Claudin-18: A Dominant Tight Junction Protein in Barrett’ s Esophagus and Likely
Contributor to its Acid Resistance
Biljana Jovov1, Christina M. Van Itallie1, Nicholas J. Shaheen1, Johnny L. Carson2,3,
Todd M. Gambling3, James M. Anderson4and Roy C. Orlando1
1Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
2The Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599, USA
3Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
4Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill,
NC 27599, USA
Running Heads: Claudin-18 and Barrett’ s esophagus
Biljana Jovov, M.D.
Department of Medicine,
University of North Carolina at Chapel Hill
Ph: 919 843-4598
Fax: 919 966-6413
E mail: email@example.com
Page 1 of 33
Articles in PresS. Am J Physiol Gastrointest Liver Physiol (October 11, 2007). doi:10.1152/ajpgi.00158.2007
Copyright © 2007 by the American Physiological Society.
Barrett's esophagus (BE) is a specialized columnar epithelium (SCE) that
develops as replacement for damaged squamous epithelium (SqE) in subjects with reflux
disease, and as such it is apparently more acid resistant than SqE. How SCE resists acid
injury is poorly understood; but one means may involve altered tight junctions (TJs) since
the TJ in SqE is an early target of attack and damage by acid in reflux disease. To assess
this possibility, quantitative RT-PCR for 21 claudins was performed on endoscopic
biopsies on SCE of BE and from healthy SqE from subjects without esophageal disease.
In SCE, Cldn-18 was the most highly expressed at the mRNA level and this finding
paralleled by marked elevation in protein expression on immunoblots. In contrast in SqE,
Cldn-18 was minimally expressed at the mRNA level and undetectable at the protein
level. Immunofluorescence studies showed membrane localization of Cldn-18 and co-
localization with the TJ protein, ZO-1. When Cldn-18 was overexpressed in MDCK II
cells and mounted as monolayers in Ussing chambers, it raised electrical resistance and,
as shown by lower dilution potentials to a NaCl gradient and lower diffusion potentials to
acidic gradients, selectively reduced paracellular permeability to both Na+and H+
compared to parental MDCK cells. We conclude that Cldn-18 is the dominant claudin in
the TJ of SCE and propose that the change from a Cldn-18 deficient TJ in SqE to a Cldn-
18 rich TJ in SCE contributes to the greater acid resistance of BE.
Claudin profiling; quantitative RT-PCR; paracellular permeability; stratified squamous
Page 2 of 33
Barrett's esophagus (BE) is defined as the presence of a metaplastic specialized
columnar epithelium (SCE) within the tubular esophagus. It is readily identified on
endoscopy by its reddish appearance macroscopically compared to the paler esophageal
stratified squamous epithelium (SqE). The diagnosis of BE is confirmed on esophageal
biopsy by the presence of goblet cells that stain positive with Alcian blue at pH 2.5 for
acidic mucins (29). Biochemically BE is characterized by abundant expression of the
intestinal protein villin and mucin 2 (20, 21, 31). The metaplastic SCE of BE arises in the
setting of gastroesophageal reflux disease (GERD) likely as replacement for acid-
damaged SqE, and as such, SCE is more acid resistant than SqE and represents a form of
‘ adaptive protection’ against a hostile luminal environment (28). Clinical observations
that support this concept include the fact that BE may be found in asymptomatic subjects
without treatment for GERD and when stressed by esophageal acid perfusion as part of
the Bernstein test experience either no symptoms or symptoms far less severe than those
with GERD without BE (12, 17). In addition, BE is clinically stable for long periods of
time, and this is the case irrespective of type or effectiveness of anti-reflux therapy (7,
What contributes to the relative acid resistance of SCE in BE compared with
native SqE remains unknown. However, candidate functions include the ability of SCE to
secrete from its surface cells both mucins that form a viscoelastic surface layer (8) and
bicarbonate that forms a more effective lumen-to-surface buffer zone for neutralization of
hydrogen ions (H+) (1, 38). Another possible candidate may be the structure and function
of the tight junction (TJ) in SCE since evidence suggests that in GERD the TJ of SqE is
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an early target for attack and damage by luminal acid. The result of this attack on SqE is
an increase in paracellular permeability and the development of a lesion known as
‘ dilated intercellular spaces’ . Indeed dilated intercellular spaces in SqE are now
recognized as an early and important feature of both the erosive and non-erosive forms of
GERD (3, 4, 36, 40, 46). Since the TJs in SqE appear vulnerable to acid damage and the
SCE of BE relatively more resistant to such injury, we hypothesized that the TJ of SCE
may account, at least in part, to the greater acid resistance of BE.
