Molecular physiology and pathophysiology of tight junctions. I. Biogenesis of tight junctions and epithelial polarity

The tight junction (TJ) was first noticed through its ability to control permeation across the paracellular route, but the homologies of its molecular components with peptides that participate in tumor suppression, nuclear addressing, and cell proliferation indicate that it may be involved in many other fundamental functions. TJs are formed by a dozen molecular species that assemble through PDZ and other protein-protein clustering promoting sequences, in response to the activation of E-cadherin. The TJ occupies a highly specific position between the apical and the basolateral domains. Its first molecular components seem to be delivered to such a position by addressing signals in their molecule and, once anchored, serve as a clustering nucleus for further TJ-associated molecules. Although in mature epithelial cells TJs and E-cadherin do not colocalize, a complex chain of reactions goes from one to the other that involves alpha-, beta-, and gamma-catenins, two different G proteins, phospholipase C, protein kinase C, calmodulin, mitogen-activated protein kinase, and molecules pertaining to the cytoskeleton, which keep the TJ sensitive to physiological requirements and local conditions (notably to Ca(2+)-dependent cell-cell contacts) throughout the life of the epithelium.     

Cell-matrix interactions and cell-cell junctions during epithelial histo-morphogenesis in the developing mouse incisor.

The continuously growing rodent incisor develops mainly along its antero-posterior axis. The labio-lingual asymmetry which characterizes this tooth is initiated at the cap stage and increases further during the cap to bell transition (ED14 to ED16) when histogenesis of the enamel organ proceeds. Histology, transmission electron microscopy (TEM), and immunostaining were used to document the changes in the basement membrane (BM) as well as the modifications of epithelial cell-matrix and cell-cell interactions during this period. The expression of plakoglobin, desmoglein and E-cadherin at ED14 suggested that the main cell-cell junctional complexes were adherens junctions. The expression of desmoglein and TEM observations suggested a progressive antero-posterior stabilization of the enamel organ by means of desmosomes from ED14 to ED18. alpha6 integrin, BP 230 and laminin gamma2 chain were all expressed in the developing incisor but were not always co-distributed. Immunostaining and TEM suggested that only primitive type II hemidesmosomes were present. At ED14, cells of the enamel knot (EK) did not show any specific expression for antigens involved in cell-cell interaction. However, strong staining for the laminin gamma2 chain characterized the BM in contact with EK cells. The BM in the labial part of the cervical loop demonstrated ultrastructural changes: the presence of loops of the lamina densa in this region preceeded the differential expression of the integrin alpha6 subunit and that of the laminin gamma2 chain in the labial/lingual parts of the cervical loop. Apoptosis was transiently observed in the contiguous mesenchyme. This affected osteoblasts and also nerve cells close to the labial part of the cervical loop.     

Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin

Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell-cell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1-induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.     

Nephrocystin-conserved domains involved in targeting to epithelial cell-cell junctions,interaction with filamins,and establishing cell polarity

Nephrocystin is the protein product of the gene mutated in juvenile nephronophthisis, an autosomal recessive cystic kidney disease afflicting children and young adults. Because the normal cellular function of nephrocystin is largely unknown, the molecular defects underlying disease pathogenesis remain obscure. Analysis of nephrocystin amino acid sequences from human and other species revealed three distinct conserved domains including Src homology 3 and coil-coil domains in the N-terminal region, as well as a large highly conserved C-terminal region bearing no obvious homology to other proteins and hence referred to as the "nephrocystin homology domain" (NHD). The objective of this study was to gain insight into nephrocystin function by defining functional properties of the conserved domains. We analyzed a series of nephrocystin deletion mutants expressed in Madin-Darby canine kidney and COS-7 cells. This analysis revealed previously unrecognized functional attributes of the NHD, including abilities to promote both self-association and epithelial cell-cell junctional targeting. We further observed that Madin-Darby canine kidney cell lines stably expressing a nephrocystin mutant with a deletion of the Src homology 3 domain have reduced ability to establish tight junctions as measured by transepithelial electrical resistance. Finally, from a two-hybrid screen and coimmunoprecipitation studies we identified members of the filamin family of actin-binding proteins as having the capacity to interact with the NHD. These findings support a functional role for nephrocystin as a docking protein involved in organizing a protein complex to regulate the actin cytoskeleton at sites of epithelial cell-cell adhesion and further suggest that these properties are important for establishing epithelial cell polarity.     

