regulation of intercellular tight junctions by zonula occludens toxin and its eukaryotic analogue...

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Regulation of Intercellular Tight Junctions by Zonula Occludens Toxin and Its Eukaryotic Analogue Zonulin

ALESSIO FASANOa

Division of Pediatric Gastroenterology and Nutrition, Gastrointestinal Pathophysiology Section, Center for Vaccine Development, and Department of Physiology,University of Maryland School of Medicine, Baltimore, Maryland 21201, USA

ABSTRACT: The intestinal epithelium represents the largest interface betweenthe external environment and the internal host milieu and constitutes the ma-jor barrier through which molecules can either be absorbed or secreted. Thereis now substantial evidence that tight junctions (tj) play a major role in regu-lating epithelial permeability by influencing paracellular flow of fluid and sol-utes. Tj are one of the hallmarks of absorptive and secretory epithelia.Evidence now exists that tj are dynamic rather than static structures and readi-ly adapt to a variety of developmental, physiological, and pathological circum-stances. These adaptive mechanisms are still incompletely understood.Activation of PKC either by Zonula occludens toxin (Zot) or by phorbol estersincreases paracellular permeability. Alteration of epithelial tj is a recently de-scribed property for infectious agents. Clostridium difficile toxin A and B andinfluenza and vesicular stomatitis viruses have been shown to loosen tj in tissueculture monolayers. Unlike what occurs after the Zot stimulus, these changesappear to be irreversible and are associated with destruction of the tj complex.On the basis of this observation, we postulated that Zot may mimic the effectof a functionally and immunologically related endogenous modulator of epithe-lial tj. We were able to identify an intestinal Zot analogue, which we namedzonulin. It is conceivable that the zonulins participate in the physiological reg-ulation of intercellular tj not only in the small intestine, but also throughout awide range of extraintestinal epithelia as well as the ubiquitous vascular endot-helium, including the blood-brain barrier. Disregulation of this hypotheticalzonulin model may contribute to disease states that involve disordered intercel-lular communication, including developmental and intestinal disorders, tissueinflammation, malignant transformation, and metastasis.

The intestinal epithelium represents the largest interface (more then 2,000,000 cm2)between the external environment and the internal host milieu and constitutes themajor barrier through which molecules can either be absorbed or secreted. There is

aAddress for correspondence: Alessio Fasano, M.D., Division of Pediatric Gastroenterologyand Nutrition, University of Maryland School of Medicine, 685 W. Baltimore St., HSF Building,Room 465, Baltimore, MD 21201. Voice: 410-328-0812; fax 410-328-1072.

[email protected]

215FASANO: REGULATION OF INTERCELLULAR TIGHT JUNCTIONS

now substantial evidence that tight junctions (tj) play a major role in regulating epi-thelial permeability by influencing paracellular flow of fluid and solutes. Tj is oneof the hallmarks of absorptive and secretory epithelia. As a barrier between apicaland basolateral compartments, it selectively controls the passive diffusion of ionsand small water-soluble solutes through the paracellular pathway, thereby counter-regulating any gradients generated by transcellular pathways.1 Variations in transep-ithelial conductance can usually be attributed to changes in the permeability of theparacellular pathway, since the resistance of the enterocyte plasma membrane is rel-atively high.2 The tj represent the major barrier within this paracellular pathway, andthe electrical resistance of epithelial tissues seems to depend on the number of trans-membrane protein strands and their complexity within the tj as observed by freeze-fracture electron microscopy.3 Evidence now exists that tj, once regarded as staticstructures, are in fact dynamic and readily adapt to a variety of developmental,4–6

physiological,7–10 and pathological11–13 circumstances. These adaptive mechanismsare still incompletely understood.

In the presence of Ca2+, tj assembly is the result of a complex cascade of bio-chemical events that ultimately lead to the formation of an organized network of tjelements, the composition of which has been only partially characterized. Two can-didates for the transmembrane protein strands, occludin and clandins, have recentlybeen identified14 (FIG. 1). Several proteins have been identified in a cytoplasmic sub-membraneous plaque underlying membrane contacts (FIG.1), but their function re-mains to be established. ZO-1 and ZO-2 each exists as a heterodimer15 in adetergent-stable complex with an uncharacterized 130-kDa protein (ZO-3). Mostimmunoelectron microscopic studies have localized ZO-1 to precisely beneathmembrane contacts.16 Both ZO-1 and ZO-2 belong to the membrane-associated gua-nylate kinase (MAGUK) family of proteins.17 Several other peripheral membrane pro-teins have been localized to the tj, including cingulin,18 7H6,19 rab 13,20 Gαi–2,

