Download Intercellular adhesion, signalling and the cytoskeleton

review
Intercellular adhesion, signalling and
the cytoskeleton
Colin Jamora and Elaine Fuchs*
Department of Molecular Genetics and Cell Biology, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637, USA
*e-mail: [email protected]
Connections between the cytoskeleton and intercellular junctions profoundly influence cell shape and motility. It is
becoming increasingly clear that in addition to structural functions, components of the adhesion apparatus also
possess signalling capabilities. Recent studies suggest that their dual function may provide the means to integrate
changes in morphology and gene expression during tissue and organ development.
uring morphogenesis, mechanical forces generated by the
dynamic rearrangements of cell–cell contacts and the
cytoskeleton modulate the changes in cell shape and motility that transform uniform sheets of cells into specialized threedimensional structures. As morphogenesis proceeds, groups of
cells must remain cohesive, while selectively disassembling other
intercellular and substratum connections. This remodelling of
epithelial sheets is initiated and maintained by the instructive
cues of growth factors, such as Wnt, Bmp/Noggin, Notch/Delta,
fibroblast growth factors (FGF), epidermal growth factors (EGF)
and Hedgehog1.
D
Transmembrane members of the cadherin superfamily of intercellular adhesion proteins have a pivotal function in these morphogenetic processes. E-cadherin, the classical cadherin, forms cell–cell
contacts through homotypic interactions, which results in the formation of stable junctions. The ectodomains of E-cadherins dimerize
and cluster in a calcium-dependent manner, triggering an association
of the cytoplasmic tails of cadherins to the actin cytoskeletal network.
The recruitment of α- and β-catenin is required for this cytoskeletal
linkage, which in turn is essential for the stabilization and formation
of E-cadherin-mediated cell–cell junctions, referred to as adherens
junctions (AJs)2.
a
b
c
Keratin intermediate
filament
α-actinin
*
Figure 1 Structure of the adherens junction (AJ) and desmosomes. a,
Ultrastructure of the AJ and desmosomes in keratinocytes examined by transmission electron microscopy, 16 h after calcium switch. AJs (arrow) alternate with
desmosomes (asterisk) along the border of two cells. A schematic diagram of the
proteins that constitute the AJ (b) and the desmosome (c) is shown.
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a
0
13
26
39
b
52
c
Figure 2 Intermediate structures during the early formation of cell–cell contacts. a, GFP–E-cadherin distribution examined by time lapse confocal microscopy
in MDCK cells. Elapsed time between images is shown on the bottom left in minutes. Arrows point to ‘plaques’ and an aggregate of E-cadherin–GFP plaques is circled. (Image kindly provided by C. Adams, J. Ehrlich, and W. J. Nelson (Stanford
University)). b, Ultrastructure of keratinocytes 2.5 h after calcium switch, showing
filopodial-like projections filled with actin bundles (see ref. 6). c, Anti-E-cadherin
immunofluorescence microscopy of keratinocytes after 2.5 h of calcium-induced AJ
formation. E-cadherin is organized in double rows of puncta, known as ‘adhesion
zippers’ (A. Vaezi and E. Fuchs).
The assembly of the AJ complex is a highly regulated process
and begins when β-catenin binds to the carboxyl terminus of Ecadherin and promotes its transport through the secretory pathway3. When the cadherin–β-catenin complex reaches the plasma
membrane, α-catenin is recruited from the cytosol and binds to the
complex through β-catenin4–5. α-catenin can bind directly to actin
filaments, or indirectly, through the linker protein vinculin, which
in turn binds VASP6. Vinculin can bind directly to actin and is also
a component of cell-substratum contacts, known as focal adhesions7. VASP has been implicated in both actin polymerization and
in directing growing actin filaments to the sites of cell adhesion8.
Individual cadherin–actin units can be clustered and stabilized by
α-actinin, which crosslinks adjacent actin filaments9. Another
catenin protein that directly binds to E-cadherin is p120 (ref. 10).
However, the functional consequence of the binding of p120 to the
juxtamembrane region of the cadherin has been controversial, as
there is conflicting evidence as to whether p120 behaves as a positive or negative regulator of adhesion. These seemingly inconsistent
results have generated a model in which the trans binding of cadherins results in the activation of p120 and the strengthening of
adhesion, whereas intracellular signalling induces the inhibitory
effects of p120 (ref. 11).