RhoGTPase signalling at epithelial tight junctions: Bridging the GAP between polarity and cancer

The establishment and maintenance of epithelial polarity must be correctly controlled for normal development and homeostasis. Tight junctions (TJ) in vertebrates define apical and basolateral membrane domains in polarized epithelia via bi-directional, complex signalling pathways between TJ themselves and the cytoskeleton they are associated with. RhoGTPases are central to these processes and evidence suggests that their regulation is coordinated by interactions between GEFs and GAPs with junctional, cytoplasmic adapter proteins. In this InFocus review we determine that the expression, localization or stability of a variety of these adaptor proteins is altered in various cancers, potentially representing an important mechanistic link between loss of polarity and cancer. We focus here, on two well characterized RhoGTPases Cdc42 and RhoA who's GEFs and GAPs are predominantly localized to TJ via cytoplasmic adaptor proteins.     

RalA and RalB differentially regulate development of epithelial tight junctions

Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity. Although many interactions between TJ constituents have been identified, questions remain concerning how specific functions of TJs are established and regulated. Here we investigated the roles of Ral GTPases and their common effector exocyst complex in the formation of nascent TJs. Unexpectedly, RNA interference-mediated suppression of RalA or RalB caused opposing changes in TJ development. RalA reduction increased paracellular permeability and decreased incorporation of components into TJs, whereas RalB reduction decreased paracellular permeability and increased incorporation of components into TJs. Activities of both Ral GTPases were mediated through the exocyst. Finally, we show that TJ-mediated separation of apical-basal membrane domains is established prior to equilibration of barrier function and that it is unaffected by Ral knockdown or specific composition of TJs.     

Assembly of tight junctions during early vertebrate development

Tight junction formation during development is critical for embryonic patterning and organization. We consider mechanisms of junction biogenesis in cleaving mouse and Xenopus eggs. Junction assembly follows the establishment of cell polarity at 8-cell (mouse) or 2-cell (Xenopus) stages, characterized by sequential membrane delivery of constituents, coordinated by embryonic (mouse) or maternal (Xenopus) expression programmes. Cadherin adhesion is permissive for tight junction construction only in the mouse. Occludin post-translational modification and membrane delivery, mediated by delayed ZO-1 alpha(+)isoform expression in the mouse, provides a mechanism for completion of tight junction biogenesis and sealing, regulating the timing of blastocoel cavitation.     

EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins β1‐integrin and Na⁺/K⁺ ATPase to the apical membrane of host cells. The Crumbs (Crb) polarity complex (Crb3/Pals1/Patj) is essential for epithelial cell polarisation and tight junction (TJ) assembly. Here, we demonstrate that EPEC displaces Crb3 and Pals1 from the apical membrane to the cytoplasm of cultured intestinal epithelial cells and colonocytes of infected mice. In vitro studies show that EspF, but not Map, alters Crb3, whereas both effectors modulate Pals1. EspF perturbs polarity formation in cyst morphogenesis assays and induces endocytosis and apical redistribution of Na⁺/K⁺ ATPase. EspF binds to sorting nexin 9 (SNX9) causing membrane remodelling in host cells. Infection with ΔespF/pespFD3, a mutant strain that ablates EspF binding to SNX9, or inhibition of dynamin, attenuates Crb3 endocytosis caused by EPEC. In addition, infection with ΔespF/pespFD3 has no impact on Na⁺/K⁺ ATPase endocytosis. These data support the hypothesis that EPEC perturbs apical–basal polarity in an EspF‐dependent manner, which would contribute to EPEC‐associated diarrhoea by disruption of TJ and altering the crucial positioning of membrane transporters involved in the absorption of ions and solutes.     

Regulation of epithelial cell shape and polarity by cell-cell adhesion (Review)

Among all cell types that exhibit a polarized phenotype, epithelial cells are unique in that their polarity depends on the integration of the cell into a tissue, the epithelium. In recent years, the analysis of epithelial cell polarity in different epithelia and organisms has contributed to an understanding of the components involved and has further demonstrated that cell polarity and cell adhesion are intimately related to each other. Therefore, processes that mediate and modulate cell adhesion and coordinate adhesion and cell shape are fundamental for the function of epithelia. Recent results obtained in Drosophila melanogaster and Caenorhabditis elegans have provided further insight into the complex circuits regulating these processes, and have laid the direction for future analysis.     

Assembly of epithelial tight junctions is negatively regulated by Par6

Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts-claudins, occludin, and junctional adhesion molecule (JAM)-plus peripheral proteins such as ZO-1. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs and more generally in metazoan cell polarity establishment. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Here, we show that Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca(2+) depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCzeta, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs, but neither a dominant-negative Cdc42 mutant nor the CRIB domain of gammaPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells.     

Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity

The development of polarized epithelial cells from unpolarized precursor cells follows induction of cell-cell contacts and requires resorting of proteins into different membrane domains. We show that in MDCK cells the distributions of two membrane proteins, Dg-1 and E-cadherin, become restricted to the basal-lateral membrane domain within 8 h of cell-cell contact. During this time, however, 60-80% of newly synthesized Dg-1 and E-cadherin is delivered directly to the forming apical membrane and then rapidly removed, while the remainder is delivered to the basal-lateral membrane and has a longer residence time. Direct delivery of greater than 95% of these proteins from the Golgi complex to the basal-lateral membrane occurs greater than 48 h later. In contrast, we show that two apical proteins are efficiently delivered and restricted to the apical cell surface within 2 h after cell-cell contact. These results provide insight into mechanisms involved in the development of epithelial cell surface polarity, and the establishment of protein sorting pathways in polarized cells.     

PAR3beta,a novel homologue of the cell polarity protein PAR3, localizes to tight junctions

The cell polarity protein PAR3, conserved from the nematode to the vertebrate, forms a complex with PAR6 and atypical protein kinase C (aPKC), and the protein complex occurs at the tight junctions in mammalian epithelial cells. Here we have cloned human cDNA for a novel PAR3 homologue, designated PAR3beta, whose messages are present in a variety of tissues and most abundantly expressed in the adult and fetal kidneys. The encoded protein of 1,205 amino acids contains a region homologous to the aPKC-binding domain of PAR3alpha, another human homologue previously identified, and three PDZ domains; the first PDZ domain of PAR3alpha is considered to interact with PAR6. Unexpectedly, in contrast to other PAR3s found in various species, PAR3beta is incapable of binding to any isotypes of PAR6 or aPKC. Nevertheless PAR3beta, expressed intrinsically or extrinsically, localizes to the tight junctions, indicating that the localization does not require the ternary complex formation.     

A novel monoclonal antibody against the second extracellular loop of occludin disrupts epithelial cell polarity.

The tight junction (TJ) regulates epithelial cell polarity and paracellular permeability. In the present study, to investigate whether the second extracellular loop of occludin affects the localization of carcinoembryonic antigen (CEA) and CD26 expressed on apical membranes, and the fence function of the TJ, the human intestinal epithelial cell line T84 was treated with the monoclonal anti-occludin antibody (MAb) 1H8, corresponding to the second extracellular loop of occludin. In T84 cells treated with MAb 1H8, occludin disappeared, and CEA and CD26 were observed to diffuse from the apical membrane to the basolateral membrane. Furthermore, a decrease in the fence function of TJ was observed without changes in the TJ strands and barrier function. When T84 cells precultured in low calcium (Ca) medium were recultured in normal Ca medium in the presence of MAb 1H8, recruitment of occludin to the apical-most membranes and recovery in distribution of CEA and CD26 were markedly retarded compared with the control. These results suggested that MAb 1H8 against the second extracellular loop of occludin selectively affected formation of the apical/basolateral intramembrane diffusion barrier and that the second extracellular loop of occludin plays a crucial role in the maintenance of epithelial cell polarity by the TJ.     

Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction

We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1alpha- isoform, was co-localized with both alpha- and beta-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1alpha- and other TJ proteins (ZO-1alpha+ isoform, occludin, cingulin) co-localized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy confirmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the specification of the AJC site and the formation and segregation of the TJ.     

Glucocorticoid down-regulation of fascin protein expression is required for the steroid-induced formation of tight junctions and cell-cell interactions in rat mammary epithelial tumor cells

Glucocorticoid hormones, which are physiological regulators of mammary epithelium development, induce the formation of tight junctions in rat Con8 mammary epithelial tumor cells. We have discovered that, as part of this process, the synthetic glucocorticoid dexamethasone strongly and reversibly down-regulated the expression of fascin, an actin-bundling protein that also interacts with the adherens junction component beta-catenin. Ectopic constitutive expression of full-length mouse fascin containing a Myc epitope tag (Myc-fascin) in Con8 cells inhibited the dexamethasone stimulation of transepithelial electrical resistance, disrupted the induced localization of the tight junction protein occludin and the adherens junction protein beta-catenin to the cell periphery, and prevented the rearrangement of the actin cytoskeleton. Ectopic expression of either the carboxyl-terminal 213 amino acids of fascin, which includes the actin and beta-catenin-binding sites, or the amino-terminal 313 amino acids of fascin failed to disrupt the glucocorticoid induction of tight junction formation. Mammary tumor cells expressing the full-length Myc-fascin remained generally glucocorticoid responsive and displayed no changes in the levels or protein-protein interactions of junctional proteins or the amount of cytoskeletal associated actin filaments. However, a cell aggregation assay demonstrated that the expression of Myc-fascin abrogated the dexamethasone induction of cell-cell adhesion. Our results implicate the down-regulation of fascin as a key intermediate step that directly links glucocorticoid receptor signaling to the coordinate control of junctional complex formation and cell-cell interactions in mammary tumor epithelial cells.     

