1. MOLECULAR STRUCTURE AND FUNCTION OF THE TIGHT JUNCTION
Cadherin-Mediated Regulation of Tight
Junctions in Stratifying Epithelia
Christian Michels,a,b
Saeed Yadranji Aghdam,a,b,c
and Carien M. Niessena,b,d
a
Department of Dermatology
b
Center for Molecular Medicine Cologne
c
International Graduate School for Genetics and Functional Genomics
d
Cologne 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 not
only regulate barrier properties of simple epithelial cells but also play crucial functions
in the regulation of the largest barrier of the organism, the stratifying epidermis of the
skin. Here we will address the importance of tight junctions for the skin barrier function
and 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 and
serves as its first-line structural and functional
defense against dehydration, chemical sub-
stances, and microorganisms. An important as-
pect in the formation and maintenance of the
barrier is the tight intercellular adhesion be-
tween keratinocytes, which is mediated by in-
tercellular junctions, such as adherens junctions
and desmosomes. This results in the forma-
tion of strong cohesive cell sheets that, through
a complex differentiation process, ultimately
form the cornified layer, the outermost protec-
tive layer of the skin. For keratinizing epithe-
lia, it was originally thought that the secretion
and deposition of this cross-linked protein–lipid
barrier obviated the need for a tight junction
Address for correspondence: Carien M. Niessen, Department of
Dermatology, Center for Molecular Medicine Cologne, University
of Cologne, Joseph Stelzmannstrasse 9, 50931 Cologne, Germany.
carien.niessen@uni-koeln.de
barrier in such tissues. However, ultrastructural
analysis combined with electron dense tracer
studies 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 stratum
granulosum was mainly attributed to the in-
tercellular deposition of lamellar bodies.3
This
view remained unchallenged despite the con-
tinued localization/identification of tight junc-
tion components in stratifying epithelia.4,5
A
seminal breakthrough came with the observa-
tion that, in the absence of claudin-1, mice die
of massive epidermal water loss due to impaired
barrier function of the stratum granulosum.6
This provided the first functional evidence that
epidermal barrier function required a tight
junction component. Subsequently, ultrastruc-
tural analysis revealed the presence of a dense
network of continuous strands resembling tight
junctions in the stratum granulosum of hu-
man epidermis,7
thus indicating the formation
of 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.
163

2. 164 Annals of the New York Academy of Sciences
in human skin barrier function was recently
underscored by the identification of claudin-
1 mutations in an ichthyosis syndrome addi-
tionally characterized by sclerosing cholangitis
and other features.8
It is now widely accepted
that tight junctions form a critical part of the
barrier in the upper viable layer of stratifying
epithelia.
Classical Cadherins
and the Epidermis
The cadherin adhesion family of Ca2+
-dependent intercellular adhesion molecules is
a key determinant of morphogenesis and tissue
architecture.31
Cadherins do not only mediate
intercellular adhesion but also regulate a wide
spectrum of other cellular functions, such as
polarity, 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 filament
system, classical cadherins regulate the actin cy-
toskeleton via linker molecules called catenins.
P120ctn regulates cadherin cell surface stability
and potentially connects adhesion to regulation
of Rho GTpases, whereas β-catenin links the
cadherin to the actin regulator α-catenin.10
Two types of classical cadherins are ex-
pressed in the epidermis: P-cadherin, expressed
in the basal layer mainly around and in hair fol-
licles, and E-cadherin, found in all layers of the
epidermis. 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-specific
deletion of E-cadherin resulted in hair loss11,12
and disturbed barrier function from abnormal
tight junction function13
(see below). It was re-
cently reported that E-cadherin is a target of
autoantibodies in pemphigus, a skin blistering
disease.14
Mutations in human P-cadherin are
associated with a hair disorder “hypotrichosis
with 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 any
obvious phenotypes,17
suggesting that E- and
P-cadherin either serve partially differential
functions or have a different functional over-
lap in human and mouse.
