Morphology,cell-cell interactions,and migratory activity of IAR-2 epithelial cells transformed with the <Emphasis Type="Italic">RAS</Emphasis> oncogene: Contribution of cell adhesion protein E-Cadherin

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphological and migration characteristics of epithelial cells of IAR1162 and IAR1170 clones derived from a mixed culture of N-RasV12 oncogene-transformed IAR-2 cell line. It was found that the oncogenic RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, assembled E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the oncogenic RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of cell motility in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the motility of transformed epithelial cells and plays an important role in their collective migration.     

Rearrangements of the Actin Cytoskeleton and E-Cadherin–Based Adherens Junctions Caused by Neoplasic Transformation Change Cell–Cell Interactions

E-cadherin–mediated cell–cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell–cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer. Fluorescence staining, however, showed that in IAR1170 and IAR-6-1 cells the marginal actin bundle, which is typical of nontransformed IAR-2 cells, disappeared, and the continuous adhesion belt (tangential adherens junctions (AJs)) was replaced by radially oriented E-cadherin–based AJs. Time-lapse imaging of IAR-6-1 cells stably transfected with GFP-E-cadherin revealed that AJs in transformed cells are very dynamic and unstable. The regulation of AJ assembly by Rho family small GTPases was different in nontransformed and in transformed IAR epithelial cells. As our experiments with the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin have shown, the formation and maintenance of radial AJs critically depend on myosin II-mediated contractility. Using the RNAi technique for the depletion of mDia1 and loading cells with N17Rac, we established that mDia1 and Rac are involved in the assembly of tangential AJs in nontransformed epithelial cells but not in radial AJs in transformed cells. Neoplastic transformation changed cell–cell interactions, preventing contact paralysis after the establishment of cell–cell contact and promoting dynamic cell–cell adhesion and motile behavior of cells. It is suggested that the disappearance of the marginal actin bundle and rearrangements of AJs may change the adhesive function of E-cadherin and play an active role in migratory activity of carcinoma cells.     

Role of nectin in formation of E-cadherin-based adherens junctions in keratinocytes: analysis with the N-cadherin dominant negative mutant

E-cadherin is a Ca(2+)-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin-based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin-based AJs in keratinocytes.     

Regulation by nectin of the velocity of the formation of adherens junctions and tight junctions

Cadherins are key Ca(2+)-dependent cell-cell adhesion molecules at adherens junctions (AJs) in fibroblasts and epithelial cells, whereas claudins are key Ca(2+)-independent cell-cell adhesion molecules at tight junctions (TJs) in epithelial cells. The formation and maintenance of TJs are dependent on the formation and maintenance of AJs. Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules which comprise a family of four members, nectin-1, -2, -3, and -4, and are involved in the formation of AJs in cooperation with cadherins, and the subsequent formation of TJs. We show here that the velocity of the formation of the E-cadherin-based AJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in L cells stably expressing E-cadherin and Madin-Darby canine kidney cells. Moreover, the velocity of the formation of the claudin-based TJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in Madin-Darby canine kidney cells. These results indicate that nectins regulate the velocity of the formation of the E-cadherin-based AJs and the subsequent formation of the claudin-based TJs.     

A novel role of nectins in inhibition of the E-cadherin-induced activation of Rac and formation of cell-cell adherens junctions

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules. The trans-interactions of nectins recruit cadherins to the nectin-based cell-cell adhesion, resulting in formation of cell-cell adherens junctions (AJs) in epithelial cells and fibroblasts. The trans-interaction of E-cadherin induces activation of Rac small G protein, whereas the trans-interactions of nectins induce activation of not only Rac but also Cdc42 small G protein. We showed by the fluorescent resonance energy transfer (FRET) imaging that the trans-interaction of E-cadherin induced dynamic activation and inactivation of Rac, which led to dynamic formation and retraction of lamellipodia. Moreover, we found here that the nectins, which did not trans-interact with other nectins (non-trans-interacting nectins), inhibited the E-cadherin-induced activation of Rac and reduced the velocity of the formation of the E-cadherin-based cell-cell AJs. The inhibitory effect of non-trans-interacting nectins was suppressed by the activation of Cdc42 induced by the trans-interactions of nectins. These results indicate a novel role of nectins in regulation of the E-cadherin-induced activation of Rac and formation of cell-cell AJs.     

