RESUMO
Tumor hypoxia has been associated with cancer progression, angiogenesis, and metastasis via modifications in the release and cargo composition of extracellular vesicles secreted by tumor cells. Indeed, hypoxic extracellular vesicles are known to trigger a variety of angiogenic responses via different mechanisms. We recently showed that hypoxia promotes endosomal signaling in tumor cells via HIF-1α-dependent induction of the guanine exchange factor ALS2, which activates Rab5, leading to downstream events involved in cell migration and invasion. Since Rab5-dependent signaling is required for endothelial cell migration and angiogenesis, we explored the possibility that hypoxia promotes the release of small extracellular vesicles containing ALS2, which in turn activate Rab5 in recipient endothelial cells leading to pro-angiogenic properties. In doing so, we found that hypoxia promoted ALS2 expression and incorporation as cargo within small extracellular vesicles, leading to subsequent transfer to recipient endothelial cells and promoting cell migration, tube formation, and downstream Rab5 activation. Consequently, ALS2-containing small extracellular vesicles increased early endosome size and number in recipient endothelial cells, which was followed by subsequent sequestration of components of the ß-catenin destruction complex within endosomal compartments, leading to stabilization and nuclear localization of ß-catenin. These events converged in the expression of ß-catenin target genes involved in angiogenesis. Knockdown of ALS2 in donor tumor cells precluded its incorporation into small extracellular vesicles, preventing Rab5-downstream events and endothelial cell responses, which depended on Rab5 activity and guanine exchange factor activity of ALS2. These findings indicate that vesicular ALS2, secreted in hypoxia, promotes endothelial cell events leading to angiogenesis. Finally, these events might explain how tumor angiogenesis proceeds in hypoxic conditions.
Assuntos
Movimento Celular , Vesículas Extracelulares , Fatores de Troca do Nucleotídeo Guanina , Transdução de Sinais , beta Catenina , Proteínas rab5 de Ligação ao GTP , Humanos , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , beta Catenina/metabolismo , Vesículas Extracelulares/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Linhagem Celular TumoralRESUMO
Tumor hypoxia and the hypoxia inducible factor-1, HIF-1, play critical roles in cancer progression and metastasis. We previously showed that hypoxia activates the endosomal GTPase Rab5, leading to tumor cell migration and invasion, and that these events do not involve changes in Rab protein expression, suggesting the participation of intermediate activators. Here, we identified ALS2, a guanine nucleotide exchange factor that is upregulated in cancer, as responsible for increased Rab5-GTP loading, cell migration and metastasis in hypoxia. Specifically, hypoxia augmented ALS2 mRNA and protein levels, and these events involved HIF-1α-dependent transcription, as shown by RNAi, pharmacological inhibition, chromatin immunoprecipitation and bioinformatics analyses, which identified a functional HIF-1α-binding site in the proximal promoter region of ALS2. Moreover, ALS2 and Rab5 activity were elevated both in a model of endogenous HIF-1α stabilization (renal cell carcinoma) and by following expression of stable non-hydroxylatable HIF-1α. Strikingly, ALS2 upregulation in hypoxia was required for Rab5 activation, tumor cell migration and invasion, as well as experimental metastasis in C57BL/6 mice. Finally, immunohistochemical analyses in patient biopsies with renal cell carcinoma showed that elevated HIF-1α correlates with increased ALS2 expression. Hence, this study identifies ALS2 as a novel hypoxia-inducible gene associated with tumor progression and metastasis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Carcinoma de Células Renais/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Ativação Transcricional/genética , Hipóxia Tumoral , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Cells expressing eGFP-tagged Rab5 (wild-type or the GDP-Rab5 mutant) and the DsRed-tagged α1B-adrenergic receptors were employed and the roles of GRK2 were studied utilizing paroxetine and the dominant-negative mutant of GRK2 (DN-GRK2). The following parameters were studied: a) FRET (as an index of α1B-adrenergic receptor-Rab5 interaction): b) intracellular accumulation of DsRed fluorescence (receptor internalization); c) α1B-adrenergic receptor phosphorylation, and d) noradrenaline-induced increase in intracellular calcium concentration. Noradrenaline increased α1B-adrenergic receptor-Rab5 interaction, which was blocked by paroxetine and by expression of the dominant-negative GRK2 mutant. Similarly, paroxetine and expression of the DN-GRK2 or the GDP-Rab5 mutants markedly decreased receptor internalization, α1B-adrenergic receptor phosphorylation, and attenuated the ability of the adrenergic agonist to induce homologous desensitization (calcium signaling). The S406, 410,412A α1B-adrenergic receptor mutant did not reproduce the actions of GRK2 inhibition. The data indicate that GRK2 and Rab5 play key roles in α1B-adrenergic receptor phosphorylation, internalization, and desensitization. The possibility that Rab5 might form part of a signaling complex is suggested, as well as that GDP-Rab5 might interfere with the ability of GRK2 to catalyze α1B-adrenergic receptor phosphorylation.