RESUMO
In this paper we report that the integrin complex alpha 1/beta 1, a laminin/collagen receptor, is expressed on cultured foreskin microvascular endothelium, but is absent on endothelial cells from large vessels such as the aorta and umbilical and femoral veins. The restricted expression of integrin alpha 1/beta 1 to microvascular endothelium was also demonstrated in vivo, by immunohistochemical staining of human tissue sections. Alpha 1 specific antibodies reacted strongly with endothelial cells of small blood vessels and capillaries in several tissues, but not with endothelium of vein and arteries of umbilical cord. Expression of integrin alpha 1 can be induced in cultured umbilical vein endothelial cells by treatment with 5 ng/ml tumor necrosis factor alpha (TNF alpha). Induction of alpha 1 subunit expression also occurred after treatment of umbilical vein endothelium with 10(-5) M retinoic acid or with 10 nM PMA; Maximal induction of alpha 1 integrin was reached after 48 h of treatment and costimulation with TNF alpha and PMA resulted in a synergistic effect. The induction of alpha 1 integrin changed the adhesive properties of umbilical vein endothelial cells, by increasing the adhesiveness to collagen, laminin, and laminin fragment P1, while adhesion to fibronectin and laminin fragment E8 remained constant. The alpha 1 integrin is thus a marker of a specific population of endothelial cells and its expression confers distinctive properties of interaction with the underlying basal membrane.
Assuntos
Endotélio Vascular/fisiologia , Integrinas/biossíntese , Sequência de Aminoácidos , Adesão Celular , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Integrinas/análise , Integrinas/isolamento & purificação , Cinética , Masculino , Dados de Sequência Molecular , Músculos/citologia , Músculos/fisiologia , Peptídeos/síntese química , Peptídeos/imunologia , Pele/irrigação sanguínea , Acetato de Tetradecanoilforbol/farmacologia , Tretinoína/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Veias UmbilicaisRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/genética , Genes fos , Integrinas/metabolismo , Proteínas do Leite , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Transativadores/metabolismo , Células 3T3 , Animais , Adesão Celular , Linhagem Celular , Ativação Enzimática/genética , Proteínas da Matriz Extracelular/metabolismo , Imunofluorescência , Humanos , Janus Quinase 2 , Camundongos , Proteínas Nucleares/metabolismo , Fosforilação , Fosfotirosina/análise , RNA Mensageiro/metabolismo , Fator de Transcrição STAT5 , Transdução de Sinais , Transfecção , Proteínas Supressoras de TumorRESUMO
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.
Assuntos
Actinas/fisiologia , Citoesqueleto/fisiologia , Oncogenes , Proteínas Oncogênicas de Retroviridae/genética , Células 3T3 , Animais , Adesão Celular , Divisão Celular , Linhagem Celular Transformada , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Matriz Extracelular , Fatores de Troca do Nucleotídeo Guanina , Integrinas/biossíntese , Camundongos , Microinjeções , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Oncogênicas de Retroviridae/biossíntese , Proteínas Oncogênicas de Retroviridae/farmacologia , TransfecçãoRESUMO
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.
Assuntos
Actinas/fisiologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Proteínas Oncogênicas de Retroviridae/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Células 3T3 , Animais , Células COS , Linhagem Celular Transformada , Inibição de Migração Celular , Movimento Celular/genética , Tamanho Celular , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Ativação Enzimática , Fibronectinas/fisiologia , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Oncogênicas de Retroviridae/biossíntese , Proteínas Oncogênicas de Retroviridae/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Moléculas de Adesão Celular/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Fator de Ativação de Plaquetas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Tirosina/metabolismo , Sequência de Aminoácidos , Citoesqueleto/efeitos dos fármacos , Ativação Enzimática , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Dados de Sequência Molecular , Fosforilação , Glicoproteínas da Membrana de Plaquetas/fisiologia , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidoresRESUMO
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.
Assuntos
Espinhas Dendríticas/fisiologia , Guanilato Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfogênese , Plasticidade Neuronal/fisiologia , Sinapses/metabolismo , TransfecçãoRESUMO
2-5A[ppp(A2'p)n5'A] has been implicated as a mediator in the antiviral action of interferon. Its direct evaluation as an indicator of virus replication is hampered by two limitations: its inability to penetrate intact cells, and its rapid intracellular degradation by (2'-5')phosphodiesterase. These problems could be overcome by using a microinjection technique whereby a phosphodiesterase-resistant analog of 2-A, in which the 2'-terminals adenosine residue is replaced by 2-(9-adenyl)-6-hydroxy-methyl-4-hexylmorpholine, was injected into individual HeLa cells before infection with mengovirus or vesicular stomatitis virus (VSV). This comparative assay with two representatives of different virus classes in a single experimental system pointed to the high sensitivity of VSV to inhibition by 2-5A oligonucleotides, in contrast with the low sensitivity of mengovirus. Microinjection of the hexylmorpholine 2-5A analog led to a much greater reduction in mengovirus yield than did microinjection of 2-5A itself.
