role of tight junctions in cell proliferation and cancer

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PROGRESS IN HISTOCHEMISTRY

AND CYTOCHEMISTRYProgress in Histochemistry and Cytochemistry 42 (2007) 1–57

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Role of tight junctions in cell proliferation and cancer

Lorenza Gonzalez-Mariscal�, Susana Lechuga1, Erika Garay1

Center for Research and Advanced Studies (Cinvestav), Department of Physiology, Biophysics

and Neuroscience, Ave. Instituto Politecnico Nacional 2508, Mexico, D.F. 07360, Mexico

Abstract

The acquisition of a cancerous phenotype by epithelial cells involves the disruption of

intercellular adhesions. The reorganization of the E-cadherin/b-catenin complex in adherens

junctions during cell transformation is widely recognized. Instead the implication of tight

junctions (TJs) in this process is starting to be unraveled. The aim of this article is to review the

role of TJ proteins in cell proliferation and cancer.

r 2007 Elsevier GmbH. All rights reserved.

Keywords: Cancer; Tight junctions; Claudins; Occludin; ZO-1; PDZ; Oncoproteins

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. TJs a characteristic feature of epithelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3. The molecular composition of TJs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.1. Integral proteins of the TJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.2. Peripheral proteins of the TJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

.

n

d

e

3.2.1. Peripheral TJ proteins with PDZ domains . . . . . . . . . . . . . . . . . . . 16

3.2.2. Peripheral TJ proteins without PDZ domains . . . . . . . . . . . . . . . . . 20

4. TJ and related structures in stratified epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . 21

see front matter r 2007 Elsevier GmbH. All rights reserved.

proghi.2007.01.001

ding author. Tel.: +5255 5061 3966; fax: +5255 5061 3754.

ress: [email protected] (L. Gonzalez-Mariscal).

d equally.

www.elsevier.de/proghi

dx.doi.org/10.1016/j.proghi.2007.01.001

mailto:[email protected]

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L. Gonzalez-Mariscal et al. / Progress in Histochemistry and Cytochemistry 42 (2007) 1–572

5. TJ proteins not always concentrate at the plasma membrane . . . . . . . . . . . . . . . . 22

6. TJ proteins play a role in the regulation of cell proliferation, differentiation

and gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

6.1. ZO proteins are homologous to the tumor suppressor protein Dlg . . . . . . . 23

6.2. Adaptor proteins of the TJ that associate to tumor suppressors . . . . . . . . . 24

6.2.1. Interaction of MAGIs with the tumor suppressor PTEN/MMAC . . . 24

6.2.2. The tumor suppressor hScrib associates to ZO-2 . . . . . . . . . . . . . . . 24

6.3. Nuclear factors that localize at TJ or associate with TJ proteins at

the nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

6.3.1. huASH, a protein involved in chromatin remodeling . . . . . . . . . . . . 25

6.3.2. Scaffold attachment factor (SAF-B), a chromatin component

involved in the assembly of transcriptosome complexes,

associates with ZO-2 at the nucleus . . . . . . . . . . . . . . . . . . . . . . . . 25

6.3.3. At the TJ ZO-1 sequesters ZONAB, a transcription factor

involved in cell proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

6.3.4. ZO-2 associates at the plasma membrane and the nucleus with

Jun, Fos and

C/EBP transcription factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

6.4. Interaction of cingulin to the nucleotide exchange factor GEF-H1/Lfc. . . . . 29

7. TJ proteins with PDZ domains are targets of different viral oncoproteins. . . . . . . 29

7.1. Human papilloma virus (HPV). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

7.2. Adenovirus (Ad) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

7.3. T-cell leukemia virus-1 (HTLV-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

7.4. Simian virus 40 (SV40) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

8. In cancerous epithelia, the expression of TJ proteins is altered. . . . . . . . . . . . . . . 31

9. Transcription factors and epigenetic changes that regulate TJ protein expression. . 37

10. Effect of cytokines and growth factors on the expression of TJ proteins . . . . . . . . 38

10.1. Interferon-g (IFNg) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

10.2. Hepatocyte growth factor (HGF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

10.3. Tumor necrosis factor-a (TNFa) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

10.4. Epithelial growth factor (EGF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

10.5. Transforming growth factor-b1 (TGF-b1) . . . . . . . . . . . . . . . . . . . . . . . . . 40

11. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

1. Introduction

The term cancer refers to a broad category of diseases that arise as a result of theaccumulation of mutations, chromosomal instabilities and epigenetic changes thattogether impair the cell’s system of cell growth and death. Carcinomas aremalignancies that originate in epithelial tissues. Epithelial cells cover the externalsurface of the body, and line the internal cavities and ducts of the organism. Cancersthat originate from epithelial cells include those of the breast, lung, prostate andcolon. In humans, more than 90% of all neoplasm are carcinomas, although amongchildren, particularly before the adolescent years, carcinomas are very rare andinstead leukemias, central nervous system cancer, lymphomas, sarcomas and

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L. Gonzalez-Mariscal et al. / Progress in Histochemistry and Cytochemistry 42 (2007) 1–57 3

embryonal cancers represent a greater burden (National Cancer Institute and USNational Institutes of Health, 2003). It is no surprise that in adults most cancers areepithelial in origin, since epithelial cells are in more pronounced contact withenvironmental carcinogenic agents than other somatic cells.

During development and in cancer progression, a phenotype transition fromepithelial to mesenchymal (EMT) takes place (Hay, 1995). Epithelial cells display twoparticular phenotypic characteristics: (1) the formation of layers integrated by polygonalcells that are closely joined by membrane structures named tight junctions (TJs), and (2)an apical-basolateral polarization (Cereijido et al., 2004) (Fig. 1A). In contrast,mesenchymal cells do not form organized cell layers, are not polarized, contact theneighboring cells only focally and are not associated with the basal lamina. They displaya fusiform or spindle-like morphology and tend to be highly mobile (Fig. 1B).

Mesenchymal cells are an invention of metazoans that according to the fossilrecord appeared 560 to 600 million years ago, although evidence from molecularbiology suggests they were existing as early as 800 to 1000 million years ago (ConwayMorris, 1993). However, the metazoans of the Phylum Porifera (sponges) have nodistinct body tissues, no cavity between the layers of cells and no recognizable bodysymmetry (Fig. 2A). These animals probably arose from protists whose cells remainedtogether after division forming a multicellular colony. Metazoans in all other Phyla

have distinct cell layers and symmetrical bodies. The members of the Phyla Cnidaria

(anemones, corals and hydroids) and Ctenophora (sea gooseberries) are diploblastic,which means that they have only two cell layers, the ectoderm and endoderm,separated by a thick gelatinous mesoglea. These organisms have no enclosed bodycavity, are radially symmetrical (Fig. 2B) and appear to have evolved from protistsforming a lineage independent from all other animals (Purves et al., 2004b).

The rest of the metazoans are triploblastic as their embryos have a third layer, themesoderm between the ectoderm and the endoderm. It is pertinent to mention thatalthough Cnidaria lack obvious mesoderm, they express in the entocodon, a tissuethat separates from the ectoderm and gives rise the musculature, certain genesinvolved in mesoderm and muscle differentiation in triploblastic animals, such astwist, mef2 and snail (Ball et al., 2004).

During embryogenesis of triploblastic metazoans, the EMT is indispensable fortissues and organs to be formed. In its absence, development cannot proceed past theblastula stage. During gastrulation, the blastoderm invaginates to form a pocket ofan inner germ layer named the endoderm, leaving an outer germ layer, the ectoderm.The cells that bulge into the blastocoel are the beginning of the mesoderm, the thirdgerm layer that forms between the ectoderm and the endoderm (Fig. 2C) (Purveset al., 2004a). In vertebrates, the importance of EMT in morphogenesis isconsiderably augmented as it allows the formation of vertebrae, the cardiac valves,the disappearance of the male mullerian ducts and the ontogeny of the neural crest,crucial for the formation of most of the peripheral nervous system, the craneo-facialskeleton and the pigmented cells of the body (Thiery and Sleeman, 2006). The reverseprocess named mesenchymal–epithelial transition (MET) also occurs and is crucialfor somitogenesis, kidney development and coelomic cavity formation (Thiery andSleeman, 2006).

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(B) Mesenchymal cells

(A) Epithelial cells

BasolateralMembrane

ApicalMembrane

Tightjunction

Fig. 1. Morphological appearance of epithelial and mesenchymal cells. (A, upper) Epithelial

cells form sheets of polygonal cells closely joined together. (A, lower) A lateral scheme of an

epithelial monolayer is shown for identification of the apical and basolateral plasma

membranes. The TJ localizes at the limit between the apical and the basolateral membranes.

(B) Mesenchymal cells have a fusiform or spindle like shape, are not polarized and only

establish focal contacts with their neighbors.


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Phylum Echinodermata

(Triploblast)

Sea urchinSea urchin Gastrulation

Mesoderm

Ectoderm

Endoderm

Phylum Cnidaria

(Diploblast)

Jellyfish Medusa Polyp

Epidermis(ectoderm)

Enteron

Mesoglea

Mouth

Gastrodermis

(endoderm)

Choanocyte

Spicule

W ater flow

W ater

flow

Spongocoel Amoebocyte

Porocyte

Epidermis

Phylum Porifera

Sponge

(A)

(B)

(C)

Fig. 2. Mesenchymal cells are an invention of metazoans. (A) The sponges (Phylum Porifera)

are simple multicellular animals that have little differentiation or coordination among their

cells. (B) The Cnidaria like the jellyfish have symmetrical bodies and embryos with two cell

layers: The ectoderm and endoderm separated by a thick gel called the mesoglea. The two

body forms of cnidaria are illustrated: the polyp (asexual) and the medusa (sexual).

(c) Appearance of the mesoderm during gastrulation in a sea urchin. All metazoans except

Porifera, Cnidaria and Ctenofora are triploblatic which means that they have a third layer

called the mesoderm which appears during gastrulation between the ectoderm and endoderm.


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The EMT and MET encompass a wide spectrum of changes including precisemolecular differences. For example, while mesenchymal cells express vimentin,different types of cytokeratins are present in epithelial cells (Gilles et al., 2003).Moreover, mesenchymal cells do not express E-cadherin, a calcium-dependentcell–cell adhesion molecule that in epithelial cells, concentrates at the adherensjunction (AJ) and initiates cell contact between neighboring cells (Peinado et al.,2004). The expression of several TJ proteins is also deregulated during EMT. Theloss of proteins involved in epithelial cell–cell contact is crucial for the developmentof a fibroblastic phenotype with invasive properties in cancerous tissue, and is insharp contrast to tissue remodeling occurring during morphogenesis, tuboligenesisand tissue regeneration, where cell–cell adhesion proteins are only transientlydown modulated or relocalized (Gumbiner, 2000; Zegers et al., 2003). For example,E-cadherin suffers a reduction in its adhesive activity upon convergent extension, atissue elongation process that takes place in Xenopus gastrulation (Brieher andGumbiner, 1994), and E-cadherin is transiently distributed at random around thecell surface while ZO-1 remains localized at sites of cell–cell contact, when thescatter/hepatocyte growth factor (HGF) is employed to induce tuboligenesis inepithelial MDCK cells (Pollack et al., 1998).

In carcinomas, the initial step of metastasic dissemination includes the detachmentof epithelial cells from the extracellular matrix and cell rounding due to disruption ofthe actin cytoskeleton. In normal cells, these processes respectively induce two typesof apotosis: anoikis, termed from the Greek word for homelessness, and amorphosis.Both prevent normal cells from colonizing elsewhere when detached. In contrast,cancerous cells survive by using autocrine and paracrine mechanism that suppressprogrammed cell death, stimulate tissue invasion and promote the growth of newblood vessels to supply oxygen (Frisch and Ruoslahti, 1997; Liotta and Kohn, 2004;Mehlen and Puisieux, 2006).

The arrival of cDNA microarray technology has allowed the analysis of thousandsof genes in order to identify those involved in carcinogenesis (Al Moustafa et al.,2002). Among the genes that suffer a significant change in expression are as expected,those associated with signal transduction (e.g. growth factors), cell cycle progression,transcription and apoptosis. cDNA microarrays have also revealed how genesinvolved in cell–cell adhesion, including those of the TJ, are under- or over-expressedin different carcinomas. In the present review, we will analyze the correlationbetween the loss of functional TJs in cancer progression and metastasis and willexplore the role of TJs in the control of cellular proliferation and differentiation. Wewill start by explaining the function of TJs in epithelia cells and will then give anoverview of the molecular constituents of the TJ.

2. TJs a characteristic feature of epithelial cells

Epithelial cells are the boundary between the organism and the externalenvironment. While the epithelial cells of the skin cover the body, other epithelia

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surround the ducts and compartments of the organism. From the point of view ofthe individual, the external environment is not only constituted but the milieusurrounding the body but also by the content found in the interior of cavities andducts of the organism such as the stomach, uterus, urinary bladder, intestine, urinarytract, etc. (Fig. 3).

Epithelial cells are able to function as the body frontier, since they are capable ofrestricting the passage of ions and molecules through them. Two pathways areavailable for transepithelial transport (Fig. 4): the first one is the transcellularpathway that requires the transit of substances through the cell crossing the apicalmembrane facing the external milieu or the lumen of a cavity or a duct, and thebasolateral plasma membrane in contact with the interior of the organism. Toaccomplish this purpose it is necessary for the element to be transported either to belipophilic or to count with specific transport mechanism such as carriers, pumps,channels or co-transporters. An alternative route of transit across epithelial sheets isconstituted by the paracellular pathway located at the intercellular space betweenadjacent cells. Transport through this route is regulated by a seal established at theuppermost portion of the lateral membrane by the TJs.

