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The Rockefeller University Press, 0021-9525/2002/3/1099/13 $5.00
The Journal of Cell Biology, Volume 156, Number 6, March 18, 2002 1099–1111
http://www.jcb.org/cgi/doi/10.1083/jcb.200110122
JCB
Article
1099
Claudin-based tight junctions are crucial for
the mammalian epidermal barrier: a lesson
from claudin-1–deficient mice
Mikio Furuse,
1
Masaki Hata,
2
Kyoko Furuse,
2
Yoko Yoshida,
3
Akinori Haratake,
4
Yoshinobu Sugitani,
5
Tetsuo Noda,
5,6
Akiharu Kubo,
1
and Shoichiro Tsukita
1
1
Department of Cell Biology, Kyoto University Faculty of Medicine, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan
2
KAN Research Institute, Inc., Kyoto Research Park, Chudoji, Shimogyo-ku, Kyoto 600-8317, Japan
3
Department of Dermatology, Kyoto University Faculty of Medicine, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
4
Basic Research Laboratory, Kanebo, Ltd., Kotobuki-cho, Odawara, Kanagawa 250-0002, Japan
5
Department of Cell Biology, Japanese Foundation for Cancer Research-Cancer Institute, Kami-Ikebukuro, Toshima-ku,
Tokyo 170-8455, Japan
6
Department of Molecular Genetics, Tohoku University School of Medicine, Seryo-cho, Aoba-ku, Sendai 980-8575, Japan
he tight junction (TJ) and its adhesion molecules,
claudins, are responsible for the barrier function of
simple epithelia, but TJs have not been thought to
play an important role in the barrier function of mammalian
stratified epithelia, including the epidermis. Here we
generated claudin-1–deficient mice and found that the
animals died within 1 d of birth with wrinkled skin. Dehy-
dration assay and transepidermal water loss measurements
revealed that in these mice the epidermal barrier was severely
affected, although the layered organization of keratino-
cytes appeared to be normal. These unexpected findings
prompted us to reexamine TJs in the epidermis of wild-type
mice. Close inspection by immunofluorescence microscopy
with an antioccludin monoclonal antibody, a TJ-specific
T
marker, identified continuous TJs in the stratum granulosum,
where claudin-1 and -4 were concentrated. The occurrence
of TJs was also confirmed by ultrathin section EM. In
claudin-1–deficient mice, claudin-1 appeared to have
simply been removed from these TJs, leaving occludin-
positive (and also claudin-4–positive) TJs. Interestingly, in
the wild-type epidermis these occludin-positive TJs efficiently
prevented the diffusion of subcutaneously injected tracer
(
600 D) toward the skin surface, whereas in the claudin-1–
deficient epidermis the tracer appeared to pass through
these TJs. These findings provide the first evidence that
continuous claudin-based TJs occur in the epidermis and
that these TJs are crucial for the barrier function of the
mammalian skin.
Introduction
For homeostasis within multicellular organisms, the internal
environment must be isolated and buffered against the vagaries
and extremes of the external environment and further divided
into various compositionally distinct fluid compartments.
This isolation/compartmentalization is established by cellular
sheets of epithelia (including endothelia and mesothelia) delin-
eating the body surface and cavities. The epithelia are classified
into two types according to the number of cell layers, simple
and stratified: the former consists of a single layer of polarized
cells, whereas the latter, including the epidermis, is character-
ized by multiple layers of less polarized cells.
In vertebrate simple epithelial cells, tight junctions (TJs),*
one mode of intercellular adhesion, occur in the most apical
region of the lateral membranes and have been thought to
create a primary barrier to the diffusion of solutes through
the paracellular route: this TJ-based intercellular sealing is
indispensable for the compartmentalization within the body
(Schneeberger and Lynch, 1992; Gumbiner, 1993; Anderson
and van Itallie, 1995). TJs were first identified by EM. On
ultrathin section EM, TJs appear as a zone where plasma
Address correspondence to Shoichiro Tsukita, Dept. of Cell Biology,
Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501,
Japan. Tel.: 81-75-753-4372. Fax: 81-75-753-4660. E-mail:
htsukita@mfour.med.kyoto-u.ac.jp
Key words: claudin-1; tight junction; skin; epidermis; barrier
*Abbreviations used in this paper: CE, cell envelope; ES, embryonic
stem; mAb, monoclonal antibody; pAb, polyclonal antibody; RT, reverse
transcription; TJ, tight junction; TEWL, transepidermal water loss.
on November 14, 2019jcb.rupress.org Downloaded from http://doi.org/10.1083/jcb.200110122Published Online: 11 March, 2002 | Supp Info:
1100 The Journal of Cell Biology
|
Volume 156, Number 6, 2002
membranes of neighboring cells focally make complete con-
tact (Farquhar and Palade, 1963). On freeze-fracture EM,
TJs are visualized as a continuous anastomosing network of
intramembranous particle strands (TJ strands or fibrils) and
complementary grooves (Staehelin, 1974). These observa-
tions led to our current understanding of the three-dimen-
sional structure of TJs; each TJ strand associates laterally
with another TJ strand in apposing membranes of adjacent
cells to form “paired” TJ strands, where the intercellular
space is completely obliterated (Tsukita et al., 2001).
The molecular architecture of TJ strands has been unrav-
eled in recent years. Occludin with four transmembrane do-
mains was identified as the first integral membrane protein
localizing at TJs (Furuse et al., 1993;
Ando-Akatsuka et al.,
1996). Immunoreplica EM and immunofluorescence mi-
croscopy revealed that occludin is a bona fide component of
TJ strands in most types of cells, that is, occludin is a very
good marker for TJ strands themselves (Fujimoto, 1995; Fu-
ruse et al., 1996; Saitou et al., 1997). However, gene knock-
out analyses at the mouse embryonic stem (ES) cell and
whole body level showed that occludin is not indispensable
for the formation of TJ strands (Saitou et al., 1998, 2000).
Although several lines of evidence suggested some important
role for occludin in TJ strands, our knowledge on its physio-
logical function is still fragmentary (Balda et al., 1996; Mc-
Carthy et al., 1996; Chen et al., 1997; Wong and Gum-
biner, 1997).
