www.elsevier.com/locate/yexcr
Experimental Cell Research 298 (2004) 1–8
Connexin 26-mediated gap junctional intercellular communication
suppresses paracellular permeability of human intestinal epithelial
cell monolayers
Hidekazu Morita, Tatsuro Katsuno,* Aihiro Hoshimoto, Noriaki Hirano,Yasushi Saito, and Yasuo Suzuki
Clinical Cell Biology (F5), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
Received 10 November 2003, revised version received 16 March 2004
Available online 6 May 2004
Abstract
In some cell types, gap junctional intercellular communication (GJIC) is associated with tight junctions. The present study was performed
to determine the roles of GJIC in regulation of the barrier function of tight junctions. Caco-2 human colonic cells were used as a monolayer
model, and barrier function was monitored by measuring mannitol permeability and transepithelial electrical resistance (TER). The
monolayers were chemically disrupted by treatment with oleic acid and taurocholic acid. Western blotting analyses were performed to
evaluate the protein levels of connexins, which are components of gap junctional intercellular channels. Cx26 expression was detected in
preconfluent Caco-2 cells, and its level increased gradually after the monolayer reached confluency. These results prompted us to examine
whether overexpression of Cx26 affects barrier function. Monolayers of Caco-2 cells stably expressing Cx26 showed significantly lower
mannitol permeability and higher TER than mock transfectants when the monolayers were chemically disrupted. The levels of claudin-4, an
important component of tight junctions, were significantly increased in the stable Cx26 transfectant. These results suggest that Cx26-
mediated GJIC may play a crucial role in enhancing the barrier function of Caco-2 cell monolayers.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Gap junctional intercellular communication (GJIC); Tight junctions; Caco-2 cells; Paracellular permeability; Connexin 26; Claudin-4
Introduction
The intestinal epithelium performs an important barrier
function, selectively restricting the permeation of ions and
nonelectrolytes. It also prevents macromolecules from
accessing the internal milieu as well as losing cells and
extracellular proteins into the intestinal lumen. Macromo-
lecules have been reported to permeate the intestinal
epithelium mainly via the paracellular pathway regulated
by intercellular tight junctions between adjacent cells [1–
3]. Previous studies in experimental animals and clinical
studies of human disease have demonstrated an association
0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2004.03.046
* Corresponding author. Division of Clinical Cell Biology (F5),
Department of Internal Medicine, Graduate School of Medicine, Chiba
University, 1-8-1 Inohana, Chuo-Ward, Chiba 260-8670, Japan. Fax: +81-
43-226-2095.
E-mail address: [email protected] (T. Katsuno).
between increased epithelial paracellular permeability and
intestinal mucosal inflammation [4–8]. Although tight
junctions have been shown to consist of at least a dozen
molecular species, including occludin, claudins, cingulin,
ZO-1, ZO-2, ZO-3 etc., that extend from their lips to the
cytoskeleton [9,10], the mechanism by which the perme-
ability of tight junctions is regulated has yet to be fully
elucidated [11–18].
Gap junctional intercellular communication (GJIC)
channels allow rapid exchange of ions and metabolites
up to approximately 1 kDa in size, including second
messengers such as cyclic AMP, IP3, and Ca2+ between
adjacent cells. Gap junctions are plasma membrane spa-
tial microdomains constructed of assemblies of channel
proteins called connexins. Approximately 20 types of
connexins have been identified in the human and mouse
genomes. Most cell types express multiple connexin
isoforms providing a structural basis for the charge and
size selectivity of these intercellular channels. However,
H. Morita et al. / Experimental Cell Research 298 (2004) 1–82
the precise nature of the GJIC channel remains unclear
[19,20].
Tight junction strands as well as the integral tight
junction proteins have been shown to be induced in Cx32-
transfected hepatocytes [21,22]. In fibroblasts and cardiac
myocytes, Cx43 was shown to interact with ZO-1 [23,24].
In the present study, we examined the roles of GJIC in the
regulation of protein expression and the function of tight
junctions using human intestinal epithelial cells (Caco-2
cells) overexpressing human Cx26 protein. Our results
indicated that GJIC regulates claudin-4 protein expression
and the paracellular permeability of Caco-2 human intestinal
epithelial cells.
