RESUMEN
We have shown previously that phosphorylation of Mdm2 by ATM and c-Abl regulates Mdm2-p53 signaling and alters the effects of DNA damage in mice, including bone marrow failure and tumorigenesis induced by ionizing radiation. Here, we examine the physiological effects of Mdm2 phosphorylation by Akt, another DNA damage effector kinase. Surprisingly, Akt phosphorylation of Mdm2 does not alter the p53-mediated effects of ionizing radiation in cells or mice but regulates the p53 response to oxidative stress. Akt phosphorylation of Mdm2 serine residue 183 increases nuclear Mdm2 stability, decreases p53 levels, and prevents senescence in primary cells exposed to reactive oxidative species (ROS). Using multiple mouse models of ROS-induced cancer, we show that Mdm2 phosphorylation by Akt reduces senescence to promote KrasG12D-driven lung cancers and carcinogen-induced papilloma and hepatocellular carcinomas. Collectively, we document a unique physiologic role for Akt-Mdm2-p53 signaling in regulating cell growth and tumorigenesis in response to oxidative stress.
Asunto(s)
Neoplasias Inducidas por Radiación/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/efectos de la radiación , Carcinógenos/toxicidad , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , Proliferación Celular/genética , Senescencia Celular/efectos de los fármacos , Senescencia Celular/efectos de la radiación , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Modelos Animales de Enfermedad , Humanos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Neoplasias Inducidas por Radiación/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Papillomaviridae/patogenicidad , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Radiación Ionizante , Especies Reactivas de Oxígeno/metabolismoRESUMEN
During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.
Asunto(s)
Células Madre Embrionarias de Ratones/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Inactivación del Cromosoma X/genética , Animales , Técnicas de Cultivo de Célula , Femenino , Ratones , Proteínas Mutantes/metabolismoRESUMEN
The oncoprotein Mdm2 is a RING domain-containing E3 ubiquitin ligase that ubiquitinates G protein-coupled receptor kinase 2 (GRK2) and ß-arrestin2, thereby regulating ß-adrenergic receptor (ßAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects ßAR signaling and cardiac function in adult mice. Using Mdm2/p53-KO mice, which survive for 9-12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac ß1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53-KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53-KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53-KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53-KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53-KO was impaired. Mdm2/p53-KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53-KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable ßAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued ß1AR-induced cardiac contractility in Mdm2/p53-KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of ßAR signaling.
Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Contracción Miocárdica/fisiología , Proteínas Proto-Oncogénicas c-mdm2/fisiología , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal , Agonistas Adrenérgicos beta/farmacología , Animales , Ecocardiografía , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Corazón/diagnóstico por imagen , Corazón/fisiología , Hemodinámica/efectos de los fármacos , Isoproterenol/farmacología , Ratones , Ratones Noqueados , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , UbiquitinaciónRESUMEN
The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2S394A mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2Y393F) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/química , Tolerancia a Radiación , Proteína p53 Supresora de Tumor/metabolismo , Alelos , Animales , Apoptosis , Daño del ADN , Exones , Femenino , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/genética , Neoplasias/radioterapia , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de SeñalRESUMEN
Fidelity of histone gene expression is important for normal cell growth and differentiation that is stringently controlled during development but is compromised during tumorigenesis. Efficient production of histones for packaging newly replicated DNA is particularly important for proper cell division and epigenetic control during the initial pre-implantation stages of embryonic development. Here, we addressed the unresolved question of when the machinery for histone gene transcription is activated in the developing zygote to accommodate temporal demands for histone gene expression. We examined induction of Histone Nuclear Factor P (HINFP), the only known transcription factor required for histone H4 gene expression, that binds directly to a unique H4 promoter-specific element to regulate histone H4 transcription. We show that Hinfp gene transcripts are stored in oocytes and maternally transmitted to the zygote. Transcripts from the paternal Hinfp gene, which reflect induction of zygotic gene expression, are apparent at the 4- to 8-cell stage, when most maternal mRNA pools are depleted. Loss of Hinfp expression due to gene ablation reduces cell numbers in E3.5 stage embryos and compromises implantation. Reduced cell proliferation is attributable to severe reduction in histone mRNA levels accompanied by reduced cell survival and genomic damage as measured by cleaved Caspase 3 and phospho-H2AX staining, respectively. We conclude that transmission of maternal Hinfp transcripts and zygotic activation of the Hinfp gene together are necessary to control H4 gene expression in early pre-implantation embryos in order to support normal embryonic development.
