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Review Article
Tight junction-related human diseases
Norimasa Sawada
Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan
Tight junctions are intercellular junctions adjacent to the
apical ends of paracellular spaces. They have two classical
functions, the barrier function and the fence function. The
former regulates the passage of ions, water and various
molecules through paracellular spaces, and is thus related
to edema, jaundice, diarrhea and blood-borne metastasis.
The latter function maintains cell polarity by forming a fence
to prevent intermixing of molecules in the apical membrane
with those in the lateral membrane. This function is deeply
involved in cancer cell properties in terms of loss of cell
polarity. Recently, two novel aspects of tight junctions have
been reported. One is their involvement in signal transduc-
tion. The other is that fact that tight junctions are consid-
ered to be a crucial component of innate immunity. In
addition, since some proteins comprising tight junctions
work as receptors for viruses and extracellular stimuli,
pathogenic bacteria and viruses target and affect the tight
junction functions, leading to diseases. In this review, the
relationship between tight junctions and human diseases
will be described.
Key words: barrier function, cancer, claudin, fence function,
human diseases, immunity, occludin, tight junctions
Establishment of a distinct internal environment is absolutely
required for multicellular organisms to maintain life. For this
purpose, all of their surfaces, the skin, gastrointestinal tract,
respiratory tract, etc., are covered by various kinds of epithe-
lia. In particular, for epithelial and endothelial sheets to work
efficiently as a barrier,1–5 paracellular spaces must be strictly
sealed by tight junctions, which are characterized as a set of
continuous and anastomosing strands at the apicalmost
regions of the lateral cell membranes (Fig. 1).
The strict regulation of diffusion of solutes through para-
cellular spaces by tight junctions is referred to as the barrier
function of the tight junction. When tight junctions of epithelial
cells that cover the biliary tree and gastrointestinal tract
become disordered, jaundice and diarrhea occur, respec-
tively. Although vascular permeability depends on both the
paracellular pathway and transcellular pathway of endothelial
sheets, edema develops mainly as a result of the dysfunction
of tight junctions between cells. Thus, dysfunction of tight
junctions is considered to be intimately related to various
pathological conditions.6
Epithelial cells have two distinct domains of the cell
surface; the apical and basolateral cell membranes. Since
the two domains play different roles, the compositions of
proteins and lipids in the respective membrane domains are
different. To prevent intermixing of molecules in the apical
membrane with those in the lateral membrane, tight junctions
continuously surrounding the apical pole work as a fence.
This function of the tight junction is referred as the fence
function. When the function is impaired, cells fail to perform
their vectorial work, in terms of loss of cell polarity, and this is
presumably deeply involved in cancer cell biology.
In addition to the above-mentioned functions of tight junc-
tions, two properties of tight junctions are becoming clear.
One is their involvement in signal transduction and the other
is their participation in innate immunity. In this review, we will
describe the properties of tight junctions, changes in tight
junctions under pathological conditions, and the possible
clinical application of tight junction research.
MOLECULAR COMPONENTS OF THE TIGHT
JUNCTION (TABLE 1)
Integral membrane proteins
Of the proteins comprising tight junctions, the integral mem-
brane proteins are the claudin family,2,3,7 tight junction-
associated MARVEL proteins (TAMPs)5,8 composed of
occludin, tricellulin9and MARVELD3, the immunoglobulin
Correspondence: Norimasa Sawada, MD, PhD, Department of
Pathology, Sapporo Medical University School of Medicine, S1, W17,
Sapporo 060-8556, Japan. Email: sawadan@sapmed.ac.jp
Received 10 October 2012. Accepted for publication 19 November
2012.
