Pervanadate stabilizes desmosomes

Desmosomes are intercellular junctions responsible for strong cell-cell adhesion in epithelia and cardiac muscle. Numerous studies have shown that the other major type of epithelial cell adhesion, the adherens junction, is destabilized by src-induced tyrosine phosphorylation of two of its principal components, E-cadherin and β-catenin. Here we show that treatment of epithelial cells with the potent tyrosine phosphatase inhibitor sodium pervanadate causes tyrosine phosphorylation of the major desmosomal components desmoglein 2 and plakoglobin in both the non-ionic detergent soluble and insoluble cell fractions and, surprisingly, stabilizes desmosomal adhesion, inducing the hyper-adhesive form normally found in tissues and confluent cell sheets. Taken together with the few other studies on desmosomes these results suggest that the effects of tyrosine phosphorylation on desmosomal adhesion are complex.Key words: desmosome, cell-cell adhesion, intercellular junction, tyrosine phosphorylation, pervanadate, desmoglein, plakoglobin     

Pervanadate stabilizes desmosomes

Desmosomes are intercellular junctions responsible for strong cell-cell adhesion in epithelia and cardiac muscle. Numerous studies have shown that the other major type of epithelial cell adhesion, the adherens junction, is destabilized by src-induced tyrosine phosphorylation of two of its principal components, E-cadherin and β-catenin. Here we show that treatment of epithelial cells with the potent tyrosine phosphatase inhibitor sodium pervanadate causes tyrosine phosphorylation of the major desmosomal components desmoglein 2 and plakoglobin in both the non-ionic detergent soluble and insoluble cell fractions and, surprisingly, stabilizes desmosomal adhesion, inducing the hyper-adhesive form normally found in tissues and confluent cell sheets. Taken together with the few other studies on desmosomes these results suggest that the effects of tyrosine phosphorylation on desmosomal adhesion are complex.     

Real-time monitoring of epithelial cell-cell and cell-substrate interactions by infrared surface plasmon spectroscopy

The development of novel technologies capable of monitoring the dynamics of cell-cell and cell-substrate interactions in real time and a label-free manner is vital for gaining deeper insights into these most fundamental cellular processes. However, the label-free technologies available today provide only limited information on these processes. Here, we report a new (to our knowledge) infrared surface plasmon resonance (SPR)-based methodology that can resolve distinct phases of cell-cell and cell-substrate adhesion of polarized Madin Darby canine kidney epithelial cells. Due to the extended penetration depth of the infrared SP wave, the dynamics of cell adhesion can be detected with high accuracy and high temporal resolution. Analysis of the temporal variation of the SPR reflectivity spectrum revealed the existence of multiple phases in epithelial cell adhesion: initial contact of the cells with the substrate (cell deposition), cell spreading, formation of intercellular contacts, and subsequent generation of cell clusters. The final formation of a continuous cell monolayer could also be sensed. The SPR measurements were validated by optical microscopy imaging. However, in contrast to the SPR method, the optical analyses were laborious and less quantitative, and hence provided only limited information on the dynamics and phases of cell adhesion.     

Cell-cell contact preserves cell viability via plakoglobin

Control over cell viability is a fundamental property underlying numerous physiological processes. Cell spreading on a substrate was previously demonstrated to be a major factor in determining the viability of individual cells. In multicellular organisms, cell-cell contact is likely to play a significant role in regulating cell vitality, but its function is easily masked by cell-substrate interactions, thus remains incompletely characterized. In this study, we show that suspended immortalized human keratinocyte sheets with persisting intercellular contacts exhibited significant contraction, junctional actin localization, and reinforcement of cell-cell adhesion strength. Further, cells within these sheets remain viable, in contrast to trypsinized cells suspended without either cell-cell or cell-substrate contact, which underwent apoptosis at high rates. Suppression of plakoglobin weakened cell-cell adhesion in cell sheets and suppressed apoptosis in suspended, trypsinized cells. These results demonstrate that cell-cell contact may be a fundamental control mechanism governing cell viability and that the junctional protein plakoglobin is a key regulator of this process. Given the near-ubiquity of plakoglobin in multicellular organisms, these findings could have significant implications for understanding cell adhesion, modeling disease progression, developing therapeutics and improving the viability of tissue engineering protocols.     

