Role of extracellular matrix-cell interaction and epidermal growth factor (EGF) on EGF-receptors and actin cytoskeleton arrangement in infantile pituitary cells

Epidermal growth factor (EGF) induces changes in cell morphology, actin cytoskeleton, and adhesion processes in cultured infantile pituitary cells. The extracellular matrix, through integrin engagement, collaborates with growth factors in cell signaling. We have examined the participation of collagen I/III and collagen plus fibronectin in the EGF response of infantile pituitary cells with respect to their cell morphology and actin cytoskeleton. As a comparison, we have used poly-lysine as a substrate. Infantile cells elicit the EGF response when they are associated with extracellular matrix proteins, but no response can be obtained with poly-lysine as the substrate. Cells acquire a flattened shape and organize their actin filaments and vinculin as in focal adhesions. Because the EGF receptor (EGFR) is linked to the actin cytoskeleton in other cells structuring a microdomain in cell signaling, we have investigated this association and substrate adhesion participation in infantile pituitary cells. The proportion of EGFR associated with the actin cytoskeleton is approximately 31%; no difference has been observed between the substrates used. Cells in suspension show actin-associated EGFR, suggesting an association independent of cell adhesion. However, no colocalization of EGFRs with actin fibers has been observed, suggesting an indirect association. Compared with β₁-integrin, which is linked to actin fibers through structural proteins, EGFR binds more strongly with the actin cytoskeleton. This study thus shows cell adhesion dependence on the EGF effect in the actin cytoskeleton arrangement; this is probably favored by the actin fiber/EGFR association that facilitates the cell signaling pathways for actin cytoskeleton organization in infantile pituitary cells.This work was supported by the National Council of Science and Technology of México (grant 44619, and a fellowship to C.T.).     

Monitoring the Cytoskeletal EGF Response in Live Gastric Carcinoma Cells

Altered cell motility is considered to be a key factor in determining tumor invasion and metastasis. Epidermal growth factor (EGF) signaling has been implicated in this process by affecting cytoskeletal organization and dynamics in multiple ways. To sort the temporal and spatial regulation of EGF-dependent cytoskeletal re-organization in relation to a cell’s motile behavior time-lapse microscopy was performed on EGF-responsive gastric carcinoma-derived MKN1 cells co-expressing different fluorescently labeled cytoskeletal filaments and focal adhesion components in various combinations. The experiments showed that EGF almost instantaneously induces a considerable increase in membrane ruffling and lamellipodial activity that can be inhibited by Cetuximab EGF receptor antibodies and is not elicited in non-responsive gastric carcinoma Hs746T cells. The transient cell extensions are rich in actin but lack microtubules and keratin intermediate filaments. We show that this EGF-induced increase in membrane motility can be measured by a simple image processing routine. Microtubule plus-ends subsequently invade growing cell extensions, which start to accumulate focal complexes at the lamellipodium-lamellum junction. Such paxillin-positive complexes mature into focal adhesions by tyrosine phosphorylation and recruitment of zyxin. These adhesions then serve as nucleation sites for keratin filaments which are used to enlarge the neighboring peripheral keratin network. Focal adhesions are either disassembled or give rise to stable zyxin-rich fibrillar adhesions which disassemble in the presence of EGF to support formation of new focal adhesion sites in the cell periphery. Taken together the results serve as a basis for modeling the early cytoskeletal EGF response as a tightly coordinated and step-wise process which is relevant for the prediction of the effectiveness of anti-EGF receptor-based tumor therapy.     

Sustained Formation of Focal Adhesions with Paxillin in Morphological Differentiation of PC12 Cells

Differentiation of PC12 cells triggered by nerve growth factor (NGF) is characterized by several well-defined events including induction of a set of neuron-specific genes, gain of membrane excitability, and morphological changes such as neurite outgrowth. Here we report that K252a, a protein kinase inhibitor, converts the proliferation signal of epidermal growth factor (EGF) into the morphological differentiation signal without inducing the sustained activation of ERK and the expression of neurofilament. Major effects of EGF/K252a, found also in the NGF-treated cells, are the sustained mobility shift of paxillin in SDS-PAGE and the promoted association of Crk-II with paxillin. These effects explain the prominent and robust development of peripheral focal adhesion assembly and stress fiber-like structures observed in the early stages of PC12 cell differentiation. These results suggest a model that cytoskeletal reorganization via focal adhesion assembly triggered by NGF provides a signal required for the morphological differentiation of PC12 cells.     

