RESUMO
Leukocyte entry into the central nervous system (CNS) is essential for immune surveillance but is also the basis for the development of pathologic inflammatory conditions within the CNS, such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). The actin-binding protein, cortactin, in endothelial cells is an important player in regulating the interaction of immune cells with the vascular endothelium. Cortactin has been shown to control the integrity of the endothelial barrier and to support neutrophil transendothelial migration in vitro and in vivo in the skin. Here we use cortactin gene-inactivated male and female mice to study the role of this protein in EAE. Inducing EAE by immunization with a myelin oligodendrocyte glycoprotein peptide (MOG35-55) revealed an ameliorated disease course in cortactin gene-deficient female mice compared with WT mice. However, proliferation capacity and expression of IL-17A and IFNγ by cortactin-deficient and WT splenocytes did not differ, suggesting that the lack of cortactin does not affect induction of the immune response. Rather, cortactin deficiency caused decreased vascular permeability and reduced leukocyte infiltration into the brains and spinal cords of EAE mice. Accordingly, cortactin gene-deficient mice had smaller numbers of proinflammatory cuffs, less extensive demyelination, and reduced expression levels of proinflammatory cytokines within the neural tissue compared with WT littermates. Thus, cortactin contributes to the development of neural inflammation by supporting leukocyte transmigration through the blood-brain barrier and, therefore, represents a potential candidate for targeting CNS autoimmunity.SIGNIFICANCE STATEMENT Multiple sclerosis is an autoimmune neuroinflammatory disorder, based on the entry of inflammatory leukocytes into the CNS where these cells cause demyelination and neurodegeneration. Here, we use a mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis, and show that gene inactivation of cortactin, an actin binding protein that modulates actin dynamics and branching, protects against neuroinflammation in experimental autoimmune encephalomyelitis. Leukocyte infiltration into the CNS was inhibited in cortactin-deficient mice, and lack of cortactin in cultured primary brain endothelial cells inhibited leukocyte transmigration. Expression levels of proinflammatory cytokines in the CNS and induction of vascular permeability were reduced. We conclude that cortactin represents a novel potential target for the treatment of multiple sclerosis.
Assuntos
Barreira Hematoencefálica , Quimiotaxia de Leucócito/fisiologia , Cortactina/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Leucócitos/imunologia , Migração Transendotelial e Transepitelial/fisiologia , Animais , Encéfalo/imunologia , Encéfalo/patologia , Cortactina/deficiência , Cortactina/genética , Citocinas/biossíntese , Citocinas/genética , Encefalomielite Autoimune Experimental/patologia , Feminino , Linfonodos/imunologia , Linfonodos/patologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/imunologia , Infiltração de Neutrófilos , Fragmentos de Peptídeos/imunologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Medula Espinal/imunologia , Medula Espinal/patologia , Baço/imunologia , Baço/patologiaRESUMO
Cortactin (CTTN) is a substrate of the Src kinase Lyn that is known to play an actin cytoskeletal regulatory role involved in cell migration and cancer progression following its phosphorylation at Y421. We recently demonstrated that Cortactin is overexpressed in patients with chronic lymphocytic leukaemia (CLL). This work was aimed at defining the functional role of Cortactin in these patients. We found that Cortactin is variably expressed in CLL patients both in the peripheral blood and lymph nodes and that its expression correlates with the release of matrix metalloproteinase 9 (MMP-9) and the motility of neoplastic cells. Cortactin knockdown, by siRNA, induced a reduction in MMP-9 release as well as a decrease of migration capability of leukaemic B cells in vitro, also after chemotactic stimulus. Furthermore, Cortactin phosphorylation was lowered by the Src kinase-inhibitor PP2 with a consequent decrease of MMP-9 release in culture medium. An impaired migration, as compared to control experiments without Cortactin knockdown, was observed following CXCL12 triggering. Reduced Cortactin expression and phosphorylation were also detected both in vivo and in vitro after treatment with Ibrutinib, a Btk inhibitor. Our results highlight the role of Cortactin in CLL as a check-point molecule between the BCR and CXCR4 signalling pathways.