Calcium also stimulates desmosomal cadherins (desmogleins
and desmocollins) to assemble desmosomes. In contrast to E-cadherin, desmosomal cadherins form heterotypic interactions that are
linked to intermediate filaments (IFs; for a review see ref. 12). The
link between desmosomal cadherins and the IF network can occur
in a number of ways, all of which appear to utilize the C-terminal
domain of desmoplakin (DP) to bind to IFs13,14. A member of the
plakin family of cytoskeletal linker proteins, DP can function as a
bridge between IFs by associating with the juxtamembrane domain
of desmocollin15. Alternatively, it can join IFs to desmogleins by
binding to plakoglobin (PG), which in turn binds to the cytoplasmic
tail of the cadherin. PG shares sequence similarities with β-catenin
and the two can substitute for one another in the formation of AJs16.
In contrast, α-catenin and DP are distinct, providing specificity to
the cytoskeletal linkages to the two types of intercellular junctions.
DP can also indirectly associate with desmocollin and desmoglein
by binding plakophilin, another relative of β-catenin17.
Fig. 1 depicts these two types of specialized cadherin-mediated
intercellular junctions. Both AJs and desmosomes are fundamental
features of epithelial cells, and their central function in establishing
epithelial sheets and polarity is the foundation on which higher
ordered structures are built.
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Adhesion and the cytoskeleton
The assembly and disassembly of intercellular junctions during
morphogenesis are accompanied by dramatic cytoskeletal
rearrangements that promote changes in cell shape and motility.
These rearrangements can also affect mitotic spindle orientation
and epithelial polarity. A number of insights into the molecular
mechanisms that couple cytoskeletal and adhesive dynamics have
been gained through in vitro studies that exploit the ability to
induce intercellular junction formation through calcium stimulation or cadherin-activating antibodies.
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Insights into actin dynamics and intercellular adhesion have also
come from the use of green fluorescent protein (GFP)-tagged E-cadherin and real time microscopy to study the mechanism of adhesion
in Madin Darby Canine Kidney (MDCK) epithelial cells18. At the initial stages of calcium-induced intercellular adhesion, E-cadherin, βcatenin and α-catenin organize into distinct aggregates, referred to as
‘puncta’18,19. These cadherin–catenin complexes are linked to the
cytoskeleton through thin bundles of actin that forms a bridge
between the puncta and cortical actin belt. In the second ‘maturation’
step of intercellular adhesion, cortical actin disappears and is
replaced by a continuous line of actin fibres at the interface of adhering cells, arranged in parallel with the plasma membrane (Fig. 2a).
Recently, Vasioukhin et al.6,20 added to these pioneering studies
using cultured primary epidermal keratinocytes from transgenic
and α-catenin conditional knockout mice to explore the consequences of severing connections to the actin cytoskeleton on intercellular adhesion. In wild-type keratinocytes, calcium induced the
formation of filopodia-like projections packed with bundles of
actin (Fig. 2b). These projections were postulated to promote cells
to seek, find and literally grasp onto their neighbours. The use of
filopodial projections seems to be a conserved intermediate stage in
the process of joining sheets of epithelia to seal off the interior of
an organism from its external environment. In Caenorhabditis elegans embryogenesis, for example, filopodia are used to prime the
membranes for the rapid sealing of epithelia during ventral closure21. In Drosophila melanogaster, filopodial extensions are used to
fuse developing branches during tracheal morphogenesis and to
seal imaginal discs during dorsal closure of the thorax22,23.
The outcome of these transient cell contacts is a recognizable
intermediate stage in intercellular adhesion, referred to as an adhesion zipper (Fig. 2c). The structure derives it name from the fact that
it consists of a double row of E-cadherin–catenin puncta, each
anchored to radial actin fibres, and that it is required for the sealing
of the two membranes together. The adhesion zipper is probably
analogous to the less organized arrays of puncta seen in epithelial
cells, such as MDCK cells. In keratinocytes, both nascent and maturation steps of intercellular adhesion require AJ assembly, whereas desmosomes seem to be involved primarily in the second step24.