Evidence for a Role of the Epithelial Glycoprotein 40 (Ep-CAM) in Epithelial Cell-Cell Adhesion

Recently we have demonstrated that a 40kD human epithelium-specific glycoprotein exhibits the features of a homophilic cell-cell adhesion molecule, when expressed in transfected murine cells. We suggested the name Ep-CAM for this molecule (Litvinov et al., J. Cell Biol., 125: 437–446). Here we investigate the possible biological function of Ep-CAM in its natural environment—cells of epithelial origin. Immunolocalization of Ep-CAM in tissues and in cultures of epithelial/carcinoma cells showed that the majority of the Ep-CAM molecules are localized at cell-cell boundaries, predominantly along the whole lateral domain of polarized cells. In vitro, on single cells in suspension, the Ep-CAM molecules are present on the entire cell surface, and when the single cells grow attached, Ep-CAM is present at their pseudo-apical domain. During formation of intercellular contacts by such single cells, the majority of the Ep-CAM molecules are redistributed from the pseudoapical to the lateral domain of the cell membrane. Attachment of cells to the substrate does not cause redistribution of the molecules to the site of substrate attachment irrespective of the adhesive substrate (fibronectin, collagens, laminin, EHS-matrigel were tested). The monoclonal antibody 323/A3, reactive with the extracellular domain of the Ep-CAM molecule, has a strong negative effect on the aggregating behaviour of COV362 ovarian carcinoma cells and RC-6 immortalized mammary epithelial cells. The mAb affected cell aggregation in both cell lines in the presence of Ca⁺⁺, but with RC-6 cells the effect was more pronounced in low-calcium medium. The effects of the 323/A3 mAb on the already established intercellular contacts was not significant. The data presented demonstrate that the Ep-CAM molecules are functionally active in the epithelial and carcinoma cells tested, are capable of mediating Ca¹⁺-independent intercellular adhesions, and are not likely to be involved in cell-substrate adhesion.     

Evidence for a Role of the Epithelial Glycoprotein 40 (Ep-CAM) in Epithelial Cell-Cell Adhesion

Recently we have demonstrated that a 40kD human epithelium-specific glycoprotein exhibits the features of a homophilic cell-cell adhesion molecule, when expressed in transfected murine cells. We suggested the name Ep-CAM for this molecule (Litvinov et al., J. Cell Biol., 125: 437-446). Here we investigate the possible biological function of Ep-CAM in its natural environment—cells of epithelial origin. Immunolocalization of Ep-CAM in tissues and in cultures of epithelial/carcinoma cells showed that the majority of the Ep-CAM molecules are localized at cell-cell boundaries, predominantly along the whole lateral domain of polarized cells. In vitro, on single cells in suspension, the Ep-CAM molecules are present on the entire cell surface, and when the single cells grow attached, Ep-CAM is present at their pseudo-apical domain. During formation of intercellular contacts by such single cells, the majority of the Ep-CAM molecules are redistributed from the pseudoapical to the lateral domain of the cell membrane. Attachment of cells to the substrate does not cause redistribution of the molecules to the site of substrate attachment irrespective of the adhesive substrate (fibronectin, collagens, laminin, EHS-matrigel were tested). The monoclonal antibody 323/A3, reactive with the extracellular domain of the Ep-CAM molecule, has a strong negative effect on the aggregating behaviour of COV362 ovarian carcinoma cells and RC-6 immortalized mammary epithelial cells. The mAb affected cell aggregation in both cell lines in the presence of Ca⁺⁺, but with RC-6 cells the effect was more pronounced in low-calcium medium. The effects of the 323/A3 mAb on the already established intercellular contacts was not significant. The data presented demonstrate that the Ep-CAM molecules are functionally active in the epithelial and carcinoma cells tested, are capable of mediating Ca¹⁺-independent intercellular adhesions, and are not likely to be involved in cell-substrate adhesion.     

Cell-cell interactions mediate the response of vascular smooth muscle cells to substrate stiffness

The vessel wall experiences progressive stiffening with age and the development of cardiovascular disease, which alters the micromechanical environment experienced by resident vascular smooth muscle cells (VSMCs). In vitro studies have shown that VSMCs are sensitive to substrate stiffness, but the exact molecular mechanisms of their response to stiffness remains unknown. Studies have also shown that cell-cell interactions can affect mechanotransduction at the cell-substrate interface. Using flexible substrates, we show that the expression of proteins associated with cell-matrix adhesion and cytoskeletal tension is regulated by substrate stiffness, and that an increase in cell density selectively attenuates some of these effects. We also show that cell-cell interactions exert a strong effect on cell morphology in a substrate-stiffness dependent manner. Collectively, the data suggest that as VSMCs form cell-cell contacts, substrate stiffness becomes a less potent regulator of focal adhesion signaling. This study provides insight into the mechanisms by which VSMCs respond to the mechanical environment of the blood vessel wall, and point to cell-cell interactions as critical mediators of VSMC response to vascular injury.     