Fig. 1. Western blotting analysis for Cx26 and h-actin in Caco-2 cells. Cell
lysates were prepared from parental Caco-2 cells on days �1, 0, 3, and 6
post-confluency. Western blotting analysis was performed using anti-Cx26
and anti-h-actin antibodies. Data shown are from one representative of five
independent experiments.
Materials and methods
Cell culture
Caco-2 cells were cultured at 37jC in an atmosphere of
5% CO2/95% air. The cells were maintained in DMEM with
4.5 g/l glucose, 2 mM L-glutamine, 50 units/ml penicillin,
50 Ag/ml streptomycin, 10 mM HEPES, 1% essential and
nonessential amino acids, and 15% FBS, unless otherwise
indicated.
Western blotting
Parental and transfected Caco-2 cells were lysed by
boiling in PBS and 1% SDS containing 100 Ag/ml
phenylmethylsulfonyl fluoride and 1 mM sodium ortho-
vanadate at the indicated times after plating. Proteins
were assayed with bicinchoninic acid. Aliquots of 20 Agof proteins were separated by SDS-PAGE (10–20%
resolving gels) and electroblotted onto nitrocellulose
membranes (NENk Life Science Products, Inc., Boston,
MA). The membranes were saturated for 30 min at room
temperature with blocking buffer (0.6% Tween 20, 1%
bovine serum albumin (BSA), 10% skimmed milk) and
incubated with anti-Cx26, anti-Cx32, anti-claudin-1 (Cat
#71-7800), anti-claudin-4, anti-occludin (Zymed Labora-
tories, Inc., San Francisco, CA), anti-Cx45 (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), or anti-h actin
(Sigma-Aldrich Corp., St. Louis, MO) antibodies at room
temperature overnight. The membranes were incubated
with horseradish peroxidase (HRP)-conjugated anti-rabbit
or mouse IgG (Vector Laboratories, Burlingame, CA) at
room temperature for 45 min, and detection was carried
out using an enhanced chemiluminescence (ECL) West-
ern blotting system (Bio-Rad Laboratories, Hercules,
CA).
RNA isolation and reverse transcription-PCR (RT-PCR)
RT-PCR was performed on total RNA extracted from the
cells using RNAzol (Tel-Test Inc., Friendswood, TX).
Aliquots of 1 Ag of total RNA were reverse-transcribed
into cDNA using a mixture of oligo(dT) and AMV reverse
transcriptase under the recommended conditions. cDNA
synthesis was performed in a total volume of 20 Al for 20min at 50jC and terminated by incubation for 5 min at
99jC. PCR was performed in mixtures containing 20 pM
of the appropriate primer pair and 2.5 U of Taq DNA
polymerase (Takara Bio Inc., Tokyo, Japan). Reaction
mixtures (total volume, 100 Al) were subjected to 40 cycles
of PCR, with a profile of 30 s at 94jC, 30 s at 58jC, and60 s at 72jC with a final elongation step of 7 min at 72jC,using a GeneAmp PCR system 9700 (Applied Biosystems,
Foster City, CA). Aliquots of 3 Al of the PCR products
were analyzed by 1% agarose gel electrophoresis with
staining with ethidium bromide. The primers used to detect
Cx26 by RT-PCR were sense, 5V-CCG CCC AGA GTA
GAA GA-3V; and anti-sense, 5V-CGG GTT GCC TCA TCC-
3V.
cDNA construction and transfection
Human Cx26 cDNA [51] was subcloned into the
HindIII–XbaI restriction sites of the expression vector
pcDNA3.1. For transient transfection, parental cells were
transfected with 4 Ag of Cx26 cDNA using Lipofect-
AMINE (Invitrogen Corp., Carlsbad, CA). After 5 h of
incubation, the cells were transferred to DMEM containing
15% FBS. All cell cultures were maintained for 48 h after
transfection and then examined by immunocytochemistry.
For stable transfection, parental cells were transfected with
4 Ag of Cx26 cDNA using LipofectAMINE. After 48 h,
the cells were transferred to selection medium containing
400 Ag/dl G418 (Sigma). When surviving colonies had
grown sufficiently to allow detection visually, they were
individually picked and propagated separately. Following
initial screening of 20 clones for Cx26 by Western
blotting, we chose one clone (termed #1) for further
analysis.