The dazzling actin rearrangements seen in calcium- and antibody-stimulated intercellular adhesion are at least, in part, regulated by the Rho family of small GTPases. This family includes the
Rho subfamily members (which promote stress fibre formation),
Rac (which mediates lamellipodia formation), and Cdc42 (which
can generate filopodia)25. Although all three of these subfamilies of
Rho GTPases have been implicated in intercellular adhesion, a
function for activated Rac is the most firmly established.
Antibodies used to stimulate E-cadherin signalling recruit cytosolic GFP-tagged Rac to sites of cell–cell contact in MDCK cells26.
Activated Rac seems to enhance adhesion by binding to and
inhibiting IQGAP, a regulatory molecule that can bind to and dissociate α- and β-catenin from AJs27. That said, activated Rac also
promotes cell motility when AJs are few or absent. Although at first
glance this seems incompatible, directed cell motility is a prerequisite for intercellular adhesion and epithelial sheet formation, as
reflected by the need to physically bring cells close to one another.
The cell has resolved this problem by regulating the localization of
the guanine nucleotide exchange factors (GEFs), which are
upstream activators of Rho proteins. In this regard, it was recently
discovered that under conditions that favoured motility, the protein
Tiam1 (T-lymphoma invasion and metastasis gene 1), a GEF for
Rac, is localized at lamellae and ruffles, but in a nonmotile cell,
Tiam1 is found at AJs28. The localization of active Rac at AJs may
also be regulated by phosphatidylinositol-3-OH kinase (PI(3)K),
which is activated by E-cadherin29. Conversely, treatment of cells
with wortmannin, a potent inhibitor of PI(3)K and a regulator of
phospholipid dynamics, blocks the recruitment of Rac and disrupts
cell–cell adhesion26. Interestingly, most GEFs have a pleckstrin
homology (PH) domain that binds phosphatidylinositol lipids and
enables proteins to be targeted to specific membrane subdomains.
E-cadherin-mediated intercellular adhesion also seems to result
in Cdc42 activation. This was first observed in in vitro studies with
mutants of Cdc42 (ref. 30), and was recently confirmed with a GFPtagged substrate for Cdc42 (ref. 31). These cell culture studies are
also supported by genetic studies showing that a dominant-negative
Cdc42 mutant blocks filopodial extension and epithelial adhesion in
Drosophila dorsal closure32. Although evidence is lacking for a direct
interaction between E-cadherin and Cdc42, the activation of Cdc42
by E-cadherin is particularly noteworthy, because it suggests a possible molecular explanation of how these adhesion proteins might
establish cell polarity. The PAR/atypical protein kinase C (aPKC)
kinase complex is activated by Cdc42 and translocates to sites of
cell–cell adhesion after calcium stimulation33. The PAR complex,
composed of aPKC, PAR3/ASIP (aPKC specific interacting protein)
and PAR6, is an evolutionarily conserved ternary complex with a
fundamental function in cell polarity in metazoans34. The postulated function of activated Cdc42 is to induce a structural change in
the PAR complex that allows for the binding of junctional adhesion
molecules (JAMs). JAMs are membrane proteins that facilitate the
formation of tight junctions, which are often adjacent to AJs. By
generating a scaffold for the formation of a tight junction, the Ecadherin/Cdc42/PAR/aPKC pathway may facilitate the establishment of apical and basolateral membranes of a polarized cell.
The ability of E-cadherin to activate both Cdc42 and PI(3)K
may also provide another avenue to promote actin polymerization.
Both Cdc42 and phosphatidylinositol-4,5-bisphosphate (PIP2; the
lipid metabolite produced by PI(3)K) function as cofactors for the
activation of the Wilcott-Aldrich Syndrome protein (WASP;
reviewed in ref. 35). WASP, in turn, activates a complex of proteins
called Arp2/3 (actin-related protein), which can initiate the formation of actin branches from pre-existing filaments. The formation of
these actin branches are reminiscent of the thin actin filaments that
emanate from the cortical actin belt to the puncta of E-cadherin
during the initial stages of AJ formation in MDCK cells18.