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.     

The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin

Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified beta-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of beta-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including beta-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion.     

Modulation of intercellular adherens-type junctions and tyrosine phosphorylation of their components in RSV-transformed cultured chick lens cells.

Transformation of cultured chick lens epithelial cells with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV) leads to radical changes in cell shape and interactions. When cultured at the restrictive temperature (42 degrees C), the transformed cells largely retained epithelial morphology and intercellular adherens junctions (AJ), whereas on switch to the permissive temperature (37 degrees C) they rapidly became fibroblastoid, their AJ deteriorated, and cell adhesion molecules (A-CAM) (N-cadherin) largely disappeared from intercellular contact sites. The microfilament system that was primarily associated with these junctions was markedly rearranged on shift to 37 degrees C and remained associated mainly with cell-substrate focal contacts. These apparent changes in intercellular AJ were not accompanied by significant alterations in the cellular content of several junction-associated molecules, including A-CAM, vinculin, and talin. Immunolabeling with phosphotyrosine-specific antibodies indicated that both cell-substrate and intercellular AJ were the major cellular targets for the pp60v-src tyrosine-specific protein kinase. It was further shown that intercellular AJ components serve as substrates to tyrosine kinases also in nontransformed lens cells, because the addition of a combination of vanadate and H2O2--which are potent inhibitors of protein tyrosine phosphatases--leads to a remarkable accumulation of immunoreactive phosphotyrosine-containing proteins in these junctions. This finding suggests that intercellular junctions are major sites of action of protein tyrosine kinases and that protein tyrosine phosphatases play a major role in the regulation of phosphotyrosine levels in AJ of both normal and RSV-transformed cells.     

The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

Plakoglobin (PG) is an armadillo protein that associates with both classic and desmosomal cadherins, but is primarily concentrated in mature desmosomes in epithelia. While reduced levels of PG have been reported in localized and hormone refractory prostate tumors, the functional significance of these changes is unknown. Here we report that PG expression is reduced in samples of a prostate tumor tissue array and inversely correlated with advancing tumor potential in 7 PCa cell lines. Ectopically expressed PG enhanced intercellular adhesive strength, and attenuated the motility and invasion of aggressive cell lines, whereas silencing PG in less tumorigenic cells had the opposite effect. PG also regulated cell-substrate adhesion and motility through extracellular matrix (ECM)-dependent inhibition of Src kinase, suggesting that PG's effects were not due solely to increased intercellular adhesion. PG silencing resulted in elevated levels of the ECM protein vitronectin (VN), and exposing PG-expressing cells to VN induced Src activity. Furthermore, increased VN levels and Src activation correlated with diminished expression of PG in patient tissues. Thus, PG may inhibit Src by keeping VN low. Our results suggest that loss of intercellular adhesion due to reduced PG expression might be exacerbated by activation of Src through a PG-dependent mechanism. Furthermore, PG down-regulation during PCa progression could contribute to the known VN-dependent promotion of PCa invasion and metastasis, demonstrating a novel functional interaction between desmosomal cell-cell adhesion and cell-substrate adhesion signaling axes in prostate cancer.     

Effects of the extracellular matrix on fetal choroid plexus epithelial cells: changes in morphology and multicellular organization do not affect gene expression.

We have developed a primary culture system for fetal mouse choroid plexus epithelial cells which maintains their differentiated phenotype. When grown on a reconstituted basement membrane substrate (Matrigel) epithelial cells formed aggregates which became embedded in the matrix and developed into characteristic and highly reproducible multicellular vesicular structures. These vesicles consisted of a squamous layer of epithelial cells with extensive attachment to the matrix substrate, surrounding a fluid-filled lumen. Electron microscopy showed that cells comprising these vesicles had a high degree of membrane specialization and polarized morphology which in many respects mimicked the in vivo morphology. Biochemical analyses demonstrated that under these culture conditions the tissue-specific pattern of gene expression of fetal choroid plexus epithelium was maintained. After 6 days in culture these cells contained approximately the same amount of transthyretin mRNA as the 12.5-day choroid plexus in vivo, and the level of total RNA per cell, which is proportional to the protein synthetic capability of the cells, was also maintained. The pattern of protein secretion was also very similar to that generated by fetal mouse choroid plexus cells in vivo. In contrast choroid plexus epithelial cells attached poorly to collagen I gels. Heterogeneous aggregates were formed in which cell-cell interactions were more extensive than cell-substrate interactions, and in no cases was a central lumen observed. Cells on the surface of large aggregates showed some evidence of membrane polarization, while the majority of cells in the cultures exhibited little evidence of polarized morphology. Despite the striking difference in morphology and multicellular organization these cells still expressed high levels of transthyretin mRNA and maintained the same pattern of protein synthesis as cells cultured on Matrigel. These results indicate that the basement membrane is important for the organization of choroid plexus epithelial cells into a functional epithelium in vitro and thus presumably the maintenance of the integrity of the blood-brain barrier in vivo. In contrast to several other epithelial systems which have been studied, the type of extracellular matrix does not appear to directly influence tissue-specific gene expression by choroid plexus epithelial cells. Thus the level of gene expression is not dependent on the cytoarchitecture and multicellular organization of this cell type.     