21,22

and PKC.22 Recently, a novel protein (symplekin) has been described that not onlyassociates with tj but can also be localized to the nucleus.23 Similar to ZO-1, sym-plekin is also expressed by cells that do not form tj, where it appears to be only inthe nucleus. ZO-1 also can be localized to the nucleus, but unlike symplekin, only ingrowing but not in differentiated epithelial cells.24 This dual localization for these tjcomponents suggests that tj might also be involved in the regulation of gene expres-sion, cell growth, and differentiation.25 Beside rab 13, other small GTP-binding pro-teins are known to regulate the cortical cytoskeleton; Rho regulates actinpolymerization and focal adhesion formation.26 In polarized epithelial cells, Rhoalso regulates tj organization and permeability.27 Other proteins, such as Rac and fo-cal adhesion kinase (FAK), play a role in plasma membrane ruffling and focal adhe-sion formation.28 Whether these molecules also participate in tj regulation isunknown.29 On the basis of the bidirectional signaling that is transduced across focaladhesions30 and the zonula adherens,31 it is conceivable that tj-associated proteinsare similarly involved in transducing signals in one or more directions across the cellmembrane and in regulating links to the cortical actin cytoskeleton. In eukaryoticcells, junctional complex proteins, actin filaments, microtubules, and intermediatefilaments interact to form the cytoskeleton network involved in determination of cellarchitecture, intracellular transport, modulation of surface receptors, paracellularpermeability, mitosis, cell motility, and differentiation.32

216 ANNALS NEW YORK ACADEMY OF SCIENCES

There is now a large body of evidence that structural and functional linkage existsbetween the actin cytoskeleton and the tj complex of absorptive cells.33–35 The actincytoskeleton is composed of a complicated meshwork of microfilaments the precisegeometry of which is regulated by a large cadre of actin binding proteins. The archi-tecture of the actin cytoskeleton appears to be critical for tj function. Most of the ac-tin is positioned under the apical junctional complex where myosin II and severalactin binding proteins, including α-catenin, vinculin, and radixin, have been identi-fied.29 Myosin movement along actin filaments is regulated by ATP and phosphory-lation of the regulatory light chain by Ca2+-calmodulin–activated myosin light-chainkinase.36 In several systems, increases in intracellular Ca2+ can affect phosphoryla-tion of myosin regulatory light chain contraction of perijunctional actin and causeincreased paracellular permeability.37 We have recently demonstrated PKC-depen-dent actin polymerization associated with increments in paracellular permeability.38

FIGURE 1. Model for components of the tight junction. Occludin, the transmembraneprotein strand, is anatomically and functionally connected with the cell cytoskeleton via thejunctional complex. This complex comprises a series of proteins, including ZO-1, ZO-2, andp130 (ZO-3). Other proteins, such as cingulin, 7H6, rab13, rho, and ras, are located furtherfrom the cell membrane. However, they also seem involved in the regulation of tight junctionpermeability.


A variety of intracellular mediators have been shown to alter tj function and/orstructure. Tj within the amphibian gallbladder39 and goldfish40 and flounder41 intes-tine display enhanced resistance to passive ion flow as intracellular cAMP is elevat-ed. In addition, exposure of amphibian gallbladder to Ca2+ ionophore appears toenhance tj resistance and induce alterations in tj structure.42 Last, activation of PKCeither by Zonula occludens toxin (Zot)38 or by phorbol esters43–45 increases paracel-lular permeability. Alteration of epithelial tj is a recently described property of in-fectious agents. Clostridium difficile toxin A46 and B47 and influenza and vesicularstomatitis viruses48 have been shown to loosen tj in tissue culture monolayers. How-ever, unlike what occurs after the Zot stimulus, these changes appear to be irrevers-ible and are associated with destruction of the tj complex.46,47

To meet the many diverse physiological challenges to which both epithelia andendothelia are subjected, tj must be capable of rapid and coordinated responses. Thisrequires the presence of a complex regulatory system that orchestrates the state ofassembly of the tj multiprotein complex. While it is well accepted that tj are dynamicstructures, surprisingly little is known about their regulation. The discovery of Zot,a protein elaborated by Vibrio cholerae, has shed some light on the intricate mecha-nisms involved in the modulation of the intestinal paracellular pathway. Zot actionis mediated by a cascade of intracellular events that lead to a protein kinase C(PKC)α–dependent polymerization of actin microfilaments strategically localized toregulate the paracellular pathway.38 These changes are a prerequisite to opening oftj and are evident at a toxin concentration as low as 1.1 × 10−13 M.49 The toxin exertsits effect by interacting with the surface of enteric cells. By using immunofluores-cence binding studies, we have shown that Zot binding varies within the intestine,being more detectable in the jejunum and distal ileum, and decreasing along the vil-lous-crypt axis.49 This binding distribution coincides with the regional effect of Zoton intestinal permeability49 and with the preferential F-actin redistribution inducedby Zot in the mature cells of the villi,38 suggesting that the regional effect of Zot isassociated to its binding of a surface receptor (r) the distribution of which varieswithin the intestine and along the villous-crypt axis. These data showed that Zot reg-ulates tj in a rapid, reversible, and reproducible fashion and probably activates intra-cellular signals, which are operative during the physiologic modulation of theparacellular pathway (FIG. 2).

