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.     

Activation of Both MAP Kinase and Phosphatidylinositide 3-Kinase by Ras Is Required for Hepatocyte Growth Factor/Scatter Factor–induced Adherens Junction Disassembly

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell–cell junctions and subsequent cell scattering. In Madin–Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induced the loss of the adherens junction proteins E-cadherin and β-catenin from intercellular junctions during cell spreading, and the HGF/SF response was blocked by dominant-negative N17Ras. Desmosomes and tight junctions were regulated separately from adherens junctions, because they were not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac were required downstream of Ras, because loss of adherens junctions was blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of MAP kinase kinase 1 or Rac1. All of these inhibitors also prevented HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110α subunit of PI 3-kinase alone did not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras is required for adherens junction disassembly and that this is essential for the motile response to HGF/SF.     

Stimulation of β1-Integrin Function by Epidermal Growth Factor and Heregulin-β Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

Integrins and growth factor receptors are important participants in cellular adhesion and migration. The EGF receptor (EGFR) family of tyrosine kinases and the β1-integrin adhesion receptors are of particular interest, given the implication for their involvement in the initiation and progression of tumorigenesis. We used adhesion and chemotaxis assays to further elucidate the relationship between these two families of transmembrane signaling molecules. Specifically, we examined integrin-mediated adhesive and migratory characteristics of the metastatic breast carcinoma cell line MDA-MB-435 in response to stimulation with growth factors that bind to and activate the EGFR or erbB3 in these cells. Although ligand engagement of the EGFR stimulated modest β1-dependent increases in cell adhesion and motility, heregulin-β (HRGβ) binding to the erbB3 receptor initiated rapid and potent induction of breast carcinoma cell adhesion and migration and required dimerization of erbB3 with erbB2. Pharmacologic inhibitors of phosphoinositide 3-OH kinase (PI 3-K) or transient expression of dominant negative forms of PI 3-K inhibited both EGF- and HRGβ-mediated adhesion and potently blocked HRGβ- and EGF-induced cell motility. Our results illustrate the critical role of PI 3-K activity in signaling pathways initiated by the EGFR or erbB3 to up-regulate β1-integrin function.     

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.     

Sertoli-germ cell anchoring junction dynamics in the testis are regulated by an interplay of lipid and protein kinases

When Sertoli and germ cells were co-cultured in vitro in serum-free chemically defined medium, functional anchoring junctions such as cell-cell intermediate filament-based desmosome-like junctions and cell-cell actin-based adherens junctions (e.g. ectoplasmic specialization (ES)) were formed within 1-2 days. This event was marked by the induction of several protein kinases such as phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (PKB; also known as Akt), p21-activated kinase-2 (PAK-2), and their downstream effector (ERK) as well as an increase in PKB intrinsic activity. PI3K, phospho (p)-PKB, and PAK were co-localized to the site of apical ES in the seminiferous epithelium of the rat testis in immunohistochemistry studies. Furthermore, PI3K also co-localized with p-PKB to the same site in the epithelium as determined by fluorescence microscopy, consistent with their localization at the ES. These kinases were shown to associate with ES-associated proteins such as beta1-integrin, phosphorylated focal adhesion kinase, and c-Src by co-immunoprecipitation, suggesting that the integrin.laminin protein complex at the apical ES likely utilizes these protein kinases as regulatory proteins to modulate Sertoli-germ cell adherens junction dynamics via the ERK signaling pathway. To validate this hypothesis further, an in vivo model using AF-2364 (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) to perturb Sertoli-germ cell anchoring junction function, inducing germ cell loss from the epithelium in adult rats, was used in conjunction with specific inhibitors. Interestingly, the event of germ cell loss induced by AF-2364 in vivo was also associated with induction of PI3K, p-PKB, PAK-2, and p-ERK as well as a surge in intrinsic PKB activity. Perhaps the most important of all, pretreatment of rats with wortmannin (a PI3K inhibitor) or anti-beta1-integrin antibody via intratesticular injection indeed delayed AF-2364-induced spermatid loss from the epithelium. In summary, these results illustrate that Sertoli-germ cell anchoring junction dynamics in the testis are regulated, at least in part, via the beta1-integrin/PI3K/PKB/ERK signaling pathway.     

