Med Electron Microsc (2003) 36:147–156 DOI 10.1007/s00795-003-0219-y

© The Clinical Electron Microscopy Society of Japan 2003

REVIEW Norimasa Sawada · Masaki Murata · Keisuke Kikuchi Makoto Osanai · Hirotoshi Tobioka · Takashi Kojima Hideki Chiba

Tight junctions and human diseases

Received: March 11, 2003 / Accepted: March 18, 2003

Abstract Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the barrier (or gate) function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as 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 biology, in terms of loss of cell polarity. Of the proteins comprising tight junctions, integral membrane proteins occludin, claudins, and JAMs have been recently discovered. Of these molecules, claudins are exclusively responsible for the formation of tight-junction strands and are connected with the actin cytoskeleton mediated by ZO-1. Thus, both functions of tight junctions are dependent on the integrity of the actin cytoskeleton as well as ATP. Mutations in the claudin14 and the claudin16 genes result in hereditary deafness and hereditary hypomagnesemia, respectively. Some pathogenic bacteria and viruses target and affect the tight-junction function, leading to diseases. In this review, the relationship between tight junctions and human diseases is summarized. Key words Tight junctions · Barrier function · Fence function · Claudin · Occludin · ZO-1 · Human diseases · Drug delivery

N. Sawada (*) · M. Murata · K. Kikuchi · M. Osanai · H. Tobioka · T. Kojima · H. Chiba Department of Pathology, Sapporo Medical University School of Medicine, S1, W17, Sapporo 060-8556, Japan Tel. ⫹81-11-611-2111; Fax ⫹81-11-613-5665 e-mail: [email protected]

Introduction Multicellular organisms are primarily required to establish a distinct internal environment to maintain their life. For this purpose, all their surfaces, the skin, gastrointestinal tract, respiratory tract, etc., are covered by various kinds of epithelia. More importantly, for epithelial and endothelial sheets to work efficiently as a barrier,1–3 intercellular spaces must be strictly sealed by tight junctions, which are characterized as a set of continuous and anastomosing strands at the apicalmost region of the lateral cell membranes (Fig. 1A,B).4,5 The prevention of free diffusion of solutes through paracellular spaces by tight junctions is referred to as the barrier function of the tight junction (Fig. 1C). When tight junctions of epithelial cells that cover the biliary tree and gastrointestinal tract become disordered, jaundice and diarrhea, respectively, occur. Although vascular permeability depends on both the paracellular pathway and caveolar transcellular pathway of endothelial sheets, edema developes mainly as a result of dysfunction of tight junctions between the cells. Even in one multicellular organism, several organs are relatively independent of its internal homeostasis and are wrapped by endothelial cell sheets. One example is the blood–brain barrier, composed of highly specialized endothelial cells with well-developed tight junctions that protect the central nervous system.6 Recently, it has become clear that the integral membrane proteins of tight junctions claudin-2, claudin-4, and claudin-16 form selective channels through the tight-junction barrier (Fig. 1C).6–12 Epithelial cells have two distinct domains of cell membranes, the apical and basolateral membrane. Because these 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 (Fig. 1C). When the function is impaired,

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Fig. 1. Morphology and functions of tight junctions. A Schematic diagram of tight junction. B Tight junction strands on freeze-fracture replica. C The fence and barrier functions of tight junctions

cells fail to do vectorial work, in terms of loss of cell polarity. Thus, this function is deeply involved in cancer cell biology.

Molecular components of the tight junction Integral membrane proteins of tight junctions On electron micrographs of freeze-fracture replicas, tight junctions are sets of continuous anastomosing strands formed by intramembranous particles (Fig. 1B). Thus, much effort has been expended to identify integral membrane proteins specific for tight-junction strands. Recently, the molecular composition of tight junctions has begun to be clarified in detail. To date, three kinds of integral membrane proteins, occludin,13 claudins,14,15 and members of the Ig superfamilies JAM and CAR,16–18 have been discovered. Of these proteins, the claudin family, consisting of more than 20 members, is solely responsible for forming tightjunction strands.2,19,20 Two or more different claudin species are generally expressed in single cells in various tissues.

Each claudin has two extracellular loops: the first loop is larger and more hydrophobic than the second loop. Claudins bind to ZO-1, ZO-2, ZO-3, MUPP1, and PATJ.21–23 Of the claudins, claudin-5 is predominantly expressed in endothelial cells.24 Claudin-3 and -4 are receptors for Clostridium perfringens enterotoxin.25,26 Claudin-11, previously termed oligodendrocyte-specific protein (OSP), is expressed in oligodendrocytes and Sertoli cells.27 Tight junctions of myelinating glia are found between membrane lamellae of the same cells and are named autotypic or reflexive junctions. In this context, Schwann cells, a kind of myelinating glia for peripheral nerves, express claudin-1, -2, and -5.28 Claudins are necessary and sufficient for the formation of tight junctions by their homophilic and heterophilic binding of adjacent cells.29,30 Some of them are expected to form exracellular aqueous pores in paracellular spaces.7–12 A distinct example is claudin-16/paracellin-1, responsible for hereditary hypomagnesemia.8 Claudin-16 forms paracellular magnesium channels in the thick ascending limb of Henle. Similarly, claudin-2 and -4 are considered to form selective channels in tight junctions.9–11 Claudins have recently been reported to recruit and promote the activation of pro-

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matrix metalloproteinase 2 (pro-MMP-2),31 suggesting that they may play as yet unknown roles in cellular processes other than tight-junction functions. Occludin was the first integral membrane protein of tight junctions to be discovered13 and can be clearly detected in tight-junction strands by immunolabeling freeze-fracture replicas.32 However, introduction of occludin cDNA into L cells fails to result in formation of continuous anastomosing strands,20 but the occludin-deficient embryonic stem (ES) cells have the capability to develop tight-junction strands indistinguishable from the normal strands.33 Nevertheless, the immunohistochemical intensity of occludin in various tissues correlates well with the number of the strands.34 Of the tight-junction proteins, occludin is the most ubiquitously expressed at the apicalmost basolateral membranes, being the most reliable immunohistochemical marker for tight junctions.35 Compared to claudins, occludin has a relatively long cytoplasmic C-terminus containing several phosphorylation sites and a coiled-coil domain, which probably interact with phosphokinase C (PKC)-ζ, c-Yes, connexin26, and the regulatory subunit of phosphatidylinositol 3kinase,36,37 as well as occludin itself, ZO-1,38 and ZO-3.39,40 The roles of occludin in the regulation of tight junctions remain to be clarified, although actin filaments seems to have a deeper association with occludin than with claudins.41 JAM (junctional adhersion molecule)-1, JAM-2, JAM-3, CAR (coxsackievirus and adenovirus receptor), and ESAM (endothelial cell-selective adhesion molecule) belong to the CTX family in the immunoglobulin superfamily.16–18,42,43 All the members have extracellular V-type and C2-type immunoglobulin domains, a single transmembrane region, and a cytoplasmic tail. JAM lacks the capability to form tightjunction strands. The cytoplasmic C-terminus of JAM binds to cingulin, occludin, ZO-1, AF-6, PAR-3, CASK/LIN-2, and MUPP1.22,44–48 Particularly after binding to PAR-3,46,47 PAR-6 and aPKC are recruited to establish tight junctions.49 JAM is a ligand of β2-integrin lymphocyte functionassociated antigen 1 (LFA-1)50 and also a receptor for reovirus.51 This group seems to play roles in inflammatory reactions, including infection and extravasation of inflammatory cells.

