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EVOLUTIONARY HOMOLOGS

Table of contents

E-cadherin and signaling pathways

The observed in vivo expression patterns of alpha-catenin, beta-catenin, and plakoglobin suggest that this process occurs as these proteins are directly linked with the developmental regulation of cell junctions; cardiac cells become stably committed and phenotypically differentiated to eventually form a mature myocardium. Perturbation of N-cadherin using a function perturbing N-cadherin antibody (NCD-2) inhibits normal early heart development and myogenesis in a cephalocaudal, stage-dependent manner. A model is proposed whereby myocardial cell compartmentalization also defines the endocardial population. The presence of beta-catenin suggests that a similar signaling pathway involving Wnt (wingless)-mediated events may function in myocardial cell compartmentalization during early vertebrate heart development, as in Drosophila contractile vessel development. In Drosophila, contractile vessel development requires wingless. Elimination of wingless function for a short time period after gastrulation in Drosophila results in the selective loss of heart precursors. The data presented here are consistent with the likelihood that Wnt signaling may be involved in the regulation of the N-cadherin/ß-catenin-mediated events associated with vertebrate heart development. The Wnt2 gene is expressed in the early mouse heart field at 7.5-8 days of gestation. This coincides with the period of events described here (Linask, 1997).

The alpha-catenin molecule links E-cadherin/ beta-catenin or E-cadherin/plakoglobin complexes to the actin cytoskeleton. Several invasive human colon carcinoma cell lines were studied that lack alpha-catenin. They showed a solitary and rounded morphotype that correlates with increased invasiveness. These round cell variants acquire a more normal epithelial phenotype upon transfection with an alpha-catenin expression plasmid, and also upon treatment with the protein kinase C (See Drosophila PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Video registrations show that the cells start to establish elaborated intercellular junctions within 30 min after addition of TPA. Interestingly, this normalizing TPA effect is not associated with alpha-catenin induction. There are only minor TPA-induced changes in E-cadherin staining. In contrast, desmosomal and tight junctional proteins are dramatically rearranged, with a conversion from cytoplasmic clusters to obvious concentration at cell-cell contacts and exposition at the exterior cell surface. TPA induces the appearance of typical desmosomal plaques. TPA-restored cell-cell adhesion is E-cadherin dependent, as demonstrated by a blocking antibody in a cell aggregation assay. Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too. Remarkably, the combination of anti-E-cadherin and anti-desmoglein antibodies synergistically inhibits the TPA effect. These studies show that it is possible to bypass the need for normal alpha-catenin expression to establish tight intercellular adhesion by epithelial cells. Apparently, the underlying mechanism comprises upregulation of desmosomes and tight junctions by activation of the PKC signaling pathway, whereas E-cadherin remains essential for basic cell-cell adhesion, even in the absence of alpha-catenin (van Hengel, 1997).

In mitogenic signaling pathways, Shc participates in the growth factor activation of Ras by interacting with activated receptors and/or the Grb-2.Sos complex. Shc, through its SH2 domain, forms a complex with the cytoplasmic domain of cadherin, a transmembrane protein involved in the Ca2+-dependent regulation of cell-cell adhesion. This interaction is demonstrated in a yeast two-hybrid assay, by co-precipitation from mammalian cells, and by direct biochemical analysis in vitro. The Shc-cadherin association is phosphotyrosine-dependent and is abrogated by addition of epidermal growth factor to A-431 cells maintained in Ca2+-free medium, a condition that promotes changes in cell shape. Shc may therefore participate in the control of cell-cell adhesion as well as mitogenic signaling through Ras (Xu, 1997).

Cadherins are calcium-dependent cell-cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, whether or not members of the Rho family of small GTPases are necessary for cadherin adhesion was investigated. In fibroblasts, the Rho family members Rho and Rac (See Drosophila Rac) regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, Rho and Rac are required for the establishment of cadherin-mediated cell-cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removes cadherin complexes from junctions induced for up to 3 h, while desmosomes are not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, both microfilament-associated receptors. The concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac is not sufficient to stabilize cadherin dependent cell-cell contacts when endogenous Rho is inhibited. Upon induction of calcium-dependent intercellular adhesion, there is a rapid accumulation of actin at sites of cell-cell contacts, which is prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors is perturbed by inhibiting Rac but not Rho. These results provide new insights into the role of the small GTPases in the cadherin-dependent cell-cell contact formation and the remodeling of actin filaments in epithelial cells (Braga, 1997).