The permeability of ions and uncharged molecules across the TJ are highly
dependent upon the nature of its bridging proteins – and these proteins are predominantly
members of the multigene family known as the claudins. Consequently, we initially
approached the hypothesis posed that the TJ of SCE is more acid resistant than that of
SqE by performing a claudin gene expression profile on SCE in BE and comparing it to
that of healthy SqE from subjects without esophageal disease. Not surprisingly, given the
differences in phenotype, the profile for SCE differed dramatically from that of SqE.
What was surprising, however, was that the claudin profile of SCE was quantitatively
dominated by a qualitatively unique claudin - Claudin-18 (Cldn-18). Therefore, we
performed immunoblots and immunofluorescence microscopy (IF) to identify and
characterize the distribution of Cldn-18 protein in SCE and overexpressed Cldn-18 in
MDCK II cells to assess in Ussing chambers its effect on TJ function.
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Material and Methods
Two to three esophageal biopsies were obtained using large cup biopsy forceps
from 20 adult subjects, ages 18-75 years old, with either non-dysplastic BE or history of
BE with transient low grade dysplasia. These subjects were undergoing upper endoscopic
surveillance for cancer. Similarly healthy human SqE was obtained using large cup
biopsy forceps from 12 subjects being endoscoped for clinical reasons. These subjects
had no history of reflux symptoms or esophageal disease and a grossly-normal esophagus
on endoscopy. Patients gave written informed consent prior to the procedure, and the
study was approved by the Human Research Ethics Committee of UNC Chapel Hill.
Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)
qRT-PCR was performed on endoscopic biopsies using previously described
methods (15). In brief biopsies underwent total RNA isolation using RNeasy kits
(Qiagen, Inc., Valencia, CA) per the manufacturer’ s recommended protocols. RNA was
treated with TURBO DNase (TURBO DNA-free kit, Ambion, Inc., Austin, TX) to
remove contamination by genomic DNA. cDNA was synthesized from 2.5 µg of treated
RNA for each tissue sample using Superscript III reverse transcriptase (Invitrogen
Corporation, Carlsbad, CA) with an equal amount of RNA included in a No-RT control
for each separate RNA sample. Real-time PCR primers used in this study were validated
primer sets (QuantiTect Primers Assays, Qiagen, Inc., Valencia, CA). Primer-specific
details such as assay location, transcript detected, ensemble transcript ID and amplicon
length can be found at www.qiagen.com/GeneGlobe. Real-time PCR was performed
with 1:25 dilutions of the cDNA (in triplicate) with and No-RT control for each sample
Page 5 of 33
and as well as no template reaction controls. Reactions consisted of SYBR Green
JumpStartTaq ReadyMix for Quantitative PCR (Sigma– Aldrich Co., St. Louis, MO), pre-
made primers, and 5 µL of sample (cDNA or control). Amplification was performed in a
Rotor-Gene 3000 (Corbett Research, Mortlake, Australia) thermal cycler at 95°C for
3 min followed by 37 cycles of 94°C for 15 seconds, 54°C for 20 seconds, and 72°C for
25 seconds. Following amplification, a melting curve analysis was performed by heating
the reactions from 50 to 99°C in 0.2°C intervals while monitoring fluorescence. The
cycle at which each sample crossed a fluorescence threshold, Ct, was determined and
triplicate values for each cDNA averaged.
Eef1a1 served as a control gene for normalization between samples. It was
included in each cycling run and demonstrated consistency among runs (14). ZO-1
showed a constant relationship to Eef1a1 in all samples and was also considered a non-
changing control. To simplify reporting, gene expression was normalized to ZO-1
expression by calculating a ∆Ct= (Ctof ZO-1 − Ctof gene). Relative expression values
were calculated as 2 ∆Ct, setting the expression value of ZO-1 to 1.0. Experimental error
was estimated for each gene in each tissue by comparing the coefficient variation (CV) of
the average Ctvalue for four samples, error = [(2%CV)/100] × [relative expression value].