Phosphotyrosine signalling as a regulator of neural crest cell adhesion and motility

We demonstrate that neural crest cell-cell adhesion, cell-substrate adhesion, and ultimately cell motility, are highly dependent on the balanced action of tyrosine kinases and tyrosine phosphatases. Neural crest cell migration on fibronectin is diminished in the presence of the tyrosine phosphatase inhibitor vanadate or tyrosine kinase inhibitor herbimycin A, while cadherin-rich cell-cell adhesions are significantly increased. In contrast, cells treated with the kinase inhibitor genistein have decreased motility, rearrange rapidly and reversibly into a pavement-like monolayer, but have no increase in cadherin interactions. Genistein-sensitive tyrosine kinases may therefore abrogate a latent sensitivity of neural crest cells to contact-mediated inhibition of movement. Furthermore, we show that the activity of herbimycin A-sensitive kinases is necessary for focal adhesion formation in these cells. Moreover, the size and distribution of these adhesions are acutely sensitive to the actions of tyrosine phosphatases and genistein-sensitive kinases. We propose that in migrating neural crest cells there is a balance in phosphotyrosine signalling which minimises both cell-cell adhesion and contact inhibition of movement, while enhancing dynamic cell-substrate interactions and thus the conditions for motility.     

ADAM-15/metargidin mediates homotypic aggregation of human T lymphocytes and heterotypic interactions of T lymphocytes with intestinal epithelial cells

Intestinal epithelial cells (IEC) play an immunoregulatory role in the intestine. This role involves cell-cell interactions with intraepithelial lymphocytes that may also play a role in some enteropathies. The discovery of the RGD motif-containing Protein ADAM-15 (a disintegrin and metalloprotease-15) raises the question of its involvement in these cell-cell interactions. Cell adhesion assays were performed using the Jurkat E6.1 T cell line as a model of T lymphocytes and Caco2-BBE monolayers as a model of intestinal epithelia. Our results show that an anti-ADAM-15 ectodomain antibody inhibited the attachment of Jurkat cells on Caco2-BBE monolayers. Overexpression of ADAM-15 in Caco2-BBE cells enhanced Jurkat cell binding, and overexpression of ADAM-15 in Jurkat cells enhanced their aggregation. Mutagenesis experiments showed that both the mutation of ADAM-15 RGD domain or the deletion of its cytoplasmic tail decreased these cell-cell interactions. Moreover, wound-healing experiments showed that epithelial ADAM-15-mediated Jurkat cell adhesion to Caco2-BBE cells enhances the mechanisms of wound repair. We also found that ADAM-15-mediated aggregation of Jurkat cells increases the expression of tumor necrosis factor-alpha mRNA. These results demonstrate the following: 1) ADAM-15 is involved in heterotypic adhesion of intraepithelial lymphocytes to IEC as well as in homotypic aggregation of T cells; 2) both the RGD motif and the cytoplasmic tail of ADAM-15 are involved for these cell-cell interactions; and 3) ADAM-15-mediated cell-cell interactions are involved in mechanisms of epithelial restitution and production of pro-inflammatory mediators. Altogether these findings point to ADAM-15 as a possible therapeutic target for prevention of inappropriate T cell activation involved in some pathologies.     

Morphology, cell-cell interactions, and migratory activity of IAR-2 epithelial cells transformed with the RAS 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 morphologic and mi gration characteristics of epithelial cells of Iar1162 and IAR1170 clones derived from a mixed culture of on cogene N-RasV12-transformed cell line IAR-2. It was found that the mutant oncogene 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, formed E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the mutant oncogene 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. IAR 1170 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 locomotor activity in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the locomotor activity of transformed epithelial cells and plays an important role in their collective migration.     

Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members

To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.     

