Adhesive interactions between cells are dynamic and regulated during tissue development
and homeostasis. Cadherins are major cell–cell adhesion molecules involved in the
development and maintenance of all solid tissues (Takeichi 1991; Gumbiner 1996). Therefore,
regulation of cadherin-mediated adhesion and the associated adherens junctions is
thought to underlie the dynamics of the adhesive interactions between cells. A major
form of regulation occurs at the level of cadherin gene expression. The level of cadherin
expression influences the strength of adhesion (Steinberg and Takeichi 1994), and
the type of cadherin expressed determines the specificity of cell interactions (Nose
et al. 1988) and properties of the interactions. However, there is accumulating evidence
that posttranscriptional regulation of cadherin adhesive activity is responsible for
many of the dynamic, rapid changes in cell interactions that underlie tissue morphogenesis
and homeostasis. The mechanisms underlying the regulation of cadherin adhesive activity
is the topic of this Mini-Review.
Types of Cadherin Regulation
Several different mechanisms have been proposed for cadherin regulation. However,
before discussing these in detail, it is worth first considering what exactly is meant
by cadherin regulation. Many different cellular processes can affect cell adhesion
and the state of adherens junctions, which are formed by the cadherins. During certain
morphogenetic processes, for example, the strength of cell adhesion is modulated rapidly
in response to growth factors or other signals without gross changes in the presence
of adhesive complexes or junctions at cell contacts (Fig. 1, top). This direct response
to cellular signals is analogous to the rapid regulation of integrin function, called
inside-out signaling, which occurs in leukocytes and platelets (Ginsberg et al. 1992).
On the other hand, dramatic changes in the assembly or disassembly of adherens junctions
also seem to occur, usually in association with major changes in cell state or differentiation,
such as the epithelial–mesenchymal or mesenchymal–epithelial transitions (Fig. 1,
middle). Such transitions involve major changes in cell differentiation, and are likely
to affect the state of assembly of cell contacts and adherens junctions directly and
indirectly in complex ways. In addition, the normal assembly and turnover of cadherin-based
junctions in cells under steady-state conditions is likely to be under regulatory
control (Fig. 1, bottom). The biogenesis of junctions from newly synthesized components
and their coordinated turnover are complex multistep processes that, like formation
of all subcellular organelles and compartments, are subject to cellular control mechanisms
(Adams et al. 1996; Le et al. 1999). The 5–10-h half-life for E-cadherin in confluent
epithelial cells (McCrea and Gumbiner 1991; Shore and Nelson 1991) make cell contacts
or junctions susceptible to rapid alteration or remodeling by a number of mechanisms,
including changes in gene expression. Clearly, these different cellular processes
affect cell adhesion and adherens junctions at different levels and, therefore, could
be regulated by different mechanisms. In tumor cells, in which cadherins are often
found to be dysfunctional, any of the above regulatory processes could be perturbed.
Cadherin regulation has been examined in numerous model systems. Regulation of cadherin
activity in real tissues or organisms has been well documented in a few cases. Compaction
of the early mouse embryo, leading to the formation of the trophectodermal epithelium,
is a striking example (Fleming and Johnson 1988). It results from the rapid activation
of E-cadherin–mediated adhesion in response to a cellular signal (Vestweber et al.
1987). In this case, preexisting E-cadherin at the cell surface is recruited to regions
of cell contact concomitant with activation of adhesion. A more subtle form of cadherin
regulation at the cell surface occurs during tissue elongation in the Xenopus embryo,
which contributes to the morphogenetic movements of gastrulation. This form of tissue
morphogenesis, called convergent extension, results from local cell rearrangements,
which requires that cells continually break and remake adhesive contacts (Gerhart
and Keller 1986). When Xenopus animal cap explants are stimulated to undergo convergent
extension–driven elongation by treatment with the mesoderm inducing factor activin,
the adhesive activity of the major cadherin, C-cadherin (or EP-cadherin), at the cell
surface is significantly reduced (Brieher and Gumbiner 1994). Restoration of high
adhesive activity with a C-cadherin activating mAb inhibits tissue elongation, demonstrating
that regulation is required for normal morphogenesis (Zhong et al. 1999).
