Molecular Physiology and Pathophysiology of Tight Junctions I ...

Am J Physiol Gastrointest Liver Physiol 279: G477–G482, 2000.

themes Molecular Physiology and Pathophysiology of Tight Junctions I. Biogenesis of tight junctions and epithelial polarity M. CEREIJIDO, L. SHOSHANI, AND R. G. CONTRERAS Center for Research and Advanced Studies, 07000 Mexico City, Mexico Cereijido, M., L. Shoshani, and R. G. Contreras. Molecular Physiology and Pathophysiology of Tight Junctions. I. Biogenesis of tight junctions and epithelial polarity. Am J Physiol Gastrointest Liver Physiol 279: G477–G482, 2000.—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 ␣-, ␤-, and ␥-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 Ca2⫹-dependent cell-cell contacts) throughout the life of the epithelium. apical membrane; basolateral membrane; polarity; E-cadherin; zonula occludens-1; occludin

(TJs) were so belittled that, more than a century after they were first described, they were not even represented in the model of Koefoed-Johnsen and Ussing (20) that covered four decades of epithelial cell physiology. The renaissance of interest in this structure was caused by the discovery that fluxes through some epithelia proceed mainly through a paracellular route limited by the TJ. However, even then, given its poor ability to discriminate between diverse permeatTIGHT JUNCTIONS

Address for reprint requests and other correspondence: M. Cereijido, Dept. of Physiology, Biophysics & Neurosciences, CINVESTAV, Apdo. Postal 14–740, 07000 Me´xico, D.F., Me´xico (E-mail: [email protected]). http://www.ajpgi.org

ing species compared with the plasma membrane, it was expected that the TJ would consist of a couple of rather inconspicuous molecules placed in neighboring cells and bridged by a mere Ca2⫹ salt link (6). The scope has since drastically changed because of a series of observations. 1) Transepithelial electrical resistance (TER) ranges from 10 (e.g., proximal kidney tubule) to ⬎10,000 (e.g., urinary bladder) ⍀ 䡠 cm2, indicating that TJs can adjust the degree of tightness to physiological needs (6). 2) The TJ has been found to consist of a cluster of protein species [zonula occludens (ZO)-1, ZO-2, ZO-3, cingulin, occludin, claudins, etc.; see Fig. 1] that stretches from its lips to the cytoskeleton (2, 10, 14). 3) Some of these proteins contain nuclear addressing signals (10, 18). 4) TJ molecules change their degree of phosphorylation in response to physiological conditions and pharmacological challenge (1, 2, 10, 14). 5) In any given moment, TJs may relax their tightness to such an extent that they allow the passage of macrophages toward an infected site or spermatozoa in their travel from the Sertoli cell to the lumen of the seminiferous tube (14). 6) An increasing number of diseases are associated with TJ molecules (14, 28). 7) Some TJ-associated molecules contain consensus segments with tumor suppressor proteins (10). 8) Proteins are not the only component of the TJ, because this structure also contains lipids that seem to have structural and physiological relevance (5). Furthermore, the TJ acts as a barrier that prevents lipids in the apical and the basolateral membrane from mixing (13). We now face the challenge to 1) find the remaining molecular pieces of the TJ, 2) figure out how all of the TJ molecules are assembled, 3) explain the role of each protein and how the whole machinery works, 4) explain how it is controlled by cytoplasmic and extracellular signals, 5) predict and find the pathological conditions that result from a failure in a given TJ molecule or in overall functioning, and 6) explore the relationship between the TJ and apical/basolateral polarity, which is the other prominent feature of the so-called “transepithelial transporting phenotype.” The aim of the

0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society

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TIGHT JUNCTION ASSEMBLY

Fig. 1. Molecular structure of the tight junction (TJ). Molecules like zonula occludens (ZO)-1, ZO-2, ZO-3, claudins, and occludins are represented twice to indicate their association to diverse molecules while maintaining the clarity of the figure. Actually, when observed with light microscopes, these molecules are observed to colocalize. For the same reason, the cytoskeleton of actin, spectrin, and fodrin is represented at the far left, although most of the molecules are directly or indirectly attached to this structure. Rab2b, Rab13, 7H6, and AF.6 are depicted at the bottom because their location and molecular relationships are still undetermined. ZAK, ZO-associated kinase; VAMP, vesicle-associated membrane protein; VAP-33, VAMP-associated protein of 33 kDa; ASIP, atypical protein kinase C isotype-specific interacting protein; ZONAB, ZO-1 nucleic acid binding protein; 4.1R, isoform of the red blood cell protein 4.1; JAM, junction adhesion molecule. Red sun symbols indicate phosphorylation.