Although the association of microtubules and adhesive structures has long been known, the function of this interaction is only
recently being elucidated. Intriguingly, the addition of stimulatory
anti-E-cadherin antibodies to mammalian cytoplasts resulted in
the stabilization of microtubules36. In this centrosome-free system,
stabilization occurred at the minus ends of microtubules, which
otherwise would have exhibited dynamic instability. Examples
where microtubule arrays naturally lack an association with centrosomes include polarized epithelial cells and axonal outgrowths
of neurons. Furthermore, it was recently shown that Drosophila
cadherin regulates the orientation of asymmetric division by influencing the localization of the mitotic spindle37. The molecular
nature of the connection between microtubules and AJs remains
unknown, but candidates include β-catenin, known to associate
with the microtubule binding protein APC (adenomatous polyposis
coli), as well as with microtubules38. Other proteins in the microtubule–adhesion connection include the microtubule interacting
proteins EB1 and ACF (actin crosslinking family)-7. APC, EB1 and
ACF-7 are all recruited to the sites of intercellular adhesion and they
function as possible means by which intercellular junctions control
planar cell polarity and/or spindle pole orientation39,40.
An additional function for the recruitment of microtubules to the
sites of cell–cell adhesion could be to facilitate retrograde transport
from the plus ends of microtubules (at the cell periphery) to the
minus ends at the centrosome (adjacent to the nucleus). To this end,
it was recently demonstrated that β-catenin could recruit and bind
the molecular motor protein dynein to nascent adhesion junctions41.
The function of microtubule-crosslinking proteins at this site could
be to capture and tether the microtubules that project to cell–cell contacts, and to use dynein to load cargo from the cell periphery and
transport it to the cell centre. Altogether, β-catenin may have a critical
function in enabling the microtubule network to sense and react to
dynamic rearrangements of the actin cytoskeleton.
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a
b
First signal : adopt a hair
follicle fate
Placode
Hair germ
Second signal: form a dermal
papilla (DP)
Hair
shaft
Matrix
DP
Third signal: matrix cells
proliferate & differentiate
Figure 3 Adhesion dynamics during hair follicle morphogenesis. a, A model
of the 3 signals exchanged between the ectoderm and underlying mesenchyme
during hair follicle morphogenesis. b, Dynamic expression of E-cadherin (green) and
P-cadherin (red) during hair follicle morphogenesis. Adapted from ref. 60 © (2001)
with permission from Elsevier Science.
Adhesion dynamics during development
The importance of adhesion molecules in normal development is
illustrated by the early embryonic lethality seen in mice harbouring
null mutations in different genes of the adhesion apparatus. For
example, mouse embryos lacking E-cadherin and α-catenin die at
the blastocyst stage, because of the failure to form a trophectodermal epithelium42,43. Mouse embryos that lack β-catenin die just
after gastrulation44. The ability of β-catenin-null embryos to survive longer than those lacking E-cadherin has been ascribed to the
functional redundancy of β-catenin and PG in AJ formation16. The
lethality at gastrulation has been attributed to the unique function
of β-catenin as a transcription cofactor in canonical Wnt signalling
(see below; ref. 45). Later stages of development also require cadherins, as judged by the deleterious effects of inhibitory antibodies
that prevent homophilic cadherin interactions. For example, blocking antibodies to N-cadherin injected into a chick embryo caused a
defect in the establishment of left–right asymmetry46. Conditional
gene disruption of β-catenin in the epidermis showed defects in
cell-fate determination47. Additionally, conditional ablation of αcatenin in skin epithelium results in a loss of hair follicle morphogenesis, a partial loss of epithelial polarity, marked hyperproliferation and epithelial invaginations20.
Desmosomes also have an important function in development,
as judged by the fact that targeted ablation of PG results in cardiovascular defects48 and desmoglein-3 ablation gives rise to mice with
abnormalities in their skin49. Likewise, targeted disruption of
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desmocollin-1 in the skin results in hyperproliferation and compromised barrier function50. Additionally, DP-null embryos fail to
develop past embryonic stage (E) 6.5, because of compromised
function in the extra-embryonic tissues51. Tetraploid rescue of the
DP knockout to examine embryonic defects resulted in post-gastrulation defects in the heart and neuro-epithelium and vacular
perturbations, and conditional DP knockout in the skin resulted in
severe blistering24,52. In most of these situations, the defects
observed involved primarily cell degeneration. However, recent
studies have also revealed a function for desmosomes in cell sorting, demonstrated by the segregation of lumenal and myoepithelial
cells in an aveolar morphogenesis assay53. The requirement for programmed fluctuations in adhesion protein levels during morphogenesis is illustrated by the detrimental effect that their forced
expression often has on development. Overexpression of E-cadherin in early Xenopus laevis embryos prevented β-catenin from
translocating into the nucleus, and this in turn inhibited the induction of dorsal mesoderm and axis formation54,55. Given that
Wnt/Wingless signalling is a prerequisite for the stabilization of
β-catenin and the promotion of its nuclear translocation, it is not
surprising that these features parallel those seen when
Wnt/Wingless signalling is impaired56. Further studies of Xenopus
embryogenesis demonstrated that inhibiting the downregulation of
C-cadherin with activating antibodies in animal cap explants effectively prevented tissue elongation, apparently by blocking convergent extension in these cells57,58.
