Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
Author for correspondence (e-mail:
htsukita{at}mfour.med.kyoto-u.ac.jp)
Accepted 27 January 2003
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Summary |
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Key words: Snail, Claudin, Occludin, Tight junction, Epitheliummesenchyme transition
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Introduction |
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The tight junction (TJ) is an important structure that determines
epithelial cell polarity and disappears during EMT. TJs constitute the
epithelial junctional complex, together with adherens junctions (AJs) and
desmosomes, and are located at the most apical part of the complex
(Farquhar and Palade, 1963).
TJs create the primary barrier to the diffusion of solutes through the
paracellular pathway and maintain cell polarity as a boundary between the
apical and basolateral plasma membrane domains
(Schneeberger and Lynch, 1992
;
Gumbiner, 1993
;
Anderson and van Itallie, 1995
;
Tsukita et al., 2001
). On
ultrathin section electron microscopy, TJs appear as a series of discrete
sites of apparent fusion, involving the outer leaflet of the plasma membranes
of adjacent cells (Farquhar and Palade,
1963
). On freeze-fracture replica electron microscopy, TJs appear
as a set of continuous, anastomosing intramembranous particle strands (TJ
strands) (Staehelin,
1974
).
The molecular architecture of TJs has been unraveled rapidly in recent
years. Three closely related PDZ-domain-containing proteins (ZO-1, ZO-2 and
ZO-3) constitute the undercoat structure of TJs together with other peripheral
membrane proteins such as cinglin, 7H6 antigen and symplekin
(Stevenson et al., 1986;
Gumbiner et al., 1991
;
Balda et al., 1993
;
Citi et al., 1988
;
Zhong et al., 1993
;
Keon et al., 1996
;
Mitic and Anderson, 1998
;
Tsukita and Furuse, 1999a
). As
constituents of TJ strands themselves, two distinct types of integral membrane
proteins have been identified: occludin and claudins
(Furuse et al., 1993
;
Furuse et al., 1998a
;
Furuse et al., 1998b
). Both
occludin and claudins bear four transmembrane domains but do not show any
sequence similarity with each other. Claudins and occludin are thought to
constitute the backbone of TJ strands and to modulate some functions of TJs,
respectively (Tsukita et al.,
2001
). Claudins compose a multi-gene family consisting of more
than 20 members (Morita et al.,
1999
; Tsukita and Furuse,
1999b
).
The question has naturally arisen of how TJs with such a complicated
molecular architecture disappear during EMT. Recently, a zinc-finger
transcription factor, Snail, has been implicated in the switching mechanism
for EMT (Nieto, 2002). This
gene was initially identified in Drosophila to be responsible for
gastrulation (Grau et al.,
1984
; Nusslein-Volhard et al.,
1984
; Alberga et al.,
1991
). When chick embryos were treated with antisense
oligonucleotides against Slug, the functional homolog of
Snail in chicks, their mesoderm formation was affected
(Nieto et al., 1994
).
Furthermore, Snail-deficient mice died at the gastrulation stage
because of incomplete EMT (Carver et al.,
2001
). These mice developed a mesodermal layer that expressed some
mesoderm-specific genes, but the mesoderm layer possessed some epithelial
characteristics such as apical-basal polarity, retaining the expression of
E-cadherin, a major cell adhesion molecule at AJs. This was consistent with
our previous observation that a Drosophila Snail mutant failed to
down-regulate E-cadherin expression at the ectoderm prior to
gastrulation (Oda et al.,
1998
).
Recently, Snail has been shown to bind directly to E-boxes in the
E-cadherin promoter and to repress E-cadherin expression
directly, resulting in the destruction of AJs
(Cano et al., 2000;
Batlle et al., 2000
). In this
study, from the viewpoint of epithelial cell polarity, we examined the
molecular mechanism for the destruction of TJs during EMT. We first
established an in vitro mouse Snail-induced EMT using mouse cultured
epithelial cells, and found that Snail directly suppressed the gene expression
of claudins and occludin. Furthermore, we showed that E-boxes in the
claudin and occludin promoters were responsible for this
Snail-induced repression of their promoter activities. We believe that these
findings provide a new insight into the molecular mechanism of EMT.
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Materials and Methods |
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SDS-PAGE and immunoblotting
The whole cell lysates of cultured cells were subjected to one-dimensional
SDS-PAGE (12.5%), according to the method of Laemmli
(Laemmli, 1970), and gels were
stained with Coomassie brilliant blue R-250. For immunoblotting, proteins were
electrophoretically transferred from gels onto nitrocellulose membranes, which
were then incubated with the first antibody. Bound antibodies were detected
with biotinylated second antibodies and streptavidin-conjugated alkaline
phosphatase (Amersham, Arlington Heights, IL). Nitroblue tetrazolium and
bromochloroindolyl phosphate were used as substrates for the detection of
alkaline phosphatase.
