfield3
1 Division of Gastroenterology, Department of Medicine,
Department of Veterans Affairs Medical Center, and
3 Department of Biochemistry, California State University,
Long Beach 90822; and 2 Division of Gastroenterology, Department
of Medicine, University of California, Irvine, California 92717
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ABSTRACT |
The intracellular mechanisms that
mediate cytochalasin-induced increase in intestinal epithelial tight
junction (TJ) permeability are unclear. In this study, we examined the
involvement of myosin light chain kinase (MLCK) in this process, using
the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin
B (Cyto B) (5 µg/ml) produced an increase in Caco-2 MLCK activity,
which correlated with the increase in Caco-2 TJ permeability. The
inhibition of Cyto B-induced MLCK activation prevented the increase in
Caco-2 TJ permeability. Additionally, myosin-Mg2+-ATPase
inhibitor and metabolic inhibitors (which inhibit MLCK induced
actin-myosin contraction) also prevented the Cyto B-induced increase in
Caco-2 TJ permeability. Cyto B caused a late-phase (15-30 min)
aggregation of actin fragments into large actin clumps, which was also
inhibited by MLCK inhibitors. Cyto B produced a morphological
disturbance of the ZO-1 TJ proteins, visually correlating with the
functional increase in Caco-2 TJ permeability. The MLCK and
myosin-Mg2+-ATPase inhibitors prevented both the functional
increase in TJ permeability and disruption of ZO-1 proteins. These
findings suggested that Cyto B-induced increase in Caco-2 TJ
permeability is regulated by MLCK activation.
paracellular permeability; myosin light chain kinase; actin
filaments; ZO-1 protein.
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INTRODUCTION |
A MAJOR FUNCTION OF
INTESTINAL epithelial cells is to provide a physical
barrier between the hostile intestinal lumen and the subepithelial
tissue. The apicolaterally located tight junctions (TJs) form a
paracellular seal between the lateral membranes of the adjacent cells
and act as a structural barrier against the paracellular penetration of
hydrophilic molecules (2, 31). Disruption of the
intestinal epithelial TJ complexes results in a "leaky gut" with an
increase in intestinal paracellular permeability (18, 25,
31). It had been proposed in some diseases that a defective
intestinal epithelial TJ barrier allows the paracellular permeation of
toxic luminal substances, which leads to intestinal inflammation and
mucosal injury (3, 11, 14, 18, 25, 40). Specifically,
evidence had been presented suggesting that an altered intestinal
epithelial TJ permeability may be an important pathogenic factor in
intestinal diseases such as Crohn's disease (18, 19, 25,
36), nonsteroidal anti-inflammatory drug-associated enteritis
(3), and in diarrheal syndromes caused by
Clostridial difficile, Vibrio
cholera, and enteropathogenic Escherichia coli (11, 14, 40). The precise intracellular processes that
regulate intestinal epithelial TJ permeability in pathological and
normal physiological conditions remain poorly understood.
The intercellular TJs encircle the intestinal epithelial cells in a
belt-like manner at the apical cellular borders at the level of zonula
occludens. The TJs make homotypic contact across the intercellular
spaces between the adjacent cells (2). The lateral
contacts, which may be visualized by electron microscopy and
freeze-fracture analysis, act as a structural barrier against the
paracellular permeation (31, 34). There is also a high density of cytoskeletal elements and actin and myosin filaments, which
encircle the intestinal epithelial cells near the apical cellular
borders at the level of zonula adherens (31-34).
Previous studies (29, 33) have shown that disruption of
the perijunctional actin filaments with cytochalasins (specific
actin-disrupting agents) causes an increase in intestinal epithelial TJ
permeability. Cytochalasins disrupt actin microfilaments by a direct
severing effect, interfering with actin subunit polymerization and
inducing reactive cellular response (4, 5, 12, 33, 39). In this regard, cytochalasins have been widely used as probes for studying
actin-mediated cell activities.
The cytochalasin disruption of perijunctional actin filaments
culminates in morphological and functional disturbance of intestinal TJs (29, 33). The intracellular mechanisms that modulate
this actin filament-mediated increase in intestinal TJ permeability have not been delineated. Because actin function is closely dependent on its interaction with myosins, we hypothesized that cytochalasin modulation of TJ permeability is mediated by regulation of myosin light
chain kinase (MLCK) activity. Specifically, we tested the hypothesis
that cytochalasin-induced increase in intestinal TJ permeability was
mediated by MLCK activation and perijunctional actin-myosin interaction
and that MLCK activation was an important triggering event leading to
the increase in intestinal TJ permeability. We used the filter-grown
Caco-2 intestinal epithelial monolayers as the in vitro model system to
study the effects of Cyto B on intestinal epithelial TJ permeability.
The human colon cancer-derived Caco-2 intestinal epithelial cell system
has been widely used as an in vitro model of intestinal epithelia
(15, 16, 26, 38). When confluent and allowed to mature on
permeable inserts, Caco-2 cells form TJs and attain many of the
morphological and functional characteristics of the enterocytes
(15, 16, 38). Our results provide new insight into the
intracellular mechanism of cytochalasin modulation of intestinal
epithelial TJ permeability.
