RESUMO
BACKGROUND: Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. METHODS: We purified and characterized sEVs from post-MI hearts and cultured cMSCs. Then, we analyzed cMSC-EV cargo and proneoplastic effects on several lines of cancer cells, macrophages, and endothelial cells. Next, we modeled heterotopic and orthotopic lung and breast cancer tumors in mice with post-MI LVD. We transferred cMSC-sEVs to assess sEV biodistribution and its effect on tumor growth. Finally, we tested the effects of sEV depletion and spironolactone treatment on cMSC-EV release and tumor growth. RESULTS: Post-MI hearts, particularly cMSCs, produced more sEVs with proneoplastic cargo than nonfailing hearts did. Proteomic analysis revealed unique protein profiles and higher quantities of tumor-promoting cytokines, proteins, and microRNAs in cMSC-sEVs from post-MI hearts. The proneoplastic effects of cMSC-sEVs varied with different types of cancer, with lung and colon cancers being more affected than melanoma and breast cancer cell lines. Post-MI cMSC-sEVs also activated resting macrophages into proangiogenic and protumorigenic states in vitro. At 28-day follow-up, mice with post-MI LVD developed larger heterotopic and orthotopic lung tumors than did sham-MI mice. Adoptive transfer of cMSC-sEVs from post-MI hearts accelerated the growth of heterotopic and orthotopic lung tumors, and biodistribution analysis revealed accumulating cMSC-sEVs in tumor cells along with accelerated tumor cell proliferation. sEV depletion reduced the tumor-promoting effects of MI, and adoptive transfer of cMSC-sEVs from post-MI hearts partially restored these effects. Finally, spironolactone treatment reduced the number of cMSC-sEVs and suppressed tumor growth during post-MI LVD. CONCLUSIONS: Cardiac sEVs, specifically cMSC-sEVs from post-MI hearts, carry multiple protumorigenic factors. Uptake of cMSC-sEVs by cancer cells accelerates tumor growth. Treatment with spironolactone significantly reduces accelerated tumor growth after MI. Our results provide new insight into the mechanism connecting post-MI LVD to cancer and propose a translational option to mitigate this deadly association.
Assuntos
Vesículas Extracelulares , Insuficiência Cardíaca , Infarto do Miocárdio , Animais , Vesículas Extracelulares/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/etiologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Camundongos , Humanos , Feminino , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Linhagem Celular Tumoral , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Masculino , Proliferação de Células/efeitos dos fármacosRESUMO
Invasive melanoma is an aggressive form of skin cancer with high incidence of mortality. The process of tumor invasion is a crucial primary step in the metastatic cascade, yet the mechanisms involved are still under investigation. Here we document a critical role for MLK3 (MAP3K11) in the regulation of melanoma cell invasion. We report the unexpected finding that cellular loss of MLK3 in melanoma cells promotes cell invasion. Cellular depletion of MLK3 expression results in the hyperactivation of ERK, which is linked to the formation of a BRAF/Hsp90/Cdc37 protein complex. ERK hyperactivation leads to enhanced phosphorylation and inactivation of GSK3ß and the stabilization of c-Jun and JNK activity. Blocking of ERK and JNK signaling as well as Hsp90 activity downstream of MLK3-silencing significantly reduces melanoma invasion. Furthermore, ERK activation in the aforementioned context is coupled to MT1-MMP transcription as well as the TOM1L1-dependent localization of the membrane protease to invadopodia at the invasive front. These studies provide critical insight into the mechanisms that couple MLK3 loss with BRAF hyperactivation and its consequence on melanoma invasion.
Assuntos
MAP Quinase Quinase Quinases , Melanoma , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Melanoma/genética , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismoRESUMO
Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound vesicles released by cells that contain bioactive cargoes including proteins, lipids and nucleic acids. Multiple subpopulations of EVs have now been recognized and these include exosomes and microvesicles. EVs have been thought to facilitate intercellular and distal communication to bring about various processes that enable tumor progression and metastases. Here, we describe the current knowledge of the functional cargo contained within EVs, with a focus on tumor microvesicles, and review the emerging theory of how EVs support immune suppression in cancer.
