RESUMEN
The neurotrophin-3 (NT-3) receptor tropomyosin receptor kinase C (TrkC/NTRK3) has been described as a dependence receptor and, as such, triggers apoptosis in the absence of its ligand NT-3. This proapoptotic activity has been proposed to confer a tumor suppressor activity to this classic tyrosine kinase receptor (RTK). By investigating interacting partners that might facilitate TrkC-induced cell death, we have identified the basic helix-loop-helix (bHLH) transcription factor Hey1 and importin-α3 (karyopherin alpha 4 [KPNA4]) as direct interactors of TrkC intracellular domain, and we show that Hey1 is required for TrkC-induced apoptosis. We propose here that the cleaved proapoptotic portion of TrkC intracellular domain (called TrkC killer-fragment [TrkC-KF]) is translocated to the nucleus by importins and interacts there with Hey1. We also demonstrate that Hey1 and TrkC-KF transcriptionally silence mouse double minute 2 homolog (MDM2), thus contributing to p53 stabilization. p53 transcriptionally regulates the expression of TrkC-KF cytoplasmic and mitochondrial interactors cofactor of breast cancer 1 (COBRA1) and B cell lymphoma 2-associated X (BAX), which will subsequently trigger the intrinsic pathway of apoptosis. Of interest, TrkC was proposed to constrain tumor progression in neuroblastoma (NB), and we demonstrate in an avian model that TrkC tumor suppressor activity requires Hey1 and p53.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neuroblastoma/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Receptor trkC/metabolismo , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Embrión de Pollo , Cromatina/metabolismo , Regulación de la Expresión Génica , Células HCT116 , Células HEK293 , Humanos , Carioferinas/metabolismo , RatonesRESUMEN
The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. The molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic "killer" fragment (TrkC KF). Here, we show that TrkC KF interacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. We also show that, in the developing chick neural tube, NT-3 silencing is associated with neuroepithelial cell death that is rescued by Cobra1 silencing. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes Bax activation, cytochrome c release, and apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1.
Asunto(s)
Apoptosis/fisiología , Mitocondrias/metabolismo , Proteínas Nucleares/metabolismo , Receptor trkC/metabolismo , Animales , Embrión de Pollo/metabolismo , Citocromos c/metabolismo , Citosol/metabolismo , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Silenciador del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Neuronas/metabolismo , Neurotrofina 3/metabolismo , Neurotrofina 3/farmacología , Proteínas Nucleares/genética , Fragmentos de Péptidos/metabolismo , Proteínas de Unión al ARN , Receptor trkC/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The development of the sensory nervous system is the result of fine-tuned waves of neurogenesis and apoptosis which control the appropriate number of precursors and newly generated neurons and orient them toward a specific lineage. Neurotrophins and their tyrosine-kinase receptors (RTK) orchestrate this process. They have long been in the scope of the neurotrophic theory which established that a neuron is committed to die unless a trophic factor generated by its target provides it with a survival signal. The neural death has thus always been described as a "default" program, survival being the major player to control the number of cells. New insights have been brought by the gain of function studies which recently demonstrated that TrkC (NTRK3) is a "dependence receptor" able to actively trigger apoptosis in absence of its ligand NT-3. In order to address the role of TrkC pro-apoptotic activity in the control of sensory neurons number, we generated a TrkC gene-trap mutant mice. We found out that this new murine model recapitulates the sensory phenotype of TrkC constitutive mutants, with reduced DRG size and reduced number of DRG neurons. We engineered these mice strain with a lacZ reporter in order to follow the fate of neurons committed to a TrkC lineage and observed that they are specifically protected from NT-3 mediated apoptosis in NT-3/TrkC double knock-out embryos. Finally, using a chicken model we demonstrated that silencing NT-3 emanating from the ventral neural tube induced apoptosis in the DRG anlage. This apoptosis was inhibited by silencing TrkC. This work thus demonstrates that, during in vivo DRG development, TrkC behaves as a two-sided receptor transducing positive signals of neuronal survival in response to NT-3, but actively inducing neuronal cell death when unbound. This functional duality sets adequate number of neurons committed to a TrkC identity in the forming DRG.
