RESUMO
Migration of T cells is essential for their ability to mount immune responses. Chemokine-induced T cell migration requires WNK1, a kinase that regulates ion influx into the cell. However, it is not known why ion entry is necessary for T cell movement. Here we show that signaling from the chemokine receptor CCR7 leads to activation of WNK1 and its downstream pathway at the leading edge of migrating CD4+ T cells, resulting in ion influx and water entry by osmosis. We propose that WNK1-induced water entry is required to swell the membrane at the leading edge, generating space into which actin filaments can polymerize, thereby facilitating forward movement of the cell. Given the broad expression of WNK1 pathway proteins, our study suggests that ion and water influx are likely to be essential for migration in many cell types, including leukocytes and metastatic tumor cells.
Assuntos
Citoesqueleto de Actina , Actinas , Actinas/metabolismo , Polimerização , Movimento Celular/fisiologia , Citoesqueleto de Actina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Migration and adhesion play critical roles in B cells, regulating recirculation between lymphoid organs, migration within lymphoid tissue, and interaction with CD4+ T cells. However, there is limited knowledge of how B cells integrate chemokine receptor and integrin signaling with B cell activation to generate efficient humoral responses. Here, we show that the WNK1 kinase, a regulator of migration and adhesion, is essential in B cells for T-dependent and -independent antibody responses. We demonstrate that WNK1 transduces signals from the BCR, CXCR5, and CD40, and using intravital imaging, we show that WNK1 regulates migration of naive and activated B cells, and their interactions with T cells. Unexpectedly, we show that WNK1 is required for BCR- and CD40-induced proliferation, acting through the OXSR1 and STK39 kinases, and for efficient B cell-T cell collaboration in vivo. Thus, WNK1 is critical for humoral immune responses, by regulating B cell migration, adhesion, and T cell-dependent activation.
Assuntos
Formação de Anticorpos , Linfócitos B , Camundongos , Animais , Tecido Linfoide , Transdução de Sinais , Linfócitos T CD4-Positivos , Antígenos CD40/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismoRESUMO
Memory B cells (MBCs) are long-lived cells that form a critical part of immunological memory, providing rapid antibody responses to recurring infections. However, very little is known about signals controlling MBC survival. Previous work has shown that antigen is not required for MBC survival, but a requirement for the B cell antigen receptor (BCR) has not been tested. Other studies have shown that, unlike naive B cells, MBCs do not express BAFFR and their survival is independent of BAFF, the ligand for BAFFR. Here, using inducible genetic ablation, we show that survival of MBCs is critically dependent on the BCR and on signaling through the associated CD79A protein. Unexpectedly, we found that MBCs express BAFFR and that their survival requires BAFF and BAFFR; hence, loss of BAFF or BAFFR impairs recall responses. Finally, we show that MBC survival requires IKK2, a kinase that transduces BAFFR signals. Thus, MBC survival is critically dependent on signaling from BCR and BAFFR.
Assuntos
Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Memória Imunológica/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Receptor do Fator Ativador de Células B/imunologia , Sobrevivência Celular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologiaRESUMO
WNK1, a kinase that controls kidney salt homeostasis, also regulates adhesion and migration in CD4+ T cells. Wnk1 is highly expressed in thymocytes, and since migration is important for thymocyte maturation, we investigated a role for WNK1 in mouse thymocyte development. We find that WNK1 is required for the transition of double negative (DN) thymocytes through the ß-selection checkpoint and subsequent proliferation and differentiation into double positive (DP) thymocytes. Furthermore, we show that WNK1 negatively regulates LFA1-mediated adhesion and positively regulates CXCL12-induced migration in DN thymocytes. Despite this, migration defects of WNK1-deficient thymocytes do not account for the developmental arrest. Instead, we show that in DN thymocytes WNK1 transduces pre-TCR signals via OXSR1 and STK39 kinases, and the SLC12A2 ion co-transporter that are required for post-transcriptional upregulation of MYC and subsequent proliferation and differentiation into DP thymocytes. Thus, a pathway regulating ion homeostasis is a critical regulator of thymocyte development.