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Cortactina/fisiologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas Proto-Oncogênicas c-bcr/fisiologia , Receptores CXCR4/fisiologia , Adenina/análogos & derivados , Adulto , Tirosina Quinase da Agamaglobulinemia , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Movimento Celular/fisiologia , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Proteínas de Neoplasias/fisiologia , Fosforilação/efeitos dos fármacos , Piperidinas , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Quinases da Família src/fisiologiaRESUMO
BACKGROUND: Cell migration and invasion through interstitial tissues are dependent upon several specialized characteristics of the migratory cell notably generation of proteolytic membranous protrusions or invadopodia. Ameloblastoma is a benign odontogenic epithelial neoplasm with a locally infiltrative behaviour. Cortactin and MMT1-MMP are two invadopodia proteins implicated in its local invasiveness. Other invadopodia regulators, namely N-WASP, WIP and Src kinase remain unclarified. This study addresses their roles in ameloblastoma. MATERIALS AND METHOD: Eighty-seven paraffin-embedded ameloblastoma cases (20 unicystic, 47 solid/multicystic, 3 desmoplastic and 17 recurrent) were subjected to immunohistochemistry for expression of cortactin, N-WASP, WIP, Src kinase and F-actin, and findings correlated with clinicopathological parameters. RESULTS: Invadopodia proteins (except Src kinase) and F-actin were widely detected in ameloblastoma (cortactin: n = 73/87, 83.9%; N-WASP: n = 59/87; 67.8%; WIP: n = 77/87; 88.5%; and F-actin: n = 87/87, 100%). Protein localization was mainly cytoplasmic and/or membranous, and occasionally nuclear for F-actin. Cortactin, which functions as an actin-scaffolding protein, demonstrated significantly higher expression levels within ameloblastoma tumoral epithelium than in stroma (P < 0.05). N-WASP, which coordinates actin polymerization and invadopodia-mediated extracellular matrix degradation, was overexpressed in the solid/multicystic subtype (P < 0.05). WIP, an upstream regulator of N-WASP, and F-actin were significantly upregulated along the tumour invasive front compared to tumour centres (P < 0.05). Except for males with cortactin overexpression, other clinical parameters (age, ethnicity and anatomical site) showed no significant correlations. CONCLUSIONS: Present results suggest that local invasiveness of ameloblastoma is dependent upon the migratory potential of its tumour cells as defined by their distribution of cortactin, N-WASP and WIP in correlation with F-actin cytoskeletal dynamics.
Assuntos
Ameloblastoma/metabolismo , Cortactina/fisiologia , Proteínas do Citoesqueleto/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neoplasias Maxilomandibulares/metabolismo , Podossomos/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologia , Actinas/análise , Actinas/biossíntese , Actinas/fisiologia , Adolescente , Adulto , Idoso , Ameloblastoma/patologia , Movimento Celular/fisiologia , Criança , Cortactina/biossíntese , Proteínas do Citoesqueleto/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Neoplasias Maxilomandibulares/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/biossíntese , Adulto Jovem , Quinases da Família src/análise , Quinases da Família src/fisiologiaRESUMO
In the present study, we wanted to examine the predominant factor/s in the initiation of metastasis. We used samples of advanced grades of renal clear cell carcinoma with documented clinical history of vena caval spread as the experimental group. The major rationale for this selection is the fact that renal cell carcinoma metastasize extensively through the inferior vena cava up to the pulmonary bed and often exist as a continuous mass of metastatic tissue. As cortactin plays a significant role in invadopodia formation during initiation of metastasis, in the present study, we tested expression of cortactin and phospho(tyr421)-cortactin in different grades of renal cell clear carcinoma and examined its property to bind to actin. The findings of the present study suggest that the variations of the local physiological milieu are the driving forces for metastasis by enhancing molecular mechanisms for lamellipodia formation. We conclude that localization of cortactin in cancer cells and interaction between actin and its nucleators are crucial for cancer progression.
