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1.
BMC Cell Biol ; 18(1): 28, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28851287

RESUMO

BACKGROUND: Eph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/macrophage cell lines. Although mature mononuclear leukocytes express several members of the EphA/ephrin-A subclass, their expression has not been examined in monocytes undergoing during differentiation and maturation. RESULTS: Using RT-PCR, we have shown that EphA2, ephrin-A1, and ephrin-A2 expression was upregulated in murine bone marrow mononuclear cells during monocyte maturation. Moreover, EphA2 and EphA4 expression was induced, and ephrin-A4 expression was upregulated, in a human promyelocytic leukemia cell line, HL60, along with monocyte differentiation toward the classical CD14++CD16- monocyte subset. Using RT-PCR and flow cytometry, we have also shown that expression levels of αL, αM, αX, and ß2 integrin subunits were upregulated in HL60 cells along with monocyte differentiation while those of α4, α5, α6, and ß1 subunits were unchanged. Using a cell attachment stripe assay, we have shown that stimulation by EphA as well as ephrin-A, likely promoted adhesion to an integrin ligand-coated surface in HL60 monocytes. Moreover, EphA and ephrin-A stimulation likely promoted the formation of protrusions in HL60 monocytes. CONCLUSIONS: Notably, this study is the first analysis of EphA/ephrin-A expression during monocytic differentiation/maturation and of ephrin-A stimulation affecting monocyte adhesion to an integrin ligand-coated surface. Thus, we propose that monocyte adhesion via integrin activation and the formation of protrusions is likely promoted by stimulation of EphA as well as of ephrin-A.


Assuntos
Diferenciação Celular/fisiologia , Efrinas/genética , Efrinas/metabolismo , Monócitos , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Animais , Células da Medula Óssea/citologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/enzimologia , Extensões da Superfície Celular/metabolismo , Células Cultivadas , Efrina-A1/genética , Efrina-A1/metabolismo , Efrina-A1/farmacologia , Células HL-60 , Humanos , Integrinas/genética , Integrinas/metabolismo , Ligantes , Masculino , Camundongos , Monócitos/citologia , Monócitos/enzimologia , Monócitos/metabolismo , Receptores da Família Eph/farmacologia , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacos
2.
J Cell Sci ; 129(4): 743-56, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26769900

RESUMO

During angiogenesis, endothelial cells must coordinate matrix proteolysis with migration. Here, we tested whether the focal adhesion scaffold protein Hic-5 (also known as TGFB1I1) regulated endothelial sprouting in three dimensions. Hic-5 silencing reduced endothelial sprouting and lumen formation, and sprouting defects were rescued by the return of Hic-5 expression. Pro-angiogenic factors enhanced colocalization and complex formation between membrane type-1 matrix metalloproteinase (MT1-MMP, also known as MMP14) and Hic-5, but not between paxillin and MT1-MMP. The LIM2 and LIM3 domains of Hic-5 were necessary and sufficient for Hic-5 to form a complex with MT1-MMP. The degree of interaction between MT1-MMP and Hic-5 and the localization of the complex within detergent-resistant membrane fractions were enhanced during endothelial sprouting, and Hic-5 depletion lowered the surface levels of MT1-MMP. In addition, we observed that loss of Hic-5 partially reduced complex formation between MT1-MMP and focal adhesion kinase (FAK, also known as PTK2), suggesting that Hic-5 bridges MT1-MMP and FAK. Finally, Hic-5 LIM2-LIM3 deletion mutants reduced sprout initiation. Hic-5, MT1-MMP and FAK colocalized in angiogenic vessels during porcine pregnancy, supporting that this complex assembles during angiogenesis in vivo. Collectively, Hic-5 appears to enhance complex formation between MT1-MMP and FAK in activated endothelial cells, which likely coordinates matrix proteolysis and cell motility.