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell-cell junctions and subsequent cell scattering. In Madin-Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induce the loss of the adherens junction proteins E-cadherin and beta-catenin from intercellular junctions during cell spreading, and the HGF/SF response is blocked by dominant-negative N17Ras. Desmosomes and tight junctions are regulated separately from adherens junctions, because the adherens junctions are not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac are required downstream of Ras, because loss of adherens junctions is blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of either or both MAP kinase kinase 1 or Rac1. All of these inhibitors also prevent HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110alpha subunit of PI 3-kinase alone does not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras are required for adherens junction disassembly and that the disassembly process is essential for the motile response to HGF/SF (Potempa, 1998).

ß-Catenin bonds to the HMG-type transcription factor lymphoid enhancer factor-1 (LEF-1). The Xenopus LEF-1/ß-catenin complex, which undergoes nuclear translocation during Wnt signaling, binds to an E-cadherin promoter fragment. In mouse embryos during primitive streak formation, embryonic ectodermal cells, which represent a true epithelial cell layer, give rise to mesoderm. During primitive streak formation, some ectodermal cells loose E-cadherin expression and express LEF-1. From the Xenopus results, it is tempting to speculate that during this process, a complex of LEF-1 and ß-catenin is involved in down-regulating E-cadherin transcription. The observed interaction of LEF-1 with ß-catenin raises the possibility that LEF-1 might be involved in dorsal mesoderm formation. To test for this possibility, LEF-1 mRNA was overexpressed in Xenopus embryos. Overexpression of LEF-1 mRNA causes secondary axis formation, and this effect is enhanced with overexpression of ß-catenin (Huber, 1996).

Recent studies have demonstrated the importance of E-cadherin, a homophilic cell-cell adhesion molecule, in the contact inhibition of the growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell-cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, these cells were transfected with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells results in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell-cell adhesion, E-cadherin expression also results in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27(kip1) and a late reduction in cyclin D1 protein. Tightly adherent spheroids also show increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin-neutralizing antibodies in three-dimensional culture simultaneously prevents adhesion and stimulates proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin-dependent growth inhibition, E-cadherin-positive cells were engineered to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin-neutralizing antibodies on proliferation can be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27 (St. Croix, 1998).

The insulin-like growth factors (IGFs) are well known mitogens, both in vivo and in vitro, while functions in cellular differentiation have also been indicated. A new role for the IGF pathway in regulating head formation has been demonstrated in Xenopus embryos. Both IGF-1 and IGF-2, along with their receptor IGF-1R, are expressed early during embryogenesis, and the IGF-1R is present particularly in anterior and dorsal structures. Overexpression of IGF-1 leads to anterior expansion of head neural tissue as well as formation of ectopic eyes and cement gland, while IGF-1 receptor depletion using antisense morpholino oligonucleotides drastically reduces head structures. Furthermore, IGF signaling exerts this effect by antagonizing the activity of the Wnt signal transduction pathway in the early embryo, at the level of ß-catenin. Thus, the IGF pathway is required for head formation during embryogenesis (Richard-Parpaillon, 2002).

Wnt signaling is involved in numerous developmental processes, such as dorsal axis formation, patterning of the central nervous system, and establishment of cell polarity. The pathway is tightly regulated during embryogenesis and it is becoming increasingly clear that crossregulation between Wnt and other signaling pathways contributes to the complexity and specificity of Wnt activity. For example, retinoid signaling and a specific MAP kinase pathway (TAK/NLK) can both inhibit Wnt activity. However, previous evidence for an interaction between the IGF and the Wnt pathways is limited. IGF-1 stimulation induces a rapid tyrosine-phosphorylation of ß-catenin in a cell line derived from a human colonic adenocarcinoma. It has also been shown that the phosphorylation of ß-catenin induced by IGF-1 leads to a dissociation of the pool of ß-catenin, which is bound to E-cadherin at the plasma membrane, resulting in its relocation to the cytoplasm (Richard-Parpaillon, 2002 and references therein).