Assuntos
Nucleotídeos de Adenina/farmacologia , Antivirais/farmacologia , Oligorribonucleotídeos/farmacologia , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Interferons/farmacologia , Mengovirus/efeitos dos fármacos , Microinjeções , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Interferon-induced 2-5A synthetases are probably involved in some antiviral actions of interferon. In human cells two different mRNAs (1.6, 1.8 kb long) coding for this protein are transcribed from the same gene and are produced by differential splicing. The relationship between the two mRNAs of different size and the active enzyme is not clear, nor is the cellular localization of the latter known. We have cloned a cDNA corresponding to the 1.6 kb RNA. This cDNA was sequenced and its complete coding region was subcloned into pSP64. The resulting plasmid was used to direct the synthesis of micrograms of capped RNA transcript after linearization in the 3'-non-coding region. A 39 kDa protein was synthesized when this RNA was translated in rabbit reticulocyte lysate. When this capped RNA was introduced by microinjection into Xenopus oocytes, production of 2-5A synthetase was clearly observed in the cytoplasm and 10-30% of the enzyme accumulated with time in the nucleoplasm. Analysis of cytoplasmic homogenates of these oocytes on a glycerol gradient revealed that the enzyme is fully active in the monomeric form.
Assuntos
2',5'-Oligoadenilato Sintetase/genética , Interferon Tipo I/farmacologia , 2',5'-Oligoadenilato Sintetase/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/análise , Sistema Livre de Células , Citoplasma/análise , DNA/análise , DNA Recombinante , Indução Enzimática/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Microinjeções , Oócitos/metabolismo , Splicing de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Xenopus laevisRESUMO
Cells derived from skin biopsies from two Kaposi's sarcoma patients, an elderly female with a sporadic non-AIDS form, and an AIDS-affected homosexual male, were established in culture. The classic patient had a few small lesions, while the epidemic case presented-large, disseminated, cutaneous and oral mucosa lesions. The cells obtained from both patients, termed IST-KS2 and AIDS-IST-KS3 respectively, had the characteristic spindle shape reported for Kaposi's sarcoma-derived cells. By immunocytochemistry they were both found to express the smooth muscle specific isoform of alpha actin. The KS cells expressed the fibroblastic antigen TE-7, which is not expressed in endothelial cells. Furthermore both KS cultures were negative for the endothelium associated markers Factor VIII, EN4 and PAL-E. They were also negative for the leukocyte antigen CD45, but were positive for vimentin. Immunocytochemistry studies were therefore suggestive of a primitive mesenchymal cell. When the KS-derived cells were grown on a gel of reconstituted basement membrane, both cultures formed large branching colonies characteristic of malignant cells of mesenchymal origin. No differences were observed between HIV-related and the sporadic KS-derived cultures studied. Fibroblasts and smooth muscle cells did not form branching colonies, while endothelial cells on matrigel differentiated forming tube-like structures. Supernatants from both sporadic and AIDS-related KS cell cultures had similar effects on endothelial cell growth in vitro and were also found to stimulate chemotaxis and chemoinvasion of normal vascular endothelial cells in the Boyden chamber assay, showing angiogenic potential in vitro. Our results demonstrate that long term cultures of spindle shaped cells derived from either HIV-associated and classic KS show the same histocytochemical phenotype, have invasiveness in matrigel similar to that of malignant sarcomas, and share in vitro angiogenic properties. Therefore, factors from the host are likely to be responsible for the divergent clinical picture of the classic and epidemic Kaposi's patients studied here.
RESUMO
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.
Assuntos
Actinas/fisiologia , Adesão Celular/efeitos dos fármacos , Citoesqueleto/química , Integrinas/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Células 3T3 , Actinas/química , Animais , Ativação Enzimática , Matriz Extracelular/fisiologia , Fibronectinas/fisiologia , GTP Fosfo-Hidrolases/fisiologia , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Faloidina , Proteína Quinase C/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt , Pseudópodes/fisiologiaRESUMO
Analysis of five personal cases of the intravertebral vacuum cleft phenomenon and a close examination of the literature, suggest that it is rather more frequent than it first appears. Intravertebral vacuum cleft tends to occur more readily in the vertebrae of the dorsolumbar hinge and mainly in the elderly, slightly more common in women than men. The main factors, from the pathogenetic point of view, are osteoporosis, a history of trauma and frequent occurrence of rheumatic problems which are often complicated by vasculitis. The comparison between excessive uptake of calcium-mimetic technetium and uptake deficit of technetium phytate by the vertebra with a vacuum cleft, leads us to consider it as an expression of osteo and bone marrow necrosis.
