Mammalian tight junctions in the regulation of epithelial differentiation and proliferation Karl Matter, Saima Aijaz, Anna Tsapara and Maria S Balda 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. Addresses Division of Cell Biology, Institute of Ophthalmology, University College London, Bath Street, London, EC1V 9EL, UK Corresponding authors: Matter, Karl ([email protected]); Balda, Maria S ([email protected])

the intermixing of apical and basolateral lipids in the exoplasmic membrane leaflet of the plasma membrane. TJs have a similar molecular architecture to other adhesion complexes (Figure 1) [3–5]. They consist of transmembrane proteins such as claudins, occludin and JAMs, which mediate adhesion and barrier formation as well as selective paracellular diffusion. These membrane proteins interact with a cytoplasmic plaque consisting of junctional adaptors, such as the ZO proteins, that contain multiple protein–protein interaction domains. These junctional adaptors form a protein network that links the junction to the actin cytoskeleton and recruits different types of signalling proteins that regulate junction assembly and function as well as epithelial proliferation and differentiation. The interactions with the actin cytoskeleton are thought to be important for regulation of the permeability properties of the junction and for processes requiring junctional reorganisation.

Regulation of epithelial polarisation Current Opinion in Cell Biology 2005, 17:453–458 This review comes from a themed issue on Cell-to-cell contact and extracellular matrix Edited by Inke S Na¨thke and W James Nelson Available online 10th August 2005 0955-0674/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2005.08.003

Introduction Epithelia form continuous cellular sheets composed of polarised cells that adhere to each other so that diffusion along the paracellular space becomes restricted. Epithelial intercellular adhesion is primarily mediated by the apical junctional complex, which consists of tight and adherens junctions as well as desmosomes. This review focuses on tight junctions (TJs), which contribute to epithelial biogenesis and function by regulating cell proliferation and differentiation, as well as by forming a regulated and semipermeable paracellular diffusion barrier. We will focus on the role of TJs in the regulation of cell proliferation and differentiation as comprehensive reviews have recently been written on their barrier properties and the molecular mechanisms that permit selective paracellular diffusion [1,2]. TJs form a morphological and functional border between the apical and basolateral cell surface domains, restricting www.sciencedirect.com

TJs harbour two evolutionarily conserved signalling complexes that regulate cell polarisation: the CRB3/Pals1/ PATJ complex and the Cdc42-interacting Par3/Par6/ aPKC (atypical protein kinase C) complex. Components of both complexes are important for the generation of polarity in different cell types and for the regulation of epithelial junction assembly. The CRB3/Pals1/PATJ or crumbs complex was first identified in Drosophila, where it consists of crumbs, stardust and Drosophila PATJ, and localises to the subapical complex (marginal zone), the most apical junctional structure in flies [6]. Crumbs plays a role in the morphogenesis of ectodermal epithelia and the regulation of apical membrane biogenesis. Mammals express three different crumbs homologues; one of these, CRB3, is expressed on the apical membrane of epithelial cells and associates with TJs, where it forms a complex with Pals1, the homologue of stardust, and PATJ [7,8]. Experiments based on overexpression and depletion by RNA interference (RNAi) of the three proteins suggest that the CRB3/Pals1/PATJ complex regulates junction assembly when studied by Ca2+ switch in confluent monolayers [7,9,10,11]. Defects in polarisation were only observed in collagen gel experiments, which follow the formation of polarised cysts, but not in monolayers, suggesting that the kinetics of junction assembly might be a critical determinant for epithelial polarisation in culture systems that require a more stringent coordination between proliferation, junction assembly and differentiation. Current Opinion in Cell Biology 2005, 17:453–458

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Figure 1

Composition of tight junctions (TJs). The schematic drawing of a TJ indicates the four major classes of TJ-associated proteins: transmembrane proteins, adaptors, signalling proteins, and transcriptional and posttranscriptional regulators. Actin filaments are also indicated as many TJ proteins interact with F-actin. The boxes show examples of the four classes of TJ proteins and, in parenthesis, direct interactions they are known to engage in. Note, only proteins mentioned in this review are indicated.

The Par3/Par6/aPKC complex was originally identified in C. elegans because of its role in asymmetric cell division [12]. In mammalian epithelia, the Par3/Par6/aPKC complex associates with TJs and regulates junction assembly and polarisation [13,14]. Par3 interacts with the cytoplasmic domains of JAMs and hence mediates junctional recruitment of the complex [15,16]. Par6 interacts with GTP-bound Cdc42 (cell-division control protein 42, a Rho GTPase), a conserved regulator of cell polarity that becomes activated upon the induction of cell–cell adhesion [14]. Binding of Par6 to Cdc42 results in the activation of aPKC, which is required for the formation of distinct tight and adherens junctions (Figure 2) [17]. The components of the Par3/Par6/aPKC complex seem to influence several signalling mechanisms that regulate epithelial polarisation. One of the substrates of aPKC is Par1b/MARK2/EMK1, a protein kinase that regulates epithelial polarity by organising the microtubule network and polarised membrane traffic [18,19,20]. Par3 also regulates TJ assembly via a Par6/aPKC-independent mechanism by regulating Rac1 activation via TIAM1, a guanine nucleotide exchange factor (GEF) [21]. By contrast, Par6 has been linked to the loss of the epithelial Current Opinion in Cell Biology 2005, 17:453–458