Cytoplasmic proteins These tight-junction proteins can be divided into two groups.2 One group consists of PDZ domain-containing proteins: ZO-1, ZO-2, ZO-3, ASIP/PAR-3, PAR-6, MAGI-1, MAGI-2, MAGI-3, AF-6/s-afadin,52 MUPP1,22 and PATJ.53 The other group comprises cingulin, 7H6 antigen, symplekin, heterotrimeric G proteins, aPKC, ZONAB, huASH1, Rab-3b, rab-13, PTEN, Pilt,54 JEAP,55 and protein phosphatase 2A.56 Of these proteins, ZO-1 and MUPP1 may work as scaffolds of tight junctions, because the former interacts with all the integral membrane proteins21,38,44 and the latter interacts with claudins and JAM.22 ZO-2 and ZO-3 also interact with claudins.21 APKC and PP 2A may regulate phosphorylation levels of the tight-junction proteins to es-

tablish cell polarity or to regulate tight-junction functions. Of these proteins, ZO-1, ZO-2, and cingulin57 can bind to actin filaments. PTEN, a tumor suppressor, is lipid phosphatase, which antagonizes PI3K/Akt signaling and binds to MAGI-1 and MAGI-2.58,59 Sec6/Sec8 homologues and VAP-33, which are involved in vesicular transport, colocalize with ZO-1 and/or occludin,60,61 although their localization at tight junctions has not been demonstrated by immunogold labeling. These findings, coupled with localization of rab3b and rab13, strongly suggest that microdomains around tight junctions are pivotal sites for polarized vesicular transport including tight-junction components.

Regulation of tight-junction functions Tight junctions, the apicalmost component of intercellular junctional complexes, separate the apical from the basolateral 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 fence and barrier functions of the tight junction 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, respectively. Although formation and maintenance of tight junctions require ATP62 and integrity of the actin cytoskeleton,63 the barrier function is more deeply dependent than the fence function on ATP64 and actin.65 In this context, Na,K-ATPasemediated regulation of RhoA GTPase activity is crucial for formation of tight junctions as well as the other intercellular junctions of polarized epithelial cells.66 Upon the introduction of occludin cDNA into L cells, gap junctions are occasionally associated with short strandlike structures on freeze-fracture replicas,19 suggesting a close relationship between gap and tight junctions. Some gap-junction proteins such as Cx43 and Cx45 are known to bind to ZO-1.67–69 Similarly, Cx32 colocalizes at tight junctions and probably interacts with tight-junction proteins.70 Consistently, intercellular communication via gap junctions composed of Cx32 enhances tight-junction functions.71 Effects of cytokines and growth factors that serve as extracellular stimuli on tight junctions are listed in Table 1. Interestingly, transforming growth factor-beta (TGF-β) and II-10 prevent cytokine-induced decrease in the barrier function,82,85 although many factors cause the barrier function to deteriorate. Tumor necrosis factor-alpha (TNF-α) and interferon-γ downregulate occludin expression at the transcriptional level.87 Other factors may regulate tight-junction barrier function via various signal transduction systems such as the mitogen-activated protein kinase (MAPK) pathway. For example, estrogen receptor kinase (ERK) activation is required for claudin-2 expression, but not that of claudin-1, in colonic cells.86 At present, information about transcriptional regulation of tight-junction proteins is very fragmentary.88–93 A transcriptional factor hepatocyte nuclear factor (HNF)-4α is demonstrated to initiate expression of at least three tight-junction molecules, occludin,

150 Table 1. Cytokines, growth factors, and tight-junction function Decrease the barrier function: IFN-γ,72 TNF-α,73 HGF,74 TGF-α,75 IGF-I and IGF-II,76 VEGF,77 IL-1,78 IL-4,79 IL-1380 Increase or protect the barrier function: EGF,81 TGF-β,82 GDNF,83 neurturin,84 IL-10,85 IL-1786

claudin-6, and claudin-7, as well as biogenesis of functional tight junctions and the acquisition of polarized epithelial morphology in F9 cells.94 Zonulin, the human intestinal analogue of Zonula occludens toxin derived from Vibrio cholerae, has a receptor on the surface of the small intestine.95,96 Zonulin causes PKC-dependent polymerization of actin filaments. Once the small intestine is exposed to enterobacteria, zonulin is secreted to disassemble tight junctions of the small intestine,97 resulting in diarrhea. Tight junctions of endothelial sheets in vivo are leaky in general, because a wide variety of substances must be exchanged between blood and organs through paracellular 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–tissue barrier.98 Interestingly, the endothelial cells forming the blood–tissue barrier in the brain,83,99 retina,84 and testis100 express receptors for glia cell line-derived neurotrophic factor (GDNF), which enhances the barrier function of the endothelial cells when coupled with cAMP in vitro. In addition, the blood–thymus barrier, the blood–air barrier in the lung, the blood–follicle barrier in the ovary, and the placental (or maternofetal) barrier are often referred to in textbooks, although the physiological regulation of each barrier is not fully understood. Regarding the biogenesis of tight junctions, it is speculated that formation of adherens junctions precedes formation of tight junctions.101–103 To establish cell polarity of epithelial cells, in terms of development of the junctional complexes, E-cadherin or nectin first binds to the respective molecules on the surfaces of adjacent cells in a homophilic manner. Immediately, ZO-1 and JAM are recruited at spotlike primordial adherens junctions, and then Par-3, Par6, and aPKC are recruited to JAM, and occludin and claudin are also recruited to ZO-1 complexes. Finally, by unknown mechanisms probably involving the aPKC–Par complex and delivery of the integral membrane proteins, tight junctions are segregated from adherens junctions. Consistently, ZO-1, a marker for tight junctions in wellpolarized epithelia, is found at adherens junctions as well as at tight junctions in epithelial cells showing not so well developed tight junctions, i.e., hepatocytes, and in cadherinbased adhesion sites in cadherin-transfected fibroblasts.104 This finding suggests that establishment of mature intercellular junctional complexes requires segregation of tight junctions from adherens junctions. The degree of segregation of tight junctions from adherens junctions, in other words, maturation of tight junctions, might result in distinct dependency on Rho, Rac, and Cdc42 in different cell types, i.e., epithelial105 versus endothelial106 cells.