Assuntos
Comunicação Celular/imunologia , Micropartículas Derivadas de Células/imunologia , Vesículas Extracelulares/imunologia , Neoplasias/imunologia , Animais , Micropartículas Derivadas de Células/patologia , Vesículas Extracelulares/patologia , Humanos , Metástase Neoplásica , Neoplasias/patologiaRESUMO
The study of extracellular vesicles (EVs) is a rapidly evolving field, owing in large part to recent advances in the realization of their significant contributions to normal physiology and disease. Once discredited as cell debris, these membrane vesicles have now emerged as mediators of intercellular communication by interaction with target cells, drug and gene delivery, and as potentially versatile platforms of clinical biomarkers as a result of their distinctive protein, nucleic acid and lipid cargoes. While there are multiple classes of EVs released from almost all cell types, here we focus primarily on the biogenesis, fate and functional cargoes of microvesicles (MVs). MVs regulate many important cellular processes including facilitating cell invasion, cell growth, evasion of immune response, stimulating angiogenesis, drug resistance and many others.
Assuntos
Vesículas Extracelulares/metabolismo , Animais , Transporte Biológico , Vesículas Extracelulares/ultraestrutura , Humanos , Biogênese de OrganelasRESUMO
Extracellular vesicle (EV) shedding is a biologically conserved cellular process across virtually every cell type. In cancer, EVs shed from tumor and stromal cells to the tumor microenvironment play a major role in determining tumor fate, which to a large extent is dictated by the biologically active cargo contained in EVs. Current understanding of various cancer-associated EVs has enabled the outlining of mechanistic connections between cargo and tumor-promoting functions. In this chapter, we describe examples of EV-mediated communication between tumor cells and stromal cells, highlighting the molecular constituents responsible for pro-tumorigenic effects. Furthermore, we discuss the roles of matrix-degrading EVs in cell invasion. Finally, we summarize research on the potential use of EVs as a novel approach to cancer therapeutics.
Assuntos
Vesículas Extracelulares , Neoplasias/patologia , Microambiente Tumoral , Humanos , Neoplasias/terapia , Células EstromaisRESUMO
Recent advances in the study of tumor-derived microvesicles reveal new insights into the cellular basis of disease progression and the potential to translate this knowledge into innovative approaches for cancer diagnostics and personalized therapy. Tumor-derived microvesicles are heterogeneous membrane-bound sacs that are shed from the surfaces of tumor cells into the extracellular environment. They have been thought to deposit paracrine information and create paths of least resistance, as well as be taken up by cells in the tumor microenvironment to modulate the molecular makeup and behavior of recipient cells. The complexity of their bioactive cargo-which includes proteins, RNA, microRNA, and DNA-suggests multipronged mechanisms by which microvesicles can condition the extracellular milieu to facilitate disease progression. The formation of these shed vesicles likely involves both a redistribution of surface lipids and the vertical trafficking of cargo to sites of microvesicle biogenesis at the cell surface. Current research also suggests that molecular profiling of these structures could unleash their potential as circulating biomarkers as well as platforms for personalized medicine. Thus, new and improved strategies for microvesicle identification, isolation, and capture will have marked implications in point-of-care diagnostics for cancer patients.