© 2012 The Author
Pathology International © 2012 Japanese Society of Pathology and
Wiley Publishing Asia Pty Ltd
Pathology International 2012 doi:10.1111/pin.12021
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superfamily composed of the JAM family, CAR, and ESAM,
Bves10 and LSR (lypolysis-stimulated lipoprotein receptor).11
The claudin family, comprised of 27 members, is exclusively
responsible for the formation of tight junction strands. Clau-
dins are necessary and sufficient for the formation of tight
junctions by homophilic and heterophilic binding to adjacent
cells.12,13 Regarding hereditary diseases, mutations of the
claudin-1 gene cause neonatal sclerosing cholangitis with
ichthyosis.14 Those of claudin-14 cause deafness.15 Some
claudins are expected to form extracellular aqueous pores in
paracellular spaces.16,17 Distinct examples are claudin-16/
paracellin-118 and claudin-19,19 which are responsible for
hereditary hypomagnesemia. Similarly, claudins are consid-
ered to form selective channels in tight junctions because of
the existence of charged amino acids on the first extracellular
loop.17 Several claudins are expressed in one cell.1,2 Their
regulation is very complicated, as it is both claudin-specific
and cell-specific. Claudin-1, -6 and -9 are reported to be
required for the entry of hepatitis C virus (HCV) into hepato-
cytes after binding of HCV to CD81.20 Claudins have been
reported to recruit and promote the activation of pro-MMP-
2.21,22 Claudin-7, however, is reported to suppress expression
of MMP-3.23 In addition, claudin-7 interacts with Ep-CAM24
and, in claudin-7-deficient mice, expression of integrin a2is
reduced and its localization altered in the intestinal epithelia.23
These findings show that some claudins play as yet unknown
roles in cellular processes other than tight junction functions.
Occludin was the first integral membrane protein of tight
junctions discovered,25 and can be clearly detected in tight
junction strands by immunolabeling freeze-fracture replicas.
However, occludin-deficient ES cells have the capability to
develop tight junction strands indistinguishable from the
normal strands.26 Nevertheless, the immunohistochemical
intensity of occludin in various tissues correlates well with the
number of strands.27 Of the tight junction proteins, occludin is
the most ubiquitously expressed at the apicalmost basolat-
eral membranes, and is the most reliable immunohistoche-
mical marker for tight junctions.28 Compared to claudins,
occludin has a relatively long cytoplasmic c-terminus contain-
ing several phosphorylation sites and a coiled-coil domain
that probably interacts with PKC-z, c-Yes, connexin26, and
the regulatory subunit of phosphatidylinositol 3-kinase (PI3K)
among others,29 as well as occludin itself, ZO-1 and ZO-3.1,2
Thus roles of occludin in signal transduction have been pro-
posed (Fig. 2), and its involvement in apoptosis reported.30,31
It, like claudin, is required for HCV infection.20 However, the
roles of occludin in regulation of tight junctions remain to be
clarified.
Tricellulin is a member of the MARVELD3 subfamily. It is
concentrated at tricellular contacts and is regulated by the
c-JNK pathway in both normal and malignant pancreatic duct
cells.32 The C-terminus of tricellulin is highly similar to that of
occludin, and mutations of tricelluilin cause deafness.33
JAM (junctional adhesion molecule)-A, JAM-B, JAM-D,
CAR (coxsackievirus and adenovirus receptor) and ESAM
(endothelial cell-selective adhesion molecule) belong to the
immunoglobulin superfamily.34 All of the members have extra-
cellular V-type and C2-type immunoglobulin domains, a
Figure 1 Morphology of tight junctions.
(a) Schematic diagram of tight junction. (b)
Tight junction strands on freeze-fracture
replica.
Table 1 Tight junction-related proteins
1. Transmembrane proteins
Claudin family (cldn-1, ~27)
TAMPs (occludin, tricellulin, MarvelD3)
Immunoglobulin superfamily (JAM family, ESAM, CAR)
LSR(lypolysis-stimulated lipoprotein receptor)
2. Cytoplasmic proteins
a) PDZ domain-containing proteins
ZO-1, -2, -3, MAGI-1, -2, -3, MUPP-1, PAR-3, PAR-6, PALS-1,
PATJ, mDlg, Scrib, afadin
b) Other proteins
Cingulin, Symplekin, heterotrimeric G protein, Rab3b, Rab13,
ZONAB, huASH1, GEF-H1, aPKC, PP2A, PTEN, Pilt,
CRB3, LYRIC, CASK/LIN-2, Merlin, Angiomotin/JEAP,
TAZ/YAP, etc.
2 N. Sawada
© 2012 The Author
Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
single transmembrane region and a cytoplasmic tail. JAM-A
lacks the capability to form tight junction strands. It is a ligand
of lymphocyte function-associated antigen 1 (LFA-1)35 and
also a receptor for reovirus.36 This group is considered to play
roles in inflammatory reactions, particularly extravasation of
inflammatory cells.37
Cytoplasmic proteins1,2,6
These tight junction proteins can be divided into two groups.
