MOLECULAR STRUCTURE AND FUNCTION OF THE TIGHT JUNCTION

Cadherin-Mediated Regulation of TightJunctions in Stratifying Epithelia

Christian Michels,a,b Saeed Yadranji Aghdam,a,b,c

and Carien M. Niessena,b,d

aDepartment of DermatologybCenter for Molecular Medicine Cologne

cInternational Graduate School for Genetics and Functional GenomicsdCologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases,

University of Cologne, Cologne, Germany

In recent years several seminal breakthroughs have revealed that tight junctions notonly regulate barrier properties of simple epithelial cells but also play crucial functionsin the regulation of the largest barrier of the organism, the stratifying epidermis of theskin. Here we will address the importance of tight junctions for the skin barrier functionand discuss data from our studies and from others that indicate how cadherins, polarity,and other pathways may regulate these junctions in stratifying epithelia.

Key words: tight junctions; skin barrier; cadherin; polarity

Introduction

The stratifying epidermis of the skin sepa-rates the organism from its environment andserves as its first-line structural and functionaldefense against dehydration, chemical sub-stances, and microorganisms. An important as-pect in the formation and maintenance of thebarrier is the tight intercellular adhesion be-tween keratinocytes, which is mediated by in-tercellular junctions, such as adherens junctionsand desmosomes. This results in the forma-tion of strong cohesive cell sheets that, througha complex differentiation process, ultimatelyform the cornified layer, the outermost protec-tive layer of the skin. For keratinizing epithe-lia, it was originally thought that the secretionand deposition of this cross-linked protein–lipidbarrier obviated the need for a tight junction

Address for correspondence: Carien M. Niessen, Department ofDermatology, Center for Molecular Medicine Cologne, Universityof Cologne, Joseph Stelzmannstrasse 9, 50931 Cologne, Germany.carien.niessen@uni-koeln.de

barrier in such tissues. However, ultrastructuralanalysis combined with electron dense tracerstudies revealed restricted diffusion to the up-permost viable layer, the stratum granulosum,suggesting the existence of a tight junction bar-rier.1,2 Although tight junction strands were ob-served by freeze fracture, they appeared incom-plete, and thus barrier function in the stratumgranulosum was mainly attributed to the in-tercellular deposition of lamellar bodies.3 Thisview remained unchallenged despite the con-tinued localization/identification of tight junc-tion components in stratifying epithelia.4,5 Aseminal breakthrough came with the observa-tion that, in the absence of claudin-1, mice dieof massive epidermal water loss due to impairedbarrier function of the stratum granulosum.6

This provided the first functional evidence thatepidermal barrier function required a tightjunction component. Subsequently, ultrastruc-tural analysis revealed the presence of a densenetwork of continuous strands resembling tightjunctions in the stratum granulosum of hu-man epidermis,7 thus indicating the formationof a seal. The importance of tight junctions

Molecular Structure and Function of the Tight Junction: Ann. N.Y. Acad. Sci. 1165: 163–168 (2009).doi: 10.1111/j.1749-6632.2009.04443.x c© 2009 New York Academy of Sciences.

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in human skin barrier function was recentlyunderscored by the identification of claudin-1 mutations in an ichthyosis syndrome addi-tionally characterized by sclerosing cholangitisand other features.8 It is now widely acceptedthat tight junctions form a critical part of thebarrier in the upper viable layer of stratifyingepithelia.