Assuntos
Biomarcadores Tumorais/metabolismo , Micropartículas Derivadas de Células/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Microambiente Tumoral , Espaço Extracelular/metabolismo , Humanos , Medicina de PrecisãoRESUMO
Epithelial cells form tissues with many functions, including secretion and environmental separation and protection. Glandular epithelial tissues comprise cysts and tubules that are formed from a polarized, single-epithelial cell layer surrounding a central, fluid-filled lumen. The pathways regulating key processes in epithelial tissue morphogenesis such as mitotic spindle formation are incompletely understood, but are important to investigate, as their dysregulation is a signature of epithelial tumors. Here, we describe a signaling axis that manifests in a defect in mitotic spindle orientation during epithelial growth and cystogenesis. We found that activation of the small GTPase ADP-ribosylation factor 6 (ARF6) results in the sustained internalization of cell-surface components such as the cMet receptor and the cell-adhesion molecule E-cadherin. The spindle orientation defect arising from elevated levels of ARF6-GTP required an increase in cMet endocytosis, but was independent of E-cadherin internalization or elevated extracellular signal-regulated kinase (ERK) activity resulting from internalized receptor signaling on endosomes. Misorientation of the mitotic spindle resulted in the development of epithelial cysts with structural abnormalities, the most conspicuous of which was the presence of multiple intercellular lumens. Abnormal mitotic spindle orientation was necessary but insufficient to disrupt glandular development, as blocking the strong prosurvival signal resulting from ERK hyperactivation yielded structurally normal cysts despite continued manifestation of spindle orientation defects. Our findings highlight a previously unknown link between ARF6 activation, cMet receptor internalization, and mitotic spindle orientation during epithelial glandular morphogenesis.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Cistos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Organoides/metabolismo , Fuso Acromático/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Animais , Caderinas/genética , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Divisão Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Polaridade Celular/efeitos dos fármacos , Cistos/ultraestrutura , Cães , Endocitose , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/ultraestrutura , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Regulação da Expressão Gênica , Células Madin Darby de Rim Canino , Proteínas Associadas aos Microtúbulos/genética , Morfogênese/genética , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/ultraestrutura , Técnicas de Cultura de TecidosRESUMO
Aberrant cellular cholesterol accumulation contributes to the pathophysiology of many diseases including neurodegenerative disorders such as Niemann-Pick Type C (NPC) and Alzheimer's Disease1-4. Many aspects of cholesterol efflux from cells remain elusive. Here we describe the utility of cholesterol-rich giant plasma membrane vesicles (GPMVs) as a means to monitor cholesterol that is translocated to the plasma membrane for secretion. We demonstrate that small molecules known to enhance lipid efflux, including those in clinical trials for lipid storage disorders, enhance this GPMV formation. Conversely, pharmacological inhibition of cholesterol efflux blocks GPMV formation. We show that microtubule stabilization via paclitaxel treatment and increased tubulin acetylation via HDAC6 inhibition promotes the formation of GPMVs with concomitant reduction in cellular cholesterol in a cell model of NPC disease. The pan-deacetylase inhibitor panobinostat, which has been shown to reduce the severity of cholesterol storage in NPC, elicited a similar response. Further, the disruption of actin polymerization inhibits the formation of GPMVs, whereas the small GTP-binding protein Arl4c promotes actin remodeling at sites overlapping with GPMV formation. Thus, monitoring the formation of GPMVs provides a new avenue to better understand diseases whose pathology may be sensitive to alterations in cellular cholesterol.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Exocitose/fisiologia , Vesículas Extracelulares/metabolismo , Tamanho das Organelas/fisiologia , Transporte Biológico , Linhagem Celular , Membrana Celular/patologia , Vesículas Extracelulares/patologia , Humanos , Metabolismo dos Lipídeos/fisiologia , Imagem ÓpticaRESUMO
Information transmission from tumor cells to non-tumor cells in the surrounding microenvironment via microvesicles is a more recently studied form of intercellular signaling that can have a marked impact on the tumor microenvironment. Tumor-derived microvesicles (TMVs) are packed with information including signaling proteins and nucleic acids, and can be taken up by target cells, enabling paracrine signaling. While previous research has focused on how vesicles released from pathologic cells differ from normal cells, the heterogeneity that exists within the TMV population itself is not fully characterized, and only beginning to be appreciated. In this review, we summarize current understanding of the biogenesis and roles of shed TMVs in the tumor microenvironment, and speculate on the consequences for tumor cell signaling in light of the hypothesis that there exists variance within the TMV population. The analysis of differential signaling upon cell-TMV interactions provides insights into potential mechanisms of intercellular communication.