Assuntos
Células Endoteliais da Veia Umbilical Humana/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas com Domínio LIM/fisiologia , Metaloproteinase 14 da Matriz/metabolismo , Animais , Movimento Celular , Extensões da Superfície Celular/enzimologia , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Neovascularização Fisiológica , Gravidez , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Sus scrofa
3.
J Cell Physiol ; 231(8): 1695-708, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26599499

RESUMO

Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over-expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over-expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over-expressing cells, Rab7 and LC3B-II co-localized and co-localization in large circular structures occurred only in Rab7 over-expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co-localization of Rab7 with cortactin and f-actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over-expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP-stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over-expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695-1708, 2016. © 2015 Wiley Periodicals, Inc.


Assuntos
Caderinas/metabolismo , Extensões da Superfície Celular/enzimologia , Endocitose , Tireoglobulina/metabolismo , Glândula Tireoide/enzimologia , Vacúolos/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Autofagia , Linhagem Celular , Extensões da Superfície Celular/efeitos dos fármacos , Cortactina/metabolismo , AMP Cíclico/metabolismo , Endocitose/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Pinocitose , Interferência de RNA , Ratos Endogâmicos F344 , Sistemas do Segundo Mensageiro , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Fatores de Tempo , Transfecção , Vacúolos/efeitos dos fármacos , Quinases Ativadas por p21/metabolismo , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7 , Proteínas rac1 de Ligação ao GTP/metabolismo
4.
Cell Signal ; 27(8): 1643-51, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25889896

RESUMO

Rho GTPases function as molecular switches that connect changes of the external environment to intracellular signaling pathways. They are active at various subcellular sites and require fast and tight regulation to fulfill their role as transducers of extracellular stimuli. New imaging technologies visualizing the active states of Rho proteins in living cells elucidated the necessity of precise spatiotemporal activation of the GTPases. The local regulation of Rho proteins is coordinated by the interaction with different guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that turn on and off GTPase signaling to downstream effectors. GEFs and GAPs thus serve as critical signaling nodes that specify the amplitude and duration of a particular Rho signaling pathway. Despite their importance in Rho regulation, the molecular aspects underlying the spatiotemporal control of the regulators themselves are still largely elusive. In this review we will focus on the Deleted in Liver Cancer (DLC) family of RhoGAP proteins and summarize the evidence gathered over the past years revealing their different subcellular localizations that might account for isoform-specific functions. We will also highlight the importance of their tightly controlled expression in the context of neoplastic transformation.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Membrana Celular/enzimologia , Núcleo Celular/enzimologia , Extensões da Superfície Celular/enzimologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Estruturas Citoplasmáticas/enzimologia , Proteínas Ativadoras de GTPase/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Fatores de Tempo , Proteínas Supressoras de Tumor/genética
5.
Pancreas ; 44(2): 331-40, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25426613

RESUMO

OBJECTIVE: The aim of this study was to investigate the role of peroxiredoxin 1 (Prdx1) in the invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells. METHODS: Immunohistochemistry was used to determine overexpression of Prdx1 in human PDAC tissues. Immunoprecipitation and immunocytochemistry were used to determine the interaction and intracellular distribution of Prdx1 and a member of the mitogen-activated protein kinase (MAPK) family protein, p38 MAPK, in PDAC cells. Finally, immunocytochemistry and Matrigel invasion assay were used to examine the effects of Prdx1 and p38 MAPK on the formation of cell protrusions and PDAC cell invasion. RESULTS: Prdx1 is overexpressed in human PDAC tissues. Peroxiredoxin 1 interacts with active forms of p38 MAPK, and complexes of Prdx1 and phosphorylated p38 MAPK localize at the leading edges of migrating PDAC cells. Suppression of Prdx1 decreases active p38 MAPK localized in cell protrusions and inhibits the invasiveness of PDAC cells. Consequently, suppression of Prdx1 inhibits membrane ruffling and protrusions. The p38 MAPK inhibitor SB203580 also decreases the formation of membrane protrusions and inhibits invasiveness. CONCLUSIONS: Prdx1 associates with the formation of membrane protrusions through modulation of the activity of p38 MAPK, which in turn promotes PDAC cell invasion.