Recently, two related integral membrane proteins with
molecular masses of
23 kD, claudin-1 and -2, were identi-
fied as components of TJ strands (Furuse et al., 1998a). Fur-
thermore, claudins were shown to comprise a multigene
family consisting of
20 members (Furuse et al., 1998a;
Morita et al., 1999a,b,c; Simon et al., 1999; Tsukita and Fu-
ruse, 1999). Claudin molecules also bore four transmem-
brane domains but showed no sequence similarity to occlu-
din. Interestingly, when an individual claudin species was
singly introduced into mouse L fibroblasts lacking TJs clau-
din molecules were concentrated at cell–cell contact planes
and polymerized within apposing plasma membranes to re-
constitute paired TJ strands (Furuse et al., 1998b). Through
further detailed transfection experiments and immunolabel-
ing studies, it is now widely accepted that heterogeneous
claudin species constitute the backbone of TJ strands in situ,
that is, TJ strands are copolymers of heterogeneous claudin
species (and also occludin) (Furuse et al., 1999).
When MDCK I cells, in which TJ strands were prima-
rily composed of claudin-1 and -4, were incubated with a
claudin-4–binding peptide (the COOH-terminal half of
Clostridium perfringens
enterotoxin), claudin-4 was specifi-
cally removed from TJ strands, resulting in a significant in-
crease in TJ permeability (Sonoda et al., 1999). Further-
more, positional cloning recently identified claudin-14 as
the gene responsible for human hereditary deafness (Wil-
cox et al., 2001). Mutations in this gene were interpreted
to cause an increase in the TJ permeability of the Corti or-
gan, affecting the compartmentalization in the cochlea.
These findings indicated that claudins are directly involved
not only in the formation of TJ strands but also in their
barrier function in simple epithelia. On the other hand,
morphological and physiological studies so far have re-
vealed that TJs are not a simple barrier: they show ion and
size selectivity, and their barrier function varies signifi-
cantly in tightness depending on the cell type and physio-
logical requirements, and to explain these characteristics
aqueous pores (or paracellular channels) have been postu-
lated to exist within paired TJ strands (Diamond, 1977;
Claude, 1978; Reuss 1992; Gumbiner, 1993). Recent anal-
yses of human hereditary hypomagnesemia, in which the
claudin-16 gene is mutated (Simon et al., 1999), and de-
tailed transfection experiments using MDCK cells (Furuse
Figure 1.
Generation of claudin-1–
deficient mice.
(A) Restriction maps of
the wild-type allele, the targeting vector,
and the targeted allele of the mouse
claudin-1 gene. The first ATG codon
was located in the putative exon 1,
which encoded the NH
2
-terminal
portion (amino acids 1–75) of the
claudin-1 molecule containing the first
transmembrane domain and the first
extracellular loop. The targeting vector
contained the pgk-neo cassette in its
middle portion to delete most of exon 1
in the targeted allele. The position of the
probe for Southern blotting is indicated
as a bar. E, EcoRI; H, HindIII; S, SacI. (B)
Genotype analyses by Southern blotting
of HindIII-digested genomic DNA from
wild-type (
/
), heterozygous (
/
),
and homozygous (
/
) mice for the
mutant claudin-1 gene allele. Southern
blotting with the probe indicated in A
yielded a 6.8- and 3.9-kb band from the
wild-type and targeted allele, respectively. (C) Loss of claudin-1 mRNA in the liver of claudin-1
/
mice examined by RT-PCR. As a control, the
hypoxanthine phosphoribosyl transferase gene was equally amplified in all samples. (D) Loss of the claudin-1 protein in the liver of claudin-1
/
mice examined by immunofluorescence microscopy. Frozen sections of the liver were stained with anti–claudin-1 pAb. In the wild-type liver,
claudin-1 was concentrated at TJs along bile canaliculi, whereas in the claudin-1
/
liver these signals became undetectable. Bar, 20
m.
Claudin-1 and the mammalian epidermal barrier |
Furuse et al. 1101
et al., 2001; Van Itallie et al., 2001) suggested that the
density and nature of aqueous pores are determined by the
combination and the mixing ratio of claudins in individual
paired TJ strands (Tsukita and Furuse, 2000). Thus, clau-
dins are thought to play crucial roles in homeostasis within
multicellular organisms at least in vertebrates: (a) claudins
confer the barrier function on simple epithelia by consti-
tuting TJ strands, and (b) claudins are directly involved in
the transport of materials across simple epithelia through
the paracellular pathway by tuning the tightness and the
selectivity of TJ strands.
In this study, as a first step to elucidating the role of
claudins at the whole body level we generated mice lacking
claudin-1, which is expressed in most organs but in espe-
cially large amounts in the liver and kidney (Furuse et
al., 1998a). Interestingly, claudin-1–deficient mice died
within 1 d of birth and showed severe defects in the perme-
ability barrier of the epidermis. This finding was unex-
pected because in the past several decades the roles of TJs
in the barrier function of mammalian stratified epithelia,
especially of the epidermis, have been mostly ignored
(Squier, 1973; Elias and Friend, 1975; Elias et al., 1977).
This study not only provides a completely new insight into
the barrier mechanism of stratified epithelia, especially the
skin, but also implicates claudins, that is, TJs, in the com-
partmentalization of mammals in more general ways than
ever expected.
Results
Generation of claudin-1–deficient mice showing rapid
postnatal lethality
To explore the function of claudin-1 in vivo, we produced
mice unable to express it. Nucleotide sequencing and restric-
tion mapping identified four exons that cover the whole ORF
of claudin-1: the putative exon 1 contained the first ATG and
encoded the NH
2
-terminal portion (amino acid 1–75) of the
claudin-1 molecule containing the first transmembrane do-
main and the first extracellular loop (Fig. 1 A). We con-
structed a targeting vector, which was designed to disrupt the
claudin-1 gene by replacing most of exon 1 with the neomy-
cin resistance gene (Fig. 1 A). Two distinct lines of mice were
generated from distinct ES cell clones in which the claudin-1
gene was disrupted by homologous recombination. Southern
blotting confirmed the disruption of the claudin-1 gene in
heterozygous and homozygous mutant mice (Fig. 1 B), and
reverse transcription (RT)-PCR detected no claudin-1 mRNA
from the liver of homozygous mutant mice (Fig. 1 C). Consis-
tently, the claudin-1 protein, which was concentrated at TJs
along bile canaliculi in the liver of wild-type newborn mice,
was not detected in the liver of homozygous mutant mice by
immunofluorescence microscopy (Fig. 1 D). Because both
lines of mice showed the same phenotypes, we will mainly
present data obtained from one line.