Assuntos
Micropartículas Derivadas de Células/fisiologia , Microambiente Tumoral/fisiologia , Comunicação Celular/fisiologia , Humanos , Transdução de Sinais/fisiologiaRESUMO
Extracellular membrane vesicles have recently emerged as versatile mediators of intercellular communication, pathogenesis, drug and gene delivery and as potentially rich reservoirs of clinical biomarkers. Channeling their properties toward patient care is dependent on technological progress in approaches used for their analysis and molecular profiling.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Animais , Biomarcadores/metabolismo , Comunicação Celular , Exossomos/metabolismo , Humanos , Imunoterapia , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Medicina de Precisão , Proteoma/metabolismoRESUMO
A wide range of cellular activities depends upon endocytic recycling. ARF6, a small molecular weight GTPase, regulates the processes of endocytosis and endocytic recycling in concert with various effector molecules and other small GTPases. This review highlights three critical processes that involve ARF6-mediated endosomal membrane trafficking-cell motility, cytokinesis, and cholesterol homeostasis. In each case, the function of ARF6-mediated trafficking varies-including localization of specific protein and lipid cargo, regulation of bulk membrane movement, and modulation of intracellular signaling. As described in this review, mis-regulation of endocytic traffic can result in human disease when it compromises the cell's ability to regulate cell movement and invasion, cell division, and lipid homeostasis.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Divisão Celular , Movimento Celular , Endocitose , Homeostase , Lipídeos/fisiologia , Fator 6 de Ribosilação do ADP , Animais , HumanosRESUMO
Oncosomes are tumor-derived microvesicles that transmit signaling complexes between cell and tissue compartments. Herein, we show that amoeboid tumor cells export large (1- to 10-µm diameter) vesicles, derived from bulky cellular protrusions, that contain metalloproteinases, RNA, caveolin-1, and the GTPase ADP-ribosylation factor 6, and are biologically active toward tumor cells, endothelial cells, and fibroblasts. We describe methods by which large oncosomes can be selectively sorted by flow cytometry and analyzed independently of vesicles <1 µm. Structures resembling large oncosomes were identified in the circulation of different mouse models of prostate cancer, and their abundance correlated with tumor progression. Similar large vesicles were also identified in human tumor tissues, but they were not detected in the benign compartment. They were more abundant in metastases. Our results suggest that tumor microvesicles substantially larger than exosome-sized particles can be visualized and quantified in tissues and in the circulation, and isolated and characterized using clinically adaptable methods. These findings also suggest a mechanism by which migrating tumor cells condition the tumor microenvironment and distant sites, thereby potentiating advanced disease.
Assuntos
Micropartículas Derivadas de Células/patologia , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Fator 6 de Ribosilação do ADP , Animais , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/ultraestrutura , Citometria de Fluxo , Humanos , Masculino , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias da Próstata/ultraestruturaRESUMO
Tumor progression, from early-stage invasion to the formation of distal metastases, relies on the capacity of tumor cells to modify the extracellular matrix (ECM) and communicate with the surrounding stroma. Extracellular vesicles (EVs) provide an important means to regulate cell invasion due to the selective inclusion of cargoes such as proteases and matrix proteins into EVs that can degrade or modify the ECM. EVs have also been shown to facilitate intercellular communication in the tumor microenvironment through paracrine signaling, which can impact ECM invasion by cancer cells. Here, we describe the current knowledge of EVs as facilitators of tumor invasion by virtue of their effects on proteolytic degradation and modification of the ECM, their ability to educate the stromal cells in the tumor microenvironment, and their role as mediators of long-range communication aiding in cell invasion and matrix remodeling at secondary sites.