Assuntos
Diferenciação Celular/genética , Proteínas Proto-Oncogênicas c-myc/genética , Timócitos/metabolismo , Timo/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/genética , Animais , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismoRESUMO
Toll-like receptors (TLRs) play an important role in immune responses to pathogens by transducing signals in innate immune cells in response to microbial products. TLRs are also expressed on B cells, and TLR signaling in B cells contributes to antibody-mediated immunity and autoimmunity. The SYK tyrosine kinase is essential for signaling from the B cell antigen receptor (BCR), and thus for antibody responses. Surprisingly, we find that it is also required for B cell survival, proliferation, and cytokine secretion in response to signaling through several TLRs. We show that treatment of B cells with lipopolysaccharide, the ligand for TLR4, results in SYK activation and that this is dependent on the BCR. Furthermore, we show that B cells lacking the BCR are also defective in TLR-induced B cell activation. Our results demonstrate that TLR4 signals through two distinct pathways, one via the BCR leading to activation of SYK, ERK, and AKT and the other through MYD88 leading to activation of NF-κB.
Assuntos
Linfócitos B/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Quinase Syk/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Feminino , Lipopolissacarídeos/metabolismo , Ativação Linfocitária/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Fungal infections are major causes of morbidity and mortality, especially in immunocompromised individuals. The innate immune system senses fungal pathogens through Syk-coupled C-type lectin receptors (CLRs), which signal through the conserved immune adaptor Card9. Although Card9 is essential for antifungal defense, the mechanisms that couple CLR-proximal events to Card9 control are not well defined. Here, we identify Vav proteins as key activators of the Card9 pathway. Vav1, Vav2, and Vav3 cooperate downstream of Dectin-1, Dectin-2, and Mincle to engage Card9 for NF-κB control and proinflammatory gene transcription. Although Vav family members show functional redundancy, Vav1/2/3-/- mice phenocopy Card9-/- animals with extreme susceptibility to fungi. In this context, Vav3 is the single most important Vav in mice, and a polymorphism in human VAV3 is associated with susceptibility to candidemia in patients. Our results reveal a molecular mechanism for CLR-mediated Card9 regulation that controls innate immunity to fungal infections.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Candida/metabolismo , Candidemia/genética , Imunidade Inata/genética , Lectinas Tipo C/genética , Animais , Antifúngicos/administração & dosagem , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Candida/genética , Candida/patogenicidade , Candidemia/microbiologia , Candidemia/patologia , Humanos , Lectinas Tipo C/metabolismo , Proteínas de Membrana/genética , Camundongos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Adhesion and migration of T cells are controlled by chemokines and by adhesion molecules, especially integrins, and have critical roles in the normal physiological function of T lymphocytes. Using an RNA-mediated interference screen, we identified the WNK1 kinase as a regulator of both integrin-mediated adhesion and T cell migration. We found that WNK1 is a negative regulator of integrin-mediated adhesion, whereas it acts as a positive regulator of migration via the kinases OXSR1 and STK39 and the ion co-transporter SLC12A2. WNK1-deficient T cells home less efficiently to lymphoid organs and migrate more slowly through them. Our results reveal that a pathway previously known only to regulate salt homeostasis in the kidney functions to balance T cell adhesion and migration.
Assuntos
Adesão Celular/genética , Movimento Celular/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Linfócitos T/fisiologia , Animais , Células Cultivadas , Homeostase , Transporte de Íons , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Receptores de Retorno de Linfócitos/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
Follicular B cell survival requires signaling from BAFFR, a receptor for BAFF and the B cell antigen receptor (BCR). This "tonic" BCR survival signal is distinct from that induced by antigen binding and may be ligand-independent. We show that inducible inactivation of the Syk tyrosine kinase, a key signal transducer from the BCR following antigen binding, resulted in the death of most follicular B cells because Syk-deficient cells were unable to survive in response to BAFF. Genetic rescue studies demonstrated that Syk transduces BAFFR survival signals via ERK and PI3 kinase. Surprisingly, BAFFR signaling directly induced phosphorylation of both Syk and the BCR-associated Igα signaling subunit, and this Syk phosphorylation required the BCR. We conclude that the BCR and Igα may be required for B cell survival because they function as adaptor proteins in a BAFFR signaling pathway leading to activation of Syk, demonstrating previously unrecognized crosstalk between the two receptors.
