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2.
Proc Natl Acad Sci U S A ; 112(23): 7201-6, 2015 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-26015568

RESUMO

B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) is a BCL-2 family protein with high homology to the multidomain proapoptotic proteins BAX and BAK, yet Bok(-/-) and even Bax(-/-)Bok(-/-) and Bak(-/-)Bok(-/-) mice were reported to have no overt phenotype or apoptotic defects in response to a host of classical stress stimuli. These surprising findings were interpreted to reflect functional compensation among the BAX, BAK, and BOK proteins. However, BOK cannot compensate for the severe apoptotic defects of Bax(-/-)Bak(-/-) mice despite its widespread expression. Here, we independently developed Bok(-/-) mice and found that Bok(-/-) cells are selectively defective in their response to endoplasmic reticulum (ER) stress stimuli, consistent with the predominant subcellular localization of BOK at the ER. Whereas Bok(-/-) mouse embryonic fibroblasts exposed to thapsigargin, A23187, brefeldin A, DTT, geldanamycin, or bortezomib manifested reduced activation of the mitochondrial apoptotic pathway, the death response to other stimuli such as etoposide, staurosporine, or UV remained fully intact. Multiple organs in Bok(-/-) mice exhibited resistance to thapsigargin-induced apoptosis in vivo. Although the ER stress agents activated the unfolded protein response, both ATF4 and CHOP activation were diminished in Bok(-/-) cells and mice. Importantly, BAX and BAK were unable to compensate for the defective apoptotic response to ER stress observed in SV40-transformed and primary Bok(-/-) cells, and in vivo. These findings support a selective and distinguishing role for BOK in regulating the apoptotic response to ER stress, revealing--to our knowledge--the first bona fide apoptotic defect linked to Bok deletion.


Assuntos
Apoptose/fisiologia , Retículo Endoplasmático/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Fator 4 Ativador da Transcrição/metabolismo , Animais , Anexina A5/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Cultivadas , Retículo Endoplasmático/enzimologia , Ativação Enzimática , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fator de Transcrição CHOP/metabolismo
3.
Cell Metab ; 19(2): 272-84, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24506868

RESUMO

The homeostatic balance of hepatic glucose utilization, storage, and production is exquisitely controlled by hormonal signals and hepatic carbon metabolism during fed and fasted states. How the liver senses extracellular glucose to cue glucose utilization versus production is not fully understood. We show that the physiologic balance of hepatic glycolysis and gluconeogenesis is regulated by Bcl-2-associated agonist of cell death (BAD), a protein with roles in apoptosis and metabolism. BAD deficiency reprograms hepatic substrate and energy metabolism toward diminished glycolysis, excess fatty acid oxidation, and exaggerated glucose production that escapes suppression by insulin. Genetic and biochemical evidence suggests that BAD's suppression of gluconeogenesis is actuated by phosphorylation of its BCL-2 homology (BH)-3 domain and subsequent activation of glucokinase. The physiologic relevance of these findings is evident from the ability of a BAD phosphomimic variant to counteract unrestrained gluconeogenesis and improve glycemia in leptin-resistant and high-fat diet models of diabetes and insulin resistance.


Assuntos
Metabolismo Energético/fisiologia , Gluconeogênese/fisiologia , Fígado/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Animais , Metabolismo Energético/genética , Gluconeogênese/genética , Camundongos , Camundongos Mutantes , Fosforilação , Proteína de Morte Celular Associada a bcl/genética
4.
Blood ; 123(6): 884-93, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24352880

RESUMO

Mantle cell lymphoma (MCL) is a highly aggressive B-cell lymphoma resistant to conventional chemotherapy. Although defined by the characteristic t(11;14) translocation, MCL has not been recapitulated in transgenic mouse models of cyclin D1 overexpression alone. Indeed, several genetic aberrations have been identified in MCL that may contribute to its pathogenesis and chemoresistance. Of particular interest is the frequent biallelic deletion of the proapoptotic BCL-2 family protein BIM. BIM exerts its pro-death function via its α-helical BH3 death domain that has the dual capacity to inhibit antiapoptotic proteins such as BCL-2 and MCL-1 and directly trigger proapoptotic proteins such as the mitochondrial executioner protein BAX. To evaluate a functional role for Bim deletion in the pathogenesis of MCL, we generated cyclin D1-transgenic mice harboring Bim-deficient B cells. In response to immunization, Eµ(CycD1)CD19(CRE)Bim(fl/fl) mice manifested selective expansion of their splenic mantle zone compartment. Three distinct immune stimulation regimens induced lymphomas with histopathologic and molecular features of human MCL in a subset of mice. Thus, deletion of Bim in B cells, in the context of cyclin D1 overexpression, disrupts a critical control point in lymphoid maturation and predisposes to the development of MCL. This genetic proof of concept for MCL pathogenesis suggests an opportunity to reactivate the death pathway by pharmacologic mimicry of proapoptotic BIM.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Linfócitos B/patologia , Ciclina D1/fisiologia , Linfoma de Célula do Manto/patologia , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Linfócitos B/metabolismo , Proteína 11 Semelhante a Bcl-2 , Ciclo Celular , Feminino , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Imunofenotipagem , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
5.
Neuron ; 74(4): 719-30, 2012 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-22632729