present themes article is to summarize the information available on points 2 and 6. Our main source of information has been established cell lines that retain the ability to make TJs and polarize in vitro (7). When harvested with trypsinEDTA, these cells lose TJs and polarity but recover them in a few hours, in particular if plating is made at confluence. Fortunately, the information collected in the last couple of decades indicates that cells handled in this way form TJs and polarize with the same mechanism that they would use spontaneously in natural epithelia. Cells deprived of Ca2⫹ and maintained in suspension with a stirrer do not form TJs or polarize and have relatively few, nonspecific membrane sites for attachment. The addition of soluble forms of collagen prompts cells to expose more and more specific sites, but they still remain unpolarized and fail to form TJs (27). Ca2⫹ promotes the formation of clumps, in which suspended cells take each other as substrate, form TJs, and polarize, orienting the apical domain toward the outer side of the clump. Collagen added under this condition binds to the apical membrane and causes an inversion of polarity and a relocation of TJs (4). When Ca2⫹ is removed soon after plating (30–40 min), cells do not form TJs or polarize, indicating that although attachment is a clearly asymmetric condition, cells cannot process the information or, rather, they can only process it to a certain point. Thus cells incubated without Ca2⫹ retain apical markers and basolateral markers (e.g., Na⫹-K⫹-ATPase; Ref. 11), as well as components of the TJ, in intracellular membrane vesicles (26). Ca2⫹ added at this time triggers polarization and junction formation in a few hours (“Ca2⫹ switch”;

Ref. 16). This ion is also important for the maintenance of the TJs, because these can be opened and resealed by removal and restoration of Ca2⫹ in diverse experimental conditions (21). EXTRACELLULAR EVENTS

Ca2⫹ acts primarily on the extracellular side (12), as indicated by the following. 1) An extracellular Ca2⫹ concentration of 0.1 mM is high enough to trigger junction formation and polarization yet is not sufficient to increase the level of cytosolic Ca2⫹ [14 ⫾ 8 nM (n ⫽ 8)] that was reduced as a consequence of the preincubation without this ion [20 ⫾ 8 nM (n ⫽ 7)]. 2) La3⫹ blocks Ca2⫹ penetration (Fig. 2) but does not prevent this ion from triggering junction formation or polarization. 3) Cd2⫹, in contrast, blocks both Ca2⫹ influx and the development of the transporting phenotype (12), because it interacts with the extracellular part of Ecadherin, more specifically with a motif of 13 amino acids (25). Extracellular Ca2⫹ activates E-cadherin, which can now aggregate with other E-cadherin molecules on the same cell, an arrangement that favors binding to E-cadherin of a neighboring cell (31). When the outermost E-cadherin repeat is blocked with the specific antibody ECCD-1, TJs do not form. If already established, another antibody, DECMA-1, promotes their reopening. Yet the outermost repeat cannot act by itself when the other four cadherin repeats are deleted (29). The observation that extracellular Ca2⫹ is sufficient to trigger junction formation and polarization does not exclude the possibility that its penetration into the cytoplasm would produce a series of additional phe-