In the late XIX and early XX centuries, the anatomical formations sealing theoutermost end of the intercellular space were detected by light microscopy(Anderson and Cereijido, 2001). The arrival of electron microscopy, allowedFarquhar and Palade in 1963 to distinguish different types of specialized intercellularjunctions. TJs were initially described as membrane ‘‘kissing points’’ since theexternal leaflets of the lateral plasma membrane observed by thin section electronmicroscopy, appeared to fuse at precise spots of intercellular contact. Further studiesinvolving freeze–fracture analysis revealed that in contrast to desmosomes and gapjunctions that constitute isolated spots of cell–cell adhesion, TJ form a network ofstrands that encircle epithelial cells at the limit between the apical and the basolateralmembrane (Fig. 5).

The permeability through the paracellular pathway can be evaluated by twomethods, the measurement of transepithelial electrical resistance (TER) and the fluxof tracer solutes that can be detected either for being electron-dense (e.g. lanthanum,ruthenium red) and hence detectable by the electron microscope or for having afluorescent (e.g. FITC-dextran) or radioactive label (3H-mannitol). Several decadesago when the electrophysiologists explored the transepithelial transport acrossdiverse natural epithelia, it became clear that while some epithelia like the distalconvoluted tubule, the urinary bladder and the stomach duct exhibit a TER in theorder of several hundreds or even thousand of O cm2, others like the proximal tubuleof the kidney or the small intestine reach only a few O cm2 (Claude andGoodenough, 1973; Powell, 1981). At the beginning, the low resistance attainedby leaky epithelia was thought to arise by the fragility and/or improper handling ofthese preparations. However, later studies demonstrated that this is not the case, andthat leaky epithelia are in fact present in several tissues of the body and owe their lowTER to the intense flux of ions and molecules that constantly flows through theirparacellular pathway (Diamond, 1962a, b; Diamond and wright, 1969; Moreno,1975a, b). Therefore, the name TJ is misleading since not all are particularly tight.

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External

stratified

epithelium

milieuDucts Cavities

Stomach content(external milieu)

Kidney

simple cudoidal

epithelium

Urine

(external milieu)

Gastric pits

Stomach

Gastric mucosa

(simple columnar epithelium)

}

Fig. 3. In multicellular organisms, the external environment comprises the milieu that

surrounds the body and the gas and liquid content found inside the cavities and ducts of the

organism. The scheme illustrates the presence of epithelia at the frontier with the external

media surrounding the organism and lining the cavities and ducts of the body here exemplified

by the stomach and the kidney tubules.


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ApicalMembrane

BasolateralMembrane

TJ

ParacellularPathway

TranscellularPathway

Aqueous pore

Paracellular Pathway

Cell1

Intercellular space

TJ transmembraneproteins

Cell2

CC

CC

CC

C

Transcellular Pathway

Pump Channel Co-transporter

Fig. 4. Transport routes across epithelial cell sheets. Two routes are available for ions or

molecules to traverse an epithelial monolayer: the transcellular and the paracellular pathways.

The latter is controlled by the TJ.


TJs have also been assigned the role of a fence that blocks the free diffusion oflipids and proteins within the plasma membrane from the apical to the basolateralsurface and vice-versa (Dragsten et al., 1981; van Meer et al., 1986; van Meer andSimons, 1986). This property of TJs apparently allows epithelial cells to maintain apolarized distribution of proteins in their plasma membrane and hence permits avectorial transport of ion and molecules across epithelial sheets. This concept has,however, been recently challenged, as individual epithelial cells can be induced to

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Fig. 5. Appearance of tight junctions in freeze–fracture replicas. Freeze–fracture replicas of

the epithelium of mouse intestine (A, courtesy of A. Martinez-Palomo and V. Chavez) and

MDCK cells (B). (A) Network of TJ strands that encircles the cells at the limit between the

lateral membrane and the apical surface, distinguished by the presence of microvilli (M). (B)

Region of tricellular contacts, where three cells join together. This area characterized for

concentrating tricellulin, an integral protein of the TJ, can be distinguished in freeze–fracture

by the appearance of central sealing elements (arrow) oriented vertically and connected to

numerous perpendicular TJ strands (arrowhead).


polarize in the absence of cell–cell contacts (Baas et al., 2004) and epithelial cellshaving no TJs have been found to display a normal segregation of membraneproteins, suggesting that TJ might after all, not be as crucial as previously thoughtfor maintaining epithelial polarization (Umeda et al., 2006).

3. The molecular composition of TJs

The observation that in tight epithelia the TJ severely restricts the paracellularflow of ions and molecules, while in leaky epithelia a strong paracellular flux isencountered, suggested a differential composition of TJs among tight and leakyepithelia. However, this issue remained unresolved until the molecular constituentsof the TJ started to be identified in the late 1980s. Now the TJ is regarded as acomplex array of integral and peripheral proteins. The latter have also been named

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adaptor proteins since they connect the transmembrane proteins of the TJ with theactin cytoskeleton and to numerous proteins involved in cell signaling and vesicletrafficking. In order to be able to describe the changes in TJ composition in tissuesafflicted with cancer, we will next provide an overview of the molecular constituentsof the TJ.

3.1. Integral proteins of the TJ

At the TJ, five types of integral proteins are found (Table 1): occludin, tricellulin,claudins, junctional adhesion molecules (JAMs) and Crumbs (Fig. 6). Occludin,tricellulin and claudins are tetraspan proteins that expose their amino and carboxylterminal domains to the cytoplasm and exhibit two extracellular loops, involved inestablishing contact with a homotypic molecule located in the TJ region of theadjacent cell. The extracellular loops of occludin are of approximately the samelength (45 and 44 amino acids in human, 52 and 44 in mouse), while in tricellulin andclaudins the first loop is longer (tricelullin, 41 vs. 30 residues; claudins, 41 to 55 vs. 10to 21 amino acids). In occludin and tricellulin, the first loop is enriched with tyrosineand glycine residues (occludin, 58%; tricellulin, 41%) while the second loop ofoccludin is rich in tyrosines. The extracellular loops of different claudins displayinstead, variability in the distribution and number of charged residues which areimportant in influencing the passage of ions through the paracellular space (Miticand Van Itallie, 2001). Another particular characteristic of claudins is the presence ofthe WGLWCC motif in their first extracellular loop (Van Itallie and Anderson,2006). The carboxyl terminal sequence of tricellulin and occludin are conserved (last130 residues are 32% identical) and the genes encoding these two proteins arelocated in tandem on mouse chromosome 13 and human chromosome 5, suggestingthat they may have duplicated during evolution (Ikenouchi et al., 2005). Claudinsdisplay the shortest carboxyl tail of the TJ tetraspan proteins (occludin 259 aa,tricellulin 193 aa, claudins 21 to 44 aa) and are the only ones to end in PDZ bindingmotifs, which bind the PDZ domains present in the cytoplasmic scaffolding proteinsof the TJ (see below). Instead the long carboxyl terminal domain of occludininteracts with a wide diversity of cytoplasmic proteins including the TJ adaptorproteins cingulin (Cordenonsi et al., 1999), ZO-1 (Furuse et al., 1994), ZO-2 (Penget al., 2003) and ZO-3 (Haskins et al., 1998), connexins (Kojima et al., 1999, 2001)and signaling molecules like PKC (Andreeva et al., 2001; Nusrat et al., 2000), c-Yes(Chen et al., 2002; Nusrat et al., 2000), c-Src (Kale et al., 2003), and CK2 (Smaleset al., 2003). To date, only the E3 ubiquitin protein ligase Itch has been found to bindto the amino terminal portion of occludin (Traweger et al., 2002).

JAMs are type I proteins of the TJ characterized by two extracellular Ig-likedomains with intramolecular disulfide bonds, a single transmembrane region and acytoplasmic domain that ends with a canonical PDZ binding motif. The length of thecytoplasmic tail differs among JAMs, those that have a short tail of 45–50 residues,like JAM-A, -B and -C exhibit a type II PDZ binding motif, while others like CAR,ESAM, JAM4 and CLMP that have a tail of 105 to 165 residues display type I PDZbinding motif. This difference suggests their association to different sets of molecules

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Table

1.Generalcharacteristics

ofthetransm

embraneproteinsoftheTJ

Protein

Extracellularloops

Distinctive

motifs

Involved

in:

Loops

length

Aminoacid

composition

Polarity

determination

BiogenesisofTJ

Constitutionof

TJfilaments

Regulation

of

paracellular

permeability

Chargeand

size

selectivity

of

paracellular

pathway

Leukocyte

adhesionand

transendotelial

migration

Tet

rasp

an

Occludin

1st¼

2nd

1st

enriched

withY

andG

(58%)

No

No

Yes

Yes

No

No

2ndrich

inY

Claudins

1st4

2nd

Charge

residues

WGLWCC

No

No

Yes

Yes

Yes

No

PDZ

binding

motif

N-

glycosylation

sites

Tricellulin

1stE

2nd

1st

enriched

withY

andG

(41%)

No

No

Yes

Yes

No

No

Type

I

JAMs

Extracellular

region

TwoIg-like

domains

R(V

/I/L)E

dim

erization

motif

No

Yes

No

Yes

No

Yes

PDZ

binding

motif

CRB3

PDZ

binding

motif

Yes

Yes

No

No

No

No


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41-55a

21-44a

259a187a

193a

10-21a

52a 44a30a

41a

N

N

NC

C

C

TricellulinOccludinClaudin

Signature residuesGlycine residuesTyrosine residues

Glycosylation sitesCharged residues

PDZ binding motif

37a40-165a

JAM

CRB3

S

S

N

N

C C

S

S

Fig. 6. Schematic representation of the integral proteins of the TJ. Scheme of the three

tetraspan (claudin, occludin and tricellulin) and two type I (JAM and CRB3) proteins of the

TJ. The number of residues present in the extracellular loops and carboxyl tails is indicated.

Signature residues, PDZ binding motifs, glycosylation sites and charged amino acids are

indicated with different colors.


containing PDZ domains. Another common feature for JAM family proteins is thepresence of two or more N-glycosylation sites in the extracellular domain. Withregards to the Ig-like domains, these are frequently classified as C1, C2, V and I typebased on their similarity to the variable and constant regions of antibodies (Williamsand Barclay, 1988). In JAM proteins, there is no general pattern regarding theirresemblance to the canonical Ig-like structures as they harbor different combinationsof V-type and C-type Ig domains (Kostrewa et al., 2001; Liu et al., 2000; Malergueet al., 1998; Martin-Padura et al., 1998; Palmeri et al., 2000; Prota et al., 2003) .JAMs A, B and C have a R(V/I/L)E dimerization motif at the membrane distalIg-like domain (Kostrewa et al., 2001).

The role played by occludin, tricellulin, claudins and JAMs, began to be unraveledin recent years. In the case of claudins, the evidence obtained with (a) knock-outanimals (Furuse et al., 2002), (b) fibroblasts and epithelial cell lines transfected withdifferent claudin constructs (Alexandre et al., 2005; Van Itallie et al., 2001, 2003;

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Yu et al., 2003), (c) hereditary human diseases where claudin genes are mutated(Muller et al., 2003; Simon et al., 1999; Wilcox et al., 2001), as well as (d) tissues thatlike the mammalian nephron display a varying degree of epithelial tightness (Kiuchi-Saishin et al., 2002; Li et al., 2004a; Reyes et al., 2002), indicates that claudinsconstitute the molecular backbone of TJ filaments (Tsukita and Furuse, 2002)responsible for integrating the large aqueous pores or channels of the paracellularpathway capable of size and charge selectivity. Hence, the combination of claudinsexpressed in a given tissue determines in great measure the particular characteristicof its paracellular pathway (Tsukita and Furuse, 2000).

In contrast, the role of occludin, on TJ structure and function remainscontroversial as occludin knock-out mice are viable and exhibit TJs with anapparent normal morphology (Saitou et al., 2000). Yet other evidence indicates thatthe over-expression of occludin increases the TER of transfected cells (Balda et al.,1996b; McCarthy et al., 1996), and that a higher amount of occludin is present in thetighter than in the leakier portions of the mammalian nephron (Gonzalez-Mariscalet al., 2000).

Tricellulin is the latest integral protein to be identified at the TJ. It is concentratedat the vertically oriented TJ strands of tricellular contacts (Fig. 5B). Tricellulinappears to play a crucial role in the paracellular barrier mechanism since its silencingby RNAi inhibits the development of the TER and severely affects in a size selectivemanner, the paracellular transit of molecules (Ikenouchi et al., 2005).

In contrast to occludin and claudins, JAMs transfection does not induce theappearance of strands at the plasma membrane that resemble those found in the TJof epithelial cells. Instead JAM transfection induces the appearance in freeze–fracurereplicas of areas devoid of intramembrane particles (IMPs) (Itoh et al., 2001). Thispattern is remarkable since similar IMP-free areas are observed during the beginningof TJ assembly in epithelia (Montesano et al., 1975). In endothelial cells, JAM-Clocalizes within minutes to newly formed cell–cell contact (Aurrand-Lions et al.,2001), and during embryonic development JAMs appears at the cellular bordersearlier than any other integral TJ protein. For example, in mouse JAM-A is presentat the plasma membrane at the 8-cell stage (Thomas et al., 2004) while claudin-1and occludin arrive until the 32-cell stage just prior to blastocele cavitation (Fleminget al., 2001; Sheth et al., 2000). In mammals, the recruitment of JAM to the cellularborders appears to be a crucial step, as it allows protein Par3 to interact with thecytoplasmic domain of JAM. This event triggers the junctional recruitment of thePar3/Par6/aPKC complex. The latter regulates late stages of junction biogenesisthat allows the maturation of the junctional complex into distinct TJ and AJs (Ebnetet al., 2003; Matter and Balda, 2003).