Immunofluorescence microscopy
Cells grown on glass coverslips were fixed with cold
50% acetone/50% ethanol for 5 min. Immunocytochemis-
Fig. 2. Western blotting analysis for Cx26 and h-actin in mock-
transfected cells (A) and the stable Cx26 transfectant (B). Cx26 cDNA,
obtained by RT-PCR on Caco-2 cells, was subcloned into the expression
vector pcDNA3.1. Then, parental Caco-2 cells were stably transfected
with Cx26 cDNA. Mock transfectants received only the expression
vector pcDNA3.1. Cell lysates were prepared from the stable Cx26
transfectant and mock transfectant at 3 days post-confluency. Western
blotting analysis was performed using anti-Cx26 and anti-h-actinantibodies. Data shown are from one representative of five independent
experiments.
H. Morita et al. / Experimental Cell Research 298 (2004) 1–8 3
try was performed with polyclonal anti-Cx26 and anti-
claudin-4 (Zymed). They were visualized using Alexia 488
(green)-conjugated anti-rabbit IgG (Molecular Probes Inc.,
Eugene, OR). The specimens were examined with an
Axiovision fluorescence microscope (Carl Zeiss, Oberko-
chen, Germany).
Models of epithelial monolayer injury and determination of
monolayer barrier function
Approximately 105 cells (mock or stable Cx26 cDNA
transfectants) were seeded per well in the presence of
100 Al media in the apical compartment of a Transwell
apparatus (Corning Life Sciences, Corning, NY) made
Fig. 3. Immunocytochemistry for Cx26 in the mock cells (A) and the stable Cx
3 days post-confluency and reacted with anti-Cx26 antibody. Cx26 was visuali
methods. The specimens were examined with a fluorescence microscope
experiments.
of polycarbonate with 6.5 mm wells and a pore size of
5.0 Am, with 600 Al of medium in the basal compart-
ment. Twenty-four hours before addition of agents that
damage the monolayer, 100 Al of 10�6 M 18a-glycyr-
rhetinic acid (AGA) (ICN Biomedicals Inc., Costa Mesa,
CA), which interferes with gap junctional intercellular
communication (GJIC), was added to the apical well
[25–28]. Subsequently, solutions (100 Al/well) contain-
ing 3 � 10�3 M oleic acid plus 4.5 � 10�3 M
taurocholic acid, which causes monolayer damage as
reported previously [29–31], were added to the apical
compartment and the monolayers were incubated at
37jC for 60 min.
Determination of [3H]-mannitol flux through the
monolayers was assessed by monitoring the ability to
prevent penetration of the inert compound mannitol into
the basal compartment after addition to the apical com-
partment. After incubation with agents that damage the
monolayer, the medium in the apical compartment was
removed and replaced with 250 Al of fresh medium
supplemented with 125 Al of medium containing D-
[1-3H]-mannitol (Daiichi Pharmaceuticals, Tokyo, Japan).
Incubation was then continued at 37jC on a rotary
shaker at 30 rpm for 4 h. Subsequently, samples were
obtained from the basal compartments, added to scintil-
lation fluid, and the amount of [3H]-mannitol was deter-
mined. Inert probe penetration (permeability) was
expressed as the total content of [3H]-mannitol in the
basal compartment divided by that present in the 125
Al of supplemented medium added.
Transepithelial electrical resistance (TER) was measured
using a Millipore electrical resistance system with or with-
out agents that damage the monolayer. Two Transwell
chambers were left blank to determine the intrinsic resis-
tance of the membrane. Final values were obtained by
subtracting the mean blank value and the results are
expressed as V cm2.
26 transfectant (B). Cells were fixed in cold 50% acetone/50% ethanol at
zed using Alexia 488-conjugated antibody as described in Materials and
(�40). Data shown are from one representative of five independent
Fig. 4. (a) Effect of stable expression of Cx26 on the paracellular
permeability of Caco-2 monolayers. Paracellular permeability of the
monolayer was evaluated as the ratio of the concentration of the mannitol
marker in the basal compartment to that in the apical compartment. (A)
Mock transfectant (no treatment). (B) Stable Cx26 transfectant (no
treatment). (C) Mock transfectant (chemically disrupted by treatment with
3 � 10�3 M oleic acid plus 4.5 � 10�3 M taurocholic acid). (D) Stable
Cx26 transfectant (chemically disrupted). (E) Stable Cx26 transfectant
(chemically disrupted in the presence of 18a-glycyrrhetinic acid, an agent
that interferes with GJIC). Data are expressed as means F SEM, n = 3 for
each time point and group. *P < 0.05 compared with the stable Cx26
transfectant monolayer (chemically disrupted). (b) Effect of stable
expression of Cx26 on the transepithelial electrical resistance (TER) of
Caco-2 monolayers. Values of TER were obtained by subtracting the blank
value and the results are expressed as V cm2. (A) Mock transfectant (no
treatment). (B) Stable Cx26 transfectant (no treatment). (C) Mock
transfectant (chemically disrupted by treatment with 3 � 10�3 M oleic
acid plus 4.5 � 10�3 M taurocholic acid). (D) Stable Cx26 transfectant
(chemically disrupted). (E) Stable Cx26 transfectant (chemically disrupted
in the presence of 18a-glycyrrhetinic acid, an agent that interferes with
GJIC). Data are expressed as means F SEM, n = 3 for each time point and
group. *P < 0.05 compared with the stable Cx26 transfectant monolayer
(chemically disrupted).