Assuntos
Receptor do Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Fator Ativador de Células B/imunologia , Fator Ativador de Células B/farmacologia , Receptor do Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Antígenos CD79/imunologia , Antígenos CD79/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Imunológicos , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas/genética , Proteínas/imunologia , Proteínas/metabolismo , RNA não Traduzido , Receptor Cross-Talk/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk , Tamoxifeno/farmacologiaRESUMO
Trisomy of human chromosome 21 (Hsa21) results in Down syndrome (DS), a disorder that affects many aspects of physiology, including hematopoiesis. DS children have greatly increased rates of acute lymphoblastic leukemia and acute megakaryoblastic leukemia (AMKL); DS newborns present with transient myeloproliferative disorder (TMD), a preleukemic form of AMKL. TMD and DS-AMKL almost always carry an acquired mutation in GATA1 resulting in exclusive synthesis of a truncated protein (GATA1s), suggesting that both trisomy 21 and GATA1 mutations are required for leukemogenesis. To gain further understanding of how Hsa21 contributes to hematopoietic abnormalities, we examined the Tc1 mouse model of DS, which carries an almost complete freely segregating copy of Hsa21, and is the most complete model of DS available. We show that although Tc1 mice do not develop leukemia, they have macrocytic anemia and increased extramedullary hematopoiesis. Introduction of GATA1s into Tc1 mice resulted in a synergistic increase in megakaryopoiesis, but did not result in leukemia or a TMD-like phenotype, demonstrating that GATA1s and trisomy of approximately 80% of Hsa21 perturb megakaryopoiesis but are insufficient to induce leukemia.
Assuntos
Cromossomos Humanos Par 21/metabolismo , Síndrome de Down/metabolismo , Mielopoese , Anemia Macrocítica/genética , Anemia Macrocítica/metabolismo , Anemia Macrocítica/fisiopatologia , Animais , Cromossomos Humanos Par 21/genética , Modelos Animais de Doenças , Síndrome de Down/genética , Síndrome de Down/fisiopatologia , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Humanos , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/metabolismo , Leucemia Megacarioblástica Aguda/fisiopatologia , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologiaRESUMO
The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal-regulated kinase and protein kinase D1, and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas Proto-Oncogênicas c-vav/fisiologia , Linfócitos T/citologia , Animais , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas c-vav/genéticaRESUMO
Down syndrome (DS) is a genetic disorder arising from the presence of a third copy of human chromosome 21 (Hsa21). Recently, O'Doherty et al. [An aneuploid mouse strain carrying human chromosome 21 with Down syndrome phenotypes. Science 309 (2005) 2033-2037] generated a trans-species aneuploid mouse line (Tc1) that carries an almost complete Hsa21. The Tc1 mouse is the most complete animal model for DS currently available. Tc1 mice show many features that relate to human DS, including alterations in memory, synaptic plasticity, cerebellar neuronal number, heart development and mandible size. Because motor deficits are one of the most frequently occurring features of DS, we have undertaken a detailed analysis of motor behaviour in cerebellum-dependent learning tasks that require high motor coordination and balance. In addition, basic electrophysiological properties of cerebellar circuitry and synaptic plasticity have been investigated. Our results reveal that, compared with controls, Tc1 mice exhibit a higher spontaneous locomotor activity, a reduced ability to habituate to their environments, a different gait and major deficits on several measures of motor coordination and balance in the rota rod and static rod tests. Moreover, cerebellar long-term depression is essentially normal in Tc1 mice, with only a slight difference in time course. Our observations provide further evidence that support the validity of the Tc1 mouse as a model for DS, which will help us to provide insights into the causal factors responsible for motor deficits observed in persons with DS.
