RESUMEN
Spatial restriction of mRNA to distinct subcellular locations enables local regulation and synthesis of proteins. However, the organizing principles of mRNA localization remain poorly understood. Here we analyzed subcellular transcriptomes of neural projections and soma of primary mouse cortical neurons and two neuronal cell lines and found that alternative last exons (ALEs) often confer isoform-specific localization. Surprisingly, gene-distal ALE isoforms were four times more often localized to neurites than gene-proximal isoforms. Localized isoforms were induced during neuronal differentiation and enriched for motifs associated with muscleblind-like (Mbnl) family RNA-binding proteins. Depletion of Mbnl1 and/or Mbnl2 reduced localization of hundreds of transcripts, implicating Mbnls in localization of mRNAs to neurites. We provide evidence supporting a model in which the linkage between genomic position of ALEs and subcellular localization enables coordinated induction of localization-competent mRNA isoforms through a post-transcriptional regulatory program that is induced during differentiation and reversed in cellular reprogramming and cancer.
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Proteínas de Unión al ADN/genética , Neuritas/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Empalme Alternativo/genética , Animales , Diferenciación Celular/genética , Reprogramación Celular/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Exones , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Isoformas de Proteínas , Estructura Terciaria de Proteína , Procesamiento Postranscripcional del ARN/genética , Proteínas de Unión al ARN/antagonistas & inhibidores , Transcriptoma/genéticaRESUMEN
Despite the fact that the majority of lung cancer deaths are due to metastasis, the molecular mechanisms driving metastatic progression are poorly understood. Here, we present evidence that loss of Foxa2 and Cdx2 synergizes with loss of Nkx2-1 to fully activate the metastatic program. These three lineage-specific transcription factors are consistently down-regulated in metastatic cells compared with nonmetastatic cells. Knockdown of these three factors acts synergistically and is sufficient to promote the metastatic potential of nonmetastatic cells to that of naturally arising metastatic cells in vivo. Furthermore, silencing of these three transcription factors is sufficient to account for a significant fraction of the gene expression differences between the nonmetastatic and metastatic states in lung adenocarcinoma, including up-regulated expression of the invadopodia component Tks5long, the embryonal proto-oncogene Hmga2, and the epithelial-to-mesenchymal mediator Snail. Finally, analyses of tumors from a genetically engineered mouse model and patients show that low expression of Nkx2-1, Foxa2, and Cdx2 strongly correlates with more advanced tumors and worse survival. Our findings reveal that a large part of the complex transcriptional network in metastasis can be controlled by a small number of regulatory nodes that function redundantly, and loss of multiple nodes is required to fully activate the metastatic program.
Asunto(s)
Adenocarcinoma/fisiopatología , Factor Nuclear 3-beta del Hepatocito/metabolismo , Proteínas de Homeodominio/metabolismo , Neoplasias Pulmonares/fisiopatología , Metástasis de la Neoplasia/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Adenocarcinoma del Pulmón , Animales , Animales Modificados Genéticamente , Factor de Transcripción CDX2 , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Factor Nuclear 3-beta del Hepatocito/genética , Proteínas de Homeodominio/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Ratones , Ratones Desnudos , Proteínas Nucleares/genética , Proto-Oncogenes Mas , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genéticaRESUMEN
Therapeutic outcomes in oncology may be aided by precision diagnostics that offer early detection, localization and the opportunity to monitor response to therapy. Here, we report a multimodal nanosensor engineered to target tumours through acidosis, respond to proteases in the microenvironment to release urinary reporters and (optionally) carry positron emission tomography probes to enable localization of primary and metastatic cancers in mouse models of colorectal cancer. We present a paradigm wherein this multimodal sensor can be employed longitudinally to assess burden of disease non-invasively, including tumour progression and response to chemotherapy. Specifically, we showed that acidosis-mediated tumour insertion enhanced on-target release of matrix metalloproteinase-responsive reporters in urine. Subsequent on-demand loading of the radiotracer 64Cu allowed pH-dependent tumour visualization, enabling enriched microenvironmental characterization when compared with the conventional metabolic tracer 18F-fluorodeoxyglucose. Through tailored target specificities, this modular platform has the capacity to be engineered as a pan-cancer test that may guide treatment decisions for numerous tumour types.
