RESUMEN
Isocitrate dehydrogenase-1 (Idh1) is an important metabolic enzyme that produces NADPH by converting isocitrate to α-ketoglutarate. Idh1 is known to reduce reactive oxygen species (ROS) induced in cells by treatment with lipopolysaccharide (LPS) in vitro. Here, we used Idh1-deficient knockout (Idh1 KO) mice to investigate the role of Idh1 in antioxidant defense in vivo. Idh1 KO mice showed heightened susceptibility to death induced by LPS and exhibited increased serum levels of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The serum of LPS-injected Idh1 KO mice also contained elevated levels of AST, a marker of inflammatory liver damage. Furthermore, after LPS injection, livers of Idh1 KO mice showed histological evidence of elevated oxidative DNA damage compared with livers of wild-type (WT) mice. Idh1 KO livers showed a faster and more pronounced oxidative stress than WT livers. In line with that, Idh1 KO hepatocytes showed higher ROS levels and an increase in the NADP(+)/NADPH ratio when compared with hepatocytes isolated from WT mice. These results suggest that Idh1 has a physiological function in protecting cells from oxidative stress by regulating the intracellular NADP(+)/NADPH ratio. Our findings suggest that stimulation of Idh1 activity may be an effective therapeutic strategy for reducing oxidative stress during inflammatory responses, including the early stages of septic shock.
Asunto(s)
Endotoxinas/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Isocitrato Deshidrogenasa/metabolismo , NADP/metabolismo , Animales , Células Cultivadas , Citometría de Flujo , Isocitrato Deshidrogenasa/genética , Ratones , Ratones Noqueados , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
It is well known that protein tyrosine phosphatases (PTPs) that become oxidized due to exposure to reactive oxygen species (ROS) undergo a conformational change and are inactivated. However, whether PTPs can actively regulate ROS levels in order to prevent PTP inhibition has yet to be investigated. Here, we demonstrate that PTP non-receptor type 12 (PTPN12) protects cells against aberrant ROS accumulation and death induced by oxidative stress. Murine embryonic fibroblasts (MEFs) deficient in PTPN12 underwent increased ROS-induced apoptosis under conditions of antioxidant depletion. Cells lacking PTPN12 also showed defective activation of FOXO1/3a, transcription factors required for the upregulation of several antioxidant genes. PTPN12-mediated regulation of ROS appeared to be mediated by phosphoinositide-dependent kinase-1 (PDK1), which was hyperstimulated in the absence of PTPN12. As tight regulation of ROS to sustain survival is a key feature of cancer cells, we examined PTPN12 levels in tumors from a cohort of breast cancer patients. Patients whose tumors showed high levels of PTPN12 transcripts had a significantly poorer prognosis. Analysis of tissues from patients with various breast cancer subtypes revealed that more triple-negative breast cancers, the most aggressive breast cancer subtype, showed high PTPN12 expression than any other subtype. Furthermore, both human breast cancer cells and mouse mammary epithelial tumor cells engineered to lack PTPN12 exhibited reduced tumorigenic and metastatic potential in vivo that correlated with their elevated ROS levels. The involvement of PTPN12 in the antioxidant response of breast cancer cells suggests that PTPN12 may represent a novel therapeutic target for this disease.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Estrés Oxidativo , Proteína Tirosina Fosfatasa no Receptora Tipo 12/fisiología , Transducción de Señal , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , División Celular , Células Cultivadas , Femenino , Humanos , Ratones , Pronóstico , Proteína Tirosina Fosfatasa no Receptora Tipo 12/genética , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Breast cancer is the most common solid tumor and the second most common cause of death in women. Despite a large body of literature and progress in breast cancer research, many molecular aspects of this complex disease are still poorly understood, hindering the design of specific and effective therapeutic strategies. To identify the molecules important in breast cancer progression and metastasis, we tested the in vivo effects of inhibiting the functions of various kinases and genes involved in the regulation/modulation of the cytoskeleton by downregulating them in mouse PyMT mammary tumor cells and human breast cancer cell lines. These kinases and cytoskeletal regulators were selected based on their prognostic values for breast cancer patient survival. PyMT tumor cells, in which a selected gene was stably knocked down were injected into the tail veins of mice, and the formation of tumors in the lungs was monitored. One of the several genes found to be important for tumor growth in the lungs was NIMA-related kinases 2 (Nek2), a cell cycle-related protein kinase. Furthermore, Nek2 was also important for tumor growth in the mammary fat pad. In various human breast cancer cell lines, Nek2 knockdown induced aneuploidy and cell cycle arrest that led to cell death. Significantly, the breast cancer cell line most sensitive to Nek2 depletion was of the triple negative breast cancer subtype. Our data indicate that Nek2 has a pivotal role in breast cancer growth at primary and secondary sites, and thus may be an attractive and novel therapeutic target for this disease.