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Transferência Ressonante de Energia de Fluorescência , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/genética , Células HEK293 , Humanos , Mutação , Norepinefrina/farmacologia , Paroxetina/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Axonal transport is required for neuronal development and survival. Transport from the axon to the soma is driven by the molecular motor cytoplasmic dynein, yet it remains unclear how dynein is spatially and temporally regulated. We find that the dynein effector Hook1 mediates transport of TrkB-BDNF-signaling endosomes in primary hippocampal neurons. Hook1 comigrates with a subpopulation of Rab5 endosomes positive for TrkB and BDNF, which exhibit processive retrograde motility with faster velocities than the overall Rab5 population. Knockdown of Hook1 significantly reduced the motility of BDNF-signaling endosomes without affecting the motility of other organelles. In microfluidic chambers, Hook1 depletion resulted in a significant decrease in the flux and processivity of BDNF-Qdots along the mid-axon, an effect specific for Hook1 but not Hook3. Hook1 depletion inhibited BDNF trafficking to the soma and blocked downstream BDNF- and TrkB-dependent signaling to the nucleus. Together, these studies support a model in which differential association with cargo-specific effectors efficiently regulates dynein in neurons.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dineínas do Citoplasma/metabolismo , Endossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Receptor trkB/metabolismo , Animais , Transporte Axonal , Sítios de Ligação , Fator Neurotrófico Derivado do Encéfalo/genética , Núcleo Celular/metabolismo , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Endossomos/ultraestrutura , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Neurônios/ultraestrutura , Cultura Primária de Células , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptor trkB/genética , Transdução de Sinais , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteína Vermelha FluorescenteRESUMO
The Rab protein family belongs to a superfamily of ras-like GTP-binding proteins. Rab proteins regulate many steps of membrane trafficking. In this study, three Rab family members, Rab5B, Rab6A, and Rab7, designated LvRab5B, LvRab6A, and LvRab7, were cloned from Litopenaeus vannamei. The full-length cDNA sequences of LvRab5B, LvRab6A, and LvRab7 were 1383, 873, and 767 nucleotides in length and they encoded proteins of 211, 212, and 205 amino acids, respectively. Using qRT-PCR, the mRNA expression levels of the three proteins were determined in the hepatopancreas of L. vannamei at different stages after infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus challenge. The results indicated that the mRNA expression levels of LvRab5B, LvRab6A, and LvRab7 were all significantly up-regulated after virus injection, suggesting that these genes may play essential roles in the immune response to viral infection in shrimp.
Assuntos
Regulação da Expressão Gênica , Penaeidae/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , Densovirinae , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Vírus da Síndrome da Mancha Branca 1 , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
The small GTPase Rab5 has been extensively studied in the context of endocytic trafficking because it is critical in the regulation of early endosome dynamics. In addition to this canonical role, evidence obtained in recent years implicates Rab5 in the regulation of cell migration. This novel role of Rab5 is based not only on an indirect relationship between cell migration and endosomal trafficking as separate processes, but also on the direct regulation of signaling proteins implicated in cell migration. However, the precise mechanisms underlying this connection have remained elusive. Recent studies have shown that the activation of Rab5 is a critical event for maintaining the dynamics of focal adhesions, which is fundamental in regulating not only cell migration but also tumor cell invasion.
Assuntos
Movimento Celular/genética , Adesões Focais/genética , Invasividade Neoplásica/genética , Neoplasias/genética , Proteínas rab5 de Ligação ao GTP/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Humanos , Neoplasias/patologia , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Migration and invasion are essential steps associated with tumor cell metastasis and increasing evidence points towards endosome trafficking being essential in this process. Indeed, the small GTPase Rab5, a crucial regulator of early endosome dynamics, promotes cell migration in vitro and in vivo. Precisely how Rab5 participates in these events remains to be determined. Considering that focal adhesions represent structures crucial to cell migration, we specifically asked whether Rab5 activation promoted focal adhesion disassembly and thereby facilitated migration and invasion of metastatic cancer cells. Pulldown and biosensor assays revealed that Rab5-GTP loading increased at the leading edge of migrating tumor cells. Additionally, targeting of Rab5 by different shRNA sequences, but not control shRNA, decreased Rab5-GTP levels, leading to reduced cell spreading, migration and invasiveness. Re-expression in knockdown cells of wild-type Rab5, but not the S34N mutant (GDP-bound), restored these properties. Importantly, Rab5 association with the focal adhesion proteins vinculin and paxillin increased during migration, and expression of wild-type, but not GDP-bound Rab5, accelerated focal adhesion disassembly, as well as FAK dephosphorylation on tyrosine 397. Finally, Rab5-driven invasiveness required focal adhesion disassembly, as treatment with the FAK inhibitor number 14 prevented Matrigel invasion and matrix metalloproteinase release. Taken together, these observations show that Rab5 activation is required to enhance cancer cell migration and invasion by promoting focal adhesion disassembly.
