1 Department of Clinical Physiology, Benjamin Franklin Medical School, Freie
Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
2 Department of Gastroenterology, Infectiology and Rheumatology, Benjamin
Franklin Medical School, Freie Universität Berlin, Hindenburgdamm 30,
12200 Berlin, Germany
3 Department of Pharmacology, Benjamin Franklin Medical School, Freie
Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany
* Author for correspondence (e-mail: michael.fromm{at}medizin.fu-berlin.de)
Accepted 11 September 2002
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Summary |
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Monolayers of C7 cells exhibited a high transepithelial resistance (>1
k cm2), compared with C11 cells (<100
cm2). Genuine expression of claudin-1 and claudin-2, but not of
occludin or claudin-3, was reciprocal to transepithelial resistance. However,
confocal microscopy revealed a marked subjunctional localization of claudin-1
in C11 cells, indicating that claudin-1 is not functionally related to the low
tight junctional resistance of C11 cells.
Strain MDCK-C7, which endogenously does not express junctional claudin-2,
was transfected with claudin-2 cDNA. In transfected cells, but not in vector
controls, the protein was detected in colocalization with junctional occludin
by means of immunohistochemical analyses. Overexpression of claudin-2 in the
originally tight epithelium with claudin-2 cDNA resulted in a 5.6-fold higher
paracellular conductivity and relative ion permeabilities of
Na+1, K+=1.02, NMDG+=0.79,
choline+=0.71, Cl-=0.12, Br-=0.10 (vector
control, 1:1.04:0.95:0.94:0.85:0.83). By contrast, fluxes of (radioactively
labeled) mannitol and lactulose and (fluorescence labeled) 4 kDa dextran were
not changed. Hence, with regular Ringer's, Na+ conductivity was 0.2
mS cm-2 in vector controls and 1.7 mS cm-2 in
claudin-2-transfected cells, while Cl- conductivity was 0.2 mS
cm-2 in both cells. Thus, presence of junctional claudin-2 causes
the formation of cation-selective channels sufficient to transform a `tight'
tight junction into a leaky one.
Key words: Zonula occludens, Claudins, Occludin, Transepithelial resistance, Impedance analysis
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Introduction |
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The claudin family shows an organ- and tissue-specific expression of
individual members. Deficiency or aberrant expression of distinct claudins has
been reported to be associated with severe pathophysiological consequences
(for a review, see Anderson,
2001; Tsukita et al.,
2001
). Claudin-1-deficient mice die within one day of birth
because of loss of epidermal barrier function
(Furuse et al., 2002
). Further
defects were autosomal recessive deafness in the case of claudin-14
(Wilcox et al., 2001
),
hypomagnesaemia, which is associated with mutations of claudin-16
(Simon et al., 1999
), and the
fact that CNS myelin and sertoli cell TJ strands are absent in Osp/claudin-11
null mice (Gow et al., 1999
).
Details concerning functional properties of single claudins have been
described for claudin 2, 4 and 15. Furuse et al. demonstrated that expression
of claudin-2 is correlated with a decrease of transepithelial resistance
(Furuse et al., 2001
).
However, the first direct demonstration of the ability of a claudin to
influence paracellular ion selectivity was contributed by Van Itallie et al.
for claudin-4 (Van Itallie et al.,
2001
). In addition, Colegio et al. showed that the first
extracellular domain of claudin-4 is responsible for this property and that
expression of wild-type claudin-15 leads to an increase in transepithelial
resistance (Colegio et al.,
2002
).
There are two twin strains of Madin-Darby canine kidney (MDCK) cells with
markedly different transepithelial resistance, MDCK I and II
(Richardson et al., 1981) and
MDCK-C7 and -C11 (Gekle et al.,
1994
). Recently, Furuse et al. reported that those with a high
resistance (MDCK I) lack claudin-2, and that expression of claudin-2 lowers
the transepithelial resistance (Furuse et
al., 2001
). This is a most important finding, although a decrease
in transepithelial resistance does not prove that paracellular, rather than
transcellular, pathways were affected.
Beyond the approach of Tsukita et al., we employed confocal laser scanning microscopy, impedance analysis, biionic/dilution potential measurements, and tracer flux experiments. The results demonstrate that claudin-2 is responsible for the formation of a paracellular, cation-selective pore.