RESUMO

Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phosphoregulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive K(ATP) channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the K(ATP) channel, implicating the BAD-K(ATP) axis in metabolic control of neuronal excitation and seizure responses.


Assuntos
Metabolismo Energético/fisiologia , Hipocampo/metabolismo , Canais KATP/metabolismo , Convulsões/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Animais , Apoptose/fisiologia , Astrócitos/metabolismo , Células Cultivadas , Eletroencefalografia , Hipocampo/fisiopatologia , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Consumo de Oxigênio/fisiologia , Fosforilação , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Proteína de Morte Celular Associada a bcl/genética
6.
J Clin Invest ; 122(6): 2018-31, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22622039

RESUMO

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2-interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence-specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.


Assuntos
Proteínas Reguladoras de Apoptose/farmacologia , Apoptose/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteínas de Membrana/farmacologia , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , Animais , Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Transplante de Neoplasias , Estrutura Secundária de Proteína , Transplante Heterólogo
7.
J Clin Invest ; 120(10): 3673-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20890041

RESUMO

Apoptosis of motor neurons is a well-documented feature in amyotrophic lateral sclerosis (ALS) and related motor neuron diseases (MNDs). However, the role of apoptosis in the pathogenesis of these diseases remains unresolved. One possibility is that the affected motor neurons only succumb to apoptosis once they have exhausted functional capacity. If true, blocking apoptosis should confer no therapeutic benefit. To directly investigate this idea, we tested whether tissue-specific deletion in the mouse CNS of BCL2-associated X protein (BAX) and BCL2-homologous antagonist/killer (BAK), 2 proapoptotic BCL-2 family proteins that together represent an essential gateway to the mitochondrial apoptotic pathway, would protect against motor neuron degeneration. We found that neuronal deletion of Bax and Bak in a mouse model of familial ALS not only halted neuronal loss, but prevented axonal degeneration, symptom onset, weight loss, and paralysis and extended survival. These results show that motor neurons damaged in ALS activate the mitochondrial apoptotic pathway early in the disease process and that apoptotic signaling directly contributes to neuromuscular degeneration and neuronal dysfunction. Hence, inhibiting apoptosis upstream of mitochondrial permeabilization represents a possible therapeutic strategy for preserving functional motor neurons in ALS and other MNDs.


Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Apoptose , Mitocôndrias/fisiologia , Neurônios Motores/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Proteína X Associada a bcl-2/fisiologia , Esclerose Lateral Amiotrófica/terapia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Superóxido Dismutase/fisiologia , Superóxido Dismutase-1 , Proteína Killer-Antagonista Homóloga a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/antagonistas & inibidores
8.
Sci Signal ; 2(85): ra48, 2009 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-19706873

RESUMO

The proapoptotic proteins BAX and BAK constitute the mitochondrial apoptotic gateway that executes cellular demise after integrating death signals. The lethal BAK is kept in check by voltage-dependent anion channel 2 (VDAC2), a mammalian-restricted VDAC isoform. Here, we provide evidence showing a critical role for the VADC2-BAK complex in determining thymocyte survival in vivo. Genetic depletion of Vdac2 in the thymus resulted in excessive cell death and hypersensitivity to diverse death stimuli including engagement of the T cell receptor. These phenotypes were completely rescued by the concurrent deletion of Bak but not that of Bax. Thus, the VDAC2-BAK axis provides a mechanism that governs the homeostasis of thymocytes. Our study reveals a sophisticated built-in rheostat that likely fine-tunes immune competence to balance autoimmunity and immunodeficiency.


