Reduced SNAP-25 increases PSD-95 mobility and impairs spine morphogenesis.

Impairment of synaptic function can lead to neuropsychiatric disorders collectively referred to as synaptopathies. The SNARE protein SNAP-25 is implicated in several brain pathologies and, indeed, brain areas of psychiatric patients often display reduced SNAP-25 expression. It has been recently found that acute downregulation of SNAP-25 in brain slices impairs long-term potentiation; however, the processes through which this occurs are still poorly defined. We show that in vivo acute downregulation of SNAP-25 in CA1 hippocampal region affects spine number. Consistently, hippocampal neurons from SNAP-25 heterozygous mice show reduced densities of dendritic spines and defective PSD-95 dynamics. Finally, we show that, in brain, SNAP-25 is part of a molecular complex including PSD-95 and p140Cap, with p140Cap being capable to bind to both SNAP-25 and PSD-95. These data demonstrate an unexpected role of SNAP-25 in controlling PSD-95 clustering and open the possibility that genetic reductions of the protein levels - as occurring in schizophrenia - may contribute to the pathology through an effect on postsynaptic function and plasticity.

p140Cap suppresses the invasive properties of highly metastatic MTLn3-EGFR cells via impaired cortactin phosphorylation.

We have recently shown that the adaptor protein p140Cap regulates tumor properties in terms of cell motility and growth. Here, by using the highly metastatic rat adenocarcinoma cell line MTLn3-epidermal growth factor receptor (EGFR), we assess the role of p140Cap in metastasis formation. Orthotopic transplantation of MTLn3-EGFR cells over-expressing p140Cap in Rag2(-/-)γ(c)(-/-) mice resulted in normal primary tumor growth compared with the controls. Strikingly, p140Cap over-expression causes an 80% inhibition in the number of lung metastases. p140Cap over-expressing cells display a 50% reduction in directional cell migration, an increased number and size of focal adhesions, and a strong impairment in the ability to invade in a 3D matrix. p140Cap over-expression affects EGFR signaling and tyrosine phosphorylation of cortactin in response to EGF stimulation. Intriguingly, p140Cap associates with cortactin via interaction with its second proline-rich domain to the cortactin SH3 domain. The phosphomimetic cortactin tyrosine 421 mutant rescues migration and invasive properties in p140Cap over-expressing cells. Taken together, these data demonstrate that p140Cap suppresses the invasive properties of highly metastatic breast carcinoma cells by inhibiting cortactin-dependent cell motility.

ß1 integrin controls EGFR signaling and tumorigenic properties of lung cancer cells.

Lung cancer is the leading cause of cancer death worldwide. The epidermal growth factor receptor (EGFR) represents the main target for non-small cell lung cancer (NSCLC) therapy, as its overexpression or constitutive activation contributes to malignancy and correlates with poor prognosis. Our previous work demonstrated that in epithelial cells ß1 integrin is required for propagating EGFR signaling from the plasma membrane to the nucleus. In this study, we silenced ß1 integrin in human NSCLC A549 cells. The ß1 integrin-silenced cells show a defective activation of the EGFR signaling cascade, leading to decreased in vitro proliferation, enhanced sensitivity to cisplatin and Gefitinib, impaired migration and invasive behavior. Inhibitory effects on tumor growth and on the EGFR pathway were also observed in in vivo experiments. Moreover, ß1 integrin silencing increases the amount of EGFR on the cell surface, suggesting that ß1 integrin is required for efficient constitutive EGFR turnover at the cell membrane. Although the rate of EGF internalization and recycling is not affected in silenced cells, EGFR signaling is recovered only by expression of the Rab-coupling protein RCP, indicating that ß1 integrin sustains the endocytic machinery required for EGFR signaling. Overall, these results show that ß1 integrin is an essential regulator of EGFR signaling and tumorigenic properties of lung cancer cells, and that its silencing might represent an adjuvant approach to anti-EGFR therapy.

Inhibition of ß1 integrin and IL-3Rß common subunit interaction hinders tumour angiogenesis.