Asunto(s)
Aneuploidia , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Centrosoma/patología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Línea Celular Tumoral , Segregación Cromosómica/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/secundario , Ratones , Quinasas Relacionadas con NIMA , Trasplante de Neoplasias , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Flotillin microdomains, specialized lipid raft domains in cell membranes, serve as physical platforms for many different molecules important in crucial intracellular signaling pathways. Flotillin-2 (Flot2), together with flotillin-1, is a marker for lipid raft microdomains distinct from caveolar lipid rafts, and has been implicated in the progression of cancer and metastasis formation. Based largely on studies in xenograft models, flotillin-2 has been implicated in the progression of multiple types of human tumors, including breast cancer. In our studies, we identified flotillin-2 as highly amplified in a high-throughput comparative genomic hybridization screen of human breast cancer cell lines and breast tumor samples. Short hairpin RNA-mediated reduction of flotillin-2 protein levels significantly reduced the tumorigenicity and metastatic capability of a human breast cancer cell line in vivo. We generated mice deficient for flotillin-2 and also found a reduction of flotillin-1 protein levels and complete absence of flotillin-specific membrane microdomains in these mice. To examine the role of Flot2 in mammary tumorigenesis and lung metastasis, we used an in vivo molecular genetics approach, crossing a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with gene-targeted Flot2(-/-) mice. Flotillin-2 deficiency lead to a striking reduction in the number of lung metastasis observed, but had no influence on primary tumor formation in this model. Our results indicate, using a novel in vivo animal model approach, that Flot2 is an important regulator of mammary tumor-derived lung metastasis.
Asunto(s)
Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Hibridación Genómica Comparativa , Modelos Animales de Enfermedad , Femenino , Silenciador del Gen , Humanos , RatonesRESUMEN
WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors. The physiological role of WSX-1, which is expressed mainly in T cells, was investigated in gene-targeted WSX-1-deficient mice. IFN-gamma production was reduced in isolated WSX-1(-/-) T cells subjected to primary stimulation in vitro to induce Th1 differentiation but was normal in fully differentiated and activated WSX-1(-/-) Th1 cells that had received secondary stimulation. WSX-1(-/-) mice were remarkably susceptible to Leishmania major infection, showing impaired IFN-gamma production early in the infection. However, IFN-gamma production during the later phases of the infection was not impaired in the knockout. WSX-1(-/-) mice also showed poorly differentiated granulomas with dispersed accumulations of mononuclear cells when infected with bacillus Calmette-Guerin (BCG). Thus, WSX-1 is essential for the initial mounting of Th1 responses but dispensable for their maintenance.