One group consists of the PDZ domain-containing proteins
ZO-1, ZO-2, ZO-3, ASIP/Par3, Par6, MAGI-1, MAGI-2,
MAGI-3, AF-6/afadin, MUPP1 and PATJ. The other group is
comprised of cingulin, 7H6 antigen, symplekin, heterotrimeric
G proteins, aPKC, ZONAB, huASH1, Rab-3b, rab-13, PTEN,
Pilt, angiomotin/JEAP38 and protein phosphatase 2A.
Of these proteins, ZO-1, ZO-2 and cingulin can bind to
actin filaments. ZO-1 and ZO-2 are essential for formation of
tight junctions39 because both interact with all of the integral
membrane proteins and the latter interacts with claudins and
JAM-A.22 ZO-2 and ZO-3 also interact with claudins.21 Muta-
tion of ZO-2 causes hypercholanemia.13 aPKC and PP 2A
may regulate phosphorylation levels of the tight junction pro-
teins to establish cell polarity and/or to regulate tight junction
functions. PTEN lipid phosphatase antagonizes PI3K/Akt sig-
naling and binds to MAGI-1 and MAGI-2. Recently, the Hippo
pathway-related proteins YAP, TAZ, angiomotin/JEAP and
Merlin were reported to localize at tight junctions.40,41 Since
tight junctions are functional multiprotein assemblies, each of
the components and the related proteins may affect their
functions. In this sense, understanding of life and the func-
tions of each protein of tight junctions is required.
Regarding the biogenesis of tight junctions, in terms of
polarization42 it is speculated that formation of adherens junc-
tions precedes formation of tight junctions. To establish the
cell polarity of epithelial cells, in terms of development of the
junctional complexes, E-cadherin and/or nectin first bind to
the respective molecules on the surfaces of adjacent cells
in a homophilic manner. Immediately thereafter, ZO-1 and
JAM-A are recruited at spot-like primordial adherens junc-
tions, and then Par3, Par6 and aPKC are recruited to JAM-A,
and occludin and claudin are also recruited to ZO-1 com-
plexes. Lastly, by unknown mechanisms probably involving
an aPKC/Par complex and delivery of the integral membrane
proteins, tight junctions are segregated from adherens junc-
tions. Consistently, during polarization of mouse teratoma
cell line F9, HNF-4aplays crucial roles in the formation of
tight junctions43 as well as adherens junctions.44 Furthermore
HNF-4ainduces formation of microvilli on the apical sur-
face.45 These findings show that formation of tight junctions is
very closely related to the establishment of cell polarity.
Roles of tight junctions in biology
Tight junctions, the apicalmost component of intercellular
junctional complexes, separate the apical from the basolat-
eral cell surface domains to maintain cell polarity (the fence
function), and also regulate solute and water flow through the
paracellular space (the barrier function). The formation and
maintenance of tight junctions require ATP46 and integrity of
the actin cytoskeleton.47 The barrier function is more deeply
dependent on ATP48 and actin49 than the fence function. In
addition, gap junctional intercellular communication strength-
ens tight junctions.50 These properties of tight junctions
suggest that the fence and barrier functions of the tight junc-
tion have a common feature of compartmentalization; the
fence function is performed at the subcellular level and the
barrier function is performed at the organ level.
Under physiological conditions, epithelial cells maintain
polarity and the functional multicellular sheet of epithelium
maintains individual homeostasis via the fence function and
the barrier function, respectively (Fig. 3). The fence function
segregates growth factors that exist in the apical surface from
their receptors on the basolateral cell surface, establishing
Figure 2 Possible participation of occludin in signal transduction.
Figure 3 Schematic diagram of the fence and barrier functions of
tight junctions. Fence function: tight junctions prevent intermixing of
molecules in the apical membrane with those in the lateral mem-
brane. Barrier function: tight junctions regulate diffusion of solutes
through paracellular spaces.