Assuntos
Carcinoma de Células Renais/patologia , Cortactina/fisiologia , Neoplasias Renais/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Quinase de Cadeia Leve de Miosina/metabolismo , Metástase NeoplásicaRESUMO
The maintenance of endothelial barrier is critical for the vascular homeostasis and is maintained by the interaction of adherens junction (AJ) and tight junction (TJ) proteins between adjacent cells. This interaction is stabilized by actin cytoskeleton forming cortical actin ring. Here, we developed a novel vascular leakage blocker, Sac-1004 and investigated its mechanism of action in endothelial cells (ECs). Sac-1004 inhibited endothelial hyperpermeability induced by vascular endothelial growth factor, histamine and thrombin via stabilization of cortical actin ring and AJ proteins at the cell-cell junction. Treatment of Sac-1004 in ECs increased cAMP levels and activated Rac, both of which are known to strengthen endothelial barrier. Furthermore, Sac-1004 induced phosphorylation of cortactin and its localization at cell membrane that is essential for the stabilization of cortical actin ring. These effects of Sac-1004 on ECs were significantly abrogated by dideoxyadenosine (adenylyl cyclase inhibitor) and NSC23766 (Rac inhibitor). Taken together, our findings indicate that Sac-1004 blocks vascular leakage by enhancing endothelial integrity via the cAMP/Rac/cortactin pathway and imply the potential usefulness of Sac-1004 in the development of therapeutic means for vascular leakage-related diseases.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , AMP Cíclico/fisiologia , Endotélio Vascular/efeitos dos fármacos , Pregnenolona/análogos & derivados , Saponinas/farmacologia , Transdução de Sinais/fisiologia , Proteínas rac de Ligação ao GTP/fisiologia , Células Cultivadas , Cortactina/fisiologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Pregnenolona/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Neuronal growth cone (GC) migration and targeting are essential processes for the formation of a neural network during embryonic development. Currently, the mechanisms that support directed motility of GCs are not fully defined. The large GTPase dynamin and an interacting actin-binding protein, cortactin, have been localized to GCs, although the function performed by this complex is unclear. We have found that cortactin and the ubiquitous form of dynamin (Dyn) 2 exhibit a striking co-localization at the base of the transition zone of advancing GCs of embryonic hippocampal neurons. Confocal and total internal reflection fluorescence microscopies demonstrate that this basal localization represents point contacts. Exogenous expression of wild-type Dyn2 and cortactin leads to large, exceptionally flat, and static GCs, whereas disrupting this complex has no such effect. We find that excessive GC spreading is induced by Dyn2 and cortactin over-expression and substantial recruitment of the point contact-associated, actin-binding protein α-actinin1 to the ventral GC membrane. The distributions of other point contact proteins such as vinculin or paxillin appear unchanged. Immunoprecipitation experiments show that both Dyn2 and cortactin reside in a complex with α-actinin1. These findings provide new insights into the role of Dyn2 and the actin cytoskeleton in GC adhesion and motility.
Assuntos
Cortactina/fisiologia , Dinamina II/fisiologia , Cones de Crescimento/fisiologia , Cones de Crescimento/ultraestrutura , Hipocampo/fisiologia , Neurônios/fisiologia , Animais , Comunicação Celular/fisiologia , Células Cultivadas , Hipocampo/citologia , Hipocampo/embriologia , Neurogênese/fisiologia , Neurônios/citologia , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Cbl proteins are regulators of signal transduction through many pathways and, consequently, regulate cell function and development. They are ubiquitin ligases that ubiquitinate and target many signaling molecules for degradation. The Cbl proteins themselves are regulated by an increasingly complex network of interactions that fine-tune the effects that Cbl proteins have on signaling. The negative regulation of Cbl protein function can occur via cis-acting structural elements that prevent inappropriate ubiquitin ligase activity, degradation of the Cbl proteins, inhibition without degradation owing to interaction with other signaling proteins, deubiquitination of Cbl substrates, and regulation of assembly of the endosomal ESCRT-I complex. Defects in the regulatory mechanisms that control Cbl function are implicated in the development of immunological and malignant diseases.
Assuntos
Regulação Enzimológica da Expressão Gênica , Proteínas Proto-Oncogênicas c-cbl/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Doenças Autoimunes/fisiopatologia , Antígenos CD28/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/fisiologia , Cortactina/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Proteínas de Membrana , Neoplasias/fisiopatologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Oncogênicas Virais/fisiologia , Proteínas Tirosina Fosfatases , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteína cdc42 de Ligação ao GTP/fisiologia , Quinases da Família src/metabolismoRESUMO
It has long been recognized that adhesion receptors cooperate with the cytoskeleton during morphogenesis, tissue remodeling and homeostasis. But how this occurs is less well-understood. A host of cytoskeletal regulators have been reported to have functional and biochemical linkage with adhesion receptors. The challenge remains to find functionally-coherent patterns within this increasingly large corpus of molecular information. In this review we discuss one approach, to identify distinctive functional modules that contribute to different adhesive processes. We illustrate this by considering Arp2/3-driven surface protrusion, which is utilized at both integrin-based cell-matrix adhesions and cadherin-based cell-cell adhesions. We further argue that regulatory proteins, such as cortactin, serve to coordinate the molecular components of this protrusive apparatus into a cohesive module.