If a sample’ s signal did not rise above threshold within 37 cycles, it was considered not
detectable (ND). Amplification efficiency for each individual reaction was monitored by
the Rotor-Gene software (v.5) comparative quantification function. Ctvalues were not
adjusted for differences in amplification efficiencies, because efficiencies were
consistently close to 1.9 for all reactions (2.00 is the value of a theoretically 100%
efficient reaction i.e., doubling each cycle). Determination of expression for villin and
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mucin 2 served as biochemical markers to confirm the presence of SCE in biopsies from
subjects with BE (20, 21, 31).
Immunofluorescence (IF) and immunoblotting
Methods for indirect IF and digital image processing have been described before
(19, 30). Immuno-labeled fluorescent MDCK cells were imaged using a Zeiss 510 Meta
confocal microscope with a 63x 1.4 NA oil immersion objective with scanning in the x-y
or x-z planes using a pinhole size of 1.0 Airy unit. Filters were setup for simultaneous
scanning of Cy 2 (emission 505-530 nm) and Cy 3 (LP 585 nm). Lack of bleed through
was confirmed by transiently cutting off the 543 nm excitation and noting a lack of signal
in the Texas Red channel.
Biopsies for IF and immunoblots were flash frozen in liquid N2and stored at -80o
C. Tissue lysates were prepared by homogenizing tissue with the TissueLyser bead mill
(Qiagen, Inc., Valencia, CA) in 20 volumes of a 50 mM Hepes buffer (pH 7.4) with 1%
Triton X-100, 0.05% SDS, 0.2% DOC, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA,
and Complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics Corporation,
Indianapolis, IN). Cell debris was removed by a short centrifugation at 5000 rpm. An
aliquot of cleared lysate was kept for protein quantitation using the BCA Protein Assay
Kit (Pierce Biotechnology, Inc., Rockford IL), and the rest diluted with SDS-Laemmli
sample buffer. Methods for electrophoresis and immunoblotting were standard and have
been described previously (18, 45). Antibodies against claudins 1, 4, 18 and ZO-1 were
purchased from Zymed Laboratories (San Francisco, CA) and antibodies against actin
from Sigma (St. Louis, MO). Secondary antibodies for immunoblots were goat anti-rabbit
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IRDye 800 (Rockland, Gilbertsville, PA) and goat anti-mouse Alexa680 (Molecular
Probes, Carlsbad, CA). Signals were detected using an Odyssey Infrared Imaging System
(LI-COR, Inc., Lincoln, NE).
Cldn-18 isoform A2.1 was cloned by RT-PCR from mouse duodenum RNA using
the following primers: 5’ GAATTCGCCGCCATGTCGGTGACCGCCTGC and 3’
GAATTCGCCTACACATAGTCATACTTGGTAGGATGAG, into pCR2.1 TOPO
(Invitrogene); the cDNA was digested and cloned into the EcoRI site of pTRE. The Cldn-
18 plasmid was cotransfected with pSVZeo into the tet-off MDCK II cell line (Clontech)
and stable cell lines were selected with 1 mg/ml zeocin. Plasmid structure for Cldn-2 was
previously described (5).
Electrophysiology using Madin-Darby Canine Kidney (MDCK) II cell line
Electrophysiological characterization of MDCK II monolayers was carried out
according to published methods (43). Stable MDCK II tet-off cell lines (Clontech
Laboratories) transfected with Cldn-18 or Cldn-2 were grown on Snapwell filters (Costar;
Corning Life Sciences, Acton, MA) for 4 days, induced without doxycycline or
noninduced with doxycycline (50 ng/ml). Transmonolayer resistance was measured by
using a modified Ussing chamber with a microcomputer-controlled voltage-current clamp
(Harvard Apparatus, Holliston, MA) with buffer A (120 mM NaCl, 10 mM HEPES, pH
7.4, 5 mM KCl, 10 mM NaHCO3,1.2 mM CaCl2, and 1 mM MgSO4) in the apical and
basolateral chambers. Transmonolayer dilution potentials were measured upon dilution of
Page 8 of 33
the apical chamber (buffer B: 60 mM NaCl, 120 mM mannitol, 10 mM HEPES, pH
7.4, 5 mM KCl, 10 mM NaHCO3, 1.2 mM CaCl2, and 1 mM MgSO4) relative to the
basolateral chamber (buffer A) (43). Dilution potentials were immediately stable and
repeatedly measured (every 6 s) for at least 30 s after buffer A had been replaced with
buffer B in the apical chamber. Voltage and current electrodes consisted of a Ag-AgCl
wire in 3M KCl saturated with AgCl housed in a glass barrel with a microporous ceramic
tip (Harvard Apparatus). Liquid junction potentials were calculated by using the
Henderson diffusion equation for univalent ions as previously described (43).
