Junctional Adhesion Molecule,a Novel Member of the Immunoglobulin Superfamily That Distributes at Intercellular Junctions and Modulates Monocyte Transmigration

Tight junctions are the most apical components of endothelial and epithelial intercellular cleft. In the endothelium these structures play an important role in the control of paracellular permeability to circulating cells and solutes. The only known integral membrane protein localized at sites of membrane–membrane interaction of tight junctions is occludin, which is linked inside the cells to a complex network of cytoskeletal and signaling proteins. We report here the identification of a novel protein (junctional adhesion molecule [JAM]) that is selectively concentrated at intercellular junctions of endothelial and epithelial cells of different origins. Confocal and immunoelectron microscopy shows that JAM codistributes with tight junction components at the apical region of the intercellular cleft. A cDNA clone encoding JAM defines a novel immunoglobulin gene superfamily member that consists of two V-type Ig domains. An mAb directed to JAM (BV11) was found to inhibit spontaneous and chemokine-induced monocyte transmigration through an endothelial cell monolayer in vitro. Systemic treatment of mice with BV11 mAb blocked monocyte infiltration upon chemokine administration in subcutaneous air pouches. Thus, JAM is a new component of endothelial and epithelial junctions that play a role in regulating monocyte transmigration.     

Reciprocal influence of connexins and apical junction proteins on their expressions and functions

Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another.     

Tight junctions and the modulation of barrier function in disease

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease.     

Structure, regulation, and pathophysiology of tight junctions in the gastrointestinal tract.

The tight junction, or zonula occludens, forms an intercellular barrier between epithelial cells within the gastrointestinal tract and liver and, by limiting the movement of water and solutes through the intercellular space, maintains the physicochemical separation of tissue compartments. The paracellular barrier properties of junctions are regulated and quite different among epithelia. The junction also forms an intramembrane barrier between the apical and basolateral membrane domains, contributing to segregation of biochemically distinct components of these plasma membrane surfaces. Here we briefly review three rapidly developing areas of medically relevant basic knowledge about the tight junction. First, we describe the presently incomplete knowledge of the molecular structure of the tight junction as a framework for understanding its functional properties. Second, we consider experimental evidence defining how the barrier properties of junctions are physiologically regulated and, third, how barrier properties are specifically altered in, and contribute to, pathologic processes affecting epithelia.     

A freeze-fracture and lanthanum tracer study of the complex junction between Sertoli cells of the canine testis

What appear to be true septate junctions by all techniques currently available for the cytological identification of intercellular junctions are part of a complex junction that interconnects the Sertoli cells of the canine testis. In the seminiferous epithelium, septate junctions are located basal to belts of tight junctions. In thin sections, septate junctions appear as double, parallel, transverse connections or septa spanning an approximately 90-A intercellular space between adjacent Sertoli cells. In en face sections of lanthanum-aldehyde-perfused specimens, the septa themselves exclude lanthanum and appear as electron-lucent lines arranged in a series of double, parallel rows on a background of electron-dense lanthanum. In freeze-fracture replicas this vertebrate septate junction appears as double, parallel rows of individual or fused particles which conform to the distribution of the intercellular septa. Septate junctions can be clearly distinguished from tight junctions as tight junctions prevent the movement of lanthanum tracer toward the lumen, appear as single rows of individual or fused particles in interlacing patterns within freeze-fracture replicas, and are seen as areas of close membrane apposition in thin sections. Both the septate junction and the tight junction are associated with specializations of the Sertoli cell cytoplasm. This is the first demonstration in a vertebrate tissue of a true septate junction.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.     

A novel four transmembrane spanning protein, CLP24. A hypoxically regulated cell junction protein.

A novel hypoxically regulated intercellular junction protein (claudin-like protein of 24 kDa, CLP24) has been identified that shows homology to the myelin protein 22/epithelial membrane protein 1/claudin family of cell junction proteins, which are involved in the modulation of paracellular permeability. The CLP24 protein contains four predicted transmembrane domains and a C-terminal protein-protein interaction domain. These domains are characteristic of the four transmembrane spanning (tetraspan) family of proteins, which includes myelin protein 22, and are involved in cell adhesion at tight, gap and adherens junctions. Expression profiling analyses show that CLP24 is highly expressed in lung, heart, kidney and placental tissues. Cellular studies confirm that the CLP24 protein localizes to cell-cell junctions and co-localizes with the beta-catenin adherens junction-associated protein but not with tight junctions. Over-expression of CLP24 results in decreased adhesion between cells, and functional paracellular flux studies confirm that over-expression of the CLP24 protein modulates the junctional barrier function. These data therefore suggest that CLP24 is a novel, hypoxically regulated tetraspan adherens junction protein that modulates cell adhesion, paracellular permeability and angiogenesis.     