Integrin/cytokine receptor interaction provides permissive signals leading to neoangiogenesis, and integrins are crucial for differentiation of endothelial progenitor cells (EPCs). It is known that the inflammatory interleukin-3 (IL-3), released in the tumoral microenvironment, contributes to both angiogenesis and vasculogenic processes. Herein, we generated IL-3 receptor beta common (IL-3Rßc) extracellular domain-derived fusion proteins (Fc) to elucidate the molecular mechanisms regulating these processes. Three different Fc were generated, containing the entire extracellular domain of IL-3Rßc (Fc1.4), a fragment corresponding to domains 1-3 (Fc1.3) and a fragment corresponding to domain 4 (Fc4), respectively. The ability of the fusion proteins to interfere with IL-3Rßc/ß1 integrin interaction was assessed on endothelial cells (ECs), EPCs and murine-derived ECs. Pull-down experiments showed that Fc1.4 and Fc4 fusion proteins specifically interacted with ß1 integrin. Fc4 and Fc1.4 fragments prevented IL-3-mediated EPC expansion, arterial morphogenesis and tumour-derived EC migration, without affecting cell adhesion. Fc4 in vivo inhibited the IL-3-mediated vasculogenic process, as well as inflammatory and tumour vascular growth. In conclusion, these data identify the ß1 integrin-interacting domain in the juxta-membrane IL-3Rßc extracellular domain, and provide the rational for targeting this interaction to impair vascular growth.

p140Cap dual regulation of E-cadherin/EGFR cross-talk and Ras signalling in tumour cell scatter and proliferation.

The adaptor protein p140Cap/SNIP is a novel Src-binding protein that regulates Src activation through C-terminal Src kinase (Csk). Here, by gain and loss of function approaches in breast and colon cancer cells, we report that p140Cap immobilizes E-cadherin at the cell membrane and inhibits EGFR and Erk1/2 signalling, blocking scatter and proliferation of cancer cells. p140Cap-dependent regulation of E-cadherin/EGFR cross-talk and cell motility is due to the inhibition of Src kinase. However, rescue of Src activity is not sufficient to restore Erk1/2 phosphorylation and proliferation. Indeed, p140Cap also impairs Erk1/2 phosphorylation by affecting Ras activity, downstream to the EGFR. In conclusion, p140Cap stabilizes adherens junctions and inhibits EGFR and Ras signalling through the dual control of both Src and Ras activities, thus affecting crucial cancer properties such as invasion and growth. Interestingly, p140Cap expression is lost in more aggressive human breast cancers, showing an inverse correlation with EGFR expression. Therefore, p140Cap mechanistically behaves as a tumour suppressor that inhibits signalling pathways leading to aggressive phenotypes.

Physical and functional interaction between integrins and hERG potassium channels.

Integrins are adhesion receptors capable of transmitting intracellular signals that regulate many different cellular functions. Among integrin-mediated signals, the activation of ion channels can be included. We demonstrated that a long-lasting activation of hERG (human ether-a-go-go-related gene) potassium channels occurs in both human neuroblastoma and leukaemia cells after the activation of the beta1 integrin subunit. This activation is apparently a determining factor inducing neurite extension and osteoclastic differentiation in both the cell types. More recently, we provided evidences that beta1 integrins and hERG channels co-precipitate in both the cell types. Preliminary results suggest that a macromolecular signalling complex indeed occurs between integrins and the hERG1 protein and that hERG channel activity can modulate integrin downstream signalling.

Integrin regulation of epidermal growth factor (EGF) receptor and of EGF-dependent responses.

Integrin signalling co-ordinates with signalling originating from growth factor receptors in the co-operative control of cell proliferation, survival and migration. Increasing evidence suggests that integrins form physical complexes at the cell membrane with growth factor receptors, giving rise to signalling platforms at the adhesive sites. It is probable that at these sites integrins regulate adhesion and at the same time physically constrain and direct the response to soluble growth factors towards proliferation or survival stimuli. These co-operative effects might depend on integrin ability to activate growth factor receptors. In the present paper, we summarize our recent study showing that integrin-dependent adhesion triggers ligand-independent EGFR (epidermal growth factor receptor) activation to transduce downstream signalling. In addition, we also show that integrin-induced signalling pathways are necessary for EGF-dependent transcriptional response, demonstrating the requirement of the co-operation between cell-matrix adhesion and EGFR to achieve full biological responses.

HERG K+ channels activation during beta(1) integrin-mediated adhesion to fibronectin induces an up-regulation of alpha(v)beta(3) integrin in the preosteoclastic leukemia cell line FLG 29.1.