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MATERIALS AND METHODS |
DMEM, trypsin, and fetal bovine serum were purchased from Life
Technologies (Gaithersburg, MD). Glutamine, penicillin, streptomycin, and PBS solution were purchased from Irvine Scientific (Santa Ana, CA).
Cyto B was purchased from Sigma Chemical (St. Louis, MO). Millicell-HA
0.4-µm permeable filters (12 mm) were purchased from Millipore
(Bedford, MA). Anti-ZO-1 antibody and FITC-strepavidin were obtained
from Zymed Laboratories (San Francisco, CA), and fluorescein-conjugated
rabbit anti-rat antibodies were obtained from Boehringer Mannheim
(Indianapolis, IN). [14C]mannitol was obtained from NEN
Research Products (Wilmington, DE). All other chemicals were of a
reagent grade.
Cell cultures.
Caco-2 cells were purchased from American Type Culture Collection
(Rockville, MD). The stock cultures were grown in a culture medium
composed of DMEM with 4.5 mg/ml glucose, 50 U/ml penicillin, 50 U/ml
streptomycin, 4 mmol/l glutamine, and 10% fetal bovine serum
(16, 38). Culture medium was changed every 1-2 days. The cells were subcultured by partial digestion with 0.25% trypsin and
0.9 mmol/l EDTA in Ca2+-free and Mg2+-free PBS
solution. For growth on filters, high-density Caco-2 cells (5 × 105 cells) were plated on nitrocellulose-based Millicell-HA
filters and monitored regularly by measuring epithelial resistance.
Determination of epithelial monolayer resistance and paracellular
permeability.
The electrical resistance of the filter-grown intestinal monolayers was
measured with an epithelial voltohmmeter (World Precision Instruments,
Sarasota, FL) as previously reported (27). For resistance
measurements, both apical and basolateral sides of the epithelium were
bathed with same buffer solution. Electrical resistance was measured
until similar values were recorded on three consecutive measurements.
The resistances of monolayers in this study are reported after
subtraction of the resistance value of the filters alone. The effect of
Cyto B on Caco-2 monolayer paracellular permeability was examined using
the established paracellular marker mannitol (24, 28, 33,
33). For determination of mucosal-to-serosal flux rates of the
paracellular probe mannitol, only Caco-2-plated filters having
epithelial resistance of 340-420
· cm2
were used. The filter-grown Caco-2 monolayers reached epithelial resistance of 340-420
· cm2 by 3-4 wk
post plating (16, 29). Unless specified otherwise, Krebs-phosphate saline buffer (pH 7.4) was used as the incubation solution during the experiments. Buffered solution (300 µl)
was added to the apical compartment, and 450 µl were added to the basolateral compartment to ensure equal hydrostatic pressure as recommended by the manufacturer. Known concentrations of mannitol (10 µmol/l) and its radioactive tracer ([14C]mannitol) were
added to the apical solution. Low concentrations of mannitol were used
to ensure that negligible osmotic or concentration gradient was
introduced. The test reagent was added to both the apical and the
basolateral compartments as indicated. All flux studies were carried
out at 37°C. All of the experiments were repeated three to five times
to ensure reproducibility.
Fluorescein labeling of cytoskeletal structures and TJ proteins.
Distribution of actin microfilaments was assessed using fluorescent
labeling techniques as previously described (29). Caco-2 monolayers grown on coverslips were fixed with 3.75% formaldehyde solution in PBS for 20 min at room temperature and were permeabilized in acetone at
20°C for 5 min and washed with 1 M PBS solution. Subsequently, 10 microunits of fluorescein-labeled phalloidin (Molecular Probes, Eugene, OR) dissolved in 200 microliter of PBS were
placed on the coverslips for 40 min. After the PBS rinse, coverslips
were mounted on a slide with the cell side down in a 1:1 solution of
PBS and glycerol.
The TJ protein ZO-1 and myosin II filaments were labeled with anti-ZO-1
and anti-myosin II antibodies, respectively (29, 41).
Caco-2 monolayers were fixed with 2.0% formaldehyde and permeabilized
in acetone as described above. The Caco-2 monolayers were labeled with
appropriate primary antibody. This was followed by incubation with 1:30
diluted Tris-buffered saline solution containing secondary anti-rabbit
IgG biotinylated antibody (Zymed Laboratories) and incubation with 1:20
diluted Tris-buffered saline solution containing FITC-labeled
strepavidin (Zymed Laboratories). Coverslips were mounted in 60%
glycerol, Tris-buffered saline solution, and 0.4% n-propyl
gallate. The fluorescein-labeled structures were viewed by a
"blinded" person unaware of the coded experimental conditions,
using either Nikon-PCM 2000 confocal imaging system attached to a Nikon
Eclipse 800 microscope or Optronics DEI-750 CE digital output imaging
system (Goleta, CA) attached to Nikon Labophot epifluorescence
microscope. The photomicrographic images of fluorescein-labeled ZO-1
proteins and the perijunctional actin and myosin filaments were
obtained at focal levels corresponding to the region of zonula
occludens (2-3 µm below the apical brush-border membrane) and
zonula adherens (3-4 µm below the apical brush-border membrane),
respectively, using the Optronics imaging system. All of the
fluorescent labeling experiments were repeated four to six times in
duplicates to ensure reproducibility.