Tight junction claudins and the kidney in sickness and in health

The epithelial cell tight junction has several functions including the control of paracellular transport between epithelial cells. Renal paracellular transport has been long recognized to exhibit unique characteristics within different segments of the nephron, functions as an important component of normal renal physiology and has been speculated to contribute to renal related pathology if functioning abnormally. The discovery of a large family of tight junction associated 4-transmembrane spanning domain proteins named claudins has advanced our understanding on how the paracellular permeability properties of tight junctions are determined. In the kidney, claudins are expressed in a nephron-specific pattern and are major determinants of the paracellular permeability of tight junctions in different nephron segments. The combination of nephron segment claudin expression patterns, inherited renal diseases, and renal epithelial cell culture models is providing important clues about how tight junction claudin molecules function in different segments of the nephron under normal and pathological conditions. This review discusses early observations of renal tubule paracellular transport and more recent information on the discovery of the claudin family of tight junction associated membrane proteins and how they relate to normal renal function as well as diseases of the human kidney.     

Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells

The epithelial lining of the gastrointestinal tract forms a regulated, selectively permeable barrier between luminal contents and the underlying tissue compartments. Permeability across the epithelium is, in part, determined by the rate-limiting barrier of the paracellular pathway-the most apical intercellular junction referred to as the tight junction (TJ). The TJ is composed of a multiprotein complex that affiliates with the underlying apical actomyosin ring. TJ structure and function, and therefore epithelial permeability, are influenced by diverse physiological and pathological stimuli; here we review examples of such stimuli that are detected at the cell surface. For example, luminal glucose induces an increase in paracellular permeability to small molecules. Similarly, but by other means, cytokines and leukocytes in the vicinity of the epithelium also regulate TJ structure and paracellular permeability by influencing the TJ protein complex and/or its association with the underlying actin cytoskeleton.     

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background  

Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction.     

Claudins--key pieces in the tight junction puzzle

Tight junctions restrict the flow of ions and aqueous molecules between cells by forming a selective barrier to the paracellular pathway. Permeability of the tight junction barrier is determined by a class of transmembrane proteins known as claudins. The relationship between claudins and paracellular permeability is complex and determined not only by the profile of claudin expression but also by the arrangement of claudins and other proteins into tight junction strands. This review summarizes progress in understanding how claudins are assembled into tight junctions and how they interact with other tight junction proteins.     

Structural organization of the tight junctions

Tight junctions are the most apical organelle of the apical junctional complex and are primarily involved in the regulation of paracellular permeability and membrane polarity. Extensive research in the past two decades has identified not only the individual molecules of the tight junctions but also their mutual interactions, which are the focus of the present review article. While a complete map of the interactions among the tight junction molecules is probably far from being complete, the available evidence already allows outlining the general molecular architecture of the tight junctions. Here, with the aim of gaining deeper mechanistic understanding of tight junction assembly, regulation and function, we have subdivided the known molecular interactions into four major clusters that are centered on cell surface, polarity, cytoskeletal and signaling molecules.     

Morphologic characteristics of intercellular junctions in early embryogenesis of mice

Dynamics concerning certain intercellular junctions have been followed during the preimplantation period of development in mouse embryos. The morphological analysis of the preimplantational embryos has demonstrated, that at the initial stages of cleavage (2-4 blastomeres) the cells make contacts by means of nonspecific junctions. Specialized intercellular junctions appear at the stage of 8 blastomeres and are presented as dotted tight and gap junctions. When the embryo is developing from the stage of 8 up to the stage of 16 blastomeres, certain connective complexes appear, consisting of dotted or cord-like tight and gap junctions. At the late morula stage, the external blastomeres in the apical part have contacts with each other by means of cingular tight junctions. In this place a connective complex might emerge; it is displayed as a tight junction and one or two gap junctions. At the blastocyst stage desmosomes and adhision zones appear. Between trophectodermal cells a connective complex arises; it is presented in the slice as a tight cingular junction, desmosomes (as a rule two) and an adhision zone. Between cells of the internal cellular mass the intercellular junctions are presented as dotted tight and gap junctions. Cells of the polar trophoectoderm and cells of the internal cellular mass could have contacts by means of gap and dotted tight junctions.     