Integrin receptors have been demonstrated to mediate either "inside-to-out" and "outside-to-in" signals, and by this way are capable of regulating many cellular functions, such as cell growth and differentiation, cell migration, and activation. Among the various integrin-centered signaling pathways discovered so far, we demonstrated that the modulation of the electrical potential of the plasma membrane (V(REST)) is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells. This modulation is sustained by the activation of HERG K(+) channels, encoded by the ether-à-go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in the hemopoietic system: in particular, osteoclasts are generated as well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin receptors. We studied the effects of cell interaction with the extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that this adhesion induces an osteoclast phenotype in these cells, as evidenced by the appearance of tartrate-resistant acid phosphatase, as well as by the increased expression of CD51/alpha(v)beta(3) integrin and calcitonin receptor. An early activation of HERG current (I(HERG)), without any increase in herg RNA or modifications of HERG protein was also observed in FN-adhering cells. This activation is apparently sustained by the beta(1) integrin subunit activation, through the involvement of a pertussis-toxin sensitive G(i) protein, and appears to be a determinant signal for the up-regulation of alpha(v)beta(3) integrin, as well as for the increased expression of calcitonin receptor.

Distinct involvement of cdc42 and RhoA GTPases in actin organization and cell shape in untransformed and Dbl oncogene transformed NIH3T3 cells.

The Dbl oncogene is a putative exchange factor for the small GTPases RhoA and Cdc42, which are involved in actin polymerization into stress fibers and filopodia, respectively. We report here that, upon adhesion to fibronectin, Dbl-transformed NIH3T3 cells display a contracted, polygonal shape with a high number of short stress fibers. In contrast, untransformed NIH3T3 cells acquire the characteristic fibroblast morphology and organize a regular mesh of long stress fibers. We show that in Dbl-transformed and in untransformed NIH3T3 cells the different shape and actin cytoskeleton organization observed in the early steps of adhesion involves activation of distinct GTPases. Upon adhesion to fibronectin, cell morphology of Dbl-transformed NIH3T3 cells depends on activation of RhoA and not of Cdc42. In contrast Cdc42 activation is necessary to untransfected NIH3T3 cells to acquire their fibroblast shape. In both Dbl-transformed and in untransformed NIH3T3 cells a basal Rac activation is necessary to support stress fiber organization, while constitutive Rac activation promotes ruffles and lamellipodia formation. As a consequence of RhoA activation, Dbl-transformed cells show high activity of ROCK-alpha and CRIK kinases, two known RhoA effectors. In addition Dbl-transformed and NIH3T3 cells expressing the constitutive active form of RhoA are less motile on fibronectin than cells expressing constitutive active Cdc42. We conclude that in NIH3T3 cells in response to fibronectin the expression of the Dbl oncogene leads to a predominant activation of RhoA which both supports the peculiar cell shape and actin cytoskeleton organization in stress fibers and regulates cell motility.

Actin cytoskeleton organization in response to integrin-mediated adhesion.

Cell matrix adhesion regulates actin cytoskeleton organization through distinct steps, from formation of filopodia and lamellipodia in the early phases of cell adhesion to organization of focal adhesions and stress fibers in fully adherent cells. In this review, we follow the events induced by integrin-mediated adhesion, such as activation of GTPases Cdc42 and Rac and their effectors and their role in actin polymerization leading to formation of lamellipodia and filopodia and cell spreading. We also show that actin stress fiber and focal adhesion formation following adhesion requires cooperation between integrin-mediated signaling and additional stimuli, including activation of PKC, Rho GTPases, and PTKs such as p125Fak and Src.

Integrin-mediated adhesion of endothelial cells induces JAK2 and STAT5A activation: role in the control of c-fos gene expression.

Integrin-mediated adhesion induces several signaling pathways leading to regulation of gene transcription, control of cell cycle entry and survival from apoptosis. Here we investigate the involvement of the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in integrin-mediated signaling. Plating primary human endothelial cells from umbilical cord and the human endothelial cell line ECV304 on matrix proteins or on antibody to beta1- or alphav-integrin subunits induces transient tyrosine phosphorylation of JAK2 and STAT5A. Consistent with a role for the JAK/STAT pathway in regulation of gene transcription, adhesion to matrix proteins leads to the formation of STAT5A-containing complexes with the serum-inducible element of c-fos promoter. Stable expression of a dominant negative form of STAT5A in NIH3T3 cells reduces fibronectin-induced c-fos mRNA expression, indicating the involvement of STAT5A in integrin-mediated c-fos transcription. Thus these data present a new integrin-dependent signaling mechanism involving the JAK/STAT pathway in response to cell-matrix interaction.

