Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E- cadherin

Gap junctional intercellular communication (GJIC) of cultured mouse epidermal cells is mediated by a gap junction protein, connexin 43, and is dependent on the calcium concentration in the medium, with higher GJIC in a high-calcium (1.2 mM) medium. In several mouse epidermal cell lines, we found a good correlation between the level of GJIC and that of immunohistochemical staining of E-cadherin, a calcium-dependent cell adhesion molecule, at cell-cell contact areas. The variant cell line P3/22 showed both low GJIC and E-cadherin protein expression in low- and high-Ca2+ media. P3/22 cells showed very low E-cadherin mRNA expression. To test directly whether E-cadherin is involved in the Ca(2+)-dependent regulation of GJIC, we transfected the E-cadherin expression vector into P3/22 cells and obtained several stable clones which expressed high levels of E-cadherin mRNA. All transfectants expressed E-cadherin molecules at cell-cell contact areas in a calcium-dependent manner. GJIC was also observed in these transfectants and was calcium dependent. These results suggest that Ca(2+)-dependent regulation of GJIC in mouse epidermal cells is directly controlled by a calcium-dependent cell adhesion molecule, E-cadherin. Furthermore, several lines of evidence suggest that GJIC control by E-cadherin involves posttranslational regulation (assembly and/or function) of the gap junction protein connexin 43.     

DDR1 triggers epithelial cell differentiation by promoting cell adhesion through stabilization of E-cadherin

Discoidin domain receptor 1 (DDR1) promotes E-cadherin-mediated adhesion. The underlying mechanism and its significance, however, have not been elucidated. Here we show that DDR1 overexpression augmented, whereas dominant negative mutant (DN-DDR1) or knockdown of DDR1 inhibited E-cadherin localized in cell-cell junctions in epithelial cells. DDR1 changed the localization and abundance of E-cadherin, as well as epithelial plasticity, as manifested by enhancement of microvilli formation and alteration of cytoskeletal organization. DDR1 also reduced protein abundance of mesenchymal markers, whereas DN-DDR1 and sh-DDR1 showed opposite effects. These results suggest that expression of DDR1 increases epithelial plasticity. Expression of DDR1 augmented E-cadherin protein levels by decreasing its degradation rate. Photobleaching and photoconversion of E-cadherin conjugated with Eos fluorescence protein demonstrated that DDR1 increased the stability of E-cadherin on the cell membrane, whereas sh-DDR1 decreased it. Pull-down assay and expression of constitutively active or dominant-negative Cdc42 showed that DDR1 stabilized E-cadherin through inactivation of Cdc42. Altogether, our results show that DDR1 promotes cell-cell adhesion and differentiation through stabilization of E-cadherin, which is mediated by Cdc42 inactivation.     

Aberrant O-glycosylation inhibits stable expression of dysadherin,a carcinoma-associated antigen,and facilitates cell-cell adhesion

Recently, we identified dysadherin, a novel carcinoma-associated glycoprotein, and showed that overexpression of dysadherin in human hepatocarcinoma PLC/PRF/5 cells could suppress E-cadherin-mediated cell-cell adhesion and promote tumor metastasis. The present study shows evidence that dysadherin is actually O-glycosylated. This was based on a direct carbohydrate composition analysis of a chimera protein of an extracellular domain of dysadherin fused to an Fc fragment of immunoglobulin. To assess the importance of O-glycosylation in dysadherin function, dysadherin-transfected hepatocarcinoma cells were cultured in a medium containing benzyl-alpha-GalNAc, a modulator of O-glycosylation. This treatment facilitated homotypic cell adhesion among dysadherin transfectants accompanied with morphological changes, indicating that the anti-adhesive effect of dysadherin was weakened. Modification of O-glycan synthesis also resulted in down-regulation of dysadherin expression and up-regulation of E-cadherin expression in dysadherin transfectants but did not affect E-cadherin expression in mock transfectants. Structural analysis of O-glycans released from the dysadherin chimera proteins indicated that a series of O-glycans with core 1 and 2 structures are attached to dysadherin, and their sialylation is remarkably inhibited by benzyl-alpha-GalNAc treatment. However, sialidase treatment of the cells did not affect calcium-dependent cell aggregation, which excluded the possibility that sialic acid itself is directly involved in cell-cell adhesion. We suggest that aberrant O-glycosylation in carcinoma cells inhibits stable expression of dysadherin and leads to the up-regulation of E-cadherin expression by an unknown mechanism, resulting in increased cell-cell adhesion. The carbohydrate-directed approach to the regulation of dysadherin expression might be a new strategy for cancer therapy.     