Structural organization of the tight junctions

Tight junctions are the most apical organelle of the apical junctional complex and are primarily involved in the regulation of paracellular permeability and membrane polarity. Extensive research in the past two decades has identified not only the individual molecules of the tight junctions but also their mutual interactions, which are the focus of the present review article. While a complete map of the interactions among the tight junction molecules is probably far from being complete, the available evidence already allows outlining the general molecular architecture of the tight junctions. Here, with the aim of gaining deeper mechanistic understanding of tight junction assembly, regulation and function, we have subdivided the known molecular interactions into four major clusters that are centered on cell surface, polarity, cytoskeletal and signaling molecules.     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

Inverse correlation between the extent of N-glycan branching and intercellular adhesion in epithelia. Contribution of the Na,K-ATPase beta1 subunit

The majority of cell adhesion molecules are N-glycosylated, but the role of N-glycans in intercellular adhesion in epithelia remains ill-defined. Reducing N-glycan branching of cellular glycoproteins by swainsonine, the inhibitor of N-glycan processing, tightens and stabilizes cell-cell junctions as detected by a 3-fold decrease in the paracellular permeability and a 2-3-fold increase in the resistance of the adherens junction proteins to extraction by non-ionic detergent. In addition, exposure of cells to swainsonine inhibits motility of MDCK cells. Mutagenic removal of N-glycosylation sites from the Na,K-ATPase beta(1) subunit impairs cell-cell adhesion and decreases the effect of swainsonine on the paracellular permeability of the cell monolayer and also on detergent resistance of adherens junction proteins, indicating that the extent of N-glycan branching of this subunit is important for intercellular adhesion. The N-glycans of the Na,K-ATPase beta(1) subunit and E-cadherin are less complex in tight renal epithelia than in the leakier intestinal epithelium. The complexity of the N-glycans linked to these proteins gradually decreases upon the formation of a tight monolayer from dispersed MDCK cells. This correlates with a cell-cell adhesion-induced increase in expression of GnT-III (stops N-glycan branching) and a decrease in expression of GnTs IVC and V (promote N-glycan branching) as detected by real-time quantitative PCR. Consistent with these results, partial silencing of the gene encoding GnT-III increases branching of N-glycans linked to the Na,K-ATPase beta(1) subunit and other glycoproteins and results in a 2-fold increase in the paracellular permeability of MDCK cell monolayers. These results suggest epithelial cells can regulate tightness of cell junctions via remodeling of N-glycans, including those linked to the Na,K-ATPase beta(1)-subunit.     

PERMEABILITY OF SERTOLI CELL TIGHT JUNCTIONS TO LANTHANUM AFTER LIGATION OF DUCTUS DEFERENS AND DUCTULI EFFERENTES

The permeability of Sertoli cell tight junctions to lanthanum administered during fixation has been compared in rats after ligation of the ductus deferens and after ligation of the ductuli efferentes. In both control and vasoligated testes, lanthanum penetrated only short distances into the Sertoli cell tight junctions before stopping abruptly. The tight junction, consisting of numerous pentalaminar fusions of contiguous Sertoli cell membranes, prevented diffusion of lanthanum into the adluminal compartment of the seminiferous epithelium. In rats with ligated ductuli efferentes, lanthanum completely permeated many Sertoli cell tight junctions and occupied intercellular spaces of the adluminal compartment. In spite of their newly acquired permeability to lanthanum, tight junctions retained characteristic ultrastructural features, including numerous membrane fusions. When lanthanum-filled tight junctions were sectioned en face, membrane fusions appeared as pale lines in lakes of electron-opaque tracer. These linearly extensive fasciae occludentes occasionally ended blindly, suggesting that lanthanum may have traversed the junction by diffusing around such incomplete barriers. The increased permeability of Sertoli cell tight junctions after efferent ductule ligation, which caused rapid testicular weight gain followed by atrophy, indicates that tight junctions are sensitive to enforced retention of testicular secretions inside the seminiferous tubules. The apparent normalcy of Sertoli cell tight junctions after vasoligation, which had no effect on testis weight, supports the view that blockage of testicular secretions distal to the epididymis is relatively innocuous.     

封闭蛋白的结构、功能及其与人类疾病

紧密连接（tight junction,TJ）是脊椎动物细胞间连接的一种主要形式,对介导上皮细胞间的黏合、维持上皮细胞的功能具有重要作用。TJ是由一系列跨膜蛋白和外周蛋白相互作用而形成的一个复杂的蛋白质体系,封闭蛋白（occludin）是构成TJ的主要成分之一。目前,已发现封闭蛋白与许多人类疾病有关。本文仅就封闭蛋白的结构、功能及其与人类疾病的关系做一综述。     

Functional analysis of tight junctions

Epithelial and endothelial cells are joined to each other via a set of intercellular junctions that differ in their morphological appearance, composition, and function. The tight junction or zonula occludens is the intercellular junction that regulates diffusion between cells and therefore allows endothelia and epithelia to form cellular barriers that separate compartments of different composition. This intercellular gate formed by tight junctions is not only highly regulated but is size- and ion-selective and, hence, represents a semipermeable diffusion barrier. In epithelia, tight junctions form a morphological and functional border between the apical and basolateral cell surface domains. They directly contribute to the maintenance of cell surface polarity by forming a fence that prevents apical/basolateral diffusion of lipids in the outer leaflet of the plasma membrane. Here we describe a set of assays that allow the analysis of tight junctions to determine their integrity and functional state.     

The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium

The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.