SnapShot: Epithelial tight junctions.

Tight junctions form a morphological and functional border between the apical and basolateral cell surface domains that serves as a paracellular diffusion barrier, enabling epithelial cells to separate compartments of different composition. Tight junctions also contribute to the generation and maintenance of cell polarity and regulate signaling mechanisms that guide cell behavior, shape, and gene expression. This SnapShot illustrates their components, organization, and functions.

Tight junctions: from simple barriers to multifunctional molecular gates.

Epithelia and endothelia separate different tissue compartments and protect multicellular organisms from the outside world. This requires the formation of tight junctions, selective gates that control paracellular diffusion of ions and solutes. Tight junctions also form the border between the apical and basolateral plasma-membrane domains and are linked to the machinery that controls apicobasal polarization. Additionally, signalling networks that guide diverse cell behaviours and functions are connected to tight junctions, transmitting information to and from the cytoskeleton, nucleus and different cell adhesion complexes. Recent advances have broadened our understanding of the molecular architecture and cellular functions of tight junctions.

Proper E-cadherin membrane location in colon requires Dab2 and it modifies by inflammation and cancer.

We reported that Disabled-2 (Dab2) is located at the apical membrane in suckling rat intestine. Here, we discovered that, in colon of suckling and adult mouse and of adult human, Dab2 is only at lateral crypt cell membrane and colocalized with E-cadherin. Dab2 depletion in Caco-2 cells led to E-cadherin internalization indicating that its membrane location requires Dab2. In mice, we found that 3 days of dextran sulfate sodium-induced colitis increased Dab2/E-cadherin colocalization, which was decreased as colitis progressed to 6 and 9 days. In agreement, Dab2/E-cadherin colocalization increased in human mild and severe ulcerative colitis and in polyps, being reduced in colon adenocarcinomas, which even showed epithelial Dab2 absence and E-cadherin delocalization. Epithelial Dab2 decrement preceded that of E-cadherin. We suggest that Dab2, by inhibiting E-cadherin internalization, stabilizes adherens junctions, and its absence from the epithelium may contribute to development of colon inflammation and cancer.

Biallelic Mutation of ARHGEF18, Involved in the Determination of Epithelial Apicobasal Polarity, Causes Adult-Onset Retinal Degeneration.

Mutations in more than 250 genes are implicated in inherited retinal dystrophy; the encoded proteins are involved in a broad spectrum of pathways. The presence of unsolved families after highly parallel sequencing strategies suggests that further genes remain to be identified. Whole-exome and -genome sequencing studies employed here in large cohorts of affected individuals revealed biallelic mutations in ARHGEF18 in three such individuals. ARHGEF18 encodes ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein that is a key component of tight junctions and adherens junctions. This biological pathway is known to be important for retinal development and function, as mutation of CRB1, encoding another component, causes retinal dystrophy. The retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs∗63), c.1996C>T (p.Arg666∗), c.2632G>T (p.Glu878∗), and c.2738_2761del (p.Arg913_Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA. Previously, knock-out of Arhgef18 in the medaka fish has been shown to cause larval lethality which is preceded by retinal defects that resemble those seen in zebrafish Crumbs complex knock-outs. The findings described here emphasize the peculiar sensitivity of the retina to perturbations of this pathway, which is highlighted as a target for potential therapeutic strategies.

Small and large intestine express a truncated Dab1 isoform that assembles in cell-cell junctions and co-localizes with proteins involved in endocytosis.

Disabled-1 (Dab1) is an essential intracellular adaptor protein in the reelin pathway. Our previous studies in mice intestine showed that Dab1 transmits the reelin signal to cytosolic signalling pathways. Here, we determine the Dab1 isoform expressed in rodent small and large intestine, its subcellular location and co-localization with clathrin, caveolin-1 and N-Wasp. PCR and sequencing analysis reveal that rodent small and large intestine express a Dab1 isoform that misses three (Y198, Y200 and Y220) of the five tyrosine phosphorylation sites present in brain Dab1 isoform (canonical) and contains nuclear localization and export signals. Western blot assays show that both, crypts, which shelter progenitor cells, and enterocytes express the same Dab1 isoform, suggesting that epithelial cell differentiation does not regulate intestinal generation of alternatively spliced Dab1 variants. They also reveal that the canonical and the intestinal Dab1 isoforms differ in their total degree of phosphorylation. Immunostaining assays show that in enterocytes Dab1 localizes at the apical and lateral membranes, apical vesicles, close to adherens junctions and desmosomes, as well as in the nucleus; co-localizes with clathrin and with N-Wasp but not with caveolin-1, and in Caco-2 cells Dab1 localizes at cell-to-cell junctions by a Ca2+-dependent process. In conclusion, the results indicate that in rodent intestine a truncated Dab1 variant transmits the reelin signal and may play a role in clathrin-mediated apical endocytosis and in the control of cell-to-cell junction assembly. A function of intestinal Dab1 variant as a nucleocytoplasmic shuttling protein is also inferred from its sequence and nuclear location.