The morphogenesis of the hair follicle is a prime example of
how changing patterns of cell adhesion molecules can shape a
developing organ. The hair follicle develops as one of two cell fates
that can be taken by the multipotent epithelial cells that comprise the
basal layer of the embryonic ectoderm. This layer of cells is attached
through cell-substratum adhesion molecules to the basement membrane, which separates the epithelium from the underlying mesenchyme (the developing dermis). In the mouse, hair follicles are
established in synchronous waves, each derived from a series of
sequential signals exchanged between the ectoderm and mesoderm
(for a review see ref. 59).
Classic tissue transplantation experiments have uncovered three
key mesenchymal–epithelial exchanges that orchestrate follicle formation. The first message originates from the mesenchyme,
instructing the overlying ectodermal cells to cluster into localized
thickenings (placodes), which grow downward to form hair germs
or immature follicles. The hair placodes then transmit a signal that
causes the underlying mesenchymal cells to cluster into dermal
condensates, or dermal papillae, which are then engulfed by the
base of growing hair germs. Finally, each dermal papilla transmits
signals to its closely-associated epithelial hair germ cells (matrix
cells) to proliferate and differentiate59,60 (Fig. 3a).
From the initiation of follicle development in the ectoderm and
throughout its differentiation, the patterns of intercellular adhesion
molecule expression change concomitantly with the organization
of the cells (Fig. 3b). Striking among these changes is a reduction in
the level of E-cadherin and an induction of P-cadherin in the
embryonic ectoderm, concomitant with the receipt of the first dermal message and the organization of cells to form the placode61. As
the hair germ grows downward into the mesenchymal layer, P-cadherin remains expressed at the leading front, while elsewhere, the
epithelium adopts a seemingly uniform pattern of E-cadherin
expression. As the proliferating matrix cells differentiate, the cells
again convert from a P-cadherin- to an E-cadherin-based adhesion
system.
The differential expression patterns of E- and P-cadherin during
skin morphogenesis are mimicked in tooth and feather morphogenesis, raising the question as to whether such changes in cadherin
expression patterns might have a broad function in the cell polarization associated with tissue/organ morphogenesis. Despite these
intriguing changes in cadherin expression, ablation of P-cadherin
through gene targeting did not generate obvious changes in hair or
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tooth development62. Although these knockout studies complicate
our understanding of the function of the E-/P-cadherin switch, hair
and tooth formation provide beautiful examples of how adhesiondependent morphogenesis is intricately coordinated with proliferation and differentiation to create an appendage from a homogenous
sheet of embryonic cells.
Regulation of adhesion dynamics
The tight spatiotemporal expression of cadherin proteins reflects
new complexities in AJ dynamics. Indeed, the rate-limiting step in
the formation of the adhesion junction complex seems to be the
level of cadherin expression63. Increasing evidence suggests that Wnt
signalling affects the stability of AJs in diverse ways. The canonical
Wnt/Wingless signalling pathway results in the stabilization of
β-catenin by inhibiting the serine-threonine kinase glycogen synthase kinase-3 (GSK-3), which normally marks it for degradation.
The increased cytosolic pool of β-catenin can either enhance AJ formation or translocate into the nucleus and transcriptionally activate
Lef1/Tcf target-genes (Fig. 4a). Increasing the intracellular pool of
β-catenin by transfection of cells with Wnts, dishevelled (Dsh), or
stabilized β-catenin itself, prolonged the half-life of AJs64–67.