Human diseases relevant to tight junctions Disturbance of the fence function In general, cancer cells lose their specific functions and polarity. The Ras oncogene downregulates tight-junction functions by regulating phosphorylation of occludin and ZO-1 but not claudin-1.107 Oncogenic raf-1 disrupts tight junctions by downregulating expression of occludin and claudin-1, resulting in phenotypic malignant changes.108 A decrease in occludin expression seems to be correlated to loss of cell polarity of human colonic glandular epithelium, in terms of dedifferentiation.109 MUPP1 and the MAGI family are targets of viral oncoproteins of human papillomavirus (HPV) and adenovirus.110–112 c-Kit binds to MUPP1.113 AF-6/s-afadin, involved in acute myeloid leukemia as an ALL-1 fusion partner, and MAGI-1 are substrates of Ras.114,115 On the other hand, claudin-1, -2, -3, and -5 promote activation of pro-MMP-2,31 and claudin-4 is overexpressed in ovarian cancer.116 However, very few reports have shown genetic changes in tight-junction proteins117 except for PTEN, a tumor suppressor.118 In welldifferentiated adenocarcinomas developed from human colon and endometrium, comparable amounts of occludin were detected by a morphometrical technique combined with immunohistochemistry of their original epithelium, and with further dedifferentiation cancer cells lose tight junctions (unpublished observation). Collectively, these findings may show that loss of tight junctions in cancer cells is a secondary or late event, although tight junctions are considered to be deeply involved in tumorigenesis and metastasis. Disturbance of the barrier function Because 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 pathological conditions where the physiological regulation of passage of ions, molecules, and inflammatory cells may be affected. In Table 2, human diseases involving dysfunction of tight junctions are listed. It is particularly clear that tight junctions are targets of pathogens such as bacteria, viruses, and allergens that enter the human body. These pathogens affect the barrier function of tight junctions by the following three mechanisms (summarized in Table 3). The first mechanism is functional changes of tight junction proteins. JAM is a receptor for reovirus51 and CAR is a receptor for coxsackievirus and adenovirus.18 JAM is required for reovirus-induced activation of NF-kB.51 Claudin-

151 Table 2. Human diseases relevant to tight-junction functions I. Disturbance of the fence function (maintenance of cell polarity): Cancer cells107–109 Oncogenic papillomavirus infection110–112 II. Disturbance of the barrier function (regulation of paracellular pathway): 1. Vascular system: Edema Endotoxinemia Cytokinemia78 Diabetic retinopathy138,140 Multiple sclerosis6 Blood-borne metastasis143,157 2. Gastrointestinal tract158: Bacterial gastritis127,128 Pseudomembranous colitis121 Crohn’s disease136,137 Ulcerative colitis135–137 Celiac disease95 Collagenous colitis159 3. Liver: Jaundice160,161 Primary biliary cirrhosis132 Primary sclerosing cholangitis132 4. Respiratory tract: Asthma120 Adult (or acute) respiratory distress syndrome (ARDS)162 5. Viral infections: Reovirus,51 adenovirus,18 coxsackievirus,18 rotavirus,126 HIV6 6. Hereditary diseases: Hypomagnesemia8 Deafness163 Cystic fibrosis164 7. Miscellaneous: Ovarian hyperstimulation syndrome165

3 and -4 are receptors for enterotoxin of Clostridium perfringens (CPE), which is a common cause of food poisoning.25,26 When CPE binds claudin-4 expressed in colonic surface cells, the complexes internalize as do other ligand– receptor complexes, and then the function of tight junctions becomes disordered.119 Very interestingly, this phenomenon is observed only when the basolateral surface of the cell is exposed to CPE. Hemagglutinin/protease (HA/P) produced by Vibrio cholerae digests occludin.120 V. cholerae cause severe diarrhea by the combination of cholerae toxin, zonula occludens toxin (ZOT), and HA/P. The cysteine protease allergen Der p 1 of fecal pellets of Dermatophagoides pteronyssinus also digests occludin, presumably resulting in opening tight junctions of the airway to enter inside the body and cause asthma.121 The second mechanism is a change in actin organization. Although actin organization is affected by a wide variety of cellular conditions, only direct involvement of Rho and myosin light chain kinase activity are listed. Toxins from Clostridium diphtheriae, Clostridium difficile, and enteropathogenic Escherichia coli change Rho activity by modification of amino acids122 to cause severe colitis, the former two causing psuedomembranous colitis. Enteropathogenic E. coli also induces myosin light chain phosphorylations,123 resulting in diarrhea. The last group contains many kinds of indirect mechanisms including unknown ones. ZOT of V. cholerae

Table 3. Mechanisms of dysfunction of the barrier function caused by pathogenic agents 1.

2.

3.

Functional changes of tight junction proteins (target molecules) Clostridium perfringens (claudin-3, -4) Vibrio cholerae (occludin) Reovirus (JAM) Coxsackievirus and adenovirus (CAR) Dermatophagoides pteronyssinus (occludin, claudin-1) Change in actin organization by modulation of Rho and myosin light chain kinase activity Clostridium diphtheriae Clostridium difficile Enteropathogenic Escherichia coli Indirect mechanisms such as PKC activation, including unknown ones Bacteroides fragilis Helicobacter pylori Rotavirus

activates PKC,124 resulting in loss of tight junctions. Bacteroides fragilis digests E-cadherin by its metalloprotease,125 causing disruption of tight junctions. Rotavirus and Helicobacter pylori vacuolating toxin cause an increase in paracellular permeability of intestinal cells by unknown mechanisms.126–128 Endotoxin directly induces permeability of colonic cells129 and dysfunction of hepatocyte tight junctions, causing hyperbilirubinemia.130 Experimental colitis, a model of inflammatory bowel diseases, Crohn’s disease, and ulcerative colitis, is often accompanied by intrahepatic cholestasis,131 presumably resulting from not only cytokinemia but endotoxinemia. Although tight junctions of hepatocytes are not fully segregated from adherens junctions,104 they prevent bile leakage to the blood flow (blood– biliary barrier132). In chronic cholestatic liver disease, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC), tight junctions in the liver are impaired.133 In the early phase of PBC, tight junctions of biliary epithelim are altered, whereas tight junctions of hepatocytes are altered in PSC. All kinds of pathological conditions involve cytokine production. The proinflammatory cytokines TNF-α and interferon-γ downregulate the expression of occludin, causing dysfunction of tight junctions.87 Both factors also cause redistribution of JAM in endothelial cells.134 In inflammatory bowel diseases, intestinal permeability increases, probably because of downregulation of occludin.135–137 Zonulin95–97 is secreted from the small intestine upon exposure of the intestine to enterobacteria, resulting in opening tight junctions of the intestine. During the acute phase of celiac disease, zonulin is overexpressed, accompanied by an increase in the intestinal permeability. Inflammation is always accompanied by an increase in vascular permeability, in part caused by vascular endothelial growth factor (VEGF), which primarily affects the barrier function of tight junctions by both phosphorylation and downregulation of occludin.138,139 Cancer cells also secrete VEGF to induce angiogenesis, and presumably to intravasate and extravasate, in other words, to pass forcibly through the tight junctions of vascular endothelium and metastasize. Thus, various kinds of diseases that involve