No obvious phenotype was apparent in heterozygous mu-
tant mice, and when these were interbred wild-type, het-
Figure 2. Impairment of the epidermal
barrier in claudin-1–deficient mice. (A)
12-h-old claudin-1/, claudin-1/, and
claudin-1/ mice. Claudin-1/ mice
were characterized by wrinkled skin and
died within 1 d of birth. (B) Dehydration
assay. After Cln1
/
intercross litter-
mates obtained by Caesarian section at
embryonic day 18.5 were resuscitated,
their weights were monitored hourly
without feeding. Genotyping was per-
formed afterwards. Only Cln1
/
mice
showed rapid and steady weight loss.
(C) TEWL measurements in two sets of
Cln1
/
intercross newborns. Genotyping
identified two Cln1
/
newborns, and
only these mice showed excessive TEWL.
1102 The Journal of Cell Biology
|
Volume 156, Number 6, 2002
erozygous, and homozygous mutant mice were produced in
the expected Mendelian ratios (Table I). At birth, homozy-
gotes were macroscopically indistinguishable from wild-type
and heterozygous littermates. However, their skin soon be-
gan to show a wrinkled appearance (Fig. 2 A), and their
movements ceased. All homozygotes died within 1 d of birth.
Impairment of the epidermal barrier in
claudin-1–deficient mice
TJs, that is, claudins, were not thought to be involved directly
in the barrier function of stratified epithelial cells, especially of
the skin, but the abnormal skin appearance of claudin-1
/
(
Cln1
/
) mice led us to examine their epidermal barrier in
detail. First, to simply check the loss of water across the skin,
according to the method described previously (Segre et al.,
1999), we obtained
Cln1
/
intercross littermates by Cae-
sarian section at embryonic day 18.5, resuscitated them, mon-
itored their weights hourly without feeding, and genotyped
them. In a representative experiment, two mice showed rapid
and steady weight loss (down to
85% of the initial body
weight after 12 h), whereas the others maintained weight (Fig.
2 B). Interestingly, only the two littermates showing weight
loss were identified as
Cln1
/
mice. This perfect correlation
between
Cln1
/
genotype and weight loss was reproducibly
obtained in different series of measurements. Since urination
was not detected in most littermates examined, the rapid
weight loss observed in
Cln1
/
mice may be attributed to the
evaporation of water through the epidermis. To confirm this
interpretation, we directly measured the transepidermal water
loss (TEWL) in two sets of
Cln1
/
intercross newborns,
which were afterwards genotyped. As shown in Fig. 2 C,
in good agreement with the data of body weight loss only
Cln1
/
mice showed excessive TEWL compared with
Cln1
/
and
Cln1
/
mice. We then concluded that the epidermal bar-
rier of
Cln1
/
mice is severely affected.
Morphology of the epidermis of claudin-1–deficient mice
The unexpected findings on defects in the epidermal barrier
of
Cln1
/
mice prompted us to examine the morphology of
their skin after confirming the presence and absence of clau-
din-1 in the skin of
Cln1
/
/
Cln1
/
and
Cln1
/
mice, re-
spectively, by immunoblotting (Fig. 3 A). In spite of its severe
defects of barrier function, the epidermis of newborn
Cln1
/
mice did not exhibit overt abnormalities in the layered organi-
zation of keratinocytes at the level of hematoxylin-eosin–
stained paraffin section images (Fig. 3 B). The epidermis of
newborn
Cln1
/
mice consists of a single cell layer of stratum
basale and 2–3 cell layers each of strata spinosum and granulo-
sum, which are covered with stratum corneum. Strata basale,
spinosum, and granulosum of newborn
Cln1
/
mice were in-
distinguishable from those of
Cln1
/
mice. The only differ-
ence was detected in stratum corneum: under conventional
fixation/embedding conditions, flattened cells in the upper
portion of the stratum corneum of
Cln1
/
mice appeared to
peel off, showing a loose basket-weave pattern, whereas in the
Cln1
/
skin cornified cells were compacted more tightly. Ul-
trathin section EM confirmed these findings at higher resolu-
tion (Fig. 3 C): the appearance of individual cells from stra-
tum basale to granulosum was indistinguishable between the
Cln1
/
and
Cln1
/
skin.
Occludin, claudins, and TJs in the epidermis of
wild-type and claudin-1–deficient mice
As a deficiency of claudin-1 appeared to make the epidermis
leaky without disorganizing the layered cellular architecture,
we were led to reexamine the occurrence and distribution of
TJs in the mouse epidermis. Occludin is known to be highly
Table I.
Genotypic analysis of offsprings from
heterozygous/heterozygous breeding pairs
Wild-type Heterozygous Homozygous
27 55 34
Offsprings from 15 breeding pairs were analyzed.
Figure 3. Morphology of the epidermis of claudin-1–deficient
mice. (A) Anti–claudin-1 pAb immunoblotting of the whole lysate
of the skin obtained from Cln1
/
, Cln1
/
, and Cln1
/
newborn
mice. As a control, the cell lysate of L fibroblast transfectants
exogenously expressing mouse claudin-1 (C1L cells) was used.
Claudin-1 was detected in the Cln1
/
and Cln1
/
skin but not in
the Cln1
/
skin. Bars indicate molecular sizes as 31, 21, and 14 kD
from the top. (B) Hematoxylin-eosin–stained sectional images of
skin obtained from the back of Cln1
/
and Cln1
/
newborns. The
epidermis of Cln1
/
newborn mice did not exhibit overt abnormal-
ities in the layered organization of keratinocytes except that stratum
corneum appeared to be thicker and compacted tighter than that of
the Cln1
/
epidermis. SC, stratum corneum; GR, stratum granulosum;
SP, stratum spinosum; BL, stratum basale. Bar, 20 m. (C) Ultrathin
section electron microscopic images of skin obtained from the back
of Cln1
/
and Cln1
/
newborns. The appearance of individual
keratinocytes was indistinguishable between the Cln1
/
and Cln1
/
skin. Bars, 0.5 m.
Claudin-1 and the mammalian epidermal barrier | Furuse et al. 1103
concentrated at TJ strands in most simple epithelial cells,
that is, occludin is the best general marker for TJ strands
identified to date. Therefore, we first reexamined the distri-
bution of occludin in the skin of wild-type newborn mice.