Assuntos
Carcinoma Ductal Pancreático/enzimologia , Movimento Celular , Neoplasias Pancreáticas/enzimologia , Peroxirredoxinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Extensões da Superfície Celular/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Peroxirredoxinas/genética , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Transdução de Sinais , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
6.
Nat Cell Biol ; 16(6): 574-86, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24859002

RESUMO

Rho family GTPases control cell migration and participate in the regulation of cancer metastasis. Invadopodia, associated with invasive tumour cells, are crucial for cellular invasion and metastasis. To study Rac1 GTPase in invadopodia dynamics, we developed a genetically encoded, single-chain Rac1 fluorescence resonance energy (FRET) transfer biosensor. The biosensor shows Rac1 activity exclusion from the core of invadopodia, and higher activity when invadopodia disappear, suggesting that reduced Rac1 activity is necessary for their stability, and Rac1 activation is involved in disassembly. Photoactivating Rac1 at invadopodia confirmed this previously unknown Rac1 function. We describe here an invadopodia disassembly model, where a signalling axis involving TrioGEF, Rac1, Pak1, and phosphorylation of cortactin, causes invadopodia dissolution. This mechanism is critical for the proper turnover of invasive structures during tumour cell invasion, where a balance of proteolytic activity and locomotory protrusions must be carefully coordinated to achieve a maximally invasive phenotype.


Assuntos
Neoplasias da Mama/enzimologia , Movimento Celular , Extensões da Superfície Celular/enzimologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Técnicas Biossensoriais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Extensões da Superfície Celular/patologia , Cortactina/metabolismo , Matriz Extracelular/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Ratos , Fatores de Tempo , Transfecção , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genética
7.
Mol Biol Cell ; 25(13): 2061-70, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24807903

RESUMO

Movement through the extracellular matrix (ECM) requires cells to degrade ECM components, primarily through the action of matrix metalloproteinases (MMPs). Membrane type 1-matrix metalloproteinase (MT1-MMP) has an essential role in matrix degradation and cell invasion and localizes to subcellular degradative structures termed invadopodia. Trafficking of MT1-MMP to invadopodia is required for the function of these structures, and here we examine the role of N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE)-mediated membrane traffic in the transport of MT1-MMP to invadopodia. During invadopodium formation in MDA-MB-231 human breast cancer cells, increased association of SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) is detected by coimmunoprecipitation. Blocking the function of these SNAREs perturbs invadopodium-based ECM degradation and cell invasion. Increased level of SNAP23-Syntaxin4-VAMP7 interaction correlates with decreased Syntaxin4 phosphorylation. These results reveal an important role for SNARE-regulated trafficking of MT1-MMP to invadopodia during cellular invasion of ECM.


Assuntos
Extensões da Superfície Celular/enzimologia , Metaloproteinase 14 da Matriz/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Humanos , Invasividade Neoplásica , Transporte Proteico
9.
Cell Death Dis ; 4: e726, 2013 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-23868059

RESUMO

Lymphocytes form cell-cell connections by various mechanisms, including intercellular networks through actin-supported long-range plasma membrane (PM) extensions, termed tunneling nanotubes (TNTs). In this study, we tested in vitro whether TNTs form between human antigen-presenting B cells and T cells following cell contact and whether they enable the transfer of PM-associated proteins, such as green fluorescent protein (GFP)-tagged H-Ras (GFP-H-Ras). To address this question, we employed advanced techniques, including cell trapping by optical tweezers and live-cell imaging by 4D spinning-disk confocal microscopy. First, we showed that TNTs can form after optically trapped conjugated B and T cells are being pulled apart. Next, we determined by measuring fluorescence recovery after photobleaching that GFP-H-Ras diffuses freely in the membrane of TNTs that form spontaneously between B and T cells during coculturing. Importantly, by 4D time-lapse imaging, we showed that GFP-H-Ras-enriched PM patches accumulate at the junction between TNTs and the T-cell body and subsequently transfer to the T-cell surface. Furthermore, the PM patches adopted by T cells were enriched for another B-cell-derived transmembrane receptor, CD86. As predicted, the capacity of GFP-H-Ras to transfer between B and T cells, during coculturing, was dependent on its normal post-transcriptional lipidation and consequent PM anchorage. In summary, our data indicate that TNTs connecting B and T cells provide a hitherto undescribed route for the transfer of PM patches containing, for example, H-Ras from B to T cells.