However, despite this accumulation of cytoplasmic ß-catenin, no enhancement of Wnt activity is observed after stimulation by IGF-1 alone, as determined by using the Wnt-responsive luciferase reporter construct TOP-FLASH. Recent structural studies might provide an explanation for this paradox. It has been shown that the charged residues involved in this interaction between ß-catenin/E-cadherin are the same as those required for the ß-catenin/ TCF interaction. Thus, this raises the interesting possibility that tyrosine-phosphorylation of ß-catenin, which blocks its association with E-cadherin may also prevent interaction between this molecule and its downstream effector Tcf. This is a potential point at which IGF signaling may inhibit the Wnt pathway. In the future, it will be interesting to investigate this hypothesis further, and also to determine whether the PI3K or the MAPK activated by IGF-1R may be involved in this process (Richard-Parpaillon, 2002).

The leucocyte common antigen-related phosphatase (LAR) has been implicated in receptor tyrosine kinase signalling pathways while also displaying cell-density-dependency and localization to adherens junctions. Whereas physiological substrates for LAR have not been identified unequivocally, beta-catenin associates with LAR and is a substrate in vitro. With the implication that LAR may play a role in regulating E-cadherin-dependent cell-cell communication and contact inhibition, the relationship of LAR with E-cadherin was investigated. LAR expression increases with cell density in the human breast cancer cell line MCF-7 and in Ln 3 cells derived from the 13762NF rat mammary adenocarcinoma. LAR protein levels decrease rapidly when cells are replated at a low density after attaining high expression of LAR at high cell density. COS-7 cells display comparable density-dependent regulation of LAR expression when transiently expressing exogenous LAR under the control of a constitutively active promoter, indicating that the regulation of expression is not at the level of gene regulation. Disrupting homophilic E-cadherin complexes by chelating extracellular calcium causes a marked decrease in LAR protein levels. Similarly, blocking E-cadherin interactions with saturating amounts of E-cadherin antibody (HECD-1) also leads to a rapid and pronounced loss of cellular LAR. In contrast, mimicking cell-surface E-cadherin engagement by plating cells at low density on to dishes coated with HECD-1 results in a 2-fold increase in LAR expression compared with controls. These results suggest that density-dependent regulation of LAR expression is mediated by functional E-cadherin and may play a role in density-dependent contact inhibition by regulating tyrosine phosphorylation in E-cadherin complexes (Symons, 2002).

Cadherins are critically involved in tissue development and tissue homeostasis. Neuronal cadherin (N-cadherin) is cleaved specifically by the disintegrin and metalloproteinase ADAM10 in its ectodomain. ADAM10 is not only responsible for the constitutive, but also for the regulated, shedding of this adhesion molecule in fibroblasts and neuronal cells directly regulating the overall levels of N-cadherin expression at the cell surface. The ADAM10-induced N-cadherin cleavage results in changes in the adhesive behaviour of cells and also in a dramatic redistribution of ß-catenin from the cell surface to the cytoplasmic pool, thereby influencing the expression of ß-catenin target genes. These data therefore demonstrate a crucial role of ADAM10 in the regulation of cell-cell adhesion and on ß-catenin signalling, leading to the conclusion that this protease constitutes a central switch in the signalling pathway from N-cadherin at the cell surface to ß-catenin/LEF-1-regulated gene expression in the nucleus (Reiss, 2005).

Bone morphogenetic proteins (Bmps) are required for the specification of ventrolateral cell fates during embryonic dorsoventral patterning and for proper convergence and extension gastrulation movements, but the mechanisms underlying the latter role remained elusive. Via bead implantations, this study shows that the Bmp gradient determines the direction of lateral mesodermal cell migration during dorsal convergence in the zebrafish gastrula. This effect is independent of its role during dorsoventral patterning and of noncanonical Wnt signaling. However, it requires Bmp signal transduction through Alk8 and Smad5 to negatively regulate Ca2+/Cadherin-dependent cell-cell adhesiveness. In vivo, converging mesodermal cells form lamellipodia that attach to adjacent cells. Bmp signaling diminishes the Cadherin-dependent stability of such contact points, thereby abrogating subsequent cell displacement during lamellipodial retraction. It is proposed that the ventral-to-dorsal Bmp gradient has an instructive role to establish a reverse gradient of cell-cell adhesiveness, thereby defining different migratory zones and directing lamellipodia-driven cell migrations during dorsal convergence in lateral regions of the zebrafish gastrula (von der Hardt, 2007).

