Interactions of tight junctions with membrane channels and transporters

Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.     

Interactions of tight junctions with membrane channels and transporters

Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.     

Comparison of the function of the tight junctions of endothelial cells and epithelial cells in regulating the movement of electrolytes and macromolecules across the cell monolayer

In cell culture, both endothelial and epithelial cell monolayers have been found to generate structurally similar tight junctional complexes, as assessed by thin complexes of the two cell types are, at least in part, responsible for the very different permeability characteristics of native endothelial and epithelial cell monolayers. The purpose of this work was to compare cultured endothelial and epithelial cells with respect to the function of their tight junctional complexes in regulating the movement of macromolecules and ions across the cell monolayers, and define functional parameters to characterize the tight junctional complexes. Bovine aorta endothelial cells and T84 colonic carcinoma epithelial cells were cultured on a microporous membrane support. The permeability coefficients of inulin, albumin, and insulin were determined with the cell monolayers and compared with the permeability coefficients obtained with 3T3-C2 fibroblasts, a cell line that does not generate tight junctions. Electrical resistance measurements across the monolayer-filter systems were also compared. The permeability coefficient of albumin across the endothelial cell monolayer compared favorably with other reported values. Likewise, the electrical resistance across the T84 cell monolayer was in good agreement with published values. Utilizing permeability coefficients for macromolecules as an index of tight junction function, we found that a distinction between a lack of tight junctions (fibroblasts), the presence of endothelial tight junctions, and the presence of epithelial tight junctions was readily made. However, when utilizing electrical resistance as an index of tight junction function, identical measurements were obtained with fibroblasts and endothelial cells. This indicates that more than one index of tight junction function is necessary to characterize the junctional complexes. Although structurally similar, epithelial cell and endothelial cell tight junctions perform very different functions, and, from our data, we conclude that the demonstration of tight junctional structures by electron microscopy is not relevant to the functional nature of the junction: structure does not imply function. A minimal assessment of tight junction function should rely on both the determination of the electrical resistance across the cell monolayer, and the determination of the permeability coefficients of selected macromolecules.     

Multiple domains of occludin are involved in the regulation of paracellular permeability

Tight junctions form selective paracellular diffusion barriers that regulate the diffusion of solutes across epithelia and constitute intramembrane diffusion barriers that prevent the intermixing of apical and basolateral lipids in the extracytoplasmic leaflet of the plasma membrane. In MDCK cells, previous expression experiments demonstrated that occludin, a tight junction protein with four transmembrane domains, is critically involved in both of these tight junction functions and that its COOH-terminal cytoplasmic domain is of functional importance. By expressing mutant and chimeric occludin that exert a dominant negative effect on selective paracellular diffusion, we now demonstrate that the extracytoplasmic domains and at least one of the transmembrane domains are also critically involved in selective paracellular permeability. Multiple domains of occludin are thus important for the regulation of paracellular permeability. Expression of chimeras containing at least one transmembrane domain of occludin also resulted in an enhanced intracellular accumulation of claudin-4, another transmembrane protein of tight junctions, suggesting that the two proteins may cooperate in the regulation of paracellular permeability.     

Organization and signaling of endothelial cell-to-cell junctions in various regions of the blood and lymphatic vascular trees

Adhesive intercellular junctions between endothelial cells are formed by tight junctions and adherens junctions. In addition to promoting cell-to-cell adhesion, these structures regulate paracellular permeability, contact inhibition of endothelial cell growth, cell survival, and maintenance of cell polarity. Furthermore, adherens junctions are required for the correct organization of new vessels during embryo development or during tissue proliferation in the adult. Extensive research on cultured epithelial and endothelial cells has resulted in the identification of many molecular components of tight junctions and adherens junctions. Such studies have revealed the complexity of these structures, which are formed by membrane-associated adhesion proteins and a network of several intracellular signaling partners. This review focuses on the structural organization of junctional structures and their functional interactions in the endothelium of blood vessels and lymphatics. We emphasize the way that these structures regulate endothelial cell homeostasis by transferring specific intracellular signals and by modulating activation and signaling of growth factor receptors. This work was supported by the Associazione Italiana per la Ricerca sul Cancro, Association for International Cancer Research, European Community (Integrated Project Contract no. LSHG-CT-2004–503573; NoE MAIN 502935; NoE EVGN 503254; EUSTROKE consortium; Angioscaff consortium; Optistem consortium), Istituto Superiore di Sanità, Italian Ministry of Health, MIUR (COFIN prot: 2006058482_002), and Fondation Leducq Transatlantic Network of Excellence (E.D.). Additional support came from US National Institutes of Health grants HL24136 and HL59157 from the National Heart, Lung, and Blood Institute and CA82923 from the National Cancer Institute and AngelWorks Foundation (D.McD.).     