As reported previously, occludin was detected in stratum
granulosum consisting of 2–3 layers of granular cells, but
close inspection in transverse sections revealed that occludin
was concentrated as dots in the most apical region of the lat-
eral membranes of the granular cells in the second layer (Fig.
4 A). This dot-like concentration of occludin was also de-
tected in some but not all of the granular cells in the first
(uppermost) and/or third layer. However, as shown in Fig. 6
A, when this immunostaining was done for thicker tangen-
tial sections it became clear that occludin is concentrated not
as dots but as lines circumscribing individual granular cells
continuously. Interestingly, these honeycomb networks of
continuous TJs were occasionally observed in two layers.
In contrast, intense signals for claudin-1 were detected
throughout plasma membranes of epidermal keratinocytes
from stratum basale to granulosum of the wild-type epider-
mis, although the signal from the first (uppermost) layer of
granular cells was fairly weak (Fig. 4 A). However, in the
lateral membranes of granular cells in the second layer
claudin-1 appeared to be concentrated as dots in the most
apical regions. When sections were double stained with an-
tioccludin monoclonal antibody (mAb) and anti–claudin-1
polyclonal antibody (pAb), these claudin-1 concentrations
coincided precisely with the occludin concentrations. In
thicker oblique sections, claudin-1 was coconcentrated pre-
cisely at the occludin-positive lines. Furthermore, in addi-
tion to claudin-1 we found that claudin-4 was also ex-
pressed in the epidermis (Fig. 4 B). Interestingly, distinct
from claudin-1, claudin-4 was distributed mainly in the
second/third layers of stratum granulosum. Claudin-4 was
Figure 4. Occludin, claudin-1, and
claudin-4 in the wild-type epidermis.
(A) Double immunofluorescence
microscopy of frozen sections of skin
from the back of wild-type mice with
antioccludin mAb and anti–claudin-1
pAb. In transverse sections (a and b),
occludin was concentrated as dots in
the most apical region of the lateral
membranes of the granular cells in the
second layer (arrowheads). This dot-like
concentration of occludin was also
found in some but not all of the granular
cells in the first (uppermost) and/or third
layer. Claudin-1 was distributed diffusely
throughout plasma membranes of
keratinocytes from stratum basale to
granulosum, although the signal from
the first layer of stratum granulosum was
fairly weak (a). At higher magnification
(b), in the lateral membranes of granular
cells in the second layer claudin-1 was
coconcentrated as dots together with
occludin in the most apical regions
(arrowheads). In thicker oblique sections
(c), claudin-1 was coconcentrated pre-
cisely at the occludin-positive lines
(arrowhead). SC, stratum corneum. The
broken line represents the dermis/
epidermis border. Bars, 10 m. (B) Dou-
ble immunofluorescence microscopy of
frozen sections of skin from the back of
wild-type mice with antioccludin mAb
and anti–claudin-4 pAb. Distinct from
claudin-1, claudin-4 was distributed
mainly in the second/third layers of
stratum granulosum (a). Claudin-4 was
also concentrated at the occludin-positive
lines circumscribing granular cells
(arrowhead) (b). SC, stratum corneum.
The broken line represents the dermis/
epidermis border. Bars, 20 m.
1104 The Journal of Cell Biology | Volume 156, Number 6, 2002
also concentrated at the occludin-positive lines circum-
scribing granular cells.
We then observed the epidermis of wild-type newborn
mice by ultrathin section EM. As reported previously, it
was difficult to detect typical TJs when we simply ob-
served the wild-type epidermis. However, to our surprise
when we focused on the most apical region of the lateral
membranes of the granular cells in the second layer, with-
out exception we detected typical TJs just above the des-
mosomes (Fig. 5).
In the epidermis of Cln1
/
newborn mice, occludin was
still concentrated in the most apical region of lateral mem-
branes of granular cells, although the intensity of occludin
signals appeared to be slightly weaker than that in the
Cln1
/
epidermis. The continuity of the occludin-positive
lines circumscribing individual granular cells did not ap-
pear to be affected at least at the light microscopic level by
claudin-1 deficiency (Fig. 6 A). As expected, claudin-1 was
undetectable in the Cln1
/
epidermis, but the subcellular
distribution of claudin-4 and the intensity of its signal
were also indistinguishable from those in the Cln1
/
epi-
dermis (Fig. 6 B). EM of the Cln1
/
epidermis revealed
that at the most apical region of the lateral membranes of
granular cells in the second layer TJs were observed fre-
quently. However, it was technically difficult to quantita-
tively evaluate the difference in the extent of development
of TJs between the Cln1
/
and Cln1
/
epidermis by ul-
trathin section EM (Fig. 7).
These findings favored the notion that continuous TJs con-
sisting of at least occludin, claudin-1, and claudin-4 occur in
the stratum granulosum of the wild-type epidermis and that
in Cln1
/
mice claudin-1 was simply removed from TJs and
other nonjunctional plasma membranes, whereas the layered
organization of keratinocytes was unaffected.
Barrier functions of TJs in the epidermis
Then questions have arisen whether TJs in the wild-type
epidermis can create a primary barrier to the diffusion of
materials through the paracellular pathway and whether the
barrier function of TJs is affected in the Cln1
/
epidermis.
To address these questions, we performed a tracer experi-
ment according to the method developed by Chen et al.
(1997). We injected an isotonic solution containing a pri-
mary amine-reactive biotinylation reagent (mol wt. 557 D),
which covalently cross-links to an accessible cell surface, sub-
cutaneously into the back of Cln1
/
and Cln1
/
new-
borns, and after 30 min incubation the skin was dissected
out and frozen. Frozen sections were double labeled with an-
tioccludin mAb in green and streptavidin in red to detect
TJs and bound biotin, respectively (Fig. 8). In the Cln1
/
epidermis, the biotinylation reagent appeared to diffuse
through the paracellular spaces from stratum basale to the
second layer of stratum granulosum, but this diffusion was
abruptly prevented at the points where occludin was concen-
trated, that is, TJs (Fig. 8, a–c). In sharp contrast, in the
Cln1
/
epidermis the diffusion of injected biotinylation re-
agent was not stopped at the occludin-positive TJs, but the
reagent appeared to pass through TJs to reach the border be-
tween the stratum granulosum and corneum (Fig. 8, d–f).