Assuntos
Deleção Clonal/fisiologia , Linfócitos T/citologia , Canal de Ânion 2 Dependente de Voltagem/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Apoptose/genética , Apoptose/fisiologia , Autoimunidade/genética , Autoimunidade/fisiologia , Complexo CD3/imunologia , Deleção Clonal/genética , Dimerização , Feminino , Técnicas de Inativação de Genes , Genótipo , Transporte de Íons/genética , Transporte de Íons/fisiologia , Masculino , Camundongos , Camundongos Knockout , Membranas Mitocondriais/fisiologia , Timo/citologia , Canal de Ânion 2 Dependente de Voltagem/deficiência , Canal de Ânion 2 Dependente de Voltagem/genética , Proteína Killer-Antagonista Homóloga a bcl-2/deficiência , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/fisiologia
9.
Blood ; 113(12): 2805-15, 2009 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-19064728

RESUMO

During hematopoiesis, myeloid cell leukemia-1 (MCL-1) mediates the survival of bone marrow progenitors and lymphocytes. However, its requirement during myeloid cell differentiation, development, and effector function is less clear. Lineage-specific deletion of MCL-1 in myeloid precursors results in neutropenia due to death during differentiation. The loss of mature neutrophils induced by Mcl-1 deletion was not rescued by genetic deletion of proapoptotic Bim and Puma or by exogenous cytokine treatment. However, blockade of intrinsic apoptosis by lineage-specific deletion of both multidomain proapoptotics Bax and Bak was capable of rescuing the neutropenia associated with Mcl-1 deletion. In the monocytic lineage, despite efficient Mcl-1 deletion, monocytes and macrophages undergo normal development. During the phagocytosis of extracellular bacteria, macrophages concomitantly increase the expression of both MCL-1 and BIM. However, Mcl-1-deficient macrophages exhibit increased sensitivity to death during bacterial phagocytosis that can be abolished by codeletion of Bim. These data suggest that MCL-1 may be necessary to antagonize BIM during macrophage effector responses. Thus, MCL-1 plays selective roles in myeloid development, being required for neutrophil development and setting the threshold for apoptosis during a macrophage effector response.


Assuntos
Granulócitos/citologia , Ativação de Macrófagos/fisiologia , Macrófagos Peritoneais/citologia , Mielopoese/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2 , Células da Medula Óssea/citologia , Escherichia coli , Filgrastim , Deleção de Genes , Fator Estimulador de Colônias de Granulócitos/farmacologia , Macrófagos Peritoneais/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Sequência 1 de Leucemia de Células Mieloides , Mielopoese/efeitos dos fármacos , Especificidade de Órgãos , Fagocitose , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Recombinantes , Organismos Livres de Patógenos Específicos , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/deficiência , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Proteína X Associada a bcl-2/deficiência , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/fisiologia
10.
Nat Med ; 14(2): 144-53, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18223655

RESUMO

The proapoptotic BCL-2 family member BAD resides in a glucokinase-containing complex that regulates glucose-driven mitochondrial respiration. Here, we present genetic evidence of a physiologic role for BAD in glucose-stimulated insulin secretion by beta cells. This novel function of BAD is specifically dependent upon the phosphorylation of its BH3 sequence, previously defined as an essential death domain. We highlight the pharmacologic relevance of phosphorylated BAD BH3 by using cell-permeable, hydrocarbon-stapled BAD BH3 helices that target glucokinase, restore glucose-driven mitochondrial respiration and correct the insulin secretory response in Bad-deficient islets. Our studies uncover an alternative target and function for the BAD BH3 domain and emphasize the therapeutic potential of phosphorylated BAD BH3 mimetics in selectively restoring beta cell function. Furthermore, we show that BAD regulates the physiologic adaptation of beta cell mass during high-fat feeding. Our findings provide genetic proof of the bifunctional activities of BAD in both beta cell survival and insulin secretion.