Experimentally induced murine Rhabdomyosarcomas-correlation between cellular contacts, matrix formation and cellular differentiation

Rhabdomyosarcomas (RMSs) consist of a mixture of primitive mesenchymal cells as well as cells showing various stages of rhabdomyomatous differentiation. The qualitative and quantitative degree of the rhabdomyomatous differentiation of the cells, evaluated by their morphology and expression of defined structural and functional proteins, is accepted as the basis of diagnosis and is considered to be related to the biological behaviour of RMSs. Therefore we investigated solid experimentally induced murine RMSs, adherent (subconfluent, confluent) cell cultures obtained therefrom, and also suspension cultures and studied the expression of muscular differentiation markers (vimentin, desmin, myoglobin) and the formation of extracellular matrix components (fibronectin, laminin). When we compared solid tumours with adherent cell cultures of decreasing cell densities (confluent up to single cells) and with cells grown in suspension, we found a gradual decline of differentiation ("dedifferentiation"). This decline paralleled the decrease of cell-cell and cell-substrate contacts. In suspension cultures, cells were prevented from interacting with each other and the substratum, no rhabdomyomatous differentiation of the cells took place. If restoration of cellular contacts was allowed, either by adherent growth or by reinoculation into nude mice, the process of dedifferentiation was completely reversible. Consequently, it was demonstrated that the increase of cell-cell and cell-substrate contacts was strongly associated with the appearance or increasing expression of the desmin intermediate filament cytoskeleton and with formation of the extracellular matrix components fibronectin and laminin. The microfilament (F-actin) system was modulated from an impressive stress-fiber system in subconfluent to a dense network in confluent monolayers. The extent of cell-substrate contacts, mediated by extracellular matrix components, and the number of cell-cell interactions are responsible for the capability of a malignant mesenchymal cell, which is able to undergo rhabdomyomatous differentiation, to achieve the various stages of maturation.     

Epidermal growth factor receptor inhibition promotes desmosome assembly and strengthens intercellular adhesion in squamous cell carcinoma cells

The epidermal growth factor receptor (EGFR) has been proposed as a key modulator of cadherin-containing intercellular junctions, particularly in tumors that overexpress this tyrosine kinase. Here the EGFR tyrosine kinase inhibitor PKI166 and EGFR blocking antibody C225, both of which are used clinically to treat head and neck cancers, were used to determine the effects of EGFR inhibition on intercellular junction assembly and adhesion in oral squamous cell carcinoma cells. EGFR inhibition resulted in a transition from a fibroblastic morphology to a more epithelial phenotype in cells grown in low calcium; under these conditions cadherin-mediated cell-cell adhesion is normally reduced, and desmosomes are absent. The accumulated levels of desmoglein 2 (Dsg2) and desmocollin 2 increased 1.7-2.0-fold, and both desmosomal cadherin and plaque components were recruited to cell-cell borders. This redistribution was paralleled by an increase in Dsg2 and desmoplakin in the Triton-insoluble cell fraction, suggesting that EGFR blockade promotes desmosome assembly. Importantly, E-cadherin expression and solubility were unchanged. Furthermore, PKI166 blocked tyrosine phosphorylation of Dsg2 and plakoglobin following epidermal growth factor stimulation, whereas no change in phosphorylation was detected for E-cadherin and beta-catenin. The increase in Dsg2 protein was in part due to the inhibition of matrix metalloproteinase-dependent proteolysis of this desmosomal cadherin. These morphological and biochemical changes were accompanied by an increase in intercellular adhesion based on functional assays at all calcium concentrations tested. Our results suggest that EGFR inhibition promotes desmosome assembly in oral squamous cell carcinoma cells, resulting in increased cell-cell adhesion.     

Tissue architecture: the ultimate regulator of epithelial function?

The architecture of a tissue is defined by the nature and the integrity of its cellular and extracellular compartments, and is based on proper adhesive cell-cell and cell-extracellular matrix interactions. Cadherins and integrins are major adhesion-mediators that assemble epithelial cells together laterally and attach them basally to a subepithelial basement membrane, respectively. Because cell adhesion complexes are linked to the cytoskeleton and to the cellular signalling pathways, they represent checkpoints for regulation of cell shape and gene expression and thus are instructive for cell behaviour and function. This organization allows a reciprocal flow of mechanical and biochemical information between the cell and its microenvironment, and necessitates that cells actively maintain a state of homeostasis within a given tissue context. The loss of the ability of tumour cells to establish correct adhesive interactions with their microenvironment results in disruption of tissue architecture with often fatal consequences for the host organism. This review discusses the role of cell adhesion in the maintenance of tissue structure and analyses how tissue structure regulates epithelial function.