phenotype: TGFb-induced epithelial–mesenchymal transition (EMT) requires Par6 phosphorylation by the TGFb receptor type II [22]. Phosphorylation triggers an interaction with the ubiquitin ligase Smurf1, which has been proposed to target junction-associated RhoA for degradation and, hence, to induce disintegration of the junctional complex (Figure 2). The CRB3/Pals1/PATJ and Par3/Par6/aPKC complexes are not independent but interacting pathways. Par6 binds Pals1 and CRB3, and the interaction between Par6 and Pals1 is regulated by Cdc42 [10,23]. In Drosophila, aPKC phosphorylates and thereby activates crumbs [24]. Thus, the two TJ-associated polarity complexes seem to function as a signalling module that coordinates junction assembly and polarisation.

Regulation of epithelial proliferation and gene expression TJs regulate epithelial proliferation by different molecular mechanisms, which generally suppress proliferation as cell density (and hence TJ assembly) increases. Expression of several TJ proteins is affected in certain carcinomas; however, whether these alterations are a cause or a www.sciencedirect.com

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Figure 2

RhoGTPases and tight junctions (TJs). The schematic drawing shows the principal pathways by which Cdc42, Rac1 and RhoA regulate tight junctions as well as how TJ-associated proteins regulate activation of these GTPases. These pathways regulate TJ assembly and disassembly, cell proliferation, paracellular permeability and modulation of the actin cytoskeleton during expulsion of apoptotic cells from epithelia. Double-headed arrows indicate interactions of which the functional consequence is not known.

consequence of carcinogenesis is not clear. Nevertheless, the TJ-associated adaptor proteins ZO-2, MAGI-1 and MUPP1 can bind and inactivate viral oncogenes, and oncogenes and tumour suppressors localize to TJs, suggesting that modulation of TJ protein expression contributes to carcinogenesis [25]. TJs affect proliferation and gene expression by two different types of mechanisms: modulation of signalling cascades, and sequestration of transcription factors and cell cycle regulators. Regulation of signalling cascades

In a large proportion of cancers, loss of epithelial differentiation correlates with deregulation of Ras signalling. The best-known effectors of Ras are the Raf kinases, which stimulate cell cycle entry via ERK/MAP kinase activation. TJs are known to be connected to Raf-1 signalwww.sciencedirect.com

ling, as overexpression of the junctional membrane protein occludin reverses Raf-1-mediated transformation of a salivary gland cell line [26]. Suppression of Raf-1 signalling requires the second extracellular loop of occludin, which also modulates TGFb-induced EMT by binding TGFb receptor type I [27,28]. As activation of the ERK pathway is important for TGFb-induced EMT [29], it is possible that occludin not only suppresses Raf-1 but is also important for its activation and may hence coordinate Raf signalling at TJs. Deletion of the occludin gene in mice affects the differentiation of some epithelial cell types, which would be compatible with occludin playing a role in the modulation of intracellular signalling pathways [30]. Occludin has also been linked to lipid signalling as it interacts with phosphatidylinositol-3 kinase [31,32]. The Current Opinion in Cell Biology 2005, 17:453–458

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product of phosphatidylinositol-3 kinase is hydrolysed by the tumour suppresser PTEN (phosphatase and tensin homolog), which binds the TJ adaptors MAGI-2 and -3 [33,34]. This interaction has been proposed to mediate the recruitment of PTEN to junctions, resulting in the dephosphorylation of phosphatidylinositol phosphates and, hence, the inhibition of signalling by protein kinase B/Akt, a key regulator of signalling pathways activated by growth factors and integrins [35,36]. Regulation of RhoA has also been linked to occludin. MDCK cells in which occludin has been depleted by RNAi fail to activate RhoA in response to certain stimuli (Figure 2) [37]. It is not known how occludin stimulates RhoA activation, but the process may involve the Dbl oncogene family member GEF-H1/Lfc, a GEF for RhoA that localises to TJs and regulates G1/S-phase progression of MDCK cells [38,39]. GEF-H1/Lfc is down-regulated by the KIT inhibitor and anti-tumour drug Gleevec, suggesting that this GEF is a physiologically and pathologically important regulator of cell cycle progression [40]. GEF-H1/Lfc interacts with the TJ protein cingulin, which inhibits its GEF activity and, consequently, cell cycle progression (Figure 2). Therefore, the observed effects that partial deletion of the cingulin gene has on the expression of some TJ proteins and transcription factors specifying endodermal differentiation might in part be due to effects on RhoA signalling [41]. Because up-regulation of cingulin correlates with cell cycle arrest and differentiation [39,42], TJ formation contributes to the down-regulation of RhoA activation and signalling in high-density epithelial cells by inhibiting GEF-H1/Lfc. It will be important to determine which types of proliferation-regulating RhoA effector pathway(s) are stimulated by GEF-H1/Lfc and whether its activation involves other signalling pathways. Direct regulation of transcription factors