Assuntos
Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Sequência de Aminoácidos , Animais , Glicemia , Cálcio/metabolismo , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Dieta , Glucoquinase/metabolismo , Glucose/farmacologia , Humanos , Hidrocarbonetos/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/enzimologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Peptídeos/farmacologia , Fosfosserina/metabolismo , Estrutura Terciária de Proteína , Proteína de Morte Celular Associada a bcl/química , Proteína de Morte Celular Associada a bcl/deficiência
11.
Cell Stem Cell ; 1(3): 324-37, 2007 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-18371366

RESUMO

The Mixed Lineage Leukemia (MLL) gene is essential for embryonic hematopoietic stem cell (HSC) development, but its role during adult hematopoiesis is unknown. Using an inducible knockout model, we demonstrate that Mll is essential for the maintenance of adult HSCs and progenitors, with fatal bone marrow failure occurring within 3 weeks of Mll deletion. Mll-deficient cells are selectively lost from mixed bone marrow chimeras, demonstrating their failure to self-renew even in an intact bone marrow environment. Surprisingly, HSCs lacking Mll exhibit ectopic cell-cycle entry, resulting in the depletion of quiescent HSCs. In contrast, Mll deletion in myelo-erythroid progenitors results in reduced proliferation and reduced response to cytokine-induced cell-cycle entry. Committed lymphoid and myeloid cells no longer require Mll, defining the early multipotent stages of hematopoiesis as Mll dependent. These studies demonstrate that Mll plays selective and independent roles within the hematopoietic system, maintaining quiescence in HSCs and promoting proliferation in progenitors.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Alelos , Animais , Medula Óssea/patologia , Contagem de Células , Morte Celular , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Quimera , Células Precursoras Eritroides/citologia , Éxons/genética , Células-Tronco Hematopoéticas/citologia , Homeostase , Humanos , Camundongos , Células Mieloides/citologia , Células Progenitoras Mieloides/citologia , Deleção de Sequência , Homologia de Sequência de Aminoácidos
12.
Genes Dev ; 20(17): 2397-409, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16951254

RESUMO

Taspase1 was identified as the threonine endopeptidase that cleaves mixed-lineage leukemia (MLL) for proper Hox gene expression in vitro. To investigate its functions in vivo, we generated Taspase1(-/-) mice. Taspase1 deficiency results in noncleavage (nc) of MLL and MLL2 and homeotic transformations. Remarkably, our in vivo studies uncover an unexpected role of Taspase1 in the cell cycle. Taspase1(-/-) animals are smaller in size. Taspase1(-/-) mouse embryonic fibroblasts (MEFs) exhibit impaired proliferation, and acute deletion of Taspase1 leads to a marked reduction of thymocytes. Taspase1 deficiency incurs down-regulation of Cyclin Es, As, and Bs and up-regulation of p16(Ink4a) . We show that MLL and MLL2 directly target E2Fs for Cyclin expression. The uncleaved precursor MLL displays a reduced histone H3 methyl transferase activity in vitro. Accordingly, chromatin immunoprecipitation assays demonstrate a markedly decreased histone H3 K4 trimethylation at Cyclin E1 and E2 genes in Taspase1(-/-) cells. Furthermore, MLL(nc/nc;2nc/nc) MEFs are also impaired in proliferation. Our data are consistent with a model in which precursor MLLs, activated by Taspase1, target to Cyclins through E2Fs to methylate histone H3 at K4, leading to activation. Lastly, Taspase1(-/-) cells are resistant to oncogenic transformation, and Taspase1 is overexpressed in many cancer cell lines. Thus, Taspase1 may serve as a target for cancer therapeutics.


Assuntos
Ciclo Celular/fisiologia , Endopeptidases/fisiologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Endopeptidases/biossíntese , Endopeptidases/deficiência , Endopeptidases/genética , Hidrólise , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide/genética
13.
Nat Genet ; 37(11): 1210-2, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16200063

RESUMO

Stratifin (Sfn, also called 14-3-3sigma) is highly expressed in differentiating epidermis and mediates cell cycle arrest. Sfn is repressed in cancer, but its function during development is uncharacterized. We identified an insertion mutation in the gene Sfn in repeated epilation (Er) mutant mice by positional cloning. Er/+ mice expressed a truncated Sfn protein, which probably contributes to the defects in Er/Er and Er/+ epidermis and to cancer development in Er/+ mice.