Differential regulation of endogenous cadherin expression in Madin-Darby canine kidney cells by cell-cell adhesion and activation of beta -catenin signaling

Cadherins mediate cell-cell adhesion, but little is known about how their expression is regulated. In Madin-Darby canine kidney (MDCK) cells, the cadherin-associated cytoplasmic proteins alpha- and beta-catenin form high molecular weight protein complexes with two glycoproteins (Stewart, D. B., and Nelson, W. J. (1997) J. Biol. Chem. 272, 29652-29662), one of which is E-cadherin and the other we show here is the type II cadherin, cadherin-6 (K-cadherin). In low density, motile MDCK cells, the steady-state level of cadherin-6 is low, but protein is synthesized. However, following cell-cell adhesion, cadherin-6 becomes stabilized and accumulates by >50-fold at cell-cell contacts while the E-cadherin level increases only 5-fold during the same period. To investigate a role of beta-catenin in regulation of cadherin expression in MDCK cells, we examined the effects of expressing signaling-active beta-catenin mutants (DeltaGSK, DeltaN90, and DeltaN131). In these cells, while levels of E-cadherin, alpha- and beta-catenin are similar to those in control cells, levels of cadherin-6 are significantly reduced due to rapid degradation of newly synthesized protein. Additionally, these cells appeared more motile and less cohesive, as expression of DeltaGSK-beta-catenin delayed the establishment of tight confluent cell monolayers compared with control cells. These results indicate that the level of cadherin-6, but not that of E-cadherin, is strictly regulated post-translationally in response to Wnt signaling, and that E-cadherin and cadherin-6 may contribute different properties to cell-cell adhesion and the epithelial phenotype.     

E-cadherin decreased human breast cancer cells sensitivity to staurosporine by up-regulating Bcl-2 expression

E-cadherin, a well-characterized cell-cell adhesion molecule, executes multifunction roles on cell behaviors. However, its effect on chemo-resistance remains controversial. In this study, we found that E-cadherin positive breast cell lines were less sensitive to staurosporine compared to E-cadherin negative ones. Next, we substantiated that the expression of E-cadherin in MDA-MB-435 cells could partly counteract the cytotoxic effect induced by staurosporine through a series of apoptosis assay. The resistance of E-cadherin over-expressing cells to staurosporine may due to the up-regulation of Bcl-2/Bax ratio. When E-cadherin interference plasmids were transfected into MCF-7 cells, Bcl-2 expression was down-regulated. Moreover, perturbation of E-cadherin function by blocking the cell-cell contact resulted in decreased cellular levels of Bcl-2 protein expression. All these results demonstrated the chemo-resistance function of E-cadherin in the condition of staurosporine treatment, therefore, might contribute effective therapeutic strategies in breast carcinoma.     

E-cadherin affects MAP activation by growth factors stimulation in human carcinoma cells

Met and EGF receptor (EGFR) activation is correlated with dissociation of cell-cell adhesion and with increase in mobility of cancer cells. E-cadherin is a major protein of adhesion junctions. Using different approaches we have shown that EGF receptors intracellular localization depends of E-cadherin function. It was found that EGFR localized on the membrane in HT-29 cells which formed mature cell-cell contacts. Moreover, EGFR was colocalized with E-cadherin at the site of cell-cell adhesion in Triton-insoluble fraction. EGFR was accumulated preliminary in cytosol in E-cadherin negative HBL-100 cells. Study of signal transduction mediated by EGF and HGF in cells with different state of cell adhesion demonstrated that E-cadherin could affect ERK-signal-duration. Our preliminary studies proposed that mislocalization of Met and EGFR in E-cadherin negative cells altered receptors downstream signaling.     

Poorly Differentiated Colon Carcinoma Cell Lines Deficient in α-Catenin Expression Express High Levels of Surface E-cadherin but Lack Ca2+-Dependent Cell-Cell Adhesion