SH2-containing 5'-inositol phosphatase, SHIP2, regulates cytoskeleton organization and ligand-dependent down-regulation of the epidermal growth factor receptor

Phosphoinositide lipid second messengers are integral components of signaling pathways mediated by insulin, growth factors, and integrins. SHIP2 dephosphorylates phosphatidylinositol 3,4,5-trisphosphate generated by the activated phosphatidylinositol 3'-kinase. SHIP2 down-regulates insulin signaling and is present at higher levels in diabetes and obesity. SHIP2 associates with p130Cas and filamin, regulators of cell adhesion/migration and cytoskeleton, influencing cell adhesion/spreading. Type I collagen specifically induces Src-mediated tyrosine phosphorylation of SHIP2. To better understand SHIP2 function, we employed RNA interference (RNAi) approach to silence the expression of the endogenous SHIP2 in HeLa cells. Suppression of SHIP2 levels caused severe F-actin deformities characterized by weak cortical actin and peripheral actin spikes. SHIP2 RNAi cells displayed cell-spreading defects involving a notable absence of focal contact structures and the formation of multiple slender membrane protrusions capped by actin spikes. Furthermore, decreased SHIP2 levels altered distribution of early endocytic antigen 1 (EEA1)-positive endocytic vesicles and of vesicles containing internalized epidermal growth factor (EGF) and transferrin. EGF treatment of SHIP2 RNAi cells led to the following: enhanced EGF receptor (EGFR) degradation; increased EGFR ubiquitination; and increased association of EGFR with c-Cbl ubiquitin ligase. Taken together, these experiments demonstrate that SHIP2 functions in the maintenance and dynamic remodeling of actin structures as well as in endocytosis, having a major impact on ligand-induced EGFR internalization and degradation. Accordingly, we suggest that, in HeLa cells, SHIP2 plays a distinct role in signaling pathways mediated by integrins and growth factor receptors.     

Focal contacts and the cytoskeleton

Cultured cells attach to the substratum by means of specialized domains of cell surface, called focal contacts. The inner side of the cell membrane is associated in these structures with cytoskeletal elements, while the outer side is connected with extracellular matrix. The present review describes both light and electron microscopic methods of studying the focal contacts and ultrastructure of adhesion plaque, that is the cytoskeletal domain of focal contact. The proteins of adhesion plaque and focal contact membranes are also characterized. The processes of the formation of focal contacts and their association with the bundles of actin microfilaments in normal cultured fibroblasts are described in detail. Association of focal contacts with other cytoskeletal elements microtubules and intermediate filaments is discussed. The neoplastic transformation induced changes of focal contact system and cytoskeletal structures associated with contact sites are described.     

Actin Polymerization Is Required for Negative Feedback Regulation of Epidermal Growth Factor-Induced Signal Transduction

Epidermal growth factor (EGF) induces rapid actin filament assembly in the membrane skeleton of a variety of cells. To investigate the significance of this process for signal transduction, actin polymerization is inhibited by dihydrocytochalasin B (CB). CB almost completely abolishes EGF-induced actin polymerization, as assessed by quantitative confocal laser scanning microscopy. Under these conditions, EGF induces enhanced EGF receptor (EGFR) tyrosine kinase activity, as well as superinduction of the c-fosproto-oncogene. These data suggest that EGF-induced actin polymerization may be important for negative feedback regulation of signal transduction by the EGFR. The phosphorylation of Thr⁶⁵⁴by protein kinase C (PKC) is a well-characterized negative feedback control mechanism for signal transduction by the EGFR tyrosine kinase. A synthetic peptide, corresponding to the regions flanking Thr⁶⁵⁴of the EGFR, is used to analyze EGF stimulated PKC activity by incorporation of³²P into the peptide. Cotreatment of cells with CB and EGF results in a complete loss of EGF-induced phosphorylation of the peptide. These data suggest that actin polymerization is obligatory for negative feedback regulation of the EGFR tyrosine kinase through the C-kinase pathway.     