However, when a stable form of β-catenin was introduced into the
epithelial tissues of transgenic mice, some of the cells aberrantly
switched on Lef1/Tcf target-genes68–70. Intriguingly, the phenotypic
outcome of expressing stabilized β-catenin was hair follicle downgrowth when an epidermal keratin promoter was used, and mammary gland morphogenesis when the MMTV promoter was used.
What might account for the different adhesive responses of
MDCK, keratinocytes and breast epithelial cells to canonical Wnt
signalling? Although several explanations are possible, it is tempting to speculate that the factor determining whether a cell will
respond to stabilized β-catenin by enhancing or destabilizing and
remodelling AJs is the degree of nuclear translocation of β-catenin
and/or the concentration of its transcription partners. If so, a corollary to this prediction would be that canonical Wnt-induced
changes in transcription might include genes encoding AJ-destabilizing and/or remodelling factors. Although future experiments will
be necessary to determine the extent to which this hypothesis is
correct, it is intriguing that the E-cadherin promoter can bind proteins from the Lef/Tcf family of proteins in vitro71. If genes such as
E-cadherin are regulated in vivo by canonical Wnt signalling, then
proteins and factors which influence Lef1/Tcf levels may also have
a direct impact on AJ dynamics in cells and tissues.
In the last five years, researchers have witnessed the discovery of
an increasing number of nuclear factors that adversely affect the
transcription of the E-cadherin gene by binding the E-boxes within its promoter. One is the Smad Interacting Protein (SIP), which is
induced by the engagement of transforming growth factor-β
(TGFβ) with its receptor. SIP catalyses a dramatic reduction in
E-cadherin gene expression, and correspondingly, a SIP transgene
bestowed a marked invasive capacity on transfected MDCK cells72.
Other transcriptional regulators that bind to the E-box of the
E-cadherin promoter include the transcription factors E12/E47
(ref. 73) and the Snail family of proteins, which can be induced by
FGF-receptor 1 (FGFR1)74. Snail proteins were originally identified
as crucial factors for gastrulation and mesoderm induction in
developing mouse embryos. Snail localizes to regions where E-cadherin levels are low, such as the primitive streak and the neural
crest75,76, as well as to sites of epithelial–mesenchymal transitions
(EMTs). When overexpressed in epithelial cells, both Snail and
E12/E47 induce EMTs. EMTs are another interesting example of
morphogenesis, in which subpopulations of epithelial cells downregulate their cell–cell adhesion apparatus to leave their site and
move into a new microenvironment. However, not all members of
the Snail family of proteins adversely affect E-cadherin expression.
For example, Escargot, a close cousin of Snail, upregulates the transcription of shotgun, the Drosophila gene for E-cadherin23. Other
transcription factors enhance E-cadherin expression, including the
vitamin D3/vitamin D receptor (VDR) and the tumour suppressor
protein Wt1, whose absence results in the abnormal kidney development associated with Wilms’ tumour.
Although transcriptional changes in the genes encoding AJ proteins seem to have profound regulatory functions during morphogenesis, post-translational modifications of these proteins can also
have a major impact on the status of intercellular adhesion. In this
scenario, Wnt signalling resurfaces, but this time involving routes
not yet explored in this review. In mammals, there are at least 18
different Wnt proteins, which interact with one of ten different frizzled receptors (Fzs). Often this occurs in combination with other
coreceptors, such as LDL-related proteins (LRPs), to activate four
possible signalling pathways77. Post-translational modifications
seem to underlie the AJ dynamics that are induced by the noncanonical Wnt/calcium pathway. This pathway seems to activate
heterotrimeric G proteins, which in turn activate two calcium-regulated serine-threonine kinases: PKC and casein kinase II (CKII;
Fig. 4b). PKC is involved in a broad range of developmental
processes, including the regulation of cadherin complexes (for a
review, see ref. 2). For example, pharmacological inhibitors identify a function for calcium and PKC in the disassembly of vascular
E-cadherin-based junctions78. Additionally, during Xenopus gastrulation, Fz7-dependent PKC signalling stimulates developing
mesodermal and ectodermal cells to sort into distinct layers79, and
overexpression of Fz7 decreases intercellular adhesion in a PKCdependent manner80. CKII can directly phosphorylate the cytoplasmic tail of E-cadherin81, which increases its binding to
β-catenin in vitro82. Furthermore, it was recently observed that
casein kinase I phosphorylates and destabilizes the β-catenin
degradation complex, resulting in an increase in β-catenin levels83.