Studies on several different types of carcinomas, with the notable exception of colon carcinoma, have shown that poorly differentiated tumors are frequently deficient in E-cadherin dependent cell-cell adhesion. In this study, we examined Ca²⁺-dependent cell-cell adhesion in colon carcinoma cell lines. Five poorly differentiated (Clone A, MIP 101, RKO, CCL 222, CCL 228) and four moderately-well differentiated (CX-1, CCL 235, DLD-2, CCL 187) colon carcinoma cell lines were assayed for their ability to form cell-cell aggregates and for their levels of E-cadherin expression. All of the poorly differentiated cell lines exhibited low levels of Ca²⁺-dependent cell-cell aggregation, in contrast to the moderately-well differentiated cell lines. Contrary to most previous studies, however, we observed that three of the five poorly differentiated cell lines examined expressed E-cadherin by FACS analysis and immunoprecipitation using an E-cadherin mAb. In fact, two of these cell lines expressed a 3- to 4-fold higher level of E-cadherin than that found in the moderately-well differentiated cell lines. mRNA levels for E-cadherin, as evaluated by both RT-PCR and Northern hybridization, corresponded to the levels of protein expression in each of the cell lines. Immunoprecipitation with an E-cadherin mAb, which is known to co-precipitate the catenins, demonstrated that the three poorly differentiated cell lines expressing E-cadherin did not co-precipitate α-catenin, although all of the moderately-well differentiated cell lines expressed both α- and β-catenin. RT-PCR confirmed the absence of the α-catenin mRNA from two of these cell lines. Stable expression of an α-catenin cDNA in one of the poorly differentiated cell lines lacking α-catenin expression resulted in a 5-fold increase in its level of Ca²⁺-dependent cell-cell aggregation, providing evidence that α-catenin is directly responsible for the loss of cell-cell adhesion in some cell lines. The α-catenin transfectants also exhibited a marked reduction in migration on collagen I. These data indicate that loss of α-catenin expression, as well as E-cadherin expression, can lead to a phenotype associated with poorly differentiated colon carcinomas.     

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.     

PKC-δ binds to E-cadherin and mediates EGF-induced cell scattering

EGF is known to affect adherens junctions and disrupt cell-cell adhesion in a variety of carcinomas but the underlying mechanisms are not completely understood. Using human tumor epithelial cells overexpressing EGFR we demonstrated that EGF-induced cell scattering was mediated by protein kinase C-delta (PKC-δ). PKC-δ knockdown by siRNA significantly inhibited EGF-induced internalization of E-cadherin into the cytoplasm and blocked cell scattering. EGF phosphorylated PKC-δ at Y311 and ectopic expression of the mutant Y311F prevented PKC-δ binding to E-cadherin and EGF-induced cell scattering. Moreover, depletion of Src using siRNA decreased EGF-induced phosphorylation of PKC-δ at Y311 and blocked scattering. Finally, EGF reduced expression of the tight junction protein, occludin, and this effect was also mediated by PKC-δ through Src. In summary, PKC-δ mediated the effects of EGF on adherens and tight junctions thereby playing an important role in cell-cell adhesion with possible wider implications in tumor metastasis or epithelial-to-mesenchymal transition.     

Effect of tumor-associated mutant E-cadherin variants with defects in exons 8 or 9 on matrix metalloproteinase 3

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. The cell adhesion molecule E-cadherin is frequently down-regulated or mutated in tumors. In addition to down-regulation of cell adhesion, degradation of the extracellular matrix by matrix metalloproteinases is necessary for tumor cell spread. To investigate a possible link between E-cadherin and matrix metalloproteinase 3 (MMP-3), we examined expression of MMP-3 in human MDA-MB-435S cells transfected with wild-type (wt) or three different tumor-associated mutant E-cadherin variants with alterations in exons 8 or 9, originally identified in gastric carcinoma patients. In the presence of wt E-cadherin, the MMP-3 protein level was decreased in cellular lysates and in the supernatant where a secreted form of the protein is detectable. Down-regulation of MMP-3 was not found in MDA-MB-435S transfectants expressing mutant E-cadherin variants which indicates that E-cadherin mutations interfere with the MMP-3 suppressing function of E-cadherin. The mechanism of regulation of MMP-3 by E-cadherin is presently not clear. We have previously found that cell motility is enhanced by expression of the mutant E-cadherin variants used in this study. Here, we found that application of the synthetic inhibitor of MMP-3 NNGH and small interfering RNA (siRNA) directed against MMP-3 reduce mutant E-cadherin-enhanced cell motility. Taken together, our results point to a functional link between MMP-3 and E-cadherin. MMP-3 is differentially regulated by expression of wt or mutant E-cadherin. On the other hand, MMP-3 plays a role in the enhancement of cell motility by mutant E-cadherin. Both observations may be highly relevant for tumor progression since they concern degradation of the extracellular matrix and tumor cell spread.     