Cadherins and long-term potentiation

The cadherins are a family of cell-cell adhesion molecules that mediate Ca2+-dependent homophilic interactions between cells and transduce signals by interacting with cytoplasmic proteins. In the hippocampus, immunostaining combined with confocal microscopy has revealed that both neural- (N-) and epithelial- (E-) cadherin are present at synaptic sites, implying a role in synaptic function. Pretreatment of hippocampal slices with antibodies (Abs) raised against the extracellular domain of either N-cad or E-cad had no effect on basal synaptic properties but significantly reduces long-term potentiation (LTP). Infusion of antagonistic peptides containing the His-Ala-Val (HAV) consensus sequence for cadherin dimerization also attenuate LTP induction without affecting previously established LTP. Because the intense synaptic stimulation associated with LTP induction might transiently deplete extracellular Ca2+ and hence potentially destabilize cadherin-cadherin interactions, an examination was carried out to determine if slices could be protected from inhibition by N-cad Abs or HAV peptides by raising the extracellular Ca2+ concentration. Indeed, high extracellular Ca2+ prevents the block of LTP by these agents. Taken together, these results indicate that cadherins are involved in synaptic plasticity, and the stability of cadherin-cadherin bonds may be regulated by synaptic stimulation (Tang, 1998).

Cadherins, cell survival, cancer and apoptosis

Growth factor deprivation of endothelial cells induces apoptosis, which is characterized by membrane blebbing, cell rounding, and subsequent loss of cell-matrix and cell-cell contacts. In this study, it is shown that initiation of endothelial apoptosis correlates with cleavage and disassembly of intracellular and extracellular components of adherens junctions. beta-Catenin and plakoglobin, which form intracellular links between vascular endothelial cadherin (VE-cadherin) and actin-binding alpha-catenin in adherens junctions, are cleaved in apoptotic cells. In vitro incubations of cell lysates and immunoprecipitates with recombinant caspases indicate that CPP32 and Mch2 are involved, possibly by initiating proteolytic processing. Cleaved beta-catenin from lysates of apoptotic cells does not bind to endogenous alpha-catenin, whereas plakoglobin retains its binding capacity. The extracellular portion of the adherens junctions is also altered during apoptosis because VE-cadherin, which mediates endothelial cell-cell interactions, dramatically decreases on the surface of cells. An extracellular fragment of VE-cadherin can be detected in the conditioned medium, and this "shedding" of VE-cadherin can be blocked by an inhibitor of metalloproteinases. Thus, cleavage of beta-catenin and plakoglobin and shedding of VE-cadherin may act in concert to disrupt structural and signaling properties of adherens junctions and may actively interrupt extracellular signals required for endothelial cell survival (Herren, 1998).

Epithelial (E)-cadherin and its associated cytoplasmic proteins (alpha-, beta-, and gamma-catenins) are important mediators of epithelial cell-cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor- and invasion-suppressor role for E-cadherin, and loss of cadherin expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, 49 human colon carcinoma cell lines from 43 patients were screened by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, frameshift single-base deletions were detected in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhances intercellular adhesion, induces differentiation, retards proliferation, inhibits tumorigenicity, and restores responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide, a proteolysis resistent peptide implicated in the promotion of wound healing, stimulation of epithelial cell migration, and protection of the intestinal epithelial barrier). These results indicate that, although mutations that inactivate E-cadherin occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin-catenin-mediated adhesion and signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2 (Efstathiou, 1999).