Growing evidence indicates that JAM proteins contribute to the regulation ofparacellular permeability. Thus, JAM-A antibodies and a soluble extracellularfragment of JAM-A fused to the IgG Fc domain, inhibit the TER recovery ofepithelial monolayers after disruption of intercellular junctions by transient calciumdepletion (Liu et al., 2000), and the over-expression of JAM-A, JAM-C, CAR andJAM-4 diminish the permeability of fluorescent paracellular tracers in epithelialmonolayers (Aurrand-Lions et al., 2001; Cohen et al., 2001; Hirabayashi et al., 2003).

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JAMs also play a critical role in leukocyte adhesion and migration. JAMspromotion of leukocyte adhesion appears to be mediated by heterophilic interactionswith integrins and by association to other JAM proteins. For example,integrin LFA-1 mediates neutrophil and T-cell adhesion to JAM-A in endothelialcells (Ostermann et al., 2002), while the adhesion of endothelial JAM-B withJAM-C found in the surface of T cells, enables a posterior engagement of theleukocyte integrin a4b1 to endothelial JAM-B (Cunningham et al., 2002).Transfection of JAM-C promotes transendothelial migration of lymphocytes(Johnson-Leger et al., 2002) and JAM-C antibodies decrease neutrophil migrationacross intestinal epithelial cells (Zen et al., 2004). In contrast, the participationof JAM-A in leukocyte migration appears to be limited to specific types ofinflammation and varies according to the species. Thus antibodies to JAM-A blockchemokine induced monocytes transmigration through a mouse endothelial cellmonolayer in vitro, and the systemic treatment of mice with the antibody blocksneutrophil infiltration upon chemokine administration in subcutaneous air pouches(Martin-Padura et al., 1998). Moreover, JAM-A antibodies inhibit leukocyteinfiltration in a cytokine induced murine model of meningitis (Del Maschio et al.,1999). In contrast JAM-A antibodies do not affect leukocyte influx into the centralnervous system in bacterial and viral models of meningitis (Lechner et al., 2000)nor in experiments performed with human epithelial or endothelial monolayers(Liu et al., 2000; Shaw et al., 2001).

Crumbs is another transmembrane protein of the TJ. It was first identifiedin Drosophila, as a protein with 30 EGF-like repeats and four laminin A G-domain-like repeats in its extracellular domain. It localized at the top of the lateralmembrane, in a subapical complex with stardust (Sdt) and Disc lost (Dlt).Fly embryos the lack crumbs and Sdt fail to form a zonula adherens and displaya disruption of the apico-basal polarity (Knust et al., 1993; Tepass and Knust, 1993).Over-expression of crumbs results in an enormous expansion of the apicalplasma membrane and the concomitant reduction of the basolateral domain(Wodarz et al., 1995). In mammals, three homologs of crumbs are expressed, oneof them named CRB3 is present at TJs where it forms a complex with Pals1,the homolog of Sdt, and PATJ the homolog of Dlt (Lemmers et al., 2002; Roh et al.,2002b). CRB3 shares the conserved cytoplasmic domain with other crumbsbut exhibits a very short extracellular domain without the EGF- and lamininA-like G repeats present in the other crumbs. In mammals, the over-expression ofCRB3 leads to delayed TJ formation in epithelial monolayers and disruptionof polarity in epithelial cells cultured in collagen (Roh et al., 2003). The CRB3/Pals1/PATJ complex interacts with the submembranous complex of Par3–Par6–aPKCand together regulates TJ assembly. The interaction between these two com-plexes requires the amino terminus of Pals1 and the PDZ domain of Par6(Hurd et al., 2003). It is important to notice that while the Par3–Par6–aPKCcomplex is involved in polarity determination of a wide variety of tissuesincluding epithelia, neuroblasts (Horne-Badovinac et al., 2001) and astrocytes(Etienne-Manneville and Hall, 2001), the CRB3–Pals1–PATJ is specific forepithelia.

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3.2. Peripheral proteins of the TJ

At the cytoplasmic side of the TJ, a wide spectrum of proteins is found (Table 2).We will start their description classifying them according to the presence of PDZdomains in their sequence. This feature gives them the possibility of establishingparticular protein–protein interactions. Due to the presence of this domain and toothers also involved in protein–protein associations, the peripheral proteins of the TJare considered scaffold proteins, as they cluster in a particular subcellular location awide array of molecules. In this segment we will also describe another subset of TJperipheral proteins that lack PDZ domains.

3.2.1. Peripheral TJ proteins with PDZ domains

PDZ domains are modules of 80–90 residues, named after the proteins in whichthey were initially identified: the postsynaptic density protein PSD-95, the Drosophila

lethal disc large tumor suppressor protein Dlg and the TJ scaffold protein zonulaoccludens 1 (ZO-1). These modules mediate intermolecular homotypic interactionsbetween PDZ domains, like the ones established between ZO-1 and ZO-2 (Fanninget al., 1998; Itoh et al., 1999b), ZO-1 and ZO-3 (Wittchen et al., 1999), and PATJ andZO-3 (Roh et al., 2002a), as well as heterotypic interactions by association to specificmotifs (e.g. S/TXV, fXf) (Songyang et al., 1997) found at the carboxyl terminalends of several proteins, including gap junction connexins (Ciolofan et al., 2006;Giepmans and Moolenaar, 1998; Kausalya et al., 2001; Li et al., 2004b), claudins(Itoh et al., 1999a), JAMs (Ebnet et al., 2000, 2003) and the peripheral TJ proteinsZO-2 (Metais et al., 2005) and ZO-3 (Roh et al., 2002a). The interaction of PDZdomains with PDZ binding motifs has a complex specificity, as some particular PDZdomains and not other within the same protein are selected for these particularinteractions. For example, the PDZ binding motif of claudins associates with the firstPDZ domain of ZO proteins (Itoh et al., 1999a) , the tenth PDZ domain of MUPP1(Hamazaki et al., 2002) and PDZ-8 of PATJ (Roh et al., 2002a), while the interactionof the PDZ binding motif of JAMs takes place with the second and third PDZdomains of ZO-1 (Bazzoni et al., 2000; Ebnet et al., 2000), PDZ-2 of Par-3 (Ebnetet al., 2001; Itoh et al., 2001) and PDZ-9 of MUPP-1 (Hamazaki et al., 2002).Similarly, the PDZ binding motif of ZO-3 associates PDZ-6 of PATJ (Roh et al.,2002a), and carboxyl terminal residues of several connexins associate with the firstand second PDZ domains of ZO proteins (Giepmans and Moolenaar, 1998;Kausalya et al., 2001; Li et al., 2004b; Nielsen et al., 2003).

3.2.1.1. The membrane-associated guanylate kinase (MAGUK) proteins of theTJ. Within the PDZ proteins of the TJ several subgroups can be distinguished. Thefirst one corresponds to the MAGUK protein family that includes TJ proteins ZO-1,ZO-2, ZO-3 and Pals1. MAGUK proteins are characterized by the additionalpresence of a SH3 module, homologous to a non-catalytic region present in thetyrosine kinase product of the v-Src oncogene (Dalgarno et al., 1997), as well as aguanylate kinase (GUK) domain, homologous to the enzyme that catalyzes theconversion of GMP to GDP at the expense of ATP. The TJ MAGUK proteins are

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Table

2.Characteristics

oftheadaptorproteinsoftheTJ

Protein

Generalcharacteristics

Associationwith

No.of

PDZ

Family

Homologousto

tumor

suppressors

Nuclear

localization

NLS

NES

Viral

oncoproteins

Tumor

suppressors

Transcription

factors

Factors

that

promote

cell

proliferation

Wit

hP

DZ

dom

ain

s

ZO–1

3MAGUK

Yes

Yes

Yes

Yes

N.D

.N.D

.ZONAB

ZONAB/

CDK4Apg-2

ZO–2

3MAGUK

Yes

Yes

Yes

Yes

AdE4-O

RF1

hScrib

and

SAF-B

Jun,Fos,C/

EBP,Kyot2

N.D

.

ZO–3

3MAGUK

Yes

Yes

Yes

Yes

N.D

.N.D

.N.D

.N.D

.

Pals1

1MAGUK

N.D

.N.D

.Yes

Yes

N.D

.N.D

.N.D

.N.D

.

HPV

E6

MAGI-1

6MAGI

N.D

.Yes

Yes

Yes

AdE4-O

RF1

PTEN

N.D

.N.D

.

MAGI-2

6MAGI

N.D

.N.D

.Yes

Yes

HPV

E6

MAGI-3

6MAGI

N.D

.N.D

.Yes

Yes

HPV

E6

PTEN

N.D

.N.D

.

HTLV

Tax1

PTEN

N.D

.N.D

.

PAR-3

3N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.

PAR-6

6N.D

.N.D

.Yes

Yes

Yes

N.D

.N.D

.N.D

.N.D

.

PATJ

10

N.D

.N.D

.N.D

.N.D

.N.D

.HPV

E6

N.D

.N.D

.N.D

.

AdE4-O

RF1

MUPP1

13

N.D

.N.D

.N.D

.N.D

.N.D

.HPV

E6

N.D

.N.D

.N.D

.

AdE4-O

RF1

AF-6

1N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.Ras

Wit

hout

PD

Z

Cingulin

No

N.D

.N.D

.Yes

Yes

Yes

N.D

.N.D

.N.D

.GEF-H

1

Symplekin

No

N.D

.N.D

.Yes

Yes

Yes

N.D

.N.D

.N.D

.N.D

.

7H6

No

N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.

Pilt

No

N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.

JEAP

No

N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.N.D

.

N.D

.¼

Nondetermined.


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predicted to bind neither GMP nor ATP since they lack critical amino acidsresponsible for nucleotide binding and catalysis and are therefore consideredenzymatically inactive (Haskins et al., 1998; Kistner et al., 1995).

The GUK domains instead have been found to mediate protein–proteininteractions. For example, the neuronal proteins GKAP/DAP (Kim et al., 1997;Satoh et al., 1997; Takeuchi et al., 1997) and BEGAIN (Deguchi et al., 1998) bind tothe GUK domain of PSD-95. Some GUK domains have been found to interact withMAGUK SH3 modules. So is the case of the PSD-95 GUK domain that binds thePSD-95 SH3 region, suggesting the establishment of an intramolecular interaction(McGee and Bredt, 1999). The SH3 and GUK interactions also generateheterodimers. Thus the GUK domain of PSD-95 binds to the SH3 region ofNE-dlg (Masuko et al., 1999) a MAGUK expressed in neuronal and endocrinetissue, and the GUK domain of CASK, a MAGUK protein widely expressed inhuman tissues and characterized for having an amino terminal region homologous tocalmodulin, binds the SH3 region of Dlg (Kuwahara et al., 1999). In vitro theintramolecular binding between SH3 and GUK modules appears to predominateover the intermolecular interactions (Nix et al., 2000). The SH3–GUK interaction isunusual in the sense that the GUK domain lacks the PXXP motifs, typically requiredfor interaction with SH3 modules (Dalgarno et al., 1997), and that mutations in thecanonical peptide binding sites of SH3 do not disrupt the SH3–GUK interaction.Furthermore, recent evidence indicates that this interaction includes the hinge regioninterconnecting the SH3 and GUK domains, suggesting that in MAGUK proteinsthese discrete protein domains have evolved into an integrated functional unit(McGee et al., 2001). The hinge region is proposed to modulate inter- versusintramolecular associations in the SH3–GUK unit. Hence, the MAGUK proteinmonomer is in a condition where intramolecular SH3–GUK interactions arefavored. However, binding of calmodulin or other unknown factors to the hingeregion, triggers a 3D domain swapping that promotes the interaction of SH3domains with GUK modules present in different molecules generating the formationof homodimers and/or heteromultimers (McGee et al., 2001). In ZO-1, the hingeregion has recently been found to function as another site of association for theoccludin C-terminus, besides the GUK domain. This interaction is both helical andionic since basic residues in ZO-1 and acidic residues in occludin, and coiled-coilhelix motifs in occludin and in the hinge region of ZO-1 are essential (Schmidt et al.,2004). Furthermore, occludin and a-catenin both appear to bind to the same sites inZO-1, the hinge region and the GUK domain. This observation generates theconcept that ZO-1 successively associates with a-catenin at the AJs and occludin atthe TJ (Muller et al., 2005).

The establishment of cultured epithelial cells in which the expression of ZO-1 andZO-2 is suppressed by homologous recombination and RNA interferencerespectively, has given crucial clues for understanding the role of these proteins inTJ strand formation (Umeda et al., 2006). These ZO-1(ko)/ZO-2(kd) cells lack TJsand upon exogenous expression of either ZO-1 or ZO-2, restore the appearance of TJstrands at the TJ region. This led to conclude that ZO-1 and ZO-2 are essential forthe polymerization of proteins that constitute TJ strands. Since the first PDZ domain

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of ZO proteins is known to bind to claudins it was speculated that a truncated ZO-1containing only the three PDZ domains of the molecule (NZO-1) will be sufficient totrigger TJ formation. Instead it was observed that NZO-1 localized at the cytoplasmand claudins did not polymerize. When a longer construct that included theSH3–GUK region was introduced in the ZO-1(ko)/ZO-2(kd) cells, claudinspolymerized at the border between the apical and the lateral membrane. To furtheranalyze the role of the SH3–GUK domain in claudin polymerization, NZO-1 wasforcibly recruited to the membrane by the addition of a myristoylation sequenceand another signal was added to induce its dimerization by a homodimerizer. Whenthe latter was not added, NZO-1 was recruited to the plasma membrane due to themyristoylation signal but no claudin polymerization was observed, instead theaddition of the homodimerizer induced the appearance of prominent TJ strandsthroughout the lateral membrane. Taken together these results indicate thatdimerized ZO-1 and probably also ZO-2, are needed to initiate the polymerizationof claudins and to determine their correct localization at the uppermost portion ofthe lateral membrane.