Tight junction-related human diseases 3
© 2012 The Author
Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
cell polarity.51 On the other hand, the barrier function prevents
growth factors localized on the apical side from diffusing into
paracellular spaces. Upon dysfunction of tight junctions,
growth factors bind to their receptors on basolateral mem-
branes though permeable tight junctions, resulting in quick
responses to stimuli. This ligand-receptor segregation by
tight junctions may be critical to maintain the physiological
condition.51
The fence and the barrier functions of tight junctions are
well recognized. Recently, two other roles of tight junctions
have become clear. One is regulation of signal transduc-
tion.52 Actually, occludin is capable of binding to many signal
transduction-related molecules such as TGF-b,53 and partici-
pates in apoptosis.30 It has become clear that tight junctions
tether some of the important molecules of the Hippo pathway,
which is a growth suppressive pathway activated by unknown
factors. In addition, Merlin (neurofibromatosis type II gene) is
a tumor suppressor gene that shuttles between the nucleus
and tight junctions.54 Since the Hippo pathway is considered
to be deeply related to contact inhibition, the question of how
tight junctions are involved needs to be clarified, as do the
stimulants of the pathway.
The other role is participation of tight junctions in the
immune system (Fig. 4). This relationship has been inves-
tigated in mucosal immunity, though it is not fully clarified. It
has been considered that tight junctions are very static
components of innate immunity and a physical barrier
against allergens, pollutants and bacteria. In other words,
tight junctions have been thought to participate in innate
immunity as a fort and immune cells are soldiers against
various agents. In this context, dendritic cells residing
between epithelial cells express occludin, claudin-1 and
ZO-1, sending their dendrites outside through paracellular
spaces in the colonic epithelium, probably to sample anti-
gens in the intestine.55 A similar relationship between
dendritic cells and epithelial cells has been observed in the
nasal mucosa,56 and the epidermis.57 These findings imply
that dendritic cells may pass through intercellular spaces by
using tight junction molecules to sense extrinsic noxious
molecules.
In the human nasal mucosal epithelium, a key molecule
of allergic rhinitis, thymic stromal lymphopoietin (TSLP),
upregulates the functions of tight junctions with an increase
of proteins, including claudin-7.58 TSLP was also reported to
upregulate claudin-7 expression in dendritic cells.59 Regard-
ing Toll-like receptor (TLR) signals, the TLR3 ligand poly (I:C)
reduces JAM-A in human nasal epithelial cells.60 In cutane-
ous tissue, several types of TLR, including TLR3, enhance
tight junction functions in keratinocytes.61 These observations
show that tight junctions may play unknown but distinct roles
in regulating innate immunity.
JAM-A and occludin seem to be involved in transendothe-
lial diapedesis of inflammatory cells. JAM-A is a ligand of
LFA-1 on the surface of the lymphocyte,35 Leukocytes
derived from JAM-A-null mice fail to pass between endothe-
lial cells.37,62 Occludin is expressed in activated T lympho-
cytes to make the diapedesis smooth with minimal effects on
the tight junction function.63 Occludin is also involved in tran-
sepithelial migration of neutrophils.64 Recently, occludin was
reported to be required for migration of delta/gamma lympho-
cytes between epithelial cells.65 Furthermore, the relationship
between claudin-4 and T cell maturation66 shows involvement
of tight junctions and the proteins in establishment of the
immune system.
Thus, tight junctions seem to be integrated into the immune
system. These findings might result from that fact that tight
junction proteins are able to interact between heterotypic
cells, i.e. epithelial cells and dendritic cells,55,56 like a glue
and/or a lubricant. In the future, the roles of tight junctions in
immunity will be clarified and how the tight junctional barrier
Figure 4 Schematic diagram of involve-
ment of tight junctions in innate immunity.
PRRs, pattern recognition receptors; PAR,
protease-activated receptor.
4 N. Sawada
© 2012 The Author
Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
is integrated into the immune system, coping with all kinds of
agents, should be elucidated.