Caco-2 MLCK-kinase activity determination.
Caco-2 MLCK activity was determined by measuring in vitro kinase
activity of the immunoprecipitated MLCK obtained from the Caco-2 cells
after treatment with various experimental reagents. For MLCK
immunoprecipitation, Caco-2 monolayers were serum-deprived overnight.
The Caco-2 cells were then exposed to appropriate experimental conditions. At the completion of the experiments, Caco-2 cells were
immediately rinsed with ice-cold Hanks' balanced salt solution. Cells
were then lysed using 0.8 ml lysis buffer (50 mM HEPES, 100 mM NaCl, 2 mM EDTA, 1 µM pepstatin, 1 µg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, 2 mM sodium vanadate, 2 µg/ml
aprotinin, and 40 mM para-nitrophenol phosphate
di-cyclohexylammonium salt) and scraped, and lysates were placed in
Microfuge tubes (tube A) and microfuged for 5 min to yield a
clear lysate.
Anti-MLCK antibody (5 µl/200 µl lysis buffer) obtained from Sigma
Chemical was added to a separate Microfuge tube (tube B) containing protein A beads and incubated end-over-end for 1 h at
4°C. Then 100 µl of each cleared lysate (tube A) were
added to the microvial (tube B) containing the pelleted
protein A-Sepharose bead coupled with anti-MLCK antibodies and
incubated end-over-end for 2 h at 4°C. The microvial containing
the immunoprecipitates was microfuged, and the supernatant was
aspirated. Immunoprecipitates were washed sequentially with lysis
buffer and a solution of 10 mM HEPES and 10 mM Mg acetate at 4°C.
Immunoprecipitated MLCK was then used in an in vitro kinase reaction in
Microfuge tubes to determine the MLCK activity by measuring the rate of
MLC phosphorylation by the immunoprecipitated MLCK. For this, 20 µl
purified chicken gizzard MLC protein (2 mg/ml), 20 µl of three times
hot mix {150 µM ATP, 10 µl [32P]ATP (5 µCi/reaction), 30 mM magnesium acetate, and 30 mM HEPES} were added
and mixed with the immunoprecipitated MLCK, for a 10-min reaction
period at 30°C. The MLCK catalyzed phosphorylation reaction was
terminated by addition of 20 µl stop buffer solution (1 ml 2 M Tris
buffer, pH 6.8, 2 ml 20% SDS, 4 ml glycerol, 3 ml water, 308 mg
dithiothreitol, and trace of bromophenol blue). Subsequently, the
reaction mixture was boiled for 3 min and microfuged for 10 s, and
then the supernatant (40-50 µl) was separated on 10% SDS-PAGE. The gel was fixed in 40% MeOH and 10% acetic acid overnight and stained with Coomassie blue solution, dried, and autoradiographed, and
the MLC band at 19.5 kDa was identified. The experiments were repeated
three times to ensure reproducibility. It should be noted that similar
results were also obtained when Ca2+ (0.2 mM) and
calmodulin (1 µM) were added to the kinase reaction mixture.
Intracellular MLC phosphorylation assay.
After cell-cycle synchronization in serum-free buffer solution
overnight, Caco-2 monolayers were incubated for 1 h at 37°C in
phosphate-free medium containing 5% dialyzed fetal bovine
serum. At the end of the incubation period, monolayers were
washed and labeled with 32Pi (final
concentration, 0.2 mCi/ml) for 2 h at 37°C. Subsequently, monolayers were exposed to various experimental conditions. At the end
of the experimental period, monolayers were washed with iced-cold PBS,
then lysed with lysis buffer for 30 min at 4°C. The lysates were
microcentrifuged, and MLC was immunoprecipitated from the supernatant
with anti-MLC antibody (Sigma Chemical) at 4°C. After centrifugation
and rinse, the immunoprecipitated MLC was resolved by SDS-PAGE on a
12% gel, followed by an autoradiography.
 |
RESULTS |
Effect of Cyto B on Caco-2 actin filaments and TJ permeability.
The Cyto B effect on Caco-2 actin microfilaments and TJ permeability
was determined by fluorescein labeling of Caco-2 actin filaments and
measurements of epithelial resistance and mucosal-to-serosal flux of
paracellular marker mannitol across the filter-grown Caco-2 monolayers.
Consistent with the native intestinal epithelia (32), Caco-2 actin filaments were localized at the apical perijunctional area
at the level of zonula adherens just below the zonula occludens and
appeared as a continuous band encircling the cells at the cellular
borders (Fig. 1A). Cyto B (5 µg/ml) produced a progressive disruption of the Caco-2 actin
filaments with breakage, displacement and clumping of the
perijunctional actin filaments (Fig. 1,
B-D). Cyto B (5 µg/ml) treatment resulted
in a drop in Caco-2 epithelial resistance (Fig.
2A) and an increase in
mucosal-to-serosal flux of mannitol (Fig. 2B), indicating an
increase in Caco-2 epithelial TJ permeability.

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Fig. 1.