Involvement of the annexin II-S100A10 complex in the formation of E-cadherin-based adherens junctions in Madin-Darby canine kidney cells

E-cadherin and nectins are major cell-cell adhesion molecules at adherens junctions (AJs) in epithelial cells. When Madin-Darby canine kidney (MDCK) cells stably expressing nectin-1 (nectin-1-MDCK cells) are cultured at normal Ca(2+), E-cadherin and nectin-1 are concentrated at the cell-cell contact sites. When these cells are cultured at low Ca(2+), E-cadherin disappears from the cell-cell contact sites, but nectin-1 persists there. When these cells are re-cultured at normal Ca(2+), E-cadherin is recruited to the nectin-based cell-cell contact sites. We found here that this recruitment was dependent on protein synthesis, because a protein synthesis inhibitor, cycloheximide, prevented the accumulation of E-cadherin. When nectin-1-MDCK cells, precultured at low Ca(2+) in the presence of a proteasome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal), were re-cultured at normal Ca(2+), E-cadherin was recruited to the nectin-based cell-cell contact sites but the level of E-cadherin was reduced. Similar results were obtained when wild-type MDCK cells were used instead of nectin-1-MDCK cells. These results suggest that degradation of one or more protein factors and de novo synthesis of the same or different protein factor(s) are needed for the formation of the E-cadherin-based AJs. We biochemically identified the annexin II-S100A10 complex as such a candidate. Depletion of plasma membrane cholesterol, which abolished the localization of the annexin II-S100A10 complex at the plasma membrane, inhibited the re-concentration of E-cadherin at the nectin-based cell-cell contact sites in the Ca(2+) switch experiment. Knockdown of annexin II by RNA interference also inhibited the re-concentration of E-cadherin. These results indicate that the annexin II-S100A10 complex is involved in the formation of the E-cadherin-based AJs in MDCK cells.     

p120ctn and P-Cadherin but Not E-Cadherin Regulate Cell Motility and Invasion of DU145 Prostate Cancer Cells

Background

Adherens junctions consist of transmembrane cadherins, which interact intracellularly with p120ctn, ß-catenin and α-catenin. p120ctn is known to regulate cell-cell adhesion by increasing cadherin stability, but the effects of other adherens junction components on cell-cell adhesion have not been compared with that of p120ctn.

Methodology/Principal Findings

We show that depletion of p120ctn by small interfering RNA (siRNA) in DU145 prostate cancer and MCF10A breast epithelial cells reduces the expression levels of the adherens junction proteins, E-cadherin, P-cadherin, ß-catenin and α-catenin, and induces loss of cell-cell adhesion. p120ctn-depleted cells also have increased migration speed and invasion, which correlates with increased Rap1 but not Rac1 or RhoA activity. Downregulation of P-cadherin, β-catenin and α-catenin but not E-cadherin induces a loss of cell-cell adhesion, increased migration and enhanced invasion similar to p120ctn depletion. However, only p120ctn depletion leads to a decrease in the levels of other adherens junction proteins.

Conclusions/Significance

Our data indicate that P-cadherin but not E-cadherin is important for maintaining adherens junctions in DU145 and MCF10A cells, and that depletion of any of the cadherin-associated proteins, p120ctn, ß-catenin or α-catenin, is sufficient to disrupt adherens junctions in DU145 cells and increase migration and cancer cell invasion.     