Keratinocyte junctions and the epidermal barrier: how to make a skin-tight dress

Although intercellular junctions are known to be the major regulators of permeability of simple epithelia, they had not been thought to be important in regulating the permeability of stratified mammalian epithelia. Furuse et al. (2002)(this issue) demonstrate that functional tight junctions may indeed be a necessary part of the permeability barrier of the skin.     

Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis

Endothelial cells lining the vessel wall are connected by adherens, tight and gap junctions. These junctional complexes are related to those found at epithelial junctions but with notable changes in terms of specific molecules and organization. Endothelial junctional proteins play important roles in tissue integrity but also in vascular permeability, leukocyte extravasation and angiogenesis. In this review, we will focus on specific mechanisms of endothelial tight and adherens junctions.     

Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis

Endothelial cells lining the vessel wall are connected by adherens, tight and gap junctions. These junctional complexes are related to those found at epithelial junctions but with notable changes in terms of specific molecules and organization. Endothelial junctional proteins play important roles in tissue integrity but also in vascular permeability, leukocyte extravasation and angiogenesis. In this review, we will focus on specific mechanisms of endothelial tight and adherens junctions.     

The role of junctional adhesion molecules in cell-cell interactions

Cell-cell-interactions are important for the regulation of tissue integrity, the generation of barriers between different tissues and body compartments thereby providing an effective defence against toxic or pathogenic agents, as well as for the regulation of inflammatory cell recruitment. Intercellular interactions are regulated by adhesion receptors on adjacent cells which upon extracellular ligand binding mediate intracellular signals. In the vasculature, neighbouring endothelial cells interact with each other through various adhesion molecules leading to the generation of junctional complexes like tight junctions (TJs) and adherens junctions (AJs) which regulate both leukocyte endothelial interactions and paracellular permeability. In this context, emerging evidence points to the importance of the family of junctional adhesion molecules (JAMs), which are localized in tight junctions of endothelial and epithelial cells and are implicated in the regulation of both leukocyte extravasation as well as junction formation and permeability.     

Occludin modulates transepithelial migration of neutrophils

Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the paracellular route. To avoid breakdown of the epithelial barrier, this process requires coordinated opening and closing of tight junctions, the most apical intercellular junctions in epithelia. To determine the function of epithelial tight junction proteins in this process, we analyzed neutrophil migration across monolayers formed by stably transfected epithelial cells expressing wild-type and mutant occludin, a membrane protein of tight junctions with four transmembrane domains and both termini in the cytosol. We found that expression of mutants with a modified N-terminal cytoplasmic domain up-regulated migration, whereas deletion of the C-terminal cytoplasmic domain did not have an effect. The N-terminal cytosolic domain was also found to be important for the linear arrangement of occludin within tight junctions but not for the permeability barrier. Moreover, expression of mutant occludin bearing a mutation in one of the two extracellular domains inhibited neutrophil migration. The effects of transfected occludin mutants on neutrophil migration did not correlate with their effects on selective paracellular permeability and transepithelial electrical resistance. Hence, specific domains and functional properties of occludin modulate transepithelial migration of neutrophils.     