Effect of cytochalasin B (Cyto B) (5 µg/ml) on perijunctional
Caco-2 actin microfilaments. Caco-2 F-actin filaments were labeled with
fluorescein-conjugated phalloidin as described in MATERIALS AND
METHODS. The sequential effect of Cyto B (5 µg/ml) on Caco-2
actin microfilaments at time 0 (A) and 1 (B), 15 (C), and 30 min (D) is shown
in the photomicrographs (original magnification, ×80). By 1 min of
Cyto B exposure, perijunctional actin filaments were fragmented and
present diffusely throughout the cytoplasm. By 15-30 min of Cyto B
exposure, actin fragments coalesced to form large actin clumps or
"foci" near the perijunctional areas.
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Fig. 2.
Effect of Cyto B (5 µg/ml) on Caco-2 epithelial
resistance and paracellular permeability. A: Cyto B (5 µg/ml) effect on Caco-2 epithelial resistance expressed as
· cm2. Inset: magnified view of the
early time course. B: Cyto B (5 µg/ml) effect on
mucosal-to-serosal flux of paracellular marker mannitol expressed in
nmol/cm2. Values are means ± SE; n = 4.
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Cytochalasins disrupt actin filaments by direct breakage of actin
filament network as well as through secondary cellular response (39). After initial breakage or severing of actin
filaments into smaller fragments, the actin fragments later combine to
form large cytoskeletal clumps or "foci" (33, 39).
Similarly, in our studies, two distinct time-related alterations in
perijunctional actin filaments were visible after Cyto B treatment of
the Caco-2 cells (Fig. 1). The earliest changes, which occurred within
the first minute of the Cyto B exposure, were characterized by the breakage of the perijunctional actin filaments into smaller actin fragments (Fig. 1B). The small fragments were seen
distributed diffusely throughout the cytoplasm at the level of zonula
adherens, giving a hazy "fluffy" appearance in the cytoplasm with
some actin filaments remaining localized at the cellular borders. On
longer exposure, the smaller actin fragments coalesced into large actin clumps or foci (Fig. 1, C and D). This
"late phase" formation of actin clumps was inhibited by metabolic
inhibitors 2,4-dinitrophenol (1 mM) and sodium azide (30 mM) (Fig.
3B). In contrast, the early phase changes in actin filaments (breakage into smaller fragments) were
not inhibited by the metabolic inhibitors (Fig. 3A). In
fact, there appeared to be a slight accentuation of actin fragment
formation after pretreatment with metabolic inhibitors (Fig.
3B).

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Fig. 3.
Effect of metabolic inhibitors on Cyto B induced
modulation of perijunctional Caco-2 actin microfilaments (en face
views). Caco-2 cells were energy depleted by incubation with
2-deoxy-D-glucose (2 mM) and 2,4-dinitrophenol (1 mM) for
1 h. Subsequently, the energy-depleted Caco-2 monolayers were
treated with Cyto B (5 µg/ml) for 1 (A) and 30 min
(B), respectively (original magnification, ×80).
2,4-Dinitrophenol did not affect the Cyto B fragmentation of actin
filaments at the 1-min time period, but prevented the actin clump
formation and further accentuated the actin fragmentation at the 30-min
time period. Similar results were also obtained with sodium azide.
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Role of MLCK in Cyto B-induced increase in Caco-2 TJ permeability.
In the following studies, we examined the involvement of Caco-2 MLCK in
Cyto B-induced increase in Caco-2 TJ permeability. First, the effect of
Cyto B on Caco-2 MLCK activity was examined by immunoprecipitation of
Caco-2 MLCK. After Cyto B (5 µg/ml) treatment, Caco-2 MLCK was
isolated by immunoprecipitation with anti-MLCK antibody. The kinase
activity of the immunoprecipitated MLCK was then determined by
measuring in vitro MLC phosphorylation. MLCK obtained from the Cyto
B-treated cells produced a significant increase in in vitro MLC
phosphorylation compared with that of control or untreated cells,
indicating activation of Caco-2 MLCK (Fig.
4). The time course of Cyto B effect on
Caco-2 MLCK activation indicated that the peak MLCK activation occurred
between 5 and 10 min after Cyto B exposure (Fig. 4). Direct addition of
Cyto B to the immunoprecipitated MLCK did not have significant effect on MLCK activity, indicating that Cyto B does not directly activate MLCK under in vitro conditions.

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Fig. 4.
Effect of Cyto B on Caco-2 myosin light chain kinase
(MLCK) activity. Caco-2 MLCK activity was determined as described in
MATERIALS AND METHODS. Caco-2 monolayers were exposed to
Cyto B for increasing time periods (0-30 min). Subsequently,
Caco-2 monolayers were lysed, and Caco-2 MLCK was immunoprecipitated.
The activity of the immunoprecipitated MLCK was determined by in vitro
kinetic measurement of MLC phosphorylation. Phosphorylated MLC (P-MLC;
~19.5 kDa) was separated by 10% SDS-PAGE, stained with Coomassie
blue solution, and autoradiographed as described in MATERIALS AND
METHODS. Cyto B produced a time-dependent activation of Caco-2
MLCK with the peak activation occurring between 5 and 10 min after Cyto
B exposure.