Asunto(s)
Leishmania major , Leishmaniasis Cutánea/inmunología , Receptores de Citocinas/fisiología , Células TH1/inmunología , Animales , Diferenciación Celular , División Celular , Células Cultivadas , Granuloma/patología , Sistema Hematopoyético/fisiología , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-4/biosíntesis , Interleucina-4/genética , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/patología , Sistema Linfático/inmunología , Ratones , Ratones Noqueados , Mycobacterium bovis , ARN Mensajero/biosíntesis , Receptores de Citocinas/genética , Receptores de Interleucina , Tuberculosis/patologíaRESUMEN
Tissue inhibitors of metalloproteinases regulate ECM degradation by matrix metalloproteinases (MMPs). We have developed a mouse line deficient for tissue inhibitor of metalloproteinases-3 (TIMP-3), the only TIMP known to reside within the ECM. Homozygous Timp-3-null animals develop spontaneous air space enlargement in the lung that is evident at 2 weeks after birth and progresses with age of the animal. As early as 13 months of age animals become moribund. Lung function, measured by carbon monoxide uptake, is impaired in aged null animals. Lungs from aged null animals have reduced abundance of collagen, enhanced degradation of collagen in the peribronchiolar space, and disorganization of collagen fibrils in the alveolar interstitium, but no increase in inflammatory cell infiltration or evidence of fibrosis in comparison with controls. Using in situ zymography, we show that lungs from aged null animals have heightened MMP activity over wild-type and heterozygotic animals. Finally, TIMP-3-null fibroblast cultures demonstrate enhanced destruction of ECM molecules in vitro. We propose that the deletion of TIMP-3 results in a shift of the TIMP/MMP balance in the lung to favor ECM degradation, culminating in incapacitating illness and a shorter life span.
Asunto(s)
Pulmón/patología , Inhibidor Tisular de Metaloproteinasa-3/fisiología , Aire , Animales , Colágeno/metabolismo , Matriz Extracelular/fisiología , Pulmón/fisiopatología , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Noqueados , Alveolos Pulmonares/patología , Inhibidor Tisular de Metaloproteinasa-3/deficiencia , Inhibidor Tisular de Metaloproteinasa-3/genéticaRESUMEN
The dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7(-/-) mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7(-/-) mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor-driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.
Asunto(s)
Linfocitos B/citología , MAP Quinasa Quinasa 4 , Mastocitos/citología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Linfocitos T/citología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , División Celular , Activación Enzimática , Marcación de Gen , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Interleucina-3/metabolismo , Interleucina-3/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 7 , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis , Factor de Células Madre/metabolismo , Factor de Células Madre/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Timo/citologíaRESUMEN
The molecular adapter Fyb/Slap regulates signaling downstream of the T cell receptor (TCR), but whether it plays a positive or negative role is controversial. We demonstrate that Fyb/Slap-deficient T cells exhibit defective proliferation and cytokine production in response to TCR stimulation. Fyb/Slap is also required in vivo for T cell-dependent immune responses. Functionally, Fyb/Slap has no apparent role in the activation of known TCR signaling pathways, F-actin polymerization, or TCR clustering. Rather, Fyb/Slap regulates TCR-induced integrin clustering and adhesion. Thus, Fyb/Slap is the first molecular adapter to be identified that couples TCR stimulation to the avidity modulation of integrins governing T cell adhesion.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/fisiología , Integrinas/metabolismo , Activación de Linfocitos , Fosfoproteínas/fisiología , Linfocitos T/fisiología , Actinas/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos B/inmunología , Complejo CD3/metabolismo , Proteínas Portadoras/genética , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Quimera , Marcación de Gen , Humanos , Inmunización , Inmunoglobulina G/biosíntesis , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Interleucina-2/farmacología , Lectinas Tipo C , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Fosfoproteínas/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