HUMAN DISEASES RELATED TO TIGHT JUNCTIONS
Infectious diseases
Claudin-3 and -4 are receptors for the enterotoxin of
Clostridium perfringens (CPE), which is a common cause of
food poisoning.67,68 When CPE binds claudin-4 expressed in
MDCK cells, the complexes internalize like other ligand-
receptor complexes, and then the function of tight junctions
becomes disordered.69 Very interestingly, this phenomenon
is observed only when the basolateral surface of the cell
is exposed to CPE.70 The Helicobacter pylori toxin CagA
causes an increase in paracellular permeability of intestinal
cells by inactivating Par1/MARK kinase.71 As a result, the
barrier function of gastric foveolar epithelium deteriorates
and microvilli disappear.72
Hemagglutinin/protease (HA/P) produced by Vibrio chol-
erae digests occluding.73 Vibrio cholerae causes severe
diarrhea via the combination of cholerea toxin, zonula
occludens toxin (ZOT) and HA/P. The second mechanism is
a change in actin organization. This organization takes
place in a wide variety of cellular conditions, including regu-
lation of tight junctions. In particular, some kinds of bacteria
directly affect Rho and myosin light chain kinase activities
in tight junction functions.6Toxins from Clostridium diphthe-
riae,Clostridium difficile and enteropathogenic Escherichia
coli change Rho activity by modification of amino acids to
cause severe colitis, the former two causing psuedomem-
branous colitis. Enteropathogenic Escherichia coli also
induces myosin light chain phosphorylation, resulting in
diarrhea. ZOT of V. cholerae activates PKC, resulting in
loss of tight junctions.
In viral infection, occludin and claudins are coreceptors of
hepatitis C virus, CAR is a receptor of adenoviruses and
Coxsackie viruses and JAM-A is a reovirus receptor.34 In
addition, pathogenic viruses such as those causing influenza
target the PDZ domain of cytoplasmic tight junction-related
proteins (Fig. 5).74
Malabsorption of Ca ions due to vitamin D deficiency
Calcium plays a fundamental role in various physiological
functions such as bone mineralization, blood coagulation,
neuromuscular transmission and muscle contraction, as well
as cell–cell adhesion and intracellular signaling. Ca2+is
absorbed in the intestinal mucosa via two distinct routes, the
transcellular and paracellular pathways.75,76 The molecular
basis for paracellular Ca2+absorption, which occurs through-
out the intestine, is largely unknown. Recent studies have
disclosed that claudins are the major determinant of the
barrier function of tight junctions. Importantly, the first extra-
cellular loop, in which there is a wide variation in the position
and number of charged amino acids depending on each
claudin, is known to create paracellular pores (channels) for
cations or anions between neighboring cells.13 The expres-
sion of putative cation-permissive claudin-2, -7, -12 and -15
in the intestine of vitamin D receptor-deficient mice was com-
pared to that of wild mice,77 clearly showing a decrease of
claudin-2 and -12. This was confirmed by approaches using
RNAi and overexpression in the vitamin D-responsive intes-
tinal cell line Caco-2.78 These two claudins are essential for
Ca2+absorption between intestinal epithelial cells, providing
a novel mechanism underlying vitamin D-dependent intesti-
nal Ca2+transport. Thus, vitamin D deficiency rickets affects
Ca2+absorption in the intestine via the paracellular pathway
as well as the transcellular pathway.
Figure 5 Human viruses and tight
junction-related proteins. HCV, hepatitis
C virus; HIV, human immunodeficiency
virus; HPV, human papillomavirus; HTLV,
human lymphotropic virus; SARS, severe
acute respiratory syndrome; TBEV, tick-
borne encephalitis virus.
Tight junction-related human diseases 5
© 2012 The Author
Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Diabetic retinopathy
Tight junctions of endothelial sheets in vivo are leaky in
general, because a wide variety of substances must be
exchanged between the blood and organs through paracel-
lular pathways as well as transcellular pathways of
the sheets. In certain organs such as the brain and retina,
however, endothelial cells possessing well-developed tight
junctions form a blood-brain barrier (BBB),79 Interestingly, the
endothelial cells forming the blood-tissue barrier in the
brain,80,81 retina82 and testis83 express receptors for glial cell
line-derived neurotrophic factor (GDNF), which is secreted
from astrocytes.84 In diabetic retinopathy, the blood-retinal
barrier (BRB) is leaky due to VEGF.85 Glyceraldehyde-
conjugated advanced glycation end-products (AGEs) induce
VEGF, making tight junctions of the BBB-forming endothelial
cells highly permeable and also decease GDNF, making the
BBB less permeable in astrocytes.86 Inversely, retinoic acid
and RAR-ashow opposite effects on astrocytes. In the early
stage of diabetic retinopathy, VEGF makes the BRB highly
permeable. RAR-areduces VEGF expression in astrocytes,
resulting in a marked decrease in the leakage of dye from the
BRB.87 It is very clear that dysfunction of tight junctions of the
BRB initially causes diabetic retinopathy. However, these
results suggest that it is possible to indirectly treat diabetic
retinopathy using a paracrine mechanism. In this case, astro-
cytes may be the most suitable target for treatment in the
early phase of diabetic retinopathy in terms of maintaining the
impermeability of the BRB.