Epidermal inactivation in mice revealed
overlapping and specific functions for the
cadherin-associated catenins. Loss of β-catenin
in the epidermis confirmed its importance in
the Wnt signaling pathway and its role in hair
follicle morphogenesis and stem cell regulation.
However, no obvious defects in intercellular ad-
hesion and junction formation were observed,
most likely because plakoglobin substituted for
β-catenin in the cadherin complex. Inactiva-
tion of p120ctn
in the epidermis resulted in
reduced adherens junctions and skin inflam-
mation associated with activation of nuclear
factor kappa β (NF-κβ).18
An almost complete
loss of adherens junctions, reduced desmo-
somes, and subsequent skin blistering were ob-
served when α-catenin was deleted from the
epidermis.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. This
is in agreement with studies in human ker-
atinocytes showing that antibodies to both
E- and P-cadherin inhibit adherens junctions
and desmosomes and interfere with stratifi-
cation. Studies by the Fuchs laboratory and
our group using knockout/knockdown strate-
gies confirmed that classical cadherins are cru-
cial and cooperate in the regulation of junctions
and barrier function20
(Michels et al., submitted
manuscript).
E-Cadherin Regulates
Epidermal Tight Junctions
To examine the role of E-cadherin in strat-
ifying epithelia, such as the epidermis, and to
address if loss of E-cadherin contributes to the
phenotypes observed in the absence of one of

3. Michels et al.: Tight Junctions in Skin 165
the catenins, we used Cre-Lox-P technology
to 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 an
adhesion molecule such as E-cadherin. Mice
with epidermal loss of E-cadherin did have a
red parchment paper-like appearance of the
skin and died from enhanced epidermal wa-
ter loss. The most obvious explanation for
the observed water loss was a disturbed stra-
tum corneum barrier function. However, lu-
cifer yellow penetration assays failed to detect
breaches in the “outside-in” stratum corneum
barrier. In addition, cornified envelopes had
a regular shape and size, and toluidine blue
dye penetration assays revealed no difference
in the temporal development of the stratum
corneum barrier.13
These results resembled ob-
servations for the claudin-1 knockout mice,
which showed a normal outside-in barrier
function but disturbed inside-out barrier func-
tion.6
This prompted us to test the inside-
out barrier function. Whereas control mice
showed 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, such
as zonula occludens (ZO)-1, claudin-1, and
claudin-4.13
The Polarity Protein
Atypical-Protein Kinase C
Regulates Tight Junctions in
Stratifying Epithelia
In simple epithelia, a close relationship exists
between 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 to
tight 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 the
membrane as we recently obtained evidence for
an interaction between classical cadherins and
aPKC.30
To test the functional involvement of if aPKC
in epidermal tight junction barrier function,
we followed barrier formation over time, us-
ing transepithelial resistance (TER) measure-
ments. Primary keratinocytes are switched to
high calcium medium (1.8 mmol/L Ca2+
) to
induce intercellular junction and thus barrier
formation. Blocking aPKC function, either by
pharmacological inhibition or by overexpres-
sion of a dominant negative aPKCζ mutant,
inhibited the formation of the barrier, whereas
overexpression of a wild-type aPKCζ enhanced
and accelerated barrier formation, indicating
that aPKC is indeed a regulator for epidermal
tight junctions, as was also found by others.23
Interestingly, unlike what is observed in sim-
ple epithelial cell cultures, inhibition of aPKC
function did not result in altered localization
of claudin-1, occludin, and ZO-1.24
Thus, un-
like most systems, our system allows us to sep-
arate initial tight junction formation from its
function and suggests that aPKCs regulate a
late step in the biogenesis of tight junctions.
In mammalians two isoforms of aPKC exist,
aPKCι/λ and aPKCζ, which are encoded by
different genes but share 76% sequence iden-
tity. Although their overlapping and separate
functions are less clear, in vitro studies suggest
that 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 the
basal layer, aPKCι/λ appears more promi-
nently enriched at tight junctional contacts,
suggesting that aPKCι/λ may regulate skin
barrier function. If aPKC and Rac are indeed
intermediates in E-cadherin mediated regula-
tion of tight junction function remains to be
answered.