Assuntos
Síndrome de Down/fisiopatologia , Desempenho Psicomotor , Animais , Cerebelo/fisiologia , Síndrome de Down/genética , Feminino , Marcha , Depressão Sináptica de Longo Prazo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Força Muscular , Plasticidade NeuronalRESUMO
Homologous sets of transcription factors direct conserved tissue-specific gene expression, yet transcription factor-binding events diverge rapidly between closely related species. We used hepatocytes from an aneuploid mouse strain carrying human chromosome 21 to determine, on a chromosomal scale, whether interspecies differences in transcriptional regulation are primarily directed by human genetic sequence or mouse nuclear environment. Virtually all transcription factor-binding locations, landmarks of transcription initiation, and the resulting gene expression observed in human hepatocytes were recapitulated across the entire human chromosome 21 in the mouse hepatocyte nucleus. Thus, in homologous tissues, genetic sequence is largely responsible for directing transcriptional programs; interspecies differences in epigenetic machinery, cellular environment, and transcription factors themselves play secondary roles.
Assuntos
Cromossomos Humanos Par 21/genética , Regulação da Expressão Gênica , Fatores Nucleares de Hepatócito/metabolismo , Hepatócitos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Cromossomos Humanos Par 21/metabolismo , Modelos Animais de Doenças , Síndrome de Down/genética , Histonas/metabolismo , Humanos , Metilação , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade da Espécie , Sítio de Iniciação de TranscriçãoRESUMO
Down syndrome (DS) is a genetic disorder arising from the presence of a third copy of the human chromosome 21 (Hsa21). Recently, O'Doherty and colleagues in an earlier study generated a new genetic mouse model of DS (Tc1) that carries an almost complete Hsa21. Since DS is the most common genetic cause of mental retardation, we have undertaken a detailed analysis of cognitive function and synaptic plasticity in Tc1 mice. Here we show that Tc1 mice have impaired spatial working memory (WM) but spared long-term spatial reference memory (RM) in the Morris watermaze. Similarly, Tc1 mice are selectively impaired in short-term memory (STM) but have intact long-term memory (LTM) in the novel object recognition task. The pattern of impaired STM and normal LTM is paralleled by a corresponding phenotype in long-term potentiation (LTP). Freely-moving Tc1 mice exhibit reduced LTP 1 h after induction but normal maintenance over days in the dentate gyrus of the hippocampal formation. Biochemical analysis revealed a reduction in membrane surface expression of the AMPAR (alpha-amino-3-hydroxy-5-methyl-4-propionic acid receptor) subunit GluR1 in the hippocampus of Tc1 mice, suggesting a potential mechanism for the impairment in early LTP. Our observations also provide further evidence that STM and LTM for hippocampus-dependent tasks are subserved by parallel processing streams.
Assuntos
Síndrome de Down , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo/fisiologia , Plasticidade Neuronal/fisiologia , Análise de Variância , Animais , Cromossomos Humanos Par 21 , Aprendizagem por Discriminação/fisiologia , Modelos Animais de Doenças , Síndrome de Down/genética , Síndrome de Down/patologia , Síndrome de Down/fisiopatologia , Eletrofisiologia , Comportamento Exploratório/fisiologia , Hipocampo/fisiopatologia , Hipocampo/efeitos da radiação , Humanos , Técnicas In Vitro , Potenciação de Longa Duração/genética , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Reconhecimento Fisiológico de Modelo/fisiologia , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Comportamento Espacial/fisiologia , TrissomiaRESUMO
Expression of the Syk family tyrosine kinase Zap70 is strongly correlated with poor clinical outcome in chronic lymphocytic leukemia, the most common human leukemia characterized by B cell accumulation. The expression of Zap70 may reflect the specific cell of origin of the tumor or may contribute to pathology. Thus, the normal role of Zap70 in B cell physiology is of great interest. While initial studies reported that Zap70 expression in the mouse was limited to T and NK cells, more recent work has shown expression in early B cell progenitors and in splenic B cells, suggesting that the kinase may play a role in the development or activation of B cells. In this study, we show that Zap70 is expressed in all developing subsets of B cells as well as in recirculating B cells, marginal zone B cells and peritoneal B1 cells. Analysis of Zap70-deficient mice shows no unique role for Zap70 in either the development of B cells or in their in vitro and in vivo activation. However, we show that Zap70 can rescue the defective positive selection of immature B cells into the recirculating pool in Syk-deficient mice, demonstrating functional redundancy between these two kinases.