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Acidosis/diagnóstico , Neoplasias Colorrectales/diagnóstico , Imagen Multimodal , Medicina de Precisión , Microambiente Tumoral , Acidosis/complicaciones , Animales , Neoplasias Colorrectales/complicaciones , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Femenino , Fluorodesoxiglucosa F18 , Ratones , Ratones Endogámicos BALB C , Tomografía de Emisión de PositronesRESUMEN
Rap1-interacting adaptor molecule (RIAM) is a Rap1 effector that mediates the recruitment of talin to integrins, thereby supporting their activation. In this study, we investigated the role of RIAM in an adoptive transfer model for type I diabetes and report that RIAM expression in T cells is necessary for diabetes development. Loss of RIAM did not prevent lymphocyte recruitment to draining lymph nodes 24 h after transfer, but it was required for Ag-driven proliferation and cytotoxic killing. RIAM is recruited to immune synapses along with talin and LFA-1, and loss of RIAM profoundly suppresses Ag-dependent conjugate formation in primary naive and effector T cells. These data identify the requirement of RIAM for formation of immunological synapses and in resulting T cell functions in autoimmunity. Moreover, because RIAM-null mice are healthy, fertile, and display no bleeding abnormalities, our results identify RIAM and its regulators as potential targets for therapies of T cell-mediated autoimmunity.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Presentadoras de Antígenos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Proteínas de la Membrana/metabolismo , Linfocitos T/inmunología , Talina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Traslado Adoptivo , Animales , Proliferación Celular/genética , Células Cultivadas , Citotoxicidad Inmunológica/genética , Humanos , Sinapsis Inmunológicas/inmunología , Proteínas de la Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/trasplanteRESUMEN
Altered cell polarity and migration are hallmarks of cancer and metastases. Here we show that inactivation of the retinoblastoma gene (Rb) tumor suppressor causes defects in tissue closure that reflect the inability of Rb null epithelial cells to efficiently migrate and polarize. These defects occur independently of pRB's anti-proliferative role and instead correlate with upregulation of RhoA signaling and mislocalization of apical-basal polarity proteins. Notably, concomitant inactivation of tp53 specifically overrides the motility defect, and not the aberrant polarity, thereby uncovering previously unappreciated mechanisms by which Rb and tp53 mutations cooperate to promote cancer development and metastases.
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Movimiento Celular/genética , Polaridad Celular/genética , Células Epiteliales/metabolismo , Proteína de Retinoblastoma/genética , Proteínas Supresoras de Tumor/genética , Proteínas de Fase Aguda/metabolismo , Animales , Silenciador del Gen , Humanos , Ratones , Mutación , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Talin is an integrin adaptor, which controls integrin activity in all hematopoietic cells. How intracellular signals promote talin binding to the integrin tail leading to integrin activation is still poorly understood, especially in leukocytes. In vitro studies identified an integrin activation complex whose formation is initiated by the interaction of active, guanosine triphosphate (GTP)-bound Ras-related protein 1 (Rap1) with the adapter protein Rap1-GTP-interacting adapter molecule (RIAM) followed by the recruitment of talin to the plasma membrane. Unexpectedly, loss-of-function studies in mice have shown that the talin-activating role of RIAM is neither required for development nor for integrin activation in platelets. In this study, we show that leukocyte integrin activation critically depends on RIAM both in vitro and in vivo. RIAM deficiency results in a loss of ß2 integrin activation in multiple leukocyte populations, impaired leukocyte adhesion to inflamed vessels, and accumulation in the circulation. Surprisingly, however, the major leukocyte ß1 integrin family member, α4ß1, was only partially affected by RIAM deficiency in leukocytes. Thus, although talin is an essential, shared regulator of all integrin classes expressed by leukocytes, we report that ß2 and α4 integrins use different RIAM-dependent and -independent pathways to undergo activation by talin.