Involvement of β1-Integrin Up-regulation in Basic Fibroblast Growth Factor- and Epidermal Growth Factor-induced Proliferation of Mouse Neuroepithelial Cells

In neural stem cells, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote cell proliferation and self-renewal. In the bFGF- and EGF-responsive neural stem cells, β1-integrin also plays important roles in crucial cellular processes, including proliferation, migration, and apoptosis. The cross-talk of the signaling pathways mediated by these growth factors and β1-integrin, however, has not been fully elucidated. Here we report a novel molecular mechanism through which bFGF or EGF promotes the proliferation of mouse neuroepithelial cells (NECs). In the NECs, total β1-integrin expression levels and proliferation were dose-dependently increased by bFGF but not by EGF. EGF rather than bFGF strongly induced the increase of β1-integrin localization on the NEC surface. bFGF- and EGF-induced β1-integrin up-regulation and proliferation were inhibited after treatment with a mitogen-activated protein kinase kinase inhibitor, U0126, which indicates the dependence on the mitogen-activated protein kinase pathway. Involvement of β1-integrin in bFGF- and EGF-induced proliferation was confirmed by the finding that NEC proliferation and adhesion to fibronectin-coated dishes were inhibited by knockdown of β1-integrin using small interfering RNA. On the other hand, apoptosis was induced in NECs treated with RGD peptide, a small β1-integrin inhibitor peptide with the Arg-Gly-Asp motif, but it was independent of β1-integrin expression levels. Those results suggest that regulation of β1-integrin expression/localization is involved in cellular processes, such as proliferation, induced by bFGF and EGF in NECs. The mechanism underlying the proliferation through β1-integrin would not be expected to be completely identical, however, for bFGF and EGF.     

Epidermal growth factor increased the expression of alpha2beta1-integrin and modulated integrin-mediated signaling in human cervical adenocarcinoma cells

Epidermal growth factor (EGF) receptor (EGFR) is involved in various basic biochemical pathways and is thus thought to play an important role in cell migration. We examined the effect of EGF on motility, migration, and morphology of a human adenocarcinoma cell line CAC-1. EGF treatment increased the motility of cervical adenocarcinoma cells and promoted migration of the cells on fibronectin and type IV collagen. EGF induced morphological changes with lamellipodia during EGFR-mediated motility. The results of an immunoprecipitation study showed that EGF up-regulated the expression of alpha2beta1-integrin in a dose-dependent manner. EGF-induced cell migration was blocked by alpha2beta1-integrin antibody. Our results also showed that EGF treatment stimulated the level of tyrosine dephosphorylation of FAK, which is required for EGF-induced changes in motility, migration, and cell morphology. A tyrosine kinase inhibitor (ZD1839) blocked EGF-induced changes in cervical adenocarcinoma cells. The results suggest that EGF promotes cell motility and migration and increases the expression of alpha2beta1-integrin, possibly by decreasing FAK phosphorylation.     

Matrix-dependent Tiam1/Rac Signaling in Epithelial Cells Promotes Either Cell–Cell Adhesion or Cell Migration and Is Regulated by Phosphatidylinositol 3-Kinase

We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin–mediated cell–cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin–mediated cell–cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin–mediated cell– cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase– PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin–mediated cell– cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.     

Crk synergizes with epidermal growth factor for epithelial invasion and morphogenesis and is required for the met morphogenic program

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.     

Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation.

The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brush-border formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation.     

Epithelial cell cluster size affects force distribution in response to EGF-induced collective contractility

Several factors present in the extracellular environment regulate epithelial cell adhesion and dynamics. Among them, growth factors such as EGF, upon binding to their receptors at the cell surface, get internalized and directly activate the acto-myosin machinery. In this study we present the effects of EGF on the contractility of epithelial cancer cell colonies in confined geometry of different sizes. We show that the extent to which EGF triggers contractility scales with the cluster size and thus the number of cells. Moreover, the collective contractility results in a radial distribution of traction forces, which are dependent on integrin β1 peripheral adhesions and transmitted to neighboring cells through adherens junctions. Taken together, EGF-induced contractility acts on the mechanical crosstalk and linkage between the cell-cell and cell-matrix compartments, regulating collective responses.     

Epidermal growth factor-induced DNA synthesis. Key role for Src phosphorylation of the docking protein Gab2