While the PDZ domains appear to be critical for interconnecting gap and TJproteins as well as for establishing a link between integral and peripheral TJ proteins,the SH3 module of MAGUK proteins seems to be important for clustering signaltransduction molecules at specific membrane domains. SH3 domains are present invarious molecules involved in enzymatic and structural functions. In the c-Ablprotooncogenic protein, the SH3 region exerts a negative regulation of transforma-tion (Cicchetti et al., 1992) and in Drosophila dlg A, this domain is critical for thesignaling and tumor suppressor activity of the protein (Hough et al., 1997). In theMAGUK proteins of the TJ, only the SH3 binding partners of ZO-1 have beenexplored, resulting with the discovery of a serine protein kinase named ZO-1associated kinase (ZAK), that phosphorylates a region C-terminal to the SH3domain (Balda et al., 1996a), and of a transcription factor named ZO-1-associatednucleic acid-binding protein (ZONAB), homologous to Y-box proteins (Balda andMatter, 2000). Below we will describe in further detail, how the interaction of ZO-1with ZONAB is crucial for the regulation of cell differentiation and proliferation.

3.2.1.2. The MAGUK inverted (MAGI) proteins of the TJ. The MAGUKinverted proteins MAGI-1, 2 and 3 localize at the TJ of epithelial cells. The nameMAGI gives reference to the fact that in these proteins the GUK domain is locatedat the amino portion of the molecule while five PDZ domains are found at thecarboxyl region. Another PDZ was later identified at the amino terminus of MAGIproteins, before the GUK domain. A further difference with MAGUK proteinsrelies in the presence in MAGI of a WW module instead of a SH3 domain. WWmodules are the smallest protein modules in nature. Their 40 amino acids form athree-stranded antiparallel b sheet, where the two signature tryptophan (W) residuesare spaced 20–22 amino acids apart. These modules bind short proline-rich motifsthat include the following consensus: PPXY, PPLP, poly-P motifs flanked by R/Kand (phospho-S/T)P. Several results suggests that competition at overlapping targetsites exists for SH3 and WW domains (Macias et al., 2002; Sudol and Hunter, 2000).

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3.2.1.3. TJ proteins with single or multiple PDZ domains. Multi-PDZ domainprotein 1 (MUPP1) and Pals 1-associated TJ protein (PATJ) are TJ proteins that,respectively, contain 13 and 10 PDZ domains. Additionally, they exhibit at theamino terminus a protein–protein interaction module known to recruit TJ MAGUKproteins (MRE).

Two proteins with a single PDZ domain have been identified at the TJ: Par-6 andAF-6/Afadin. Par-6 is a partitioning-defective protein that forms a complex withaPKC and Par-3, another TJ protein with three PDZ domains. This complexregulates cell polarity and the maturation of the junctional complex into TJs and AJs(Matter and Balda, 2003). AF-6/afadin was originally identified as the fusion partnerof the acute lymphoblastic leukemia-1 (ALL-1) protein (Prasad et al., 1993). TheALL-1/AF6 chimeric protein is the critical product associated with acute myeloidleukemia with t(6;11) chromosome translocation. AF6/afadin has two splice variantsknown as long (L) and short (S) AF6/afadins (Mandai et al., 1997). S-AF6/afadin isrestricted to the brain and displays a dual residency at the plasma membrane andnuclear speckles (Buchert et al., 2006). L-afadin is widely expressed in cell–cellcontacts of epithelial cells displaying a distribution similar to that of ZO-1(Yamamoto et al., 1997). L-AF-6/afadin is a target of Ras (Boettner et al., 2000), aGTP-binding oncoprotein activated in numerous human cancers. ZO-1/AF-6interaction is inhibited by activated Ras, since both Ras and ZO-1 interact withthe same domain of AF-6/L-afadin located at the amino portion of the molecule.L-AF-6/afadin associates to the cytoplasmic tail of nectin, therefore ZO-1 can berecruited to nectin based cell–cell adhesion sites by its interaction with L-AF6/afadin, (Yokoyama et al., 2001). Nectins are Ca2+-independent cell adhesionmolecules of the AJs that contain three Ig-like repeats (Takai and Nakanishi, 2003).Nectins form homo-cis dimmers and then homo and hetero-trans dimmers throughthe extracellular region, causing cell–cell adhesion. Next nectins recruit the cadherin/catenin complex to the merging AJ region, through association to the peripheralmembrane proteins ponsin (Mandai et al., 1999), an afadin and vinculin bindingprotein, ADIP (Asada et al., 2003) and LMO7 (Ooshio et al., 2004), both afadin anda-actinin binding protein.

When annexin II, a Ca2+ and phospholipids binding protein, is knocked down inMDCK cells, TJs develop even though cadherin based cell–cell adhesion is notestablished (Yamada et al., 2006). In this system, the formation of TJs relies innectin-based cell–cell adhesion and in afadin capability of binding to nectin, ZO-1and the actin cytoskeleton. TJ formation in this model requires the activation ofCdc42 and Rac small G proteins and the subsequent reorganization of the IQGAP1-dependent actin cytoskeleton induced by the nectin based cell–cell adhesion.

3.2.2. Peripheral TJ proteins without PDZ domains

Here we will describe proteins like cingulin and symplekin. Other peripheral TJprotein lacking PDZ domains, like 7H6, Pilt and JEAP, will not be described sinceno relationship has so far been found between them and cancer.

Cingulin is an actin binding and bundling protein (D’Atri and Citi, 2001) thatinteracts in vitro with non-muscle myosin II (Cordenonsi et al., 1999). In chicken

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brush border cells, cingulin co-purifies with the actomyosin fraction (Citi et al.,1989), further suggesting a role of cingulin in the organization of the actincytoskeleton. In Xenopus embryos, cingulin is recruited into nascent TJ at the 2-cellstage, earlier than all the other TJ proteins. No invertebrate homolog of cinguin hasbeen identified so far, indicating that it may be a TJ component exclusive ofvertebrates. Cingulin molecules form parallel dimmers with a globular head and tail,and coiled-coil rod domains (Guillemont and Citi, 2006).

The role of symplekin at the TJ of epithelial cells remains obscure (Keon et al.,1996). This protein present in numerous cells, including those lacking stable cell–cellcontacts, is better known for its presence in the nucleoplasm and cytoplasm, where itinteracts with proteins involved in mRNA polyadenylation (Hofmann et al., 2002;Takagaki and Manley, 2000).

4. TJ and related structures in stratified epithelia

TJ constitute the paracellular seal that separates the external surface or an internalluminal space from the intersticium. Therefore, it had long been thought, that instratified epithelia, particularly within their deeper strata TJs were absent (Elias andFriend, 1975). However, recent studies have demonstrated the presence of TJproteins and TJ related structures in diverse stratified tissues (Furuse et al., 2002;Langbein et al., 2002), even in the border of specific formations that lack any lumenand that are found within stratified epithelia, such as the horn pearls of squamouscell carcinomas (Langbein et al., 2003).

In the mammalian epidermis, the expression of occludin and claudin-1 is restrictedto the most apical regions of the second layer of the stratum granulosum, whileclaudin-4 is present in deeper layers of the stratum (Furuse et al., 2002). ZO-1,cingulin and symplekin are abundant in the upper layers but can also be detectedalong the cell borders in the lower strata (Langbein et al., 2002). In claudin-1 KOmice, the epidermal barrier is compromised, allowing the passage of paracellulartracers, inducing dehydration, wrinkled skin and animal death within 1 day of birth(Furuse et al., 2002). These results suggest that the presence of claudin-1 andoccludin at the upper region of the stratum granulosum is needed for theestablishment of an effective paracellular barrier.

Electron microscopy has revealed the presence in stratified epithelia of differentkind of structures related to TJs (Langbein et al., 2002). Typical TJ identified by‘‘kissing points’’, are abundant in the stratum granulosum of the epidermis and theupper living cell layers of other stratified epithelia. Besides them, extended closecontact junctions, with a very fine dense line, often partly interrupted, andresembling the septate junctions of invertebrates, have been detected in several strataof these epithelia. Other structures include extended junctions with an electron densemidline whose width varies from 3 (lamellated junction) to 30 nm (sandwichjunction). These pentalaminar junctions adjacent to desmosomes are very similar togap junctions; however, the absence of connexin 43 and their positive stain foroccludin, identify them as TJ related structures. These junctions obviously provide

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close cell–cell contact and might therefore be important in providing architecturalstability, but whether they serve a role in regulating paracellular permeability,remains to be established. However, the results with the claudin-1 KO mice indicatethat the paracellular seal is only established at the uppermost layer of stratifiedepithelia, where typical TJs are detected.

5. TJ proteins not always concentrate at the plasma membrane

In 1996, ZO-1 was found concentrated at the nucleus of sparse epithelial cells inculture (Gottardi et al., 1996). This observation opened the search for the presence ofplaque proteins of the TJ at the nucleus. Since then nuclear staining of other TJadaptor proteins like ZO-2 (Islas et al., 2002) and cingulin (Citi and Cordenonsi,1999) has been found in subconfluent epithelial cells in culture. Many peripheral TJproteins including ZO-1, ZO-2, ZO-3, Pals1, MAGI-1, -2 and -3, Par-3 and PATJcontain nuclear localization and exportation signals (NLS and NES) in theirsequence (Lopez-Bayghen et al., 2006), although only the functionality of thosepresent in ZO-2 has been proven (Gonzalez-Mariscal et al., 2006; Jaramillo et al.,2004). Canine ZO-2 has five NLS and four NES. The transfection of ZO-2 intoMDCK cells has revealed that the mutation of any of the NESs present in themolecule is sufficient to induce nuclear accumulation of the protein. ZO-2 is presentin nuclear matrix preparations and binds to lamin B1 and nuclear actin.

The amount of ZO-2 found at the nucleus in cultured epithelial cells, increasessignificantly in response to mechanical injury (Islas et al., 2002), heat shock (42 1C)and chemical insult (CdCl2) (Traweger et al., 2003). Interestingly, the amino segmentof ZO-2 that contains the three PDZ domains and NLS of the molecule, is capableof relocalizing endogenous ZO-1 from sites of cell–cell contact to the nucleus(Kausalya et al., 2004). At the sites of initial cell–cell contact in sparse cultures,ZO-1, E-cadherin and the armadillo repeat protein deleted in velo-cardio-facialsyndrome (ARVCF), form a complex. The latter interacts via its C-terminal PDZbinding motif, with ZO-1 and ZO-2, and through its armadillo repeat domains withE-cadherin. Upon disruption of cell–cell adhesion, ARVCF is released from theplasma membrane and localizes to the nucleus. ZO-2 appears to be crucial in nucleartranslocation of ARVCF, since the latter process requires the presence of afunctional PDZ-binding motif in ARVCF and of a ZO-2 amino segment with intactNLS (Kausalya et al., 2004). The precise cellular functions of ARVCF remain to beelucidated.

In stratified epithelia, the presence of ZO-1, cingulin, occludin and claudins 1 and4 is restricted to TJs (Citi et al., 1989; Langbein et al., 2002). In contrast, symplekinexhibits a strong nuclear distribution at the proliferative compartments of the basalcell layer and at the non-proliferative strata of the suprabasal cells of the esophagus(Keon et al., 1996).

In cancerous cells, some TJ proteins have been detected at the nucleus. Such is thecase of ZO-1 in papillary thyroid carcinoma (Fluge et al., 2001) and in dissociatedpancreatic cancer cells (Takai et al., 2005), of symplekin in colon carcinoma, and of

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symplekin and cingulin in various cancerous cell lines (Keon et al., 1996; Nakamuraet al., 2000).

Taken together these observations indicate that some adaptor proteins of the TJconcentrate at the nucleus when the cells are transformed, are subjected toenvironmental stress or when the epithelial sheet does not form a continuous barrierdue to mechanical injury or to low-density seeding.

6. TJ proteins play a role in the regulation of cell proliferation,

differentiation and gene expression

Until the early nineties, TJs were mainly perceived as paracellular seals. Theacquisition of a vast amount of evidence has since then broadened our view in such away that TJs are now considered as active participants in the regulation of cellproliferation, gene transcription and the state of cellular differentiation. In thissection, we will describe the similarity existent between some TJ proteins and knowntumor suppressor proteins and then we will focus on the association of TJ proteins tofactors involved in the regulation of cell proliferation and gene expression.

6.1. ZO proteins are homologous to the tumor suppressor protein Dlg

When the full length cDNA sequence of human ZO-1 was uncovered, it becameclear that the amino terminal region that contains the characteristic MAGUKfeatures, is homologous to the product of the lethal disc large-1 (dlg) tumorsuppressor gene of Drosophila (Willott et al., 1993). This homology can now beextended to the other ZO proteins of the TJ: ZO-2 and ZO-3.