Assuntos
Alopecia/genética , Biomarcadores Tumorais/genética , Exonucleases/genética , Remoção de Cabelo , Camundongos Mutantes/anatomia & histologia , Mutação/genética , Proteínas de Neoplasias/genética , Neoplasias Cutâneas/genética , Proteínas 14-3-3 , Alopecia/patologia , Animais , Células Epidérmicas , Exorribonucleases , Heterozigoto , Masculino , Camundongos , Dados de Sequência Molecular , Fenótipo
14.
Proc Natl Acad Sci U S A ; 102(34): 12005-10, 2005 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-16103352

RESUMO

Mitochondrial permeability transition (PT) is a phenomenon induced by high levels of matrix calcium and is characterized by the opening of the PT pore (PTP). Activation of the PTP results in loss of mitochondrial membrane potential, expansion of the matrix, and rupture of the mitochondrial outer membrane. Consequently, PT has been implicated in both apoptotic and necrotic cell death. Cyclophilin D (CypD) appears to be a critical component of the PTP. To investigate the role of CypD in cell death, we created a CypD-deficient mouse. In vitro, CypD-deficient mitochondria showed an increased capacity to retain calcium and were no longer susceptible to PT induced by the addition of calcium. CypD-deficient primary mouse embryonic fibroblasts (MEFs) were as susceptible to classical apoptotic stimuli as the WT, suggesting that CypD is not a central component of cell death in response to these specific death stimuli. However, CypD-deficient MEFs were significantly less susceptible than their WT counterparts to cell death induced by hydrogen peroxide, implicating CypD in oxidative stress-induced cell death. Importantly, CypD-deficient mice displayed a dramatic reduction in brain infarct size after acute middle cerebral artery occlusion and reperfusion, strongly supporting an essential role for CypD in an ischemic injury model in which calcium overload and oxidative stress have been implicated.


Assuntos
Apoptose/fisiologia , Isquemia Encefálica/fisiopatologia , Ciclofilinas/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Neurônios/fisiologia , Canais de Ânion Dependentes de Voltagem/metabolismo , Animais , Apoptose/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Cálcio/metabolismo , Peptidil-Prolil Isomerase F , Ciclofilinas/deficiência , Peróxido de Hidrogênio/toxicidade , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Dilatação Mitocondrial/fisiologia , Neurônios/metabolismo , Permeabilidade , Espectrofotometria
15.
EMBO J ; 24(2): 368-81, 2005 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-15635450

RESUMO

Chromosomal translocations that fuse the mixed lineage leukemia (MLL) gene with multiple partners typify acute leukemias of infancy as well as therapy-related leukemias. We utilized a conditional knockin strategy to bypass the embryonic lethality caused by MLL-CBP expression and to assess the immediate effects of induced MLL-CBP expression on hematopoiesis. Within days of activating MLL-CBP, the fusion protein selectively expanded granulocyte/macrophage progenitors (GMP) and enhanced their self-renewal/proliferation. MLL-CBP altered the gene expression program of GMP, upregulating a subset of genes including Hox a9. Inhibition of Hox a9 expression by RNA interference demonstrated that MLL-CBP required Hox a9 for its enhanced cell expansion. Following exposure to sublethal gamma-irradiation or N-ethyl-N-nitrosourea (ENU), MLL-CBP mice developed myelomonocytic hyperplasia and progressed to fatal myeloproliferative disorders. These represented the spectrum of therapy-induced acute myelomonocytic leukemia/chronic myelomonocytic leukemia/myelodysplastic/myeloproliferative disorder similar to that seen in humans possessing the t(11;16). This model of MLL-CBP therapy-related myeloproliferative disease demonstrates the selectivity of this MLL fusion for GMP cells and its ability to initiate leukemogenesis in conjunction with cooperating mutations.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Transtornos Mieloproliferativos/induzido quimicamente , Proteínas Nucleares/fisiologia , Proto-Oncogenes/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Proteína de Ligação a CREB , Histona-Lisina N-Metiltransferase , Camundongos , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Dev Cell ; 6(3): 437-43, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15030765