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Materials and Methods |
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For electrophysiological measurements and molecular analyses, epithelial cell monolayers were grown on porous polycarbonate culture plate inserts (effective area 0.6 cm2, MillicellTM-HA, Millipore, Bedford, MA). On day 7, resistance and impedance analyses were performed. Inserts were mounted in Ussing chambers, and water-jacketed gas lifts were filled with 10 ml circulating fluid on each side. The standard bathing ringer solution contained: 113.6 mM NaCl, 2.4 mM Na2HPO4, 0.6 mM NaH2PO4, 21 mM NaHCO3, 5.4 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 10 mM D(+)-glucose. According to the respective experimental approach, low (20 nM), and high (12.6 mM) Ca2+ concentrations (impedance analysis) were employed, or NaCl was partially substituted during flux and dilution potential experiments. All solutions were gassed with 95% O2 and 5% CO2, to ensure a pH value of 7.4 at 37°C.
Electrophysiology
Short-circuit current (ISC, µmol h-1
cm-2) and transepithelial resistance, referring to tissue area
(Repi, cm2), were measured in Ussing
chambers specially designed for insertion of Millicell filters
(Kreusel et al., 1991
).
Resistance of bathing solution and filter support
(Rfilter) was measured prior to each single experiment and
subtracted. Impedance analysis was performed to characterize the paracellular
component of the transepithelial resistance of MDCK monolayers as described
previously (Gitter et al.,
1997a
; Gitter et al.,
1997b
). Briefly, a programmable frequency response analyzer in
combination with an electrochemical interface (models 1250 and 1286, Solartron
Schlumberger, Farnborough, UK) was employed for application of sinusoidal
currents, logarithmically spaced in frequencies from 1 kHz to 65 kHz. The
electrical equivalent circuit of the epithelium comprises an ohmic
paracellular pathway (Rpara) and a transcellular pathway
with capacitive components (representing cell membranes) resulting in a
complex impedance (Ztrans).
The circuit model is shown in Fig.
1A. Impedance (Zepi) was plotted in Nyquist
diagrams (Fig. 1B). In each
experiment with C7 monolayers, the paracellular resistance,
Rpara, was changed by lowering the extracellular
Ca2+ concentration. In the case of C11 monolayers, the reduced
Ca2+ concentration led to a complete breakdown of the epithelial
barrier and, therefore, the extracellular Ca2+ concentration was
increased resulting in a higher Rpara. In each case it was
assumed, however, that the perturbation (ctrlCa) affected
Rpara, but not Ztrans.
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|
The two sets of data, differing in Rpara, allowed subtraction of Ztrans. Hence, the fit of impedance data with the equations of the electrical model allowed evaluation of Rpara.
Flux and dilution/biionic potential measurements
Measurement of unidirectional tracer flux from the apical to the
basolateral side was performed under short-circuit conditions with 25 kBq/ml
of [3H]-mannitol or [3H]-lactulose (Biotrend, Cologne,
Germany). The medium also contained non-labeled tracer molecules (10 mM
mannitol or 10 mM lactulose, respectively). Four 15-minute flux periods were
analyzed (Schultz and Zalusky,
1964). Upon initiation and completion, a 100 µl sample was
taken from the donor (apical) side, and 900 µl Ringer's and 4 ml of Ultima
Gold high flash-point liquid scintillation cocktail (Packard Bioscience,
Groningen, The Netherlands) were added. Samples (1 ml) of the receiving
(basolateral) side, replaced with fresh Ringer's, were mixed with 4 ml of the
liquid scintillation cocktail. All 5 ml samples were subsequently analyzed
with a Tri-Carb 2100TR Liquid Scintillation counter (Packard, Meriden, CT). In
4 kDa FITC-dextran flux analyses the high molecular weight fluorescent dye was
dissolved in Ringer's at a concentration of 25 mg/ml and dialyzed against the
same buffer. This solution was employed in the basolateral compartment. After
5 hours incubation the amount of FITC-dextran in the apical compartment was
measured with a fluorometer at 520 nm (Spectramax Gemini, Molecular devices,
Sunnyvale, CA).