Cell culture systems also have been used to study cadherin regulation. Culture models
with the most obvious relevance to regulation in vivo are the responses of epithelial
cell lines to growth factors. EGF and scatter factor/HGF induce decreased cell–cell
contact without apparent loss or disruption of the E-cadherin–catenin complex (Weidner
et al. 1990; Shibamoto et al. 1994). In simple cultures, these growth factors cause
cells to completely dissociate or scatter from each other, but their physiological
roles may be more pertinent to cell rearrangements and tissue morphogenesis. In three-dimensional
matrix-embedded cultures, epithelial cells undergo tubulogenesis in response to the
scatter factor/HGF (Montesano et al. 1991). Similar morphogenetic behaviors are observed
for endothelial cells in response to their growth factors. Cell culture models also
have been used to try to examine the effects of pharmacological inhibitors or expression
of wild-type or mutant signal transduction proteins on adhesion or cell junctions.
Such models can yield valuable information about potential signaling pathways relevant
to the expression and/or functions of cadherins or cell junctions, but in many cases
their physiological roles remain to be assessed.
Mechanisms of Cadherin Regulation
Cadherins form tight complexes with catenins, which are believed to link the cadherins
functionally to the actin cytoskeleton (Fig. 2 A) (Kemler 1993). Because they are
required for strong cell–cell adhesion in tissues, the catenins have often been investigated
as potential cytoplasmic targets for regulation. Changes in the composition of the
complex, phosphorylation of components in the complex, and alterations in the interaction
of the complex with the actin cytoskeleton have all been suggested to play a role
in regulation of adhesion.
Changes in the composition of the cadherin–catenin complex have been proposed to play
a role in some cases of cadherin regulation. For example, activation of the wnt signaling
pathway, which leads to increased levels of β-catenin (or plakoglobin), has been found
to promote the formation of the complex at the plasma membrane and enhance cadherin-mediated
adhesion in certain cell lines (Bradley et al. 1993; Hinck et al. 1994). However,
in many cell types, the levels of cadherin expression, rather than catenin levels,
seem to be rate limiting for complex formation and cell adhesion (Nagafuchi et al.
1991; Kowalczyk et al. 1994; Guger and Gumbiner 1995; Yap et al. 1998). Thus, although
the wnt pathway is known to work through modulation of β-catenin levels, its role
in the physiological or developmental regulation of adhesion remains uncertain.
Typically, when cadherin adhesion activity at the cell surface is acutely and rapidly
modulated in response to developmental signals or growth factors, analogous to integrin
inside-out signaling, no detectable alterations in the composition of the cadherin–catenin
complex have been apparent (Weidner et al. 1990; Brieher and Gumbiner 1994; Shibamoto
et al. 1994). Nonetheless, disruption of the complex has been observed to occur in
a few cases as a result of the perturbation of intracellular signaling pathways; for
example, by the expression of activated Cdc42 (see below) or tyrosine phosphatase
inhibitors (Ozawa and Kemler 1998a). The physiological roles of these perturbations
remain to be established; it is not yet known whether they mediate rapid cell-surface
regulation of adhesion, control the biogenesis or turnover of cell junctions, or regulate
events associated with major changes in cell states, such as the epithelial–mesenchymal
transition.
The interaction of α-catenin with the actin cytoskeleton may also provide an important
potential locus for regulation. α-Catenin interacts with a number of actin-binding
proteins, including α-actinin, vinculin, ZO-1, as well as with actin itself (Fig.
2 B; Knudsen et al. 1995; Rimm et al. 1995; Watabe-Uchida et al. 1998; Imamura et
al. 1999). The roles of these various interactions in cadherin function are only beginning
to be analyzed. Vinculin seems to be important for organizing E-cadherin into a zonular
adherens junction typical of epithelial cells, but may not be essential for basic
adhesive functions or adhesion in nonepithelial cells (Watabe-Uchida et al. 1998).
The ZO-1 binding region of α-catenin seems to influence the strength of cadherin-mediated
adhesion in nonepithelial cells, but ZO-1 binding does not seem to be critical for
E-cadherin function or adherens junctions in epithelial cells (Imamura et al. 1999).