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Fig. 2. Initial steps in the assembly of the TJ during a Ca2⫹ switch. In the absence of Ca2⫹ the 2 molecules of E-cadherin (bottom) are inactive. The addition of this ion (small blue dots) elicits 2 main effects: it binds to the extracellular repeats and promotes complexing of E-cadherin with ␣- and ␤-catenins or with ␣- and ␥-catenins (␤ and ␥ seem to be exchangeable; right). In turn, the complex formed binds to p120, vinculin, and ␣-actinin and, indirectly, to the cytoskeleton of actin. Ca2⫹ also causes clustering of E-cadherins in the plane of the membrane, which acquire the capacity to bind to E-cadherin in the neighboring cell through their outermost repeat (brown). Antibodies that bind specifically to the extracellular domain of E-cadherin block TJ formation and open alreadysealed TJs. A putative contact receptor activates phospholipase C (PLC) through 2 different G proteins. PLC then splits phosphatidyl 4,5-bisphosphate (PiP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C (PKC). La3⫹ prevents penetration of Ca2⫹ into the cells (top right) but does not impair E-cadherin triggering of TJ formation. Cd2⫹, in contrast, has affinity both for the mechanisms that translate Ca2⫹ across the cell membrane and for the extracellular moiety of E-cadherin. The affinity of Cd2⫹ for this moiety is enough to block Ca2⫹ but not enough to replace this ion in the triggering of TJ formation.

nomena. Likewise, E-cadherin appears to have a large number of cellular roles besides that of promoting and maintaining TJs (31). INTRACELLULAR EVENTS

The events described in the previous paragraphs are followed by the activation of a putative contact receptor (Fig. 2), whose signal is transduced through a cascade involving two different G proteins controlling a phospholipase C (PLC), a protein kinase C (PKC), calmodulin (1), and mitogen-activated protein kinase (MAPK) (9). This results in the formation of junctional strands observed in freeze fracture replicas, blockade of the paracellular flux of extracellular markers such as ruthenium red and ferritin, and the development of a TER (7). All these steps in the synthesis and assembly, and even in the disassembly, of the TJ involve important phosphorylation steps (10). This led to a considerable effort to detect where and when phosphorylations are needed. It was found that ZO-1 and ZO-2 proteins become phosphorylated on Ser, Thr, and Tyr residues, cingulin is phosphorylated on Ser residues, and occludin has several levels of phos-

phorylation on Ser and Thr, which are required for its incorporation into the TJ (10). Likewise, the assembly of this structure depends on vinculin phosphorylation on Ser and Thr (24). However, we still have no dynamic model to explain the interplay of Ca2⫹ levels, small GTP-binding proteins, and TJ-associated molecules. There is ample evidence that this cascade involves the cytoskeleton, which is linked to E-cadherin through p130, ␣-, ␤-, and ␥-catenins, vinculin, ␣-actinin, fodrin, and spectrin (32). In fact, actin also combines with ZO-1, ZO-2, ZO-3, occludin, cingulin, and other TJ molecules. The cytoskeleton is likely to constitute a structural framework as well as an informative network conveying and delivering signals from the adherens junctions to the TJ and back. Drugs interfering with microfilaments and microtubules block junction formation (23) as well as polarization during a Ca2⫹ switch and reverse these processes in fully polarized cells with already-established TJs (32). Although there is ample evidence that the TJ is a structure that restricts diffusion through the paracellular permeation route, there are no data supporting its role as an anchoring structure. TJs might eventu-

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ally be discovered to be like our lips, which enable our mouth to hold water yet are not sewn together. In this respect, we know that E-cadherin is needed to trigger junction formation (19) and that TJs depend on the E-cadherin/E-cadherin link throughout the life of the epithelium, because TJs in mature monolayers can be opened by interfering with E-cadherin or when the chain of reactions starting with this molecule is interrupted by the lack of ␣-catenin (30). These observations, together with the fact that E-cadherin does not localize in the TJ, suggest that this molecule acts primarily from afar, by triggering the cascade of chemical events resulting in TJ formation. However, we cannot exclude the possibility that E-cadherin would also be needed to hold the two neighboring cells together so that TJs can be formed. The results shown in Fig. 3 would agree with the first possibility; this figure shows a monolayer of cocultured Madin-Darby canine kidney (MDCK) and LLC-PK1 (pig epithelial kidney) cells that exhibits homophilic (MDCK-MDCK and LLC-PK1-LLC-PK1) as well as heterophilic (MDCKLLC-PK1) TJs, revealed by the continuous distribution of occludin and ZO-1 molecules and by the development of a TER (not shown). Heterophilic TJs are in keeping with the observation that TJs can be established between cells from different organs of the same animal and even from different animal species (17). E-cadherin, in contrast, is only observed at homologous MDCK-MDCK borders, as indicated by DECMA-1 antibody, a result that agrees with the observation that the cell-cell contact provided by E-cadherin is highly specific (31). The antibody used has no specificity for E-cadherin in LLC-PK1 cells, but it should bind to E-cadherin of MDCK cells in heterophilic borders, if these cells express this molecule in such junctions. Together, these results suggest that E-cadherin can influence the TJ mainly through signals traveling downstream, because even in monolayers of pure MDCK cells this molecule does not codistribute with occludin, ZO-1, or any other typical TJ molecules. Despite this distance, sealing of the TJ remains sensitive to the fate of E-cadherin throughout. Nevertheless, the relationship between TJs and E-cadherin may be more complex than it appears today. Thus Troxell et al. (29) found typical junctional strands and some TJ markers, such as ZO-1 and occludin, in cells whose expression of endogenous E-cadherin had been severely reduced. In this respect, it is worth remembering that the TJ is formed by a dozen molecular species arranged in a complex manner, it has several functions that can be gauged through different parameters (TER, permeability, fluorescent lipid probes), and its presence is acknowledged when its typical membrane strands are found in freeze fracture replicas. Obviously, the presence of a marker or the detection of one of these characteristics does not ensure that the other would necessarily be present. Several TJ molecules like ZO-1, ZO-2, and ZO-3 belong to the membrane-associated guanylate kinase (MAGUK) family of proteins, characterized by having a SH3, a GuK, and up to three PDZ domains that par-