The outcome of T-cell responses after T-cell encounter with specific antigens is modulated by co-stimulatory signals, which are required for both lymphocyte activation and development of adaptive immunity. ICOS, an inducible co-stimulator with homology to CD28, is expressed on activated, but not resting T cells, and shows T-cell co-stimulatory function in vitro. ICOS binds specifically to its counter-receptor B7RP-1 (refs 5,6,7), but not to B7-1 or B7-2. Here we provide in vivo genetic evidence that ICOS delivers a co-stimulatory signal that is essential both for efficient interaction between T and B cells and for normal antibody responses to T-cell-dependent antigens. To determine the physiological function of ICOS, we generated and characterized gene-targeted ICOS-deficient mice. In vivo, a lack of ICOS results in severely deficient T-cell-dependent B-cell responses. Germinal centre formation is impaired and immunoglobulin class switching, including production of allergy-mediating IgE, is defective. ICOS-deficient T cells primed in in vivo and restimulated in vitro with specific antigen produce only low levels of interleukin-4, but remain fully competent to produce interferon-gamma.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Ficoll/análogos & derivados , Linfocitos T Colaboradores-Inductores/fisiología , Animales , Antígenos/inmunología , Antígenos de Diferenciación de Linfocitos T/genética , Linfocitos B/inmunología , Comunicación Celular , División Celular , Células Cultivadas , Femenino , Ficoll/inmunología , Citometría de Flujo , Marcación de Gen , Centro Germinal/fisiología , Hemocianinas/inmunología , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles , Interferón gamma/biosíntesis , Interferón gamma/fisiología , Interleucina-4/biosíntesis , Interleucina-4/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T Colaboradores-Inductores/inmunología , Trinitrobencenos/inmunologíaRESUMEN
PTEN, a tumor suppressor gene, is essential for embryogenesis. We used the Cre-loxP system to generate a T cell-specific deletion of the Pten gene (Pten(flox/-) mice). All Pten(flox/-) mice develop CD4+ T cell lymphomas by 17 weeks. Pten(flox/-) mice show increased thymic cellularity due in part to a defect in thymic negative selection. Pten(flox/-) mice exhibit elevated levels of B cells and CD4+ T cells in the periphery, spontaneous activation of CD4+ T cells, autoantibody production, and hypergammaglobulinemia. Pten(flox/-) T cells hyperproliferate, are autoreactive, secrete increased levels of Th1/Th2 cytokines, resist apoptosis, and show increased phosphorylation of PKB/Akt and ERK. Peripheral tolerance to SEB is also impaired in Pten(flox/-) mice. PTEN is thus an important regulator of T cell homeostasis and self-tolerance.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Genes Supresores de Tumor , Proteínas I-kappa B , Tolerancia Inmunológica , Monoéster Fosfórico Hidrolasas/inmunología , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor , Animales , Apoptosis/inmunología , Linfocitos T CD4-Positivos/citología , División Celular , Citocinas/biosíntesis , Proteínas de Unión al ADN/metabolismo , Enterotoxinas/inmunología , Femenino , Enfermedades Linfáticas/inmunología , Linfoma de Células T/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Antígenos/genética , Receptores de Antígenos/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Esplenomegalia , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Timo/anomalías , Timo/citologíaRESUMEN
Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.
Asunto(s)
Apoptosis/fisiología , Flavoproteínas/fisiología , Proteínas de la Membrana/fisiología , Mitocondrias/fisiología , Animales , Factor Inductor de la Apoptosis , Factor Apoptótico 1 Activador de Proteasas , Caspasa 9 , Caspasas/metabolismo , Diferenciación Celular , Quimera , Embrión de Mamíferos/citología , Desarrollo Embrionario y Fetal/fisiología , Femenino , Flavoproteínas/genética , Marcación de Gen , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Proteínas/fisiología , Recombinación Genética , Células MadreRESUMEN
Functional inactivation of the tumor susceptibility gene tsg101 in NIH 3T3 fibroblasts results in cellular transformation and the ability to form metastatic tumors in nude mice. The N-terminal region of tsg101 protein is structurally similar to the catalytic domain of ubiquitin-conjugating enzymes, suggesting a potential role of tsg101 in ubiquitin-mediated protein degradation. The C-terminal domain of TSG101 can function as a repressor of transcription. To investigate the physiological function of tsg101, we generated a null mutation of the mouse gene by gene targeting. Homozygous tsg101-/- embryos fail to develop past day 6.5 of embryogenesis (E6.5), are reduced in size, and do not form mesoderm. Mutant embryos show a decrease in cellular proliferation in vivo and in vitro but no increase in apoptosis. Although levels of p53 transcripts were not affected in tsg101-/- embryos, p53 protein accumulated dramatically, implying altered posttranscriptional control of p53. In addition, transcription of the p53 effector, cyclin-dependent kinase inhibitor p21(WAF-1/CIP-1), was increased 5- to 10-fold, whereas activation of MDM2 transcription secondary to p53 elevation was not observed. Introduction of a p53 null mutation into tsg101-/- embryos rescued the gastrulation defect and prolonged survival until E8.5. These results demonstrate that tsg101 is essential for the proliferative burst before the onset of gastrulation and establish a functional connection between tsg101 and the p53 pathway in vivo.