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VEGF production, such as diabetic retinopathy140 and metastasis,141–143 involve tight-junction dysfunction. Information about the relationships between the immune system and tight-junction proteins is still very fragmentary. Dendritic cells in the intestine express occludin, claudin-1, and ZO-1, sending their dendrites outside through paracellular spaces between colonic epithelium, probably to sample bacteria.144 Regarding transendothelial diapedesis of mononuclear cells, JAM and occludin seem to be invoved. JAM is a ligand of LFA-1 on the surface of the lymphocyte.50 Occludin is expressed in activated T lymphocytes to make the diapedesis smooth with minimal effects on the tight-junction function.145 Occludin is also involved in transepithelial migration of neutrophils.146 A protease of mast cells playing an important role in allergic reaction increases epithelial permeability, probably because of changes in localization of occludin.147 Furthermore, the relationship between regulation of tight junctions and the blood–thymus barrier, probably involved in establishment of the immune system, remains to be elucidated. Additionally, astrocytes playing important roles in repair of the central nervous system also express occludin and claudin-1 in vitro.148,149

Perspectives Since the discoveries of occludin in 199313 and of claudins in 1998,14 comprehension of tight junctions has been rapidly growing. Until 2001, no one accepted that squamous epithelium had functional tight junctions.150 This observation confirmed that the tight junction was a compartmentalizing apparatus to maintain homeostasis at the individual level as well as at the organ level.3 As applications of knowledge of tight junctions for medicine, drug delivery via tight junctions and cancer therapy using CPE seem promising. Development of drugs to open tight junctions will help to treat brain tumors by drugs,151 and to help to administer biologically active peptides152 or vectors carrying a specific gene per os.153 To invent new drug delivery techniques, we must follow the strategies of pathogenic agents. CPE seems to be effective for treatment of cancer-expressing claudin-3 and claudin-4.154,155 Development of agents making tight junctions close might also be highly useful as new types of antiinflammatory drugs, antimetastatic drugs, and antidiarrhea drugs.156 Such agents, in particular, are very promising for treatment of diabetic retinopathy, the most common cause of loss of sight. In the future, much better understanding of the molecular mechanisms of regulation of the functions will be required to apply the cell biology of tight junctions to medicine, to make tight junctions open or close with complete control. Acknowledgments This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labor and Welfare of Japan, and by the Kato Memorial Bioscience Foundation, the Uehara Memorial Foundation, the Suhara Memorial Foundation and the Smoking Research Foundation. We also thank Mr. K. Barrymore for help with the manuscript.

References 1. Cereijido M (ed) (1991) Tight junctions. CRC Press, Boca Raton 2. Tsukita S, Furuse M, Itoh M (2001) Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2:285–293 3. Tsukita S, Furuse M (2002) Claudin-based barrier in simple and stratified cellular sheets. Curr Opin Cell Biol 14:531–536 4. Farquhar MG, Palade GE (1963) Junctional complexes in various epithelia. J Cell Biol 17:375–412 5. Staehelin LA (1973) Further observations on the fine structure of freeze-cleaved tight junctions. J Cell Sci 13:763–786 6. Pardridge WM (ed) (1998) Introduction to the blood–brain barrier. Cambridge University Press, Cambridge 7. Tsukita S, Furuse M (2000) Pores in the wall: claudins constitute tight junction strands containing aqueous pores. J Cell Biol 149:13–16 8. Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP (1999) Paracellin-1, a renal tight junction protein required for paracellular Mg2⫹ resorption. Science 285:103–106 9. Amasheh S, Meiri N, Gitter AH, Schoneberg T, Mankertz J, Schulzke JD, Fromm M (2002) Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J Cell Sci 115:4969–4976 10. Furuse M, Furuse K, Sasaki H, Tsukita S (2001) Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells. J Cell Biol 153:263–272 11. Van Itallie C, Rahner C, Anderson JM (2001) Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest 107:1319– 1327 12. Colegio OR, Van Itallie CM, McCrea HJ, Rahner C, Anderson JM (2002) Claudins create charge-selective channels in the paracellular pathway between epithelial cells. Am J Physiol Cell Physiol 283:C142–C147 13. Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsuikta S (1993) Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123:1777–1788 14. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S (1998) Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141:1539–1555 15. Morita K, Furuse M, Fujimoto K, Tsukita S (1999) Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci U S A 96:511–516 16. Aurrand-Lions M, Johnson-Leger C, Wong C, du Pasquier L, Imhof BA (2001) Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three JAM family members. Blood 98:3699–3707 17. Martin-Padura I, Lostaglio S, Schneemann M, Williams L, Romano M, Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa A, Simmons D, Dejana E (1998) Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol 142:117–127 18. Cohen CJ, Shieh JT, Pickles RJ, Okegawa T, Hsieh JT, Bergelson JM (2001) The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction. Proc Natl Acad Sci U S A 98:15191–15196 19. Heiskala M, Peterson PA, Yang Y (2001) The roles of claudin superfamily proteins in paracellular transport. Traffic 2:93–98 20. Furuse M, Sasaki H, Fujimoto K, Tsukita S (1998) A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol 143:391–401 21. Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S (1999) Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J Cell Biol 147:1351–1363 22. Hamazaki Y, Itoh M, Sasaki H, Furuse M, Tsukita S (2002) MultiPDZ domain protein 1 (MUPP1) is concentrated at tight junc-

153

23.

24. 25.

26.

27. 28.

29. 30. 31.

32.

33.

34.

35. 36.

37.

38.

39.

40.

41.

tions through its possible interaction with claudin-1 and junctional adhesion molecule. J Biol Chem 277:455–461 Roh MH, Liu CJ, Laurinec S, Margolis B (2002) The carboxyl terminus of zona occludens-3 binds and recruits a mammalian homologue of discs lost to tight junctions. J Biol Chem 277:27501– 27509 Morita K, Sasaki H, Furuse M, Tsukita S (1999) Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol 147:185–194 Katahira J, Sugiyama H, Inoue N, Horiguchi Y, Matsuda M, Sugimoto N (1997) Clostridium perfringens enterotoxin utilizes two structurally related membrane proteins as functional receptors in vivo. J Biol Chem 272:26652–26658 Fujita K, Katahira J, Horiguchi Y, Sonoda N, Furuse M, Tsukita S (2000) Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein. FEBS Lett 476:258–261 Morita K, Sasaki H, Fujimoto K, Furuse M, Tsukita S (1999) Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis. J Cell Biol 145:579–588 Poliak S, Matlis S, Ullmer C, Scherer SS, Peles E (2002) Distinct claudins and associated PDZ proteins form different autotypic tight junctions in myelinating Schwann cells. J Cell Biol 159:361– 372 Furuse M, Sasaki H, Tsukita S (1999) Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol 147:891–903 Furuse M, Sasaki H, Fujimoto K, Tsukita S (1998) A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol 143:391–401 Miyamori H, Takino T, Kobayashi Y, Tokai H, Itoh Y, Seiki M, Sato H (2001) Claudin promotes activation of promatrix metalloproteinase-2 mediated by membrane-type matrix metalloproteinases. J Biol Chem 276:28204–28211. Fujimoto K (1995) Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes. J Cell Sci 108:3443– 3449 Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, Furuse M, Takano H, Noda T, Tsukita S (1998) Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol 141:397–408 Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Inazawa J, Fujimoto K, Tsukita S (1997) Mammalian occludin in epithelial cells: its expression and subcellular distribution. Eur J Cell Biol 73:222–231 Tsukita S, Furuse M (1999) Occludin and claudins in tightjunction strands: leading or supporting players? Trends Cell Biol 9:268–273 Nusrat A, Chen JA, Foley CS, Liang TW, Tom J, Cromwell M, Quan C, Mrsny RJ (2000) The coiled-coil domain of occludin can act to organize structural and functional elements of the epithelial tight junction. J Biol Chem 275:29816–29822 Chen YH, Lu Q, Goodenough DA, Jeansonne B (2002) Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells. Mol Biol Cell 13:1227–1237 Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S (1994) Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 127:1617–1626 Haskins J, Gu L, Wittchen ES, Hibbard J, Stevenson BR (1998) ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J Cell Biol 141:199–208 Fanning AS, Jameson BJ, Jesaitis LA, Anderson JM (1998) The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem 273:29745–29753 Kojima T, Sawada N, Yamamoto M, Kokai Y, Mori M, Mochizuki Y (1999) Disruption of circumferential actin filament causes disappearance of occludin from the cell borders of rat hepatocytes in primary culture without distinct changes of tight junction strands. Cell Struct Funct 24:11–17