The above-mentioned conditions are examples of human
diseases related to the barrier function of tight junctions.
Since tight junctions are located at the apicalmost areas
of basolateral spaces between epithelial, endothelial or
mesothelial cells, they are involved in a wide variety of patho-
logical conditions where the physiological regulation of the
passage of ions, molecules, and inflammatory cells may be
affected. In Table 2, human diseases involving dysfunction of
tight junctions are listed. Inflammation is always accompa-
nied by an increase in vascular permeability, in part caused
by VEGF, which primarily affects the barrier function of tight
junctions via both phosphorylation and down-regulation of
occluding.95,96 Cancer cells also secrete VEGF to induce
angiogenesis, presumably to intravasate and extravasate, in
other words, to metastasize. Thus, various diseases in which
VEGF production is involved may cause tight junction dys-
function. To a greater or lesser degree, many cytokines affect
the functions of tight junctions under pathological conditions.
Neoplasms
First of all, discussion of the relationship between tight junc-
tions and human cancer must be divided into two aspects.
One is changes in tight junctions as a cellular apparatus and
the other is changes in a certain components of tight junc-
tions, such as claudin.
Changes in tight junctions
In general, cancer cells lose their specific functions and
polarity with a decrease in the development of tight junctions.
In well-differentiated adenocarcinomas derived from human
colon and endometrium, comparable amounts of occludin are
detected, and with further dedifferentiation cancer cells lose
tight junctions.97,98 These findings show that cancer cells
irreversibly and progressively lose tight junctions with dedif-
ferentiation by means of genetic and epigenetic changes
(Fig. 6).
On the other hand, once malignant transformation occurs,
the degree of differentiation in cancer cells can reversibly
Table 2 Human diseases related to tight junctions
I. Disturbance of the barrier fynction
1. Hereditary diseases
Hypomagnesemia
Deafness
Neonatal sclerosing cholangitis with ichthyosis
Familial hypercholanemia
2. Vascular system37
Edema
Endotoxinemia
Cytokinemia
Diabetic retinopathy
Multiple sclerosis
Blood-borne metastasis
3. Gastrointestinal tract88,89
Bacterial gastritis
Pseudomembranous colitis
Crohn’s disease
Ulcerative colitis
Celiac disease
Collagenous Colitis
Malabsorption of Ca ions in vitamin D deficiency
4. Liver90
Jaundice
Primary biliary cirrhosis
Primary sclerosing cholangitis
5. Respiratory tract91,92
Asthma
Adult (or acute) respiratory distress syndrome (ARDS)
Nasal allergy
6. Cutaneous tissue93
Atopic dermatitis
7. Bacterial infection94
Vibrio cholerae, Helicobacter pylori,
Clostridium perfringens, Clostridium diphtheria,
Clostridium difficile, enteropathogenic Escherichia coli
8. Viral infections:74
Reovirus, adenovirus, coxsackievirus, rotavirus. HIV,
Hepatitis C virus, RS virus etc.
II. Disturbance of the fence function
Cancer cells
Oncogenic papillomavirus infection
6 N. Sawada
© 2012 The Author
Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
change in terms of structural atypia. This reversible change is
called the epithelial-mesenchymal transition (EMT).99,100 The
EMT is essential for the development of the body; i.e. normal
cells often migrate during embryogenesis. Regarding cancer
cells, however, EMT is deeply associated with the malignant
properties of invasion and metastasis. In the process of
invasion and metastasis, cancer cells detach from cell
nests and change from epithelial to mesenchymal shape.