RESUMO

The Mixed-Lineage Leukemia (MLL) gene encodes a Trithorax-related chromatin-modifying protooncogene that positively regulates Hox genes. In addition to their well-characterized roles in axial patterning, Trithorax and Polycomb family proteins perform less-understood functions in vertebrate hematopoiesis. To define the role of MLL in the development of the hematopoietic system, we examined the potential of cells lacking MLL. Mll-deficient cells could not develop into lymphocytes in adult RAG-2 chimeric animals. Similarly, in vitro differentiation of B cells required MLL. In chimeric embryos, Mll-deficient cells failed to contribute to fetal liver hematopoietic stem cell/progenitor populations. Moreover, we show that aorta-gonad-mesonephros (AGM) cells from Mll-deficient embryos lacked hematopoietic stem cell (HSC) activity despite their ability to generate hematopoietic progeny in vitro. These results demonstrate an intrinsic requirement for MLL in definitive hematopoiesis, where it is essential for the generation of HSCs in the embryo.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Proto-Oncogenes , Células-Tronco/fisiologia , Animais , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Aorta/citologia , Aorta/embriologia , Aorta/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura/métodos , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Citometria de Fluxo/métodos , Galactosídeos/metabolismo , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Histona-Lisina N-Metiltransferase , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Indóis/metabolismo , Células Matadoras Naturais/fisiologia , Antígenos Comuns de Leucócito/metabolismo , Fígado/citologia , Fígado/metabolismo , Linfopoese/efeitos dos fármacos , Linfopoese/fisiologia , Mesonefro/citologia , Mesonefro/embriologia , Mesonefro/metabolismo , Camundongos , Camundongos Transgênicos , Proteína de Leucina Linfoide-Mieloide , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Science ; 301(5632): 513-7, 2003 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-12881569

RESUMO

The multidomain proapoptotic molecules BAK or BAX are required to initiate the mitochondrial pathway of apoptosis. How cells maintain the potentially lethal proapoptotic effector BAK in a monomeric inactive conformation at mitochondria is unknown. In viable cells, we found BAK complexed with mitochondrial outer-membrane protein VDAC2, a VDAC isoform present in low abundance that interacts specifically with the inactive conformer of BAK. Cells deficient in VDAC2, but not cells lacking the more abundant VDAC1, exhibited enhanced BAK oligomerization and were more susceptible to apoptotic death. Conversely, overexpression of VDAC2 selectively prevented BAK activation and inhibited the mitochondrial apoptotic pathway. Death signals activate "BH3-only" molecules such as tBID, BIM, or BAD, which displace VDAC2 from BAK, enabling homo-oligomerization of BAK and apoptosis. Thus, VDAC2, an isoform restricted to mammals, regulates the activity of BAK and provides a connection between mitochondrial physiology and the core apoptotic pathway.


Assuntos
Apoptose , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Porinas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Biopolímeros , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Linhagem Celular , Células Cultivadas , Etoposídeo/farmacologia , Humanos , Membranas Intracelulares/metabolismo , Células Jurkat , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/metabolismo , Porinas/genética , Porinas/isolamento & purificação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/farmacologia , Estaurosporina/farmacologia , Canal de Ânion 1 Dependente de Voltagem , Canal de Ânion 2 Dependente de Voltagem , Canais de Ânion Dependentes de Voltagem , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2
18.
Mol Endocrinol ; 17(1): 93-106, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12511609

RESUMO

Somatostatin (SRIF) regulates pancreatic insulin and glucagon secretion. In the present study we describe the generation of SRIF receptor subtype 5 knockout (sst(5) KO) mice to examine the role of SRIF receptor subtypes (sst) in regulating insulin secretion and glucose homeostasis. Mice deficient in sst(5) were viable, fertile, appeared healthy, and displayed no obvious phenotypic abnormalities. Pancreatic islets isolated from sst(5) KO mice displayed increased total insulin content as compared with islets obtained from wild-type (WT) mice. Somatostatin-28 (SRIF-28) and the sst(5)/sst(1)-selective agonist compound 5/1 potently inhibited glucose-stimulated insulin secretion from WT islets. SRIF-28 inhibited insulin secretion from sst(5) KO islets with 16-fold less potency while the maximal effect of compound 5/1 was markedly diminished when compared with its effects in WT islets. sst(5) KO mice exhibited decreased blood glucose and plasma insulin levels and increased leptin and glucagon concentrations compared with WT mice. Furthermore, sst(5) KO mice displayed decreased susceptibility to high fat diet-induced insulin resistance. The results of these studies suggest sst(5) mediates SRIF inhibition of pancreatic insulin secretion and contributes to the regulation of glucose homeostasis and insulin sensitivity. Our findings suggest a potential beneficial role of sst(5) antagonists for alleviating metabolic abnormalities associated with obesity and insulin resistance.


Assuntos
Glucose/metabolismo , Insulina/metabolismo , Receptores de Somatostatina/fisiologia , Animais , Células CHO , Clonagem Molecular/métodos , Cricetinae , Feminino , Marcação de Genes/métodos , Homeostase/fisiologia , Resistência à Insulina/fisiologia , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/deficiência , Receptores de Somatostatina/genética , Somatostatina/metabolismo , Somatostatina-28 , Transfecção
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