Regulation of tight junctions and loss of barrier function in pathophysiology

The mechanism by which epithelial and endothelial cells interact to form polarized tissue is of fundamental importance to multicellular organisms. Dysregulation of these barriers occurs in a variety of diseases, destroying the normal cellular environments and leading to organ failure. Increased levels of growth factors are a common characteristic of diseases exhibiting tissue permeability, suggesting that growth factors play a direct role in elevating permeability. Of particular concern for this laboratory, increased expression of vascular endothelial growth factor may enhance vascular permeability in diabetic retinopathy, leading to vision impairment and blindness. However, the mechanism by which growth factors increase permeability is unclear. Polarized cells form strong barriers through the development of tight junctions, which are specialized regions of the junctional complex. Tight junctions are composed of three types of transmembrane proteins, a number of peripheral membrane structural proteins, and are associated with a variety of regulatory proteins. Recent data suggest that growth factor-stimulated alterations in tight junctions contribute to permeability in a variety of disease states. The goal of this review was to elucidate potential mechanisms by which elevated growth factors elicit deregulated paracellular permeability via altered regulation of tight junctions, with particular emphasis on the tight junction proteins occludin and ZO-1, protein kinase C signaling, and endocytosis of junctional proteins. Understanding the molecular mechanisms underlying growth factor-mediated regulation of tight junctions will facilitate the development of novel treatments for diseases such as brain tumors, diabetic retinopathy and other diseases with compromised tight junction barriers.     

The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis

The coxsackie- and adenovirus receptor (CAR) is a transmembrane protein belonging to the immunoglobulin superfamily. The function of CAR as a virus receptor has been extensively analyzed, while its physiological role and expression pattern in adult tissues have remained less clear. CAR associates with epithelial tight junctions in vitro and mediates cell-cell adhesion. Using a set of affinity-purified antibodies, we show that CAR is predominantly expressed in epithelial cells lining the body cavities in adult mice, where it specifically co-localizes with the tight junction components ZO-1 and occludin. Notably, CAR could not be detected in endothelial cells of the vasculature, including brain capillaries. CAR expression correlated positively with the maturity of tight junctions and inversely with permeability. With a few exceptions, the two known CAR isoforms were co-expressed in most epithelial cells analyzed. A CAR mutant lacking the intracellular tail over-expressed in transgenic mice was diffusely localized over the plasma membrane, showing the importance of this domain for correct subcellular localization in vivo. We conclude that CAR is localized to epithelial tight junctions in vivo where it may play a role in the regulation of epithelial permeability and tissue homeostasis.     

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.     

Recent Advances in Alcoholic Liver Disease I. Role of intestinal permeability and endotoxemia in alcoholic liver disease

A significant body of evidence indicates that endotoxemia and endotoxin-mediated hepatocellular damage play a crucial role in the pathogenesis of alcoholic liver disease. A close correlation between endotoxemia and the severity of alcohol-induced liver injury is supported by a number of clinical and experimental studies. Elevated intestinal permeability appears to be the major factor involved in the mechanism of alcoholic endotoxemia and the pathogenesis of alcoholic liver disease. Ethanol and its metabolic derivatives, acetaldehyde in particular, alter intracellular signal-transduction pathways leading to the disruption of epithelial tight junctions and an increase in paracellular permeability to macromolecules. Studies addressing the mechanisms of such epithelial disruption and the protective factors that prevent ethanol and acetaldehyde-mediated disruption of epithelial tight junctions are critically important in the investigations toward the search of preventive and therapeutic strategies for alcoholic liver disease.     

Lysophosphatidic Acid Increases Tight Junction Permeability in Cultured Brain Endothelial Cells

Abstract: Brain capillary endothelial cells are coupled by a continuous belt of complex high-electrical-resistance tight junctions that are largely responsible for the blood-brain barrier. We have investigated mechanisms regulating tight junction permeability in brain endothelial cells cultured to maintain high-resistance junctions. The phospholipid lysophosphatidic acid (LPA) was found to cause a rapid, reversible, and dose-dependent decrease in transcellular electrical resistance in brain endothelial cells. LPA also increased the paracellular flux of sucrose, which, together with the resistance decrease, indicated increased tight junction permeability. Activation of protein kinase C attenuated the effect of LPA, suggesting that it was mediated by activation of a signalling pathway. LPA did not cause any obvious relocalization of adherens junction- or tight junction-associated proteins. However, it did stimulate the formation of stress fibres, the recruitment of focal adhesion components, and the appearance of tyrosine phosphorylated protein at focal contacts. Our study shows that LPA is a modulator of tight junction permeability in brain endothelial cells in culture and raises the possibility that it triggers blood-brain barrier permeability changes under (patho)physiological conditions.     