Assuntos
Neoplasias da Mama/metabolismo , Adesão Celular/fisiologia , Adesões Focais/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Ativação Enzimática , Feminino , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Paxilina/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Vinculina/metabolismo , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
The small GTPase Rab5 is a key regulator of endosome/phagosome maturation and in intravesicular infections marks a phagosome stage at which decisions over pathogen replication or destruction are integrated. It is currently unclear whether Leishmania-infected phagosomes uniformly pass through a Rab5(+) stage on their intracellular path to compartments with late endosomal/early lysosomal characteristics. Differences in routes and final compartments could have consequences for accessibility to antileishmanial drugs. Here, we generated a unique gfp-rab5 transgenic mouse model to visualize Rab5 recruitment to early parasite-containing phagosomes in primary host cells. Using real-time fluorescence imaging of phagosomes carrying Leishmania mexicana, we determined that parasite-infested phagosomes follow a uniform sequence of transient Rab5 recruitment. Residence in Rab5(+) compartments was much shorter compared with phagosomes harboring latex beads. Furthermore, a comparative analysis of parasite life-cycle stages and mutants deficient in lpg1, the gene encoding the enzyme required for synthesis of the dominant surface lipophosphoglycan, indicated that parasite surface ligands and host cell receptors modulate pathogen residence times in Rab5(+) phagosomes, but, as far as tested, had no significant effect on intracellular L. mexicana survival or replication.
Assuntos
Leishmania mexicana/fisiologia , Macrófagos/metabolismo , Fagossomos/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Feminino , Proteínas de Fluorescência Verde/metabolismo , Cinética , Ligantes , Camundongos , Camundongos Transgênicos , Fatores de Tempo , Transgenes/genética , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that can cause fatal encephalitis in humans. It remains a naturally emerging disease as well as a highly developed biological weapon. VEEV is transmitted to humans in nature by mosquito vectors. Little is known about VEEV entry, especially in mosquito cells. Here, a novel luciferase-based virus entry assay is used to show that the entry of VEEV into mosquito cells requires acidification. Furthermore, mosquito homologs of key human proteins (Rab5 and Rab7) involved in endocytosis were isolated and characterized. Rab5 is found on early endosomes and Rab7 on late endosomes and both are important for VEEV entry in mammalian cells. Each was shown to have analogous function in mosquito cells to that seen in mammalian cells. The wild-type, dominant negative and constitutively active mutants were then used to demonstrate that VEEV requires passage through early and late endosomes before infection can take place. This work indicates that the infection mechanism in mosquitoes and mammals is through a common and ancient evolutionarily conserved pathway.
Assuntos
Culicidae/virologia , Vírus da Encefalite Equina Venezuelana/crescimento & desenvolvimento , Endossomos/virologia , Internalização do Vírus , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rab5 de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Culicidae/citologia , Endossomos/química , Genes Reporter , Humanos , Luciferases/biossíntese , Luciferases/genética , Microscopia Confocal , Dados de Sequência Molecular , Alinhamento de Sequência , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/análise , Proteínas rab5 de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
The etiologic agent of Q fever Coxiella burnetii, is an intracellular obligate parasite that develops large vacuoles with phagolysosomal characteristics, containing multiple replicating bacteria. We have previously shown that Phase II C. burnetii replicative vacuoles generated after 24-48 h post infection are decorated with the autophagic protein LC3. The aim of the present study was to examine, at earlier stages of infection, the distribution and roles of the small GTPases Rab5 and Rab7, markers of early and late endosomes respectively, as well as of the protein LC3 on C. burnetii trafficking. Our results indicate that: (i) Coxiella phagosomes (Cph) acquire the two Rab proteins sequentially during infection; (ii) overexpression of a dominant negative mutant form of Rab5, but not of Rab7, impaired Coxiella entry, whereas both Rab5 and Rab7 dominant negative mutants inhibited vacuole formation; (iii) Cph colocalized with the protein LC3 as early as 5 min after infection; acquisition of this protein appeared to be a bacterially driven process, because it was inhibited by the bacteriostatic antibiotic chloramphenicol and (iv) C. burnetii delayed the arrival of the typical lysosomal protease cathepsin D to the Cph, which delay is further increased by starvation-induced autophagy. Based on our results we propose that C. burnetii transits through the normal endo/phagocytic pathway but actively interacts with autophagosomes at early times after infection. This intersection with the autophagic pathway delays fusion with the lysosomal compartment possibly favouring the intracellular differentiation and survival of the bacteria.