Epithelial-mesenchymal transition is accompanied by loss of
occludin and claudins as well as E-cadherin. The genes of
these proteins contain an E-box in their promoter.101
The loss of tight junctions is visualized by the fence func-
tion. As shown in Fig. 7,102 well-differentiated adenocarcoma
cells of the pancreas still possessing the fence function phe-
notypically change into poorly differentiated adenocarcinoma
and lose the function with EMT-inducing treatments such as
TGF-b-treatment and hypoxia. Both treatments significantly
induce Slug, SIP1 and Snail in the well-differentiated adeno-
carcoma cells,102 These transcriptional factors bind to E-box
in the promoter of E-cadherin and tight junction protein
genes, probably resulting in undergoing EMT. This change is
reversible, indicating that cancer cells undergo EMT and
MET, presumably induced by cytokines such as TGF-b, not
genetic or epigenetic changes.
Release from growth suppression includes two mecha-
nisms. One is loss of the functions of tight junctions, which
lose ligand-receptor segregation.51 When the barrier function
is lost, growth factors that normally exist in the apical mucus
can bind to its receptors normally located on the basolateral
surface of the cells. As a result, cell proliferation occurs in an
Figure 6 Schematic diagram of changes
of tight junctions during carcinogenesis.
EMT, epithelial–mesenchymal transition.
Figure 7 Changes of tight junction fence
function during epithelial–mesenchymal
transition of well-differentiated adenocarci-
noma of the pancreas induced by TGF-b.
(a) TGF-b-induced expression of Snail,
Slug and SIP1. (b) Visualization of the cell
membranes. TGF-band hypoxia revers-
ibly cause the fence function of well-
differentiated adenocarcinoma (HPAC) to
deteriorate, resembling that of poorly
differentiated adenocarcinoma (PANC-1).
In well-differentiated adenocarcinoma,
tight junctions prevent basolateral mem-
branes from being labeled by BODIPY-
sphingomyelin without the stimulation.
Arrow heads show cell-cell contacts.
, Snail; , Slug; , SIP1.
Tight junction-related human diseases 7
© 2012 The Author
Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
autocrine manner.51 The other feasible mechanism is related
to signal transduction. Tight junctions work as docks of cell
cycle-regulating molecules52 and as a possible target of the
Hippo pathway suppressive of cell proliferation and crucial for
organ size regulation.40 Tight junctions tether Hippo signal-
related molecules such as TAZ/YAP, and tumor suppressor
gene Merlin (neurofibromatosis type 2) binds to the tight
junction protein PATJ.41
Changes of expression of claudin family
Changes of expression of claudins are summarized in
Table 3.4,103 Expression of some claudin family members is
significantly altered by epigenetic regulation in human
cancer,104–106 though tight junctions deteriorate in cancer. At
present, we cannot draw any conclusion about the relation-
ship between claudin expression and human cancer because
the expression occurs in a cell type-specific fashion. In addi-
tion, like claudin-7,23 claudin-3 and -8 are representatively
detected on lateral membranes as well as in tight junction
regions. These claudins localized on the basolateral surface
might have unknown roles in cell functions in addition to
forming tight junctions. Further characterization of each
member of the claudin family and immunostaining panels for
various human cancers are required to elucidate why claudin
expression is changed regardless of tight junction formation,5
Are tight junctions really suppressive of tumorigenesis?
Possibly, yes. However, the above-mentioned findings show
that loss of tight junctions in cancer cells is a secondary
and/or late event of carcinogenesis, though tight junctions
are considered to be deeply involved in tumorigenesis and
invasion. On the other hand, tight junctions of endothelial and
mesothelial cells of the host possibly act as a barrier against
disordered migration leading to metastasis and dissemina-
tion of cancer cells.107–108
PROMISING APPROACHES TO BEDSIDE MEDICINE
Grossly, there are three prospective areas of application:
drug delivery systems (DDSs), markers for cancer and target
molecules for therapy. Regarding DDSs, development of
drugs to open tight junctions will help to treat brain tumors by
drugs,109 and to administer biologically active peptides.110 ,111
To invent new drug delivery techniques, we should follow
the strategies of pathogenic agents such as allergens and
viruses. In contrast to opening of tight junctions, it is theoreti-
cally possible that closing tight junctions could result in selec-
tive uptake of materials via a transcellular pathway like the
BBB. Thus, it is also feasible that dendritic cells or M cells
could be solely stimulated by closing tight junctions of sur-
rounding epithelial cells,55,112 possibly resulting in a new
method of vaccination.