The adhesion protein E-cadherin plays a central part in the process of epithelial morphogenesis. Expression of Human Snail protein is downregulated during the acquisition of metastatic potential at late stages of epithelial tumor progression. There is evidence for a transcriptional blockage of E-cadherin gene expression in this process. Snail, which is expressed by fibroblasts and some E-cadherin-negative epithelial tumor cell lines, binds to three E-boxes present in the human E-cadherin promoter and represses transcription of E-cadherin. The repressor effect is directly associated with the presence of the Snail zinc-finger domain. The functionality of the amino-terminal SNAG domain, a short domain common to all members of the vertebrate Snail family, was examined. The SNAG domain has been shown to mediate transcriptional-repressor features of both mouse Snail and Gfi-1. Snail proteins with point mutations (Sna-P2A) or deletions of the SNAG domain do not repress E-cadherin promoter activity in reporter assays, showing that integrity of both the zinc-finger and the SNAG domains are required for Snail's repressive activity at the E-cadherin promoter. Inhibition of Snail function in epithelial cancer cell lines lacking E-cadherin protein restores the expression of the E-cadherin gene (Batlle, 2000).

The Ca++-dependent cell adhesion molecule E-cadherin is expressed throughout mouse development and in adult tissues. Classical gene targeting has demonstrated that E-cadherin-deficient embryos die at the blastocyst stage. To study the involvement of E-cadherin in organogenesis, a conditional gene inactivation scheme was undertaken using the bacteriophage P1 recombinase Cre/loxP system. Mice with homozygous loxP sites in both alleles of the E-cadherin (Cdh1) gene were generated and these mice were crossed with transgenic mice with the Cre recombinase under the control of the hormone-inducible MMTV promoter. This resulted in deletion of the E-cadherin gene in the differentiating alveolar epithelial cells of the mammary gland. The mutant mammary gland developed normally up to 16-18 days of pregnancy but exhibited a dramatic phenotype around parturition. The production of milk proteins was so drastically reduced that adult mutant mothers could not suckle their offspring. Thus, the lack of E-cadherin affects the terminal differentiation program of the lactating mammary gland. In concordance with this finding, the prolactin-dependent activation of the transcription factor Stat5a is initiated but not maintained in the mutant gland. Instead, without E-cadherin massive cell death is observed at parturition and the mutant mammary gland at this stage resembles that of the involuted gland normally seen after weaning. These results demonstrate an essential role for E-cadherin in the function of differentiated alveolar epithelial cells. No tumors were detected in mutant glands lacking E-cadherin (Boussadia, 2002).

The APC tumor suppressor gene is mutated in most colon cancers. A major role of APC is the downregulation of the beta-catenin/T-cell factor (Tcf)/lymphoid enhancer factor (LEF) signalling pathway; however, there are also suggestions that it plays a role in the organization of the cytoskeleton, and in cell adhesion and migration. Stable expression of wild-type APC has been achieved in SW480 colon cancer cells, which normally express a truncated form of APC. The ectopically expressed APC is functional, and results in the translocation of beta-catenin from the nucleus and cytoplasm to the cell periphery, and reduces beta-catenin/Tcf/LEF transcriptional signalling. E-cadherin is also translocated to the cell membrane, where it forms functional adherens junctions. Total cellular levels of E-cadherin are increased in the SW480APC cells and the altered charge distribution in the presence of full-length APC suggests that APC is involved in post-translational regulation of E-cadherin localization. Changes in the location of adherens junction proteins are associated with tighter cell-cell adhesion in SW480APC cells, with consequent changes in cell morphology, the actin cytoskeleton and cell migration in a wound assay. SW480APC cells have a reduced proliferation rate, a reduced ability to form colonies in soft agar and do not grow tumors in a xenograft mouse tumor model. By regulating the intracellular transport of junctional proteins, it is proposed that APC plays a role in cell adhesion in addition to its known role in beta-catenin transcriptional signalling (Faux, 2003).

Metastasis is a multistep process during which cancer cells disseminate from the site of primary tumors and establish secondary tumors in distant organs. In a search for key regulators of metastasis in a murine breast tumor model, it was found that the transcription factor Twist, a master regulator of embryonic morphogenesis, plays an essential role in metastasis. Suppression of Twist expression in highly metastatic mammary carcinoma cells specifically inhibits their ability to metastasize from the mammary gland to the lung. Ectopic expression of Twist results in loss of E-cadherin-mediated cell-cell adhesion, activation of mesenchymal markers, and induction of cell motility, suggesting that Twist contributes to metastasis by promoting an epithelial-mesenchymal transition (EMT). In human breast cancers, high level of Twist expression is correlated with invasive lobular carcinoma, a highly infiltrating tumor type associated with loss of E-cadherin expression. These results establish a mechanistic link between Twist, EMT, and tumor metastasis (Yang, 2004).