Actin filament assembly and actin-myosin contractility are necessary for anchorage- and EGF-dependent activation of phospholipase Cgamma

Formation of actin stress fibers and the focal adhesion complex between cell and the substratum are crucial for nonmalignant cells to achieve anchorage-dependent growth. We show here that the adhesion complex formed in normal human mammary epithelial (HME) cells which adhered to type IV collagen, involved the EGF receptor (EGFR) and phospholipase Cgamma (PLCgamma) as signaling molecules, in addition to integrin beta1, alpha-actinin, and actin even before stimulation of the cells with EGF. Stimulation of cells with EGF induced tyrosine phosphorylation of EGFR and activation of PLCgamma, as assessed by the production of a second messenger diacylglycerol (DAG), without any significant increase in the amount of EGFR-bound PLCgamma. Disruption of either actin filaments by cytochalasin D (CD) or actin-myosin contractility by ML-7, an inhibitor of myosin light chain kinase (MLCK), altered the flattened morphology of quiescent cells to a retracted one, without affecting the association between EGFR and PLCgamma. Stimulation of CD- or ML-7-treated cells with EGF failed to inhibit tyrosine phosphorylation of EGFR and its association and colocalization with PLCgamma, but inhibited the PLCgamma activation. Phosphatidylinositol 4,5-bisphosphate (PtdInsP2), substrate of PLCgamma, was tightly associated with alpha-actinin and the content of alpha-actinin-bound PtdInsP2 was reduced by treatment of cells with ML-7 but not with CD. The amount of PtdInsP2 bound to alpha-actinin was increased by the addition of EGF and this EGF-induced increase was blocked by either CD or ML-7. The present results suggest that anchorage-dependent EGF signaling in HME cells may require both actin filament assembly and actin-myosin contractility for the PLCgamma activation.     

Epidermal growth factor receptor-dependent regulation of integrin-mediated signaling and cell cycle entry in epithelial cells

Integrin-mediated adhesion of epithelial cells to extracellular matrix (ECM) proteins induces prolonged tyrosine phosphorylation and partial activation of epidermal growth factor receptor (EGFR) in an integrin-dependent and EGFR ligand-independent manner. Integrin-mediated activation of EGFR in epithelial cells is required for multiple signal transduction events previously shown to be induced by cell adhesion to matrix proteins, including tyrosine phosphorylation of Shc, Cbl, and phospholipase Cgamma, and activation of the Ras/Erk and phosphatidylinositol 3'-kinase/Akt signaling pathways. In contrast, activation of focal adhesion kinase, Src, and protein kinase C, adhesion to matrix proteins, cell spreading, migration, and actin cytoskeletal rearrangements are induced independently of EGFR kinase activity. The ability of integrins to induce the activation of EGFR and its subsequent regulation of Erk and Akt activation permitted adhesion-dependent induction of cyclin D1 and p21, Rb phosphorylation, and activation of cdk4 in epithelial cells in the absence of exogenous growth factors. Adhesion of epithelial cells to the ECM failed to efficiently induce degradation of p27, to induce cdk2 activity, or to induce Myc and cyclin A synthesis; subsequently, cells did not progress into S phase. Treatment of ECM-adherent cells with EGF, or overexpression of EGFR or Myc, resulted in restoration of late-G(1) cell cycle events and progression into S phase. These results indicate that partial activation of EGFR by integrin receptors plays an important role in mediating events triggered by epithelial cell attachment to ECM; EGFR is necessary for activation of multiple integrin-induced signaling enzymes and sufficient for early events in G(1) cell cycle progression. Furthermore, these findings suggest that EGFR or Myc overexpression may provoke ligand-independent proliferation in matrix-attached cells in vivo and could contribute to carcinoma development.     

Migratory phenotypes of HSC-3 squamous carcinoma cell line induced by EGF and PMA: relevance to migration of loosening of adhesion and vinculin-associated focal contacts with prominent filopodia

Cell migration is involved in carcinoma cell invasion and wound healing. We examined motogenic cytokines that potentiated migration of human HSC-3 carcinoma cells. To assess migratory activity, modified Boyden chambers were used. Among a variety of potential motogenic cytokines, epidermal growth factor (EGF) enhanced migration of HSC-3 cells both on collagen and fibronectin. Phorbol myristate acetate (PMA) also enhanced migration. Inhibitors of protein kinase C completely inhibited PMA-induced migration, but only partly inhibited EGF-induced migration. Protein kinase A was also involved in the EGF-induced signaling pathway for migration. Although the signaling pathways were independent, and the cell shape on collagen was different from that on fibronectin, migratory cells stimulated by EGF or PMA showed common morphology on different ligands. The cells were polygonal or round in shape and the loss of long cytoplasmic extensions was noted. Migratory HSC-3 cells stimulated by EGF or PMA became less adhesive to collagen and fibronectin. Since both EGF- and PMA-stimulated migration did not require de novo protein synthesis, the signaling pathways possibly lead to assembly and disassembly of an actin cytoskeleton. Immunofluorescence for vinculin was concentrated into focal contacts in EGF- and PMA-stimulated HSC-3 cells, whereas the fluorescence signal was hardly detected in non-stimulated cells. Talin and beta1 integrin were immunolocalized at focal contacts in non-stimulated cells, and it remained unchanged in stimulated cells. Numerous filopodia visualized with actin immunofluorescence were formed around stimulated HSC-3 cells, whereas filopodia were short and sparse around elongated cytoplasms in non-stimulated cells. Thus, shortening of cytoplasmic extensions with numerous filopodia, loosening of adhesion, and vinculin-associated focal contacts were regarded as migratory phenotypes.     