Assuntos
Disco Intervertebral/diagnóstico por imagem , Doenças da Coluna Vertebral/diagnóstico por imagem , Idoso , Feminino , Gases , Humanos , Masculino , Pessoa de Meia-Idade , Osteoporose/complicações , Radiografia , Cintilografia , Doenças da Coluna Vertebral/complicaçõesRESUMO
Monoclonal antibodies (mAbs) against cultured human ovary carcinoma cells were produced. We obtained 7 mAbs which reacted diffusely with carcinoma of the ovary but only weakly with vessels and the surface epithelial layer of normal ovary. Biochemical characterization of these mAbs indicated that 3 out of 7 were specific for the alpha 3 chain of the Vla-3 integrin, a receptor for fibronectin, collagen and laminin. Using one of these mAbs, we have studied, by immunohistochemical methods, the distribution of alpha 3 beta 1 integrin in mucinous, serous and endometrioid cystoadenocarcinoma of the ovary and in their normal equivalent: endocervical, tubal and endometrial epithelia. The results show that alpha 3 beta 1 is present in cell-cell contact areas and more abundantly at the junction between epithelial cells and basement membrane in endocervical, tubal epithelia, in epithelium lining the cavity of the uterus and in surface epithelium of the ovary. However, endometrial glands showed only weak and fragmented positivity at the basal pole of the cells. 26 out of 31 ovarian cancers studied, expressed the alpha 3 beta 1 integrin. However, basal localization, typical of normal epithelia, is not prominent or disappears in tumors and is replaced by a more diffuse reaction with variable immunohistochemical staining of the neoplastic cells. Furthermore, a comparative analysis of the expression of alpha 3 beta 1 and its ligands, laminin (LM), fibronectin (FN) and collagen IV (Coll IV), demonstrated that basal polarization of Vla-3 was always correlated with the presence of laminin and Coll IV, intrinsic components of the basement membranes.
Assuntos
Integrinas/análise , Neoplasias Ovarianas/química , Ovário/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Colágeno/análise , Epitélio/química , Feminino , Fibronectinas/análise , Humanos , Imuno-Histoquímica , Integrina alfa3beta1 , Laminina/análise , Camundongos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cortactina/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Cortactina/genética , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Microscopia de Fluorescência por Excitação Multifotônica , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Fosforilação/efeitos dos fármacos , Ligação Proteica , Interferência de RNA , Ratos , Transplante HeterólogoRESUMO
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.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/metabolismo , Integrina beta1/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Anticorpos Monoclonais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cisplatino/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Gefitinibe , Humanos , Integrina beta1/genética , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Invasividade Neoplásica , Transplante de Neoplasias , Quinazolinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Transplante HeterólogoRESUMO
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.
Assuntos
Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Integrina beta1/metabolismo , Interleucina-3/metabolismo , Neovascularização Patológica/metabolismo , Adulto , Animais , Adesão Celular/fisiologia , Movimento Celular , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Camundongos , Monócitos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Células-TroncoRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Caderinas/metabolismo , Movimento Celular , Receptores ErbB/metabolismo , Neoplasias/patologia , Receptor Cross-Talk , Transdução de Sinais , Proteínas ras/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Fator de Crescimento Epidérmico/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Estabilidade Proteica , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismoAssuntos
Endotélio Vascular/fisiologia , Integrinas/metabolismo , Interferon gama/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Anticorpos , Anticorpos Monoclonais , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Citometria de Fluxo/métodos , Humanos , Integrinas/efeitos dos fármacos , Integrinas/isolamento & purificação , Receptores de Fibronectina , Receptores Imunológicos/efeitos dos fármacos , Receptores Imunológicos/isolamento & purificação , Receptores Imunológicos/metabolismo , Veias UmbilicaisRESUMO
Endothelial cells from human umbilical vein (HEC) express several distinct integrin complexes that mediate the interaction with the basal membrane components. In this paper we show that treatment with tumor necrosis factor alpha (TNF alpha) down-regulates the expression of the laminin receptor alpha 6.beta 1 integrin in cultured HEC. After 48 h of treatment with TNF alpha, the level of expression of the alpha 6.beta 1 complex reached 20% of the control value. The down-regulation of the alpha 6.beta 1 integrin is caused by a decreased expression of the alpha 6 subunit, whereas the synthesis of the beta 1 subunit remains constant. Northern blot analysis shows that the decreased level of alpha 6 subunit synthesis is caused by down-regulation of alpha 6 mRNA in TNF alpha-treated HEC. TNF alpha treatment does not alter the expression of alpha 2, alpha 3, and alpha 5 integrins, also present on endothelial cell surface, thus showing that this cytokine has a selective action on distinct integrin complexes. Down-regulation of alpha 6.beta 1 correlates with pronounced reduction in adhesion of TNF alpha-treated HEC to laminin, but not to fibronectin-coated culture dishes. In addition to TNF alpha, interleukin-1 beta also decreases the expression of the alpha 6.beta 1 integrin and reduces adhesion to laminin, thus suggesting that this regulation plays an important role in inflammation.