Assuntos
Linfócitos B/metabolismo , Diferenciação Celular , Proteína-Tirosina Quinase ZAP-70/metabolismo , Animais , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/citologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Sinalização do Cálcio/fisiologia , Proliferação de Células , Citometria de Fluxo , Haptenos , Hemocianinas/imunologia , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Cavidade Peritoneal/citologia , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Baço/citologia , Baço/imunologia , Quinase Syk , Vacinação , Proteína-Tirosina Quinase ZAP-70/deficiência , Proteína-Tirosina Quinase ZAP-70/genéticaRESUMO
Cytokines are important in adult hematopoiesis, yet their function in embryonic hematopoiesis has been largely unexplored. During development, hematopoietic stem cells (HSCs) are found in the aorta-gonad-mesonephros (AGM) region, yolk sac (YS), and placenta and require the Runx1 transcription factor for their normal generation. Since IL-3 is a Runx1 target and this cytokine acts on adult hematopoietic cells, we examined whether IL-3 affects HSCs in the mouse embryo. Using Runx1 haploinsufficient mice, we show that IL-3 amplifies HSCs from E11 AGM, YS, and placenta. Moreover, we show that IL-3 mutant embryos are deficient in HSCs and that IL-3 reveals the presence of HSCs in the AGM and YS prior to the stage at which HSCs are normally detected. Thus, our studies support an unexpected role for IL-3 during development and strongly suggest that IL-3 functions as a proliferation and/or survival factor for the earliest HSCs in the embryo.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Desenvolvimento Embrionário , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Interleucina-3/fisiologia , Animais , Aorta/citologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/deficiência , Gônadas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Interleucina-3/genética , Interleucina-3/farmacologia , Mesonefro/citologia , Camundongos , Placenta/citologia , Saco Vitelino/citologiaRESUMO
Aneuploidies are common chromosomal defects that result in growth and developmental deficits and high levels of lethality in humans. To gain insight into the biology of aneuploidies, we manipulated mouse embryonic stem cells and generated a trans-species aneuploid mouse line that stably transmits a freely segregating, almost complete human chromosome 21 (Hsa21). This "transchromosomic" mouse line, Tc1, is a model of trisomy 21, which manifests as Down syndrome (DS) in humans, and has phenotypic alterations in behavior, synaptic plasticity, cerebellar neuronal number, heart development, and mandible size that relate to human DS. Transchromosomic mouse lines such as Tc1 may represent useful genetic tools for dissecting other human aneuploidies.