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Pérdida del Embrión/metabolismo , Proteínas Nucleares , Factores de Transcripción/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , División Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Proteínas de Unión al ADN/genética , Desarrollo Embrionario y Fetal , Endodermo/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Expresión Génica , Marcación de Gen , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Bcl10, a CARD-containing protein identified from the t(1;14)(p22;q32) breakpoint in MALT lymphomas, has been shown to induce apoptosis and activate NF-kappaB in vitro. We show that one-third of bcl10-/- embryos developed exencephaly, leading to embryonic lethality. Surprisingly, bcl10-/- cells retained susceptibility to various apoptotic stimuli in vivo and in vitro. However, surviving bcl10-/- mice were severely immunodeficient and bcl10-/- lymphocytes are defective in antigen receptor or PMA/Ionomycin-induced activation. Early tyrosine phosphorylation, MAPK and AP-1 activation, and Ca2+ signaling were normal in mutant lymphocytes, but antigen receptor-induced NF-kappaB activation was absent. Thus, Bcl10 functions as a positive regulator of lymphocyte proliferation that specifically connects antigen receptor signaling in B and T cells to NF-kappaB activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Sistema Nervioso Central/anomalías , FN-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Defectos del Tubo Neural/fisiopatología , Receptores de Antígenos/metabolismo , Animales , Anisomicina/farmacología , Formación de Anticuerpos/inmunología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Apoptosis/efectos de la radiación , Proteína 10 de la LLC-Linfoma de Células B , Linfocitos B/inmunología , División Celular/inmunología , Sistema Nervioso Central/fisiología , Cisplatino/farmacología , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Genes Letales/inmunología , Inmunidad Celular/inmunología , Activación de Linfocitos/fisiología , Ratones , Ratones Noqueados , FN-kappa B/inmunología , Proteínas de Neoplasias/metabolismo , Defectos del Tubo Neural/inmunología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Receptores de Antígenos/inmunología , Transducción de Señal/inmunología , Estaurosporina/farmacología , Linfocitos T/inmunologíaRESUMEN
Mammalian telomerase is essential for the maintenance of telomere length [1-5]. Its catalytic core comprises a reverse transcriptase component (TERT) and an RNA component. While the biochemical role of mammalian TERT is well established [6-11], it is unknown whether it is sufficient for telomere-length maintenance, chromosome stability or other cellular processes. Cells from mice in which the mTert gene had been disrupted showed progressive loss of telomere DNA, a phenotype similar to cells in which the gene encoding the telomerase RNA component (mTR) has been disrupted [1,12]. On prolonged growth, mTert-deficient embryonic stem (ES) cells exhibited genomic instability, aneuploidy and telomeric fusions. ES cells heterozygous for the mTert disruption also showed telomere attrition, a phenotype that differs from heterozygous mTR cells [12]. Thus, telomere maintenance in mammals is carried out by a single, limiting TERT.
Asunto(s)
ARN , Telomerasa/fisiología , Telómero/fisiología , Animales , Línea Celular , Proteínas de Unión al ADN , Marcación de Gen , Ratones , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.
Asunto(s)
Proteínas Portadoras/fisiología , Telómero/fisiología , Animales , Proteínas Portadoras/metabolismo , Catálisis , Embrión de Mamíferos/metabolismo , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Pruebas de Precipitina , ARN/metabolismo , Proteínas de Unión al ARN , Recombinación Genética , Bazo/citología , Células Madre/metabolismo , Telomerasa , Telómero/ultraestructura , Timo/citologíaRESUMEN
Regulators of G protein signaling (RGS) proteins accelerate the GTPase activity of Galpha protein subunits in vitro, negatively regulating G protein-coupled receptor signaling. The physiological role of mammalian RGS proteins is largely unknown. The RGS family member rgs2 was cloned as an immediate early response gene up-regulated in T lymphocytes after activation. To investigate the role of RGS2 in vivo, we generated rgs2-deficient mice. We show that targeted mutation of rgs2 in mice leads to reduced T cell proliferation and IL-2 production, which translates in an impaired antiviral immunity in vivo. Interestingly, rgs2(-/-) mice also display increased anxiety responses and decreased male aggression in the absence of cognitive or motor deficits. RGS2 also controls synaptic development and basal electrical activity in hippocampal CA1 neurons. Thus, RGS2 plays an important role in T cell activation, synapse development in the hippocampus, and emotive behaviors.