Assuntos
Adesão Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Cortactina/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/ultraestrutura , Junções Célula-Matriz/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Transdução de Sinais , Tirosina/metabolismoRESUMO
Metastatic mammary carcinoma cells, which have previously been observed to form mature, matrix degrading invadopodia on a thick ECM matrix, are able to form invadopodia with similar characteristics on glass without previously applied matrix. They form in response to epidermal growth factor (EGF), and contain the usual invadopodium core proteins N-WASP, Arp2/3, cortactin, cofilin, and F-actin. The study of invadopodia on glass allows for higher resolution analysis including the use of total internal reflection microscopy and analysis of their relationship to other cell motility events, in particular, lamellipodium extension and chemotaxis toward an EGF gradient. Invadopodium formation on glass requires N-WASP and cortactin but not microtubules. In a gradient of EGF more invadopodia form on the side of the cells facing the source of EGF. In addition, depletion of N-WASP or cortactin, which blocks invadopodium fromation, inhibits chemotaxis of cells towards EGF. This appears to be a localized defect in chemotaxis since depletion of N-WASP or cortactin via siRNA had no effect on lamellipodium protrusion or barbed end generation at the lamellipodium's leading edge. Since chemotaxis to EGF by breast tumor cells is involved in metastasis, inhibiting N-WASP activity in breast tumor cells might prevent metastasis of tumor cells while not affecting chemotaxis-dependent innate immunity which depends on WASp function in macrophages.
Assuntos
Neoplasias da Mama/patologia , Quimiotaxia , Cortactina/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Quimiotaxia/genética , Cortactina/genética , Destrina/genética , Destrina/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , RNA Interferente Pequeno/genética , Ratos , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genéticaRESUMO
The Wiskott-Aldrich Syndrome protein (WASP) is an adaptor protein that is essential for podosome formation in hematopoietic cells. Given that 80% of identified Wiskott-Aldrich Syndrome patients result from mutations in the binding site for WASP-interacting-protein (WIP), we examined the possible role of WIP in the regulation of podosome architecture and cell motility in dendritic cells (DCs). Our results show that WIP is essential both for the formation of actin cores containing WASP and cortactin and for the organization of integrin and integrin-associated proteins in circular arrays, specific characteristics of podosome structure. We also found that WIP is essential for the maintenance of the high turnover of adhesions and polarity in DCs. WIP exerts these functions by regulating calpain-mediated cleavage of WASP and by facilitating the localization of WASP to sites of actin polymerization at podosomes. Taken together, our results indicate that WIP is critical for the regulation of both the stability and localization of WASP in migrating DCs and suggest that WASP and WIP operate as a functional unit to control DC motility in response to changes in the extracellular environment.
Assuntos
Proteínas de Transporte/fisiologia , Extensões da Superfície Celular/metabolismo , Células Dendríticas/fisiologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Adesão Celular , Movimento Celular , Polaridade Celular , Cortactina/fisiologia , Proteínas do Citoesqueleto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células U937 , Proteína da Síndrome de Wiskott-Aldrich/químicaRESUMO
Cortactin is an F-actin binding protein, regulating cell movement and adhesive junction assembly. However, the function of cortactin in epithelial-mesenchymal transition (EMT) remains elusive. Here we found that during transforming growth factor-beta1 (TGF-beta1)- induced EMT in AML-12 murine hepatocytes, cortactin underwent tyrosine dephosphorylation. Inhibition of the dephosphorylation of cortactin by sodium vanadate blocked TGF-beta1-induced EMT. Knockdown of cortactin by RNAi led to decrease of intercellular junction proteins E-cadherin and Zonula occludens-1 and induced expression of mesenchymal protein fibronectin. Additionally, knockdown of cortactin further promoted TGF-beta1-induced EMT in AML-12 cells, as determined by EMT markers and cell morphological changes. Moreover, migration assay showed that cortactin knockdown promoted the migration of AML-12 cells, and also enhanced TGF-beta1-induced migration. Our study showed the involvement of cortactin in the TGFbeta1- induced EMT.