Association of the dystroglycan complex isolated from bovine brain synaptosomes with proteins involved in signal transduction.

Dystroglycan is a transmembrane heterodimeric complex of alpha and beta subunits that links the extracellular matrix to the cell cytoskeleton. It was originally identified in skeletal muscle, where it anchors dystrophin to the sarcolemma. Dystroglycan is also highly expressed in nonmuscle tissues, including brain. To investigate the molecular interactions of dystroglycan in the CNS, we fractionated a digitonin-soluble extract from bovine brain synaptosomes by laminin-affinity chromatography and characterized the protein components. The 120-kDa alpha-dystroglycan was the major 125I-laminin-labeled protein detected by overlay assay. This complex, in addition to beta-dystroglycan, was also found to contain Grb2 and focal adhesion kinase p125FAK (FAK). Anti-FAK antibodies co-immunoprecipitated Grb2 with FAK. However, no direct interaction between beta-dystroglycan and FAK was detected by co-precipitation assay. Grb2, an adaptor protein involved in signal transduction and cytoskeleton organization, has been shown to bind beta-dystroglycan. We isolated both FAK and Grb2 from synaptosomal extracts by chromatography on immobilized recombinant beta-dystroglycan. In the CNS, FAK phosphorylation has been linked to membrane depolarization and neurotransmitter receptor activation. At the synapses, the adaptor protein Grb2 may mediate FAK-beta-dystroglycan interaction, and it may play a role in transferring information between the dystroglycan complex and other signaling pathways.

Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2.

Interaction between integrin alphavbeta3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin-mediated outside-in signals co-operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between alphavbeta3 integrin and tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) in human endothelial cells. We report that tyrosine-phosphorylated VEGFR-2 co-immunoprecipitated with beta3 integrin subunit, but not with beta1 or beta5, from cells stimulated with VEGF-A165. VEGFR-2 phosphorylation and mitogenicity induced by VEGF-A165 were enhanced in cells plated on the alphavbeta3 ligand, vitronectin, compared with cells plated on the alpha5beta1 ligand, fibronectin or the alpha2beta1 ligand, collagen. BV4 anti-beta3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR-2; (ii) the activation of downstream transductor phosphoinositide 3-OH kinase; and (iii) biological effects triggered by VEGF-A165. These results indicate a new role for alphavbeta3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, alphavbeta3 integrin participates in the full activation of VEGFR-2 triggered by VEGF-A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration.

Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival.

Adhesion of human primary skin fibroblasts and ECV304 endothelial cells to immobilized matrix proteins, beta1 or alphav integrin antibodies stimulates tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30 min after adhesion, and it occurs in the absence of receptor ligands. Similar results were observed with EGF receptor-transfected NIH-3T3 cells. Use of a kinase-negative EGF receptor mutant demonstrates that the integrin-stimulated tyrosine phosphorylation is due to activation of the receptor's intrinsic kinase activity. Integrin-mediated EGF receptor activation leads to Erk-1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin AG1478 and by expression of a dominant-negative EGF receptor mutant. EGF receptor and Erk-1/MAP kinase activation by integrins does not lead per se to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular matrix-mediated cell survival. Adhesion-dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing this molecule above a threshold level (5x10(3) receptors per cell). These results demonstrate that integrin-dependent EGF receptor activation is a novel signaling mechanism involved in cell survival and proliferation in response to extracellular matrix.

Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation.

A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor. While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.

Actin cytoskeleton polymerization in Dbl-transformed NIH3T3 fibroblasts is dependent on cell adhesion to specific extracellular matrix proteins.

The Dbl oncogene is the putative exchange factor for two small GTP-binding proteins, RhoA and CDC42 which are involved in the polymerization of actin to produce stress fibers and filopodia, respectively. We report here that Dbl oncogene-transformed NIH3T3 cells show actin stress fibers only when cells are plated on fibronectin. Plating of cells on collagen I and IV as well as on poly-D-lysine and gelatin induces polymerization of actin to form filopodia, lamellipodia and membrane ruffles but not stress fibers. The putative collagen receptors, alpha1/beta1 and alpha2/beta1 integrins are expressed at reduced level in Dbl-transformed cells compared to untransformed NIH3T3 fibroblasts. Nevertheless, adhesion to collagens is not altered. Inhibitory monoclonal antibody to mouse integrin beta1 subunit blocked adhesion of both Dbl-transformed and untransformed NIH3T3 cells, demonstrating that adhesion to collagen I and IV is mediated by the beta1 family of integrins. Dbl product rapidly induces the depolymerization of actin stress fibers, rounding up of the cells, and formation of filopodia and lamellipodia when microinjected in NIH3T3 cells plated on gelatin. Thus, Dbl may exert its effect on actin cytoskeleton organization in response to extracellular proteins by altering integrin-mediated signalling pathways.