Assuntos
Linfócitos B/enzimologia , Extensões da Superfície Celular/enzimologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linfócitos B/ultraestrutura , Técnicas de Cocultura , Difusão , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Lipoilação , Microscopia Confocal , Microscopia de Fluorescência , Nanotubos , Prenilação de Proteína , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/enzimologia , Linfócitos T/ultraestrutura , Imagem com Lapso de Tempo
10.
J Dermatol Sci ; 70(3): 196-203, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23622765

RESUMO

BACKGROUND: Negative-pressure wound therapy (NPWT) is developed to facilitate wound healing at controlled subatmospheric pressures in modern medicine. Molecular mechanism for this therapy is still undefined. OBJECTIVE: This study highlights the localization and time-course of the cell division control protein 42 (Cdc42) in the cell membrane at ambient pressure (AP) and negative pressures of 75mmHg (NP75), 125mmHg (NP125) and 175mmHg (NP175). METHODS: The prepared cells were cultured in a negative pressure incubator with the same O2 and CO2 tensions at the four different pressures. The effective time, complete wound closure time, cell volume, cell viability, and the fluorescence of proliferating cell nuclear antigens (PCNA) and actins were evaluated in cells at different pressures. Wound-healing process and Cdc42 fluorescence were examined in cells with the knockdown of Cdc42. Cdc42 pathway proteins in cell membranes were analyzed after incubation at different pressures for 6 and 12h. RESULTS: The cells at NP125 had less wound closure time and obvious cell podia. Similar PCNA fluorescent intensity was observed in cells at different pressures. The Cdc42, neural Wiskott-Aldrich syndrome protein, and actin expression increased significantly (p<0.05) in plasma membranes of cells at NP125 for 12h. The knockdown of active Cdc42 resulted in the absence of Cdc42 expression at the cell leading edge. CONCLUSIONS: The activation and localization of Cdc42 pathway proteins in the cell membrane are involved in the cell podia formation in keratinocytes at NP125. NPWT may facilitate cell migration to accelerate wound healing.


Assuntos
Extensões da Superfície Celular/enzimologia , Queratinócitos/enzimologia , Tratamento de Ferimentos com Pressão Negativa , Cicatrização , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Técnicas de Cultura de Células/instrumentação , Linhagem Celular , Movimento Celular , Extensões da Superfície Celular/patologia , Sobrevivência Celular , Humanos , Incubadoras , Queratinócitos/patologia , Polimerização , Pressão , Antígeno Nuclear de Célula em Proliferação/metabolismo , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteína cdc42 de Ligação ao GTP/genética
11.
Proc Natl Acad Sci U S A ; 110(3): 912-7, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23284172

RESUMO

RAC1 is a small, Ras-related GTPase that was recently reported to harbor a recurrent UV-induced signature mutation in melanoma, resulting in substitution of P29 to serine (RAC1(P29S)), ranking this the third most frequently occurring gain-of-function mutation in melanoma. Although the Ras family GTPases are mutated in about 30% of all cancers, mutations in the Rho family GTPases have rarely been observed. In this study, we demonstrate that unlike oncogenic Ras proteins, which are primarily activated by mutations that eliminate GTPase activity, the activated melanoma RAC1(P29S) protein maintains intrinsic GTP hydrolysis and is spontaneously activated by substantially increased inherent GDP/GTP nucleotide exchange. Determination and comparison of crystal structures for activated RAC1 GTPases suggest that RAC1(F28L)--a known spontaneously activated RAC1 mutant--and RAC1(P29S) are self-activated in distinct fashions. Moreover, the mechanism of RAC1(P29S) and RAC1(F28L) activation differs from the common oncogenic mutations found in Ras-like GTPases that abrogate GTP hydrolysis. The melanoma RAC1(P29S) gain-of-function point mutation therefore represents a previously undescribed class of cancer-related GTPase activity.