Intercellular junctions between human odontoblasts. A freeze-fracture study after demineralization

Intercellular junctions in the odontoblastic layer have been studied with a freeze-fracture technique. Children's tooth germs were fixed, sliced and demineralized. Samples of the pulpodentinal border were routinely prepared for freeze-fracture. Three kinds of intercellular junctions were detected between human odontoblast cell bodies: gap junctions, desmosomes and tight junctions. Numerous gap junctions are responsible for intercellular communication at different levels of the cell bodies. Focal tight junctions, parallel to the axis of the cell, and desmosomes are sites of cell-to-cell adhesion between lateral plasma membranes. At the distal end of the cell bodies, junctional complexes consist of zonular tight junctions and gap junctions. These zonular tight junctions, never before described between odontoblasts, contribute to the pseudo-epithelial organization of the odontoblastic layer. They constitute a predentin-pulp barrier, the permeability of which must be studied to establish their role in relation to dentin formation.     

Ras mutation impairs epithelial barrier function to a wide range of nonelectrolytes

Although ras mutations have been shown to affect epithelial architecture and polarity, their role in altering tight junctions remains unclear. Transfection of a valine-12 mutated ras construct into LLC-PK1 renal epithelia produces leakiness of tight junctions to certain types of solutes. Transepithelial permeability of D-mannitol increases sixfold but transepithelial electrical resistance increases >40%. This indicates decreased paracellular permeability to NaCl but increased permeability to nonelectrolytes. Permeability increases to D-mannitol (Mr 182), polyethylene glycol (Mr 4000), and 10,000-Mr methylated dextran but not to 2,000,000-Mr methylated dextran. This implies a "ceiling" on the size of solutes that can cross a ras-mutated epithelial barrier and therefore that the increased permeability is not due to loss of cells or junctions. Although the abundance of claudin-2 declined to undetectable levels in the ras-overexpressing cells compared with vector controls, levels of occludin and claudins 1, 4, and 7 increased. The abundance of claudins-3 and -5 remained unchanged. An increase in extracellular signal-regulated kinase-2 phosphorylation suggests that the downstream effects on the tight junction may be due to changes in the mitogen-activated protein kinase signaling pathway. These selective changes in permeability may influence tumorigenesis by the types of solutes now able to cross the epithelial barrier.     

Tight junctions in the rat parotid gland

The aim of this study was to elucidate the distribution and morphological changes of tight junctions during secretion in parotid gland acinar cells. Localization of tight junction-associated polypeptide ZO-1, and of tight junction transmembrane protein Occludin, was examined in rat parotid gland by immunofluorescence and immunogold labelling of ultrathin sections. Adult male Sprague-Dawley rats were intraperitoneally injected with IPR and, after 10 and 30 minutes, parotid glands were extirpated. In control specimens, positive immunoreaction for ZO-1 and Occludin was observed on the adluminal side between adjacent cells in the form of narrow elongated profiles corresponding to intercellular canaliculi. After IPR injection, canaliculi became dilated and fluorescence was no longer seen as a continuous line but appeared as an aggregation of separate bright particles. ZO-1 was more widely distributed and was recognized in other areas of the cytoplasm as well. Concurrently, omega-shaped concavities, marked by actin fluorescence, appeared along the intercellular canaliculi. We concluded that, during exocytosis, the selective permeability barrier to the paracellular pathway, based on tight junctions, becomes more leaky, owing to segregation of Occludin caused by intracellular ZO-1 distributional changes associated with actin filaments.     

Na-K-ATPase regulates tight junction permeability through occludin phosphorylation in pancreatic epithelial cells

Tight junctions are crucial for maintaining the polarity and vectorial transport functions of epithelial cells. We and others have shown that Na-K-ATPase plays a key role in the organization and permeability of tight junctions in mammalian cells and analogous septate junctions in Drosophila. However, the mechanism by which Na-K-ATPase modulates tight junctions is not known. In this study, using a well-differentiated human pancreatic epithelial cell line HPAF-II, we demonstrate that Na-K-ATPase is present at the apical junctions and forms a complex with protein phosphatase-2A, a protein known to be present at tight junctions. Inhibition of Na-K-ATPase ion transport function reduced protein phosphatase-2A activity, hyperphosphorylated occludin, induced rearrangement of tight junction strands, and increased permeability of tight junctions to ionic and nonionic solutes. These data suggest that Na-K-ATPase is required for controlling the tight junction gate function.