Immunofluorescence microscopy
Cells cultured on cover slips were rinsed twice with PBS and fixed with
ice-cooled methanol for 10 minutes. After rinsing in PBS, the fixed cells were
blocked with 1% bovine serum albumin (BSA) in PBS for 30 minutes and then
incubated with primary antibodies for 30 minutes. They were then rinsed three
times with PBS and incubated with appropriate secondary antibodies for 30
minutes. The secondary antibodies used were FITC-conjugated donkey anti-rabbit
IgG polyclonal antibody (pAb), FITC-conjugated donkey anti-mouse IgG pAb and
FITC-conjugated donkey anti-rat IgG pAb (Jackson Immunoresearch, West Grove,
PA). After rinsing with PBS, the samples were embedded in 90% glycerol-PBS
containing 0.1% paraphenylendiamine and 1%
n-propylgalate.
Northern blotting
Aliquots of total RNA (10 µg) were separated by 1.0% agaroseformaldehyde
gel electrophoresis. Hybridization with digoxigenin (DIG)-labeled RNA probes
was performed according to the manufacturer's protocol (Roche). Briefly, RNA
was transferred onto positively-charged nylon membranes, followed by
ultraviolet (UV) cross-linking. Nylon membranes were then hybridized with
DIG-labeled RNA probes at 65°C in a buffer solution containing 50%
formamide. After thorough washing and blocking, the membranes were incubated
with alkaline-phosphatase-conjugated anti-DIG antibodies for 1 hour. After
extensive rinsing, the membranes were incubated with the 1,2-dioxetane
substrate CSPD (Tropix, Bedford, MA) and exposed to X-ray film. To obtain
DIG-labeled probes, reverse-transcription PCR was performed. Total RNA was
isolated according to the method developed previously
(Chomczynski and Sacchi,
1987).
Snail expression vector and transfection
Using a total cDNA population obtained from NIH/3T3 cells as a template,
the full-length cDNA of mouse Snail was amplified by PCR and cloned
into the pCAG vector (pCAG-mSnail). Similarly, a human Snail expression vector
(pCAG-hSnail) was constructed after its full-length cDNA was amplified by PCR
using a total cDNA library of SW480 cells as a template. A mouse
Snail mutant lacking the SNAG (Snail/Gfi) domain was created
according to the method described previously
(Cano et al., 2000).
The cultured cells were transfected with one of the above expression vectors or an empty vector in serum-free DMEM containing 50 µM CaCl2 using LipofectAmine Plus (Gibco BRL). After a 2-week selection in growth medium containing 400 µg ml1 of G418, resistant colonies were separated and then screened. Ten and six independent clones were established for Snail-expressing Eph4 and CSG1 cells, respectively.
Isolation of promoter fragments, mutagenesis and reporter assays
Mouse claudin-3 (1536 to +141), mouse claudin-4
(1669 to +105), mouse claudin-7 (2336 to +195) and
human occludin (135 to +145) promoter fragments were cloned by
screening the mouse and human genomic library with respective cDNA fragments,
and were then inserted into the pGL3 vector (Promega). These reporter
constructs (0.5 µg DNA well1) were transfected into cells
cultured on 12-well dishes as described above and, 24 hours after
transfection, firefly luciferase (Luc) and Renilla luciferase (RLluc)
activities were measured using the Dual Luciferase Reporter Assay System
(Promega) according to the manufacturer's instructions. RLluc activity was
used to normalize Luc activity. In all experiments, the total amount of
transfected DNA was standardized with an empty pCAG vector.
To mutate the E-box sequence in the mouse claudin-7 promoter, a Quickchange Site Directed Mutagenesis Kit (Stratagene) was used. The core sequence, 5'-CA(G/C)(G/C)TG-3', was mutated to 5'-AA(G/C)(G/C)TA-3'.
Electrophoretic mobility shift assay
The double-stranded oligonucleotides corresponding to the following E-box
sequences were synthesized: the mouse claudin-7 E-box (+88 to +112),
5'-GGTGCGCCGCACCTGCTCG-CCCGCA-3', and the human occludin
E-box (+14 to +42), 5'-CATCCGAGTTTCAGGTGAATTGGTCACC-3'. They were
then end-labeled with 32P--CTP using the Klenow enzyme. In
mutated double-stranded oligonucleotides, the core sequence
5'-CA(G/C)(G/C)TG-3' was mutated to
5'-AA(G/C)(G/C)TA-3'. The mRNA of hemagglutinin (HA)-tagged Snail
was synthesized in vitro with T7 RNA polymerase using pCITE-HAmSnail as a
template and then translated in rabbit reticulocyte lysate (Promega).