RESUMO
Tumor cells release extracellular vesicles (EVs) that can function as mediators of intercellular communication in the tumor microenvironment. EVs contain a host of bioactive cargo, including membrane, cytosolic, and nuclear proteins, in addition to noncoding RNAs, other RNA types, and double-stranded DNA fragments. These shed vesicles may deposit paracrine information and can also be taken up by stromal cells, causing the recipient cells to undergo phenotypic changes that profoundly impact diverse facets of cancer progression. For example, this unique form of cellular cross talk helps condition the premetastatic niche, facilitates evasion of the immune response, and promotes invasive and metastatic activity. These findings, coupled with those demonstrating that the number and content of EVs produced by tumors can vary depending on their tumor of origin, disease stage, or response to therapy, have raised the exciting possibility that EVs can be used for risk stratification, diagnostic, and even prognostic purposes. We summarize recent developments and the current knowledge of EV cargoes, their impact on disease progression, and implementation of EV-based liquid biopsies as tumor biomarkers.
Assuntos
Vesículas Extracelulares , Neoplasias , Humanos , Microambiente Tumoral , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Neoplasias/patologia , Progressão da DoençaRESUMO
Extracellular nanocarriers (extracellular vesicles (EVs), lipoproteins, and ribonucleoproteins) of protein and nucleic acids mediate intercellular communication and are clinically adaptable as distinct circulating biomarkers. However, the overlapping size and density of the nanocarriers have so far prevented their efficient physical fractionation, thus impeding independent downstream molecular assays. Here, we report a bias-free high-throughput and high-yield continuous isoelectric fractionation nanocarrier fractionation technique based on their distinct isoelectric points. This nanocarrier fractionation platform is enabled by a robust and tunable linear pH profile provided by water-splitting at a bipolar membrane and stabilized by flow without ampholytes. The linear pH profile that allows easy tuning is a result of rapid equilibration of the water dissociation reaction and stabilization by flow. The platform is automated with a machine learning procedure to allow recalibration for different physiological fluids and nanocarriers. The optimized technique has a resolution of 0.3 ΔpI, sufficient to separate all nanocarriers and even subclasses of nanocarriers. Its performance is then evaluated with several biofluids, including plasma, urine, and saliva samples. Comprehensive, high-purity (plasma: >93%, urine: >95% and saliva: >97%), high-yield (plasma: >78%, urine: >87% and saliva: >96%), and probe-free isolation of ribonucleoproteins in 0.75 mL samples of various biofluids in 30 min is demonstrated, significantly outperforming affinity-based and highly biased gold standards having low yield and day-long protocols. Binary fractionation of EVs and different lipoproteins is also achieved with similar performance.
Assuntos
Líquidos Corporais , Vesículas Extracelulares , Saliva/metabolismo , Ribonucleoproteínas , Líquidos Corporais/química , Vesículas Extracelulares/metabolismo , Lipoproteínas/análise , Lipoproteínas/metabolismoRESUMO
Microvesicles are generated by the outward budding and fission of membrane vesicles from the cell surface. Recent studies suggest that microvesicle shedding is a highly regulated process that occurs in a spectrum of cell types and, more frequently, in tumor cells. Microvesicles have been widely detected in various biological fluids including peripheral blood, urine and ascitic fluids, and their function and composition depend on the cells from which they originate. By facilitating the horizontal transfer of bioactive molecules such as proteins, RNAs and microRNAs, they are now thought to have vital roles in tumor invasion and metastases, inflammation, coagulation, and stem-cell renewal and expansion. This Commentary summarizes recent literature on the properties and biogenesis of microvesicles and their potential role in cancer progression.