These findings clearly indicate that TJs function as a barrier
at least against small molecules 600 D in the wild-type
epidermis and that this barrier function of TJs was severely
affected in the Cln1
/
epidermis.
Stratum corneum in claudin-1–deficient mice
Several lines of evidence have indicated that stratum cor-
neum, especially intercellular lipid lamellae and cornified
cell envelopes (CEs), play a central role in the mammalian
epidermal barrier (Rice and Green, 1978; Elias, 1983;
Figure 5. Ultrathin section electron
microscopic images of wild-type
epidermis. (a and b) Low power electron
micrograph (a) of the stratum corneum
(SC) and the first through third layers of
stratum granulosum (SG1, SG2, and
SG3) and a corresponding schematic
drawing (b). (c) A boxed area in a and b
where occludin was expected to be
concentrated was enlarged. Typical TJ
(TJ) was detected just above desmosome
(DS). (d) Another example of the TJ–
desmosome complex observed at the
most apical region of the lateral mem-
branes of granular cells in the second
layer. Lipid lamellar bodies (arrowheads).
Kissing points of TJs (arrows). Bars: (a)
400 nm; (c) 200 nm; (d) 100 nm.
Claudin-1 and the mammalian epidermal barrier | Furuse et al. 1105
Downing, 1992; Roop, 1995; Steinert, 2000). Therefore,
we examined these structures morphologically and biochem-
ically in the epidermis of Cln1
/
mice. First, to visualize the
lipid lamellae by ultrathin section EM the skin removed
from Cln1
/
and Cln1
/
newborn mice was fixed with
glutaraldehyde followed by ruthenium tetroxide. As shown
in Fig. 9 A, in the Cln1
/
epidermis the lamellar bodies and
the well-developed/well-organized lamellae were clearly ob-
served between granular cells and cornified cells and also be-
tween flattened cornified cells, respectively, and these struc-
tures were not distinguished from those in the Cln1
/
epidermis. Furthermore, we compared the lipid contents of
the stratum corneum between Cln1
/
and Cln1
/
mice
but found no significant difference between the lipid profiles
of the two mice (unpublished data).
Then we examined CEs in which several types of pro-
teins such as loricrin and involucrin are covalently cross-
linked by transglutaminase-1. CEs were therefore very sta-
ble and were isolated by treating the wild-type epidermis
with 2% SDS in the presence of 5% 2-mercaptoethanol
(Fig. 9 B, /). Under the same isolation conditions, CEs
were also isolated efficiently from the Cln1
/
and Cln1
/
epidermis, and they were morphologically indistinguish-
able from those of the Cln1
/
epidermis (Fig. 9 B, /
and /). Finally, we examined the expression of loricrin,
involucrin, transglutaminase-1 (Matsuki et al., 1998), and
Klf4 (Segre et al., 1999) in the Cln1
/
epidermis by im-
munoblotting or Northern blotting. As shown in Fig. 9, C
and D, a deficiency of claudin-1 did not appear to affect
their expression.
Transplantation of the claudin-1–deficient skin
to nude mice
The above observations strongly support the notion that
defects in the epidermal barrier in Cln1
/
mice are due
to the disappearance of claudin-1 from the epidermis it-
self. However, since claudin-1 is expressed in various tis-
sues of wild-type mice including the liver and kidney it is
Figure 6. Occludin, claudin-1, and
claudin-4 in the claudin-1–deficient
epidermis. (A) Immunofluorescence
microscopy of frozen sections of skin
from the back of Cln1
/
and Cln1
/
newborn mice with antioccludin mAb. In
thick tangential sections of the Cln1
/
(/) and Cln1
/
(/) skin, the
occludin signal was detected as continu-
ous lines circumscribing individual
granular cells (top). In the Cln1
/
skin,
these honeycomb networks of continuous
TJs were occasionally observed in two
layers, and this characteristic feature of
TJs was not affected by a deficiency of
claudin-1 (bottom). Bars, 20 m. (B)
Double immunofluorescence micros-
copy of frozen sections of skin from the
back of Cln1
/
newborn mice with
antioccludin mAb and anti–claudin-1 (a)
or anti–claudin-4 pAb (b). As expected,
the claudin-1 signal was undetectable,
and the distribution of claudin-4 was
indistinguishable from that in the Cln1
/
skin (Fig. 4 B). Occludin-positive TJs
(arrowheads). SC, stratum corneum.
The broken line represents the dermis/
epidermis border. Bars, 20 m.
1106 The Journal of Cell Biology | Volume 156, Number 6, 2002
theoretically possible that the epidermal barrier defects
are caused by some hormonal abnormality. To address
this issue, skin fragments were removed from the back of
Cln1
/
and Cln1
/
newborn mice and grafted onto the
back of nude mice. Interestingly, at 1 mo after transplan-
tation the Cln1
/
grafts showed abnormalities in some
respects compared with the Cln1
/
grafts: macroscopi-
cally, the Cln1
/
grafts bore numerous long hairs,
whereas the Cln1
/
grafts were associated only with a
small number of short hairs (Fig. 10 A). Furthermore, the
Cln1
/
grafts became thicker than the Cln1
/
grafts,
and in some places they appeared to be covered with infil-
trates. Histological analyses revealed that the epidermis of
the Cln1
/
grafts was unusually thick unlike the back of
Cln1
/
newborn mice and the Cln1
/
grafts, although
hair follicles appeared to be developed even in the Cln1
/
grafts (Fig. 10 B). The molecular mechanism behind the
hair abnormality in the Cln1
/
grafts remains unknown,
but this finding indicated that claudin-1 deficiency af-
fected the epidermis directly, not through some indirect
hormonal action. Furthermore, the thickening of the epi-
dermis, that is, the hyperproliferation of keratinocytes in
the epidermis, is widely recognized as a typical compensa-
tory response of an epidermis that is severely compro-
mised in its barrier function. Therefore, it would be rea-
sonable to consider that claudin-1 deficiency directly
increased the permeability of the epidermis.
Discussion
Claudins, which comprise a multigene family, have been
shown to constitute the backbone of TJ strands in simple
epithelial cells and be directly involved in their barrier func-
tion (Tsukita et al., 2001). In this study, we generated mice
lacking claudin-1 by homologous recombination. These
Cln1
/
mice were born normally but died within 1 d of
birth, showing a characteristic wrinkled skin appearance.