Dynamics of cell adhesion and motility in living cells is altered by a single amino acid change in E-cadherin fused to enhanced green fluorescent protein

E-Cadherin regulates epithelial cell adhesion and is critical for the maintenance of tissue integrity. In sporadic diffuse-type gastric carcinoma, mutations of the E-cadherin gene are frequently observed that predominantly affect putative calcium binding motifs located in the linker region between the second and third extracellular domains. A single amino acid change (D370A) as found in a gastric carcinoma patient reduces cell adhesion and up-regulates cell motility. To study the effect of this mutation on the dynamics of cell adhesion and motility in living cells, enhanced green fluorescent protein (EGFP) was C-terminally fused to E-cadherin. The resulting mutant E-cadherin-EGFP fusion protein with a point mutation in exon 8 (p8-EcadEGFP) and a wild-type E-cadherin-EGFP fusion construct (wt-EcadEGFP) were expressed in human MDA-MB-435S cells. Fluorescent images were acquired by time-lapse laser scanning microscopy and E-cadherin was visualized during contact formation and in moving cells. Spatial and temporal localization of p8- and wt-EcadEGFP differed significantly. While wt-EcadEGFP was mainly localized at lateral membranes of contacting cells and formed E-cadherin puncta and plaques, p8-EcadEGFP-expressing cells frequently formed transient cell-cell contacts. During random cell migration, p8-EcadEGFP was found in lamellipodia. In contrast, wt-EcadEGFP localized at lateral cell-cell contact sites in low or non-motile cells. Inhibition of the epidermal growth factor (EGF) receptor, which plays a major role in lamellipodia formation and cell migration, reduced the motility of p8-EcadEGFP-expressing cells and caused lateral membrane staining of p8-EcadEGFP. Conversely, EGF induced cell motility and caused formation of lamellipodia that were E-cadherin positive. In conclusion, our data show that mutant E-cadherin significantly alters the dynamics of cell adhesion and motility in living cells and interferes with the formation of stable cell-cell contacts.     

泛素连接酶LNX对上皮细胞间连接的影响

为阐明泛素连接酶LNX在维持上皮细胞之间连接的作用,构建了四环素诱导表达LNX的MDCK细胞株;以免疫荧光法观察细胞连接蛋白E-钙黏素和ZO-1在细胞内的分布,发现LNX的表达使E-钙黏素和ZO-1在细胞内的分布发生明显改变,大量E-钙黏素和ZO-1聚集在胞浆中,而在细胞膜上的分布则明显减少;透射电镜观察上皮细胞间连接的超微结构显示,LNX的表达导致紧密连接和黏附连接的正常结构消失;用钙离子转换实验检测黏附连接的形成发现,表达LNX的MDCK细胞间的黏附连接形成的速度明显滞后于正常细胞.上述结果表明,LNX的表达影响了E-钙黏素在细胞膜上的正常分布,从而延迟黏附连接复合体的形成,导致上皮细胞间连接结构的异常.     

The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness.

To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.     

Homophilic adhesion of E-cadherin occurs by a co-operative two-step interaction of N-terminal domains.

Cluster formation of E-cadherin on the cell surface is believed to be of major importance for cell-cell adhesion. To mimic this process the extracellular part of mouse E-cadherin (ECAD) was recombinantly fused to the assembly domain of rat cartilage oligomeric matrix protein (COMP), resulting in the chimeric protein ECAD-COMP. The COMP domain formed a five-stranded alpha-helical coiled-coil. This enabled the formation of a pentameric ECAD with bundled C-termini and free N-termini. The pentameric protein construct ECAD-COMP and the monomeric ECAD were expressed in human embryonal kidney 293 cells. Electron microscopy, analytical ultracentrifugation, solid phase binding and cell attachment assays revealed that pentamers showed strong self-association and cell attachment, whereas monomers exhibited no activity. At the high internal concentration in the pentamer the N-terminal EC1 domains of two E-cadherin arms interact to form a ring-like structure. Then the paired domains interact with a corresponding pair from another pentamer. None of the four other extracellular domains of E-cadherin is involved in this interaction. Based on these results, an in vivo mechanism is proposed whereby two N-terminal domains of neighbouring E-cadherins at the cell surface first form a pair, which binds with high affinity to a similar complex on another cell. The strong dependence of homophilic interactions on C-terminal clustering points towards a regulation of E-cadherin mediated cell-cell adhesion via lateral association.     