Asunto(s)
Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Receptor trkC/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Animales , Apoptosis/fisiología , Línea Celular , Supervivencia Celular/fisiología , Embrión de Pollo , Femenino , Ganglios Espinales/embriología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismoRESUMEN
The role of deleted in colorectal carcinoma (DCC) as a tumour suppressor has been a matter of debate for the past 15 years. DCC gene expression is lost or markedly reduced in the majority of advanced colorectal cancers and, by functioning as a dependence receptor, DCC has been shown to induce apoptosis unless engaged by its ligand, netrin-1 (ref. 2). However, so far no animal model has supported the view that the DCC loss-of-function is causally implicated as predisposing to aggressive cancer development. To investigate the role of DCC-induced apoptosis in the control of tumour progression, here we created a mouse model in which the pro-apoptotic activity of DCC is genetically silenced. Although the loss of DCC-induced apoptosis in this mouse model is not associated with a major disorganization of the intestines, it leads to spontaneous intestinal neoplasia at a relatively low frequency. Loss of DCC-induced apoptosis is also associated with an increase in the number and aggressiveness of intestinal tumours in a predisposing APC mutant context, resulting in the development of highly invasive adenocarcinomas. These results demonstrate that DCC functions as a tumour suppressor via its ability to trigger tumour cell apoptosis.
Asunto(s)
Progresión de la Enfermedad , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Receptores de Superficie Celular/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Apoptosis/genética , Caspasas/metabolismo , Células Cultivadas , Receptor DCC , Modelos Animales de Enfermedad , Fibroblastos , Silenciador del Gen , Genes APC , Células HEK293 , Humanos , Neoplasias Intestinales/metabolismo , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Factores de Crecimiento Nervioso/deficiencia , Factores de Crecimiento Nervioso/genética , Netrina-1 , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
The TrkC neurotrophin receptor belongs to the functional dependence receptor family, members of which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer tumor-suppressor activity. Indeed, cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC is a classic tyrosine kinase receptor and therefore generally considered to be a proto-oncogene. We show here that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Furthermore, reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation, as well as in vivo tumor growth. Finally, we provide evidence that a mutation of TrkC detected in a sporadic cancer is a loss-of-proapoptotic function mutation. Together, these data support the conclusion that TrkC is a colorectal cancer tumor suppressor.
Asunto(s)
Neoplasias del Colon/genética , Receptor trkC/genética , Apoptosis , Línea Celular Tumoral , Metilación de ADN , Regulación hacia Abajo , Genes Supresores de Tumor , Humanos , Ligandos , Regiones Promotoras Genéticas , Proto-Oncogenes MasRESUMEN
Blood-circulating monocytes migrate in tissues in response to danger stimuli and differentiate there into two major actors of the immune system: macrophages and dendritic cells. Given their migratory behavior and their pivotal role in the orchestration of immune responses, it is not surprising that cells of the monocyte lineage are the target of several viruses, including human immunodeficiency virus type 1 (HIV-1). HIV-1 replicates in monocytoid cells to an extent that is influenced by their differentiation status and modulated by exogenous stimulations. Unstimulated monocytes display a relative resistance to HIV infection mostly exerted during the early steps of the viral life cycle. Despite intensive studies, the identity of the affected step remains controversial, although it is generally assumed to take place after viral entry. We reexamine here the early steps of viral infection of unstimulated monocytes using vesicular stomatitis virus G protein-pseudotyped HIV-1 virions. Our data indicate that a first block to the early steps of infection of monocytes with these particles occurs at the level of viral entry. After entry, reverse transcription and integration proceed with extremely slow kinetics rather than being blocked. Once completed, viral DNA molecules delay entry into the nucleus and integration for up to 5 to 6 days. The inefficacy of these steps accounts for the resistance of monocytes to HIV-1 during the early steps of infection.