Asunto(s)
Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Histonas/biosíntesis , ARN Mensajero Almacenado/genética , Proteínas Represoras/fisiología , Cigoto/metabolismo , Animales , Blastocisto/fisiología , Caspasa 3/metabolismo , Implantación del Embrión/fisiología , Desarrollo Embrionario/fisiología , Femenino , Genes Reporteros , Histonas/genética , Histonas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas Represoras/deficiencia , Proteínas Represoras/genéticaRESUMEN
ATM phosphorylation of Mdm2-S394 is required for robust p53 stabilization and activation in DNA-damaged cells. We have now utilized Mdm2(S394A) knockin mice to determine that phosphorylation of Mdm2-S394 regulates p53 activity and the DNA damage response in lymphatic tissues in vivo by modulating Mdm2 stability. Mdm2-S394 phosphorylation delays lymphomagenesis in Eµ-myc transgenic mice, and preventing Mdm2-S394 phosphorylation obviates the need for p53 mutation in Myc-driven tumorigenesis. However, irradiated Mdm2(S394A) mice also have increased hematopoietic stem and progenitor cell functions, and we observed decreased lymphomagenesis in sub-lethally irradiated Mdm2(S394A) mice. These findings document contrasting effects of ATM-Mdm2 signaling on p53 tumor suppression and reveal that destabilizing Mdm2 by promoting its phosphorylation by ATM would be effective in treating oncogene-induced malignancies, while inhibiting Mdm2-S394 phosphorylation during radiation exposure or chemotherapy would ameliorate bone marrow failure and prevent the development of secondary hematological malignancies.
Asunto(s)
Carcinogénesis/metabolismo , Carcinogénesis/efectos de la radiación , Oncogenes , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Radiación Ionizante , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Médula Ósea/patología , Médula Ósea/efectos de la radiación , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/efectos de la radiación , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , Tejido Linfoide/efectos de la radiación , Ratones Transgénicos , Fosforilación/efectos de la radiación , Fosfoserina/metabolismo , Estabilidad Proteica/efectos de la radiación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Tolerancia a Radiación/efectos de la radiación , Transducción de Señal/efectos de la radiación , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The p53 tumor suppressor acts as a guardian of the genome in mammalian cells undergoing DNA double strand breaks induced by a various forms of cell stress, including inappropriate growth signals or ionizing radiation. Following damage, p53 protein levels become greatly elevated in cells and p53 functions primarily as a transcription factor to regulate the expression a wide variety of genes that coordinate this DNA damage response. In cells undergoing high amounts of DNA damage, p53 can promote apoptosis, whereas in cells undergoing less damage, p53 promotes senescence or transient cell growth arrest and the expression of genes involved in DNA repair, depending upon the cell type and level of damage. Failure of the damaged cell to undergo growth arrest or apoptosis, or to respond to the DNA damage by other p53-coordinated mechanisms, can lead to inappropriate cell growth and tumorigenesis. In cells that have successfully responded to genetic damage, the amount of p53 present in the cell must return to basal levels in order for the cell to resume normal growth and function. Although regulation of p53 levels and function is coordinated by many proteins, it is now widely accepted that the master regulator of p53 is Mdm2. In this review, we discuss the role(s) of p53 in the DNA damage response and in tumor suppression, and how post-translational modification of Mdm2 regulates the Mdm2-p53 signaling axis to govern p53 activities in the cell.