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To determine whether Cyto B-induced activation of Caco-2 MLCK was due
to an increase in MLCK protein level or an increase in the activity of
the preexisting MLCK proteins, the effects of Cyto B on MLCK protein
level and cellular localization were examined by Western blot analysis
and immunofluorescent antibody labeling. In the confluent Caco-2
monolayers, MLCK was localized mainly at the perijunctional areas (data
not shown). Cyto B did not have significant effect on either MLCK
protein level (Fig. 5A) or the
cellular localization of MLCK (data not shown), suggesting that Cyto B
stimulation of Caco-2 MLCK activity resulted from an increased activity
of the preexisting MLCK proteins.

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Fig. 5.
Effect of MLCK inhibitor
1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) on
Cyto B activation of Caco-2 MLCK activity and Caco-2 MLCK protein
level. Caco-2 monolayers were incubated with either Kreb's-phosphate
saline buffer (control) or Kreb's-phosphate saline buffer containing
either Cyto B (5 µg/ml), Cyto B (5 µg/ml) and ML-7 (15 µM), or
ML-7 (15 µM) for 10 min. Subsequently, Caco-2 cells were lysed, and
MLCK activity and protein level were determined by Western blot
analysis and in vitro kinase activity measurement as described in
MATERIALS AND METHODS. A: Western blot analysis.
B: immunoblot of phosphorylated MLC. C: corresponding
densitometry measurements (means ± SE) of phosphorylated MLC
bands expressed in pixels (n = 4).
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The Cyto B effect on Caco-2 MLCK activity was further validated by
measurement of MLC phosphorylation inside the cells by direct
immunoprecipitation of MLC. The Cyto B treatment produced a significant
increase in phosphorylation of MLC inside the cells (Fig.
6), confirming intracellular activation
of Caco-2 MLCK.

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Fig. 6.
Effect of Cyto B on Caco-2 MLC phosphorylation inside the
cells. Intracellular MLC phosphorylation was determined by direct
immunoprecipitation of 32P-labeled MLC as described in
MATERIALS AND METHODS. The immunoprecipitated MLC was
resolved by 12% SDS-PAGE. Cyto B (5 µg/ml) caused an increase in MLC
phosphorylation inside the treated cells. ML-7 inhibited the Cyto
B-induced increase in MLC phosphorylation.
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Second, to confirm that Cyto B-induced increase in Caco-2 TJ
permeability was mediated by MLCK activation, the effect of MLCK inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) on Cyto B modulation of Caco-2 TJ permeability was examined. The
pretreatment of Caco-2 monolayers with ML-7, at the dose (15 µM)
previously used by Turner et al. (42) to inhibit MLCK
activity, prevented Cyto B activation of Caco-2 MLCK (Figs. 5 and 6).
ML-7 (15 µM) also significantly prevented the Cyto B-induced drop in Caco-2 epithelial resistance and increase in paracellular permeability (Fig. 7). Similarly, other potent and
selective MLCK inhibitors, including ML-9 and KT 5926 (37), also inhibited the Cyto B-induced drop in Caco-2
epithelial resistance (data not shown). These findings confirmed that
Cyto B activation of Caco-2 MLCK was required for the Cyto
B-induced increase in Caco-2 TJ permeability.

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Fig. 7.
Effect of MLCK inhibitors on Cyto B modulation of Caco-2
epithelial resistance and paracellular permeability. A:
effect of ML-7 (15 µM) on Cyto B (5 µg/ml)-induced drop in Caco-2
epithelial resistance. B: effect of ML-7 (15 µM) on Cyto B
(5 µg/ml)-induced increase in mucosal-to-serosal flux of mannitol.
A and B: n = 4.
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Third, to further validate the involvement of MLCK in this process, the
effect of the specific myosin-Mg2+-ATPase inhibitor
2,3-butadione monoxime (BDM) on Cyto B-induced increase in Caco-2 TJ
permeability was examined (17, 20). After MLCK catalyzed
phosphorylation of MLC, myosin-Mg2+-ATPase hydrolyzes ATP
to generate the energy needed for the actin-myosin contraction
(1, 22, 23). The BDM (20 mM) treatment significantly prevented the Cyto B-induced drop in Caco-2 epithelial resistance (Fig.
8A), further supporting the
involvement of MLCK and actin-myosin interaction in this
process. Finally, the effects of the nonspecific metabolic
inhibitors 2,4-dinitrophenol and sodium azide (which prevent
actin-myosin contraction by depleting metabolic energy) on Cyto
B-induced increase in Caco-2 TJ permeability were examined. Sodium
azide (30 mM) and 2,4-dinitrophenol (1 mM) also significantly prevented
the Cyto B-induced drop in Caco-2 epithelial resistance (Fig. 8,
B and C). In contrast, protein synthesis
inhibitors, cycloheximide (70 µM) and actinomycin D (1 µg/ml) did
not have significant effect on Cyto B-induced drop in Caco-2 epithelial resistance (data not shown).

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Fig. 8.
Effect of myosin-Mg2+-ATPase, metabolic energy, and
protein synthesis inhibitors on Cyto B (5 µg/ml)-induced drop in
Caco-2 epithelial resistance. The effect of 2,3-butadione monoxime
(BDM; 20 mM; A); sodium azide (30 mM; B); and
2,4-dinitrophenol (1.0 mM; C) on Cyto B modulation of Caco-2
epithelial resistance expressed in · cm2.