Assuntos
Diferenciação Celular/fisiologia , Transdiferenciação Celular/fisiologia , Cortactina/fisiologia , Rim/citologia , Fator de Crescimento Transformador beta1/fisiologia , Células Epiteliais/fisiologia , Histona Acetiltransferases/fisiologia , Humanos , Mesoderma/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células Tumorais CultivadasRESUMO
Osteoclasts are essential for bone dynamics and calcium homeostasis. The cells form a tight seal on the bone surface, onto which they secrete acid and proteases to resorb bone. The seal is associated with a ring of actin filaments. Cortactin, a c-Src substrate known to promote Arp2/3-mediated actin assembly in vitro, is expressed in osteoclasts and localizes to the sealing ring. To address the role of cortactin and actin assembly in osteoclasts, we depleted cortactin by RNA interference. Cortactin-depleted osteoclasts displayed a complete loss of bone resorption with no formation of sealing zones. On nonosteoid surfaces, osteoclasts flatten with a dynamic, actin-rich peripheral edge that contains podosomes, filopodia, and lamellipodia. Cortactin depletion led to a specific loss of podosomes, revealing a tight spatial compartmentalization of actin assembly. Podosome formation was restored in cortactin-depleted cells by expression of wild-type cortactin or a Src homology 3 point mutant of cortactin. In contrast, expression of a cortactin mutant lacking tyrosine residues phosphorylated by Src did not restore podosome formation. Cortactin was found to be an early component of the nascent podosome belt, along with dynamin, supporting a role for cortactin in actin assembly.
Assuntos
Actinas/metabolismo , Reabsorção Óssea/metabolismo , Cortactina/fisiologia , Osteoclastos/citologia , Osteoclastos/metabolismo , Proteína 2 Relacionada a Actina/metabolismo , Actinas/análise , Animais , Diferenciação Celular , Células Cultivadas , Cortactina/análise , Cortactina/genética , Dinaminas/metabolismo , Camundongos , Mutação , Osteoclastos/química , Mutação Puntual , Interferência de RNARESUMO
Myasthenia gravis (MG) is an autoimmune disease caused by antibodies against the acetylcholine receptor (AChR), muscle-specific kinase (MuSK) or other AChR-related proteins in the postsynaptic muscle membrane. Localized or general muscle weakness is the predominant symptom and is induced by the antibodies. Patients are grouped according to the presence of antibodies, symptoms, age at onset and thymus pathology. Diagnosis is straightforward in most patients with typical symptoms and a positive antibody test, although a detailed clinical and neurophysiological examination is important in antibody-negative patients. MG therapy should be ambitious and aim for clinical remission or only mild symptoms with near-normal function and quality of life. Treatment should be based on MG subgroup and includes symptomatic treatment using acetylcholinesterase inhibitors, thymectomy and immunotherapy. Intravenous immunoglobulin and plasma exchange are fast-acting treatments used for disease exacerbations, and intensive care is necessary during exacerbations with respiratory failure. Comorbidity is frequent, particularly in elderly patients. Active physical training should be encouraged.
Assuntos
Miastenia Gravis/diagnóstico , Miastenia Gravis/terapia , Acetilcolinesterase/genética , Acetilcolinesterase/fisiologia , Corticosteroides/uso terapêutico , Agrina/genética , Agrina/fisiologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Autoanticorpos/análise , Autoanticorpos/sangue , Biomarcadores/análise , Biomarcadores/sangue , Blefaroptose/etiologia , Colágeno/genética , Colágeno/fisiologia , Cortactina/genética , Cortactina/fisiologia , Eletromiografia/métodos , Humanos , Canal de Potássio Kv1.4/genética , Canal de Potássio Kv1.4/fisiologia , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/fisiologia , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Miastenia Gravis/fisiopatologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Receptores Colinérgicos/genética , Receptores Colinérgicos/fisiologia , Receptores Nicotínicos/genética , Fatores de Risco , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologiaRESUMO
Disruption of pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event in highly morbid inflammatory conditions such as sepsis and acute respiratory disease stress syndrome. Actin cytoskeleton, an essential regulator of endothelial permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. Here, we used atomic force microscopy to characterize structural and mechanical changes in the F-actin cytoskeleton of cultured human pulmonary artery EC in response to both barrier-enhancing (induced by sphingosine 1-phosphate (S1P)) and barrier-disrupting (induced by thrombin) conditions. Atomic force microscopy elasticity measurements show differential effects: for the barrier protecting molecule S1P, the elastic modulus was elevated significantly on the periphery; for the barrier-disrupting molecule thrombin, on the other hand, it was elevated significantly in the central region of the cell. The force and elasticity maps correlate with F-actin rearrangements as identified by immunofluorescence analysis. Significantly, reduced expression (via siRNA) of cortactin, an actin-binding protein essential to EC barrier regulation, resulted in a shift in the S1P-mediated elasticity pattern to more closely resemble control, unstimulated endothelium.