We have previously demonstrated that phosphatidylinositol 3-kinase (PI3-kinase) is necessary and sufficient to account for epidermal growth factor (EGF)-induced mitogenesis in rat primary hepatocytes. A cytosolic Gab2-containing complex accounts for >80% of the total EGF-induced PI3-kinase activity (Kong, M., Mounier, C., Wu, J., and Posner, B. I. (2000) J. Biol. Chem. 275, 36035-36042), suggesting a key role for Gab2 in EGF-induced mitogenesis. Here, we demonstrate that PP1, a selective inhibitor of Src family kinases, blocks the EGF-induced Gab2 tyrosine phosphorylation without inhibiting EGF-induced phosphorylation of the EGF receptor, ErbB3, or Shc. We also show that Gab2 phosphorylation is increased in Csk knockout cells in which Src family kinases are constitutively activated. Furthermore, PP1 blocks Gab2-associated downstream events including EGF-induced PI3-kinase activation, Akt phosphorylation, and DNA synthesis. We demonstrate that Gab2 and Src are constitutively associated. Since this association involves the proline-rich sequences of Gab2, it probably involves the Src homology 3 domain of Src kinase. Mutation of the proline-rich sequences in Gab2 prevented EGF-induced Gab2 phosphorylation, PI3-kinase/Akt activation, and DNA synthesis, demonstrating that Gab2 phosphorylation is critical for EGF-induced mitogenesis and is not complemented by ErbB3 or Shc phosphorylation. We also found that overexpression of a Gab2 mutant lacking SHP2 binding sites increased EGF-induced Gab2 phosphorylation and the activation of PI3-kinase but blocked activation of MAPK. In addition, we demonstrated that the Src-induced response was down-regulated by Gab2-associated SHP2. In summary, our results have defined the role for Src activation in EGF-induced hepatic mitogenesis through the phosphorylation of Gab2 and the activation of the PI3-kinase cascade.     

Role of Phosphoinositide 3-Kinase in Activation of Ras and Mitogen-Activated Protein Kinase by Epidermal Growth Factor

The paradigm for activation of Ras and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase by extracellular stimuli via tyrosine kinases, Shc, Grb2, and Sos does not encompass an obvious role for phosphoinositide (PI) 3-kinase, and yet inhibitors of this lipid kinase family have been shown to block the ERK/MAP kinase signalling pathway under certain circumstances. Here we show that in COS cells activation of both endogenous ERK2 and Ras by low, but not high, concentrations of epidermal growth factor (EGF) is suppressed by PI 3-kinase inhibitors; since Ras activation is less susceptible than ERK2 activation, PI 3-kinase-sensitive events may occur both upstream of Ras and between Ras and ERK2. However, strong elevation of PI 3-kinase lipid product levels by expression of membrane-targeted p110alpha is by itself never sufficient to activate Ras or ERK2. PI 3-kinase inhibition does not affect EGF-induced receptor autophosphorylation or adapter protein phosphorylation or complex formation. The concentrations of EGF for which PI 3-kinase inhibitors block Ras activation induce formation of Shc-Grb2 complexes but not detectable EGF receptor phosphorylation and do not activate PI 3-kinase. The activation of Ras by low, but mitogenic, concentrations of EGF is therefore dependent on basal, rather than stimulated, PI 3-kinase activity; the inhibitory effects of LY294002 and wortmannin are due to their ability to reduce the activity of PI 3-kinase to below the level in a quiescent cell and reflect a permissive rather than an upstream regulatory role for PI 3-kinase in Ras activation in this system.     

Activation of the protein kinase Akt/PKB by the formation of E-cadherin-mediated cell-cell junctions. Evidence for the association of phosphatidylinositol 3-kinase with the E-cadherin adhesion complex.

E-cadherins are surface adhesion molecules localized at the level of adherens junctions, which play a major role in cell adhesiveness by mediating calcium-dependent homophylic interactions at sites of cell-cell contacts. Recently, E-cadherins have been also implicated in a number of biological processes, including cell growth and differentiation, cell recognition, and sorting during developmental morphogenesis, as well as in aggregation-dependent cell survival. As phosphatidylinositol (PI) 3-kinase and Akt play a critical role in survival pathways in response to both growth factors and extracellular stimuli, these observations prompted us to explore whether E-cadherins could affect intracellular molecules regulating the activity of the PI 3-kinase/Akt signaling cascade. Using Madin-Darby canine kidney cells as a model system, we show here that engagement of E-cadherins in homophylic calcium-dependent cell-cell interactions results in a rapid PI 3-kinase-dependent activation of Akt and the subsequent translocation of Akt to the nucleus. Moreover, we demonstrate that the activation of PI 3-kinase in response to cell-cell contact formation involves the phosphorylation of PI 3-kinase in tyrosine residues, and the concomitant recruitment of PI 3-kinase to E-cadherin-containing protein complexes. These findings indicate that E-cadherins can initiate outside-in signal transducing pathways that regulate the activity of PI 3-kinase and Akt, thus providing a novel molecular mechanism whereby the interaction among neighboring cells and their adhesion status may ultimately control the fate of epithelial cells.     