Fig. 5. Effect of stable expression of Cx26 on the levels of tight junction
proteins. Cell lysates were prepared from mock transfectant (A), the stable
Cx26 transfectant (B), and the stable Cx26 transfectant treated with 18a-
glycyrrhetinic acid (C) at 3 days post-confluency. Western blotting analyses
were performed for claudin-1, claudin-4, occludin, and h-actin. (a) shows theresults of one representative of three independent experiments. (b) shows the
relative levels of tight junction proteins quantified using NIH Image 1.55.
Data are expressed as means F SEM, n = 3 for each time point and group.
*P < 0.05 compared with stable transfectant monolayer without 18a-
glycyrrhetinic acid treatment.
H. Morita et al. / Experimental Cell Research 298 (2004) 1–84
Data and statistical analysis
Signals were quantified using NIH Image 1.55 (Wayne
Rasband, NIH, Bethesda, MD). All data are expressed as
means F SD. Differences between groups were analyzed by
unpaired Student’s t test. A P value less than 0.05 was
considered statistically significant.
Results
Expression of connexins in parental Caco-2 cells
Western blotting analysis was performed on Caco-2
cells to evaluate the levels of expression of gap junction-
H. Morita et al. / Experimental Cell Research 298 (2004) 1–8 5
associated proteins, such as Cx26, Cx32, and Cx45, as
these molecules have been reported to be expressed in
the intestine [32]. As shown in Fig. 1, expression of
Cx26 protein was observed in parental Caco-2 cells. No
expression of Cx32 or Cx45 was observed in either
preconfluent or confluent Caco-2 cells (data not shown).
These findings prompted us to investigate the role of
Cx26 in Caco-2 cells. Interestingly, the amount of Cx26
increased gradually during after the cells reached conflu-
ency. Based on these observations, we carried out the
following experiments using Caco-2 cells on day 3 post-
confluency [33].
Expression of Cx26 in stable Cx26 transfectants
To further investigate our hypothesis that there is a
linkage between Cx26 and paracellular permeability, we
stably transfected Caco-2 cells with the gene encoding
Cx26. Briefly, Cx26 cDNA, obtained from Caco-2 cells
by RT-PCR, was subcloned into the expression vector
pcDNA3.1. Then, the parental Caco-2 cells were stably
transfected with Cx26 cDNA. One clone (termed #1) that
expressed the highest level of Cx26 was selected as de-
scribed in Materials and methods and used for further
analysis. As shown in Fig. 2, the level of Cx26 protein
expression in clone #1 was significantly increased as com-
pared with mock-transfected controls (Fig. 2).
Localization of Cx26 in stable Cx26 transfectants
Fluorescent immunocytochemistry was carried out to
examine the localization of Cx26 in the stable Cx26 trans-
fectant on day 3 post-confluency as compared to the mock-
transfected controls. Staining for Cx26 showed a linear
distribution at regions of cell–cell adhesion in both the
stable Cx26 transfectant and the mock-transfected cells (Fig.
3). This finding suggested that the exogenous Cx26 protein
Fig. 6. Immunocytochemistry for claudin-4 in mock cells (A) and the stable Cx26 t
post-confluency and reacted with anti-claudin-4 antibody. Claudin-4 was visual
methods. The specimens were examined with a fluorescence microscope (�40).
was translocated to the cell borders in the same way as
endogenous Cx26 protein.