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD18/metabolismo , Quimiotaxis de Leucocito/fisiología , Leucocitos/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Western Blotting , Adhesión Celular/fisiología , Separación Celular , Citometría de Flujo , Integrina alfa4beta1/metabolismo , Ratones , Ratones Noqueados , Talina/metabolismo , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Regulation of integrins is critical for lymphocyte adhesion to endothelium and trafficking through secondary lymphoid organs. Inside-out signaling to integrins is mediated by the small GTPase Rap1. Two effectors of Rap1 regulate integrins, RapL and Rap1 interacting adaptor molecule (RIAM). Using mice conditionally deficient in both Rap1a and Rap1b and mice null for RIAM, we show that the Rap1/RIAM module is not required for T- or B-cell development but is essential for efficient adhesion to intercellular adhesion molecule (ICAM) 1 and vascular cell adhesion molecule (VCAM) 1 and for proper trafficking of lymphocytes to secondary lymphoid organs. Interestingly, in RIAM-deficient mice, whereas peripheral lymph nodes (pLNs) were depleted of both B and T cells and recirculating B cells were diminished in the bone barrow (BM), the spleen was hypercellular, albeit with a relative deficiency of marginal zone B cells. The abnormality in lymphocyte trafficking was accompanied by defective humoral immunity to T-cell-dependent antigens. Platelet function was intact in RIAM-deficient animals. These in vivo results confirm a role for RIAM in the regulation of some, but not all, leukocyte integrins and suggest that RIAM-regulated integrin activation is required for trafficking of lymphocytes from blood into pLNs and BM, where relatively high shear forces exist in high endothelial venules and sinusoids, respectively.
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Proteínas Adaptadoras Transductoras de Señales/inmunología , Linfocitos B/inmunología , Quimiotaxis de Leucocito/inmunología , Proteínas de la Membrana/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Adhesión Celular/inmunología , Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Linfocitos T/citología , Linfocitos T/metabolismo , Proteínas de Unión al GTP rap1/inmunología , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Listeria monocytogenes is a foodborne pathogen capable of invading a broad range of cell types and replicating within the host cell cytoplasm. This paper describes the colocalization of host cell lamellipodin (Lpd) with intracellular L. monocytogenes detectable 6 h postinfection of epithelial cells. The association was mediated via interactions between both the peckstrin homology (PH) domain in Lpd and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] on the bacterial surface and by interactions between the C-terminal EVH1 (Ena/VASP [vasodilator-stimulated phosphoprotein] homology domain 1) binding domains of Lpd and the host VASP (vasodilator-stimulated phosphoprotein) recruited to the bacterial cell surface by the listerial ActA protein. Depletion of Lpd by short interfering RNA (siRNA) resulted in reduced plaque size and number, indicating a role for Lpd in cell-to-cell spread. In contrast, overexpression of Lpd resulted in an increase in the number of L. monocytogenes-containing protrusions (listeriopods). Manipulation of the levels of Lpd within the cell also affected the intracellular velocity of L. monocytogenes, with a reduction in Lpd corresponding to an increase in intracellular velocity. These data, together with the observation that Lpd accumulated at the interface between the bacteria and the developing actin tail at the initiation of actin-based movement, indicate a possible role for Lpd in the actin-based movement and the cell-to-cell spread of L. monocytogenes.