RESUMEN
Histone Nuclear Factor P (HINFP) is essential for expression of histone H4 genes. Ablation of Hinfp and consequential depletion of histones alter nucleosome spacing and cause stalled replication and DNA damage that ultimately result in genomic instability. Faithful replication and packaging of newly replicated DNA are required for normal cell cycle control and proliferation. The tumor suppressor protein p53, the guardian of the genome, controls multiple cell cycle checkpoints and its loss leads to cellular transformation. Here we addressed whether the absence of p53 impacts the outcomes/consequences of Hinfp-mediated histone H4 deficiency. We examined mouse embryonic fibroblasts lacking both Hinfp and p53. Our data revealed that the reduced histone H4 expression caused by depletion of Hinfp persists when p53 is also inactivated. Loss of p53 enhanced the abnormalities in nuclear shape and size (i.e. multi-lobed irregularly shaped nuclei) caused by Hinfp depletion and also altered the sub-nuclear organization of Histone Locus Bodies (HLBs). In addition to the polyploid phenotype resulting from deletion of either p53 or Hinfp, inactivation of both p53 and Hinfp increased mitotic defects and generated chromosomal fragility and susceptibility to DNA damage. Thus, our study conclusively establishes that simultaneous loss of both Hinfp and the p53 checkpoint is detrimental to normal cell growth and may predispose to cellular transformation.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Daño del ADN/genética , Histonas/biosíntesis , Proteínas Represoras/genética , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Fragilidad Cromosómica/genética , Replicación del ADN/genética , Fibroblastos/citología , Inestabilidad Genómica/genética , Histonas/genética , Ratones , Ratones NoqueadosRESUMEN
TOP mRNAs encode components of the translational apparatus, and repression of their translation comprises one mechanism, by which cells encountering amino acid deprivation downregulate the biosynthesis of the protein synthesis machinery. This mode of regulation involves TSC as knockout of TSC1 or TSC2 rescued TOP mRNAs translation in amino acid-starved cells. The involvement of mTOR in translational control of TOP mRNAs is demonstrated by the ability of constitutively active mTOR to relieve the translational repression of TOP mRNA upon amino acid deprivation. Consistently, knockdown of this kinase as well as its inhibition by pharmacological means blocked amino acid-induced translational activation of these mRNAs. The signaling of amino acids to TOP mRNAs involves RagB, as overexpression of active RagB derepressed the translation of these mRNAs in amino acid-starved cells. Nonetheless, knockdown of raptor or rictor failed to suppress translational activation of TOP mRNAs by amino acids, suggesting that mTORC1 or mTORC2 plays a minor, if any, role in this mode of regulation. Finally, miR10a has previously been suggested to positively regulate the translation of TOP mRNAs. However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs. Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation. Evidently, these results are questioning the positive role of microRNAs in this mode of regulation.
Asunto(s)
MicroARNs/genética , Complejos Multiproteicos/genética , Biosíntesis de Proteínas , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Proteínas Supresoras de Tumor/genética , Aminoácidos/genética , Animales , Regulación de la Expresión Génica , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Noqueados , Fosforilación , ARN Mensajero/genética , Proteína 1 del Complejo de la Esclerosis TuberosaRESUMEN
Dicer is required for the maturation of microRNA, and loss of Dicer and miRNA processing has been found to alter numerous biological events during embryogenesis, including the development of mammalian skin and hair. We have previously examined the role of miRNA biogenesis in mouse embryonic fibroblasts and found that deletion of Dicer induces cell senescence regulated, in part, by the p53 tumor suppressor. Although Dicer and miRNA molecules are thought to have either oncogenic or tumor suppressing roles in various types of cancer, a role for Dicer and miRNAs in skin carcinogenesis has not been established. Here we show that perinatal ablation of Dicer in the skin of mice leads to loss of fur in adult mice, increased epidermal cell proliferation and apoptosis, and the accumulation of widespread DNA damage in epidermal cells. Co-ablation of Dicer and p53 did not alter the timing or extent of fur loss, but greatly reduced survival of Dicer-skin ablated mice, as these mice developed multiple and highly aggressive skin carcinomas. Our results describe a new mouse model for spontaneous basal and squamous cell tumorigenesis. Furthermore, our findings reveal that loss of Dicer in the epidermis induces extensive DNA damage, activation of the DNA damage response and p53-dependent apoptosis, and that Dicer and p53 cooperate to suppress mammalian skin carcinogenesis.