Two distinct assays demonstrated that the epidermal barrier
of Cln1
/
mice was severely affected, showing excessive
TEWL. Even when the Cln1
/
skin was transplanted onto
nude mice, the graft was still abnormal with hyperprolifera-
tion of keratinocytes, a typical compensatory response of the
barrier-defective epidermis. Taking into consideration that a
sufficient amount of claudin-1 was indeed detected in the
epidermis of wild-type newborn mice, it is safe to say that
the claudin-1 molecules expressed in the epidermis are indis-
pensable for creating and maintaining the epidermal barrier.
The question then is how is claudin-1 involved in the epi-
dermal barrier. Since the layered organization of kerati-
nocytes was maintained mostly in the Cln1
/
epidermis,
claudin-1 deficiency did not appear to affect the viability
and/or differentiation of keratinocytes. Therefore, it would
be reasonable to speculate that, also in the epidermis, clau-
din-1 contributes to barrier function by constituting TJ
strands. However, in the past several decades it has been be-
lieved that in the mammalian stratified epithelium, espe-
cially in the epidermis, TJs are not developed but occur only
in a part of the stratum granulosum as fragmented strands
(Squier, 1973; Elias and Friend, 1975; Elias et al., 1977;
Morita et al., 1998). There are two reasons why TJs have
been ignored in the mammalian epidermis. First, by ul-
trathin section and freeze-fracture replica EM it was techni-
cally difficult to evaluate the existence and continuity of TJs
occurring only in a layer of flat and large granular cells. Sec-
ond, the lack of good general markers for TJ strands has
hampered the direct assessment of the existence of TJs at the
immunofluorescence microscopic level. ZO-1, a peripheral
membrane protein, which was initially identified to be con-
centrated at TJs (Stevenson et al., 1986), is localized also at
the cadherin-based cell adhesion sites in many types of cells
(Itoh et al., 1993), making this molecule inappropriate for
use as a general marker for TJs: in the mouse epidermis, ZO-1
was distributed in most layers of keratinocytes (Morita et al.,
1998). Recently, occludin has been recognized to be very
specific to TJs, especially TJ strands (Tsukita and Furuse,
1999). Indeed, previous immunostaining of frozen sections
of mouse epidermis with antioccludin antibodies revealed
intense dot-like (sometimes linear) staining at stratum gran-
ulosum, but these findings had not thrown doubt on the
widely accepted notion that there is no continuity of TJs in
the epidermis (Morita et al., 1998). However, the phenotype
of Cln1
/
mice, that is, the impairment of the epidermal
barrier, prompted us to reexamine TJs in the epidermis.
Figure 7. Ultrathin section electron microscopic image of the
claudin-1–deficient epidermis. Similar to Fig. 5, c and d, at the
region where occludin was expected to be concentrated TJ-like
structures (arrows) were observed frequently just above the
desmosome (DS). The structures appeared to be poorly developed
compared with those in the wild-type epidermis, although it was
technically difficult to quantitatively show this difference by ultrathin
section EM. Bar, 200 nm.
Claudin-1 and the mammalian epidermal barrier | Furuse et al. 1107
As shown in this study, detailed occludin staining of fro-
zen sections of the wild-type skin revealed that the large flat
cells in the second layer of stratum granulosum were fairly
polarized and that TJs circumscribed these cells continu-
ously at the most apical level of their lateral membranes in
newborn mice. It would be difficult to show the continuity
of these TJs at the electron microscopic level, but the follow-
ing two findings strongly supported the existence of contin-
uous (and functional) TJs in the epidermis: (1) in all cell–
cell contacts at the most apical region of lateral membranes
of granular cells in the second layer, typical TJs were clearly
identified just above desmosomes by ultrathin section EM
(Fig. 5), and (2) the diffusion of subcutaneously injected bi-
otinylation reagent was sharply stopped at occludin-positive
TJs (Fig. 8); this finding was highly consistent with a previ-
ous report using a colloidal lanthanum nitrate tracer (Hash-
imoto, 1971). Interestingly, in frog skin, which has a strati-
fied epithelium with some cornification, cells in the upper
layers are characterized by the presence of well-developed
TJs, which are responsible for the unusually strong barrier
function of this skin (Farquhar and Palade, 1965). Further-
more, our preliminary observations indicated that similar
occludin-positive continuous TJs occur in noncornified
stratified epithelia such as esophageal epithelium (unpub-
lished data).
In the mouse epidermis, claudin-1 and -4 were expressed
in large amounts and coconcentrated at the occludin-posi-
tive lines, that is, TJs, circumscribing granular cells. There-
fore, based on our knowledge of TJ strands obtained in sim-
ple epithelial cells the strands in the epidermis could be
regarded as heteropolymers consisting of at least occludin,
claudin-1, and claudin-4. TJ strands in cultured simple epi-
thelial cells, MDCK I cells, were also reported to be prima-
rily composed of occludin, claudin-1, and claudin-4. As de-
scribed in the Introduction, when claudin-4 was specifically
removed from TJs of MDCK I cells using a claudin-4–bind-
ing peptide the TJ barrier was severely affected: in these
cells, although the TJ strands were decreased in number TJs
and their cellular polarity was maintained (Sonoda et al.,
1999). Similar events might occur in the epidermis of
Cln1
/
mice: claudin-1 would be specifically removed from
TJs of granular cells without the morphology of TJs or their
polarity being affected. This could explain the molecular
mechanism by which the Cln1
/
epidermis became leaky
without a change in the layered organization of kerati-
nocytes. In the Cln1
/
epidermis, the continuity of TJs ap-
peared to be maintained at least at the light microscopic
level, but it was not certain at the level of TJ strands. Indeed,
the subcutaneously injected biotinylation reagent appeared
to pass through the occludin-positive TJs in the Cln1
/
epi-
dermis (Fig. 8).
Curiously, in the epidermis of wild-type mice, in addition
to being concentrated in TJs around granular cells claudin-1
and -4 appeared to be diffusely distributed along plasma
membranes of keratinocytes in more inner layers. Judging
from previous EM and immunofluorescence microscopy
with antioccludin antibodies (Elias and Friend, 1975; Elias
et al., 1977; Morita et al., 1998), it was not likely that
these diffusely distributed claudin molecules constituted TJ
strands per se or that they were involved directly in the bar-
rier function of the epidermis. In addition to our present
tracer experiment, it was reported that tracers injected into
the dermis diffused freely through intercellular routes be-
tween keratinocytes up to stratum granulosum where there
was some diffusion barrier (Hashimoto, 1971; Elias and
Friend, 1975). Elucidation of the physiological functions of
these nonpolymerized claudins would be very important in
the future study of stratified epithelium.