Growth hormone modulation of EGF-induced PI3K-Akt pathway in mice liver

The epidermal growth factor (EGF) activates the phosphatidylinositol 3-kinase (PI3K)-Akt cascade among other signaling pathways. This route is involved in cell proliferation and survival, therefore, its dysregulation can promote cancer. Considering the relevance of the PI3K-Akt signaling in cell survival and in the pathogenesis of cancer, and that GH was reported to modulate EGFR expression and signaling, the objective of this study was to analyze the effects of increased GH levels on EGF-induced PI3K-Akt signaling.EGF-induced signaling was evaluated in the liver of GH-overexpressing transgenic mice and in their normal siblings. While Akt expression was increased in GH-overexpressing mice, EGF-induced phosphorylation of Akt, relative to its protein content, was diminished at Ser473 and inhibited at Thr308; consequently, mTOR, which is a substrate of Akt, was not activated by EGF. However, the activation of PDK1, a kinase involved in Akt phosphorylation at Thr308, was not reduced in transgenic mice. Kinetics studies of EGF-induced Akt phosphorylation showed that it is rapidly and transiently induced in GH-overexpressing mice compared with normal siblings. Thus, the expression and activity of phosphatases involved in the termination of the PI3K-Akt signaling were studied. In transgenic mice, neither PTEN nor PP2A were hyperactivated; however, EGF induced the rapid and transient association of SHP-2 to Gab1, which mediates association to EGFR and activation of PI3K. Rapid recruitment of SHP2, which would accelerate the termination of the proliferative signal induced, could be therefore contributing to the diminished EGF-induced activity of Akt in GH-overexpressing mice.     

The role of PI 3-kinase in EGF-stimulated jejunal glucose transport

Epidermal growth factor (EGF) rapidly increases jejunal glucose transport. Signal transduction mechanisms mediating EGF-induced alterations in jejunal glucose transport remain to be determined. New Zealand White rabbit (1 kg) jejunal tissue was stripped and mounted in short-circuited Ussing chambers. The transport of tritiated 3-O-methylglucose was measured in the presence of the PKC agonist 1,2-dioctanoyl-sn-glycerol (1,2-DOG) or the inactive analog 1,3-dioctanoyl-sn-glycerol (1,3-DOG). Additional experiments examined the effect of the PKC inhibitor chelerythrine, the PLC inhibitor U73122, the MAPK inhibitor PD 98059, the G-protein inhibitor GDP-betaS, the PI 3-kinase inhibitor LY294002, or the microtubule inhibitor colchicine on EGF-induced jejunal glucose transport. Net jejunal 3-O-methylglucose absorption was significantly increased following specific activation of PKC. A PKC antagonist inhibited the EGF-induced increase in net 3-O-methylglucose transport, while PI 3-kinase inhibition completely blocked the EGF-induced transport increase. Inhibition of PLC, MAPK, G-proteins, and microtubules had no effect on EGF-stimulated increases in jejunal transport. We conclude that the effect of EGF on jejunal glucose transport is mediated at least in part by PKC and PI 3-kinase.     

Mechanisms of plakoglobin-dependent adhesion: desmosome-specific functions in assembly and regulation by epidermal growth factor receptor

Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins that can be incorporated into both adherens junctions and desmosomes. Loss of PG results in defects in the mechanical integrity of heart and skin and decreased adhesive strength in keratinocyte cultures established from the skin of PG knock-out (PG-/-) mice, the latter of which cannot be compensated for by overexpressing the closely related beta-catenin. In this study, we examined the mechanisms of PG-regulated adhesion in murine keratinocytes. Biochemical and morphological analyses indicated that junctional incorporation of desmosomal, but not adherens junction, components was impaired in PG-/- cells compared with PG+/- controls. Re-expression of PG, but not beta-catenin, in PG-/- cells largely reversed these effects, indicating a key role for PG in desmosome assembly. Epidermal growth factor (EGF) receptor activation resulted in Tyr phosphorylation of PG, which was accompanied by a loss of desmoplakin from desmosomes and decreased adhesive strength following 18-h EGF treatment. Importantly, introduction of a phosphorylation-deficient PG mutant into PG null cells prevented the EGF receptor-dependent loss of desmoplakin from junctions, attenuating the effects of long term EGF treatment on cell adhesion. Therefore, PG is essential for maintaining and regulating adhesive strength in keratinocytes largely through its contributions to desmosome assembly and structure. As a target for modulation by EGF, regulation of PG-dependent adhesion may play an important role during wound healing and tumor metastasis.     

Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinases in glycine-extended gastrin-induced dissociation and migration of gastric epithelial cells

The various molecular forms of gastrin can act as promoters of proliferation and differentiation in different regions of the gastrointestinal tract. We report a novel stimulatory effect of glycine-extended gastrin(17) only on cell/cell dissociation and cell migration in a non-tumorigenic mouse gastric epithelial cell line (IMGE-5). In contrast, both amidated and glycine-extended gastrin(17) stimulated proliferation of IMGE-5 cells via distinct receptors. Glycine-extended gastrin(17)-induced dissociation preceded migration and was blocked by selective inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) but did not require mitogen-activated protein (MAP) kinase activation. Furthermore, glycine-extended gastrin(17) induced a PI3-kinase-mediated tyrosine phosphorylation of the adherens junction protein beta-catenin, partial dissociation of the complex between beta-catenin and the transmembrane protein E-cadherin, and delocalization of beta-catenin into the cytoplasm. Long lasting activation of MAP kinases by glycine-extended gastrin(17) was specifically required for the migratory response, in contrast to the involvement of a rapid and transient MAP kinase activation in the proliferative response to both amidated and glycine-extended gastrin(17). Therefore, the time course of MAP kinase activation appears to be a critical determinant of the biological effects mediated by this pathway. Together with the involvement of PI3-kinase in the dissociation of adherens junctions, long term activation of MAP kinases seems responsible for the selectivity of this novel effect of G(17)-Gly on the adhesion and migration of gastric epithelial cells.     

Arf6 regulates EGF-induced internalization of E-cadherin in breast cancer cells

E-cadherin internalization facilitates dissolution of adherens junctions and promotes tumor cell epithelial-mesenchymal transition (EMT) and migration. Our previous results have shown that Arf6 exerts pro-migratory action in breast cancer cells after EGF stimulation. Despite the fact that EGF signaling stimulates EMT of breast cancer cells, the effect of Arf6 on internalization of E-cadherin of breast cancer cells under EGF treatment remains to be determined. Here, we showed that EGF dose-dependently stimulated E-cadherin internalization by MCF-7 cells with the maximal effect at 50 ng/ml. Meanwhile, EGF treatment markedly increased Arf6 activation. Arf6 was involved in complexes of E-cadherin, and more E-cadherin was pulled down with Arf6 when the activity of the latter was increased. Immunoblotting and immunofluorescence assays showed that transfection breast cancer cells with Arf6-T27N or Arf6 siRNA suppressed EGF-induced E-cadherin internalization. Taken together, our study demonstrated that Arf6 activation plays a potential role in EGF-induced E-cadherin internalization, providing new mechanism underlying the effect of Arf6 on promoting breast cancer cell metastasis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12935-015-0159-3) contains supplementary material, which is available to authorized users.     

Proliferation and differnetiation of human squamous carcinoma cell lines and normal keratinocytes: Effects of epidermal growth factor,retinoids, and hydrocortisone

Summary Exposure of squamous carcinoma cell (SCC) lines, exhibiting high levels of epidermal growth factor (EGF) receptors, to EGF for 6 d caused a dose-dependent inhibition of cell proliferation. This EGF-induced inhibition of cell proliferation occurred under both low (0.06 mM) and normal (1.6 mM) Ca²⁺ concentrations. Furthermore, the extent of EGF-induced inhibition of cell proliferation seemed to be independent of the number of EGF-receptors. This conclusion is based on the notion that the various SCC lines exhibited an increasing number of EGF receptors accompanied by a decreasing ability to differentiate, whereas no relationship was observed with the EGF-induced inhibition of cell proliferation in these cell lines. Retinoids caused also a dose-dependent inhibition of cell proliferation. The effects of EGF and retinoids were additive, indicating that different regulatory mechanisms are involved. On the other hand, hydrocortisone caused a stimulation of SCC-proliferation, also independent of EGF. In contrast to SCC cells, EGF did not affect significantly the rate of proliferation of normal keratinocytes. However, the simultaneous addition of EGF and hydrocortisone resulted in a significant increase in the rate of keratinocyte proliferation only in cells grown under normal calcium conditions. Differentiation capacity of normal keratinocytes and SCC lines was not affected by EGF. Furthermore, the retinoid-induced decrease and hydrocortisone-induced increase of competence of cells to form cornified envelopes was not affected by EGF. These observations suggest that the action of retinoids and hydrocortisone on both cell proliferation and cell differentiation occurs independently of EGF receptors. This work was partly supported by The Netherlands Cancer Foundation (Koningin Wilhelmina Fonds), grant IKW 85–71.