Asunto(s)
Carcinogénesis/genética , Carcinoma Basocelular/genética , Carcinoma de Células Escamosas/genética , ARN Helicasas DEAD-box/genética , Regulación Neoplásica de la Expresión Génica , Ribonucleasa III/genética , Neoplasias Cutáneas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Animales Recién Nacidos , Apoptosis , Carcinogénesis/metabolismo , Carcinogénesis/patología , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/mortalidad , Carcinoma Basocelular/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , ARN Helicasas DEAD-box/metabolismo , Daño del ADN , Feto , Regulación del Desarrollo de la Expresión Génica , Cabello/metabolismo , Cabello/patología , Ratones , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Ribonucleasa III/metabolismo , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Fidelity of chromatin organization is crucial for normal cell cycle progression, and perturbations in packaging of DNA may predispose to transformation. Histone H4 protein is the most highly conserved chromatin protein, required for nucleosome assembly, with multiple histone H4 gene copies encoding identical protein. There is a long-standing recognition of the linkage of histone gene expression and DNA replication. A fundamental and unresolved question is the mechanism that couples histone biosynthesis with DNA replication and fidelity of cell cycle control. Here, we conditionally ablated the obligatory histone H4 transcription factor HINFP to cause depletion of histone H4 in mammalian cells. Deregulation of histone H4 results in catastrophic cellular and molecular defects that lead to genomic instability. Histone H4 depletion increases nucleosome spacing, impedes DNA synthesis, alters chromosome complement, and creates replicative stress. Our study provides functional evidence that the tight coupling between DNA replication and histone synthesis is reciprocal.
Asunto(s)
Replicación del ADN , Inestabilidad Genómica , Histonas/genética , Proteínas Represoras/genética , Animales , Ciclo Celular , Línea Celular , Proliferación Celular , Daño del ADN , Epigénesis Genética , Histonas/metabolismo , Ratones , Ratones Noqueados , Nucleosomas/metabolismoRESUMEN
In female mice, two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X chromosome. Later, around implantation, epiblast cells of the inner cell mass that give rise to the embryo reactivate the paternal X chromosome and undergo a random form of XCI (rXCI). Xist, a long non-coding RNA crucial for both forms of XCI, is activated by the ubiquitin ligase RLIM (also known as Rnf12). Although RLIM is required for triggering iXCI in mice, its importance for rXCI has been controversial. Here we show that RLIM levels are downregulated in embryonic cells undergoing rXCI. Using mouse genetics we demonstrate that female cells lacking RLIM from pre-implantation stages onwards show hallmarks of XCI, including Xist clouds and H3K27me3 foci, and have full embryogenic potential. These results provide evidence that RLIM is dispensable for rXCI, indicating that in mice an RLIM-independent mechanism activates Xist in the embryo proper.
Asunto(s)
Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Inactivación del Cromosoma X/genética , Animales , Regulación hacia Abajo , Implantación del Embrión , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Histonas/química , Histonas/metabolismo , Hibridación Fluorescente in Situ , Lisina/metabolismo , Metilación , Ratones , Ratones Noqueados , ARN Largo no Codificante/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Cells encountering hypoxic stress conserve resources and energy by downregulating the protein synthesis. Here we demonstrate that one mechanism in this response is the translational repression of TOP mRNAs that encode components of the translational apparatus. This mode of regulation involves TSC and Rheb, as knockout of TSC1 or TSC2 or overexpression of Rheb rescued TOP mRNA translation in oxygen-deprived cells. Stress-induced translational repression of these mRNAs closely correlates with the hypophosphorylated state of 4E-BP, a translational repressor. However, a series of 4E-BP loss- and gain-of-function experiments disprove a cause-and-effect relationship between the phosphorylation status of 4E-BP and the translational repression of TOP mRNAs under oxygen or growth factor deprivation. Furthermore, the repressive effect of anoxia is similar to that attained by the very efficient inhibition of mTOR activity by Torin 1, but much more pronounced than raptor or rictor knockout. Likewise, deficiency of raptor or rictor, even though it mildly downregulated basal translation efficiency of TOP mRNAs, failed to suppress the oxygen-mediated translational activation of TOP mRNAs. Finally, co-knockdown of TIA-1 and TIAR, two RNA-binding proteins previously implicated in translational repression of TOP mRNAs in amino acid-starved cells, failed to relieve TOP mRNA translation under other stress conditions. Thus, the nature of the proximal translational regulator of TOP mRNAs remains elusive.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Oxígeno/metabolismo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas , Secuencia de Oligopirimidina en la Región 5' Terminal del ARN/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Aminoácidos/deficiencia , Aminoácidos/metabolismo , Proteínas de Ciclo Celular , Ciclina D3/metabolismo , Factores Eucarióticos de Iniciación , Células HEK293 , Humanos , Fosforilación , Proteína Asociada al mTOR Insensible a la Rapamicina , Proteína Reguladora Asociada a mTOR , Transducción de Señal , Estrés Fisiológico , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/deficienciaRESUMEN