Classical Cadherinsand the Epidermis

The cadherin adhesion family of Ca2+

-dependent intercellular adhesion molecules isa key determinant of morphogenesis and tissuearchitecture.31 Cadherins do not only mediateintercellular adhesion but also regulate a widespectrum of other cellular functions, such aspolarity, cytoskeleton, signaling, and growth.9

Both the desmosomal cadherins and the classi-cal cadherins contribute to intercellular adhe-sion of the epidermis. Unlike desmosomal cad-herins, which link to the intermediate filamentsystem, classical cadherins regulate the actin cy-toskeleton via linker molecules called catenins.P120ctn regulates cadherin cell surface stabilityand potentially connects adhesion to regulationof Rho GTpases, whereas β-catenin links thecadherin to the actin regulator α-catenin.10

Two types of classical cadherins are ex-pressed in the epidermis: P-cadherin, expressedin the basal layer mainly around and in hair fol-licles, and E-cadherin, found in all layers of theepidermis. Next to mediating cell–cell adhe-sion, cadherins can affect a wide range of cellu-lar functions that include activation of cell sig-naling pathways, regulation of the cytoskeleton,and control of cell polarity.9 Epidermal-specificdeletion of E-cadherin resulted in hair loss11,12

and disturbed barrier function from abnormaltight junction function13 (see below). It was re-cently reported that E-cadherin is a target ofautoantibodies in pemphigus, a skin blisteringdisease.14 Mutations in human P-cadherin areassociated with a hair disorder “hypotrichosiswith juvenile macular dystrophy,”15 and with

ectodermal dysplasia associated with extro-dactyly and macular dystrophy.16 In contrast,loss of P-cadherin in mice did not reveal anyobvious phenotypes,17 suggesting that E- andP-cadherin either serve partially differentialfunctions or have a different functional over-lap in human and mouse.

Epidermal inactivation in mice revealedoverlapping and specific functions for thecadherin-associated catenins. Loss of β-cateninin the epidermis confirmed its importance inthe Wnt signaling pathway and its role in hairfollicle morphogenesis and stem cell regulation.However, no obvious defects in intercellular ad-hesion and junction formation were observed,most likely because plakoglobin substituted forβ-catenin in the cadherin complex. Inactiva-tion of p120ctn in the epidermis resulted inreduced adherens junctions and skin inflam-mation associated with activation of nuclearfactor kappa β (NF-κβ).18 An almost completeloss of adherens junctions, reduced desmo-somes, and subsequent skin blistering were ob-served when α-catenin was deleted from theepidermis.19 Because both P-cadherin and E-cadherin bind to the catenins, loss of a sin-gle cadherin may be insufficient to pheno-copy deletion of one of the catenins. Thisis in agreement with studies in human ker-atinocytes showing that antibodies to bothE- and P-cadherin inhibit adherens junctionsand desmosomes and interfere with stratifi-cation. Studies by the Fuchs laboratory andour group using knockout/knockdown strate-gies confirmed that classical cadherins are cru-cial and cooperate in the regulation of junctionsand barrier function20 (Michels et al., submittedmanuscript).

E-Cadherin RegulatesEpidermal Tight Junctions

To examine the role of E-cadherin in strat-ifying epithelia, such as the epidermis, and toaddress if loss of E-cadherin contributes to thephenotypes observed in the absence of one of

Michels et al.: Tight Junctions in Skin 165

the catenins, we used Cre-Lox-P technologyto delete E-cadherin in all layers of the epi-dermis. Surprisingly, no blistering or any ob-vious defects in intercellular contacts were ob-served, as was perhaps to be expected for anadhesion molecule such as E-cadherin. Micewith epidermal loss of E-cadherin did have ared parchment paper-like appearance of theskin and died from enhanced epidermal wa-ter loss. The most obvious explanation forthe observed water loss was a disturbed stra-tum corneum barrier function. However, lu-cifer yellow penetration assays failed to detectbreaches in the “outside-in” stratum corneumbarrier. In addition, cornified envelopes hada regular shape and size, and toluidine bluedye penetration assays revealed no differencein the temporal development of the stratumcorneum barrier.13 These results resembled ob-servations for the claudin-1 knockout mice,which showed a normal outside-in barrierfunction but disturbed inside-out barrier func-tion.6 This prompted us to test the inside-out barrier function. Whereas control miceshowed restricted flow of dermally injected bi-otin, this dye diffused past the stratum granulo-sum in the E-cadherinepi−/−, indicating impair-ment of the tight junction inside-out barrier.This was confirmed by impedance measure-ments21 and coincided with alterations in local-ization of key tight junction components, suchas zonula occludens (ZO)-1, claudin-1, andclaudin-4.13