Apical acidification protocol: Cldn-18 transfected and non-transfected MDCK II
cells were grown as described above. Three replicate filters from each group were placed
in modified Ussing chambers with Solution A in both apical and basolateral chambers.
Baseline readings of the electrical potential difference (PD) and conductance were
obtained, and then the apical solution was changed (with one wash) to Solution A (above)
that was previously equilibrated to pH 3.5 with concentrated HCl. At 120 minutes, the
apical solution was again changed (with one wash) to Solution A equilibrated to pH 2.5
with concentrated HCl. PD and conductance readings were obtained immediately after
changes into the lower pH solutions and at 15 to 30 min intervals thereafter. The values
of the junction potential were obtained by measuring the voltage across blank filters in
the pairs of experimental buffers; these values were subtracted from subsequent
measurements made on cell monolayers to determine the PD.
Page 9 of 33
Claudin Gene Expression Profiles in SCE and SqE
qRT-PCR was performed for 21 claudins on endoscopic biopsies containing SCE
from 4 subjects with Barrett’ s esophagus and containing healthy SqE from 4 subjects
without esophageal disease. The 21 claudins tested included: Cldn-1 to Cldn-12, Cldn-14
to Cldn-20, Cldn-22 and Cldn-23 [note: validated primer sets were unavailable for Cldn-
13 and Cldn-21]; and all 21 were expressed to varying degrees in SCE (Figure 1). As
shown in Figure 1A, compared to ZO-1 serving as reference standard of 1.0; Cldn-4 and
Cldn-18 were highly expressed at > 1.0, Cldn-1, Cldn-12 and Cldn-23 moderately
expressed at between 0.5-1.0, and all other claudins considered to have low expression at
levels significantly below 0.5. Notably, Cldn-18 expression was dominant in SCE and at
levels that dwarfed all others (Figure 1A, insert), including that of Cldn-4 which, while
highly expressed in SCE, was >4-fold lower than Cldn-18.
Similar to SCE, a large number, 19 of the 21 claudins tested, were expressed in
SqE. However, unlike SCE, none of the 19 claudins in SqE were considered highly
expressed since expression values in SqE were <1.0 (Figure 1A). Indeed, only Cldn-4,
the most highly expressed in SqE, reached a level compatible with moderate expression
while Cldn-1 and Cldn-23 in SqE had expression levels considered low. The remaining
claudins in SqE had very low levels of expression or, as for Cldn-2 and Cldn-6, were
essentially undetectable. Also, and in stark contrast to SCE, Cldn-18 expression in SqE
was barely detectable at the mRNA level (Figure 1A).
As noted in methods, villin and mucin 2 are established markers of SCE and their
presence by qRT-PCR used to ensure that the biopsies analyzed for claudins were derived
Page 10 of 33
from Barrett’ s esophagus and not from proximal stomach (20, 21, 31). Gastric
epithelium, like SCE, however, is reported to have high levels of Cldn-18 (27). For this
reason, we obtained endoscopic biopsies from the gastric cardia of 3 subjects and
compared the results of qRT-PCR for villin, mucin 2 and Cldn-18 to those of Barrett’ s
SCE. The results, as shown in Figure 1B, confirm the differential expression of mucin 2
and villin in these tissues, with SCE having abundant villin and mucin 2 and gastric
cardia epithelium being devoid of these genes. Despite these differences, however, both
SCE and gastric epithelia were found to exhibit very high levels of expression for Cldn-
18 (Figure 1B).
Claudin Protein expression in SCE and SqE
Given the dominance of Cldn-18 expression at the mRNA level in SCE and
minimal expression in SqE, we compared the level of protein expression in SCE and SqE
by immunoblot. In addition to Cldn-18 we also determine protein expression levels of
two other claudins (Cldn-1 and Cldn-4) readily detected at the mRNA level in SCE and
SqE (Figure 2A). As shown in Figure 2A, protein expression levels for these three
claudins paralleled their level of gene expression, with Cldn-18 protein again dominant in
SCE and undetectable in SqE. Notably, the smearing of the blot for Cldin-18 in Figure
2A was a reflection of the high level of protein expression rather than lack of antibody
specificity – a conclusion supported by the high level of protein expression for Cldn-18 in
SCE on a second blot in which we added only 20% of the protein load used in Figure 1A.