42. Hirata Ki, Ishida T, Penta K, Rezaee M, Yang E, Wohlgemuth J, Quertermous T (2001) Cloning of an immunoglobulin family adhesion molecule selectively expressed by endothelial cells. J Biol Chem 276:16223–16231 43. Aurrand-Lions M, Duncan L, Ballestrem C, Imhof BA (2001) JAM-2, a novel immunoglobulin superfamily molecule, expressed by endothelial and lymphatic cells. J Biol Chem 276:2733– 2741 44. Bazzoni G, Martinez-Estrada OM, Orsenigo F, Cordenonsi M, Citi S, Dejana E (2000) Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin. J Biol Chem 275:20520–20526 45. Ebnet K, Schulz CU, Meyer Zu Brickwedde MK, Pendl GG, Vestweber D (2000) Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1. J Biol Chem 275:27979–27988 46. Itoh M, Sasaki H, Furuse M, Ozaki H, Kita T, Tsukita S (2001) Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions. J Cell Biol 154:491–497 47. Ebnet K, Suzuki A, Horikoshi Y, Hirose T, Meyer Zu Brickwedde MK, Ohno S, Vestweber D (2001) The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). EMBO J 20:3738–3748 48. Martinez-Estrada OM, Villa A, Breviario F, Orsenigo F, Dejana E, Bazzoni G (2001) Association of junctional adhesion molecule with calcium/calmodulin-dependent serine protein kinase (CASK/LIN-2) in human epithelial caco-2 cells. J Biol Chem 276:9291–9296 49. Ohno S (2001) Intercellular junctions and cellular polarity: the PAR-aPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Curr Opin Cell Biol 13:641–648 50. Ostermann G, Weber KS, Zernecke A, Schroder A, Weber C (2002) JAM-1 is a ligand of the β2 integrin LFA-1 involved in transendothelial migration of leukocytes. Nat Immunol 3:151–158 51. Barton ES, Forrest JC, Connolly JL, Chappell JD, Liu Y, Schnell FJ, Nusrat A, Parkos CA, Dermody TS (2001) Junction adhesion molecule is a receptor for reovirus. Cell 104:441–451 52. Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH, Crompton A, Chan AC, Anderson JM, Cantley LC (1997) Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275:73–77 53. Roh MH, Makarova O, Liu CJ, Shin K, Lee S, Laurinec S, Goyal M, Wiggins R, Margolis B (2002) The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J Cell Biol 157:161–172 54. Kawabe H, Nakanishi H, Asada M, Fukuhara A, Morimoto K, Takeuchi M, Takai Y (2001) Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells. J Biol Chem 276: 48350–48355 55. Nishimura M, Kakizaki M, Ono Y, Morimoto K, Takeuchi M, Inoue Y, Imai T, Takai Y (2002) JEAP, a novel component of tight junctions in exocrine cells. J Biol Chem 277:5583–5587 56. Nunbhakdi-Craig V, Machleidt T, Ogris E, Bellotto D, White CL III, Sontag E (2002) Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex. J Cell Biol 158:967–978 57. D’Atri F, Citi S (2001) Cingulin interacts with F-actin in vitro. FEBS Lett 507:21–24 58. Wu Y, Dowbenko D, Spencer S, Laura R, Lee J, Gu Q, Lasky LA (2000) Interaction of the tumor suppressor PTEN/MMAC with a PDZ domain of MAGI3, a novel membrane-associated guanylate kinase. J Biol Chem 275:21477–21485 59. Wu X, Hepner K, Castelino-Prabhu S, Do D, Kaye MB, Yuan XJ, Wood J, Ross C, Sawyers CL, Whang YE (2000) Evidence for regulation of the PTEN tumor suppressor by a membranelocalized multi-PDZ domain containing scaffold protein MAGI2. Proc Natl Acad Sci U S A 97:4233–4238 60. Grindstaff KK, Yeaman C, Anandasabapathy N, Hsu SC, Rodriguez-Boulan E, Scheller RH, Nelson WJ (1998) Sec6/8 complex is recruited to cell–cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell 93:731–740 61. Lapierre LA, Tuma PL, Navarre J, Goldenring JR, Anderson JM (1999) VAP-33 localizes to both an intracellular vesicle popula-

154

62. 63. 64. 65.

66.

67.

68.

69. 70.

71.

72. 73. 74.

75.

76. 77.

78. 79.

80. 81.