Asunto(s)
Infecciones por VIH/fisiopatología , VIH-1 , Monocitos/virología , Internalización del Virus , Cartilla de ADN/genética , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Vpx is a non-structural protein coded by members of the SIVSM/HIV-2 lineage that is believed to have originated by duplication of the common vpr gene present in primate lentiviruses. Vpx is incorporated into virion particles and is thus present during the early steps of viral infection, where it is thought to drive nuclear import of viral nucleoprotein complexes. We have previously shown that Vpx is required for SIVMAC-derived lentiviral vectors (LVs) infection of human monocyte-derived dendritic cells (DCs). However, since the requirement for Vpx is specific for DCs and not for other non-dividing cell types, this suggests that Vpx may play a role other than nuclear import. RESULTS: Here, we show that the function of Vpx in the infection of DCs is conserved exclusively within the SIVSM/HIV-2 lineage. At a molecular level, Vpx acts by promoting the accumulation of full length viral DNA. Furthermore, when supplied in target cells prior to infection, Vpx exerts a similar effect following infection of DCs with retroviruses as divergent as primate and feline lentiviruses and gammaretroviruses. Lastly, the effect of Vpx overlaps with that of the proteasome inhibitor MG132 in DCs. CONCLUSION: Overall, our results support the notion that Vpx modifies the intracellular milieu of target DCs to facilitate lentiviral infection. The data suggest that this is achieved by promoting viral escape from a proteasome-dependent pathway especially detrimental to viral infection in DCs.
Asunto(s)
Células Dendríticas/virología , VIH-2/patogenicidad , Complejo de la Endopetidasa Proteasomal/metabolismo , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Proteínas Reguladoras y Accesorias Virales/metabolismo , Animales , Línea Celular , Células Cultivadas , ADN Viral/metabolismo , Células Dendríticas/citología , VIH-2/metabolismo , Humanos , Cinética , Monocitos/citología , Virus de la Inmunodeficiencia de los Simios/metabolismoRESUMEN
It is commonly accepted that infection of nondividing cells by gammaretroviruses such as the murine leukemia viruses is inefficient due to their inability to cross the nuclear envelope barrier. Challenging this notion, we now show that human nondividing macrophages display a specific window of susceptibility to transduction with a Friend murine leukemia virus (F-MLV)-derived vector during their differentiation from monocytes. This finding suggests that factors other than the nuclear membrane govern permissiveness to gammaretroviral infection and raises the possibility of using the macrophage tropism of F-MLV in gene therapy.
Asunto(s)
Virus de la Leucemia Murina de Friend/genética , Vectores Genéticos , Macrófagos/virología , Transducción Genética , Animales , Secuencia de Bases , Diferenciación Celular , Proliferación Celular , ADN Viral/genética , Terapia Genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células HeLa , Humanos , Técnicas In Vitro , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Virus de la Leucemia Murina de Moloney/genética , Membrana Nuclear/virología , Proteínas RecombinantesRESUMEN
Heterologous lentiviral vectors (LVs) represent a way to address safety concerns in the field of gene therapy by decreasing the possibility of genetic recombination between vector and packaging constructs and the generation of replication-competent viruses. Using described LVs based on human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus MAC251 (SIV(MAC251)), we asked whether heterologous virion particles in which trans-acting factors belonged to HIV-1 and cis elements belonged to SIV(MAC251) (HIV-siv) would behave as parental homologous vectors in all cell types. To our surprise, we found that although the heterologous HIV-siv vector was as infectious as its homologous counterpart in most human cells, it was defective in the transduction of dendritic cells (DCs) and, to a lesser extent, macrophages. In DCs, the main postentry defect was observed in the formation of two-long-terminal-repeat circles, despite the fact that full-length proviral DNA was being synthesized and was associated with the nucleus. Taken together, our data suggest that heterologous HIV-siv vectors display a cell-dependent infectivity defect, most probably at a post-nuclear entry migration step. As homologous HIV and SIV vectors do transduce DCs, we believe that these results underscore the importance of a conserved interaction between cis elements and trans-acting viral factors that is lost or suboptimal in heterologous vectors and essential only in the transduction of certain cell types. For gene therapy purposes, these findings indicate that the cellular tropism of LVs can be modulated not only through the use of distinct envelope proteins or tissue-specific promoters but also through the specific combinatorial use of packaging and transfer vector constructs.