The Polarity ProteinAtypical-Protein Kinase C

Regulates Tight Junctions inStratifying Epithelia

In simple epithelia, a close relationship existsbetween the establishment of polarity and junc-tion formation. Moreover, several polarity com-plex proteins, such as the Par3/Par6/atypical-protein kinase C (aPKC) complex, localize totight junctions where their activity contributes

to tight junction barrier function.9,22 Interest-ingly, loss of E-cadherin in the epidermis al-tered the localization of aPKC and its up-stream regulator, the small GTPase Rac.13

E-cadherin might directly recruit aPKC to themembrane as we recently obtained evidence foran interaction between classical cadherins andaPKC.30

To test the functional involvement of if aPKCin epidermal tight junction barrier function,we followed barrier formation over time, us-ing transepithelial resistance (TER) measure-ments. Primary keratinocytes are switched tohigh calcium medium (1.8 mmol/L Ca2+) toinduce intercellular junction and thus barrierformation. Blocking aPKC function, either bypharmacological inhibition or by overexpres-sion of a dominant negative aPKCζ mutant,inhibited the formation of the barrier, whereasoverexpression of a wild-type aPKCζ enhancedand accelerated barrier formation, indicatingthat aPKC is indeed a regulator for epidermaltight junctions, as was also found by others.23

Interestingly, unlike what is observed in sim-ple epithelial cell cultures, inhibition of aPKCfunction did not result in altered localizationof claudin-1, occludin, and ZO-1.24 Thus, un-like most systems, our system allows us to sep-arate initial tight junction formation from itsfunction and suggests that aPKCs regulate alate step in the biogenesis of tight junctions.In mammalians two isoforms of aPKC exist,aPKCι/λ and aPKCζ, which are encoded bydifferent genes but share 76% sequence iden-tity. Although their overlapping and separatefunctions are less clear, in vitro studies suggestthat both isoforms can regulate tight junc-tions. Both isoforms are expressed in the epi-dermis, albeit with apparently different local-ization.24 Whereas aPKCζ is confined to thebasal layer, aPKCι/λ appears more promi-nently enriched at tight junctional contacts,suggesting that aPKCι/λ may regulate skinbarrier function. If aPKC and Rac are indeedintermediates in E-cadherin mediated regula-tion of tight junction function remains to beanswered.

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Figure 1. Tight junctions (TJ) in simple and stratifying epithelia.

Phosphoinositide 3-KinaseActivation Is not Important for

Epidermal Tight Junctions

E-cadherin associates with and activatesphosphoinositide 3 (PI3)-kinase, which itselfcan activate Rac. Since epidermal loss ofE-cadherin was associated with a loss of Racfrom cell–cell contacts in vivo13 and an im-paired activation in vitro (unpublished data),E-cadherin may thus regulate Rac activityin the epidermis via PI3-kinase. To test thepossible influence of PI3-kinase, primary ker-atinocytes were allowed to form intercellularjunctions and barrier function, using the Ca2+-switch/TER assay in the presence of differentconcentrations of the PI3-kinase wortmannin.Surprisingly, no difference in resistance was ob-served in the presence of even high wortmanninconcentration (Fig. 2), suggesting that, at leastin stratifying epithelia, PI3-kinase activity doesnot regulate the formation of a functional tightjunctional barrier in the stratum granulosum ofthe epidermis.