Signalling at tight junctions during epithelial differentiation and microbial pathogenesis.

Tight junctions are a component of the epithelial junctional complex, and they form the paracellular diffusion barrier that enables epithelial cells to create cellular sheets that separate compartments with different compositions. The assembly and function of tight junctions are intimately linked to the actomyosin cytoskeleton and, hence, are under the control of signalling mechanisms that regulate cytoskeletal dynamics. Tight junctions not only receive signals that guide their assembly and function, but transmit information to the cell interior to regulate cell proliferation, migration and survival. As a crucial component of the epithelial barrier, they are often targeted by pathogenic viruses and bacteria, aiding infection and the development of disease. In this Commentary, we review recent progress in the understanding of the molecular signalling mechanisms that drive junction assembly and function, and the signalling processes by which tight junctions regulate cell behaviour and survival. We also discuss the way in which junctional components are exploited by pathogenic viruses and bacteria, and how this might affect junctional signalling mechanisms.

Stress- and Rho-activated ZO-1-associated nucleic acid binding protein binding to p21 mRNA mediates stabilization, translation, and cell survival.

A central component of the cellular stress response is p21(WAF1/CIP1), which regulates cell proliferation, survival, and differentiation. Inflammation and cell stress often up-regulate p21 posttranscriptionally by regulatory mechanisms that are poorly understood. ZO-1-associated nucleic acid binding protein (ZONAB)/DbpA is a Y-box transcription factor that is regulated by components of intercellular junctions that are affected by cytokines and tissue damage. We therefore asked whether ZONAB activation is part of the cellular stress response. Here, we demonstrate that ZONAB promotes cell survival in response to proinflammatory, hyperosmotic, and cytotoxic stress and that stress-induced ZONAB activation involves the Rho regulator GEF-H1. Unexpectedly, stress-induced ZONAB activation does not stimulate ZONAB's activity as a transcription factor but leads to the posttranscriptional up-regulation of p21 protein and mRNA. Up-regulation is mediated by ZONAB binding to specific sites in the 3'-untranslated region of the p21 mRNA, resulting in mRNA stabilization and enhanced translation. Binding of ZONAB to mRNA is activated by GEF-H1 via Rho stimulation and also mediates Ras-induced p21 expression. We thus identify a unique type of stress and Rho signaling activated pathway that drives mRNA stabilization and translation and links the cellular stress response to p21 expression and cell survival.

ZO-1 Regulates Hippo-Independent YAP Activity and Cell Proliferation via a GEF-H1- and TBK1-Regulated Signalling Network.

Tight junctions are a barrier-forming cell-cell adhesion complex and have been proposed to regulate cell proliferation. However, the underlying mechanisms are not well understood. Here, we used cells deficient in the junction scaffold ZO-1 alone or together with its paralog ZO-2, which disrupts the junctional barrier. We found that ZO-1 knockout increased cell proliferation, induced loss of cell density-dependent proliferation control, and promoted apoptosis and necrosis. These phenotypes were enhanced by double ZO-1/ZO-2 knockout. Increased proliferation was dependent on two transcriptional regulators: YAP and ZONAB. ZO-1 knockout stimulated YAP nuclear translocation and activity without changes in Hippo-dependent phosphorylation. Knockout promoted TANK-binding kinase 1 (TBK1) activation and increased expression of the RhoA activator GEF-H1. Knockdown of ZO-3, another paralog interacting with ZO1, was sufficient to induce GEF-H1 expression and YAP activity. GEF-H1, TBK1, and mechanotransduction at focal adhesions were found to cooperate to activate YAP/TEAD in ZO-1-deficient cells. Thus, ZO-1 controled cell proliferation and Hippo-independent YAP activity by activating a GEF-H1- and TBK1-regulated mechanosensitive signalling network.

Ouabain modulates epithelial cell tight junction.

Epithelial cells treated with high concentrations of ouabain (e.g., 1 microM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell-cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K(+) pumping nor disturb the K(+) balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (J(DEX)). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, J(DEX), and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell-cell contacts.

Tight junctions.