About half of patients with diffuse large B-cell lymphoma (DLBCL) do not respond to or relapse soon after the standard chemotherapy, indicating a critical need to better understand the specific pathways perturbed in DLBCL for developing effective therapeutic approaches. Mice deficient in the E3 ubiquitin ligase Smurf2 spontaneously develop B-cell lymphomas that resemble human DLBCL with molecular features of germinal centre or post-germinal centre B cells. Here we show that Smurf2 mediates ubiquitination and degradation of YY1, a key germinal centre transcription factor. Smurf2 deficiency enhances YY1-mediated transactivation of c-Myc and B-cell proliferation. Furthermore, Smurf2 expression is significantly decreased in primary human DLBCL samples, and low levels of Smurf2 expression correlate with inferior survival in DLBCL patients. The Smurf2-YY1-c-Myc regulatory axis represents a novel pathway perturbed in DLBCL that suppresses B-cell proliferation and lymphomagenesis, suggesting pharmaceutical targeting of Smurf2 as a new therapeutic paradigm for DLBCL.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/genética , Proteínas Proto-Oncogénicas c-myc/genética , Ubiquitina-Proteína Ligasas/genética , Factor de Transcripción YY1/genética , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular Tumoral , Proliferación Celular , Centro Germinal/metabolismo , Centro Germinal/patología , Humanos , Activación de Linfocitos , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Transcripción Genética , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Factor de Transcripción YY1/metabolismoRESUMEN
Expression of microRNAs (miRNAs) involves transcription of miRNA genes and maturation of the primary transcripts. Recent studies have shown that posttranscriptional processing of primary and precursor miRNAs is induced after DNA damage through regulatory RNA-binding proteins in the Drosha and Dicer complexes, such as DDX5 and KSRP. However, little is known about the regulation of nuclear export of pre-miRNAs in the DNA-damage response, a critical step in miRNA maturation. Here, we show that nuclear export of pre-miRNAs is accelerated after DNA damage in an ATM-dependent manner. The ATM-activated AKT kinase phosphorylates Nup153, a key component of the nucleopore, leading to enhanced interaction between Nup153 and Exportin-5 (XPO5) and increased nuclear export of pre-miRNAs. These findings define an important role of DNA-damage signaling in miRNA transport and maturation.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células HCT116 , Humanos , Carioferinas/genética , Carioferinas/metabolismo , MicroARNs/biosíntesis , MicroARNs/genética , Datos de Secuencia Molecular , Mutación , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3'-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.
Asunto(s)
Neoplasias Óseas/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , MicroARNs/metabolismo , Osteosarcoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Regulación hacia Abajo/genética , Regulación hacia Abajo/efectos de la radiación , Rayos gamma , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Ratones , Osteosarcoma/genética , Osteosarcoma/patología , Estabilidad Proteica/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
The p53 transcription factor regulates the expression of numerous genes whose products affect cell proliferation, senescence, cellular metabolism, apoptosis, and DNA repair. These p53-mediated effects can inhibit the growth of stressed or mutated cells and suppress tumorigenesis in the organism. However, the various growth-inhibitory properties of p53 must be kept in check in nondamaged cells in order to facilitate proper embryogenesis or the homeostatic maintenance of adult tissues. This requisite inhibition of p53 is performed primarily by the MDM oncoproteins, Mdm2 and MdmX. These p53-binding proteins limit p53 activity both in normal cells and in stressed cells seeking to promote resolution of their p53-stress response. Many mouse models bearing genetic alterations in Mdm2 or MdmX have been generated to explore the function and regulation of MDM-p53 signaling in development, in tissue homeostasis, in aging, and in cancer. These models not only have demonstrated a critical need for Mdm2 and MdmX in normal cell growth and in development but more recently have identified the MDM-p53 signaling axis as a key regulator of the cellular response to a wide variety of genetic or metabolic stresses. In this review, we discuss what has been learned from various studies of these Mdm2 and MdmX mouse models and highlight a few of the many important remaining questions.