Loss of E-cadherin appears to be critical to an EMT. One major mechanism for inhibiting E-cadherin expression involves silencing of E-cadherin transcription through three E-boxes in its promoter. An over 100-fold reduction of E-cadherin mRNA level is observed in HMEC cells expressing Twist. To test whether this transcriptional repression is achieved through the three E-boxes in the E-cadherin promoter, HMEC-Twist cells was transiently transfected with a reporter construct containing the luciferase gene (Luc) under the control of the human E-cadherin promoter. Indeed, Luc activity was efficiently suppressed in the HMEC-Twist cells compared to the HMEC-control cells. A similar degree of repression was also observed in the HMEC cells expressing Snail, a known repressor of E-cadherin expression. When two Luc reporter constructs in which the E-box elements had been mutated were introduced, the Luc activities were derepressed in the HMEC cells expressing Twist. These data indicate that Twist directly or indirectly causes the transcriptional repression of E-cadherin through the E-box elements on the E-cadherin promoter (Yang, 2004).

A betaPIX-PAK2 complex confers protection against Scrib-dependent and cadherin-mediated apoptosis

During epithelial morphogenesis, a complex comprising the βPIX (PAK-interacting exchange factor β) and class I PAKs (p21-activated kinases) is recruited to adherens junctions. Scrib, the mammalian ortholog of the Drosophila polarity determinant and tumor suppressor Scribble, binds βPIX directly. Scrib is also targeted to adherens junctions by E-cadherin, where Scrib strengthens cadherin-mediated cell-cell adhesion. Although a role for the Scrib-βPIX-PAK signaling complex in promoting membrane protrusion at wound edges has been elucidated, a function for this complex at adherens junctions remains unknown. This study establish, in cultured mammalian cells, that Scrib targets βPIX and PAK2 to adherens junctions where a βPIX-PAK2 complex counterbalances apoptotic stimuli transduced by Scrib and elicited by cadherin-mediated cell-cell adhesion. Moreover, it was shown that this signaling pathway regulates cell survival in response to osmotic stress. Finally, it was determined that in suspension cultures, the Scrib-βPIX-PAK2 complex functions to regulate anoikis elicited by cadherin engagement, with Scrib promoting and the βPIX-PAK2 complex suppressing anoikis, respectively. These findings demonstrate that the Scrib-βPIX-PAK2 signaling complex functions as an essential modulator of cell survival when localized to adherens junctions of polarized epithelia. The activity of this complex at adherens junctions is thereby essential for normal epithelial morphogenesis and tolerance of physiological stress. Furthermore, when localized to adherens junctions, the Scrib-βPIX-PAK2 signaling complex serves as a key determinant of anoikis sensitivity, a pivotal mechanism in tumor suppression. Thus, this work also reveals the need to expand the definition of anoikis to include a central role for adherens junctions (Frank, 2012).

This study has established that differentiated epithelial cells rely on a survival signaling network associated with adherens junctions that is distinct from that used at cell-matrix adhesions. Specifically, it was demonstrated that epithelial cells are critically dependent on Scrib-mediated localization of a βPIX-PAK2 complex to adherens junctions to counterbalance the apoptosis-promoting effects of E-cadherin engagement. As a result, disruption of βPIX-PAK2 signaling results in a near complete loss of epithelial viability at confluent density. Consistent with an established role in phosphorylating cellular targets involved in apoptosis, a functional kinase domain is required for PAK2-dependent survival signaling. In subconfluent cultures, where the βPIX-PAK2 complex localizes to focal adhesions, the complex is dispensable for cell survival. Thus, as epithelial cells form cell-cell junctions and polarize, they become critically dependent on a prosurvival signal provided by the βPIX-PAK2 complex at adherens junctions (Frank, 2012).