N-glycosylation affects the adhesive function of E-Cadherin through modifying the composition of adherens junctions (AJs) in human breast carcinoma cell line MDA-MB-435

E-cadherin mediates calcium-dependent cell-cell adhesion between epithelial cells. The ectodomain of human E-cadherin contains four potential N-glycosylation sites at Asn residues 554, 566, 618, and 633. In this study, the role of N-glycosylation in E-cadherin-mediated cell-cell adhesion was investigated by site-directed mutagenesis. In MDA-MB-435 cells, all four potential N-glycosylation sites of human E-cadherin were N-glycosylated. Removal of N-glycan at Asn-633 dramatically affected E-cadherin stability. In contrast, mutant E-cadherin lacking the other three N-glycans showed similar protein stability in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn-554 and Asn-566 were found to affect E-cadherin-mediated calcium-dependent cell-cell adhesion, and removal of either of the two N-glycans caused a significant decrease in calcium-dependent cell-cell adhesion accompanied with elevated cell migration. Analysis of the composition of adherens junctions (AJs) revealed that removal of N-glycans on E-cadherin resulted in elevated tyrosine phosphorylation level of beta-catenin and reduced beta- and alpha-catenins at AJs. These findings demonstrate that N-glycosylation may affect the adhesive function of E-cadherin through modifying the composition of AJs.     

Formation of E-cadherin/beta-catenin-based adherens junctions in hepatocytes requires serine-10 in p27(Kip1)

The adhesion between epithelial cells at adherens junctions is regulated by signaling pathways that mediate the intracellular trafficking and assembly of its core components. Insight into the molecular mechanisms of this is necessary to understand how adherens junctions contribute to the functional organization of epithelial tissues. Here, we demonstrate that in human hepatic HepG2 cells, oncostatin M-p42/44 mitogen-activated protein kinase signaling stimulates the phosphorylation of p27(Kip1) on Ser-10 and promotes cell-cell adhesion. The overexpression of wild-type p27 or a phospho-mimetic p27S10D mutant in HepG2 cells induces a hyper-adhesive phenotype. In contrast, the overexpression of a nonphosphorylatable p27S10A mutant prevents the mobilization of E-cadherin and beta-catenin at the cell surface, reduces basal cell-cell adhesion strength, and prevents the stimulatory effect of oncostatin M on cell-cell adhesion. As part of the underlying molecular mechanism, it is shown that in p27S10A-expressing cells beta-catenin interacts with p27 and is prevented from interacting with E-cadherin. The intracellular retention of E-cadherin and beta-catenin is also observed in hepatocytes from p27S10A knockin mice that express the p27S10A mutant instead of wild-type p27. Together, these data suggest that the formation of adherens junctions in hepatocytes requires Ser-10 in p27.     

Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia

Transformed epithelial cells often are characterized by a fibroblastic or mesenchymal morphology. These cells exhibit altered cell-cell and cell-substrate interactions. Here we have identified changes in the adhesions and cytoskeletal interactions of transformed epithelial cells that contribute to their altered morphology. Using MCF-10A human breast epithelial cells as a model system, we have found that transformation by an activated form of ras is characterized by less developed adherens- type junctions between cells but increased focal adhesions. Contributing to the modified adherens junctions of the transformed cells are decreased interactions among beta-catenin, E-cadherin, and the actin cytoskeleton. The ras-transformed cells reveal elevated phosphotyrosine in many proteins, including beta-catenin and p120 Cas. Whereas in the normal cells beta-catenin is found in association with E- cadherin, p120 Cas is not. In the ras-transformed cells, the situation is reversed; tyrosine-phosphorylated p120 Cas, but not tyrosine- phosphorylated beta-catenin, now is detected in E-cadherin complexes. The tyrosine-phosphorylated beta-catenin also shows increased detergent solubility, suggesting a decreased association with the actin cytoskeleton. p120 Cas, whether tyrosine phosphorylated or not, partitions into the detergent soluble fraction, suggesting that it is not tightly bound to the actin cytoskeleton in either the normal or ras- transformed cells. Inhibitors of tyrosine kinases decrease the level of tyrosine phosphorylation and restore a normal epithelial morphology to the ras-transformed cells. In particular, decreased tyrosine phosphorylation of beta-catenin is accompanied by increased interaction with both E-cadherin and the detergent insoluble cytoskeletal fraction. These results suggest that elevated tyrosine phosphorylation of proteins such as beta-catenin and p120 Cas contribute to the altered adherens junctions of ras-transformed epithelia.     

Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts

In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations.     

αE-catenin regulates cell-cell adhesion and membrane blebbing during zebrafish epiboly

αE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of αE-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of αE-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of αE-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of αE-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of αE-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and αE-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and αE-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.     