Asunto(s)
Proteínas Portadoras/metabolismo , Interacciones Huésped-Parásitos/fisiología , Listeria monocytogenes/patogenicidad , Listeriosis/metabolismo , Proteínas de la Membrana/metabolismo , Actinas/metabolismo , Western Blotting , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Listeria monocytogenes/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
Shigella spp. are intracellular bacterial pathogens that cause diarrhoeal disease in humans. Shigella utilize the host actin cytoskeleton to enter cells, move through the cytoplasm of cells and pass into adjacent cells. Ena/VASP family proteins are highly conserved proteins that participate in actin-dependent dynamic cellular processes. We tested whether Ena/VASP family members VASP (vasodilator-stimulated phosphoprotein), Mena (mammalian-enabled) or EVL (Ena-VASP-like) contribute to Shigella flexneri spread through cell monolayers. VASP and EVL restricted cell-to-cell spread without significantly altering actin-based motility, whereas Mena had no effect on these processes. Phosphorylation of VASP on Ser153, Ser235 and Thr274 regulated its subcellular distribution and function. VASP derivatives that lack the Ena/VASP homology 1 (EVH1) domain or contain a phosphoablative mutation of Ser153 were defective in restricting S. flexneri spread, indicating that the EVH1 domain and phosphorylation on Ser153 are required for this process. The EVH1 domain and Ser153 of VASP were required for VASP localization to focal adhesions, and localization of VASP to focal adhesions and/or the leading edge was required for restriction of spread. The contribution of the EVH1 domain was from both the donor and the recipient cell, whereas the contribution of Ser153 phosphorylation was only from the donor cell. Thus, unlike host proteins characterized in Shigella pathogenesis that promote bacterial spread, VASP and EVL function to limit it. The ability of VASP and EVL to limit spread highlights the critical role of focal adhesion complexes and/or the leading edge in bacterial passage between cells.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Fibroblastos/inmunología , Fibroblastos/microbiología , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Shigella flexneri/inmunología , Animales , Moléculas de Adhesión Celular/genética , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Análisis Mutacional de ADN , Ratones , Proteínas de Microfilamentos/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosfoproteínas/genética , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
BACKGROUND: Menacalc is an immunofluorescence-based, quantitative method in which expression of the non-invasive Mena protein isoform (Mena11a) is subtracted from total Mena protein expression. Previous work has found a significant positive association between Menacalc and risk of death from breast cancer. Our goal was to determine if Menacalc could be used as an independent prognostic marker for axillary node-negative (ANN) breast cancer. METHODS: Analysis of the association of Menacalc with overall survival (death from any cause) was performed for 403 ANN tumors using Kaplan Meier survival curves and the univariate Cox proportional hazards (PH) model with the log-rank or the likelihood ratio test. Cox PH models were used to estimate hazard ratios (HRs) for the association of Menacalc with risk of death after adjustment for HER2 status and clinicopathological tumor features. RESULTS: High Menacalc was associated with increased risk of death from any cause (P=0.0199, HR (CI)=2.18 (1.19, 4.00)). A similarly elevated risk of death was found in the subset of the Menacalc cohort which did not receive hormone or chemotherapy (n=142) (P=0.0052, HR (CI)=3.80 (1.58, 9.97)). There was a trend toward increased risk of death with relatively high Menacalc in the HER2, basal and luminal molecular subtypes. CONCLUSIONS: Menacalc may serve as an independent prognostic biomarker for the ANN breast cancer patient population.
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Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/genética , Proteínas de Microfilamentos/biosíntesis , Anciano , Biomarcadores de Tumor/genética , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Proteínas de Microfilamentos/genética , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Receptor ErbB-2/genéticaRESUMEN
Entry of tumor cells into the blood stream is a critical step in cancer metastasis. Although significant progress has been made in visualizing tumor cell motility in vivo, the underlying mechanism of cancer cell intravasation remains largely unknown. We developed a microfluidic-based assay to recreate the tumor-vascular interface in three-dimensions, allowing for high resolution, real-time imaging, and precise quantification of endothelial barrier function. Studies are aimed at testing the hypothesis that carcinoma cell intravasation is regulated by biochemical factors from the interacting cells and cellular interactions with macrophages. We developed a method to measure spatially resolved endothelial permeability and show that signaling with macrophages via secretion of tumor necrosis factor alpha results in endothelial barrier impairment. Under these conditions intravasation rates were increased as validated with live imaging. To further investigate tumor-endothelial (TC-EC) signaling, we used highly invasive fibrosarcoma cells and quantified tumor cell migration dynamics and TC-EC interactions under control and perturbed (with tumor necrosis factor alpha) barrier conditions. We found that endothelial barrier impairment was associated with a higher number and faster dynamics of TC-EC interactions, in agreement with our carcinoma intravasation results. Taken together our results provide evidence that the endothelium poses a barrier to tumor cell intravasation that can be regulated by factors present in the tumor microenvironment.