Dlg protein is localized on the cytoplasmic face of septate junctions (SJs) (Woodsand Bryant, 1991). In invertebrates, SJs perform a similar barrier function to themammalian TJ, although they are ultraestructurally different as they exhibit a15–20 nm intercellular space, cut transversely by thin ribbons that in thin section givea ladder like appearance (Lane, 2001). SJs are located at the lateral membrane, basalto the AJs. In Drosophila, SJs are constituted by a complex array of proteins thatincludes the MAGUK dlg as well as: neurexin IV, a transmembrane proteinhomologous to mammalian Caspr; coracle, an homologous to 4.1 protein; and scrib,a LAP4 protein characterized for containing leucine-rich repeats (LRRs) and multi-PDZ domains (Humbert et al., 2003).

Mutations in Drosophila dlg lead to loss of apical-basal polarity, mislocalizationof adherens and apical junctions and failure to form the zonula adherens (Woodset al., 1996, 1997). Therefore dlg has also been classified as a cell polarity gene.The link between the regulation of cell polarity and cell proliferation is confirmedby observing that dlg mutations in Drosophila generate neoplastic tumors ofbrain lobes and imaginal discs that lead to death at the larval stage (Woodsand Bryant, 1989, 1991). The imaginal discs are groups of epithelial cells, whichare retained during pupal development and give rise to the adult structures ofthe fly.

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In Drosophila, dlg genetically interacts with scribble (scrib) and lethal giant larvae(lgl), both tumor suppressor genes that function in a common genetic pathway(Bilder et al., 2000).

Dlg has a highly conserved sequence among species. This condition allows themammalian dlg to rescue the phenotype of a Drosophila dlg mutant (Thomas et al.,1997). In mammals, four Dlg members (Dlg-1/SAP-97, Dlg-2/PDS-93/Chapsyn-110,Dlg-3/SAP-102 and Dlg-4/PSD-95/SAP-90) have been found in neuronal tissues,epithelia and lymphoid cells (Humbert et al., 2003). Mammalian Dlgs are involved inthe regulation of cell proliferation, thus the over-expression of both Dlg-1 and Dlg-3in 3T3 fibroblasts impairs the G1-S phase progression and inhibits cell proliferation(Hanada et al., 2000; Ishidate et al., 2000).

With regards to cancerous tissues, Dlg-3 expression is absent in some oesophaegaltumors and tumor cell lines (Makino et al., 1997), while a decreased expression ofDlg-1 is typical in gastric cancers and Dlg-4 down-regulation correlates to the moreinvasive form of gastric cancers, the diffusive gastric carcinomas (Boussioutas et al.,2003).

6.2. Adaptor proteins of the TJ that associate to tumor suppressors

6.2.1. Interaction of MAGIs with the tumor suppressor PTEN/MMAC

PTEN/MMAC is a phosphatase that catalyzes the removal of the 3-phosphatefrom phosphatidylinositol 3,4,5-trisphosphate (Maehama and Dixon, 1998). Thisphospholipid is a product of PI3K involved in the activation of the AKTprotooncogene (Downward, 1998). The latter has a variety of downstream targetsand effects including the suppression of apoptosis (Marte and Downward, 1997).Homozygous mutations in PTEN/MMAC have been found in a high percentage ofhuman glial, prostate and endometrial tumors (Cairns et al., 1997; Rasheed et al.,1997; Tashiro et al., 1997), and give rise in mice to gastrointestinal and prostatichyperplasia/dysplasia and colon carcinomas (Di Cristofano et al., 1998). PTEN/MMAC contains a PDZ binding motif that binds to a PDZ domain of MAGIs(Kotelevets et al., 2005; Wu et al., 2000a, b). This interaction inhibits the activationof the AKT signaling pathway presumably by localizing the phosphatase to themembrane and hence facilitating its access to the phosphatidylinositol 3,4,5-trisphosphate substrate. The interaction of MAGI with PTEN/MMAC in the cellmembrane prevents PTEN/MMAC protein degradation (Subauste et al., 2005).Although MAGIs are adaptor proteins of the TJ, they also interact with the AJprotein b-catenin, and this association appears to be maintained by vinculin.

In mammalian cells, the ectopic expression of MAGI-1 potentiates the interactionof PTEN with the junctional complexes, promotes E-cadherin-dependent cell–cellaggregation and reverts Src-induced invasiveness (Kotelevets et al., 2005).

6.2.2. The tumor suppressor hScrib associates to ZO-2

At the cell–cell junctions of epithelial cells, ZO-2 interacts with hScrib, themammalian homolog of the Drosophila tumor-suppressor Scribble (Metais et al.,2005). The latter is a regulator of epithelial cell polarity that belongs to the LAP

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protein family, known for containing PDZ domains and LRR (Santoni et al., 2002).In humans, hScrib expression decreases during the progression of uterine cervixcancers and in lobular breast cancers (Navarro et al., 2005).

PDZ3 and PDZ4 domains of hScrib associate with the carboxy terminus of ZO-2,where the PDZ binding motif TEL is required for the interaction. The co-localization of hScrib and ZO-2 is only detected in non-polarized cells, thereforesuggesting that this interaction takes place at the cell–cell junctions before ZO-2 issegregated from the AJ into the TJs (Metais et al., 2005).

6.3. Nuclear factors that localize at TJ or associate with TJ proteins at the nucleus

6.3.1. huASH, a protein involved in chromatin remodeling

The transcription factor huASH1 was unexpectedly detected in two distinctcellular compartments in cancerous epithelial cell lines: in small speckles throughoutthe nucleoplasm and at the cellular border co-localizing with TJ proteins (Nakamuraet al., 2000). The human homolog of Drosophila ash1 (huASH1) acts as atranscriptional regulator of homeotic genes (Mazo et al., 1990; Shearn, 1989). Itcontains a SET domain with a histone methyl transferase activity capable ofmodulating the transcriptional state of chromatin (Beisel et al., 2002; Rea et al.,2000) and a bromodomain that is expected to recognize different patterns ofacetylated histones. ASH1 also has Cys-rich Zn finger-like motif named PHD fingerimplicated in protein–protein interactions and four AT hooks for DNA binding(Tripoulas et al., 1996).

The dual location of huASH1 could in principle be due to two unrelated functions,one at the nucleus and the other at the TJ, as is the case for proteins of the AJ-likeb-catenin (Giles et al., 2003). Alternatively the presence of huASH1 at the TJ couldbe due to a cellular effort to attenuate its role in gene transcription. This possibilityhas in fact been demonstrated for the ZONAB transcription factor.

6.3.2. Scaffold attachment factor (SAF-B), a chromatin component involved in the

assembly of transcriptosome complexes, associates with ZO-2 at the nucleus

The SAF-B, which binds to scaffold or matrix attachment regions (S/MAR) ofDNA (Nayler et al., 1998), was identified in a double-hybrid screening to associate tothe TJ protein ZO-2 (Traweger et al., 2003). In epithelial cells, SAF-B co-localizesand co-immunoprecipitates with nuclear ZO-2.

SAF-B appears to participate in the assembly of transcriptosome complexes in thevicinity of actively transcribed genes (Nayler et al., 1998), and co-localizes in nuclearspeckles with SC-35, an essential pre-mRNA splicing protein (Fu and Maniatis,1990; Sahlas et al., 1993). ZO-2 was previously found to co-localize with SC-35 innuclear speckles (Islas et al., 2002).

The physiological importance of SAF-B and ZO-2 interaction has not yet beenstudied. However, from a speculative perspective, it reinforces the idea of ZO-2 beinginvolved in the regulation of gene expression, possibly by facilitating the anchoringof transcriptosome complexes to the nuclear matrix.

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In breast cancer cells, SAF-B functions as estrogen receptor co-repressor andgrowth inhibitor. SAF-B protein expression is lost in approximately 20% of breastcancers and SAF-B mutations have been identified in breast tumors that were notpresent in adjacent normal tissue. Therefore SAF-B has been proposed as a novelbreast tumor suppressor gene (Oesterreich et al., 2001)

6.3.3. At the TJ ZO-1 sequesters ZONAB, a transcription factor involved in cell

proliferation

Since the SH3 domain of Dlg had displayed a tumor suppressor function inDrosophila (Hough et al., 1997), interest developed in studying if this region couldexert a similar function in the MAGUK proteins of the TJ. Upon screening anepithelial cell expression library, the ZONAB was identified as a binding partner ofthe SH3 domain of ZO-1 (Balda and Matter, 2000). ZONAB is homologous to Ybox transcription factors, which bind to promoters with inverted CCAAT boxsequences. Y-box transcription factors have been proposed to play a role in cellproliferation (Kohno et al., 2003), and the human homolog of ZONAB, DbpA, isupregulated in human hepatocellular carcinoma (Hayashi et al., 2002) and inpancreatic adenocarcinoma cell lines (Nakatsura et al., 2001).

The cellular content of ZO-1 and ZONAB is determined by cell density albeit inthe exact opposite ways. In proliferating low-density cells, ZO-1 is expressed at lowlevels while it becomes upregulated in confluent cultures (Balda and Matter, 2000).In contrast, ZONAB levels are high in sparse cultures with proliferating cells and lowin confluent non-proliferating cultures. ZONAB also displays a dual subcellulardistribution: nuclear and TJ localization in sparse cultures and TJ concentration inconfluent monolayers. The expression level of ZO-1 strongly impacts ZONABdistribution but not its cellular content. Hence upon ZO-1 over-expression thenuclear pool of ZONAB diminishes (Balda and Matter, 2000), yet the total amountof ZONAB remains unchanged (Balda et al., 2003). Similarly ZO-1 silencing has noeffect on the total amount of ZONAB (Sourisseau et al., 2006).

In epithelial cells, ZONAB stimulates cell proliferation and increases cell density(Sourisseau et al., 2006), while transfection of ZO-1, of a construct containing itsSH3 domain or ZONAB silencing inhibits the proliferative state of the culture(Balda et al., 2003; Sourisseau et al., 2006). Moreover, ZO-1 silencing or celltransfection with a ZO-1 construct lacking the SH3 domain, promotes cellproliferation (Balda et al., 2003; Sourisseau et al., 2006). Taken together theseresults indicate that ZONAB promotes cell proliferation in a manner negativelyregulated by ZO-1. Since ZO-1 does not have known regions of interaction withDNA and the existence of specific nuclear staining of ZO-1 is still disputed (Baldaand Matter, 2000), its proliferation suppressive function appears to reside on itscapability of junctional sequestration of ZONAB. This proposal based on thediminished expression of nuclear ZONAB upon ZO-1 over-expression is reinforcedby the observation that the SH3 domain of ZO-1 that interacts with ZONAB, isnecessary and sufficient to reduce proliferation.

How ZONAB promotes cell proliferation has been studied by two venues. Thefirst arose after observing that the ZONAB human homolog DbpA interacted in two

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hybrid screens and in vitro with cell division kinases (CDKs) 4 and 5 (Moorthameret al., 1999). The direct interaction of ZONAB with CDK4 was confirmed inpull down assays employing recombinant proteins and further demonstrated in vivowith ZONAB immunoprecipitates. CDK4 is a key regulator of cell proliferationthat gets activated upon binding to cyclin D. The cylin D/CDK4 or CDK6complex drives cells through the cell cycle, acting in G1 to initiate S phase.While ZONAB does not affect the kinase activity of CDK4, the nuclear poolsof the kinase are strongly reduced by over-expresion of ZO-1 or the SH3 domainof ZO-1, as are ZONAB levels. Therefore, the effect of ZONAB upon proliferationappears to rely on its capability of linking the kinase to a mechanism that regulatesits nuclear accumulation (Balda et al., 2003). In this respect, it should be mentionedthat we have not found NLS and NES in the sequence of CDK4, hence its movementfrom the TJ to the nucleus and viceversa could be by association to proteins that likeZONAB shuttle between these locations.

The second approach for studying the effect of ZONAB on cell proliferationwas based on its character as a transcription factor. Initially this was exploredtesting the effect of ZONAB on the inverted CAAT box sequence in the promoterof erbB-2/neu. The gene reporter assays demonstrated that ZONAB functionsas a repressor of the erbB-2/neu promoter, while ZO-1 stimulates erbB-2/neuexpression by sequestering nuclear ZONAB (Balda and Matter, 2000). This wasan unexpected result since erbB-2/neu had been associated to tumorigenesis. In factthe amplification and over-expression of ErbB-2/Neu is associated withincreased progression and metastasis in 25% of human breast carcinomas and isindicative of poor prognosis in breast, ovarian and renal collecting duct carcinomas(Mayr et al., 2006; Pegram et al., 1998; Selli et al., 1997; Verri et al., 2005; Zhanget al., 1997). However, a more detailed survey revealed that ZO-1 increasesendogenous levels of ErbB-2/neu in sparse cells 2–4 times, which corresponds to thelevel found in confluent epithelial cells (Kavanagh et al., 2006). Moreover, ErbB-2over-expression in cancerous cells is around 10–100 times higher than innon-transformed cells. ErbB-2/neu upregulation has also been associated toorgan development and cell differentiation. In fact ErbB-2/Neu is required forductal morphogenesis of the mammary gland (Troyer and Lee, 2001), and inMDCK cells promotes the conversion of cysts of polarized epithelial cellsinto branching tubules in three-dimensional collagen gels (Khoury et al., 2001).The biological consequences of activation of ErbB-2/Neu, depends on how thecell integrates this signal in the overall context of the cell. Thus, in MDCK cellsthe HGF converts ErbB-2/Neu epithelial morphogenesis to cell invasion (Khouryet al., 2005). Hence, the regulation of erbB-2 gene expression by ZONAB,suggests a role of ZO-1 in the control of cell differentiation rather than in cellproliferation. Recently RalA a member of the Ras superfamily of GTPases,was found through a reverse Ras recruitment system (rRRS) to associatewith ZONAB. In high-density cultures RalA co-localizes with ZONAB at the cellborders. The sequestration of ZONAB by RalA at the cell borders results ina relief of transcriptional repression of the erbB-2/neu promoter (Frankel et al.,2005).