Assuntos
Melanoma/enzimologia , Melanoma/genética , Mutação de Sentido Incorreto , Oncogenes , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Animais , Células COS , Extensões da Superfície Celular/enzimologia , Chlorocebus aethiops , Cristalografia por Raios X , Ativação Enzimática/genética , Estudos de Associação Genética , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Cinética , Camundongos , Microscopia de Fluorescência , Modelos Moleculares , Células NIH 3T3 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Eletricidade Estática , Proteínas rac1 de Ligação ao GTP/química
12.
Mol Cancer ; 12: 2, 2013 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-23289900

RESUMO

BACKGROUND: ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. Here, we investigated mRNA and protein levels of ADAMTS-1 in normal and neoplastic tissues using qPCR, immunohistochemistry and immunoblot analyses, and we addressed the role of ADAMTS-1 in regulating migration, invasion and invadopodia formation in breast tumor cell lines. RESULTS: In a series of primary breast tumors, we observed variable levels of ADAMTS-1 mRNA expression but lower levels of ADAMTS-1 protein expression in human breast cancers as compared to normal tissue, with a striking decrease observed in high-malignancy cases (triple-negative for estrogen, progesterone and Her-2). This result prompted us to analyze the effect of ADAMTS-1 knockdown in breast cancer cells in vitro. MDA-MB-231 cells with depleted ADAMTS-1 expression demonstrated increased migration, invasion and invadopodia formation. The regulatory mechanisms underlying the effects of ADAMTS-1 may be related to VEGF, a growth factor involved in migration and invasion. MDA-MB-231 cells with depleted ADAMTS-1 showed increased VEGF concentrations in conditioned medium capable of inducing human endothelial cells (HUVEC) tubulogenesis. Furthermore, expression of the VEGF receptor (VEGFR2) was increased in MDA-MB-231 cells as compared to MCF7 cells. To further determine the relationship between ADAMTS-1 and VEGF regulating breast cancer cells, MDA-MB-231 cells with reduced expression of ADAMTS-1 were pretreated with a function-blocking antibody against VEGF and then tested in migration and invasion assays; both were partially rescued to control levels. CONCLUSIONS: ADAMTS-1 expression was decreased in human breast tumors, and ADAMTS-1 knockdown stimulated migration, invasion and invadopodia formation in breast cancer cells in vitro. Therefore, this series of experiments suggests that VEGF is involved in the effects mediated by ADAMTS-1 in breast cancer cells.


Assuntos
Proteínas ADAM/metabolismo , Neoplasias da Mama/enzimologia , Carcinoma Ductal de Mama/enzimologia , Movimento Celular , Expressão Gênica , Proteínas ADAM/genética , Proteína ADAMTS1 , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/secundário , Extensões da Superfície Celular/enzimologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Metástase Linfática , Células MCF-7 , Pessoa de Meia-Idade , Invasividade Neoplásica , RNA Interferente Pequeno/genética , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Imagem com Lapso de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto Jovem
13.
Eur J Cell Biol ; 91(11-12): 878-88, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22823952

RESUMO

In the past decade, substantial progress has been made in understanding how Src family kinases regulate the formation and function of invadosomes. Invadosomes are organized actin-rich structures that contain an F-actin core surrounded by an adhesive ring and mediate invasive migration. Src kinases orchestrate, either directly or indirectly, each phase of the invadosome life cycle including invadosome assembly, maturation and matrix degradation and disassembly. Complex arrays of Src effector proteins are involved at different stages of invadosome maturation and their spatiotemporal activity must be tightly regulated to achieve effective invasive migration. In this review, we highlight some recent progress and the challenges of understanding how Src is regulated temporally and spatially to orchestrate the dynamics of invadosomes and mediate cell invasion.