tion and with occludin at the tight junction. J Cell Sci 112:3723– 3732 Muckter H, Ben-Shaul Y, Bacher A (1987) ATP requirement for induced tight junction formation in HT 29 adenocarcinoma cells. Eur J Cell Biol 44:258–264 Bentzel CJ, Hainau B, Edelman A, Anagnostopoulos T, Benedetti EL (1976) Effect of plant cytokinins on microfilaments and tight junction permeability. Nature (Lond) 264:666–668 Mandel LJ, Bacallao R, Zampighi G (1993) Uncoupling of the molecular “fence” and paracellular “gate” functions in epithelial tight junctions. Nature (Lond) 361:552–555 Takakuwa R, Kokai Y, Kojima T, Akatsuka T, Tobioka H, Sawada N, Mori M (2000) Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B. Exp Cell Res 257:238–244 Rajasekaran SA, Palmer LG, Moon SY, Peralta Soler A, Apodaca GL, Harper JF, Zheng Y, Rajasekaran AK (2001) Na,K-ATPase activity is required for formation of tight junctions, desmosomes, and induction of polarity in epithelial cells. Mol Biol Cell 12:3717–3732 Toyofuku T, Yabuki M, Otsu K, Kuzuya T, Hori M, Tada M (1998) Direct association of the gap junction protein connexin43 with ZO-1 in cardiac myocytes. J Biol Chem 273:12725– 12731 Laing JG, Manley-Markowski RN, Koval M, Civitelli R, Steinberg TH (2001) Connexin45 interacts with zonula occludens1 and connexin43 in osteoblastic cells. J Biol Chem 276:23051– 23055 Kausalya PJ, Reichert M, Hunziker W (2001) Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells. FEBS Lett 505:92–96 Kojima T, Kokai Y, Chiba H, Yamamoto M, Mochizuki Y, Sawada N (2001) Cx32 but not Cx26 is associated with tight junctions in primary cultures of rat hepatocytes. Exp Cell Res 263:193–201 Kojima T, Spray DC, Kokai Y, Chiba H, Mochizuki Y, Sawada N (2002) Cx32 formation and/or Cx32-mediated intercellular communication induces expression and function of tight junctions in hepatocytic cell line. Exp Cell Res 276:40–51 Madara JL, Stafford J (1989) Interferon-gamma directly affects barrier function of cultured intestinal epithelial monolayers. J Clin Invest 83:724–727 Mullin JM, Snock KV (1990) Effect of tumor necrosis factor on epithelial tight junctions and transepithelial permeability. Cancer Res 50:2172–2176 Nusrat A, Parkos CA, Bacarra AE, Godowski PJ, Delp-Archer C, Rosen EM, Madara JL (1994) Hepatocyte growth factor/scatter factor effects on epithelia. Regulation of intercellular junctions in transformed and nontransformed cell lines, basolateral polarization of c-met receptor in transformed and natural intestinal epithelia, and induction of rapid wound repair in a transformed model epithelium. J Clin Invest 93:2056–2065 Buse P, Woo PL, Alexander DB, Reza A, Firestone GL (1995) Glucocorticoid-induced functional polarity of growth factor responsiveness regulates tight junction dynamics in transformed mammary epithelial tumor cells. J Biol Chem 270:28223–28227 McRoberts JA, Riley NE (1992) Regulation of T84 cell monolayer permeability by insulin-like growth factors. Am J Physiol 262:C207–C213 Kevil CG, Payne DK, Mire E, Alexander JS (1998) Vascular permeability factor/vascular endothelial cell growth factormediated permeability occurs through disorganization of endothelial junctional proteins. J Biol Chem 273:15099–15103 Marcus BC, Wyble CW, Hynes KL, Gewertz BL (1996) Cytokineinduced increases in endothelial permeability occur after adhesion molecule expression. Surgery (St Louis) 120:411–417 Colgan SP, Resnick MB, Parkos CA, Delp-Archer C, McGuirk D, Bacarra AE, Weller PF, Madara JL (1994) IL-4 directly modulates function of a model human intestinal epithelium. J Immunol 153:2122–2129 Zund G, Madara JL, Dzus AL, Awtrey CS, Colgan SP (1996) Interleukin-4 and interleukin-13 differentially regulate epithelial chloride secretion. J Biol Chem 271:7460–7464 Soler AP, Laughlin KV, Mullin JM (1993) Effects of epidermal growth factor versus phorbol ester on kidney epithelial (LLC-

82. 83.

84.

85.

86. 87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

PK1) tight junction permeability and cell division. Exp Cell Res 207:398–406 Dignass AU, Podolsky DK (1993) Cytokine modulation of intestinal epithelial cell restitution: central role of transforming growth factor β. Gastroenterology 105:1323–1332 Igarashi Y, Utsumi H, Chiba H, Yamada-Sasamori Y, Tobioka H, Kamimura Y, Furuuchi K, Kokai Y, Nakagawa T, Mori M, Sawada N (1999) Glial cell line-derived neurotrophic factor induces barrier function of endothelial cells forming the blood– brain barrier. Biochem Biophys Res Commun 261:108–112 Igarashi Y, Chiba H, Utsumi H, Miyajima H, Ishizaki T, Gotoh T, Kuwahara K, Tobioka H, Satoh M, Mori M, Sawada N (2000) Expression of receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin in the inner blood-retinal barrier of rats. Cell Struct Funct 25:237–241 Madsen KL, Lewis SA, Tavernini MM, Hibbard J, Fedorak RN (1997) Interleukin 10 prevents cytokine-induced disruption of T84 monolayer barrier integrity and limits chloride secretion. Gastroenterology 113:151–159 Kinugasa T, Sakaguchi T, Gu X, Reinecker HC (2000) Claudins regulate the intestinal barrier in response to immune mediators. Gastroenterology 118:1001–1011 Mankertz J, Tavalali S, Schmitz H, Mankertz A, Riecken EO, Fromm M, Schulzke JD (2000) Expression from the human occludin promoter is affected by tumor necrosis factor-α and interferon-γ. J Cell Sci 113:2085–2090 Wachtel M, Bolliger MF, Ishihara H, Frei K, Bluethmann H, Gloor SM (2001) Down-regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type-1 receptor and nuclear factor-kB activation. J Neurochem 78:155–162 Ota T, Fujii M, Sugizaki T, Ishii M, Miyazawa K, Aburatani H, Miyazono K (2002) Targets of transcriptional regulation by two distinct type I receptors for transforming growth factor-β in human umbilical vein endothelial cells. J Cell Physiol 193:299–318 Miwa N, Furuse M, Tsukita S, Niikawa N, Nakamura Y, Furukawa Y (2000) Involvement of claudin-1 in the beta-catenin/ Tcf signaling pathway and its frequent upregulation in human colorectal cancers. Oncol Res 12:469–476 Sakaguchi T, Gu X, Golden HM, Suh E, Rhoads DB, Reinecker HC (2002) Cloning of the human claudin-2 5⬘-flanking region revealed a TATA-less promoter with conserved binding sites in mouse and human for caudal-related homeodomain proteins and hepatocyte nuclear factor-1α. J Biol Chem 277:21361–21370 Niimi T, Nagashima K, Ward JM, Minoo P, Zimonjic DB, Popescu NC, Kimura S (2001) Claudin-18, a novel downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing. Mol Cell Biol 21:7380–7390 Kubota H, Chiba H, Takakuwa Y, Osanai M, Tobioka H, Kohama G, Mori M, Sawada N (2001) Retinoid X receptor and retinoic acid receptor mediate expression of genes encoding tightjunction proteins and barrier function in F9 cells during visceral endodermal differentiation. Exp Cell Res 263:163–172 Chiba H, Gotoh T, Kojima T, Satohisa S, Kikuchi K, Osanai M, Sawada N (2003) Hepatocyte nuclear factor (HNF)-4α triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells. Exp Cell Res 286:288–297 Fasano A, Not T, Wang W, Uzzau S, Berti I, Tommasini A, Goldblum SE (2000) Zonulin, a newly discovered modulator of intestinal permeability, and its expression in coeliac disease. Lancet 355:1518–1519 Di Pierro M, Lu R, Uzzau S, Wang W, Margaretten K, Pazzani C, Maimone F, Fasano A (2001) Zonula occludens toxin structurefunction analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain. J Biol Chem 276:19160–19165 Asmar REI, Panigrahi P, Bamford P, Berti I, Not T, Coppa GV, Catassi C, Fasano A (2002) Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure. Gastroenterology 123:1607–1615 Huber JD, Egleton RD, Davis TP (2001) Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier. Trends Neurosci 24:719–725