Effects of stable expression of Cx26 on mannitol flux of the
Caco-2 monolayer
To evaluate whether the overexpression of Cx26 in Caco-
2 cells resulted in enhanced tight junction barrier function,
mannitol flux was measured with or without chemical
disruption by treatment with oleic acid and taurocholic acid
[29–31]. Without chemical disruption, the monolayers of
both mock transfectants and the Cx26 transfectant showed
the lowest level of mannitol flux (1.4 F 0.0% and 1.6 F0.0%, respectively). When the barrier function was chemi-
cally disrupted, the monolayer of Caco-2 cells stably
expressing Cx26 showed significantly lower mannitol flux
on day 3 post-confluency (6.1 F 2.3%) than the mock
transfectant (37.9 F 2.1%). However, mannitol flux was
markedly increased in the presence of 10�6 M AGA (28.0F0.4%) (Fig. 4a), an agent that interferes with GJIC without
influencing protein translation [25–28].
Effects of stable expression of Cx26 on transepithelial
electrical resistance (TER) of the Caco-2 monolayer
TER was measured with or without chemical disruption
by treatment with oleic acid and taurocholic acid to evaluate
whether the overexpression of Cx26 in Caco-2 cells resulted
in enhanced tight junction barrier function. Without chem-
ical disruption, the monolayers of the Cx26 transfectant
showed significantly higher TER than the mock transfec-
tants (703 F 24 and 592 F 2 V cm2, respectively). When
the barrier function was chemically disrupted, the monolay-
er of Caco-2 cells stably expressing Cx26 showed markedly
higher TER (308 F 13 V cm2) than the mock transfectant
(126 F 6 V cm2). However, TER was decreased in the
presence of 10�6 M AGA (104 F 3 V cm2) (Fig. 4b).
ransfectant (B). Cells were fixed in cold 50% acetone/50% ethanol at 3 days
ized using Alexia 488-conjugated antibody as described in Materials and
Data shown are from one representative of five independent experiments.
tal Cell Research 298 (2004) 1–8
Effect of stable expression of Cx26 on the levels of tight
junction proteins
To determine whether Cx26 transfection altered the
levels of tight junction proteins, Western blotting analyses
were performed for claudin-1, claudin-4, and occludin in
mock-transfected control cells and stable Cx26 transfectant.
The level of expression of claudin-4 protein in the stable
Cx26 transfectant was significantly increased compared to
that in control cells. Nevertheless, when 10�6 M AGA was
added to the stable Cx26 transfectant, the level of claudin-4
protein expression was decreased (Fig. 5). Levels of expres-
sion of claudin-1 and occludin proteins were similar in both
control cells and the stable Cx26 transfectant.
Localization of claudin-4 in stable Cx26 transfectants
Fluorescent immunocytochemistry was carried out to
examine the localization of claudin-4 in the stable Cx26
transfectant on day 3 post-confluency as compared to the
mock-transfected controls. Staining for claudin-4 showed a
linear distribution at regions of cell–cell adhesion in both
the stable Cx26 transfectant and the mock-transfected cells
(Fig. 6). This finding suggested that claudin-4 protein in the
stable Cx26 transfectants was located at the cell borders in
the same way as that in the mock-transfected controls.
H. Morita et al. / Experimen6
Discussion
Our results demonstrated that gap junctional intercellular
communication (GJIC) regulates the paracellular permeabil-
ity of intestinal epithelial cells. To examine the roles of
GJIC in regulating tight junction protein expression and
function, we transfected the Cx26 gene into Caco-2 human
intestinal epithelial cells. Chemically disrupted cell mono-
layers of Cx26 transfectants showed lower paracellular
permeability accompanying the upregulation of claudin-4,
one of the critical elements of tight junctions in human
intestinal epithelial cells. These results suggested that Cx26
transfection induces upregulation of claudin-4, which sup-
presses tight junctional permeability in human intestinal
epithelial cell monolayers.
We first determined which connexins are expressed in
Caco-2 human intestinal epithelial cells. Western blotting
indicated that Cx26 protein was expressed in Caco-2 cells,
as reported previously [32]. This finding prompted us to
transfect Caco-2 cells with the gene encoding Cx26. All of
the experiments in this study were performed using Caco-2
cells on day 3 post-confluency because the level of expres-
sion increased gradually during after the cells reached
confluency. Immunofluorescent staining revealed identical
Cx26 protein distribution at the cell periphery in both the
parental cells and the Cx26 gene-transfected Caco-2 cells,
although Western blotting showed that the level of Cx26
protein in the Cx26-transfected Caco-2 cells was signifi-
cantly higher than that in parental cells. This implies that
both endogenous and exogenous Cx26 proteins have the
same properties and are translocated identically in Caco-2
cells.