Phosphorylation and free pool of beta-catenin are regulated by tyrosine kinases and tyrosine phosphatases during epithelial cell migration

Cell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic. Although the integrity of cell-cell contacts, such as adherens junctions, is essential for the maintenance of functional epithelia, they need to be rapidly disassembled during migration. The transmembrane cell adhesion protein E-cadherin and the cytoplasmic catenins are molecular elements of these structures. Here we demonstrate that epithelial cell migration is accompanied by tyrosine phosphorylation of beta-catenin and an increase of its free cytoplasmic pool. We show further that the protein-tyrosine phosphatase LAR (leukocyte common antigen related) colocalizes with the cadherin-catenin complex in epithelial cells and associates with beta-catenin and plakoglobin. Interestingly, ectopic expression of protein-tyrosine phosphatase (PTP) LAR inhibits epithelial cell migration by preventing phosphorylation and the increase in the free pool of beta-catenin; moreover, it inhibits tumor formation in nude mice. These data support a function for PTP LAR in the regulation of epithelial cell-cell contacts at adherens junctions as well as in the control of beta-catenin signaling functions. Thus PTP-LAR appears to play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may contribute to malignant progression and metastasis.     

Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.     

Flotillins Directly Interact with γ-Catenin and Regulate Epithelial Cell-Cell Adhesion

Flotillin-1 and flotillin-2 are two homologous, membrane raft associated proteins. Although it has been reported that flotillins are involved in cell adhesion processes and play a role during breast cancer progression, thus making them interesting future therapeutic targets, their precise function has not been well elucidated. The present study investigates the function of these proteins in cell-cell adhesion in non-malignant cells. We have used the non-malignant epithelial MCF10A cells to study the interaction network of flotillins within cell-cell adhesion complexes. RNA interference was used to examine the effect of flotillins on the structure of adherens junctions and on the association of core proteins, such as E-cadherin, with membrane rafts. We here show that the cadherin proteins of the adherens junction associate with flotillin-2 in MCF10A cells and in various human cell lines. In vitro, flotillin-1 and flotillin-2 directly interact with γ-catenin which is so far the only protein known to be present both in the adherens junction and the desmosome. Mapping of the interaction domain within the γ-catenin sequence identified the Armadillo domains 6–8, especially ARM domain 7, to be important for the association with flotillins. Furthermore, depletion of flotillins significantly influenced the morphology of the adherens junction in human epithelial MCF10A cells and altered the association of E-cadherin and γ-catenin with membrane rafts. Taken together, these observations suggest a functional role for flotillins, especially flotillin-2, in cell-cell adhesion in non-malignant epithelial cells.     

Phosphatidylinositol 3-kinase is required for adherens junction-dependent mammary epithelial cell spheroid formation

Adherens junctions facilitate and maintain epithelial cell-cell adhesion. This is true of mammary epithelial cells, both in two dimensional monolayers and in three-dimensional basement membrane cultures. Using the immortalized, functional mouse mammary epithelial scp2 cell line, we found that pharmacological inhibition of phosphatidylinositol 3-kinase (PI3-kinase) disrupted adherens junctions. In monolayers, this disruption was associated with decreased E-cadherin and beta-catenin at sites of cell-cell contact and decreased association of both proteins with the cytoskeleton. Changes in the distribution of f-actin after PI3-kinase inhibition suggest that this disruption of adherens junctions may be mediated by alterations to the cytoskeleton. In basement membrane cultures, PI3-kinase inhibition reversibly prevented adherens junction-dependent spheroid formation and differentiative milk protein gene expression, both in scp2 cells and in a second mouse mammary epithelial cell line, EpH4. Decreasing the calcium concentration in the culture medium produced similar, although less dramatic, phenotypic effects. These data indicate that adherens junctions contribute, at least in part, to the efficient induction of basement membrane-dependent differentiation of mammary epithelial cells.     