RESUMEN
Aurora A is a mitotic kinase essential for cell proliferation. In mice, ablation of Aurora A results in mitotic arrest and pre-implantation lethality, preventing studies at later stages of development. Here we report the effects of Aurora A ablation on embryo patterning at early post-implantation stages. Inactivation of Aurora A in the epiblast or visceral endoderm layers of the conceptus leads to apoptosis and inhibition of embryo growth, causing lethality and resorption at approximately E9.5. The effects on embryo patterning, however, depend on the tissue affected by the mutation. Embryos with an epiblast ablation of Aurora A properly establish the anteroposterior axis but fail to progress through gastrulation. In contrast, mutation of Aurora A in the visceral endoderm, leads to posteriorization of the conceptus or failure to elongate the anteroposterior axis. Injection of ES cells into Aurora A epiblast knockout blastocysts reconstitutes embryonic development to E9.5, indicating that the extra-embryonic tissues in these mutant embryos can sustain development to organogenesis stages. Our results reveal new ways to induce apoptosis and to ablate cells in a tissue-specific manner in vivo. Moreover, they show that epiblast-ablated embryos can be used to test the potency of stem cells.
Asunto(s)
Tipificación del Cuerpo/genética , Embrión de Mamíferos/embriología , Endodermo/embriología , Estratos Germinativos/embriología , Proteínas Serina-Treonina Quinasas/deficiencia , Animales , Apoptosis/genética , Aurora Quinasa A , Aurora Quinasas , Cartilla de ADN/genética , Células Madre Embrionarias/metabolismo , Técnica del Anticuerpo Fluorescente , Técnicas de Inactivación de Genes , Hibridación in Situ , Ratones , Proteínas Serina-Treonina Quinasas/genética , beta-GalactosidasaRESUMEN
The E3 ubiquitin ligase Smurf2 mediates ubiquitination and degradation of several protein targets involved in tumorigenesis and induces senescence in human cells. However, the functional role of Smurf2 in tumorigenesis has not been fully evaluated. In this study, we generated a mouse model of Smurf2 deficiency to characterize the function of this E3 ligase in tumorigenesis. Smurf2 deficiency attenuated p16 expression and impaired the senescence response of primary mouse embryonic fibroblasts. In support of a functional role in controlling cancer, Smurf2 deficiency increased the susceptibility of mice to spontaneous tumorigenesis, most notably B-cell lymphoma. At a premalignant stage of tumorigenesis, we documented a defective senescence response in the spleens of Smurf2-deficient mice, consistent with a mechanistic link between impaired senescence regulation and increased tumorigenesis. Taken together, our findings offer the genetic evidence of an important tumor suppressor function for Smurf2.
Asunto(s)
Envejecimiento , Proteínas Supresoras de Tumor/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Proteína 1 Inhibidora de la Diferenciación/análisis , Linfoma de Células B/etiología , Ratones , Ratones Endogámicos C57BL , Ubiquitina-Proteína Ligasas/deficienciaRESUMEN
DNA damage induced by ionizing radiation activates the ATM kinase, which subsequently stabilizes and activates the p53 tumor suppressor protein. Although phosphorylation of p53 by ATM was found previously to modulate p53 levels and transcriptional activities in vivo, it does not appear to be a major regulator of p53 stability. We have utilized mice bearing altered Mdm2 alleles to demonstrate that ATM phosphorylation of Mdm2 serine 394 is required for robust p53 stabilization and activation after DNA damage. In addition, we demonstrate that dephosphorylation of Mdm2 Ser394 regulates attenuation of the p53-mediated response to DNA damage. Therefore, the phosphorylation status of Mdm2 Ser394 governs p53 protein levels and functions in cells undergoing DNA damage.