The cell type–specific functions of these interactions suggests that they are involved
in the control of junction assembly rather than the rapid regulation of the basic
adhesion mechanism, but much more needs to be learned about the specific functions
of these important protein interactions.
Tyrosine phosphorylation of the cadherin–catenin complex also has been implicated
in the regulation of adhesion (Daniel and Reynolds 1997). Tyrosine phosphorylation
of β-catenin correlates with inhibition of cadherin-mediated adhesion resulting from
kinase activation (Matsuyoshi et al. 1992; Behrens et al. 1993; Shibamoto et al. 1994).
Moreover, both receptor tyrosine kinases and receptor tyrosine phosphatases have been
found to coimmunoprecipitate with cadherin–catenin complexes (Fig. 2 C) (Hoschuetzky
et al. 1994; Brady-Kalnay et al. 1995). However, there are very many potential substrates
for these kinases and phosphatases in the plasma membrane and cytoskeleton, and it
has not yet been shown that β-catenin phosphorylation is required for the observed
effects on cell adhesion. Indeed, an E-cadherin–α-catenin fusion chimera, which functions
without any β-catenin in the complex, has been found to remain subject to regulation
by the v-src tyrosine kinase (Takeda et al. 1995). Whether phosphorylation of other
potential substrates associated with the complex (such as p120ctn or still unidentified
proteins) participates in the regulation of cadherin activity remains to be determined.
The protein p120ctn, which is structurally related to β-catenin (armadillo repeat-containing
proteins), is also a good candidate for a regulator of cadherin adhesion activity
(Fig. 2 A). It binds to a region of the cadherin cytoplasmic tail, the juxtamembrane
domain, which is distinct from the classical catenin-binding site (Reynolds et al.
1994; Yap et al. 1998; Thoreson et al. 2000). In some cell types, p120ctn seems to
act as an inhibitor of cadherin-mediated adhesion, because either the deletion of
the cadherin juxtamembrane domain or the expression of a mutant form of p120ctn leads
to activation of adhesion (Aono et al. 1999; Ohkubo and Ozawa 1999). In Colo 205 tumor
cells, which have an intact but inactive E-cadherin–catenin complex, adhesion can
be activated with staurosporine, a serine kinase inhibitor, which also induces an
increase in the electrophoretic gel mobility of p120ctn (Aono et al. 1999). The composition
of the cadherin complex, including the amount of p120ctn, does not seem to be altered
in these greatly different adhesive states. In other cell types, the juxtamembrane
domain of the cadherin cytoplasmic tail and p120ctn has been proposed to play a positive
role in the control of cadherin-mediated adhesion. Deletion of the distal catenin-binding
domain of two cadherins, C-cadherin and VE-cadherin, does not interfere with their
basic adhesive functions in CHO cells (Navarro et al. 1995; Yap et al. 1998). Moreover,
selective uncoupling of p120ctn from E-cadherin by mutution of the p120ctn binding
site disrupts strong adhesion in cultured cells (Thoreson et al. 2000). Thus, the
juxtamembrane domain and/or p120ctn could have both positive and negative roles in
adhesion. The mechanism by which p120ctn and the juxtamembrane domain influence cadherin
function and their relationship to the functions of the distal catenin-binding domain
and the catenins is unknown.
The small GTPases, Rac, Rho, and Cdc42, have also been implicated in cadherin-mediated
adhesion (Kaibuchi et al. 1999). This subfamily of small GTPases is well known to
be involved in regulating actin–membrane interactions (Hall 1998) and, therefore,
it is not surprising that they might influence adherens junctions or cadherin-mediated
adhesion. Overexpression of constitutively active Rac generally results in greater
accumulation of E-cadherin, β-catenin, and actin at the regions of contact between
epithelial cells, whereas dominant negative Rac has the opposite effect (Braga et
al. 1997; Takaishi et al. 1997). Similarly, Rac activity is required for actin accumulation
at the adherens junction in Drosophila cells (Eaton et al. 1995). Tiam-1, a nucleotide-exchange
factor for Rac, has been localized to the adherens junctions of MDCK cells, and overexpression
of Tiam-1 or activated Rac increases E-cadherin–mediated adhesion, as measured by
cell aggregation assays (Hordijk et al. 1997). In a few cases, Rho and Cdc42 have
been found to have similar affects as Rac, but their effects have been less consistent
(Braga et al. 1997; Takaishi et al. 1997). The physiological or developmental roles
of the small GTPases in the regulation of cadherin-mediated adhesion have not been
fully elucidated, but overall the findings suggest that they may have roles in assembly
or disassembly of adherens junctions (Fig. 1, middle or bottom).
