RESUMO
BUB-related 1 (BubR1) encoded by Budding Uninhibited by Benzimidazole 1B (BUB1B) is a crucial mitotic checkpoint protein ensuring proper segregation of chromosomes during mitosis. Mutations of BUB1B are responsible for mosaic variegated aneuploidy (MVA), a human congenital disorder characterized by extensive abnormalities in chromosome number. Although microcephaly is a prominent feature of MVA carrying the BUB1B mutation, how BubR1 deficiency disturbs neural progenitor proliferation and neuronal output and leads to microcephaly is unknown. Here we show that conditional loss of BubR1 in mouse cerebral cortex recapitulates microcephaly. BubR1-deficient cortex includes a strikingly reduced number of late-born, but not of early-born, neurons, although BubR1 expression is substantially reduced from an early stage. Importantly, absence of BubR1 decreases the proportion of neural progenitors in mitosis, specifically in metaphase, suggesting shortened mitosis owing to premature chromosome segregation. In the BubR1 mutant, massive apoptotic cell death, which is likely due to the compromised genomic integrity that results from aberrant mitosis, depletes progenitors and neurons during neurogenesis. There is no apparent alteration in centrosome number, spindle formation or primary cilia, suggesting that the major effect of BubR1 deficiency on neural progenitors is to impair the mitotic checkpoint. This finding highlights the importance of the mitotic checkpoint in the pathogenesis of microcephaly. Furthermore, the ependymal cell layer does not form in the conditional knockout, revealing an unrecognized role of BubR1 in assuring the integrity of the ventricular system, which may account for the presence of hydrocephalus in some patients.
Assuntos
Proteínas de Ciclo Celular/genética , Microcefalia/genética , Mitose/genética , Neurogênese/genética , Proteínas Serina-Treonina Quinases/genética , Alelos , Animais , Apoptose/genética , Proteínas de Ciclo Celular/deficiência , Proliferação de Células/genética , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/fisiopatologia , Segregação de Cromossomos/genética , Modelos Animais de Doenças , Humanos , Camundongos , Microcefalia/metabolismo , Microcefalia/fisiopatologia , Mosaicismo , Mutação/genética , Neurônios/metabolismo , Neurônios/patologia , Proteínas Serina-Treonina Quinases/deficiência , Fuso Acromático/genética , Fuso Acromático/patologiaRESUMO
The spindle assembly checkpoint is a cellular surveillance mechanism that functions to ensure faithful chromosome segregation during mitosis. Failure of this checkpoint can result in aneuploidy, a state of having abnormal numbers of chromosomes. Most human cancers consist of aneuploid cells, but it is unclear if the aneuploidy is a cause or a consequence of tumorigenesis. Over recent years, mouse models for spindle assembly checkpoint failure have been generated to investigate the biological relevance of the different spindle assembly checkpoint genes and the pathologies associated with chromosome number instability. Most of these models exhibit susceptibility to carcinogenesis. Moreover, one model has led to the identification of the spindle checkpoint protein BubR1 as a regulator of the normal aging process.
Assuntos
Envelhecimento/genética , Genes cdc , Mitose , Neoplasias/etiologia , Neoplasias/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Fuso Acromático/fisiologia , Ubiquitina-Proteína LigasesRESUMO
CBP can function as a tumor suppressor, but the mechanisms that govern oncogenesis in its absence are unknown. Here we show that CBP inactivation in mouse thymocytes leads to lymphoma. Although CBP has been implicated in the transactivation functions of p53, development of these tumors does not seem to involve loss of p53 activity. CBP-null tumors show reduced levels of p27Kip1 and increased levels of cyclin E and Skp2, two oncoproteins that can promote p27Kip1 proteolysis. Reduction of p27Kip1 by introduction of a p27Kip1-null allele into CBP knockout mice accelerates lymphomagenesis and seems to obviate the requirement for Skp2 and cyclin E upregulation. These data suggest that CBP loss mediates lymphomagenesis in cooperation with a mechanism that reduces p27Kip1 abundance.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Linfoma de Células T/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras , Linfócitos T/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteína de Ligação a CREB , Clonagem Molecular , Ciclina E/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Dano ao DNA/fisiologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes myc/fisiologia , Predisposição Genética para Doença , Linfoma de Células T/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch , Proteínas Quinases Associadas a Fase S/metabolismo , Linfócitos T/patologia , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/fisiologiaRESUMO
B7-H4 is an immunoglobulin superfamily molecule and shown to be inhibitory for T-cell responses. To explore physiologic roles of B7-H4, we created B7-H4-deficient (KO) mice by genetic targeting. B7-H4KO mice are healthy and their T- and B-cell responses to polyclonal antigens are in normal range. However, B7-H4KO mice are more resistant to infection by Listeria monocytogenes than their littermates. Within 3 days after infection, bacterial colonies in livers and spleens are significantly lower than the controls, suggesting a role of B7-H4 in enhancing innate immunity. Further studies demonstrate that neutrophils increase in peripheral organs of B7-H4KO mice more so than their littermates but their bactericidal functions remain unchanged. Augmented innate resistance is completely dependent on neutrophils, even in the absence of adaptive immunity. In vitro B7-H4 inhibits the growth of bone marrow-derived neutrophil progenitors, suggesting an inhibitory function of B7-H4 in neutrophil expansion. Our results identify B7-H4 as a negative regulator of the neutrophil response to infection and provide a new target for manipulation of innate immunity.