Fig. 3. A: epifluorescence image of monolayers formed by mixed populations of Madin-Darby canine kidney (MDCK) and LLC-PK1 (pig epithelial kidney) cells. LLC-PK1 cells were labeled beforehand with 6.25 ␮M CMTMR (red). Occludin (green) is present in all cell borders. B: E-cadherin (green) stained with DECMA-1 as first antibody against E-cadherin is only observed in MDCK-MDCK borders but not in MDCK borders in contact with LLC-PK1 cells. C: after a calcium switch, TJs appear at the first and only cell-cell contacts close to the glass support, as revealed by staining with antibody against ZO-1. D: TJs are subsequently pushed toward the top of the lateral space.

ticipate in specific protein-protein associations. Regardless of whether signals are transmitted from Ecadherin to TJs by chemical means, through the cytoskeleton, or both, the assembly of the TJ seems to depend on a “protein clustering” process (14). Clustering proteins may contain several different modules that specifically bind to segments in other regions of


the same polypeptide or to specific sites in a different molecule. As a result of these interactions, the shape and electronic profile adopted by proteins prompts them to bind specifically to other proteins of the same (multimerization) or different molecular species. In turn, this association induces a redistribution of charges that may enable one of the assembled proteins to then specifically combine with still other proteins that thereby become new members of the cluster. Usually, the affinity of one of the molecules for a given protein species is increased (or decreased) not only by the interaction with a third protein molecule but also by phosphorylation, combination with Ca2⫹, K⫹, or Na⫹, changes in local pH, etc. Therefore, the assembly of the TJ, and the linkage of several of its proteins in a scaffold that includes the cytoskeleton, may result from a series of interactions/inductions (8) that were initiated by Ca2⫹. The life of higher organisms would be impossible without epithelial barriers that separate the internal from the environmental milieu. Therefore, it is conceivable that the most fundamental and evolutionarily conserved role of the TJs is to act as a diffusion barrier and that finer regulation of TJ permeability, as well as their participation in the variety of functions mentioned in the introduction, would constitute a later development. In this respect, it is interesting that Furuse et al. (15) have deleted the COOH-terminal cytoplasmic domain of claudins (a membrane protein of the TJ) and observed that these molecules have lost their ability to associate with ZO-1, ZO-2, and ZO-3, yet these components of the TJ were still able to exhibit the characteristic meshwork of fibrils in freeze fracture replicas. TJS AND POLARITY