Monolayers of Cx26-transfected Caco-2 cells without
chemical disruption showed higher TER than mock-trans-
fected cells, whereas both types of cell monolayer showed
the lowest mannitol flux without such treatment. These
results indicated that overexpression of Cx26 lowered the
electrical conductance for ions across the paracellular path-
way without influencing the flux for uncharged solutes.
These findings were in line with recent studies showing
dissociation between changes in the barrier properties of
TER and mannitol flux [2,34,35]. However, it was impos-
sible to estimate in the present study that overexpression of
Cx26 on the monolayer lowers mannitol flux because
monolayers of the mock cells already showed the lowest
mannitol flux without chemical disruption.
Therefore, we subsequently treated both types of cell
monolayer with oleic acid and taurocholic acid, which
increase paracellular permeability through a Ca2+-dependent
tight junction mechanism [29–31,36]. Interestingly, mono-
layers of Cx26-transfected Caco-2 cells with chemical
disruption showed significantly higher TER and lower
mannitol flux than mock-transfected cells treated in the
same way. This result indicated that overexpression of
Cx26 has a protective effect against chemical injury of
intestinal epithelial cells. There are two possible mecha-
nisms responsible for the protective effect of Cx26 trans-
fection: Cx26 protein may directly strengthen the tight
junctions, or overexpression of Cx26 protein may enhance
GJIC, which indirectly decreases paracellular permeability.
To evaluate these hypotheses, we utilized 18a-glycyrrhe-
tinic acid (AGA), which interferes with GJIC without
influencing protein translation [25–28]. Treatment of the
Cx26-tansfected Caco-2 cell monolayer with AGA resulted
in an increase in paracellular permeability, suggesting that
reduced paracellular permeability in the Cx26 transfectant
was mainly due to enhanced GJIC.
To determine the mechanism underlying the decreased
paracellular permeability in the Cx26-transfected Caco-2
cell monolayer, we then evaluated the levels of proteins
that comprise tight junctions in these cells because they
directly control paracellular permeability. We found that
claudin-4, but not occludin, was upregulated in Cx26-trans-
fected Caco-2 cells as compared to mock-transfected control
cells. This was consistent with the results of previous studies
demonstrating that claudin, rather than occludin, is critical
in the formation of tight junction strands and maintenance of
paracellular permeability [37,38]. More than 20 members of
the claudin family have been identified and different types
of claudins are expressed in different tissues. Intriguingly,
our results showed that claudin-4, but not claudin-1, was
upregulated in Cx26-transfected Caco-2 cells decreasing
paracellular permeability, indicating that claudin-4 rather
than claudin-1 plays a critical role in the maintenance of
H. Morita et al. / Experimental Cell Research 298 (2004) 1–8 7
paracellular permeability in Caco-2 human intestinal epi-
thelial cells.
Intracellular messengers and extracellular stimuli have
been found to regulate tight junctions and affect paracellular
permeability [11–13]. Activation of protein kinase C
(PKC), an intracellular messenger, may increase the perme-
ability for large molecules, whereas activation of protein
kinase A (PKA), which induces intracellular cAMP levels,
may increase the ionic conductance of tight junctions
without changing the barrier function for large molecules.
Extracellular stimuli, such as cytokines and leukocytes, also
regulate tight junction structure and paracellular permeabil-
ity by influencing tight junctional proteins and the underly-
ing actin cytoskeleton. These findings suggested that Cx-26-
mediated GJIC influences intracellular messengers that
regulate expression of claudin-4 proteins. Further studies
to identify regulators of tight junction proteins that are
important for paracellular permeability may enable the
determination of how GJIC interacts with the expression
of tight junction proteins. Resolution of this question will
provide important new insight into the mechanism of
interaction between gap junctions and tight junctions.
In conclusion, we showed here that Cx-26-mediated
GJIC suppresses paracellular permeability by upregulating
claudin-4 protein levels in human intestinal epithelial cells.
The results of this study indicated that Cx-26-mediated
GJIC strengthens the barrier function of intestinal epithelial
cell monolayers.
Acknowledgment
This study was supported by a grant from Nippon-
Shinyaku Co., Ltd. (Kyoto, Japan).
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