Immunoelectron microscope analysis of epidermal growth factor receptor (EGFR) in isolated Mytilus galloprovincialis (Lam.) digestive gland cells: evidence for ligand-induced changes in EGFR intracellular distribution

In mammalian cells, the binding of epidermal growth factor (EGF) to its receptor (EGFR), a glycoprotein with intrinsic tyrosine kinase activity, leads to the pleiotropic responses to EGF. Among these, a negative feedback response by stimulation of receptor internalization and lysosomal degradation, this attenuating signal transduction. In this work, data are reported on the identification of specific EGFRs in isolated digestive gland cells from the marine mussel (Mytilus galloprovincialis Lam.) By immunoelectron microscopy. In control digestive cells, EGFR immunoreactivity was mainly associated with cytoplasmic membrane structures and, to a lesser extent, the cell membrane. The presence of EGFR-like receptors was confirmed by Western blotting of digestive gland cell extracts with two different monoclonal antibodies that recognize either intracellular or extracellular epitopes. The addition of mammalian EGF resulted in significant time and temperature-dependent changes in EGFR subcellular distribution in mussel cells. In cells exposed to EGF for 0-15 min at 4 degrees C, the distribution of EGFR was not significantly different from that of the control cells. On the other hand, at 18 degrees C, an increased labelling along the cell membrane was observed after 5-10 min after EGF addition, with a concomitant decrease in the cytoplasmic signal. Moreover, after 20 min of exposure to EGF, ligand binding apparently resulted in EGFR compartmentation within the lysosomes. These observations were confirmed by quantitative analysis of EGFR labelling at different times of EGF exposure. Similar results were obtained utilizing the two different monoclonal antibodies. The results indicate that, in mussel digestive cells, the binding of heterologous EGF to specific receptors induces a negative feedback response by stimulating the lysosomal degradation of EGFR, thus suggesting the presence of mechanisms responsible for receptor downregulation similar to those observed in mammalian cells.     

Attachment of A-431 cells on immobilized antibodies to the EGF receptor promotes cell spreading and reorganization of the microfilament system

EGF-like sequences, inherent in a number of extracellular matrix proteins, participate in cell adhesion. It is possible that interactions of these sequences with EGF receptors (EGFR) affect actin filament organization. It was shown previously [Khrebtukova et al., 1991: Exp. Cell Res. 194:48-55] that antibodies specific to EGFR induce capping of these receptors and redistribution of cytoskeletal proteins in A-431 cells. Here we report that A-431 cells attach and spread on solid substrata coated with antibodies to EGFR, even in the absence of serum. Thus, EGFR can act as an adhesion protein and promote microfilament reorganization. Binding of the cells to the EGFR-antibody resulted in the formation of a unique cell shape characterized by numerous, actin-based filopodia radiating from the cell body, but without membrane ruffles. There was also a conspicuous circular belt of actin-containing fibers inside the cell margin, and many irregular actin aggregates in the perinuclear area. The morphologies and actin distributions in A-431 cells spread on fibronectin or laminin 2/4 were very different. On fibronectin, cells had polygonal shapes with numerous stress-fibers and thick actin-containing fibers along the cell edges. On laminin-covered substrata, the cells became fusiform and acquired broad leading lamellae with ruffles. In these cells, there were also a few bundles of filaments running the whole length of the cell body, and shorter bundles extending through the leading lamellae towards the membrane ruffles in the cell edge. These effects and those seen with immobilized EGF suggest that different ligand/receptor complexes induce specific reorganizations of the microfilament system.     

The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.