Values are means ± SE; n = 4.
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Glucose involvement in Cyto B modulation of TJ barrier.
Previous studies (42) have indicated that activation of
glucose transport system results in an MLCK-mediated increase in TJ
permeability. Because Cyto B is known to inhibit facilitative glucose
transport (8, 9), in the following studies the effect of
cytochalasin D (Cyto D) (which has no effect on glucose transport) on
Caco-2 epithelial resistance was examined. Cyto D (10 µg/ml) caused a
progressive drop in epithelial resistance (Fig.
9A). This was also
associated with an increase in MLCK activity. Moreover, ML-7 and BDM
also prevented the Cyto D-induced drop in Caco-2 epithelial resistance
(data not shown).

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Fig. 9.
Effect of Cyto D and Cyto B in glucose-free solution on
Caco-2 epithelial resistance. A: effect of Cyto D (10 µg/ml) on Caco-2 epithelial resistance. B: effect of Cyto
B (5 µg/ml) in glucose-free solution on Caco-2 epithelial resistance.
Values are means ± SE; n = 4.
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Additionally, in separate studies, the Cyto B effect on Caco-2
epithelial resistance was examined in the absence of glucose in the
incubation solution. Cyto B (10 µg/ml), in the absence of glucose in
the incubation solution, produced a similar drop in Caco-2 epithelial
resistance (Fig. 9B). As above, ML-7 and BDM also inhibited
Cyto B-induced decrease in Caco-2 epithelial resistance (data not
shown). These findings indicated that cytochalasin-induced alteration
of Caco-2 TJ barrier function was not dependent on its modulation of
glucose transport.
Role of MLCK in Cyto B modulation of Caco-2 actin filaments.
As described above, Cyto B causes two distinct types of changes in
Caco-2 actin filaments: early (<1 min) fragmentation of actin
filaments and late (15-30 min) actin clump formation (Fig. 1). In
the following studies, the involvement of MLCK in the early and the
late-phase changes in Caco-2 actin filaments was examined. The
pretreatment of Caco-2 monolayers with ML-7 (MLCK inhibitor) did not
affect early (<1 min) Cyto B severing or fragmentation of actins (Fig.
10, A-C). On
the other hand, ML-7 (15 µM) prevented the late-phase (30 min) actin
clump formation and enhanced actin fragment formation (Fig. 10,
E and F), suggesting that MLCK activation is
necessary for the conversion of actin fragments into actin clumps.
Consistent with this, myosin-Mg2+-ATPase inhibitor (BDM)
and metabolic inhibitors also did not affect early phase actin
fragmentation, but prevented late-phase actin clump formation (Figs.
10, D and G, and 3, A and
B).

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Fig. 10.
Effect of MLCK and myosin-Mg2+-ATPase
inhibitors on Cyto B (5 µg/ml) modulation of Caco-2 actin
microfilaments (en face views). A: untreated or control
monolayers. B and E: Caco-2 monolayers treated
with Cyto B for 1 and 30 min, respectively. C and
D: the effect of ML-7 (15 µM) and BDM (20 mM),
respectively, on early (1-min exposure time) Cyto B (5 µg/ml)-induced
actin fragmentation. F and G: the effect of ML-7
and BDM, respectively, on late (30-min exposure time) actin clump
formation (original magnification, ×80).
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Cyto B modulation of Caco-2 myosin filaments.
Because MLCK activation triggers actin-myosin interaction, the effect
of Cyto B-induced MLCK activation on myosin II filaments was examined
by immunofluorescent antibody labeling. In the Caco-2 intestinal
epithelial cells, myosin II filaments were localized in a belt-like
manner near the apical perijunctional areas in the region of zonula
adherens, and mirrored actin microfilament distribution (Fig.
11A). Within 1 min of
exposure to Cyto B (5 µg/ml), perijunctional myosin filaments became
disassembled and displaced from the perijunctional regions, forming a
discrete circular pattern near the cellular borders (Fig.
11B). On longer exposure, displaced myosin filaments
reorganized into larger cytoskeletal clumps near the cellular
periphery, similar to the actin filament distribution (Fig. 11). The
pretreatment of Caco-2 monolayers with ML-7 prevented both early and
late-phase changes in perijunctional myosin filaments (Fig.
12, A and B).
Similarly, BDM (Fig. 12, C and D) and sodium
azide (Fig. 12, E and F) also prevented Cyto B-induced alteration of myosin filaments, suggesting that MLCK activation and myosin-Mg2+-ATPase activity were required
for the Cyto B modulation of myosin filaments.

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Fig. 11.
Time course of Cyto B effect on perijunctional Caco-2 myosin II
filaments. Caco-2 myosin II filaments were labeled with
immunofluorescent antibody labeling technique as described in
MATERIALS AND METHODS. The sequential effect of Cyto B (5 µg/ml) on Caco-2 myosin filaments at time 0 (A)
and 1 (B), 15 (C), and 30 min (D)
(original magnification, ×80).
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Fig. 12.