As shown in Figure 2B, the lower protein load resulted in a single clean bend of
appropriate size for Cldn-18.
Cldn-18 localization to the cell membrane and TJ in SCE
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Based on confirmation that the high level of gene expression for Cldn-18 was
mirrored by high levels of protein expression, immunolocalization and co-localization
studies were performed in SCE for Cldn-18 and ZO-1, a protein known to be localized to
the TJ (Figure 3). As shown in Figure 3A, ZO-1 was localized predominantly to the
apical cell membranes, including TJ regions, of SCE while Cldn-18 was present in both
apical and basolateral cell membranes of SCE (Figure 3B). Indeed, and supporting a role
for Cldn-18 within the TJ was its co-localization with ZO-1 in the apical membranes
Expression of Cldn-18 in MDCK II cells influences TJ function
Given the quantitative dominance of Cldn-18 in SCE and its localization in the
TJ, we sought its effects on TJ function. This was done by transfection and expression of
Cldn-18 in MDCK II cells using previously described method (44). Cldn-18 is un-
detectable in uniduced MDCK II cells (Figure 4A). Following transfection and induction,
Cldn-18 expression could be demonstrated in the cell membranes of MDCK II cells
(Figure 4A) and its presence within the TJs confirmed by showing its colocalization with
ZO1 using confocal microscopy (Figure 4B).
Electrophysiologically, induction of Cldn-18 increased transmonolayer electrical
resistance 4-fold compared to uninduced monolayers and decreased dilution potentials
from +8mV in uninduced monolayers to essentially zero for Cldn-18 expressing
monolayers (Figure 5A, B). Further, by calculation from the foregoing data, the observed
decrease in dilution potentials by Cldn-18 expression was shown to reflect a selective
decrease in paracellular permeability to cations (Na+), with no significant effect on anion
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(Cl-) permeability (Figure 5C). Note: It was shown previously that treatment of MDCK
cells with doxycyline alone had no effect on paracellular permeability (6).
In vivo, however, the cation of concern is the hydrogen ion (H+) since it is
luminal exposure to refluxed gastric acid that poses the greatest risk of damage to
esophageal epithelium irrespective of type. For this reason, we also assessed whether
Cldn-18 expression reduced paracellular permeability to H+as well as Na+. This was
done by monitoring the diffusion potentials produced when MDCK II monolayers were
apically exposed to solutions acidified with HCl to pHs 3.5 and 2.5. Following apical
acidification to pH 3.5, a diffusion potential developed in uninduced monolayers but not
in monolayers expressing Cldn-18; while apical acidification to pH 2.5 resulted in even
higher diffusion potentials in the uninduced monolayers (2-3-fold greater) than those
observed in Cldn-18 expressing monolayers. Given these observations, we next
determined if the effect of Cldn-18 on the H+diffusion potential was relatively specific
by assessing whether it could be reproduced by overexpression of another claudin in
MDCK II cells. Induction of Cldn-2, however, resulted in a H+diffusion potential at pH
3.5 that was both indistinguishable from uninduced MDCK II cells (-1.9±0.4 vs. -1.6±0.3
mV at 2 min after acidification; n=3), and similar in value to that observed in Figure 5D
for uninduced MDCK II cells.
Page 13 of 33
TJs are the most apical of the cell-cell contacts in epithelia. They are known to
function in cell-cell adhesion, membrane polarity, and regulation of ion and uncharged
(aqueous) molecule passage through the paracellular pathway (11). On freeze-fracture
replicas, TJs appear as a complex network of strands that bridge the space between the
membranes of adjacent cells, with the strands themselves composed of proteins (34).
Recent studies indicate that the majority of bridging strands are formed by members of a
multigene family known as claudins (10, 24, 25, 41, 42). Claudins are small proteins with
molecular weights ranging from 20 to 27 kDa (42). They have four transmembrane-
spanning domains, two extracellular loops, and cytoplasmic -NH2and -COOH termini
(41). Currently at least 24 claudins have been identified in mammals. Within the
epithelial TJ, homophilic and heterophilic interactions occur among the claudins as well
as with other TJ proteins such as occludin and junctional adhesion molecule (2, 41).