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cDNA arrays were also employed as a strategy for detecting the differentialexpression of genes involved in cell proliferation between cell lines over expressingZONAB and those where ZONAB function was inhibited by transfection with the SH3domain of ZO-1 (Sourisseau et al., 2006). This study detected over-expression of cyclinD1 (CD1) and of proliferating cell nuclear antigen (PCNA), a DNA replication andrepair factor associated with cell cycle regulatory proteins (Loor et al., 1997). Furtheranalysis demonstrated that ZONAB increased the protein content of PCNA and CD1and was able to increase in a dose dependent manner, the activity of their respectivepromoters containing CCAAT inverted sequences. As expected, the SH3 domain ofZO-1 decreased the amount of PCNA and CD1 and inhibited the activity of theirrespective promoters. The effect of ZONAB upon PCNA and CD1 expression, couldspeculatively be related to cancer development since oncogenesis and chemicalcarcinogenesis are known to disrupt the stoichiometry of cyclins, CDKs, PCNA andp21 complexes (Gonzales et al., 1998; Xiong et al., 1992; Xiong et al., 1993).

In summary, the nuclear displacement of ZONAB induces cell proliferation byfacilitating the nuclear accumulation of CDK4 and by promoting the transcriptionof PCNA and CD1. ZO-1 inhibits this process by sequestering ZONAB at the TJ.When an epithelial cell expression library, was screened for a binding partner of theSH3 domain of ZO-1, not only ZONAB was identified (Balda and Matter, 2000), butalso Apg-2, a member of the heat shock protein 110 (Hsp 110) family, which is over-expressed in carcinomas. Apg-2 has ATPase and peptide binding domainshomologous to Hsp-70, followed by a unique C-terminal domain. The ATPasedomain of Apg-2 is responsible for the interaction with the SH3 region of ZO-1.Heat shock induces junctional recruitment of Apg-2, enhances its co-immunopre-cipitation with ZO-1 and triggers nuclear concentration of ZONAB, suggesting thatApg-2 competes with ZONAB, for binding to the SH3 domain of ZO-1. Apg-2silencing inhibits cell proliferation, presumably by favoring the establishment ZO-1/ZONAB complexes. Instead Apg-2 over-expression or heat shock treatmentstimulates ZONAB transcriptional activity. Together these results indicate that inresponse to cellular stress Apg-2 regulates ZO-1-ZONAB signaling.

6.3.4. ZO-2 associates at the plasma membrane and the nucleus with Jun, Fos and

C/EBP transcription factors

The concentration of ZO-2 at the nuclei of sparse epithelial cells in cultureprompted the search for nuclear factors that could be associating with this protein.Gel shift assays revealed the specific interaction of ZO-2 with the transcriptionfactors Jun, Fos and C/EBP. Jun and Fos form dimers mediated by their basicleucine zipper motifs. Even though ZO-2 has a putative leucine zipper domain at itsGUK domain, its interaction with these transcription factors appears to beindependent of it. Thus, the ZO-2 segment, constituted by the acidic and proline-rich regions of the molecule, is capable of interacting with Jun and Fos, while othertranscription factors containing leucine zippers are unable to associate with ZO-2.Reporter gene assays done with constructs under the control of promoters with AP-1sites, demonstrated how ZO-2, and not ZO-1, down-regulates gene expression in adose-dependent manner. ZO-2 interact with Jun, Fos and C/EBP at the nuclei in

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sparse monolayers, and at the cellular borders in confluent cultures. Theseobservations suggests that ZO-2 might be regulating the availability of thesetranscription factors at the nuclei by keeping them sequestered at the plasmamembrane of confluent monolayers (Betanzos et al., 2004).

The genes whose transcription is in vivo being modulated by the interactions ofZO-2 and AP-1 are not yet known. However, the AP-1 complex has been reported toparticipate in the regulation of cell proliferation and transformation by altering cyclinD1 transcription (Bakiri et al., 2000; Shaulian and Karin, 2001), and by repressing thetranscription of p53 (Schreiber et al., 1999). The antimitotic activity of C/EBPappears not to depend on its effect on gene transcription and instead is proposed tobe due to the direct interaction of the factor with CDK2 and CDK4 enzymes, in amanner that prevents their binding to cyclins (McKnight, 2001; Wang et al., 2001).

6.4. Interaction of cingulin to the nucleotide exchange factor GEF-H1/Lfc

With the aim of finding novel proteins that localize at the TJ, monoclonalantibodies were generated against detergent extracts of epithelial MDCK cells. Withthis approach a monoclonal that stained the TJ region was selected and was nextemployed to screen an MDCK expression library. The resulting sequence identifiedthe molecule as GEF-H1/Lfc, a guanine nucleotide exchange factor that stimulatesthe exchange of GDP by GTP (Benais-Pont et al., 2003). GEF-H1 is an oncoproteinof the Dbl family that activates Rho A but not the other Rho GTPases Rac1 orCdc42 (Glaven et al., 1996). GEF-H1/Lfc is transcriptionally activated in cancerouscell lines expressing mutant p53 proteins (Mizuarai et al., 2006). GEF-H1/Lfcassociates to microtubules, the actin cytoskeleton, as well as to cingulin, an actinbinding protein of the TJ. The latter interaction mediated by a portion of the rodsegment of cingulin (aa782-1025) and the pleckstrin homology region of GEF-H1/Lfc, inhibits RhoA activation. Accordingly, cingulin depletion activates RhoA(Aijaz et al., 2005). Activated RhoA transduces various signals into downstreamsignaling cascades, such as cytoskeleton reorganization, cellular invasion, and cellproliferation, all of which contribute to cancer progression (Aznar et al., 2004; Sahaiand Marshall, 2002). In epithelial cell lines, the over-expression of cingulin, or ofcingulin segments capable of sequestering GEF-H1/Lfc, inhibit G1/S phasetransition (Aijaz et al., 2005). In sparse cultures the expression of cingulin is low,GEF-H1/Lfc is primarily cytoplasmic, RhoA is active and the cells are in aproliferative stage. With increasing cell density, formation of cell–cell contacts isaccelerated, allowing cingulin to sequester GEF-H1/Lfc at the TJ. As a consequenceRhoA activation is interrupted and cell proliferation is inhibited.

7. TJ proteins with PDZ domains are targets of different viral

oncoproteins

Tumor viruses have evolved multiple strategies to force the infected quiescent cellsto enter the cell cycle, since replication of their genomes depends on host enzymes

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expressed during the S phase of the cell cycle. Recent findings have shown that TJproteins with PDZ domains are targets for viral oncoproteins, further demonstratingthat TJ perturbation contributes to cancer development.

Until now these studies have concentrated on human papilloma virus (HPV),adenovirus (Ad), and T-cell leukemia virus-1 (HTLV-1), and on Simian virus 40(SV40).

7.1. Human papilloma virus (HPV)

Sexually transmitted HPV is the primary etiologic agent of greater than 90% ofcervical cancers. The E6 protein constitutes one the major oncogenic determinants of‘‘high risk’’ HPV. This protein has a functional PDZ binding motif that interactswith the TJ proteins MUPP1 (Lee et al., 2000), MAGI-1 (Glaunsinger et al., 2000;Thomas et al., 2001), MAGI-2 and MAGI-3 (Thomas et al., 2002), and PATJ(Latorre et al., 2005). E6 targets these proteins as well as the AJ molecules dlg andscribble to ubiquitin mediated degradation in the proteosome (Lee et al., 2000;Nakagawa and Huibregtse, 2000; Thomas et al., 2001, 2002).

Expression of ‘‘high risk’’ HPV E6 protein in epithelial cells destabilizesTJs, generates ZO-1 mislocalization, disrupts AJs and promotes carcinogenesis(Nakagawa and Huibregtse, 2000). This effect is dependent on the PDZ bindingmotif of E6 (Nakagawa and Huibregtse, 2000; Thomas et al., 2001). In fact thechronic down-regulation of scribble is associated to the development of invasivecervical cancer (Nakagawa et al., 2004).

7.2. Adenovirus (Ad)

In most human adenoviruses, E1A is the major oncogenic determinant(Braithwaite et al., 1983). Deletions of E1A carboxyl terminal end enhance thetransforming, tumorigenic and metastatic activities of the protein (Chinnadurai,2004). Epithelial cells expressing E1A carboxyl terminal mutants, display adiscontinuous pattern of ZO-1 at the cellular borders and increased paracellularpermeability (Fischer and Quinlan, 1998). The carboxyl terminus of E1A normallyinteracts with kinases Dyrk1A/B (Zhang et al., 2001), whose yeast homolog Yak1p,has been characterized as a negative regulator of growth (Becker and Joost, 1999),and with CtBP, a transcriptional repressor of LEF/TCF [(Schaeper et al., 1995). Thelatter is a transcription factor that together with b-catenin, and the legless (Lgs) andpygopus (pygo) factors, regulates the expression of several genes involved in cancerprogression (For review see (Giles et al., 2003).

For the human adenovirus type 9, the major oncogenic determinant is the proteinencoded in E4 open reading frame 1 (E4-ORF1). This oncoprotein has a PDZbinding motif that interacts with several PDZ proteins of the TJ such as ZO-2,MAGI-1, MUPP1 and PATJ and sequesters them in the cytoplasm of fibroblasts(Glaunsinger et al., 2000, 2001; Latorre et al., 2005; Lee et al., 2000). In epithelialMDCK cells, E4-ORF1 blocks the TJ localization of PATJ and ZO-2, and disruptsboth the TJ barrier and apico-basal polarity (Latorre et al., 2005).

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7.3. T-cell leukemia virus-1 (HTLV-1)

HTLV-1 is the causative agent of adult T-cell leukemia and is the only knownoncogenic human retrovirus. Its tumorigenic potential relies on the Tax protein(Gatza et al., 2003) that upregulates the expression of the TJ protein MAGI-3. Tax1contains a PDZ binding motif that mediates the interaction with MAGI-3 altering itssubcellular localization (Ohashi et al., 2004).

7.4. Simian virus 40 (SV40)

Epidemiological and molecular analyses have evidenced a link between thetransforming DNA tumor simian virus SV40 and human malignancies (Carboneet al., 2003; Vilchez et al., 2003). Although this virus only infects non-human primates,many people were exposed to it due to a contaminant present in the polio vaccinesadministered in the 1950s (Shah and Nathanson, 1976). The small tumor antigen (sTag)of SV40 interacts with the endogenous serine/threonine protein phosphatase 2A(PP2A) (Mungre et al., 1994; Sontag et al., 1993). PP2A exists as a heterotrimericcomplex comprising a core enzyme containing the catalytic C and structural Asubunits, which bound to a regulatory B subunit. Different B subunits contribute totarget PP2A to distinct intracellular domains (Sontag, 2001). In epithelial cells, theholoenzyme containing the Ba subunit is targeted to the TJ complex, where it inducesdeposphorylation of TJ proteins including ZO-1, occludin and claudin-1 (Nunbhakdi-Craig et al., 2002). Following SV40 infection or ectopic expression, the sTag forms acomplex with the endogenous AC dimers by displacing the Ba subunit (Pallas et al.,1992; Ruediger et al., 1992). In consequence, MDCK cells stably transfected with SV40sTag display acute morphological changes and multilayering, marked disorganizationof the cytoskeleton and severe defects in the biogenesis and barrier properties of the TJ.In contrast, sTag does not prevent the formation of AJ (Nunbhakdi-Craig et al., 2003).

8. In cancerous epithelia, the expression of TJ proteins is altered

Evidence for altered TJs in cancerous epithelia has been known for more than 30years. Observations in examined tumors include attenuation or lack of TJs (Alroy,1979), increased permeability to paracellular tracers (Martinez-Palomo, 1970),discontinuities and reductions in the number of TJ strands detected by freeze–fracture (Robenek et al., 1981; Swift et al., 1983), and decreased TER (Soler et al.,1999). Taken together these results indicate a decrease in the epithelial barrierfunction of cancerous cells. The loss of TJs appears to constitute an essential step incancer development. Hence, animal treated with carcinogens like dimethylhydrazine,experience a progressive increase in colon TJ permeability prior to the developmentof tumors (Davies et al., 1989; Soler et al., 1999), and in Crohn’s, an inflammatorybowel disease associated with an increased cancer risk, an augmented TJpermeability has been detected not only in affected patients but also in unaffectedrelatives (Hollander, 1988).

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The development in recent years, of gene expression profiling techniques, hasallowed the identification of genes that are differentially expressed in cancer. Thesestudies together with standard and immunofluorescence microscopy have identifiedchanges in the expression of several TJ proteins in numerous carcinomas (Table 3).

With regards to claudins, it is noteworthy that while a particular isoform issilenced in certain cancers the same protein is found over-expressed in othercarcinomas, suggesting that the function of claudins may be tissue specific and maydepend on the particular molecular circuitry of the cell.