An electrophoretic mobility shift assay was performed essentially according
to the method described previously (Kasai
et al., 1992). Briefly, in vitro translated HA-Snail protein (or
luciferase as a control) (1 µg protein) was incubated with
32P-labeled oligonucleotides in 50 ml gel retardation buffer
consisting of 12 mM HEPES (pH 7.8), 100 mM KCl, 15 mM ZnCl2, 1 mM
DTT, 12% (v/v) glycerol, 0.05% NP-40, 20 µg ml1 BSA, and
700 mg ml1 poly (dI-dC) for 30 minutes at room temperature.
For the competition experiments, unlabeled oligonucleotides were added 10
minutes before the labeled ones. To detect the super-shift of the band, the
solution was incubated with anti-HA mAb [or anti-green-fluorescent-protein
(anti-GFP) mAb as a control] for 15 minutes at room temperature. Samples were
then electrophoresed on 4% acrylamide gel with 0.5 M Tris-borate EDTA, and the
gels were dried, followed by autoradiography.
Biotinylated oligonucleotide precipitation assay
DNA precipitations were carried out essentially according to the method
described previously (Hata, 2000). Briefly, human 293 cells transiently
expressing mouse HA-Snail were lysed, and the lysate was pre-absorbed using
ImmunoPure streptavidin-agarose beads (Pierce) for 1 hour. The sample was then
incubated with 1 µg of biotinylated double-stranded oligonucleotides
corresponding to the E4 E-box sequence of the claudin-7 promoter, an
E-box sequence of the occludin promoter, or their mutated sequences
(see above), together with 10 µg of poly(dI-dC) for 16 hours. Biotinylated
DNA-protein complexes were recovered using streptavidin-agarose beads for 1
hour, rinsed with HKMG buffer (10 mM HEPES, pH 7.9, 100 mM KCl, 5 mM
MgCl2, 10% glycerol, 1 mM DTT and 0.5% NP-40) and separated on
SDS-polyacrylamide gels. Bound HA-Snail was detected by immunoblotting with
anti-HA mAb.
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Results |
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|
Snail alters the expression levels of AJ and TJ integral membrane
proteins
We examined the expression of AJ and TJ components in Eph4 and Eph4-mSnail
cells by immunoblotting (Fig.
2A). Consistent with previous observations in dog and human
epithelial cells (Cano et al.,
2000; Batlle et al.,
2000
), the expression of E-cadherin in Eph4-mSnail cells was
completely repressed. Furthermore, TJ integral membrane proteins such as
claudin-3 and occludin became undetectable at the protein level. However, the
undercoat proteins such as p120 (a cadherin-binding protein) and ZO-1 (an
occludin/claudin-binding protein) did not alter their expression levels
compared with the wild-type cells. This expression pattern of AJ and TJ
components in Eph4-mSnail cells is very similar to that in NIH/3T3
fibroblasts. We then compared the subcellular distribution of these proteins
between parental Eph4 and Eph4-mSnail cells by immunofluorescence microscopy
(Fig. 2B). In Eph4-mSnail
cells, E-cadherin became undetectable not only at cell-cell contact regions
but also in the cytoplasm. In these cells, an AJ undercoat component, p120,
showed no concentration at the cell-cell contact regions but was diffusely
distributed in the cytoplasm. Interestingly, claudin-3 and occludin, which
were concentrated at TJs in parental Eph4 cells, completely disappeared in
Eph4-mSnail cells, whereas ZO-1 (a TJ undercoat protein) changed its
localization from TJs to the cytoplasm. Non-junctional epithelial markers such
as cytokeratin-18 appeared to be down-regulated by the Snail overexpression,
but not completely.
|
The next question was whether the disappearance of occludin/claudins in Eph4-mSnail cells is due to direct repression of their transcription by Snail or to some indirect mechanism that facilitates their degradation. To address this question, we performed northern blotting (Fig. 3A). In Eph4-mSnail cells, the transcription of claudin-3, claudin-4, claudin7 and occludin, all of which were expressed abundantly in parental Eph4 cells, completely shut down together with the E-cadherin transcription. Consistent with the immunoblotting data, the mRNA levels of ZO-1 (and also p120; data not shown) did not alter significantly. Furthermore, the expression patterns of these mRNAs in Eph4-mSnail cells were very similar to those in NIH/3T3 fibroblasts and L fibroblasts (Fig. 3A,B). Taken together, we concluded that Snail directly and simultaneously represses the transcription of occludin and distinct species of claudins.