155 99. Utsumi H, Chiba H, Kamimura Y, Osanai M, Igarashi Y, Tobioka H, Mori M, Sawada N (2000) Expression of GFRα-1, receptor for GDNF, in rat brain capillary during postnatal development of the BBB. Am J Physiol Cell Physiol 279:C361–C368 100. Kamimura Y, Chiba H, Utsumi H, Gotoh T, Tobioka H, Sawada N (2002) Barrier function of microvessels and roles of glial cell line-derived neurotrophic factor in the rat testis. Med Electron Microsc 35:139–145 101. Takeichi M (1991) Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251:1451–1455 102. Ohno S (2001) Intercellular junctions and cellular polarity: the PAR-aPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Curr Opin Cell Biol 13:641–648 103. Takai Y, Nakanishi H (2003) Nectin and afadin: novel organizers of intercellular junctions. J Cell Sci 116:17–27 104. Itoh M, Nagafuchi A, Yonemura S, Kitani-Yasuda T, Tsukita S (1993) The 220-kD protein colocalizing with cadherins in nonepithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy. J Cell Biol 121:491–502 105. Rojas R, Ruiz WG, Leung SM, Jou TS, Apodaca G (2001) Cdc42dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells. Mol Biol Cell 12:2257–2274 106. Wojciak-Stothard B, Potempa S, Eichholtz T, Ridley AJ (2001) Rho and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci 114:1343–1355 107. Chen Y, Lu Q, Schneeberger EE, Goodenough DA (2000) Restoration of tight junction structure and barrier function by downregulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells. Mol Biol Cell 11:849–862 108. Li D, Mrsny RJ (2000) Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin. J Cell Biol 148:791–800 109. Tobioka H, Isomura H, Kokai Y, Sawada N (2002) Polarized distribution of carcinoembryonic antigen is associated with a tight junction molecule in human colorectal adenocarcinoma. J Pathol 198:207–212 110. Glaunsinger BA, Lee SS, Thomas M, Banks L, Javier R (2000) Interactions of the PDZ-protein MAGI-1 with adenovirus E4ORF1 and high-risk papillomavirus E6 oncoproteins. Oncogene 19:5270–5280 111. Thomas M, Glaunsinger B, Pim D, Javier R, Banks L (2001) HPV E6 and MAGUK protein interactions: determination of the molecular basis for specific protein recognition and degradation. Oncogene 20:5431–5439 112. Lee SS, Glaunsinger B, Mantovani F, Banks L, Javier RT (2000) Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins. J Virol 74:9680–9693 113. Mancini A, Koch A, Stefan M, Niemann H, Tamura T (2000) The direct association of the multiple PDZ domain containing proteins (MUPP-1) with the human c-Kit C-terminus is regulated by tyrosine kinase activity. FEBS Lett 482:54–58 114. Yamamoto T, Harada N, Kano K, Taya S, Canaani E, Matsuura Y, Mizoguchi A, Ide C, Kaibuchi K (1997) The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells. J Cell Biol 139:785–795 115. Dobrosotskaya I, Guy RK, James GL (1997) MAGI-1, a membrane-associated guanylate kinase with a unique arrangement of protein-protein interactiondomains. J Biol Chem 276: 28204–28211 116. Hough CD, Sherman-Baust CA, Pizer ES, Montz FJ, Im DD, Rosenshein NB, Cho KR, Riggins GJ, Morin PJ (2000) Largescale serial analysis of gene expression reveals genes differentially expressed in ovarian cancer. Cancer Res 60:6281–6287 117. Hoover KB, Liao SY, Bryant PJ (1998) Loss of the tight junction MAGUK ZO-1 in breast cancer: relationship to glandular differentiation and loss of heterozygosity. Am J Pathol 153:1767–1773 118. Mayo LD, Donner DB (2002) The PTEN, Mdm2, p53 tumor suppressor-oncoprotein network. Trends Biochem Sci 27:462– 467 119. Sonoda N, Furuse M, Sasaki H, Yonemura S, Katahira J, Horiguchi Y, Tsukita S (1999) Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction

120.

121.

122. 123.

124.

125. 126. 127.

128.

129.

130.

131.

132.

133.

134.

135.

136.

137.

strands: evidence for direct involvement of claudins in tight junction barrier. J Cell Biol 147:195–204 Wu Z, Nybom P, Magnusson KE (2000) Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1. Cell Microbiol 2:11–17 Wan H, Winton HL, Soeller C, Tovey ER, Gruenert DC, Thompson PJ, Stewart GA, Taylor GW, Garrod DR, Cannell MB, Robinson C (1999) Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions. J Clin Invest 104:123–133 Boquet P (1999) Bacterial toxins inhibiting or activating small GTP-binding proteins. Ann N Y Acad Sci 886:83–90 Yuhan R, Koutsouris A, Savkovic SD, Hecht G (1997) Enteropathogenic Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial permeability. Gastroenterology 113:1873–1882 Fasano A, Fiorentini C, Donelli G, Uzzau S, Kaper JB, Margaretten K, Ding X, Guandalini S, Comstock L, Goldblum SE (1995) Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization in vitro. J Clin Invest 96:710–720 Wu S, Lim KC, Huang J, Saidi RF, Sears CL (1998) Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, Ecadherin. Proc Natl Acad Sci U S A 95:14979–14984 Svensson L, Finlay BB, Bass D, von Bonsdorff CH, Greenberg HB (1991) Symmetric infection of rotavirus on polarized human intestinal epithelial (Caco-2) cells. J Virol 65:4190–4197 Papini E, Satin B, Norais N, de Bernard M, Telford JL, Rappuoli R, Montecucco C (1998) Selective increase of the permeability of polarized epithelial cell monolayers by Helicobacter pylori vacuolating toxin. J Clin Invest 102:813–820 Suzuki K, Kokai Y, Sawada N, Takakuwa R, Kuwahara K, Isogai E, Isogai H, Mori M (2002) SS1 Helicobacter pylori disrupts the paracellular barrier of the gastric mucosa and leads to neutrophilic gastritis in mice. Virchows Arch 440:318–324 Kimura H, Sawada N, Tobioka H, Isomura H, Kokai Y, Hirata K, Mori M (1997) Bacterial lipopolysaccharide reduced intestinal barrier function and altered localization of 7H6 antigen in IEC-6 rat intestinal crypt cells. J Cell Physiol 171:284–290 Kawaguchi T, Sakisaka S, Mitsuyama K, Harada M, Koga H, Taniguchi E, Sasatomi K, Kimura R, Ueno T, Sawada N, Mori M, Sata M (2000) Cholestasis with altered structure and function of hepatocyte tight junction and decreased expression of canalicular multispecific organic anion transporter in a rat model of colitis. Hepatology 31:1285–1295 Lora L, Mazzon E, Martines D, Fries W, Muraca M, Martin A, d’Odorico A, Naccarato R, Citi S (1997) Hepatocyte tightjunctional permeability is increased in rat experimental colitis. Gastroenterology 113:1347–1354 Kojima T, Sawada N, Duffy HS, Spray DC (2001) Gap and tight junctions in liver: composition, regulation and function. In: Arias IM, Boyer JL, Chisari FV, Fausto N, Schachter D, Shafritz DA (eds) The liver: biology and pathobiology, 4th edn. Lippincott Williams & Wilkins, Philadelphia, pp 29–46 Sakisaka S, Kawaguchi T, Taniguchi E, Hanada S, Sasatomi K, Koga H, Harada M, Kimura R, Sata M, Sawada N, Mori M, Todo S, Kurohiji T (2001) Alterations in tight junctions differ between primary biliary cirrhosis and primary sclerosing cholangitis. Hepatology 33:1460–1468 Ozaki H, Ishii K, Horiuchi H, Arai H, Kawamoto T, Okawa K, Iwamatsu A, Kita T (1999) Cutting edge: combined treatment of TNF-α and IFN-γ causes redistribution of junctional adhesion molecule in human endothelial cells. J Immunol 163:553–557 Schmitz H, Barmeyer C, Fromm M, Runkel N, Foss HD, Bentzel CJ, Riecken EO, Schulzke JD (1999) Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis. Gastroenterology 116:301–309 Gassler N, Rohr C, Schneider A, Kartenbeck J, Bach A, Obermuller N, Otto HF, Autschbach F (2001) Inflammatory bowel disease is associated with changes of enterocytic junctions. Am J Physiol Gastrointest Liver Physiol 281:G216–G228 Kucharzik T, Walsh SV, Chen J, Parkos CA, Nusrat A (2001) Neutrophil transmigration in inflammatory bowel disease is associated with differential expression of epithelial intercellular junction proteins. Am J Pathol 159:2001–2009