Assuntos
Extensões da Superfície Celular/enzimologia , Quinases da Família src/metabolismo , Actinas/metabolismo , Animais , Adesão Celular , Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Oxirredução , Transdução de Sinais
14.
Mol Cell Biol ; 32(8): 1374-86, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22331470

RESUMO

Our recent studies implicated key and distinct roles for the highly related RalA and RalB small GTPases (82% sequence identity) in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis and invasive and metastatic growth, respectively. How RalB may promote PDAC invasion and metastasis has not been determined. In light of known Ral effector functions in regulation of actin organization and secretion, we addressed a possible role for RalB in formation of invadopodia, actin-rich membrane protrusions that contribute to tissue invasion and matrix remodeling. We determined that a majority of KRAS mutant PDAC cell lines exhibited invadopodia and that expression of activated K-Ras is both necessary and sufficient for invadopodium formation. Invadopodium formation was not dependent on the canonical Raf-MEK-ERK effector pathway and was instead dependent on the Ral effector pathway. However, this process was more dependent on RalB than on RalA. Surprisingly, RalB-mediated invadopodium formation was dependent on RalBP1/RLIP76 but not Sec5 and Exo84 exocyst effector function. Unexpectedly, the requirement for RalBP1 was independent of its best known function as a GTPase-activating protein for Rho small GTPases. Instead, disruption of the ATPase function of RalBP1 impaired invadopodium formation. Our results identify a novel RalB-mediated biochemical and signaling mechanism for invadopodium formation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Extensões da Superfície Celular/enzimologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Extensões da Superfície Celular/ultraestrutura , Ativação Enzimática , Humanos , Invasividade Neoplásica/ultraestrutura , Neoplasias Pancreáticas/patologia , Transdução de Sinais
15.
Free Radic Biol Med ; 52(2): 247-56, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22033009

RESUMO

Redox signaling contributes to the regulation of cancer cell proliferation, survival, and invasion and participates in the adaptation of cancer cells to their microenvironment. NADPH oxidases are important mediators of redox signaling in normal and cancer cells. Redox signal specificity in normal cells is in part achieved by targeting enzymes that generate reactive oxygen species to specific subcellular microdomains such as focal adhesions, dorsal ruffles, lipid rafts, or caveolae. In a similar fashion, redox signal specificity during cancer cell invasion can be regulated by targeting reactive oxygen generation to invasive microdomains such as invadopodia. Here we summarize recent advances in the understanding of the redox signaling processes that control the cancer cell proinvasive program by modulating cell adhesion, migration, and proteolysis as well as the interaction of cancer cells with the tumor microenvironment. We focus on redox signaling events mediated by invadopodia NADPH oxidase complexes and their contribution to cancer cell invasion.


Assuntos
Microdomínios da Membrana/enzimologia , Neoplasias/patologia , Transdução de Sinais , Animais , Adesão Celular , Movimento Celular , Extensões da Superfície Celular/enzimologia , Extensões da Superfície Celular/metabolismo , Humanos , Microdomínios da Membrana/metabolismo , NADPH Oxidases/metabolismo , Invasividade Neoplásica , Neoplasias/enzimologia , Neoplasias/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Microambiente Tumoral
16.
Cell Signal ; 23(8): 1291-8, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21419846