The impact that the loss of claudin expression exerts on cell transformation isbeginning to be unraveled. For example, it has been observed that in breast tumorspheroids the re-expression of claudin-1 induces apoptosis while no change isdetected in 2-D cell cultures (Hoevel et al., 2004). Down-regulation of claudins mightalternatively contribute to epithelial transformation, by increasing the paracellularpermeability of nutrients and growth factors to cancerous cells. It is noteworthythat the entire epidermal growth factor protein family is present in the lumen ofthe gastrointestinal (GI) tract, coming either from the saliva or being synthesized inthe GI tract itself (Barnard et al., 1995), and that after saliva, the urinary tractdisplays the second highest concentration of EGF (Jorgensen et al., 1990). Luminalgrowth factors seem to be without effect on normal epithelial tissue, due to a nearabsence of growth factor receptors on the apical surface. Instead their penetration,bellow the epithelium due to tissue injury or TJ disruption, induces a strong responseas growth factor receptors concentrate on the lateral membrane underneath the TJ(Bishop and Wen, 1994; Mullin and McGinn, 1988; Muto et al., 1991; Schevinget al., 1989). Epithelia constitute the first line of defense of the organism, and arehence constantly being damaged by toxins, microbes and viruses. Therefore, theaccumulation of growth factors in cavities and ducts of the GI and urinary tractconstitutes an evolutionary advantage for multicellular organisms, as it allows thequick encounter of growth factors with their receptors upon epithelial disruption,triggering proliferative and migratory activities needed to restore the damagedepithelial layer.

It is intriguing to find claudin up-regulation in several carcinomas (Table 3), sincethese proteins are clearly implicated in TJ formation and function. However it ispertinent to note, that despite claudin over-expression, the barrier function of thesecancerous epithelia is altered as they display an increased permeability toparacellular markers and a disorganized arrangement of TJ strands in freeze–fracture replicas (de Oliveira et al., 2005). In some colon metastasic tumors that over-express claudin-1, even nuclear localization of claudins has been reported (Dhawanet al., 2005). The impact of claudin over-expression on transformation, is highlightedby the following observations: (1) in colon cancerous cell lines derived from primarytumors, transfection with claudin-1 induces the development of a fibroblastoidmorphology, an enhanced anchorage independence as well as and increased activityof matrix metalloproteinases MMP-2 and MMP-9 (Dhawan et al., 2005), (2) in oralsquamous cell carcinoma cells that express high levels of claudin-1, siRNA silencingof this claudin, largely suppresses their invasive activity and decreases the activationof MMP-2 (Oku et al., 2006), and (3) in human ovarian surface epithelial cells the

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Table 3. Changes in TJ protein expression in human carcinomas

Protein Cancer type Expression Reference

Claudin 1 Breast Down Hoevel et al. (2002), Kramer et al. (2000),

Tokes et al. (2005a, b)

Cervical Down Sobel et al. (2005)

Colon Up de Oliveira et al. (2005), Dhawan et al.

(2005)

Epidermis:

Keratinized (pearl) Up Morita et al. (2004)

Esophagus Up Gyorffy et al. (2005), Morita et al. (2004)

Gastric Up Resnick et al. (2005)

Claudin 2 Cervical Down Sobel et al. (2005)

Colon Up Aung et al. (2006)

Esophagus Up

Claudin 3 Breast Up Kominsky et al. (2004)

Colon Up de Oliveira et al. (2005)

Esophagus Up Gyorffy et al. (2005), Montgomery et al.

(2006)

Gastric Up Resnick et al. (2005), Montgomery et al.

(2006)

Ovary Up Rangel et al. (2003), Santin et al. (2005),

Zhu et al. (2006), Hough et al. (2000)

Pancreas Up Missiaglia et al. (2004)

Prostate Up Long et al. (2001)

Claudin 4 Biliary tract Up Lodi et al. (2006)

Breast Down Tokes et al. (2005a, b)

Up Kominsky et al. (2004)


Colon Up de Oliveira et al. (2005)

Epidermis:


Unkeratinized Down Morita et al. (2004)

Esophagus Up Gyorffy et al. (2005), Montgomery et al.

(2006)

Gastric Up Cunningham et al. (2006), Resnick et al.

(2005), Montgomery et al. (2006)

Down Lee et al. (2005)

Ovary Up Rangel et al. (2003), Santin et al. (2005),

Zhu et al. (2006), Hough et al. (2000)

Pancreas Up Michl et al. (2001), Missiaglia et al. (2004)

Prostate Up Long et al. (2001)

Claudin 5 Pancreas Up Missiaglia et al. (2004)

Claudin 7 Breast Down Kominsky et al. (2003), Tokes et al.

(2005a)


Esophagus Up Montgomery et al. (2006)

Down Usami et al. (2006)


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Table 3. (continued )

Protein Cancer type Expression Reference

Gastric Up Montgomery et al. (2006)

Head and neck Down Al Moustafa et al. (2002)

Uterus Down Tobioka et al. (2004a)

Claudin 10 Liver Up Cheung et al. (2005)

Claudin 23 Gastric Down Katoh and Katoh (2003)

Occludin Breast Down Polette et al. (2005)


Colon Down Tobioka et al. (2002)

Epidermis:



Gastrointestinal Down Kimura et al. (1997), Yin et al. (2002)

Prostate Down Busch et al. (2002)

Pulmonary large and

small cells

Down Tobioka et al. (2004b)

ZO-1 Breast Down Hoover et al. (1998), Martin et al. (2004),

Sommers et al. (1992), Sommers et al.

(1994)

Colorectal Down Kaihara et al. (2003b), Kaihara et al.

(2003a), Mann et al. (1999)

Gastrointestinal Up Resnick et al. (2005)

Down Kimura et al. (1997), Lee et al. (2005)

Melanoma Up Smalley et al. (2005)

Pancreas Up Kleeff et al. (2001)

Epidermis:



Thyroid Up Fluge et al. (2001)

ZO-2 Breast Down Chlenski et al. (2000)

Pancreas Down Chlenski et al. (1999)

ZO-3 Breast Down Martin et al. (2004)

Par-3 Liver Down Fang and Xu (2001)

MUPP-1 Breast Down Martin et al. (2004)

AF-6 Breast Down Letessier et al. (2006)


over-expression of claudins 3 and 4 increases cell invasion, motility and MMP-2activity (Agarwal et al., 2005).

The potential of claudins as targets for cancer therapy is highlighted by theobservation that treatment with Clostridium perfringens enterotoxin (CPE) elicitsa rapid and specific cytolysis of breast (Kominsky et al., 2004), ovarian (Santin et al.,2005), pancreatic (Michl et al., 2001) and prostate (Long et al., 2001), carcinomacells. This toxin responsible for the gastrointestinal symptoms associated with

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C. perfringens food poisoning is released into the intestinal lumen where it binds toclaudins 3 and 4 present in the intestinal epithelial cells. This triggers the formationof a large multiprotein membrane pore that ultimately results in cell lysis. Sinceclaudins 3 and 4 are over-expressed in numerous carcinomas, this conditionsensitizes them to CPE-mediated cytolysis.

The expression of claudins also varies according to the stage of carcinogenesis.Thus in well differentiated carcinomas of the pancreas high levels of claudin-4 aredetected, while in undifferentiated and highly invasive tumors claudin-4 is weaklyexpressed. Moreover, in highly invasive pancreatic cell lines that poorly expressclaudin-4, transfection of this claudin impairs their invasiveness and metastaticpotential (Michl et al., 2003). The expression of certain claudins in carcinomas has apredictive value on the disease outcome. Hence expression of claudin-10 inhepatocellular carcinomas predicts disease recurrence after curative hepatectomy(Cheung et al., 2005) and moderate-to-strong claudin-4 staining is associated withdecreased survival rate in gastric adenocarcinomas (Resnick et al., 2005).

With regards to occludin, knock-out mice exhibit chronic inflammation andhyperplasia in the gastric epithelium that becomes severe around the 28 week of age(Saitou et al., 2000), suggesting the participation of occludin in the control of cellproliferation. Moreover, the addition of a synthetic peptide homologous to thesecond extracellular domain of occludin, decreases the amount of occludin at the TJsand leads to the formation of multilayered an non-polarized cell clusters. Thisprocess involves a cross-talk between TJs and AJs as the addition of the occludinpeptide triggers the accumulation of soluble b-catenin, increases transcription ofconstructs regulated by the b-catenin/TCF/LEF system and up-regulates c-myc(Vietor et al., 2001). The relationship between occludin silencing and oncogenictransformation is reinforced by observing that transfection of oncogenic Raf-1 intosalivary gland epithelial cells Pa-4, generates down-regulation of occludin andclaudin-1, and triggers the acquisition of a stratified phenotype (Li and Mrsny,2000). Transfection of occludin or derived constructs that include the extracellularsecond loop of the molecule, suppress the Raf-1 induced phenotype in the cell cultureand prevents tumor growth in vivo (Wang et al., 2005).

In several cell lines derived from human cervical carcinoma, murine melanoma,murine and human breast cancer and human glioma, the forced expression ofoccludin increases the sensitivity to different factors that promote apoptosis,inhibiting as a result the invasiveness, motility and metastatic potency of thecancerous cells (Osanai et al., 2006). These results identify occludin as a likelycandidate for a tumor suppressor gene.

In cancerous tissue, the expression of occludin decreases with disease progression.Thus moderately and poorly differentiated gastric carcinomas gradually reduce theiroccludin and its corresponding mRNA when compared to well differentiated ones(Kimura et al., 1997; Yin et al., 2002), and occludin is lost in unpolarized prostatecancer cells of Gleason grades 4 and 5, but prevails in less dedifferentiated cells ofGleason grades 1–3 (Busch et al., 2002). In a similar fashion, occludin is fullyexpressed in endometrioid carcinoma grade 1, and disappears in grades 2 and 3(Tobioka et al., 2004a).

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The study of JAM proteins in cancer development is starting to be unraveled.Finding that JAM-C is constitutively expressed by endothelial cells and localizes incell–cell contacts upon VEGF stimulation (Aurrand-Lions et al., 2001; Aurrand-Lions et al., 2000; Lamagna et al., 2005), triggered the study of JAM-C mediatedinteractions between tumor and endothelial cells, and its role in the formation of newblood vessels. Cells of the lung adenocarcinoma line NCI-H522 that express JAM-C,adhere to HUVEC endothelial cells in a process that can be blocked by the presenceof soluble JAM-C, or the isolated amino terminal Ig domain of JAM-C, but that isnot affected by the presence of JAM-A or JAM-B. As this effect is not observed inlung adenocarcinoma cells lacking JAM-C (NCI-H322M), it has been concludedthat the homophilic binding of JAM-C mediates tumor cell–endothelial cellinteractions (Santoso et al., 2005). Another study revealed that monoclonals againstJAM-C inhibit the growth of lung pulmonary tumor grafts, and infiltration ofmacrophages into tumors, even when the carcinoma cells do not express JAM-C.This effect is due to a reduction in tumor vasculature. Furthermore, monoclonalsagainst JAM-C decrease angiogenesis in the model of hypoxia-induced retinalneovascularization in vivo and vessel outgrowth from aortic rings in vitro (Lamagnaet al., 2005). These results define JAM-C as a valuable tool for cancer therapy.

With regards to the adaptor proteins of the TJ, several studies have reported thattheir reduced expression correlates with tumor dedifferentiation and cancerprogression (Table 3). However, ZO-1, which is generally considered as a tumorsuppressor protein, has unexpectedly been found over-expressed in melanoma(Smalley et al., 2005), gastric adenocarcinomas (Resnick et al., 2005), primaryand metastatic pancreatic cancer (Kleeff et al., 2001) and squamous cell carcinoma(Morita et al., 2004). During oncogenesis melanocyte cells loose E-cadherinand increase N-cadherin expression. This cadherin switch enables melanomacells to associate with the surrounding dermal fibroblasts. In melanoma cells,ZO-1 expression increases and concentrates at N-cadherin-based AJs betweenmelanoma cells and fibroblasts. Down-regulation of ZO-1 through RNA inter-ference leads melanoma cells to become less dentritic and more rounded, andgenerates a less adherent and invasive phenotype. These results indicate that ZO-1can exert a pro-oncogenic function if N-cadherin replaces E-cadherin expression(Smalley et al., 2005).

The transfection of ZO-1 deletion constructs that lack the SH3–GUK region,induces in epithelial cells a dramatic EMT characterized by repression of epithelialmarkers, a restricted differentiation, a dramatic change in cell shape (Ryeom et al.,2000) and a significantly induced tumorigenicity when injected into nude mice(Reichert et al., 2000). These results thus suggest that ZO-1 once delocalized from themembrane, might be involved in signaling pathways that promote cell migration andinvasion. In this respect it has been observed that in invasive breast tumor cell lines,the expression of membrane type 1 matrix metalloproteinase (MT1-MMP) correlateswith a cytoplasmic localization of ZO-1, while non-invasive cell lines displaymembrane ZO-1 staining and do not express MT1-MMP. ZO-1 silencing by RNAinterference down-regulates the expression of this metalloproteinase and decreasesthe ability of breast tumor cells to invade. Moreover, transfecting the amino terminal

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of ZO-1 activates the b-catenin/TCF/LEF pathway, a human MT1-MMP promoterand increases invasiveness (Polette et al., 2005).