Further, through PDZ (postsynaptic protein-95/discs large/zonula occludens-1) domains
on the cytoplasmic C-terminal end, the claudins bind to members of the membrane-
associated guanylate kinase family, ZO-1, ZO-2 and ZO-3; and through interactions with
this latter group, they are connected to and communicate with the actin cytoskeleton (9,
Quantitative and qualitative differences among the claudins account for much of
the diversity in TER and (perm)selectivity of the TJ (44); and such differences also can
account for varying degrees of vulnerability of the TJ to disease. For instance, the
pathologic effects of Clostridium perfringens, a common cause of foodborne
gastroenteritis, is due to an enterotoxin with the capacity to bind to Cldn-3 and Cldn-4.
Page 14 of 33
By binding these claudins, other parts of the enterotoxin are then positioned to interact
with and create small pores within the cell membrane. It is the pores that account for loss
of cell osmoregulation and ultimately for cell death (22, 35). In similar fashion, then,
differences among claudins within the TJ may theoretically make an epithelium either
more or less vulnerable to damage from luminal acid.
In the present investigation, we compared the claudin gene profiles for two
esophageal epithelia – one, SCE in BE, that is considered significantly more acid
resistant than the other, native SqE. Moreover, it has been shown that these two
esophageal epithelial types have very different and distinctive functional characteristics
with respect to ion transport and TER (39). And so not surprisingly, given such
differences in phenotype and function, the profiles were very different. For instance, SCE
had 2 claudins, Cldn-4 and Cldn-18, that were expressed to levels that exceeded that of
the reference standard, ZO-1, and three claudins, Cldn-1, Cldn-12 and Cldn-23, that came
close to the level of expression of the reference standard. In contrast, and despite
expressing 19 different claudins, none of the claudins expressed in SqE exceeded that of
the reference standard, ZO-1, and only one, Cldn-4, was expressed at a level that was
close to that of the reference. Even more dramatic, however, was the unquestioned
quantitative dominance in SCE of a qualitatively unique claudin, i.e. Cldn-18. Cldn-18
not only exceeded the reference standard in SCE but exceeded the only other highly
expressed claudin in SCE, i.e. Cldn-4, and this by greater than 4-fold. In contrast Cldn-18
was only barely detectable at the gene level in SqE and not at all at the protein level.
Moreover, the literature indicates that Cldn-18 is abundantly expressed in mouse and
Page 15 of 33
human gastric epithelium (27), expressed to a limited extent in duodenum, and almost
non-existent throughout the rest of the small and large bowel (15).
Cldn-18 to our knowledge has not been described in BE - though other
investigators have found in BE varying levels of gene and/or protein expression for Cldns
1-5 and Cldn 7 (13, 23, 26). Indeed, mouse Cldn-18 has only recently been isolated and
characterized by Niimi et al and shown to have two isoforms, one lung specific and the
other stomach-specific; and each isoform has two transcripts, one for the full length
claudin and the other for a C-terminal truncated form of the claudin. C-terminaly
truncated isoforms were not found for human Cldn-18 (27, 33) Niimi et al, when
searching for genes involved with pulmonary morphogenesis, noted that the lung-
specific, but not stomach-specific, isoform of Cldn-18 was a downstream target gene for
T/EBP/NKX2.1, a homeodomain transcription factor (27). To date, however, there is no
known transcription factor for stomach (or SCE)-specific Cldn-18.
In the present investigation dominance of Cldn-18 expression at the mRNA level
was also mirrored by dominance of protein expression in SCE. Further IF demonstrated
that Cldn-18 was distributed within both apical and basolateral cell membranes within
SCE. Moreover, and supporting a role in the TJ, Cldn-18 co-localized with the TJ protein,
ZO-1. A similarly diffuse cell membrane pattern for Cldn-18 distribution within gastric
epithelium was also observed by Niimi et al. Additionally they documented by
immunogold staining that Cldn-18 was localized to the TJ strands on freeze fracture, and
suggested that excess Cldn-18 within the membrane served as a reservoir for its shuttling
to and from the TJ.