Actin stress fibers are one of the major cytoskeletal structures in fibroblasts and are linked to the plasma membrane at focal adhesions. rho, a ras-related GTP-binding protein, rapidly stimulated stress fiber and focal adhesion formation when microinjected into serum-starved Swiss 3T3 cells. Readdition of serum produced a similar response, detectable within 2 min. This activity was due to a lysophospholipid, most likely lysophosphatidic acid, bound to serum albumin. Other growth factors including PDGF induced actin reorganization initially to form membrane ruffles, and later, after 5 to 10 min, stress fibers. For all growth factors tested the stimulation of focal adhesion and stress fiber assembly was inhibited when endogenous rho function was blocked, whereas membrane ruffling was unaffected. These data imply that rho is essential specifically for the coordinated assembly of focal adhesions and stress fibers induced by growth factors.     

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.     

Zyxin-mediated Actin Assembly Is Required for Efficient Wound Closure

Cytoskeletal regulation of cell adhesion is vital to the organization of multicellular structures. The focal adhesion protein zyxin emerged as a key regulator of actin assembly because zyxin recruits Enabled/vasodilator-stimulated phospho-proteins (Ena/VASP) to promote actin assembly. Zyxin also localizes to the sites of cell-cell adhesion and is thought to promote actin assembly with Ena/VASP. Using shRNA targeted to zyxin, we analyzed the roles of zyxin at adhesive contacts. In zyxin-deficient cells, the actin assembly at both focal adhesion and cell-cell adhesion was limited, but their migration rate was unchanged. Cell spreading on E-cadherin-coated surfaces and the formation of cell clusters were slower for zyxin-deficient cells than wild type cells. By ablating a single cell within a cell monolayer, we quantified the rate of wound closure driven by a contractile circumferential actin ring. Zyxin-deficient cells failed to recruit VASP to cell-cell junctions at the wound edge and had a slower wound closure rate than wild type cells. Our results suggest that, by recruiting VASP, zyxin regulates actin assembly at the sites of force-bearing cell-cell adhesion.     

The Rho GTPases in Macrophage Motility and Chemotaxis

The GTP-binding proteins, Rho, Rac and Cdc42 are known to regulate actin organisation. Rho induces the assembly of contractile actin-based microfilaments such as stress fibres, Rac regulates the formation of membrane ruffles and lamellipodia, and Cdc42 activation is necessary for the formation of filopodia. In addition, all three proteins can also regulate the assembly of integrin-containing focal adhesion complexes. The orchestration of these distinct cytoskeletal changes is thought to form the basis of the co-ordination of cell motility and we have investigated the roles of Rho family proteins in migration using a model system. We have found that in the macrophage cell line Bacl, the cytokine CSF-1 rapidly induces actin reorganisation: it stimulates the formation of filopodia, lamellipodia and membrane ruffles, as well as the appearance of fine actin cables within the cell. We have shown that Cdc42, Rac and Rho regulate the CSF-1 induced formation of these distinct actin filament-based structures. Using a cell tracking procedure we found that both Rho and Rac were required for CSF-1 stimulated cell translocation. In contrast, inhibition of Cdc42 does not prevent macrophages migrating in response to CSF-1, but does prevent recognition of a CSF-1 concentration gradient, so that cells now migrate randomly rather than up the gradient of this chemotactic cytokine. This implies that Cdc42, and thus probably filopodia, are required for gradient sensing and cell polarisation in macrophages.     

P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.     

Integrin-induced epidermal growth factor (EGF) receptor activation requires c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosines.

Integrin-mediated cell adhesion cooperates with growth factor receptors in the control of cell proliferation, cell survival, and cell migration. One mechanism to explain these synergistic effects is the ability of integrins to induce phosphorylation of growth factor receptors, for instance the epidermal growth factor (EGF) receptor. Here we define some aspects of the molecular mechanisms regulating integrin-dependent EGF receptor phosphorylation. We show that in the early phases of cell adhesion integrins associate with EGF receptors on the cell membrane in a macromolecular complex including the adaptor protein p130Cas and the c-Src kinase, the latter being required for adhesion-dependent assembly of the macromolecular complex. We also show that the integrin cytoplasmic tail, c-Src kinase, and the p130Cas adaptor protein are required for phosphorylation of EGF receptor in response to integrin-mediated adhesion. We show that integrins induce phosphorylation of EGF receptor on tyrosine residues 845, 1068, 1086, and 1173, but not on residue 1148, a major site of phosphorylation in response to EGF. In addition we find that integrin-mediated adhesion increases the amount of EGF receptor expressed on the cell surface. Therefore these data indicate that integrin-mediated adhesion induces assembly of a macromolecular complex containing c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosine residues.