Dilution and biionic potentials were measured with modified Ringer's solution on the mucosal or serosal side, and the data from both conditions were pooled. In the modified Ringer's, 70 mM NaCl were replaced by KCl, NMDGCl, NaBr or choline chloride. Relative ion permeabilities were calculated by means of the Goldman-Hodgkin-Katz equation and partial ion conductivities were determined using the respective ion concentrations of normal Ringer's.
Immunofluorescence studies
Immunofluorescence analysis and photography were performed as described
(Weiske et al., 2001). Cells
were grown on coverslips (18x18 mm, Menzel, Braunschweig, Germany). For
immunological studies, cells were rinsed with PBS, fixed with methanol, and
permeabilized with PBS containing 0.5% Triton X-100. Concentrations of primary
antibody were 20 µg/ml (Ms anti-occludin, Rb anti-claudin-1, -2, -3; Zymed
Laboratories, San Francisco, CA). Secondary ABs Alexa Fluor 488 goat
anti-mouse and Alexa Fluor 594 goat anti-rabbit (both used in concentrations
of 2 µg/ml) were purchased from Molecular Probes (MoBiTec, Göttingen,
Germany). Fluorescence images were obtained with a confocal microscope (Zeiss
LSM510) using excitation wavelengths of 543 nm and 488 nm. Details of the
microscopy setup are available upon request.
PCR cloning of mouse claudin-2 from mouse colon RNA
Total RNA was obtained from mouse distal colon using RNAzol B reagent (WAK
Chemie, Bad Soden, Germany). First strand cDNA was synthesized by reverse
transcriptase reaction (M-MLV, Gibco BRL, Bethesda, MD) employing oligo(dT)
primer. Sense (5'-GTCTGCCATGGCCTCCCTTG-3') and antisense
(5'-CAGCTCTGGCCCCTGGTTCT-3') primers were synthesized according to
the mouse claudin-2 sequence (Furuse et
al., 1998) and used for PCR. The resulting 720 bp PCR product
encompassing the complete claudin-2 cDNA was cloned into pGEMT-Easy (Promega,
Madison, WI). The correctness of the cDNA was verified by sequencing and then
subcloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen,
Carlsbad, CA) further referred to as p[cld-2]. MDCK-C7 cells were stably
transfected with p[cld-2] by employing the Lipofectamine plus method (Gibco
BRL, Bethesda, MD). G418-resistant cell clones were screened for claudin-2
expression by western blot (see below). C7 and C11 cells transfected with an
empty vector (p[vec]) served as controls.
Western blotting
Cells were washed in ice cold PBS, scraped from the permeable supports in
Tris-buffer containing 20 mM Tris, 5 mM MgCl2, 1 mM EDTA, 0.3 mM
EGTA, and protease inhibitors (Complete, Boehringer, Mannheim, Germany).
Protein was obtained by freeze-thaw cycles and subsequent passage through a 26
G needle. The membrane fraction was obtained by two centrifugation
steps: first, samples were centrifuged for 5 minutes at 200 g
(4°C); then, supernatant was centrifuged for 30 minutes at 43,000
g (4°C). The pellet was resuspended in Tris-buffer and
protein content was determined using BCA Protein assay reagent (Pierce,
Rockford, IL) quantified with a plate reader (Tecan, Austria). After
measurement of total membrane protein, 2.5 µg of the samples were mixed
with SDS buffer (Laemmli), loaded on a 12.5% SDS polyacrylamide gel and
electrophoresed. Proteins were detected by immunoblotting employing antibodies
raised against human occludin and claudin-1, -2 and -3. All primary antibodies
were provided from Zymed Laboratories (San Francisco, CA). Specific signals
were quantified with luminescence imaging (LAS-1000, Fujifilm, Japan) and
quantification software (AIDA, Raytest, Germany).
Statistical analysis
Data are expressed as means±standard error of the mean. Statistical
analysis was performed using Student's t-test and the Bonferroni
correction for multiple comparisons. P<0.05 was considered
significant.