156 138. Antonetti DA, Barber AJ, Hollinger LA, Wolpert EB, Gardner TW (1999) Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occluden 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors. J Biol Chem 274:23463– 23467 139. Wang W, Dentler WL, Borchardt RT (2001) VEGF increases BMEC monolayer permeability by affecting occludin expression and tight junction assembly. Am J Physiol Heart Circ Physiol 280:H434–H440 140. Murata T, Ishibashi T, Khalil A, Hata Y, Yoshikawa H, Inomata H (1995) Vascular endothelial growth factor plays a role in hyperpermeability of diabetic retinal vessels. Ophthalmic Res 27:48–52 141. Satoh H, Zhong Y, Isomura H, Saitoh M, Enomoto K, Sawada N, Mori M (1996) Localization of 7H6 tight junction-associated antigen along the cell border of vascular endothelial cells correlates with paracellular barrier function against ions, large molecules, and cancer cells. Exp Cell Res 222:269–274 142. Tobioka H, Sawada N, Zhong Y, Mori M (1996) Enhanced paracellular barrier function of rat mesothelial cells partially protects against cancer cell penetration. Br J Cancer 74:439– 445 143. Mori M, Sawada N, Kokai Y, Satoh M (1999) Role of tight junctions on the occurrence of cancer invasion and metastasis. Med Electron Microsc 32:193–198 144. Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, Granucci F, Kraehenbuhl JP, Ricciardi-Castagnoli P (2001) Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2:361–367 145. Alexander JS, Dayton T, Davis C, Hill S, Jackson TH, Blaschuk O, Symonds M, Okayama N, Kevil CG, Laroux FS, Berney SM, Kimpel D (1998) Activated T-lymphocytes express occludin, a component of tight junctions. Inflammation 22:573–582 146. Huber D, Balda MS, Matter K (2000) Occludin modulates transepithelial migration of neutrophils. J Biol Chem 275:5773– 5778 147. Scudamore CL, Jepson MA, Hirst BH, Miller HR (1998) The rat mucosal mast cell chymase, RMCP-II, alters epithelial cell monolayer permeability in association with altered distribution of the tight junction proteins ZO-1 and occludin. Eur J Cell Biol 75:321– 330 148. Bauer H, Stelzhammer W, Fuchs R, Weiger TM, Danninger C, Probst G, Krizbai IA (1999) Astrocytes and neurons express the tight junction-specific protein occludin in vitro. Exp Cell Res 250:434–438 149. Duffy HS, John GR, Lee SC, Brosnan CF, Spray DC (2000) Reciprocal regulation of the junctional proteins claudin-1 and connexin43 by interleukin-1β in primary human fetal astrocytes. J Neurosci 20:RC114(1–6) 150. Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A, Sugitani Y, Noda T, Kubo A, Tsukita S (2002) Claudin-based tight junctions

151. 152.

153. 154.

155.

156.

157. 158. 159. 160.

161.

162. 163.

164. 165.

are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 156:1099–1111 Miller G (2002) Breaking down the barriers. Nature (Lond) 419:863 Fasano A, Uzzau S (1997) Modulation of intestinal tight junctions by Zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model. J Clin Invest 99:1158–1164 Coyne CB, Kelly MM, Boucher RC, Johnson LG (2000) Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate. Am J Respir Cell Mol Biol 23:602–609 Michl P, Buchholz M, Rolke M, Kunsch S, Lohr M, McClane B, Tsukita S, Leder G, Adler G, Gress TM (2001) Claudin-4: a new target for pancreatic cancer treatment using Clostridium perfringens enterotoxin. Gastroenterology 121:678–684 Long H, Crean CD, Lee WH, Cummings OW, Gabig TG (2001) Expression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4 in prostate cancer epithelium. Cancer Res 61:7878–7881 Zolotarevsky Y, Hecht G, Koutsouris A, Gonzalez DE, Quan C, Tom J, Mrsny RJ, Turner JR (2002) A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease. Gastroenterology 123:163–172 Martin TA, Jiang WG (2001) Tight junctions and their role in cancer metastasis. Histol Histopathol 16:1183–1195 Schulzke J-D, Fromm M, Riecken E-O, Binder HJ (eds) (2000) Epithelial transport and barrier pathomechanisms in gastrointestinal disorders. Ann N Y Acad Sci 915:1–375 Burgel N, Bojarski C, Mankertz J, Zeitz M, Fromm M, Schulzke JD (2002) Mechanisms of diarrhea in collagenous colitis. Gastroenterology 123:433–443 Rahner C, Stieger B, Landmann L (1996) Structure-function correlation of tight junctional impairment after intrahepatic and extrahepatic cholestasis in rat liver. Gastroenterology 110:1564–1578 Takakuwa Y, Kokai Y, Sasaki K, Chiba H, Tobioka H, Mori M, Sawada N (2002) Bile canalicular barrier function and expression of tight-junctional molecules in rat hepatocytes during common bile duct ligation. Cell Tissue Res 307:181–189 Dudek SM, Garcia JGN (2001) Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol 91:1487–1500 Wilcox ER, Burton QL, Naz S, Riazuddin S, Smith TN, Ploplis B, Belyantseva I, Ben-Yosef T, Liburd NA, Morell RJ, Kachar B, Wu DK, Griffith AJ, Riazuddin S, Friedman TB (2001) Mutations in the gene encoding tight junction claudin-14 cause autosomal recessive deafness DFNB29. Cell 104:165–172 Coyne CB, Vanhook MK, Gambling TM, Caeson JL, Boucher RC, Johnson LG (2002) Regulation of airway tight junctions by proinflammatory cytokines. Mol Biol Cell 13:3218–3234 McClure N, Healy DL, Rogers PA, Sullivan J, Beaton L, Haning RV Jr, Connolly DT, Robertson DM (1994) Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome. Lancet 344:235–236