Asunto(s)
Agresión/fisiología , Ansiedad/fisiopatología , Activación de Linfocitos/fisiología , Proteínas RGS/fisiología , Linfocitos T/inmunología , Animales , Secuencia de Bases , División Celular/fisiología , Cartilla de ADN , Marcación de Gen , Hipocampo/citología , Hipocampo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiología , Sinapsis/fisiologíaRESUMEN
The hexosamine pathway provides UDP-N:-acetylhexosamine donor substrates used in cytosolic and Golgi-mediated glycosylation of proteins and for formation of glycosylphosphatidylinositol (GPI) anchors, which tether proteins to the outer plasma membrane. We have recently identified the murine glucosamine-6-phosphate (GlcN6P) acetyltransferase, EMeg32, as a developmentally regulated enzyme on the route to UDP-N:-acetylglucosamine (UDP-GlcNAc). Here we describe embryos and cells that have the EMeg32 gene inactivated by homologous recombination. Homozygous mutant embryos die at around embryonic day (E) 7.5 with a general proliferative delay of development. In vitro differentiated EMeg32(-/-) ES cells show reduced proliferation. Mouse embryonic fibroblasts (MEFs) deficient for EMeg32 exhibit defects in proliferation and adhesiveness, which could be complemented by stable re-expression of EMeg32 or by nutritional restoration of intracellular UDP-GlcNAc levels. Reduced UDP-GlcNAc levels predominantly translated into decreased O-GlcNAc modifications of cytosolic and nuclear proteins. Interestingly, growth-impaired EMeg32(-/-) MEFs withstand a number of apoptotic stimuli and express activated PKB/AKT. Thus, EMeg32-dependent UDP-GlcNAc levels influence cell cycle progression and susceptibility to apoptotic stimuli.
Asunto(s)
Acetiltransferasas/metabolismo , Desarrollo Embrionario y Fetal/fisiología , Proteínas de la Membrana/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismo , Acetiltransferasas/genética , Animales , Apoptosis , División Celular , Línea Celular , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Pérdida del Embrión/enzimología , Pérdida del Embrión/metabolismo , Embrión de Mamíferos , Desarrollo Embrionario y Fetal/genética , Retículo Endoplásmico/metabolismo , Fibroblastos/citología , Fibroblastos/enzimología , Fibroblastos/metabolismo , Glucosamina 6-Fosfato N-Acetiltransferasa , Glicosilación , Glicosilfosfatidilinositoles/biosíntesis , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la PolimerasaRESUMEN
Induction of NF-kappaB-dependent transcription requires phosphorylation and subsequent degradation of I-kappaB, an inhibitor of NF-kappaB, followed by nuclear translocation and DNA binding of NF-kappaB. Tumor necrosis factor receptor-associated factor 2 (TRAF2) plays a role in NF-kappaB activation in response to cytokines such as tumor necrosis factor alpha (TNFalpha). In this study, we purified and characterized a novel kinase (T2K, also known as TBK1 or NAK), which associates with TRAF2 and exhibits kinase activity towards I-kappaBalpha in vitro. The physiological function of T2K was investigated using T2K-deficient mice. Heterozygotes appear normal, but t2k(-/-) animals die at approximately E14.5 of massive liver degeneration and apoptosis. Never theless, hematopoietic progenitors from T2K-deficient fetal liver support normal lymphocyte development. Furthermore, t2k(-/-) embryonic fibroblasts and thymocytes do not display increased sensitivity to TNFalpha-induced apoptosis. In response to either TNFalpha or IL-1 induction, t2k(-/-) embryonic fibroblasts exhibit normal degradation of I-kappaB and kappaB-binding activity. However, NF-kappaB-directed transcription is dramatically reduced. These results demonstrate that, like I-kappaB kinase beta and the RelA subunit of NF-kappaB, T2K is critical in protecting embryonic liver from apoptosis. However, T2K has a unique role in the activation of NF-kappaB-directed transcription, apparently independent of I-kappaB degradation and NF-kappaB DNA binding.