Page 16 of 33
Given the quantitative dominance of Cldn-18 within the TJ of SCE, it is likely to
play a prominent role in TJ function; and one critical function of the claudins within TJs
is their ability to regulate the ion species traversing the paracellular pathway (37). Indeed
Colegio et al have shown using site-directed mutagenesis that a change of charge from
positive to negative on one aminoacid within the extracellular domain of claudin-4 could
increase paracellular permeability to Na+; and changing the charge on three aminoacids
from negative to positive in claudin-15 could reverse the ion preference of the
paracellular pathway from Na+to Cl-(6). In the present study, Cldn-18 was expressed in
MDCK II cells and shown by IF to be localized to the cell membrane and the TJ.
Notably, and consistent with the ability of claudins to alter both the electrical resistance
and ion conductance of the TJ (5), Cldn-18 expressing monolayers in Ussing chambers
exhibited higher TER and lower NaCl dilution potentials than uninduced monolayers of
MDCK II cells. Further, since these dilution potentials were similar irrespective of
sidedness of the gradient, the increase in TER and reduction in conductance with Cldn-18
expression reflected changes in paracellular, as opposed to transcellular, permeability.
Moreover, and based on the direction of the conductance change, the reduction in
paracellular permeability reflected a selective decline in cation conductance. It is also of
interest that Cldn-4 which, like Cldn-18, is highly expressed in SCE has been shown to
increase TER and lower paracellular permeability for cations (specifically Na+) in
MDCK II cells (44). This suggests the emergence of a possible theme in which the
claudin expression profile of SCE reflects a need for enhanced protection against the
potentially noxious effects of luminal cations – see below.
Page 17 of 33
Further, and of potential clinical importance, the ability of Cldn-18 expressing
monolayers to impede paracellular permeability of cations, extended to H+- these being
the smallest of cations and the ones that pose the greatest risk of damage to esophageal
epithelia in vivo. In the present experiments, resistance to the paracellular movement of
H+was demonstrated in Cldn-18 expressing MDCK II monolayers by the absence of a
H+-induced diffusion potential upon apical acidification to pH 3.5 and lower diffusion
potentials at apical pH 2.5 than observed in uninduced MDCK II monolayers. Moreover,
this resistance to H+diffusion by Cldn-18 expression was relatively specific, since it was
not observed by expression of Cldn-2, and was long lasting rather than transient. The lack
of a diffusion potential was sustained for 2 hrs at pH 3.5 and reduced significantly below
that of uninduced monolayers for about 2 hrs at pH 2.5. These observations - coupled
with the fact that the TJ appears to be a weak link in the defense of SqE against damage
by luminal acid (29) – support the hypothesis that the presence of Cldn-18 within the TJ
of SCE contributes to the acid resistance of BE. Consistent with this conclusion is the
observation that the only other digestive epithelia, gastric and duodenal, where Cldn-18 is
noted to be prominent are those that are exposed to and so require a strong defense
against luminal acid (27).
In summary, Cldn-18 is the dominant claudin of SCE in BE. It is distributed
widely within the cell membrane and co-localizes with the TJ protein, ZO-1. Among its
properties Cldn-18 selectively decreases the permeation of cations through the
paracellular pathway and this decrease extends to H+. We propose that the change from a
Cldn-18 deficient TJ in SqE to a Cldn-18 rich TJ in SCE contributes to the greater acid
resistance of BE.
Page 18 of 33
We are thankful to Dr. McNaughton Kirk for superb cut of fresh frozen tissue used for
our immunolocalization studies.
This research was supported by following grants: NIH grants DK 036013, DK 063669
and DK 45134.
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esophagus. Gastroenterology 126: A235, 2004.
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Figure 1. (A) Claudin gene expression profiles for human esophageal specialized
columnar epithelium (SCE), i.e. Barrett’ s esophagus (BE), and for healthy human
esophageal stratified squamous epithelium (SqE). Claudin expression levels are
referenced to expression levels for ZO-1 which is set at 1.0 and the Y axis broken into
two scales to accommodate the broad range. Note the large numbers of claudins
expressed in both SCE and SqE and, as emphasized in the insert, the overriding
dominance of expression for claudin-18 over that of all other claudins in both SCE and
SqE. Error bars = [(2%CV)/100] x [relative expression]; NT = not tested; ND = not
detected. (B) Comparative gene expresson for mucin 2, villin and Cldn-18 in human
gastric cardia epithelium and Barrett’ s SCE. Villin and mucin 2 are shown to be
undetectable in stomach and to be highly expressed in SCE. In contrast, Cldn-18 is highly
expressed in both epithelial types, with stomach expressing double amount of Cldn-18
expressed in SCE.