On the basis of this observation, we postulated that Zot may mimic the effect ofa functionally and immunologically related endogenous modulator of epithelial tj.The combination of affinity-purified anti-Zot antibodies and the Ussing chamber as-say allowed us to identify an intestinal Zot analogue, which we named zonulin.50

When zonulin was studied in a nonhuman primate model, it reversibly opened intes-tinal tj. Since V. cholerae infections are strictly confined to the gastrointestinal tract,we anticipated an exclusively intraintestinal role for zonulin. Much to our surprise,this protein was detected in a wide range of extra-intestinal tissues and has now beenpurified from human intestine, heart, and brain. We have provided evidence that thezonulins comprise a family of tissue-specific regulators of tight junctions.50 Eachfamily member has an MW of ∼47 kDa, a distinct N-terminal receptor binding motifthat confers tissue specificity, and a C-terminal tau-like domain probably involvedin the cytoskeleton rearrangement. Amino acid substitution within the N-terminalbinding motif identified three amino acid residues that dictate tissue specificity, al-lowing local autocrine/paracrine regulation in response to local requirements.50 The


physiological function of the zonulins remains to be established; however, it is likelythat they are involved in tj regulation responsible for the movement of fluid, macro-molecules, and leukocytes between body compartments. It is likely that zonulin isalso involved in pathological conditions, since tj dysfunction occurs in a variety ofclinical conditions affecting the gastrointestinal tract, including food allergies,51

V. cholerae infection,52 malabsorption syndromes,53,54 and inflammatory bowel dis-eases.55 It is not surprising that tj structure may be affected when the physiologicalstate of absorptive cells is dramatically changed as it is in many of these diseasestates. In cholera, jejunal biopsies obtained during the acute phase of the diseaseshowed a marked widening of the lateral intercellular spaces that was present onlyin the upper third of the villi and was maximal at the villous tips, gradually decreas-ing towards the middle of the villus.52 In symptomatic celiac patients, small intesti-nal barrier dysfunction also has been ascribed to disorganization of the tjcomplex.53,54 These changes are more pronounced in villous enterocytes, and theyresolve once the patients are treated with a gluten-free diet.54 The tj derangementthat occurs with cholera and CD coincides with Zot/zonulin receptor distributionalong the gastrointestinal tract.38 These findings, together with our observation that

FIGURE 2. Proposed Zot intracellular signaling leading to the opening of intestinaltight junctions. Zot interacts with a specific surface receptor (1) whose distribution withinthe intestine varies. The protein is then internalized and activates phospholipase C (2),which hydrolyzes phosphatidyl inositol (3) to release inositol 1,4,5-tris phosphate (PPI-3)and diacylglycerol (DAG) (4). PKCα is then activated (5), either directly (via DAG) (4) orthrough the release of intracellular Ca++ (via PPI 3) (4a). PKCα catalyzes the phosphoryla-tion of target protein(s), with subsequent polymerization of soluble G actin in F actin (7).This polymerization causes the rearrangement of the filaments of actin and the subsequentdisplacement of proteins (including ZO-1) from the junctional complex (8). As a result, in-testinal tight junctions become looser.


zonulin is overexpressed during the acute phase of CD,56 suggest that this proteincontributes to CD pathogenesis by increasing tj permeability typical of the earlystages of this clinical condition. During intestinal inflammatory states, transient lossof the tj barrier (as revealed by diminished tissue resistance and increased permeabil-ity to macromolecules) also has been implicated in transepithelial migration ofgranulocytes.57

Although tumor cell invasion and metastasis are complex processes, a key step inboth is physical disengagement from the primary tumor.58 This step involves disrup-tion of normal cell–cell adhesion in epithelial tissues. Although much of the work inthis area has focused on the adherence junction,59 evidence now exists that tj ele-ments are similarly involved. Willott and coworkers have recently demonstrated thatthe tj protein ZO-1 is homologous to the Drosophila discs large tumor suppressorprotein of septate junctions and that dlg mutations result in loss of apical-basolateralepithelial cell polarity and in neoplastic growth.60 These data suggest that tj dysfunc-tion may be involved in carcinogenesis.

In conclusion, it is conceivable that the zonulins participate in the physiologicalregulation of intercellular tj not only in the small intestine, but also throughout a widerange of extraintestinal epithelia (e.g., the tracheobronchial tree and the renal tubule)as well as the ubiquitous vascular endothelium, including the blood-brain barrier. Dis-regulation of this hypothetical zonulin model may contribute to disease states that in-volve disordered intercellular communication, including developmental and intestinaldisorders, tissue inflammation, malignant transformation, and metastasis.

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