Various functions within our bodies require the generation and maintenance of compartments with distinct compositions, which in turn necessitate the formation of semipermeable cellular diffusion barriers. For example, the blood-brain barrier protects the brain by allowing only specific molecules to pass through. Another instance is the intestinal barrier, which allows the uptake of essential nutrients, while restricting the passage of pathogenic molecules and bacteria. Breakdown of such barriers causes various pathologies, such as brain or retinal edema, or diarrhoea. Epithelia and endothelia are the most common barrier-forming cells. Individual cells in such barriers are held together by cell-cell adhesion structures - also known as intercellular junctions - that are essential for barrier formation and maintenance. Here, we will focus on the structure and assembly of tight junctions (TJs) and their functions as barriers, but will refer to other adhesive structures crucial for barrier regulation such as adherens junctions (AJs) and focal adhesions to the extracellular matrix (ECM) (Figure 1A,B). We will also discuss additional functions of TJs in cell surface polarity and the regulation of gene expression, cell function, and cell behaviour.

Correction: The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0015730.].

Mammalian tight junctions in the regulation of epithelial differentiation and proliferation.

Tight junctions are important for the permeability properties of epithelial and endothelial barriers as they restrict diffusion along the paracellular space. Recent observations have revealed that tight junctions also function in the regulation of epithelial proliferation and differentiation. They harbour evolutionarily conserved protein complexes that regulate polarisation and junction assembly. Tight junctions also recruit signalling proteins that participate in the regulation of cell proliferation and differentiation. These signalling proteins include components that affect established signalling cascades and dual localisation proteins that can associate with junctions as well as travel to the nucleus where they regulate gene expression.

ZO-1 Guides Tight Junction Assembly and Epithelial Morphogenesis via Cytoskeletal Tension-Dependent and -Independent Functions.

Formation and maintenance of tissue barriers require the coordination of cell mechanics and cell-cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to investigate how tight junctions regulate cell mechanics and epithelial morphogenesis. We found that depletion of the tight junction adaptor ZO-1 disrupted junction assembly and morphogenesis in an ECM stiffness-dependent manner and led to a stiffness-dependant reorganisation of active myosin. Both junction formation and morphogenesis were rescued by inhibition of actomyosin contractility. ZO-1 depletion also impacted mechanical tension at cell-matrix and E-cadherin-based cell-cell adhesions. The effect on E-cadherin also depended on ECM stiffness and correlated with effects of ECM stiffness on actin cytoskeleton organisation. However, ZO-1 knockout also revealed tension-independent functions of ZO-1. ZO-1-deficient cells could assemble functional barriers at low tension, but their tight junctions remained corrupted with strongly reduced and discontinuous recruitment of junctional components. Our results thus reveal that reciprocal regulation between ZO-1 and cell mechanics controls tight junction assembly and epithelial morphogenesis, and that, in a second, tension-independent step, ZO-1 is required to assemble morphologically and structurally fully assembled and functionally normal tight junctions.

Therapeutic Validation of GEF-H1 Using a De Novo Designed Inhibitor in Models of Retinal Disease.

Inflammation and fibrosis are important components of diseases that contribute to the malfunction of epithelia and endothelia. The Rho guanine nucleotide exchange factor (GEF) GEF-H1/ARHGEF-2 is induced in disease and stimulates inflammatory and fibrotic processes, cell migration, and metastasis. Here, we have generated peptide inhibitors to block the function of GEF-H1. Inhibitors were designed using a structural in silico approach or by isolating an inhibitory sequence from the autoregulatory C-terminal domain. Candidate inhibitors were tested for their ability to block RhoA/GEF-H1 binding in vitro, and their potency and specificity in cell-based assays. Successful inhibitors were then evaluated in models of TGFß-induced fibrosis, LPS-stimulated endothelial cell-cell junction disruption, and cell migration. Finally, the most potent inhibitor was successfully tested in an experimental retinal disease mouse model, in which it inhibited blood vessel leakage and ameliorated retinal inflammation when treatment was initiated after disease diagnosis. Thus, an antagonist that blocks GEF-H1 signaling effectively inhibits disease features in in vitro and in vivo disease models, demonstrating that GEF-H1 is an effective therapeutic target and establishing a new therapeutic approach.

Spatiotemporal control of actomyosin contractility by MRCKß signaling drives phagocytosis.

Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKß/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKß/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKß is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKß signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKß is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKß signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK.

Rho signaling and tight junction functions.

Tight junctions are heteromeric protein complexes that act as signaling centers by mediating the bidirectional transmission of information between the environment and the cell interior to control paracellular permeability and differentiation. Insight into tight junction-associated signaling mechanisms is of fundamental importance for our understanding of the physiology of epithelia and endothelia in health and disease.

The Y-box factor ZONAB/DbpA associates with GEF-H1/Lfc and mediates Rho-stimulated transcription.