Figure 2. Immunoblots of Cldn-1, Cldn-4 and Cldn-18 of tissue lysate prepared from
specialized columnar epithelium (SCE) of Barrett’ s esophagus (BE), or from healthy
human esophageal stratified squamous epithelium (SqE). (A) Ten micrograms of protein
are loaded in each lane. Note: protein levels correlate with relative expression of
transcripts (see Fig.1A). Cldn-1 has similar protein expression level in SqE and BE, cldn-
4 has a higher level of expression in BE than SqE, and cldn-18 is highly and exclusively
expressed in BE. Molecular weight standards are displayed on the left in kilodaltons
(kDa). Prestained SDS-PAGE standard (wide range; Bio-Rad) was used (red bands). This
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immunoblot is representative of 3 separate experiments performed using tissue from
different patients. (B) Immunoblot of Cldn-18 from BE tissue lysate. By reducing the
total protein load to only 2 micrograms per lane, the band for Cldn-18 is now shown to be
single and clean.
Figure 3. Immunofluorescent localization of the tight junction protein, ZO-1, and Cldn-18
in esophageal specialized columnar epithelium (SCE) from Barrett’ s esophagus (BE).
(A) ZO-1 localized to the apical cell membranes containing the tight junction region in
SCE. (B) Abundantly expressed Cldn-18 localized to both apical and basolateral cell
membranes in SCE. (C) Dual immunofluorescence demonstrates that Cldn-18 coloca-
lizes with ZO-1 within the apical cell membrane and tight junction regions of SCE.
Signals are pseudo-colored green for ZO-1 and red for Cldn-18. Cell nuclei are stained
blue with DAPI. Co-localization of ZO-1 and Cldn-18 results in the yellow color. Bar, 20
Figure 4. (A) Immunofluorescent localization of ZO-1 and Cldn-18 in uninduced and
induced Madin-Darby Canine Kidney (MDCK) II cells. Note that uninduced MDCK II
cells lack immunostaining for Cldn-18, while those induced to express Cldn-18 showed
staining within the plasma membrane and in intracellular vesicular compartments. ZO-1
localization was unchanged by induction of Cldn-18 expression. Bar, 20 µm. (B) Z plane
section of immunofluorescent colocalization of ZO-1 and Cldn-18 in induced MDCK II
cells. Top panel, Dual staining for ZO1 (red) and Cldn-18 (green) and their colocalization
(yellow). Middle panel: ZO1. Bottom panel: Cldn-18.
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Figure 5. Electrophysiologic effects of Cldn-18 expression in monolayers of MDCK II
cells. (A). The resting transmonolayer epithelial resistance (TER) increases 4-fold, i.e.
from 38.5 ± 2.3 ohms.cm2in uninduced cells to 185 ± 23 ohms.cm2following induction
of Cldn-18. (B). Dilution potentials (in millivolts, mV) following apical NaCl dilution
from 120mM to 60mM declines from +8mV for uninduced cells to effectively zero in the
Cldn-18 induced matched clones. Note the positive dilution potentials for uninduced
MDCK II cells reveal their cation selectivity and that this is effectively eliminated
following expression of Cldn-18 creating a non-selective monolayer. (C). Calculation of
ion permeabilities for Na+and Cl-reveals that the change in dilution potentials following
Cldn-18 induction is due to a selective decrease in Na+permeability (filled bars,
compared with uninduced, open bars) with no change in Cl-permeability. Average of 2
clones and duplicate measurements on each; means ± SE. (D) Following apical
acidification (addition of HCl) to pH 3.5 a diffusion potential (in millivolts, mV)
develops in uninduced (■) cells for 2 hrs raising the transmonolayer potential difference
(PD) from -0.5 mV to -3.0 mV while the PD for Cldn-18 induced cells remains
essentially unchanged (▲). Further apical acidification (addition of HCl) to pH 2.5
results in further increases of PD in uninduced cells to a maximum of -4.4 mV, while the
PD for Cldn-18 induced cells increases to a maximum of -2.2 mV. At pH 2.5, the
differences in diffusion potentials for uninduced and induced cells persists for almost 2
hrs; n=3, values means ± standard error (SE), * p<0.05.
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179x97mm (600 x 600 DPI)
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101x76mm (300 x 300 DPI)
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98x124mm (72 x 72 DPI)
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103x77mm (300 x 300 DPI)
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