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Results |
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To biochemically characterize the potential mechanism of these obvious differences, expression levels of TJ proteins were quantified by immunoblotting. As shown in Fig. 2, expression of occludin (Fig. 2A) was identical in both cell lines. Occludin migrated as two main bands with apparent molecular weights ranging from 55 to 67 kDa probably due to post-translational modification or occurrence of splice variants. The most obvious difference was observed for claudin-2, which was markedly expressed in C11 but only marginally detected in C7 cells (8.9±1.9% of expression in C11, n=6). In C7 cells, lower expression was also detected for claudin-1 (21.3±3.1% of expression in C11, n=5), whereas claudin-3 did not differ significantly between the two strains C7 and C11 (73.6±18% of expression in C11, n=4). All claudins were detected as a 22 kDa band (Fig. 2).
|
Next, immunofluorescence studies were performed to characterize the subcellular distribution of occludin and claudins in MDCK cell lines C7 and C11. Confocal microscope analysis demonstrated a genuine colocalization of claudin 1 and 3 with occludin in both C7 and C11, whereas in accordance with western blot analyses claudin-2 expression was detectable only in C11 (Fig. 3A,B). Z-scans of confocal images revealed that the high expression of claudin-1 in the leaky cell strain C11 is not limited to the strand region but is concentrated in subjunctional areas (Fig. 3C,D).
|
Transfected MDCK-C7 cells stably express claudin-2
For functional analyses of claudin-2, which is endogenously expressed at
high levels in the leaky strain MDCK-C11, the protein was overexpressed in
MDCK-C7, the high resistance strain with weak genuine claudin-2
expression.
Transfection resulted in a detectable expression of claudin-2 as shown by western blot studies, whereas expression of claudin-1 did not change (Fig. 4A). As a result, a dramatic decrease of epithelial resistance was observed after transfection (Repi, 27.6±0.7% compared with 100% control Repi; n=3, Fig. 4B) while no change of paracellular 4K FITC-dextran flux was detectable (n=6, Fig. 4C). The subcellular distribution of claudin-2 in the transfected clones of C7 cells was analyzed by immunological studies in combination with confocal microscopy techniques. While no expression of claudin-2 was detected in C7 cells transfected with vector alone (C7-vec; Fig. 4D), claudin-2 was found to be colocalized with the TJ protein occludin in C7 transfected with claudin-2 cDNA (C7-cld-2; Fig. 4E,F).
|
Paracellular cation permeability
To functionally characterize the clones, impedance analyses, flux
measurements and dilution/biionic potential measurements were performed. In
leaky epithelia, such as MDCK clone C11, the transepithelial resistance
Repi mainly depends on the resistance of the paracellular
pathway, Rpara, and evaluation of Repi
(by measuring the response to DC electric current) is a good estimate of the
junctional barrier to paracellular ion movement. It is important to note,
however, that in tight epithelia, such as clone C7, Rpara
cannot be estimated from Repi because
Repi of tight epithelia is mainly determined by the
bouquet of channels and carriers within the cell membranes. We therefore
determined Rpara from impedance analysis
(Fig. 5). In monolayers of C7
cells transfected with claudin-2 (C7-cld-2), Rpara was
5.6-fold lower than in clone C7-vec without claudin-2 (485±14
cm2, n=4 vs. 2734±119
cm2,
n=4, P<0.001). However, compared with C11, cells
transfected with vector alone (C11-vec: 68±2
cm2,
n=4, P<0.001), Rpara was 7.1-fold
higher in C7-cld-2.
|
Charge selectivity was assessed with measurement of NaCl dilution potentials. The ratio of Na+ to Cl- permeability (PNa/PCl) was 1.19±0.05 (n=6) in C7-vec and increased to 8.7±0.4 (n=6, P<0.001) in C7-cld-2. Hence, expression of claudin-2 created a cation-selective passive pathway. The permeability ratio of Br- to Cl- was 0.985±0.004 (n=6) in C7-vec and decreased to 0.869±0.022 (n=6, P<0.01) in C7-cld-2. In comparison, clone C11-vec showed a ratio of 0.685±0.009 (n=6, P<0.01). Measurements of biionic potentials revealed that Na+ and K+ permeabilities were not significantly different in all three clones. The permeability ratio of Na+ to choline+ (104.2 Da), was 33% higher in C7-cld-2, and 3.6 times higher in C11, than in C7-vec. The permeability ratio of Na+ to NMDG+ was 20.5% higher in C7-cld-2, and 2.7 times higher in C11-vec. These experiments demonstrated the size discrimination of claudin-2 channels. From measurements of dilution and biionic potentials relative paracellular permeabilities were calculated according to the Goldman-Hodgkin-Katz equation (Fig. 6A). Thus, transfection of C7 cells resulted in a change of relative ion permeabilities from 1 Na+ : 1.043±0.02 K+ : 0.95±0.006 NMDG+ : 0.941±0.021 choline+ : 0.846±0.034 Cl- : 0.834±0.034 Br- to 1 Na+ : 1.022±0.006 K+ : 0.79±0.016 NMDG+ : 0.705±0.013 choline+ : 0.116±0.006 Cl- : 0.101±0.006 Br-; relative permeabilities of C11 were 1 Na+ : 1.033±0.002 K+ : 0.351±0.008 NMDG+ : 0.277±0.03 choline+ : 0.023±0.003 Cl- : 0.015±0.002 Br- (n=6, Fig. 6A).