Figure 8. TJ permeability assay of the wild-type
and claudin-1–deficient epidermis. An isotonic
solution containing freshly made biotinylation
reagent into the dermis on the back of Cln1
/
and
Cln1
/
newborns, and after 30 min incubation the
skin was dissected out and frozen. Frozen sections
were double stained with antioccludin mAb and
streptavidin to label TJs and cross-linked biotin in
green and red, respectively. In the Cln1
/
epidermis
(a–c), the biotinylation reagent diffused through the
paracellular spaces from stratum basale to the
second layer of stratum granulosum, but this
diffusion was sharply stopped at occludin-positive
TJs (arrows). In the Cln1
/
epidermis (d–f), the
diffusion of injected biotinylation reagent was not
prevented at the occludin-positive TJs (arrows).
Instead, the biotinylation reagent passed through
these TJs to reach the border between stratum
granulosum and corneum. SC, stratum corneum.
Bar, 10 m.
1108 The Journal of Cell Biology | Volume 156, Number 6, 2002
To date, the formation of the cornified CE and lipid lamel-
lae in and between terminally differentiating keratinocytes,
respectively, has been widely accepted to be crucial for the
barrier function of the mammalian epidermis (Rice and
Green, 1978; Elias, 1983; Downing, 1992; Roop, 1995;
Steinert, 2000): the CE consists of proteins (e.g., involucrin,
loricrin, and SPRRs) stabilized by covalent cross-links formed
under the action of transglutaminase-1. The CE is covered
with a monomolecular layer of N-(-hydroxyacyl)sphin-
gosine bound to protein by ester bonds, and intercellular
lipid lamellae, which are secreted from granular cells, inter-
connect the CE. The present study clearly indicated that TJs,
especially claudin-1, at the stratum granulosum are directly
involved in the barrier function of the mammalian skin, but
this conclusion is not exclusive of the notion that the CE and
intercellular lipid lamellae play a crucial role in the barrier
function. Probably, during phylogenetic evolution the mam-
malian skin has obtained at least two independent systems for
forming a strong barrier: TJs in stratum granulosum and the
CE/lipid lamellae in stratum corneum. Considering that in
the Cln1
/
skin the CE and the lipid lamellae appeared to
be normal and that the transglutaminase-1–deficient mice
showed a similar rapid postnatal lethality due to water loss
across the skin (Matsuki et al., 1998), for the establishment of
the epidermal barrier in mammals these two systems are re-
quired to work concurrently.
To date, in several knockout and transgenic mice (e.g.,
Klf4-deficient mice and mice expressing desmoglein-3 ec-
topically) the epidermal barrier but not the layered organiza-
tion of keratinocytes has been shown to be affected (Segre et
al., 1999; Elias et al., 2001). Interestingly, as in Cln1
/
mice the stratum corneum of these mice was thicker than
that of wild-type mice, although the functional relevance of
this phenotype remains elusive. These mice were analyzed
mainly from the viewpoint of the CE and the lipid lamellae,
but it would be interesting if these mice were reexamined
from the viewpoint of TJs. Also, in several skin diseases in
which barrier dysfunction is suspected TJs and claudins
should be examined in detail.
In this study, we found that the Cln1
/
newborns
demonstrated severe dehydration due to excessive TEWL
across the skin and that these animals died within 1 d of
Figure 9. Stratum corneum in claudin-1–deficient
mice. (A) Ultrathin section electron microscopic
images of ruthenium tetroxide-stained stratum corneum
of the skin. Both in the Cln1
/
and Cln1
/
epidermis,
well-organized lipid lamellae and lamellar bodies
were clearly observed between flattened cornified
cells (CC; top) and between cornified cells and granular
cells (GC; bottom), respectively. Bars, 0.1 m. (B)
Isolated cornified CEs. There was no clear difference
in appearance between the Cln1
/
, Cln1
/
, and
Cln1
/
skin. Bar, 40 m. (C) Expression levels of
major components of cornified CEs, loricrin and
involucrin, detected by immunoblotting. Whole cell
lysates of the Cln1
/
, Cln1
/
, and Cln1
/
skin were
examined using specific antibodies. (D) Expression
levels of Klf4, transglutaminase-1 (TG1), and GAPDH
(control) detected by Northern blotting.
Claudin-1 and the mammalian epidermal barrier | Furuse et al. 1109
birth. Of course, it is not easy to conclusively show a
causal sequence between dehydration and death, since
claudin-1 is rather ubiquitously expressed in various or-
gans other than the skin. At the initial stage of this study,
we wondered whether in the Cln1
/
epidermis water per-
meability increased due to a loss of TJ barrier or due to an
effect on ion permeability and subsequent dehydration
due to an osmotic effect. However, our results obtained
from the tracer experiment favored the former hypothesis.
Thus, this study is the first not only to experimentally
provide evidence for the direct involvement of TJs in the
function of the mammalian epidermal barrier but also to
show that the same mechanism, that is, the use of claudin-
based TJs, is shared by the barrier function of simple and
stratified epithelia. We are only just beginning to identify
the missing pieces linking simple and stratified epithelial
cells in molecular terms.
Materials and methods
Antibodies
Rat anti–mouse occludin mAb (MOC37), guinea pig anti–mouse claudin-1
pAb, and rabbit anti–mouse claudin-4 pAb were raised and characterized
previously (Saitou et al., 1997; Furuse et al., 1999; Morita et al., 1999a).
Rabbit anti–claudin-1 pAb and rabbit anti–mouse involucrin pAb/rabbit
anti–mouse loricrin pAb were purchased from Zymed Laboratories and
Covance, respectively.