Until a few years ago, it was thought that because the TJ marks the limit between apical and basolateral domains, it would be responsible for the polarized distribution of membrane components. Apart from the fact that the TJ cannot sort membrane components, it is becoming increasingly clear that it is just the other way around: the TJ is itself a product of an overall polarizing process. Thus Matter and Balda (22) have shown that, because of a signal in the COOH-terminal domain, occludin is first delivered to the lateral membrane of the cell and transferred later to the TJ. The possibility exists that a given membrane molecule of the TJ, with affinity for another molecule in the cell membrane of the neighboring cell (15), would form the initial contact (Fig. 3), and further molecular components would be progressively recruited thereafter by intimate and specific association to form a specific cluster. The TJ is also important in preserving the polarization of mobile membrane molecules (13). However, even in this case, its role does not seem to be that of a simple fence. Thus transfection of occludin with its COOH terminus truncated and with an epitope tag inserted in the NH2 terminus impairs the ability of the

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TJ to maintain a fluorescent lipid probe in the apical domain (3). In summary, until recently, specific cell functions (e.g., ion pumping, exchange diffusion, action potentials) were discovered and characterized decades before one had the faintest idea about the responsible molecules. The picture has since reversed, because today we know a dozen molecules that integrate the TJ but whose functions are largely unknown. This is therefore the hour of the cell physiologist. Yet the task appears formidable, because TJs have a very complex structure, are intimately associated with other structures such as the cytoskeleton, the nucleus, and cell-cell and cell-substrate attachments, and also participate in proliferation and differentiation. We express our gratitude to Drs. Marı´a Suasana Balda, Lorenza Gonza´lez-Mariscal, and Karl Matter for helpful discussion. This work was supported by research grants from the National Research Council of Mexico (CONACYT). REFERENCES 1. Balda MS, Gonzalez-Mariscal L, Contreras RG, MaciasSilva M, Torres-Marquez ME, Garcia-Sainz JA, and Cereijido M. Assembly and sealing of tight junctions: possible participation of G- proteins, phospholipase C, protein kinase C and calmodulin. J Membr Biol 122: 193–202, 1991. 2. Balda MS and Matter K. Tight junctions. J Cell Sci 111: 541–547, 1998. 3. Balda MS, Whitney JA, Flores C, Gonzalez S, Cereijido M, and Matter K. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J Cell Biol 134: 1031–1049, 1996. 4. Barriere H, Chambard M, Selzner JP, Mauchamp J, and Gabrion J. Polarity reversal of inside-out thyroid follicles cultured within collagen gel: structure of the junctions assessed by freeze-fracture and lanthanum permeability. Biol Cell 62: 133– 144, 1988. 5. Calderon V, Lazaro A, Contreras RG, Shoshani L, FloresMaldonado C, Gonzalez-Mariscal L, Zampighi G, and Cereijido M. Tight junctions and the experimental modifications of lipid content. J Membr Biol 164: 59–69, 1998. 6. Cereijido M. On the evolution on the ideas of the tight junction. In: Tight Junctions, edited by Cereijido M. Boca Raton, FL: CRC, 1992, p. 1–13. 7. Cereijido M, Robbins ES, Dolan WJ, Rotunno CA, and Sabatini DD. Polarized monolayers formed by epithelial cells on a permeable and translucent support. J Cell Biol 77: 853–880, 1978. 8. Cereijido M and Rotunno CA. An Introduction to the Study of the Biological Membranes. New York: Gordon and Breach, 1971. 9. Chen Y, Lu Q, Schneeberger EE, and Goodenough DA. Restoration of tight junction structure and barrier function by down- regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells. Mol Biol Cell 11: 849–862, 2000. 10. Citi S and Cordenonsi M. The molecular basis for the structure, function, and regulation of tight junctions. In: The Adhesive Interaction of Cells, edited by Bittar E, Garrod DR, and North AJ. Greenwich, CT: JAI, 1999, p. 203–233. (Adv Mol Cell Biol 28) 11. Contreras RG, Avila G, Gutierrez C, Bolivar JJ, GonzalezMariscal L, Darzon A, Beaty G, Rodriguez-Boulan E, and Cereijido M. Repolarization of Na⫹-K⫹ pumps during establishment of epithelial monolayers. Am J Physiol Cell Physiol 257: C896–C905, 1989. 12. Contreras RG, Miller JH, Zamora M, Gonzalez-Mariscal L, and Cereijido M. Interaction of calcium with plasma membrane of epithelial (MDCK) cells during junction formation. Am J Physiol Cell Physiol 263: C313–C318, 1992.

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