Effect of MLCK, myosin-Mg2+-ATPase, and
metabolic inhibitors on early and late progression of Cyto B (5 µg/ml) disruption of perijunctional Caco-2 myosin filaments. The
effect of ML-7 (15 µM; A), BDM (20 mM; C), and
sodium azide (30 mM; E) on early (1 min) Cyto B-induced
changes in myosin filaments (see Fig. 10B). The effect of
ML-7 (15 µM; B), BDM (20 mM; D), and sodium
azide (30 mM; F) on late progression (30 min) of Cyto
B-induced changes in myosin filaments (see Fig. 10D)
(original magnification, ×80).
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|
Association between alteration of actin-cytoskeleton and ZO-1 TJ
protein.
The increase in intestinal epithelial TJ permeability is associated
with alteration of TJ structure (11, 26, 27). In the
following studies, the structural correlation between Cyto B-induced
alteration of actin-myosin cytoskeleton and TJ proteins was examined by
immunofluorescent antibody labeling of the ZO-1 proteins. In the
confluent Caco-2 monolayers, ZO-1 proteins were localized at the apical
cellular borders and appeared as a continuous dense band (Fig.
13A). Cyto B (5 µg/ml)
produced a marked disruption of the ZO-1 proteins with a breakage in
the continuity of the ZO-1 band and separation of the ZO-1 proteins
away from the cellular borders (Fig. 13B). The Cyto B
disruption and separation of the ZO-1 proteins from the cellular
periphery visually correlated with the functional increase in Caco-2 TJ
permeability. ML-7, BDM, and sodium azide prevented the Cyto B-induced
disruption of the ZO-1 proteins (Figs. 13,
C-E), suggesting that MLCK activation and
actin-myosin interaction were required for the downstream modulation of
TJ proteins. In contrast, protein synthesis inhibitors (cycloheximide
and actinomycin D) did not affect the Cyto B modulation of ZO-1
proteins (Fig. 13F).

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Fig. 13.
Effect of Cyto B on immunofluorescent localization of Caco-2 ZO-1
proteins (en face views). Caco-2 ZO-1 proteins were labeled with
immunofluorescent antibody labeling technique as described in
MATERIALS AND METHODS. A: untreated or control
Caco-2 monolayer. B: Caco-2 cells treated with Cyto B (5 µg/ml) for 30 min. Caco-2 cells were pretreated with either ML-7 (15 µM; C), BDM (20 mM; D), sodium azide (30 mM;
E), or cycloheximide (70 µM; F) before
treatment with Cyto B (5 µg/ml) for 30 min (original magnification,
×80).
|
|
 |
DISCUSSION |
The cytochalasin disruption of actin microfilaments results in an
increase in intestinal epithelial TJ permeability (29, 33). The major aim of this study was to delineate some of the intracellular mechanisms involved in cytochalasin-induced increase in
intestinal epithelial TJ permeability and also to bridge some of the
gaps in knowledge regarding this issue. Specifically, the role of MLCK
and actin-myosin interaction on Cyto B-induced increase in intestinal
epithelial TJ permeability was investigated.
Our results suggest that Cyto B-induced increase in Caco-2 TJ
permeability is an MLCK-dependent process, requiring MLCK activation. Our studies indicate that inhibition of Cyto B-induced increase in
Caco-2 MLCK activity prevents the increase in Caco-2 TJ permeability. Because actin-myosin interaction is dependent on both MLCK (induces MLC
phosphorylation) and myosin-Mg2+-ATPase (hydrolyzes ATP to
generate energy needed for actin-myosin contraction) activation, the
inhibition of Cyto B-induced increase in Caco-2 TJ permeability by
myosin-Mg2+-ATPase and metabolic inhibitors further
supports the involvement of MLCK pathway in this process. In aggregate,
our findings suggest that Cyto B-induced activation of Caco-2 MLCK
triggers a sequence of intracellular processes including
myosin-Mg2+-ATPase activation and perijunctional
actin-myosin interaction, which culminates in the functional opening of
the Caco-2 TJ barrier.
The Cyto B stimulation of Caco-2 MLCK activity could have resulted from
either an increase in MLCK expression or an increase in the activity of
the preexisting MLCK proteins. Our findings that Cyto B does not affect
Caco-2 MLCK protein level suggest that Cyto B-induced increase in MLCK
activity was due to an increase in activity of preexisting MLCK protein
and not increased expression of MLCK proteins. In this regard, protein
synthesis inhibitors do not prevent Cyto B modulation of actin-myosin
filaments or TJ permeability.
As to the mechanism of cytochalasin action on actin filaments, two
separate processes have been previously described, an
energy-independent and an energy-dependent process. Schliwa
(39) demonstrated that cytochalasin exposure of African
green monkey kidney cells (BS1 cells) produces an immediate severing or
breakage of actin filaments into smaller fragments through an
energy-independent process. Subsequently, severed actin fragments
reorganize to form large cytoskeletal clumps consisting of actin,
myosin, and tropomysin through an energy-dependent process
(39). Similarly, in this study, Cyto B also produced an
energy-independent fragmentation of Caco-2 actin filaments within
the first minute of Cyto B exposure. The metabolic inhibitors appeared
to accentuate the formation of actin fragments (perhaps by inhibiting
the energy-dependent processing of actin fragments). The late-phase
actin clump formation was prevented by metabolic inhibitors, confirming
the requirement of metabolic energy in the cytoskeletal clump formation.