Asunto(s)
Apoptosis , Hígado/metabolismo , FN-kappa B/biosíntesis , FN-kappa B/genética , Secuencia de Aminoácidos , Animales , Southern Blotting , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/metabolismo , Citometría de Flujo , Marcación de Gen , Genes Reporteros , Genotipo , Heterocigoto , Quinasa I-kappa B , Etiquetado Corte-Fin in Situ , Interleucina-1/farmacología , Ligasas/metabolismo , Hígado/patología , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Genéticos , Datos de Secuencia Molecular , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Factor 2 Asociado a Receptor de TNF , Timo/citología , Factores de Tiempo , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Casper (c-FLIP) associates with FADD and caspase-8 in signaling complexes downstream of death receptors like Fas. We generated Casper-deficient mice and cells and noted a duality in the physiological functions of this molecule. casper-/- embryos do not survive past day 10.5 of embryogenesis and exhibit impaired heart development. This phenotype is reminiscent of that reported for FADD-/- and caspase-8-/- embryos. However, unlike FADD-/- and caspase-8-/- cells, casper-/- embryonic fibroblasts are highly sensitive to FasL- or TNF-induced apoptosis and show rapid induction of caspase activities. NF-kappaB and JNK/SAPK activation is intact in TNF-stimulated casper-/- cells. These results suggest that Casper has two distinct roles: to cooperate with FADD and caspase-8 during embryonic development and to mediate cytoprotection against death factor-induced apoptosis.
Asunto(s)
Apoptosis/inmunología , Proteínas Portadoras/fisiología , Desarrollo Embrionario y Fetal/inmunología , Péptidos y Proteínas de Señalización Intracelular , Receptores del Factor de Necrosis Tumoral/fisiología , Animales , Apoptosis/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/genética , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Línea Celular , Desarrollo Embrionario y Fetal/genética , Activación Enzimática/inmunología , Femenino , Corazón/embriología , Proteínas Quinasas JNK Activadas por Mitógenos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/enzimología , Miocardio/inmunología , Miocardio/metabolismo , Miocardio/patología , FN-kappa B/metabolismo , Células Madre/enzimología , Células Madre/inmunología , Células Madre/metabolismo , Células Madre/patología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
We describe a new microtiter immunospore trapping device (MTIST device) that uses a suction system to directly trap air particulates by impaction in microtiter wells. This device can be used for rapid detection and immunoquantification of ascospores of Mycosphaerella brassicicola and conidia of Botrytis cinerea by an enzyme-linked immunosorbent assay (ELISA) under controlled environmental conditions. For ascospores of M. brassicicola correlation coefficients (r(2)) of 0.943 and 0.9514 were observed for the number of MTIST device-impacted ascospores per microtiter well and the absorbance values determined by ELISA, respectively. These values were not affected when a mixed fungal spore population was used. There was a relationship between the number of MTIST device-trapped ascospores of M. brassicicola per liter of air sampled and the amount of disease expressed on exposed plants of Brassica oleracea (Brussels sprouts). Similarly, when the MTIST device was used to trap conidia of B. cinerea, a correlation coefficient of 0.8797 was obtained for the absorbance values generated by the ELISA and the observed number of conidia per microtiter well. The relative collection efficiency of the MTIST device in controlled plant growth chambers with limited airflow was 1.7 times greater than the relative collection efficiency of a Burkard 7-day volumetric spore trap for collection of M. brassicicola ascospores. The MTIST device can be used to rapidly differentiate, determine, and accurately quantify target organisms in a microflora. The MTIST device is a portable, robust, inexpensive system that can be used to perform multiple tests in a single sampling period, and it should be useful for monitoring airborne particulates and microorganisms in a range of environments.