In pancreatic cells, northern blot analysis revealed faint expression of ZO-1mRNA in the normal pancreas, moderate expression in chronic pancreatitis samplesand moderate to high expression in all cancer samples. However, it is important tonote that while in normal pancreas and chronic pancreatitis, ZO-1 was localized atthe apicolateral boundaries of epithelial cells in pancreatic ducts, in lymph nodemetastasis ZO-1 exhibits a more random pattern of distribution on the cell surface(Kleeff et al., 2001). A somewhat similar situation is encountered in thyroidcarcinomas where ZO-1 is weakly expressed in non-neoplastic tissues while anincreased cytoplasmic, apical and even nuclear ZO-1 staining is detected in papillarycarcinomas (Fluge et al., 2001).

Taken together, these results indicate that in certain cancers, the over-expressionof ZO-1 is accompanied by a non-membranous localization of the protein that maytrigger signaling pathways involved in cell transformation.

9. Transcription factors and epigenetic changes that regulate TJ protein

expression

Snail is a transcription factor of the zinc-finger type. Its consensus binding sitecontains a motif of six bases CAGGTG, called E-box. This motif is identical to thecore binding site of basic helix-loop-helix (bHLH) transcription factors, whichindicates that snail might compete with them for the same sequence. Upon bindingto the E box, snail acts as a transcriptional repressor (Nieto, 2002).

In epithelial cells, the over-expression of snail induces EMT with concomitantrepression of the expression of E-cadherin (Cano et al., 2000), occludin, and claudins3, 4 and 7 not only at the protein but also at the mRNA level. Snail binds directly tothe E-boxes of the promoters of E-cadherin (Cano et al., 2000), occludin andclaudins 3, 4, and 7 genes (Ikenouchi et al., 2003). In the case of claudin-1 theinhibition exerted by snail is only at the protein level (Ohkubo and Ozawa, 2004).Snail also induces down-regulation of ZO-1 together with an increased appearanceof ZO-1 a� isoform. The expression of the latter is abundant in tissues with low ornull TER like endothelia (Balda and Anderson, 1993; Underwood et al., 1999) andpodocytes (Kurihara et al., 1992) respectively, mouse blastomeres that lack TJs(Sheth et al., 1997), as well as in structurally dynamic junctions like those ofthe Sertoli cell (Balda and Anderson, 1993). The inducible expression of snaildoes not alter TJ organization observed by freeze–fracture, but increases paracellularion permeability and decreases the expression of occludin and claudin-2, togetherwith an almost total suppression of the expression of claudins 4 and 7 (Carrozzinoet al., 2005).

Other transcription factors have been reported to regulate the expression of TJproteins; however, their impact on cellular transformation has not yet been explored.For example claudin-2 promoter is activated in intestinal cells by the cooperativeaction of Cdx2, an intestine specific homeobox proteins, the hepatocyte nuclear

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factor (HNF-1) and GATA-4. Cdx1 and Cdx2 are transcription factors importantfor the early differentiation of intestinal epithelial cells (Soubeyran et al., 1999; Suhand Traber, 1996). In the human colon cancer cell line Caco-2, Cdx2 exerts a morepotent activation of claudin-2 transcription, than Cdx1, and HNF-1a enhancesCdx-2 induced claudin-2 promoter activity (Sakaguchi et al., 2002). In Caco-2 cells,the expression of claudin-2 declines with increasing time in culture, however thiseffect can be reversed by forced expression of GATA-4 (Escaffit et al., 2005).

Claudin-18 has two isoforms generated by alternative splicing that exhibit lungand stomach specific expression. The expression of the former is regulated by theT/EBP/NKX2.1 homeodomain transcription factor also known as TTf-1 (Niimiet al., 2001). This factor is expressed in lung, thyroid and parts of the brain (Kimuraet al., 1996). In the lung it is expressed in all epithelial cells during epithelialmorphogenesis, but becomes progressively restricted to alveolar type II and Claracells towards the end of gestation and in postnatal days (Yuan et al., 2000).

In ovarian cancer cells, claudin-4 expression is up-regulated. Two Sp1 sitescontained in the promoter appear to be critical for its expression. However, claudin-4 promoter is further controlled by low DNA methylation and high histone H3acetylation. The reverse situation is detected in cells that do not express claudin-4(Honda et al., 2006). Epigenetic regulation of other TJ proteins has further beenreported. For example, in several cancerous cell lines occludin loss is due tohypermethylation of CpG islands on the promoter region (Osanai et al., 2006) and inbreast cancer cells no genetic alterations in claudin-1 gene have been detected whilethe methylation of CpG islands on claudin-1 promoter explains its loss of expression(Kramer et al., 2000). In epithelial and fibroblastic cells, the histone deacetylaseinhibitor sodium butyrate significantly up-regulates the protein levels of cingulin,ZO-1 and ZO-2 (Bordin et al., 2004). This result is particularly interesting sincesodium butyrate and related compounds have been shown to inhibit cellproliferation and to induce cellular differentiation and apoptosis in several celltypes including colon (Barnard and Warwick, 1993; Litvak et al., 1998), breast(Graham and Buick, 1988) and prostate cancer cells (Maier et al., 2000).

Taken together these results indicate that the expression of particular TJ proteinsin a specific organ is regulated by epigenetic and transcriptional mechanisms that insome cases involve homeodomain proteins. Understanding these mechanisms maylead to novel strategies for cancer therapy and for a better understanding of thebiology of the disease.

10. Effect of cytokines and growth factors on the expression of TJ

proteins

TJ dysfunction has been described in inflammatory processes that end up incancerous transformation. For example, Crohn’s disease and ulcerative colitisare inflammatory illnesses involving the gastrointestinal tract in which anabnormal paracellular permeability defect appears to precede the development ofboth syndromes (Schulzke and Fromm, 2001). Mounting evidence has further

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demonstrated the central role played by cytokines and growth factors in promotingTJ disruption and epithelial inflammation.

In epithelial cells, the TJ barrier is down-regulated by cytokines and growthfactors associated to inflammation such as IL-1, IL-4, IL-13, TGFa, IGF-I and II,IFNg, TNF-a and HGF, while EGF enhances the barrier function of epithelialmonolayers. The cytokine TGF-b-1 instead generates opposite effects on theepithelial barrier function depending on the target tissue.

Here we will provide a brief description of the effects of some cytokines andgrowth factors on the TJ of epithelial cells.

10.1. Interferon-c (IFNc)

This 20–25 kDa protein produced by activated T-cells and natural killer cells,induces displacement of occludin, ZO-1 and ZO-2 away from the TJ region,decreases the total cellular concentration of ZO-1 and decreases the detergentinsoluble pools of ZO-1 and ZO-2, and disrupts the apical actin ring in polarizedT-84 intestinal epithelial monolayers (Youakim and Ahdieh, 1999). TNFa actssynergistically with IFNg to reduce the epithelial barrier function by upregulatingthe IFNg receptor (Fish et al., 1999). In MDCK cells exposure to IFNg/TNFa resultsin a paradoxical elevation of TER coupled to an increase in paracellularpermeability, increased expression of claudin-1 and occludin, diminished expressionof claudin-2 and an increased staining of the perijunctional actin ring (Patrick et al.,2006). In cultured Grave’s thyroid follicles, IFNg decreases TER, diminishesclaudin-1 expression and induces profound cell shape changes and multilayeredcellular organization (Tedelind et al., 2003).

10.2. Hepatocyte growth factor (HGF)

This disulfide link heterodimeric protein produced by mesenchymal cells decreasesTER in T84 cells and promotes cells migration and wound closure in injuredmonolayers (Nusrat et al., 1994). This factor exerts in different epithelia a broadspectrum of changes in intercellular junctions that range from no change in ZO-1distribution to a redistribution of ZO-1, occludin, claudin-1, and b-catenin from themembrane to the cytosol and an altered phosphorylation pattern of E-cadherin (Jin etal., 2002; Pasdar et al., 1997). MAP kinase and PI3-kinase activation is required forcell–cell dissociation and cell movement induced by HGF (Potempa and Ridley, 1998).

10.3. Tumor necrosis factor-a (TNFa)

This cytokine produced by mononuclear cells decreases TER in a reversible ornon-reversible fashion depending on the target epithelium. Thus, in the porcine renalepithelial cell line LLC-PK1, TNF-a induces a decrease in TER followed by a sharpincrease in TER that exceeds control values (Mullin and Snock, 1990). In contrast, inthe intestinal lines HT29 and Caco-2, TNFa decreases TER in a non-reversiblefashion. This effect appears to be mediated by NF-kB activation (Ma et al., 2004). In

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freeze–fracture EM, TJ complexity in HT29 cells is decreased by TNFa, as indicatedby a decrease in the number of strands (Schmitz et al., 1999). TNF-a inducesapoptosis in some cell types (Peralta et al., 1996) but not in others (Ma et al., 2004)and promotes the formation of intracytoplasmic aggregates of F-actin (Tabibzadehet al., 1995), membrane ruffles, filopodia and actin stress fibers (Wojciak-Stothardet al., 1998). Tyrosine kinase, protein kinase A (Schmitz et al., 1999), Rho andmyosin light chain kinase (Wojciak-Stothard et al., 1998) play a role in the processtriggered by TNF-a.

10.4. Epithelial growth factor (EGF)

This 53 amino acid polypeptide regulates diverse cell responses including mitosis,apoptosis, enhanced cell motility and differentiation of a variety of cell types. Thereceptor for EGF and related ligands EGFR, HER1 and ErbB1, are prototypicalmembers of the superfamily of receptors with intrinsic tyrosine kinase activity.EGFR is over-expressed in a wide spectrum of epithelial cancers, and its expressioncorrelates with a metastasic phenotype (Bonner et al., 2002; Mukohara et al., 2003;Shimizu et al., 2001). In cancerous cells, cellular dissociation promotes the invasionof transformed cells. In pancreatic cancer cells, cellular dissociation triggered bydissociation factor DF, induces the expression of phosphorylated EGFR, MEK 1/2and ERK 1/2 and translocates ZO-1 from cell–cell junctions to the cytoplasm andnucleus (Takai et al., 2005; Tan et al., 2004, 2005). A correlation between ZO-1translocation and EGFR activation appears to be present, since the addition of theEGFR inhibitor AG1478 to cells treated with DF, induces the relocalization of ZO-1to sites of cell–cell contact and promotes cell aggregation.

A normal expression of EGFR is however essential for regulated cell proliferationand differentiation. In alveolar epithelial cells (Chen et al., 2005) and in MDCK cells(Singh and Harris, 2004), EGF increases TER. In the former cells, claudins 4 and 7are upregulated, while claudins 3 and 5 expression is inhibited. In MDCK cells EGFtreatment inhibits claudin-2 expression and augments that of claudins 1, 3 and 4. Incontrast no effect is detected in ZO-1, occludin, E-cadherin or b-catenin. In gastriccancer cells, exposure to EGF rapidly translocates ZO-1 and occludin from thecytosol to cell–cell contacts via a protein kinase C signaling pathway (Yoshida et al.,2005). Taken together these results indicate that EGF is capable of changing thepattern of expression of different TJ proteins as well as their localization.

10.5. Transforming growth factor-b1 (TGF-b1)

This 25kDa disulfide linked homodimer produced by platelets, lymphocytes,macrophages and endothelial cells generates converse effects on the epithelial barrierfunction in different tissues. Thus, in T84 intestinal epithelial cells TGF-b1 enhances thebarrier function (Planchon et al., 1999; Planchon et al., 1994) and in prostatic basalepithelial cells stimulates luminal differentiation, junction formation and ZO-1expression (Danielpour, 1999). In contrast in MDCK cells, TGF-b1 promotesEMT by Smad-dependent and -independent signaling (Medici et al., 2006). Apparently

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TGF-b1 activates the Ras-Raf-MEK-ERK-AP-1 cascade up-regulating as a conse-quence Snail expression, that as previously described inhibits the expression of claudins,occludin and E-cadherin. TGF-b1 activates PI3 kinase signaling leading to deactivationof GSK-3b, which is responsible for degrading b-catenin and snail through the ubiquitinprotesome pathway. Finally, the endocytosis of the TGF-b1 receptor complex inducesSmad signaling, which in turn promotes transcription of LEF-1. The formation ofb-catenin/LEF-1 complexes, then acts to transcribe genes that induce EMT.

11. Conclusions

One of the crucial changes detected upon cell transformation is the loss oralteration of intercellular cell–cell contacts. In this review, we have analyzed theimplication of TJ proteins in this process.

When TJs were initially observed by light microscopy in the late XIX century, theywere regarded as mere intercellular seals. Since then we have learned how theyconstitute charge and size selective paracellular channels. Evidence gained in recentyears has further indicated the importance of TJs in cell signaling. The role played byTJs in this process is complex and involves several strategies affecting geneexpression, cell proliferation and the state of cell differentiation. For example, thescaffold nature of TJ adaptor proteins coupled to their movement from the nucleus tothe plasma membrane and vice versa, allows them to concentrate at both locations,tumor suppressor proteins, nucleotide exchange factors and diverse nuclear proteinsincluding transcription factors and chromatin remodeling molecules.

Since the loss of cell–cell adhesion is a crucial step undertaken in EMT, strategiesdesigned to overcome the altered expression of TJ proteins detected in canceroustissues, could eventually lead to therapeutical treatments for handling and preventinghuman cancers. For this purpose it is of utmost importance to understand thebiological processes that regulate TJ protein expression, such as the regulation oftheir transcription, and the effect exerted by cytokines and growth factors.

The pattern of expression of TJ proteins in normal and cancerous human tissuesmight also be employed as a tool for the clinical prognosis of the disease.

Acknowledgments

This work was supported by Grant 45691-Q from the National Council forScience and Technology of Mexico (CONACYT), and by a Cinvestav Grant forprojects with the Health sector.

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role of tight junctions in cell proliferation and cancer

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