RIFLE: a novel ring zinc finger-leucine-rich repeat containing protein, regulates select cell adhesion molecules in PC12 cells

Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger-leucine-rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) resulting in the inhibition of GSK-3 kinase activity and increase of beta-catenin levels. RIFLE expression also is associated with elevated E-cadherin protein levels but not N-cadherin. The regulation of these cell adhesion-associated molecules by RIFLE is accompanied by a significant increase in cell-cell and cell-matrix adhesion. Moreover, increase in cell-cell adhesion but not cell-matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK-3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells.     

Rap1GAP impairs cell-matrix adhesion in the absence of effects on cell-cell adhesion

The significance of the widespread downregulation of Rap1GAP in human tumors is unknown. In previous studies we demonstrated that silencing Rap1GAP expression in human colon cancer cells resulted in sustained increases in Rap activity, enhanced spreading on collagen and the weakening of cell-cell contacts. The latter finding was unexpected based on the role of Rap1 in strengthening cell-cell adhesion and reports that Rap1GAP impairs cell-cell adhesion. We now show that Rap1GAP is a more effective inhibitor of cell-matrix compared to cell-cell adhesion. Overexpression of Rap1GAP in human colon cancer cells impaired Rap2 activity and the ability of cells to spread and migrate on collagen IV. Under the same conditions, Rap1GAP had no effect on cell-cell adhesion. Overexpression of Rap1GAP did not enhance the dissociation of cell aggregates nor did it impair the accumulation of β-catenin and E-cadherin at cell-cell contacts. To further explore the role of Rap1GAP in the regulation of cell-cell adhesion, Rap1GAP was overexpressed in non-transformed thyroid epithelial cells. Although the formation of cell-cell contacts required Rap1, overexpression of Rap1GAP did not impair cell-cell adhesion. These data indicate that transient, modest expression of Rap1GAP is compatible with cell-cell adhesion and that the role of Rap1GAP in the regulation of cell-cell adhesion may be more complex than is currently appreciated.Key words: Rap1GAP, cell adhesion, matrix adhesion, Rap, E-cadherin, β-catenin     

E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.

EphA2 is a member of the Eph family of receptor tyrosine kinases, which are increasingly understood to play critical roles in disease and development. We report here the regulation of EphA2 by E-cadherin. In nonneoplastic epithelia, EphA2 was tyrosine-phosphorylated and localized to sites of cell-cell contact. These properties required the proper expression and functioning of E-cadherin. In breast cancer cells that lack E-cadherin, the phosphotyrosine content of EphA2 was decreased, and EphA2 was redistributed into membrane ruffles. Expression of E-cadherin in metastatic cells restored a more normal pattern of EphA2 phosphorylation and localization. Activation of EphA2, either by E-cadherin expression or antibody-mediated aggregation, decreased cell-extracellular matrix adhesion and cell growth. Altogether, this demonstrates that EphA2 function is dependent on E-cadherin and suggests that loss of E-cadherin function may alter neoplastic cell growth and adhesion via effects on EphA2.     

Adherens junction-dependent PI3K/Akt activation induces resistance to genotoxin-induced cell death in differentiated intestinal epithelial cells

The crypt-villi axis of intestinal mucosa maintains homeostasis by renewal of epithelia, and also exhibits different properties from undifferentiated to terminally differentiated cells. We investigated differential susceptibility to genotoxin-induced cell death, based on the degree of differentiation of epithelial cells, and its mechanism. Differentiation was induced by post-confluence culture in Caco-2 cells. Methyl methanesulfonate (MMS), a direct-acting DNA alkylating agent, was used for genotoxin-induced cell death. Compared to subconfluent Caco-2 cells, 7 days post-confluent cells showed resistance to MMS-induced cell death. With increasing expression of adherens junction components of E-cadherin and β-catenin, E-cadherin and p-Akt expression increased in 7 days post-confluent Caco-2 cells, and in human intestinal tissue, expression of E-cadherin and p-Akt also increased in the upper portion of villi, compared to the crypt. Inhibition of cell-cell adhesion using EGTA decreased Akt phosphorylation, which was reversed by calcium restoration. Akt phosphorylation by calcium-mediated cell-cell adhesion was more prominent in differentiated cells. In addition, treatment of a PI3K inhibitor, LY294002, inhibited Akt phosphorylation by calcium-mediated cell-cell adhesion. Finally, the differential sensitivity to MMS-induced cell death between subconfluent and 7 days post-confluent Caco-2 cells was eliminated by inhibiting cell-cell adhesion or PI3K. Our data demonstrated that cell adhesion-mediated PI3K/Akt activation could be one of the important mechanisms of resistance to genotoxin-induced cell death in differentiated epithelial cells.     

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.