Increasing the cytosolic pool of β-catenin would be predicted to
stabilize AJ in the same way as Wnt signalling. Sifting through these
complexities to assess whether there is a common theme to phosphorylation and intercellular adhesion must await more extensive
investigations in multiple cells and tissues.
AJ proteins can also be targets for receptor tyrosine kinases or
phosphatases, which are situated in close proximity to, and can
physically interact with, E-cadherin84,85. In vitro, hepatocyte growth
factor (HGF; also known as scatter factor) and EGF stimulation can
induce the phosphorylation of β-catenin, causing the rapid dissociation of epithelial cell aggregates86. p120, a close cousin of
β-catenin and a component of AJs, was originally identified as a
target of the tyrosine kinase src87, although there is still conflicting
evidence as to whether tyrosine phosphorylation promotes or
interferes with intercellular adhesion. Taken together, the multiplicity of kinases and signal transduction pathways that can impact
on the phosphorylation state of AJ proteins, and the strikingly different effects these post-translational modifications can elicit, begin
to paint a picture of the intricate level of fine-tuning that can govern adhesion dynamics during such diverse processes as morphogenesis, wound-healing and differentiation.
Adhesion and gene expression
Increasingly, evidence points to the view that the functions of both
desmosomes and AJs extend beyond the mere structural and
mechanical properties of adhesion. To some extent, the adhesive
properties themselves may facilitate intercellular signalling pathways by drawing together the opposing membranes of different
cells or cell groups, prompting them to make additional connections, for example, through gap junctions and/or ligand–receptor
interactions. Membrane sealing can also affect the diffusion of
extracellular growth factors and the establishment of morphogen
gradients that are critical for pattern formation. The ability of cadherins to determine polarity may also influence the availability and
localization of transmembrane receptors, and hence the sensitivity
of cells to external stimuli88.
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a
b
Wnt
LRP
Wnt
APC
Dsh
Axin
β-cat
DAG
PIP2
G
α βγ
GSK-3
β-cat
?
PLC
IP3
PKC
[Ca2+]i
CamKII
Cellular
Response
β-cat
TCF/LEF
Nucleus
Canonical Wnt/β-cat pathway
Nucleus
Non-canonical Wnt/
Calcium pathway
Figure 4 Wnt signalling. a, Components of the canonical Wnt/β-catenin signalling
pathway. b, The non-canonical Wnt/calcium pathway in Drosophila melanogaster.
In addition to the myriad of indirect ways in which membrane
sealing may alter signalling in tissues, the striking correlation
between adhesion protein dynamics and the sites of cell proliferation and differentiation suggests that AJ proteins may play an
inductive function in organogenesis. For instance, in the developing hair follicle the first E- to P-cadherin switch occurs at the placode, where multipotent ectodermal cells choose a hair follicle fate
and invaginate into the dermis. A second P- to E-cadherin switch
occurs as the proliferating hair precursor cells of the matrix withdraw from the cell cycle and begin to differentiate to form the hair
shaft and it surrounding sheath60 (Fig. 3b). Though merely correlative at present, the changes in classical cadherin expression suggest
that these cadherins may directly participate in communication
pathways that guide differentiation commitments. Likewise, the
differential expression of multiple isoforms of desmosomal cadherins in the epidermis implies a broader function for these proteins than simply as a stable clamp between two cells.
Though functional studies in the skin await further investigation, it may be relevant that overexpression of E-cadherin in mouse
intestinal epithelium slows cell migration and blocks proliferation
while inducing apoptosis89. Furthermore, it has been known for
some time that overexpression of E-cadherin can also effectively
block proliferation and invasiveness of tumour cells90. This block in
proliferation is not a consequence of increased cell adhesion, but is
instead caused by the ability of E-cadherin to sequester the transcriptionally competent pool of β-catenin and effectively shut off
expression of Lef/TCF/β-catenin-responsive genes91–93.
Although elevated E-cadherin levels can suppress β-cateninmediated signalling, the effects of E-cadherin downregulation on βcatenin transcription are less clear. When E-cadherin is downregulated during morphogenesis, what happens to all the proteins that
typically associate with AJs? A priori, liberation of potential transcription cofactors, such as β-catenin, could generate global changes
in gene expression. But without Wnt-mediated stabilization of βcatenin, excess protein would be targeted for turnover. Thus, how a
cell responds to the downregulation of E-cadherin is likely to
depend on whether or not it is receiving a canonical Wnt signal.