|
Flux measurements employing [3H]-mannitol (184 Da) or
[3H]-lactulose (342.5 Da), revealed that transfection of C7 with
claudin-2 cDNA does not lead to an increased paracellular permeability for
molecules of 184 Da, although permeability of both molecules was higher in
the C11 clone (Fig. 6B).
Partial ion conductivities were calculated from relative paracellular
permeabilities of Na+, K+, NMDG+,
Cl- and Br- (Fig.
6C). Clone C7-cld2 showed a selective increase of Na+
and K+ conductance compared with C7 control (Na+:
1.739±0.064 mS cm-2 vs. 0.208±0.014 mS
cm-2; K+: 0.069±0.003 mS cm-2 vs.
0.008±0.001 mS cm-2), whereas Cl- conductance was
not significantly changed (0.178±0.011 mS cm-2 vs.
0.155±0.012 mS cm-2).
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Discussion |
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Impedance analysis
The simultaneous fit of an electrical model to the data recorded under the
different conditions yielded values for Rpara that (1)
hardly varied with the start parameters of the fit algorithm, and (2) were
reproducible in repeated measurements. The parameters of transcellular
pathways were indeterminate and, therefore, not interpreted. Because of their
reproducibility, the values of Rpara may be considered a
measure of the junctional barrier function. In the case of the low-resistance
C11 clone, a similar value of Rpara had been found
previously with a different method [conductance scanning
(Gitter et al., 1997b)]. This
correspondence defies a systematic error and supports the validity of the
present method.
Single recordings of the transepithelial impedance under one experimental
condition cannot resolve Rpara
(Kottra and Frömter,
1984), but additional information is gained by a controlled
perturbation in only one parameter of the system. Since changes of the
extracellular Ca2+ concentration are an established tool for the
modulation of TJs [Ca2+ switch
(Contreras et al., 1992
)], we
measured the frequency dependence of transepithelial impedance in the same
epithelium at two different Ca2+ concentrations, assuming this
affected only Rpara. As expected,
Rpara decreased in the C7 clones incubated with low
Ca2+ Ringer solution. In C11 cells exposed to high Ca2+
Ringer, Rpara increased.
Claudin-2 expression leads to the presence of paracellular cation
channels
Paracellular resistance, a measure of the tight junctional barrier,
decreased after transfection with claudin-2, but the transepithelial flux of
labeled mannitol, lactulose, and 4 kDa dextran did not change. Hence,
claudin-2 induces paracellular ion channels not permeable to uncharged
molecules >182 Da. In addition, biionic and dilution potential measurements
revealed a selectivity with the permeability sequence K+
Na+>NMDG+>choline+>>Cl-=Br-.
This sequence is indicative of a pronounced cation selectivity.
The colocalization of claudin-2 and occludin in the area of the tight junctions provides evidence against an indirect intracellular effect of claudin-2 modulating the tight junctions, but there is still the possibility that claudin-2 has regulatory function only and the conductive site is located somewhere else, either in the transcellular route (transmembranal channels or conductive carriers) or in the paracellular path (other tight junction proteins). Regarding the first alternative, we have shown for the first time for any claudin-induced conductance change that it is localized directly in the paracellular pathway. With this result a regulatory effect on trans-membranal channels or carriers is excluded (Fig. 1). Thus, the only possibility of an indirect effect of claudin-2 would be that it regulates the conductivity of other claudins (or of occludin or JAM). Although we cannot strictly exclude this possibility, from the molecular structure we have no indication that claudin-2 simply by its presence alters the structure of other claudins to a cation conductive state.