Several proteins have been described that localize to TJs as well as the nucleus, which has led to the speculation that they regulate gene expression [36]. One such protein is ZO-2, which interacts with ZO-1 and belongs to the same gene family that also includes the Drosophila Discslarge tumour suppressor (DlgA). ZO-2 enters the nucleus in proliferating cells, interacts with the hnRNP protein SAF-B, and binds and inhibits the transcription factors AP-1 and C/EBP [43,44]. ZO-2 is deregulated in different types of adenocarcinomas [45], suggesting that its effect on transcription is important for the regulation of epithelial proliferation and differentiation. In response to changes in cell–cell adhesion, ZO-2 and ZO-1 also regulate the nuclear accumulation of ARVCF, a member of the p120(ctn) family that associates with adherens junctions: ZO-2 promotes recruitment to the nucleus whereas ZO-1 mediates association with the plasma membrane [46]. Whether these different ZO-2-interacting proteins reflect related functions is not known. Current Opinion in Cell Biology 2005, 17:453–458

The first identified TJ protein, ZO-1, regulates proliferation of MDCK cells [47]. This function maps to the SH3 domain, which interacts with the Y-box transcription factor ZONAB, a protein that is required for normal proliferation rates [47,48]. Binding of ZO-1 to ZONAB occurs in the cytoplasm and inhibits the transcription factor by cytoplasmic sequestration. The human homologue of canine ZONAB, DbpA, is an E2F1 target gene and is overexpressed in different types of carcinomas [49]. Overexpression and depletion of ZONAB and ZO-1 affect not only proliferation but also final cell densities in fully confluent MDCK monolayers [47] (K Matter and M S Balda, unpublished). However, there appear to be differences between different cell lines, as a mouse mammary epithelial cell line deficient in ZO-1 expression was found to proliferate at the same rate as the parental cell line [50]. These differences are unlikely to be due to differences in cell types, as manipulation of ZO-1 and ZONAB also affects proliferation in human mammary epithelial cell lines (K Matter and M S Balda, unpublished). ZONAB regulates G1/S phase progression by two different mechanisms. First, it interacts with the G1/S phase regulator CDK4; hence, cytoplasmic sequestration of ZONAB by ZO-1 results in reduced nuclear CDK4 [47]. Secondly, ZONAB functions in the transcriptional regulation of cell cycle regulators (K Matter and M S Balda, unpublished). Thus, the cytoplasmic sequestration of ZONAB and CDK4 results in co-regulation of two different mechanisms that affect G1/S phase transition. At TJs, ZONAB also interacts with the small GTPase RalA [51]. Although RalA can be activated by Ras, RalA activation in normal MDCK cells is Ras-independent. As in the case of ZO-1, binding to activated RalA inhibits the transcriptional activity of ZONAB. Even though the amount of active RalA is not affected by cell density, the interaction with ZONAB occurs only in dense cells, suggesting that RalA does not directly affect the localisation but rather the activation state of ZONAB. It is also possible that this interaction is important for the regulation of cytoplasmic functions of ZONAB, as Y-box factors have been suggested to regulate RNA turnover and translation [52].

Conclusions and perspectives TJs have two important functions in the establishment of epithelial barriers: first, they regulate formation of the barriers by modulating cell proliferation, differentiation and polarisation, and second, they control barrier function by restricting paracellular diffusion. Although many TJassociated proteins have now been identified that affect epithelial polarisation and much is known about how they interact with each other, we still know very little about how these polarity complexes actually signal and stimulate polarisation, and how they interact with other www.sciencedirect.com

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signalling pathways that regulate junction assembly, such as the rab13/PKA-based and G-protein/src-based mechanisms [53,54]. In Drosophila, many of the evolutionarily conserved polarity complexes function as tumour suppressors; however, the roles of these proteins in mammalian tumorigenesis as well as whether and how they influence TJ-associated pathways that do affect proliferation are still largely unknown. Similarly, junctional adaptor proteins have now been shown to regulate different types of transcription factors, but it is not clear how these different pathways are activated or to which external stimuli they respond, or how these different pathways are coordinated with one another or with other signalling pathways that control cell proliferation and differentiation. Moreover, the identification of TJ-associated proteins such as symplekin, which participates in nuclear as well as cytoplasmic polyadenylation [55–57], suggests that TJs might contribute to as yet unexplored functions, such as regulation of mRNA stability and localisation.

Acknowledgements Research in the authors’ laboratories is supported by the Wellcome Trust as well as the Medical Research Council and the Biotechnology and Biological Sciences Research Council.

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