To date, the only direct evidence for the function of PAKs at adherens junctions is in the regulation of cadherin adhesiveness and actin-dependent cell contractility. While invoked by prior findings, the current results demonstrate for the first time that Scrib via its association with βPIX promotes targeting of PAK2 to adherens junctions. In cultured human keratinocytes, PAK1 has been reported to augment cadherin adhesiveness in response to activated Rac. PAK1 and PAK2 have also been reported to enhance and suppress, respectively, loss of cell-cell contacts in response to hepatocyte growth factor. The current results suggest that neither PAK1 nor PAK2 play a major role in steady state junctional integrity in MDCK cells. However, because MDCK cells express both PAK1 and PAK2, the possibility cannot be excluded that they may function redundantly in regulation of E-cadherin adhesiveness and/or junctional remodeling (Frank, 2012).

βPIX and PAKs have been most extensively characterized for their role in focal adhesion dynamics, where they play a coordinated role in regulating turnover of these integrin attachment sites and promoting directional motility. PAK1 and PAK2 appear to have nonredundant functions in cell invasion and motility. Moreover, there is evidence to suggest that when localized to focal adhesions, the βPIX-PAK complex promotes mitogenic signaling and that redistribution of the complex from focal adhesions to adherens junctions contributes to the cessation of epithelial proliferation and establishment of contact inhibition. Scrib also shuttles between adherens junctions and protrusive membrane structures where it promotes βPIX-PAK complex-dependent cell motility. It will be interesting to determine whether Scrib will play a role in redistribution of the βPIX-PAK2 complex from focal adhesions to adherens junctions, which occurs as cells undergo contact inhibition. In brief, taken together with the present work, these studies suggest that PAKs have distinct roles when localized to focal adhesions and adherens junctions. When localized to focal adhesions, PAKs participate in signaling pathways that control proliferation and motility, whereas at adherens junctions PAKs regulate adhesiveness and survival. As such, the translocation of the βPIX-PAK complex from to lateral membranes upon formation of stable adherens junctions likely plays a fundamental role in the transition from motile and mitogenic states to a nonmotile and quiescent state (Frank, 2012).

E-cadherin is a potent inhibitor of multiple signaling pathways and plays a fundamental role in suppression of motility and proliferation upon establishment of cell-cell contact. The ability of Scrib to promote E-cadherin adhesiveness may underlie some of its tumor suppressive potential in mammalian cells. However, increasing evidence suggests that Scrib also plays E-cadherin-independent roles in the regulation of signaling pathways, such as inhibition of ERK and AKT, as well as activation of Hippo signaling. These Scrib-dependent effects would be predicted to sensitize cells to apoptosis; a prediction supported by the present results, which furthermore establish that Scrib-mediated apoptosis is counterbalanced by its recruitment of active PAK2. Taken together, these findings demonstrate that Scrib transduces both pro- and anti-apoptotic stimuli (Frank, 2012).

Metastasis requires that cells tolerate the loss of matrix adhesion, i.e., that they are protected against anoikis. However, in spite of seminal work suggesting a role for cell-cell adhesion in modulating anoikis, the role for epithelial architecture in anoikis remains unappreciated and poorly characterized. Nevertheless, there is growing evidence indicating that loss of E-cadherin function suffices to abrogate anoikis. The current results support a key role of adherens junctions in modulating anoikis. In simple polarized epithelia, which likely rely on the summation of survival signals emanating from both focal adhesions and adherens junctions, apoptotic stimuli emanating from E-cadherin will sensitize cells to anoikis. In contrast, suprabasal cells in stratified epithelia depend exclusively on survival signals from adherens junctions. PAK2 is highly expressed in suprabasal keratinocytes, which may suggest that upregulation of PAK2-dependent survival signals emanating from adherens junctions are required to offset loss of integrin-mediated survival signaling. In summary, the current results indicate that E-cadherin does not function simply as a prosurvival or proapoptotic factor but rather as a regulatory node to coordinate death and survival signaling. The balance between pro- and antiapoptotic signaling emanating from adherens junctions plays a key role in epithelial cell viability and is likely essential in regulating diverse processes, including epithelial morphogenesis, wound healing, physiological stress, and metastasis (Frank, 2012).

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shotgun: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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