Insights into the signalling functions of various AJ-associated
proteins have been obtained either through bypassing their normal
regulatory processes or through gene ablation. We have discussed
how these approaches identified a function for β-catenin in cell fate
decisions. Recently, a negative signalling function for α-catenin has
been uncovered20.
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Loss of α-catenin in the epidermis resulted in the sustained
activation of the Ras-MAPK pathway and the association of E-cadherin–β-catenin complexes with downstream members of tyrosine
kinase growth factor receptor pathways. The inverse correlation
between cell adhesion and proliferation seen in the α-catenin-null
state may explain why loss-of-function mutations in the α-catenin
gene have been found in some human cancers94,95. Interestingly,
Nagafuchi and colleagues recently reported that E-cadherin is
required for the efficient translation of α-catenin mRNA96.
Therefore, in cases where AJs are reduced, such as during wound
healing, the downregulation of E-cadherin might cause a concomitant decrease in α-catenin levels, generating an effective transient
‘null’ state.
Additional candidates for AJ-bound transcription factors
include p120 and members of the zyxin family. Certain isoforms of
p120 localize to the nucleus under conditions where E-cadherin
levels are low97. Yeast two hybrid studies demonstrated that p120
can interact with Kaiso, a novel BTB (broad complex, tramtrak, bric
a brac) /POZ (Pox virus and zinc finger) protein, which reportedly
functions as a transcriptional repressor98. Though Kaiso is found in
the nucleus and at cell–cell junctions, it is not clear whether p120 is
solely responsible for its subcellular localization. Members of the
zyxin family of proteins, including zyxin, lipoma preferred partner
(LPP), Ajuba and thyroid-interacting protein (Trip)6 have all been
localized not only to the nucleus, but also to sites of cell–cell and
cell–substratum junctions99,100. LPP, for example, was shown to
have transcriptional activity in a GAL4-based transactivation
assay101. The absence of a DNA-binding domain and a nuclear
localization signal (NLS) suggests that LPP may be part of a larger
transcriptional complex. Interestingly, Ajuba is translocated to the
nucleus through its NLS in response to retinoic acid, and this is
accompanied by an inhibition of proliferation and an induction of
differentiation102. Together, these results suggest that zyxin family
members may provide a link between cell adhesion and the transcriptional regulation of differentiation.
Zona-occludens 1 (ZO-1) is another protein that seems to reside
at the AJ sink for transcription factors. Although traditionally considered a marker of tight junctions, ZO-1 interacts both with
β-catenin103 and α-catenin104, making it a bona fide associate of AJs.
Support for a nuclear signalling function for ZO-1 is provided by
the finding that it binds to the transcription factor ZONAB (ZO1associated nucleic acid binding protein), which controls the expression of genes involved in cell cycle progression105. However, the precise
function for ZO-1 and ZONAB in coordinating transcription, tight
junction and AJ formation remain to be elucidated.
As for desmosomal components in signalling, PG has been shown
to inhibit the transcriptional activity of the TCF4–β-catenin transcription complex106. This finding is consistent with the observation
that overexpression of PG in the skin suppresses proliferation and
hair growth107. Furthermore, many different isoforms of plakophillin
have been localized in the nucleus of cells, independently of the presence of desmosomes. The widespread nuclear expression of these
proteins has resulted in the hypothesis that they may be components
of the basic transcriptional machinery of the cell.
The list we provide here of candidates that might link transcription and intercellular adhesion is by no means comprehensive.
However, it gives a flavour of how components of the
cadherin–catenin complex, and the multitude of proteins that associate with it, participate in nucleocytoplasmic shuffling. Although
the nuclear functions of many of these proteins remains to be
determined, an emerging theme is that the cell adhesion system
may directly coordinate the changes in cell shape and movements
of morphogenesis with changes in the cell’s behaviour and program
by exerting spatiotemporal control over signalling proteins.
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ACKNOWLEDGEMENTS
We thank members of the Fuchs lab for comments on the manuscript.
We also thank C. Bauer, S. Raghavan, A. Vaezi, C. Adams and W.J. Nelson for the use of their figures.
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