As a marker for early stages of malignant lesions in the bile
duct113 and pancreas,11 4 claudin-18 is promising. It is also
expected to be a target molecule for cancer therapy.106 Exo-
somes derived from the blood of patients with ovarian cancer
contain claudi-4.115 However, further study to complete the
profile of claudin expression in human cancer is required.
Regarding cancer therapy, CPE seems to be effective
for treatment of cancer expressing claudin-4.116 Thus, this
claudin is highlighted as a target molecule for cancer therapy.
Since normal prostate epithelial cells and pancreas duct epi-
thelial cells are highly insensitive to CPE,70,117 CPE-modified
materials are expected to be new drugs for advanced or
hormone-resistant carcinoma as well as a tight junction
opener.118,119
Development of agents making tight junctions of endothe-
lial cells close might also be highly useful as new anti-
inflammatory drugs and anti-metastatic drugs. Such agents,
in particular, are very promising for prevention of diabetic
Table 3 Tissue expression of claudins in various cancers
Claudin
1 2 3 4 5 7 10 16 18 23
Breast ↑↓ ↑↓ ↑↓ ↓
Lung
Adenocarcinoma ↓↑
SCC ↑↓
Esophagus (SCC) ↑↓ ↑ ↑ ↑↓
Stomach ↑↑↑↓↑ ↑↓↓
Colon, rectum ↑↓↑↑ ↑↓↑↑↓
Hepatocellular carcinoma ↑↓ ↑ ↓ ↑↓ ↑
Biliary duct ↑↑
Pancreas duct ↑↑↑ ↑
Bladder ↑↓
Kidney ↑↑↑↑
Prostate ↑ ↑↑↓↑
Ovary (epithelial tumor) ↓ ↓↑↑↑ ↑
Uterus, cervix ↓↓ ↓ ↓
Uterus, body ↑↑ ↓
SCC, squamous cell carcinoma; ↑, increase; ↓, decrease; ↑↓, variable.
8 N. Sawada
© 2012 The Author
Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
retinopathy, the most common cause of losing sight. In the
future, much better understanding of the molecular mecha-
nisms of regulation of the functions will be required to apply
the cell biology of tight junctions to medicine and to make
tight junctions open or close with complete control.
CONCLUDING REMARKS
Fifteen years ago, no one could have imagined the recent
advances in tight junction research. Now we are struggling
to apply the outcome of the research to clinical medicine.
The surprising advances are considerably due to the out-
standing contributions of Dr Shoichiro Tsukita and his col-
leagues who discovered occludin and tricellulin, as well as
the claudin family. He proposed the field of Barriology,
based on the barrier function of tight junctions. Until
recently, research on tight junctions was exclusively on the
fence and barrier functions at the cellular level. Now it is
becoming clear that tight junctions are deeply involved in
immunity. However, the study of this issue is still in the
early stage. With advances in this area, tight junction
research may contribute to the development of drugs, vac-
cination and drug delivery systems. Further extensive study
is required to clarify the issues concerning tight junction
functions.5On the other hand, each of the tight junction
proteins, in particular each of the claudins, may play distinct
roles in cell biology in addition to the formation of tight junc-
tions. This issue should be also clarified.
The author declares that the research was conducted in
the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
ACKNOWLEDGMENTS
It was my great honor to present a summary of our research
over the past 20 years at the Department of Pathology,
Sapporo Medical University School of Medicine, as the
Pathology Award Lecture at the 101st annual meeting of the
Japanese Society of Pathology in Tokyo in 2012. To make
this review readable, it contains more background informa-
tion than my lecture. I thank my past and present collabora-
tors for their contributions to tight junction research. I also
thank Prof. Emeritus Michio Mori of Sapporo Medical Univer-
sity for his encouragement.
This work was supported by Grants-in-Aid from the Minis-
try of Education, Culture, Sports, Science and Technology
(08265253, 08457075, 09254252, 10152252, 12877280,
14370080, 14657448, 17659474, 17390117, 19390103), and
the Ministry of Health, Labor and Welfare (14110401) of
Japan, the National Project ‘Knowledge Cluster Initiative’
(2nd stage, Sapporo Biocluster Bio-S) Program for develop-
ing the supporting system for upgrading education and
research, and by the Kato Memorial Bioscience Foundation,
the Suhara Memorial Foundation and the Smoking Research
Foundation. I also thank Mr K. Barrymore for help with the
manuscript.
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