Claudin-1 targeting construct and generation of Cln1
/
mice
Three overlapping clones encoding mouse claudin-1 were obtained by
screening a 129/Sv genomic library. Using one of them, the targeting
vector was constructed by ligating a 7.0-kb SacI fragment and a 1.6-kb
SacI/EcoRI fragment, which were located upstream and downstream of
exon 1, respectively, to the pgk-neo cassette. The diphthelia toxin A ex-
pression cassette (MC1pDT-A) was placed outside the 3 arm of homol-
ogy for negative selection. This targeting vector was designed to delete
the coding region in exon 1 and subsequent 5 sequence of the intron
(Fig. 1 A). J1 ES cells were electroporated with the targeting vector and
selected for 9 d in the presence of G418. The G418-resistant colonies
were removed and screened by Southern blotting with the 3 external
probe (Fig. 1 A). Correctly targeted ES clones were identified by an addi-
tional 3.9-kb band together with the 6.8-kb band of the wild-type allele
when digested with HindIII. The targeted ES cells obtained were injected
into C57BL/6 blastocysts, which were in turn transferred into Balb/c fos-
ter mothers to obtain chimeric mice. Male chimeras were mated with
C57BL/6 females, and agouti offsprings were genotyped to confirm the
germline transmission of the targeted allele. The littermates were geno-
typed by Southern blotting. Heterozygous mice were then interbred to
produce homozygous mice.
RT-PCR and immunoblotting
Total RNA was isolated from the liver of newborn mice as described by
Chomczynski and Sacchi (1987). RT-PCR was performed to amplify a por-
tion of claudin-1 cDNA using primers, 5-AGCCAGGAGCCTCGCCCCG-
CAGCTGCA-3 (forward) and 5-CGGGTTGCCTGCAAAGT-3 (reverse).
As a control, hypoxanthine phosphoribosyl transferase cDNA was ampli-
fied as a housekeeping gene. For immunoblotting, the skin was removed
from newborn mice, frozen, ground into powder in liquid nitrogen, ho-
mogenized in the SDS-PAGE sample buffer, and then boiled for 10 min.
Equal amounts of proteins were separated in 12.5 or 15% SDS-PAGE gels
and then processed for immunoblotting as described previously (Furuse et
al., 1999).
Morphological analyses
For conventional light microscopic observation, samples were fixed with
10% formalin in phosphate-buffered saline at 4C, dehydrated in a graded
series of ethanol, and then embedded in paraffin wax. Sections 5-m
thick were mounted on slides, dewaxed, hydrated, and then stained with
hematoxylin-eosin. For immunofluorescence microscopy, frozen sections
5–10-m thick were processed as described previously (Furuse et al.,
1993).
For ultrathin section EM, newborn mice were fixed by perfusing the fix-
ative (2.5% glutaraldehyde, 2% paraformaldehyde, 0.1 M cacodylate
buffer, pH 7.3) from the heart. The skin was removed and processed as de-
scribed previously (Furuse et al., 1993). To visualize the lipid lamella in
stratum corneum, samples were postfixed with a fixative containing 1% ru-
thenium tetroxide and 0.1 M cacodylate (pH 6.8) at room temperature for
Figure 10. Transplantation of the claudin-1–deficient
skin to nude mice. (A) Macroscopic images of the skin
grafts on nude mice at 1 mo after transplantation from
newborns. The Cln1
/
grafts bore numerous long
hairs, whereas the Cln1
/
grafts had only a small
number of short hairs (left). When hairs were cropped,
the Cln1
/
grafts appeared to be thicker than the
Cln1
/
grafts, and in some places they appeared to be
covered with infiltrates (right). (B) Hematoxylin-eosin–
stained sectional images of the skin grafts on nude
mice at 1 mo after transplantation from newborns. The
epidermis of the Cln1
/
grafts was unusually thick
compared with that of the Cln1
/
grafts, although
hair follicles appeared to be developed even in the
Cln1
/
grafts. Bar, 100 m.
1110 The Journal of Cell Biology | Volume 156, Number 6, 2002
1 h and then dehydrated with a graded series of acetone (Madison et al.,
1987).
Barrier function assays
TEWL was determined by directly measuring the water evaporation from
the dorsal skin of newborn mice using a moisture analyzer NEP-1 BRAVO
(MECCO Inc.).
TJ permeability assay using surface biotinylation technique was per-
formed according to the method developed by Chen et al. (1997). 50 l of
10 mg/ml EZ-Link™ Sulfo-NHS-LC-Biotin (Pierce Chemical Co.) in PBS
containing 1 mM CaCl2 was injected into the dermis on the back of the
Cln1
/
and Cln1
/
newborns. After 30-min incubation, the skin was
taken out and frozen in liquid nitrogen. About 5-m-thick frozen sections
were fixed in 95% ethanol at 4C for 30 min and then in 100% acetone at
room temperature for 1 min. The sections were soaked in blocking solu-
tion for 15 min, incubated with antioccludin mAb for 30 min, washed
three times with blocking solution, then incubated with a mixture of FITC
anti–rat IgG pAb (Jackson ImmunoResearch Laboratories) and Streptavidin
Texas red (Oncogene Research Products) for 30 min.
Isolation of cornified CEs
To isolate cornified CEs, the skin was removed from the back of newborn
mice, homogenized in SDS-PAGE sample buffer, and then boiled for 15
min, mainly according to a method developed previously (Hohl et al.,
1991).
We thank Drs. Y. Miyachi (Kyoto University), H. Uchiwa (Kanebo, Ltd.),
H. Yoshida (Kyoto University), M. Amagai (Keio University, Tokyo, Japan),
and W.W. Franke (German Cancer Research Center, Heidelberg, Ger-
many) for valuable discussions. We also thank Ms. K. Yoshida, K. Hirao-
Minakuchi, C. Matsui, and C. Fujiwara for their excellent technical assis-
tance.
This study was supported in part by a Grant-in-Aid for Cancer Research
and a Grant-in-Aid for Scientific Research (A) from the Ministry of Educa-
tion, Science and Culture of Japan to S. Tsukita, and by Japan Society for
the Promotion of Science Research for the Future Program to M. Furuse.
Submitted: 25 October 2001
Revised: 30 January 2002
Accepted: 31 January 2002
Note added in proof. Recently, Drs. I. Moll, W.W. Franke, and their col-
leagues also reported the occurence of continuous TJs in the human epi-
dermis (Brandner, J.M., S. Keif, C. Grund, M. Randl, P. Houdek, C. Kuhn,
E. Tsouchler, W.W. Franke, and I. Moll. Organization and formation of the
tight junction-system in human epidermis and cultured keratinocytes. Eur.
J. Cell Biol. In press).
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