|
Snail represses the promoter activities of the claudin and
occludin genes
Random selection and transfection experiments identified a core of six
bases [CA(G/C)(G/C)TG] as the consensus binding site for Snail
(Muhin et al., 1993;
Fuse et al., 1994
;
Inukai et al., 1999
;
Kataoka et al., 2000
). This
motif is identical to the E-box. Indeed, the human E-cadherin
promoter contains three E-boxes (Fig.
4) and Snail was reported to directly bind to these E-boxes to
repress E-cadherin transcription
(Cano et al., 2000
;
Batlle et al., 2000
).
Therefore, we next examined whether the gene transcription of TJ integral
membrane proteins, claudins and occludin is also directly regulated by Snail.
Then, we isolated the promoters of mouse claudin-3, claudin-4 and
claudin-7. The putative transcription start point for each promoter
was estimated according to the expressed sequence tag database
(Fig. 4). Interestingly, these
promoters contained six, eight and eight E-boxes, respectively. The human
occludin promoter was isolated previously
(Mankertz et al., 2000
) and
also contained one E-box (Fig.
4).
|
We then inserted the isolated fragments of the claudin-3,
claudin-4 and claudin-7 promoters into the pGL3 plasmid upstream
of the luciferase reporter gene, and transfected these reporter constructs
into Eph4 epithelial cells and NIH/3T3 fibroblasts
(Fig. 5A). In Eph4 cells, the
claudin promoters induced a three- to tenfold increase in relative
luciferase activity above that observed in NIH/3T3 cells, indicating that
these promoter regions were sufficient to show the epithelium-specific
activity. As previously reported (Mankertz
et al., 2000), the isolated occludin promoter also showed
similar epithelium-specific activity (data not shown). We next examined the
ability of Snail to repress the claudin promoter activities. When the
Snail expression vector and the claudin reporter constructs were
co-transfected into Eph4 cells, the promoter activities of claudin-3,
claudin-4 and claudin-7 were remarkably repressed
(Fig. 5B), and this repression
depended on the dose of Snail
(Fig. 5C). Furthermore, when
co-transfected with a Snail mutant lacking the N-terminal SNAG
domain, which is required for the repressor activity of Snail in general
(Grimes et al., 1996
;
Nakayama et al., 1998
), the
claudin-7 promoter activity was not repressed
(Fig. 5D). Similar
Snail-induced repression was also observed for the occludin promoter
in human epithelial cells (HT29) (Fig.
5E). These findings indicated that the transcription of
claudins and occludin was directly regulated by Snail by
modulating the activities of their promoters.
|
Snail directly binds to E-boxes in the claudin-7 promoter
To clarify further the molecular mechanism behind the Snail-induced
repression of the claudin transcription, we examined a shorter fragment of the
claudin-7 promoter in more detail
(Fig. 6A). This short fragment,
with five E-boxes, also showed epithelium-specific promoter activity (data not
shown). We generated a series of reporter constructs that carried various
combinations of mutated E-boxes and introduced them into Eph4 cells together
with the Snail expression vector or an empty vector
(Fig. 6B). Interestingly, when
a single E-box was mutated, no significant impairment of Snail-induced
repression was observed. However, as the number of mutated E-boxes was
increased, the claudin-7 promoter became less sensitive to Snail
(Fig. 6B,C), suggesting that
E-boxes in the claudin-7 promoter are responsible for the
Snail-induced repression.
|
|
Finally, to confirm the binding of Snail to the E-box (E4) of the claudin-7 promoters in the nuclear extract, we performed a biotinylated oligonucleotide precipitation assay (Fig. 7C, lanes 1-3). Biotin-labeled double-stranded wild-type or mutated oligonucleotides of the claudin-7 E4 sequence (Fig. 6A) were incubated with a nuclear extract prepared from 293 cells transfected with the HA-Snail expression vector or an empty vector. The biotinylated oligonucleotides were then recovered using streptavidin-conjugated agarose beads, and bound HA-Snail was detected by immunoblotting with anti-HA mAb. The HA-Snail in the nuclear extracts bound specifically to the wild-type, but not to the mutated, oligonucleotides (Fig. 7C). Similar specific binding was detected when the oligonucleotides of the occludin E-box sequence were used (Fig. 7C, lanes 4-6). Taken together, we concluded that Snail binds directly to E-boxes in the claudin and occludin promoters, and that Snail directly represses their activities.