Asunto(s)
Movimiento Celular , Células Endoteliales/citología , Microfluídica/métodos , Neoplasias/patología , Comunicación Celular/fisiología , Línea Celular Tumoral , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Imagenología Tridimensional , Macrófagos/citología , Metástasis de la Neoplasia , Neoplasias/irrigación sanguínea , Permeabilidad , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.
Asunto(s)
Corteza Cerebral/citología , Neuritas/fisiología , Neuronas/fisiología , Seudópodos/fisiología , Actinas/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/fisiología , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Laminina/fisiología , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/fisiología , Proteínas Asociadas a Microtúbulos , Microtúbulos/fisiología , Mutación , Miosina Tipo II/antagonistas & inhibidores , Miosinas/biosíntesis , NADPH Deshidrogenasa/biosíntesis , Neuronas/ultraestructura , Fosfoproteínas/genética , Fosfoproteínas/fisiologíaRESUMEN
Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA-Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT-dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell-cell junction formation, and regulation of cell migration, were enriched among EMT-associated alternatively splicing events. Our analysis suggested that most EMT-associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT-associated splicing pattern. Expression of EMT-associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT-dependent splicing changes occur commonly in human tumors. The functional significance of EMT-associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT-associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.
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Empalme Alternativo , Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Fenotipo , Factores de Empalme de ARN , Proteínas de Unión al ARN/genética , Proteínas Represoras/genéticaRESUMEN
We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (Mena(INV)) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated Mena(INV) increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by Mena(INV) is dependent on a macrophage-tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of Mena(INV) and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells.
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Neoplasias de la Mama/metabolismo , Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Migración Transendotelial y Transepitelial , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Macrófagos/metabolismo , Ratones , Ratones SCID , Proteínas de Microfilamentos , Invasividad Neoplásica , Metástasis de la Neoplasia , Isoformas de Proteínas/metabolismo , RatasRESUMEN
Mammalian enabled (Mena) is a key regulator of cytoskeletal actin dynamics, which has been implicated in heart failure (HF). We have previously demonstrated that cardiac Mena deletion produced cardiac dysfunction with conduction abnormalities and hypertrophy. Moreover, elevated Mena expression correlates with HF in human and animal models, yet the precise role of Mena in cardiac pathophysiology is unclear. In these studies, we evaluated mice with cardiac myocyte-specific Mena overexpression (TTA/TgTetMena) comparable to that observed in cardiac pathology. We found that the hearts of TTA/TgTetMena mice were functionally and morphologically comparable to wild-type littermates, except for mildly increased heart mass in the transgenic mice. Interestingly, TTA/TgTetMena mice were particularly susceptible to cardiac injury, as these animals experienced pronounced decreases in ejection fraction and fractional shortening as well as heart dilatation and hypertrophy after transverse aortic constriction (TAC). By "turning off" Mena overexpression in TTA/TgTetMena mice either immediately prior to or immediately after TAC surgery, we discovered that normalizing Mena levels eliminated cardiac hypertrophy in TTA/TgTetMena animals but did not preclude post-TAC cardiac functional deterioration. These findings indicate that hearts with increased levels of Mena fare worse when subjected to cardiac injury and suggest that Mena contributes to HF pathophysiology.