Assuntos
Actinas/fisiologia , Cortactina/fisiologia , Endotélio Vascular/fisiologia , Lisofosfolipídeos/fisiologia , Artéria Pulmonar/fisiologia , Esfingosina/análogos & derivados , Trombina/fisiologia , Actinas/química , Cortactina/química , Elasticidade , Endotélio Vascular/química , Humanos , Lisofosfolipídeos/química , Microscopia de Força Atômica/métodos , Artéria Pulmonar/química , Esfingosina/química , Esfingosina/fisiologia , Estresse Mecânico , Trombina/químicaRESUMO
Cortactin is a ubiquitous actin-binding protein that was originally identified as a substrate for the protein kinase Src. It is accumulated in peripheral, actin-enriched structures of cells, including lamellipodia and membrane ruffles, suggesting that cortactin facilitates actin network formation. In addition, recent data suggests that it regulates various aspects of cell dynamics, including integrin signaling, vesicular transport, axon guidance, and cell migration. A large body of evidence indicates that cortactin is also implicated in the pathogenesis of human neoplasia. It is over-expressed in a number of epithelial carcinomas, including breast cancer and head and neck cancer. Over-expression of cortactin in human tumors has been proposed to result in increased cell migration and metastatic potential. This review aims to focus on cortactin-mediated signaling pathways, with emphasis on its contribution to tumor progression and metastasis formation.
Assuntos
Cortactina/química , Cortactina/fisiologia , Neoplasias/fisiopatologia , Sequência de Aminoácidos , Sítios de Ligação , Progressão da Doença , Humanos , Modelos Moleculares , Metástase Neoplásica/genética , Neoplasias/patologia , Conformação Proteica , Transdução de SinaisRESUMO
Cortactin is a cytoskeletal protein and src kinase substrate that is frequently overexpressed in cancer. Animal studies suggest that cortactin overexpression increases tumor aggressiveness, possibly through promotion of tumor invasion and metastasis. Recently, many studies have documented a role for cortactin in promoting cell motility and invasion, including a critical role in invadopodia, actin rich-subcellular protrusions associated with degradation of the extracellular matrix by cancer cells. Here, I review the evidence and potential mechanisms for cortactin as a critical mediator of tumor cell invasion.
Assuntos
Movimento Celular , Cortactina/fisiologia , Actinas/química , Animais , Cortactina/genética , Matriz Extracelular/metabolismo , Previsões , Humanos , Camundongos , Invasividade Neoplásica , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína/fisiologia , Pseudópodes/químicaRESUMO
Tumor metastasis depends on cell adhesiveness, motility and deformability, resulting from quantitative alterations and rearrangement of various actin-binding cytoskeletal components, such as cortactin and fascin. To clarify the involvement of cortactin and fascin expression in tumorigenesis and progression of gastric carcinoma, we performed immunohistochemistry (IHC) on tissue microarray containing gastric carcinomas, adenomas and adjacent non-neoplastic mucosa (ANNM) using the antibodies against cortactin (Ab-466, -421) and fascin as well as a comparison of their expression with clinicopathological parameters of the tumors. Gastric carcinoma cell lines MKN28, AGS, MKN45, KATO-III and HGC-27 were studied for both proteins by IHC. Cortactin-466 was found to be highly expressed in adenoma, compared with ANNMs and carcinoma (p<0.05), and more frequently in ANNMs than in carcinoma (p<0.05). Cortactin-421 expression was higher in gastric carcinomas than in adenoma and ANNMs (p<0.05). There was increased fascin expression in gastric carcinoma and adenoma than in ANNMs (p<0.05). Most of the gastric carcinoma cell lines showed expression of cortactin and fascin at different levels. Cortactin-466 expression was inversely correlated with tumor size, depth of invasion, lymphatic and venous invasion, lymph node metastasis and UICC staging (p<0.05). The converse was observed for cortactin-421 and fascin (p<0.05). There was stronger positivity of both cortactins in intestinal- versus diffuse-type carcinomas (p<0.05). Univariate analysis indicated the cumulative survival rate of patients with positive cortactin-466 expression to be higher than without its expression even stratified according to the depth of invasion (p<0.05). However, it was the converse for fascin (p<0.05). Multivariate analysis showed that age, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren's classification were independent prognostic factors for carcinomas (p<0.05). It was suggested that aberrant expression of cortactin and fascin possibly contributes to the pathogenesis, growth, invasion and metastasis of gastric carcinomas. Thus, they may be objective and effective markers to indicate the pathobiological behaviors and prognosis of gastric carcinomas.