Assuntos
Aneuploidia , Cromossomos Humanos Par 21 , Modelos Animais de Doenças , Síndrome de Down , Engenharia Genética , Camundongos Transgênicos , Animais , Comportamento Animal , Encéfalo/patologia , Contagem de Células , Linhagem Celular , Quimera , Síndrome de Down/genética , Síndrome de Down/fisiopatologia , Embrião de Mamíferos/citologia , Ossos Faciais/patologia , Feminino , Expressão Gênica , Marcadores Genéticos , Cardiopatias Congênitas/embriologia , Hipocampo/fisiopatologia , Humanos , Potenciação de Longa Duração , Ativação Linfocitária , Masculino , Aprendizagem em Labirinto , Memória , Camundongos , Camundongos Endogâmicos , Neurônios/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Crânio/patologia , Células-Tronco , Transmissão Sináptica , Linfócitos T/imunologiaRESUMO
Vav1 is a guanine nucleotide exchange factor (GEF) for Rho-family GTPases, which is activated by tyrosine phosphorylation following TCR stimulation. Vav1-deficient mice have defects in positive and negative selection of thymocytes as well as TCR-induced proliferation in mature T cells, demonstrating a critical role for Vav1 in transducing TCR signals. Binding of phospholipids to the PH domain of Vav1 has been proposed to regulate its GEF activity in vitro. To test this model in vivo, we have generated mice carrying a point mutation in the PH domain of Vav1, and we show that they have defects in T cell development and activation. We demonstrate that the mutation affects the function of Vav1 as a GEF and perturbs PI3K-dependent pathways downstream of Vav1. Unexpectedly, the mutation selectively affects TCR-induced proliferation of CD4(+) but not CD8(+) T cells, demonstrating differences in the wiring of TCR signaling pathways between the two lineages.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Proteínas de Ciclo Celular/imunologia , Linhagem da Célula/imunologia , Ativação Linfocitária/imunologia , Proteínas Proto-Oncogênicas/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Ciclo Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células , Citometria de Fluxo , Immunoblotting , Camundongos , Camundongos Transgênicos , Mutação , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-vav , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Stimulation of FcepsilonRI, the high affinity IgE receptor of mast cells results in the rapid binding of the Syk tyrosine kinase to cytoplasmic domains of FcepsilonRI and to its subsequent activation. Syk plays an essential role in signal transduction from FcepsilonRI as shown by Syk-deficient mast cells, which are defective in receptor-induced degranulation, cytokine synthesis, and intracellular pathways. However the mechanism by which Syk activates these pathways remains unclear. Activation of Syk is associated with its phosphorylation on several tyrosine residues, including the linker tyrosines Tyr317, Tyr342, and Tyr346. These residues have been proposed to play important roles in the transduction of signals by binding to other signaling proteins. To test these hypotheses in primary murine mast cells, we used retroviral infection of Syk-deficient mast cells to generate cells expressing Syk proteins bearing mutations in the linker tyrosines. We show that Tyr342 and Tyr346 contribute positively to the function of Syk and have both overlapping as well as distinct functions. Mutations in either Tyr342 or Tyr346 alone had no effect on FcepsilonRI-induced degranulation or calcium flux, whereas mutation of both residues caused a significant reduction in both pathways. In contrast, phosphorylation of PLCgamma1, PLCgamma2, and Vav1 was strongly decreased by a mutation in Tyr342 alone, whereas phosphorylation of ERK and Akt was more dependent on Tyr346. Finally we show that Tyr317 functions as a negative regulatory site and that its mutation can partially compensate for the loss of both Tyr342 and Tyr346.
Assuntos
Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/fisiologia , Mastócitos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/fisiologia , Receptores de IgE/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Densitometria , Relação Dose-Resposta a Droga , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Mastócitos/citologia , Camundongos , Mutação , Células NIH 3T3 , Fosfolipase C gama , Fosforilação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Retroviridae/genética , Transdução de Sinais , Quinase Syk , Fatores de Tempo , Fosfolipases Tipo C/metabolismo , Tirosina/químicaRESUMO
The Rac1 guanosine triphosphatase (GTPase) has been implicated in multiple cellular functions, including actin dynamics, proliferation, apoptosis, adhesion, and migration resulting from signaling by multiple receptors, including the B cell antigen receptor (BCR). We used conditional gene targeting to generate mice with specific Rac1 deficiency in the B cell lineage. In the absence of both Rac1 and the highly related Rac2, B cell development was almost completely blocked. Both GTPases were required to transduce BCR signals leading to proliferation, survival and up-regulation of BAFF-R, a receptor for BAFF, a key survival molecule required for B cell development and maintenance.