These small GTPases could indirectly regulate cadherin-mediated adhesion or junction
formation through their well known affects on the actin cytoskeleton. However, recent
studies provide evidence that Cdc42 directly affects the cadherin complex (Kuroda
et al. 1998; Fukata et al. 1999; Kaibuchi et al. 1999). IQGAP1, an effector of both
Cdc42 and Rac, can bind to E-cadherin–β-catenin complexes and compete for its binding
to α-catenin, resulting in dissociation of α-catenin from the cadherin complex and
rendering the cells nonadhesive. Cdc42 and Rac1 were found to inhibit IQGAP1 binding
to β-catenin and to rescue adhesion, which is consistent with the hypothesis that
Cdc42 and Rac1 lead to stabilization of the cadherin–catenin complex. Which physiological
role this mechanism might play in cadherin regulation in vivo has not yet been established.
Since dissociation of α-catenin from the cadherin complex has not yet been observed
in many cases of rapid physiological regulation of adhesion (inside-out signaling),
it is possible that the small GTPases and IQGAP1 regulate the overall state of assembly
of adherens junctions. Nonetheless, the findings demonstrate a mechanism by which
small GTPases can influence the state of cell contacts at the level of the association
of α-catenin with the cadherin complex.
Genetic experiments have revealed another group of proteins that seem to participate
in some way in the cellular organization or formation of epithelial adherens junctions.
These include afadin and shroom in mammals (Hildebrand and Soriano 1999; Ikeda et
al. 1999), and Discs large, Discs lost, Bazooka, stardust (gene), and Crumbs in Drosophila
(all but the last two are known to be PDZ domain-containing proteins) (Grawe et al.
1996; Müller and Wieschaus 1996; Woods et al. 1996; Bhat et al. 1999; Wodarz et al.
1999). Bazooka, Discs lost, and Crumbs are apical or apicolateral localized proteins
that are thought to play a role in the establishment of epithelial polarity. Discs
large is a septate junction protein, whereas afadin and shroom are localized to adherens
junctions. In fact, afadin and an Ig-type adhesion molecule with which it associates,
called nectin, are more highly concentrated in the adherens junctions than E-cadherin
or the catenins (Takahashi et al. 1999). None of these proteins are thought to interact
with cadherins, and it is not known whether they directly regulate cadherin adhesive
activity. They may exert their effects either indirectly through cytoskeletal organization/polarization
of the epithelial cell or by an alternate function in the adherens junction itself.
Irrespective of the roles of intracellular signaling pathways, cytoplasmic binding
proteins, and junctional organizing proteins, the mechanisms underlying the regulation
of the homophilic binding function of the cadherin extracellular domain are very poorly
understood. For integrin inside-out signaling, both conformational changes in the
ligand-binding site and alterations in higher order organization in the membrane,
such as clustering, have been implicated (Ginsberg et al. 1992; Gumbiner 1996). So
far, there is no evidence for affinity modulation of cadherins, but higher order organization
does seem to play a role in regulating C-cadherin in Xenopus embryos (Zhong et al.
1999). The nature of the higher order of cadherins at the site of cell contact and
how it is influenced by the actin cytoskeleton is not well understood. Lateral dimerization
of cadherins appears to be required for adhesive function, and it has been suggested
that dimerization could be subject to regulation (Brieher et al. 1996; Ozawa and Kemler
1998b; Tamura et al. 1998). Also, although cadherins are often associated with adherens
junctions, they can mediate adhesion independent of their incorporation into junctions.
The functional difference between junctional and nonjunctional forms of cadherin-mediated
adhesion is not well understood. Ultimately, it will be important to learn how the
homophilic adhesive bonds between cadherin molecules at the cell surface are regulated
by the cytoplasmic mechanisms to understand how cells make and break adhesive interactions
in tissues.