In our experiments, expression of claudin-2 after transfection resembled `genuine' expression levels (as detected in C11 cells), indicating that changes can be attributed to physiological claudin-2 properties, and not necessarily to a changed expression ratio of other claudins. This is supported by the finding that after transfection other claudins did not show detectable changes of expression levels.
The concentration of claudin-1 in the subjunctional region of C11 cells indicates that elevated expression levels of a single claudin may not necessarily result in exclusive localization of that claudin within the tight junction.
The functional changes induced by expression of claudin-2 are opposite to
the changes associated with overexpression of claudin-4. Van Itallie et al.
found a decrease of paracellular sodium permeability after claudin-4
overexpression in MDCK cells (Van Itallie
et al., 2001).
The hypothesis that single claudins create ion-selective paracellular
channels has been supported by Colegio et al., demonstrating that reversing
the charge of a single amino acid in the first extracellular loop of claudin-4
dramatically changes epithelial permeability
(Colegio et al., 2002).
Claudin-1 expression
In MDCK cells claudin-1 has been shown to increase resistance
(Inai et al., 1999;
McCarthy et al., 2000
). The
most striking evidence showing responsibility of claudin-1 for barrier
function has been presented in a claudin-1-knockout study: claudin-1 molecules
expressed in the epidermis are indispensable for creating and maintaining the
epidermal barrier (Furuse et al.,
2002
). Whereas these studies unequivocally show a resistance
increase and barrier formation of claudin-1, at first sight the results of our
study indicate a resistance decreasing role of claudin-1.
Closer inspection using confocal microscopy solved the puzzle: in contrast
to claudin-2, claudin-1 was predominantly found below the tight junction. This
finding is supported by Gregory et al., who demonstrated that claudin-1
expression in epithelial cells is not localized exclusively in tight
junctions, but appears along the entire interfaces of adjacent epithelial
cells as well as along the basal plasma membrane
(Gregory et al., 2001). Hence,
claudin-1 is not necessarily colocalized with occludin in the TJ, and the
higher conductivity of the C11 clone can be explained by claudin-2 expression
alone.
In the present experimental design we selectively changed the expression of claudin-2 in the C7 cells, while the amount of claudin-1 was not changed. The functional changes described here must therefore be attributed to the effect of claudin-2.
Claudin-3 expression
Claudin-3 was found in equal amounts in both strains C7 and C11, and was
always colocalized with occludin in the tight junction. Therefore, claudin-3
appears to be a constitutive transmembranal TJ protein. Data from claudin-3
transfection experiments have been published previously
(Furuse et al., 2001). The
authors reported no change of functional properties of the TJ excluding a
functional role in the difference between the high and low resistance strains.
Nevertheless, the importance of this protein was highlighted when a specific
binding of Clostridium perfringens enterotoxin to claudin-3 and -4 was
reported. In contrast, no correlation with claudin-1 and -2 could be found
(Sonoda et al., 1999
;
Fujita et al., 2000
).
Occludin expression
Occludin has been demonstrated to be an important component of the tight
junction, as elevated expression caused strand numbers to increase, followed
by a rise of transepithelial resistance and a decrease of mannitol flux
(McCarthy et al., 1996). In
addition, truncation of the C-terminus leads to an increase of paracellular
flux (Balda et al., 1996
;
Chen et al., 1997
).
Furthermore, a loss of fence function was observed, leading to a free
diffusion of lipids from the apical to the basolateral membrane domain
(Balda et al., 1996
). Occludin
colocalization with members of the claudin family has been reported
(Tsukita and Furuse, 1998
). An
important aspect of tight junctional regulation emerged from studies comparing
occludin with claudin-4 (Balda et al.,
2000
). In this study, multiple occludin domains were demonstrated
to be involved in regulation of paracellular permeability. By contrast,
potential dispensability of the presence of occludin in TJs was demonstrated
in knockout experiments: although complex morphological changes emerged from
this approach, no effect on transepithelial resistance was observed
(Saitou et al., 2000
). These
findings suggested either that occludin is not primarily responsible for
determination of the paracellular barrier or that possible splice variants
were not afflicted in the knockout experiments. As, in our approach, the
expression did not differ in low- and high-resistance MDCK cells under all
experimental conditions, occludin turned out to be a suitable reference
molecule for TJ location.
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Conclusion |
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Acknowledgments |
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References |
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