Epithelial tight junctions recruit different types of signalling proteins that regulate cell proliferation and differentiation. Little is known about how such proteins interact functionally and biochemically with each other. Here, we focus on the Y-box transcription factor ZONAB (zonula occludens 1-associated nucleic-acid-binding protein)/DbpA (DNA-binding protein A) and the Rho GTPase activator guanine nucleotide exchange factor (GEF)-H1/Lbc's first cousin, which are two tight-junction-associated signalling proteins that regulate proliferation. Our data show that the two proteins interact and that ZONAB activity is Rho-dependent. Overexpression of GEF-H1 induces accumulation of ZONAB in the nucleus and activates transcription. Microtubule-affinity regulating kinase/partition-defective-1, another type of GEF-H1-associated signalling protein, remains in the cytoplasm and partially co-localizes with the exchange factor. GEF-H1 and ZONAB are required for expression of endogenous cyclin D1, a crucial RhoA signalling target gene, and GEF-H1-stimulated cyclin D1 promoter activity requires ZONAB. Our data thus indicate that GEF-H1 and ZONAB form a signalling module that mediates Rho-regulated cyclin D1 promoter activation and expression.

ZONAB promotes proliferation and represses differentiation of proximal tubule epithelial cells.

Epithelial polarization modulates gene expression. The transcription factor zonula occludens 1 (ZO-1)-associated nucleic acid binding protein (ZONAB) can shuttle between tight junctions and nuclei, promoting cell proliferation and expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), but whether it also represses epithelial differentiation is unknown. Here, during mouse kidney ontogeny and polarization of proximal tubular cells (OK cells), ZONAB and PCNA levels decreased in parallel and inversely correlated with increasing apical differentiation, reflected by expression of megalin/cubilin, maturation of the brush border, and extension of the primary cilium. Conversely, ZONAB reexpression and loss of apical differentiation markers provided a signature for renal clear cell carcinoma. In confluent OK cells, ZONAB overexpression increased proliferation and PCNA while repressing megalin/cubilin expression and impairing differentiation of the brush border and primary cilium. Reporter and chromatin immunoprecipitation assays demonstrated that megalin and cubilin are ZONAB target genes. Sparsely plated OK cells formed small islands composed of distinct populations: Cells on the periphery, which lacked external tight junctions, strongly expressed nuclear ZONAB, proliferated, and failed to differentiate; central cells, surrounded by continuous junctions, lost nuclear ZONAB, stopped proliferating, and engaged in apical differentiation. Taken together, these data suggest that ZONAB is an important component of the mechanisms that sense epithelial density and participates in the complex transcriptional networks that regulate the switch between proliferation and differentiation.

ARHGEF18/p114RhoGEF Coordinates PKA/CREB Signaling and Actomyosin Remodeling to Promote Trophoblast Cell-Cell Fusion During Placenta Morphogenesis.

Coordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are activated by guanine nucleotide exchange factors in a spatially and temporally controlled manner. However, the roles of these Rho GTPase activators during complex developmental processes are still poorly understood. ARHGEF18/p114RhoGEF is a tight junction-associated RhoA activator that forms complexes with myosin II, and regulates actomyosin contractility. Here we show that p114RhoGEF/ARHGEF18 is required for mouse syncytiotrophoblast differentiation and placenta development. In vitro and in vivo experiments identify that p114RhoGEF controls expression of AKAP12, a protein regulating protein kinase A (PKA) signaling, and is required for PKA-induced actomyosin remodeling, cAMP-responsive element binding protein (CREB)-driven gene expression of proteins required for trophoblast differentiation, and, hence, trophoblast cell-cell fusion. Our data thus indicate that p114RhoGEF links actomyosin dynamics and cell-cell junctions to PKA/CREB signaling, gene expression and cell-cell fusion.

Spatio-temporal expression pattern and role of the tight junction protein MarvelD3 in pancreas development and function.

Tight junction complexes are involved in the establishment and maintenance of cell polarity and the regulation of signalling pathways, controlling biological processes such as cell differentiation and cell proliferation. MarvelD3 is a tight junction protein expressed in adult epithelial and endothelial cells. In Xenopus laevis, MarvelD3 morphants present differentiation defects of several ectodermal derivatives. In vitro experiments further revealed that MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behaviour and survival. In this work, we found that MarvelD3 is expressed from early developmental stages in the exocrine and endocrine compartments of the pancreas, as well as in endothelial cells of this organ. We thoroughly characterized MarvelD3 expression pattern in developing pancreas and evaluated its function by genetic ablation. Surprisingly, inactivation of MarvelD3 in mice did not alter development and differentiation of the pancreatic tissue. Moreover, tight junction formation and organization, cell polarization, and activity of the JNK-pathway were not impacted by the deletion of MarvelD3.