Consistent with this, Madara et al. (33, 35) also reported
that Cyto D exposure of the pig intestinal epithelium for 40 to 60 min
produces a multifocal aggregation of cytoskeletal elements at various
points along the perijunctional area with contraction of the enterocyte
brush border and increase in TJ permeability. The Cyto D-induced
aggregation of cytoskeletal elements and increase in TJ permeability
were also prevented by the metabolic inhibitors (35).
Thus Cyto B disruption of Caco-2 actin appears to occur in 2 stages.
First, Cyto B produces a direct fragmentation of actin filaments
through an energy-independent process. Second, actin fragments are
reorganized into large cytoskeletal clumps through an energy-dependent
process. Our findings indicate that this energy-dependent conversion of
actin fragments into large cytoskeletal clumps is prevented by MLCK and
myosin-Mg2+-ATPase inhibitors, suggesting that MLCK
activation and subsequent myosin-Mg2+-ATPase-induced
actin-myosin interaction is required for this process. Because
actin-myosin contraction is initiated by MLCK activation and
myosin-Mg2+-ATPase activation (1, 23), our
findings support a central role for actin-myosin contraction in the
actin clump formation. Consistent with this, Colemen and Mooseker
(6) previously demonstrated that villin-induced severing
of actin filaments to smaller fragments also stimulates
myosin-Mg2+-ATPase activity.
Our results also indicate a sequential relationship between Cyto B
disruption of actin filaments and alteration of myosin filaments. The
Cyto B fragmentation of actins is associated with a rapid displacement
of myosin filaments from the perijunctional regions. Within the first
minute of Cyto B exposure, there is a rapid disassembly and
displacement of myosin filament, forming a distinct circular pattern
near the cellular borders. Subsequently, myosin filaments coalesce into
large cytoskeletal clumps correlating with changes in actin filaments.
In contrast to actins, both the early phase (<1 min) and the
late-phase changes in the myosin filaments were inhibited by MLCK and
myosin-Mg2+-ATPase inhibitors, indicating that the early
changes in myosin filaments were also dependent on MLCK activation.
Because actin fragmentation results from a primary action of Cyto B and
myosin alteration results as a secondary response to actin disruption, our findings suggest that actin fragmentation (the primary event) is
responsible for the MLCK activation. The MLCK activation then presumably leads to the disassembly and displacement of the myosins (secondary response). In aggregate, these findings suggest that Cyto
B-induced actin fragmentation produces Caco-2 MLCK activation, which in
turn triggers actin-myosin interaction, leading to the displacement of
the perijunctional myosin filaments from the cellular borders.
The Cyto B modulation of actin and myosin filaments was also associated
with the morphological disruption of ZO-1 proteins, correlating with
the functional increase in TJ permeability. The Cyto B disruption of
ZO-1 proteins was prevented by MLCK, myosin-Mg2+-ATPase and
metabolic inhibitors, indicating that the downstream alteration of ZO-1
proteins is dependent on MLCK activation and actin-myosin interaction.
These findings demonstrate a causal relationship between Cyto B
activation of Caco-2 MLCK and subsequent modulation of the Caco-2 TJ
proteins and the TJ barrier function.
As for the role of ZO-1 proteins in TJ barrier function, ZO-1 proteins
have been previously proposed as a possible candidate protein linking
TJs to the perijunctional cytoskeletal elements (2, 7,
10). In support of such a role, ZO-1 proteins have been shown to
directly bind to actin filaments and to the transmembrane TJ protein
occludin (10, 13, 21). ZO-1 proteins are a member of the
membrane-associated guanylate kinase family (2, 41, 43).
The members of this protein family are present on the cytoplasmic surface of specialized cell-to-cell contact and are involved in signal
transduction and cytoskeletal organization (21, 43). Therefore, the proposed role of ZO-1 as an intermediary protein linking
TJs to the cellular cytoskeleton is consistent with the known functions
of this family of proteins (2, 7, 10). Our data, showing
that Cyto B alteration of ZO-1 protein is linked to MLCK activation and
actin-myosin interaction, support such a proposal.
In conclusion, our results provide new insight into the mechanism of
Cyto B modulation of intestinal epithelial TJ barrier. Our results
indicate that Cyto B-induced increase in Caco-2 intestinal epithelial
TJ permeability is mediated by MLCK activation. It appears that Cyto
B-induced MLCK activation triggers the perijunctional actin-myosin
interaction leading to the downstream modulation of TJ proteins and
barrier function.
 |
ACKNOWLEDGEMENTS |
This study was supported by Veterans Affairs Merit Review and
Minority Initiative grants from the Department of Veterans Affairs (T. Y. Ma).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: T. Y. Ma, Gastroenterology Section, Dept. of Veterans Affairs Medical Center, 5901 E. Seventh St., Long Beach, CA 90822 (E-mail:
tyma{at}uci.edu).
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.
Received 11 August 1999; accepted in final form 1 May 2000.
 |
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