4. 166 Annals of the New York Academy of Sciences
Figure 1. Tight junctions (TJ) in simple and stratifying epithelia.
Phosphoinositide 3-Kinase
Activation Is not Important for
Epidermal Tight Junctions
E-cadherin associates with and activates
phosphoinositide 3 (PI3)-kinase, which itself
can activate Rac. Since epidermal loss of
E-cadherin was associated with a loss of Rac
from cell–cell contacts in vivo13
and an im-
paired activation in vitro (unpublished data),
E-cadherin may thus regulate Rac activity
in the epidermis via PI3-kinase. To test the
possible influence of PI3-kinase, primary ker-
atinocytes were allowed to form intercellular
junctions and barrier function, using the Ca2+
-
switch/TER assay in the presence of different
concentrations of the PI3-kinase wortmannin.
Surprisingly, no difference in resistance was ob-
served in the presence of even high wortmannin
concentration (Fig. 2), suggesting that, at least
in stratifying epithelia, PI3-kinase activity does
not regulate the formation of a functional tight
junctional barrier in the stratum granulosum of
the epidermis.
Cooperation in Skin Barrier
Function by Tight Junctions and
Stratum Corneum
The epidermis is not a classically polar-
ized epithelium like intestine where basolateral
and apical membrane domains are separated
by 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 mouse
keratinocytes 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 the
indicated time points, using the Millipore Millicell-ERS
device (Millipore, Billerica, MA, USA).
tablishes a form of junctional polarity along
the apical to basal axis of the tissue, with the
stratum granulosum forming the viable api-
cal boundary (Fig. 1). Because formation of
the stratum corneum depends on the fusion of
lamellar bodies and keratohyalin granules with
plasma membranes at the transition between
stratum 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” function
of tight junctions and thereby may regulate the
targeted secretion of “apical” protein and lipid
vesicles directly toward the stratum corneum.
This would imply that changes in tight

5. 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 in
the 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-anchored
serine protease (CAP)1/Prss8 in the epidermis
also disturbs both barriers.26
Although the un-
derlying mechanisms are unknown, they may
involve 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 associated
with improper epidermal lipid processing and
changes in tight junction component expres-
sion. Epidermal IKK1 function is independent
of NF-κβ signaling but regulates the expression
of retinoic acid receptor target genes, many
of which are involved in epidermal barrier
function.27
Why Are Functional Tight Junctions
only Found in the Granular Layer
of the Epidermis?
The mechanisms that restrict the assembly
of a tight junction barrier to the uppermost
layers of stratifying epithelia are unclear. Be-
cause many tight junction components are ex-
pressed throughout the epidermal layers, it is
possible that a local signal in the granular layer
triggers tight junction formation. We speculate
that the dead cell/keratin layer may initiate this
signal, similar to other simple epithelia that se-
crete and are polarized by an apical matrix
(e.g., follicular epithelia in flies that secrete an
apical cuticle). Alternatively, tight junction for-
mation in the lower layers may be actively in-
hibited by the presence of an overlying viable
cell layer. Either mechanism suggests that the
restriction of tight junctions to the apical-most
layer in stratifying epidermis or apical region
of simple epithelia may be conserved. Indeed,
our data discussed here and other data support
this 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
as
does genetic loss of epidermal E-cadherin. Sim-
ilarly, blocking αPKC inhibition interferes with
tight junctions in both simple and stratifying
epithelia.23,24,29
Thus, junctional and polarity
proteins required in simple epithelia are also
turning out to be critical for epidermal bar-
rier function, suggesting these processes may
be 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 its
easy accessibility, the visible barrier, and the op-
portunity to isolate primary cells, the skin as a
model system may also provide a unique exper-
imental model system that may provide us with
insights into how barrier function regulates ep-
ithelial tissue homeostasis.
Conflicts of Interest
The authors declare no conflicts of interest.
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