Phenotypic features of BHK-21 cells used for production of foot-and-mouth disease vaccine.

BHK-21 c13 monolayer and suspension cells were investigated with regard to some phenotypic features which could bear on the quality of foot-and-mouth disease virus (FMDV) antigen produced in them. Despite good viability, suspension cells differed from monolayer cells in fundamental features of susceptibility to FMDV. Most important, FMD virus particles grown in suspension cells at high passage levels were shown to be largely degraded following inactivation with an aziridine compound. Suspension cells were characterised by a downregulation in the surface expression of both alpha 5 and alpha V integrin chains. According to the results of binding assays, both integrins could act as FMDV receptors on BHK-21 c13 cells. Reduced surface expression of integrins was correlated with disappearance of actin stress fibres, which would play a role in regular encapsidation of viral RNA and hence in stability of virus particles. With regard to FMD vaccine production, practical suggestions are put forward to evaluate the quality of BHK-21 c13 cells and FMDV Ag, which must prove stable during downstream processing.

Focal adhesion and stress fiber formation is regulated by tyrosine phosphatase activity.

Tyrosine phosphorylation of cytoskeletal proteins plays an important role in the regulation of focal adhesions and stress fiber organization. In the present study we examined the role of tyrosine phosphatases in this process using p125FAK and paxillin as substrates. We show that tyrosine phosphatase activity in Swiss 3T3 cells was markedly increased when actin stress fibers were disassembled by cell detachment from the substratum, by serum starvation, or by cytochalasin D treatment. This activity was blocked by phenylarsine oxide, an inhibitor of a specific class of tyrosine phosphatases characterized by two vicinal thiol groups in the active site. Phenylarsine oxide treatment of serum-starved cells induced increased tyrosine phosphorylation of p125FAK and paxillin in a dose-dependent manner and induced assembly of focal adhesions and actin stress fibers, showing that inhibition of one or more phenylarsine oxide-sensitive tyrosine phosphatases is a sufficient stimulus for triggering focal adhesion and actin stress fiber formation in adherent cells.

Platelet-activating factor (PAF) induces the early tyrosine phosphorylation of focal adhesion kinase (p125FAK) in human endothelial cells.

Platelet-activating factor (PAF) is a potent activator of angiogenesis and controls the motility and the shape of vascular endothelium. The mechanism(s) whereby PAF exerts its action are in part known. Here we report that the biological active (R)PAF enantiomer administrated to cultured endothelial cells induces the early phosphorylation in tyrosine residues of focal adhesion kinase (p125FAX) and paxillin, two molecules involved in the early signaling and cytoskeleton assembly in cells that undergo integrin-mediated adhesion or are challenged by neuropeptides or lysophosphatidic acid. The phenomenon is rapidly turned on, lasts for a few minutes and is adhesion-independent indicating that the chain of events induced by (R)PAF, including p125FAK activation, precedes adhesion. The inhibitory effect of WEB2086, a PAF receptor antagonist, and the lack of activity exerted by the (S)PAF enantiomer, indicate that (R)PAF-mediated p125FAK activation, is PAF receptor-dependent. Calphostin C, an inhibitor of protein kinase C blocks the effect of (R)PAF on p125FAK phosphorylation suggesting that protein kinase C activation is up-stream the activation of this tyrosine kinase. When endothelial cells are exposed to a substratum that allows adhesion and spreading. (R)PAF-stimulated cells, change their adhesive phenotype and start migrating. Inhibitors of tyrosine kinases, like 3-(1,4,-dihydroxytetralyl) methylen-2-oxindole and herbimycin A, reduce the cells migration, the transendothelial flux of albumin and the enhancement of p125FAK activity induced by (R)PAF. The observation that increased tyrosine phosphorylation of p125FAK and its ensuring association with focal adhesion occurs rapidly upon (R)PAF challenge indicates that this signaling molecule has a primary and independent role also in the signaling cascade initiated by (R)PAF.