The receptor tyrosine phosphatase Lar regulates adhesion between Drosophila male germline stem cells and the niche

The stem cell niche provides a supportive microenvironment to maintain adult stem cells in their undifferentiated state. Adhesion between adult stem cells and niche cells or the local basement membrane ensures retention of stem cells in the niche environment. Drosophila male germline stem cells (GSCs) attach to somatic hub cells, a component of their niche, through E-cadherin-mediated adherens junctions, and orient their centrosomes toward these localized junctional complexes to carry out asymmetric divisions. Here we show that the transmembrane receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar), which is best known for its function in axonal migration and synapse morphogenesis in the nervous system, helps maintain GSCs at the hub by promoting E-cadherin-based adhesion between hub cells and GSCs. Lar is expressed in GSCs and early spermatogonial cells and localizes to the hub-GSC interface. Loss of Lar function resulted in a reduced number of GSCs at the hub. Lar function was required cell-autonomously in germ cells for proper localization of Adenomatous polyposis coli 2 and E-cadherin at the hub-GSC interface and for the proper orientation of centrosomes in GSCs. Ultrastructural analysis revealed that in Lar mutants the adherens junctions between hub cells and GSCs lack the characteristic dense staining seen in wild-type controls. Thus, the Lar receptor tyrosine phosphatase appears to polarize and retain GSCs through maintenance of localized E-cadherin-based adherens junctions.     

Early enterocytic differentiation of HT-29 cells: biochemical changes and strength increases of adherens junctions

We have characterized the modulation of cell-cell adhesion and the structure of adherens junctions in the human colon adenocarcinoma HT-29 cell line that differentiates into enterocytes after glucose substitution for galactose in the medium. We demonstrate that differentiated cells (HT-29 Gal) rapidly established E-cadherin-mediated interactions in aggregation assays. This effect is not due to an increase in E-cadherin expression during this early stage of cell differentiation, but rather results from the maturation of preexisting adherens junctions. These junctions are characterized by the redistribution of E-cadherin to the basolateral membrane and its co-localization with the actin cytoskeleton. Subcellular fractionation studies indicate that actin-associated E-cadherins bind beta-catenin and p120ctn. Furthermore, the p120ctn/E-cadherin association is upregulated. These data reveal a cooperative interaction between p120ctn and E-cadherin that corresponds to mature functional adherens junctions able to initiate tight cell-cell adhesion required for epithelium architecture and further affirm the gatekeeper role of p120ctn.     

Inducible expression of p120Cas1B isoform corroborates the role for p120-catenin as a positive regulator of E-cadherin function in intestinal cancer cells

Over the past decade, the exact function of p120-catenin in regulation of E-cadherin/catenins complex has remained particularly controversial. We have previously reported that E-cadherin-mediated adhesion is tightly regulated by tyrosine phosphorylation of catenins. However, this effect is not observed in human colon carcinoma cell line Caco-2. Here, we have generated inducible Caco-2 clones that display p120Cas1B, a p120-catenin isoform poorly expressed by these cells. As a result, neither expression of the transgene nor tyrosine phosphorylation of catenins induces redistribution of E-cadherin to the cytosol and disassembly of adherens and tight junctions. In contrast, E-cadherin appears markedly increased reinforcing cell-cell adhesion. Interestingly, a substantial decrease in p120-catenin levels is found in MDCK cells expressing Snail, in which E-cadherin expression is strongly inhibited. Additionally, we show that the specific depletion of p120-catenin decreases cell-cell contacts, and increases cell motility and scattering of colonies established by HT-29 M6 cells. Together our results corroborate that p120-catenin plays an essential role in the maintenance of the required E-cadherin protein levels that prevent the loss of epithelial characteristics occurred during tumorigenesis.     

Differential modulation of cadherin-mediated cell-cell adhesion by platelet endothelial cell adhesion molecule-1 isoforms through activation of extracellular regulated kinases

The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell-cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell-cell interactions. MDCK cells lack endogenous PECAM-1 but form cell-cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Delta15 and Delta14&15, the predominant isoforms expressed in vivo. Expression of the Delta15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, alpha- and beta-catenin expression, and sustained activation of extracellular regulated kinases. The Delta15 isoform was not concentrated at cell-cell contacts. In contrast, the Delta14&15 isoform localized to sites of cell-cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.