Cooperation in Skin BarrierFunction by Tight Junctions and

Stratum Corneum

The epidermis is not a classically polar-ized epithelium like intestine where basolateraland apical membrane domains are separatedby tight junctions. Instead the epidermis es-

Figure 2. In vitro barrier formation in ker-atinocytes is not disturbed by activates phospho-inositide 3 (PI3)-kinase inhibition. Primary mousekeratinocytes were plated on collagen-coated filters(0.4 μm pore size) in low-Ca2+ medium (50 μmol/L).Junction formation was induced by switching Ca2+

concentration to 1.8 mmol/L in the absence or pres-ence of wortmannin at the indicated concentrations.Transepithelial resistance (TER) was measured at theindicated time points, using the Millipore Millicell-ERSdevice (Millipore, Billerica, MA, USA).

tablishes a form of junctional polarity alongthe apical to basal axis of the tissue, with thestratum granulosum forming the viable api-cal boundary (Fig. 1). Because formation ofthe stratum corneum depends on the fusion oflamellar bodies and keratohyalin granules withplasma membranes at the transition betweenstratum granulosum and stratum corneum lay-ers, it is tempting to speculate that the spe-cific occurrence of tight junctions in the stra-tum granulosum relates to the “fence” functionof tight junctions and thereby may regulate thetargeted secretion of “apical” protein and lipidvesicles directly toward the stratum corneum.This would imply that changes in tight

Michels et al.: Tight Junctions in Skin 167

junctions affect the stratum corneum barrier.Forced expression of claudin-6 in the upper lay-ers of the epidermis results in defects not only inthe tight junction but also stratum corneum.25

Tight junctions and stratum corneum may co-operate in barrier function at other levels be-cause inactivation of the membrane-anchoredserine protease (CAP)1/Prss8 in the epidermisalso disturbs both barriers.26 Although the un-derlying mechanisms are unknown, they mayinvolve the coordinated regulation of both bar-riers by signal molecules, such as I-κ-β-kinase 1(IKK1). Inactivation of IKK1 in the epider-mis severely impairs barrier function associatedwith improper epidermal lipid processing andchanges in tight junction component expres-sion. Epidermal IKK1 function is independentof NF-κβ signaling but regulates the expressionof retinoic acid receptor target genes, manyof which are involved in epidermal barrierfunction.27

Why Are Functional Tight Junctionsonly Found in the Granular Layer

of the Epidermis?

The mechanisms that restrict the assemblyof a tight junction barrier to the uppermostlayers of stratifying epithelia are unclear. Be-cause many tight junction components are ex-pressed throughout the epidermal layers, it ispossible that a local signal in the granular layertriggers tight junction formation. We speculatethat the dead cell/keratin layer may initiate thissignal, similar to other simple epithelia that se-crete and are polarized by an apical matrix(e.g., follicular epithelia in flies that secrete anapical cuticle). Alternatively, tight junction for-mation in the lower layers may be actively in-hibited by the presence of an overlying viablecell layer. Either mechanism suggests that therestriction of tight junctions to the apical-mostlayer in stratifying epidermis or apical regionof simple epithelia may be conserved. Indeed,our data discussed here and other data supportthis argument: E-cadherin is required for tight

junction formation in both simple and strati-fying epithelia. Blocking E-cadherin in vitro in-hibits tight junctions in simple epithelia,28 asdoes genetic loss of epidermal E-cadherin. Sim-ilarly, blocking αPKC inhibition interferes withtight junctions in both simple and stratifyingepithelia.23,24,29 Thus, junctional and polarityproteins required in simple epithelia are alsoturning out to be critical for epidermal bar-rier function, suggesting these processes maybe mechanistically related.

Concluding Remarks

Research in the last decade has provided ex-citing new insights into how tight junctions con-tribute to skin barrier function. Because of itseasy accessibility, the visible barrier, and the op-portunity to isolate primary cells, the skin as amodel system may also provide a unique exper-imental model system that may provide us withinsights into how barrier function regulates ep-ithelial tissue homeostasis.

Conflicts of Interest

The authors declare no conflicts of interest.

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