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Transporte/análise , Cortactina/análise , Proteínas dos Microfilamentos/análise , Neoplasias Gástricas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Transporte/fisiologia , Linhagem Celular Tumoral , Cortactina/fisiologia , Feminino , Humanos , Masculino , Proteínas dos Microfilamentos/fisiologia , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico , Neoplasias Gástricas/química , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/mortalidadeRESUMO
Internalization and subsequent trafficking of receptor tyrosine kinases (RTKs) play an important role in the modulation of growth factor-stimulated signaling events that affect different cellular processes, from cell growth and mitosis to motility and invasion. The intracellular transport of these receptors has traditionally been viewed as being initiated via clathrin-coated pits. However, nonclathrin pathways have been implicated as well, although these remain poorly understood. Most recently, the formation of dynamic, transient endocytic membrane structures termed circular dorsal ruffles or "dorsal waves" have been reported to selectively sequester and internalize a large percentage of a specific RTK from the surface of growth factor-stimulated cells. This process is dependent on dynamin and cortactin, two endocytic proteins that are also associated with the actin cytoskeleton, whereas it is independent of traditional coat proteins, such as clathrin and caveolin. Additionally, dorsal wave formation requires the participation and remodeling of a dynamic actin cytoskeleton. Most importantly, the formation of these structures may be less frequent in tumor cells and thereby have significant effects on receptor signaling and cell growth.
Assuntos
Endocitose/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Membrana Celular/metabolismo , Cortactina/fisiologia , Dinaminas/fisiologia , Receptores ErbB/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Transdução de Sinais/fisiologiaRESUMO
Abl family nonreceptor tyrosine kinases regulate changes in cell shape and migration. Abl2 localizes to dynamic actin-rich protrusions, such as lamellipodia in fibroblasts and dendritic spines in neurons. Abl2 interactions with cortactin, an actin filament stabilizer, are crucial for the formation and stability of actin-rich structures, but Abl2:cortactin-positive structures have not been characterized with high spatiotemporal resolution in cells. Using total internal reflection fluorescence microscopy, we demonstrate that Abl2 colocalizes with cortactin at wave-like structures within lamellum and lamellipodium tips. Abl2 and cortactin within waves are focal and transient, extend to the outer edge of lamella, and serve as the base for lamellipodia protrusions. Abl2-positive foci colocalize with integrin ß3 and paxillin, adhesive markers of the lamellum-lamellipodium interface. Cortactin-positive waves still form in Abl2 knockout cells, but the lamellipodium size is significantly reduced. This deficiency is restored following Abl2 reexpression. Complementation analyses revealed that the Abl2 C-terminal half, which contains domains that bind actin and microtubules, is necessary and sufficient for recruitment to the wave-like structures and to support normal lamellipodium size, while the kinase domain-containing N-terminal half does not impact lamellipodium size. Together, this work demonstrates that Abl2 is recruited with cortactin to actin waves through cytoskeletal interactions to promote lamellipodium extension.
Assuntos
Cortactina/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Células COS , Movimento Celular , Forma Celular , Chlorocebus aethiops , Cortactina/fisiologia , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Camundongos , Ligação Proteica , Pseudópodes/metabolismo , Pseudópodes/fisiologiaRESUMO
Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFkappaB mechanism, since pharmacological inhibition of IkappaKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).