Regulation of cell polarity during epithelial morphogenesis

Epithelial cells have an apical surface facing a lumen or outside of the organism, and a basolateral surface facing other cells and extracellular matrix. The identity of the apical surface is determined by phosphatidylinositol 4,5-bisphosphate, while phosphatidylinositol 3,4,5-trisphophosphate determines the identity of the basolateral surface. The Par3/Par6/atypical protein kinase C complex, as well as the Crumbs and Scribble complexes, controls epithelial polarity. Par4 and AMP kinase regulate polarity during conditions of energy depletion. Lumens are formed in hollow cysts and tubules by fusions of apical vesicles, such as the vacuolar apical compartment, with the plasma membrane. The polarity of individual cells is oriented and coordinated with other cells by interactions with the extracellular matrix.     

Formation of the apical pole of epithelial (Madin-Darby canine kidney) cells: polarity of an apical protein is independent of tight junctions while segregation of a basolateral marker requires cell-cell interactions

The time course of development of polarity of an apical (184-kD) and a basolateral (63-kD) plasma membrane protein of Madin-Darby canine kidney cells was followed using semiquantitative immunofluorescence on semithin (approximately 0.5-micron) frozen sections and monoclonal antibody probes. The 184-kD protein became highly polarized to the apical pole within the initial 24 h both in normal medium and in 1-5 microM Ca2+, which results in well-spread, dome-shaped cells, lacking tight junctions and other lateral membrane interactions. In contrast, the basolateral 63-kD membrane protein developed full polarity only after incubation in normal Ca2+ concentrations for greater than 72 h, a time much longer than that required for the formation of tight junctions (approximately 18 h) and failed to polarize in 1-5 microM Ca2+. These results demonstrate that intradomain restriction mechanisms independent of tight junctions, such as self-aggregation or specific interactions with the submembrane cytoskeleton, participate in the regionalization of at least some epithelial plasma membrane proteins. The full operation of these mechanisms depends on the presence of normal cell-cell interactions in the case of the basolateral 63-kD antigen but not in the case of the apical 184-kD protein.     

Role of protein tyrosine phosphorylation in acetaldehyde-induced disruption of epithelial tight junctions

Acetaldehyde-induced cytotoxicity is an important factor in pathogenesis of alcohol-related diseases; however, the mechanism of this toxicity is unknown. We recently showed that acetaldehyde increases epithelial paracellular permeability. We asked whether protein tyrosine phosphorylation via modulation of tyrosine kinases and/or PTPases is a mechanism involved in acetaldehyde-induced disruption of the tight junctions in the Caco-2 cell monolayer. Immunofluorescence localization of occludin and ZO-1 showed disruption of the tight junctions in acetaldehyde-treated cell monolayer. Administration of genistein prevented acetaldehyde-induced permeability. Acetaldehyde increased tyrosine phosphorylation of three clusters of proteins with molecular masses of 30-50, 60-90, and 110-150 kDa; three of these proteins were ZO-1, E-cadherin, and beta-catenin. Acetaldehyde reduced PTPase activity in plasma membrane and soluble fractions, whereas tyrosine kinase activity remained unaffected. Treatment with acetaldehyde resulted in a 97% loss of protein tyrosine phosphatase (PTP)1B activity and a partial reduction of PTP1C and PTP1D activities. These results strongly suggest that acetaldehyde inhibits PTPases to increase protein tyrosine phosphorylation, which may result in disruption of the tight junctions.     

TNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions

Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.     

Cell polarity and cell diversification during early mouse embryogenesis.

At the eight-cell stage of mouse development, the organization of blastomeres changes from radially symmetrical to polarized. This acquisition of cell polarity, followed by asymmetric divisions, leads to the formation of two phenotypically different cell types, which give rise to the first two cell lineages of the mouse blastocyst embryo, trophectoderm and the inner cell mass. Cell fate, controlled by positional information, is not irreversibly fixed during differentiation, providing the embryo with considerable developmental flexibility.