![]() |
Discussion |
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The promoter of E-cadherin has been identified as a direct target
for Snail. Snail directly represses E-cadherin promoter activity
(Cano et al., 2000;
Batlle et al., 2000
). During
EMT, the epithelial phenotypes of cells are converted into mesenchymal
phenotypes: various gene products involved in the epithelial phenotypes must
alter in their distribution and expression. Many of these alterations could be
explained as secondary events induced by the down-regulation of E-cadherin.
Indeed, several lines of evidence have shown that the disappearance or
dysfunction of E-cadherin results in the loss of epithelial cell polarity
(Takeichi, 1991
;
Rodriguez-Boulan and Nelson,
1989
). For example, when epithelial cells were cultured at low
Ca2+ concentration (to cause E-cadherin dysfunction), the
junctional complex, including TJs, was destroyed and epithelial cell polarity
was lost. By contrast, it was reported that the Snail-induced phenotypic
changes could not simply be attributed to the loss of E-cadherin. When
E-cadherin was exogenously expressed in Snail-expressing epithelial cultured
cells, which lost the expression of endogenous E-cadherin showing mesenchymal
phenotypes, the epithelial phenotypes were not completely restored
(Cano et al., 2000
). Therefore,
we must search for other direct targets for Snail for a better understanding
of the molecular mechanism behind EMT.
In this study, we examined the behavior of claudins and occludin, major
constituents of TJ strands, using the in vitro Snail-induced EMT system.
Because TJs are the key structures responsible for establishing and
maintaining epithelial cell polarity, claudins and occludin were expected to
show a remarkable change in their distribution during EMT. Surprisingly,
however, their expression completely shut down at the transcription level.
Furthermore, we found that, similar to the Snail-based regulation of
E-cadherin transcription (Cano et
al., 2000; Batlle et al.,
2000
), Snail completely repressed the claudin and
occludin promoter activities through its direct binding to E-boxes in
these promoters. These findings unraveled the very sophisticated mechanism by
which Snail caused EMT. Snail directly and simultaneously represses the
expression of two distinct groups of important intercellular adhesion
molecules, E-cadherin and claudins/occludin, which function at AJs and TJs,
respectively.
The regulatory mechanism of the formation and destruction of TJs has been
examined extensively, but promoter analyses of claudins and occludin are only
just beginning. The expression of occludin was shown to be regulated by
several factors at the transcription level
(Mankertz et al., 2000;
Chen et al., 2000
;
Li and Mrsny, 2000
), and
several transcription factors (such as the ß-catenin/Tcf complex and Cdx
homeodomain proteins/hepatocyte nuclear factor 1) were reported to bind
directly to claudin-1 and claudin-2 promoters, respectively
(Miwa et al., 2001
;
Sakaguchi et al., 2002
). These
findings indicated that the expression of claudins and occludin is finely
controlled depending on the physiological and pathological conditions, but our
data revealed that Snail eliminates these fine regulations to shut off the
expression of distinct claudin species and occludin completely and
simultaneously. It remains unclear why the expression of claudins and occludin
must be repressed so completely in mesenchymal cells but, conversely, these
data favored the notion that claudins and occludin, as well as E-cadherin, are
key determinants of the epithelial phenotype, including epithelial cell
polarity.
Finally, we should briefly discuss the relationship between Snail and TJs,
in particular the expression of claudins, in malignant tumors. In some types
of tumors of epithelial origin, Snail expression has been reported, when
cancer cells acquired an invasive phenotype
(Cano et al., 2000;
Batlle et al., 2000
). However,
the expression patterns of claudins varied significantly depending on the type
of tumor. In some breast cancers and squamous adenocarcinomas, claudins were
frequently down-regulated (Kramer et al.,
2000
; Al Moustafa et al.,
2002
). It is thus possible that this repression is due to the
upregulation of Snail expression, although this has not yet been
examined in detail. Recently, E12/47 and SIP1 were also found to down-regulate
E-cadherin expression through direct binding to single or paired
E-boxes (Perez-Moreno et al.,
2001
; Comijn et al.,
2001
; Bolos et al.,
2003
). Therefore, the possibility could not be excluded that, in
tumors, down-regulation of claudins occur via upregulation of these
non-Snail-related silencers. In future studies, we must further clarify the
pathological relevance of down-regulated claudin expression as well
as the possible involvement of Snail in the alteration of claudin
expression in malignant tumors.
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Acknowledgments |
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![]() |
Footnotes |
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