RESUMO

Membrane ruffling is the formation of actin rich membrane protrusions, essential for cell motility. The exact mechanism of ruffling is not fully known. Using YFP and CFP fluorescent chimeras, we show for the first time a co-localization of Phospholipase D2 (PLD2) and Growth factor Receptor Bound protein-2 (Grb2) with actin-rich membrane protrusions of macrophages. Grb2 cooperates with PLD2 in enhancing membrane ruffling, whether in resting cells or in cells stimulated with the growth factor M-CSF, although in the latter an increase in dorsal ruffles was observed, consistent with receptor-ligand internalization. Cells transfected with PLD2 mutated in the PH domain (Y169F) or with Grb2 mutated in the SH2 site (R86K) negate this effect, indicating an association PLD2(Y169)-SH2-Grb2 that was confirmed by immunoprecipitation and Western blotting. The association results in enhanced PLD activity, but the lipase activity can only partially explain the formation of membrane ruffles in vivo. A third component involves the Rho-GTPase Rac2 and it is only when Rac2 is overexpressed along with PLD2 and Rac2 that a full biological effect, including actin polymerization in vivo, is obtained. The mechanism involved is, then, as follows: PLD enzymatic action, after having been increased due to the binding to Grb2-SH2 via Y169, cooperates with Rac2, and the three molecules stimulate actin polymerization and consequently, membrane ruffle formation. Since membrane ruffling precedes cell migration, the results herein provide a novel mechanism for control of membrane dynamics, crucial for the physiology of leukocytes.


Assuntos
Membrana Celular/metabolismo , Proteína Adaptadora GRB2/metabolismo , Fosfolipase D/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Actinas/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/enzimologia , Movimento Celular , Extensões da Superfície Celular/enzimologia , Extensões da Superfície Celular/metabolismo , Chlorocebus aethiops , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Mutação , Fosfolipase D/química , Fosfolipase D/genética , Estrutura Terciária de Proteína , Proteína RAC2 de Ligação ao GTP
17.
Arthritis Rheum ; 63(6): 1591-602, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21337539

RESUMO

OBJECTIVE: Invasive synovial fibroblasts are suggested to be the major effectors of cartilage and bone destruction, and this aggressive phenotype can lead to irreversible damage. In cancer cells, invasion across tissue boundaries and metastasis have recently been shown to depend on the capacity of the cells to breach the basement membrane, a process that was linked to the formation of the actin-rich cell protrusions called invadopodia. This study was undertaken to investigate whether arthritic synovial cells use invadopodia to invade and degrade cartilage components. METHODS: Fibroblast-like synoviocytes (FLS) from control rats or rats with collagen-induced arthritis (CIA) were cultured on fluorescent matrix in the presence of Src inhibitors or were transfected with wild-type or variants of Src kinases. The in vivo effect of Src inhibition on cartilage degradation and invasion was studied in a rat model of CIA. RESULTS: FLS from rats with CIA produced more invadopodia-like structures than did FLS from control rats, leading to increased extracellular matrix degradation. Furthermore, c-Src activation was increased in synovial cells from rats with CIA, and Src activity was found to mediate the formation of invadopodia. Pharmacologic blockade of Src activity by PP2 or intraarticular expression of a c-Src-specific short hairpin RNA in the CIA model reduced synovial membrane hyperplasia and cartilage degradation, an event linked to decreased invadopodia formation by synovial fibroblasts. CONCLUSION: This study demonstrates that inhibition of invadopodia formation in arthritic synovial cells leads to a direct effect on extracellular matrix degradation in vitro and in vivo, making invadopodia a relevant therapeutic target for interfering with this process.


Assuntos
Artrite Experimental/enzimologia , Cartilagem/enzimologia , Extensões da Superfície Celular/enzimologia , Líquido Sinovial/enzimologia , Quinases da Família src/metabolismo , Animais , Artrite Experimental/patologia , Artrite Experimental/terapia , Cartilagem/patologia , Extensões da Superfície Celular/patologia , Células Cultivadas , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Endogâmicos Lew , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética
18.
Cell Signal ; 23(8): 1225-34, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21342664

RESUMO

Podosomes are ventral adhesion structures prominent in cells of the myeloid lineage. A common aspect of these cells is that they are highly motile and must to traverse multiple tissue barriers in order to perform their functions. Recently podosomes have gathered attention from researchers as important cellular structures that can influence cell adhesion, motility and matrix remodeling. Adhesive and soluble ligands act via transmembrane receptors and propagate signals to the leukocyte cytoskeleton via small G proteins of the Rho family, tyrosine kinases and scaffold proteins and are able to induce podosome formation and rearrangements. Manipulation of the signals that regulate podosome formation and dynamics can therefore be a strategy to interfere with leukocyte functions in a multitude of pathological settings, such as infections, atherosclerosis and arthritis. Here, we review the major signaling molecules that act in the formation and regulation of podosomes.