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Proteínas del Citoesqueleto/metabolismo , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/fisiopatología , Miocardio/metabolismo , Disfunción Ventricular Izquierda/fisiopatología , Animales , Insuficiencia Cardíaca/complicaciones , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos , Tamaño de los Órganos , Regulación hacia Arriba , Disfunción Ventricular Izquierda/etiologíaRESUMEN
Epithelial-mesenchymal transition (EMT), whether in developmental morphogenesis or malignant transformation, prominently involves modified cell motility behavior. Although major advances have transpired in understanding the molecular pathways regulating the process of EMT induction per se by certain environmental stimuli, an important outstanding question is how the activities of signaling pathways governing motility yield the diverse movement behaviors characteristic of pre-induction versus postinduction states across a broad landscape of growth factor contexts. For the particular case of EMT induction in human mammary cells by ectopic expression of the transcription factor Twist, we found the migration responses to a panel of growth factors (EGF, HRG, IGF, HGF) dramatically disparate between confluent pre-Twist epithelial cells and sparsely distributed post-Twist mesenchymal cells-but that a computational model quantitatively integrating multiple key signaling node activities could nonetheless account for this full range of behavior. Moreover, motility in both conditions was successfully predicted a priori for an additional growth factor (PDGF) treatment. Although this signaling network state model could comprehend motility behavior globally, modulation of the network interactions underlying the altered pathway activities was identified by ascertaining differences in quantitative topological influences among the nodes between the two conditions.
Asunto(s)
Mama/citología , Movimiento Celular , Péptidos y Proteínas de Señalización Intercelular/fisiología , Proteínas Nucleares/metabolismo , Transducción de Señal , Proteína 1 Relacionada con Twist/metabolismo , Línea Celular , Simulación por Computador , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal , Receptores ErbB/metabolismo , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Análisis de los Mínimos Cuadrados , Modelos Biológicos , Análisis Multivariante , Fenotipo , Fosforilación , Mapas de Interacción de ProteínasRESUMEN
Profilin1, a ubiquitously expressed actin-binding protein, plays a critical role in cell migration through actin cytoskeletal regulation. Given the traditional view of profilin1 as a promigratory molecule, it is difficult to reconcile observations that profilin1 is down-regulated in various invasive adenocarcinomas and that reduced profilin1 expression actually confers increased motility to certain adenocarcinoma cells. In this study, we show that profilin1 negatively regulates lamellipodin targeting to the leading edge in MDA-MB-231 breast cancer cells and normal cells; profilin1 depletion increases lamellipodin concentration at the lamellipodial tip (where it binds Ena/VASP), and this mediates the hypermotility. We report that the molecular mechanism underlying profilin1's modulation of lamellipodin localization relates to phosphoinositide control. Specifically, we show that phosphoinositide binding of profilin1 inhibits the motility of MDA-MB-231 cells by negatively regulating PI(3,4)P(2) at the membrane and thereby limiting recruitment of lamellipodin [a PI(3,4)P(2)-binding protein] and Ena/VASP to the leading edge. In summary, this study uncovers a unique biological consequence of profilin1-phosphoinositide interaction, thus providing direct evidence of profilin1's regulation of cell migration independent of its actin-related activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Proteínas de la Membrana/metabolismo , Fosfatidilinositoles/metabolismo , Profilinas/metabolismo , Proteínas Portadoras/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Humanos , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Profilinas/genéticaRESUMEN
Huntington disease is caused by the expansion of a polyglutamine repeat in the Huntingtin protein (Htt) that leads to degeneration of neurons in the central nervous system and the appearance of visible aggregates within neurons. We have developed and tested suppressor polypeptides that bind mutant Htt and interfere with the process of aggregation in cell culture. In a Drosophila model, the most potent suppressor inhibits both adult lethality and photoreceptor neuron degeneration. The appearance of aggregates in photoreceptor neurons correlates strongly with the occurrence of pathology, and expression of suppressor polypeptides delays and limits the appearance of aggregates and protects photoreceptor neurons. These results suggest that targeting the protein interactions leading to aggregate formation may be beneficial for the design and development of therapeutic agents for Huntington disease.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Péptidos/química , Animales , Western Blotting , Células COS , Línea Celular , Células Cultivadas , ADN Complementario/metabolismo , Drosophila , Epítopos , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Cinética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Microscopía por Video , Datos de Secuencia Molecular , Mutación , Neuronas/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Plásmidos , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Aminoácido , Supresión Genética , Factores de Tiempo , TransfecciónRESUMEN
RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.