Assuntos
Comunicação Celular , Micropartículas Derivadas de Células/metabolismo , Neoplasias/patologia , Neoplasias/fisiopatologia , Animais , Biomarcadores Tumorais/metabolismo , Coagulação Sanguínea , Transformação Celular Neoplásica , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/patologia , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Humanos , Metabolismo dos Lipídeos , Invasividade Neoplásica , Neoplasias/irrigação sanguínea , Neovascularização Patológica , Oncogenes , Evasão TumoralRESUMO
The shedding of extracellular vesicles (EVs) represents an important but understudied means of cell-cell communication in cancer. Among the currently described classes of EVs, tumor-derived microvesicles (TMVs) comprise a class of vesicles released directly from the cell surface. TMVs contain abundant cargo, including functional proteins and miRNA, which can be transferred to and alter the behavior of recipient cells. Here, we document that a fraction of extracellular double-stranded DNA (dsDNA) is enclosed within TMVs and protected from nuclease degradation. dsDNA inclusion in TMVs is regulated by ARF6 cycling and occurs with the cytosolic DNA sensor, cGAS, but independent of amphisome or micronuclei components. Our studies suggest that dsDNA is trafficked to TMVs via a mechanism distinct from the multivesicular body-dependent secretion reported for the extracellular release of cytosolic DNA. Furthermore, TMV dsDNA can be transferred to recipient cells with consequences to recipient cell behavior, reinforcing its relevance in mediating cell-cell communication.
Assuntos
Micropartículas Derivadas de Células , Vesículas Extracelulares , MicroRNAs , Neoplasias , Micropartículas Derivadas de Células/metabolismo , DNA/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , MicroRNAs/metabolismo , Neoplasias/metabolismoRESUMO
The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.
RESUMO
ARF6-regulated endocytosis of E-cadherin is essential during the disassembly of adherens junctions in epithelial cells. Here, we show that activation of ARF6 promotes clathrin-dependent internalization of E-cadherin and caveolae at the basolateral cell surface. Furthermore, we demonstrate that ARF6-GTP, a constitutively activate form of ARF6, interacts with and recruits Nm23-H1, a nucleoside diphosphate (NDP) kinase that provides a source of GTP for dynamin-dependent fission of coated vesicles during endocytosis. Finally, we show that ARF6-mediated recruitment of Nm-23-H1 to cell junctions is accompanied by a decrease in the cellular levels of Rac1-GTP, consistent with previous findings that Nm23-H1 down-regulates activation of Rac1. These studies provide a molecular basis for ARF6 function in polarized epithelia during adherens junction disassembly.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Junções Aderentes/fisiologia , Endocitose/fisiologia , Células Epiteliais/fisiologia , Guanosina Trifosfato/fisiologia , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Núcleosídeo-Difosfato Quinase , Fatores de Transcrição/fisiologia , Fator 6 de Ribosilação do ADP , Animais , Linhagem Celular , Movimento Celular , Polaridade Celular , Cães , Células Epiteliais/citologia , Nucleosídeo NM23 Difosfato QuinasesRESUMO
Huntington's disease (HD) is an inherited neurodegenerative disorder. Here we demonstrate that expression of arfaptin 2/POR1 (partner of Rac1) in cultured cells induces the formation of pericentriolar and nuclear aggregates, which morphologically resemble mutant huntingtin aggregates characteristic of HD. Endogenous arfaptin 2 localizes to aggregates induced by expression of an abnormal amino-terminal fragment of huntingtin that contains polyglutamine (polyQ) expansions. A dominant inhibitory mutant of arfaptin 2 inhibits aggregation of mutant huntingtin, but not in the presence of proteasome inhibitors. Using cell-free biochemical assays, we show that arfaptin 2 inhibits proteasome activity. Finally, we show that expression of arfaptin 2 is increased at sites of neurodegeneration and the protein localizes to huntingtin aggregates in HD transgenic mouse brains. Our data suggest that arfaptin 2 is involved in regulating huntingtin protein aggregation, possibly by impairing proteasome function.