Assuntos
Extensões da Superfície Celular/metabolismo , Leucócitos/metabolismo , Transdução de Sinais , Actinas/metabolismo , Actinas/fisiologia , Adesão Celular/fisiologia , Extensões da Superfície Celular/enzimologia , Leucócitos/imunologia , Leucócitos/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
19.
Eur J Cell Biol ; 90(2-3): 143-56, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-20719402

RESUMO

Cell adhesion to the extracellular matrix is mediated by adhesion receptors, mainly integrins, which upon interaction with the extracellular matrix, bind to the actin cytoskeleton via their cytoplasmic domains. This association is mediated by a variety of scaffold and signaling proteins, which control the mechanical and signaling activities of the adhesion site. Upon transformation of fibroblasts with active forms of Src (e.g., v-Src), focal adhesions are disrupted, and transformed into dot-like contacts known as podosomes, and consisting of a central actin core surrounded by an adhesion ring. To clarify the mechanism underlying Src-dependent modulation of the adhesive phenotype, and its influence on podosome organization, we screened for the effect of siRNA-mediated knockdown of tyrosine kinases, MAP kinases and phosphatases on the reorganization of the adhesion-cytoskeleton complex, induced by a constitutively active Src mutant (SrcY527F). In this screen, we discovered several genes that are involved in Src-induced remodeling of the actin cytoskeleton. We further showed that knockdown of Src in osteoclasts abolishes the formation of the podosome-based rings and impairs cell spreading, without inducing stress fiber development. Our work points to several genes that are involved in this process, and sheds new light on the molecular plasticity of integrin adhesions.


Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Quinases da Família src/metabolismo , Adesão Celular/fisiologia , Extensões da Superfície Celular/enzimologia , Extensões da Superfície Celular/metabolismo , Citoesqueleto/enzimologia , Técnicas de Silenciamento de Genes , Humanos , Integrinas/metabolismo , Osteoclastos/enzimologia , Osteoclastos/metabolismo , Transdução de Sinais
20.
Eur J Cell Biol ; 90(2-3): 128-35, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-20970878

RESUMO

Invasion across tissue boundaries by metastatic tumor cells depends on the proteolytic degradation of the extracellular matrix, initiated by the formation of invadopodia, actin-driven membrane protrusions with matrix-degradative activity. Yet, mechanisms underlying invadopodia formation remain largely unknown. In this report, we examined the role of the histone deacetylase HDAC6 in invadopodia formation and invasion by breast cancer cells. Using small interfering RNA silencing of protein expression in highly invasive MDA-MB-231 breast adenocarcinoma cells, we show that HDAC6 is required for two-dimensional matrix proteolysis. In addition, we demonstrate that HDAC6 acts as a tubulin and cortactin deacetylase. We also report that the inhibition of HDAC6 by siRNA or treatment with HDAC inhibitor TSA results in a decreased invasion capacity of a three-dimensional type I collagen matrix by MDA-MB-231 cells. These data identify HDAC6 as a critical component of the invasive apparatus of tumor cells, in both two- and three-dimensional matrices.


Assuntos
Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Extensões da Superfície Celular/enzimologia , Histona Desacetilases/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/ultraestrutura , Membrana Basal/enzimologia , Membrana Basal/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/ultraestrutura , Linhagem Celular Tumoral , Extensões da Superfície Celular/patologia , Colágeno Tipo I/metabolismo , Cortactina/metabolismo , Matriz Extracelular/enzimologia , Matriz Extracelular/patologia , Feminino , Desacetilase 6 de Histona , Histona Desacetilases/biossíntese , Histona Desacetilases/genética , Humanos , Invasividade Neoplásica , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção
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