Assuntos
Antígeno B7-1/genética , Antígeno B7-1/imunologia , Listeriose/imunologia , Neutrófilos/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/microbiologia , Células da Medula Óssea/citologia , Antígeno CD11b/metabolismo , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Neutrófilos/microbiologia , Fagocitose/imunologia , Receptores de Quimiocinas/metabolismo , Explosão Respiratória/imunologia , Linfócitos T/imunologia , Linfócitos T/microbiologia , Inibidor 1 da Ativação de Células T com Domínio V-SetRESUMO
Aging is a highly complex biological process that is believed to involve multiple mechanisms. Mice that have small amounts of the mitotic checkpoint protein BubR1 age much faster than normal mice, but whether other mitotic checkpoint genes function to prevent the early onset of aging is unknown. In this study, we show that several aging-associated phenotypes appear early in mice that are double haploinsufficient for the mitotic checkpoint genes Bub3 and Rae1 but not in mice that are single haploinsufficient for these genes. Mouse embryonic fibroblasts (MEFs) from Bub3/Rae1 haploinsufficient mice undergo premature senescence and accumulate high levels of p19, p53, p21, and p16, whereas MEFs from single haploinsufficient mice do not. Furthermore, although BubR1 hypomorphic mice have less aneuploidy than Bub3/Rae1 haploinsufficient mice, they age much faster. Our findings suggest that early onset of aging-associated phenotypes in mice with mitotic checkpoint gene defects is linked to cellular senescence and activation of the p53 and p16 pathways rather than to aneuploidy.
Assuntos
Senilidade Prematura/genética , Proteínas de Ciclo Celular/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Aneuploidia , Animais , Proteínas Cromossômicas não Histona , Haplótipos , Camundongos , Camundongos Knockout , Camundongos Mutantes , Mutação , Neoplasias/fisiopatologia , Fenótipo , Proteínas de Ligação a Poli-ADP-RiboseRESUMO
The global transcriptional coactivators CREB-binding protein (CBP) and the closely related p300 interact with over 312 proteins, making them among the most heavily connected hubs in the known mammalian protein-protein interactome. It is largely uncertain, however, if these interactions are important in specific cell lineages of adult animals, as homozygous null mutations in either CBP or p300 result in early embryonic lethality in mice. Here we describe a Cre/LoxP conditional p300 null allele (p300flox) that allows for the temporal and tissue-specific inactivation of p300. We used mice carrying p300flox and a CBP conditional knockout allele (CBPflox) in conjunction with an Lck-Cre transgene to delete CBP and p300 starting at the CD4- CD8- double-negative thymocyte stage of T-cell development. Loss of either p300 or CBP led to a decrease in CD4+ CD8+ double-positive thymocytes, but an increase in the percentage of CD8+ single-positive thymocytes seen in CBP mutant mice was not observed in p300 mutants. T cells completely lacking both CBP and p300 did not develop normally and were nonexistent or very rare in the periphery, however. T cells lacking CBP or p300 had reduced tumor necrosis factor alpha gene expression in response to phorbol ester and ionophore, while signal-responsive gene expression in CBP- or p300-deficient macrophages was largely intact. Thus, CBP and p300 each supply a surprising degree of redundant coactivation capacity in T cells and macrophages, although each gene has also unique properties in thymocyte development.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Proteína de Ligação a CREB/fisiologia , Proteína p300 Associada a E1A/fisiologia , Timo/crescimento & desenvolvimento , Alelos , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Proteína de Ligação a CREB/genética , Diferenciação Celular , Proteína p300 Associada a E1A/genética , Deleção de Genes , Expressão Gênica , Ionóforos/farmacologia , Contagem de Linfócitos , Linfoma de Células T/genética , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Ésteres de Forbol/farmacologia , Receptores de Antígenos de Linfócitos T , Baço/citologia , Baço/crescimento & desenvolvimento , Timo/citologia , Transcrição Gênica , Fator de Necrose Tumoral alfa/genéticaRESUMO
Transforming growth factor beta-inducible early gene 1 (TIEG1) is a member of the Kruppel-like transcription factor family. To understand the physiological role of TIEG1, we generated TIEG(-/-) (null) mice and found that the TIEG(-/-) mice had increased osteoblast numbers with no increased bone formation parameters. However, when calvarial osteoblasts (OBs) were isolated from neonatal TIEG(-/-) and TIEG(+/+) mice and cultured in vitro, the TIEG(-/-) cells displayed reduced expression of important OB differentiation markers. When the OBs were differentiated in vitro by treatment with bone morphogenic protein 2, the OBs from TIEG(+/+) calvaria displayed several mineralized nodules in culture, whereas those from TIEG(-/-) mice showed no nodules. To characterize the OBs' ability to support osteoclast differentiation, the OBs from TIEG(+/+) and TIEG(-/-) mice were cultured with marrow and spleen cells from TIEG(+/+) mice. Significantly fewer osteoclasts developed when TIEG(-/-) OBs were used to support osteoclast differentiation than when TIEG(+/+) OBs were used. Examination of gene expression in the TIEG(-/-) OBs revealed decreased RANKL and increased OPG expression compared to TIEG(+/+) OBs. The addition of RANKL to these cocultures only partially restored the ability of TIEG(-/-) OBs to support osteoclast differentiation, whereas M-CSF alone or combined with RANKL had no additional effect on osteoclast differentiation. We conclude from these data that TIEG1 expression in OBs is critical for both osteoblast-mediated mineralization and osteoblast support of osteoclast differentiation.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Osteoblastos/citologia , Osteoclastos/citologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2 , Calcificação Fisiológica/fisiologia , Proteínas de Transporte/metabolismo , Proliferação de Células , Técnicas de Cocultura , Proteínas de Ligação a DNA/genética , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoprotegerina , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral , Baço/citologia , Baço/metabolismo , Fatores de Transcrição/genéticaRESUMO
gly96/IEX 1 is a growth- and apoptosis-regulating, immediate early gene that is widely expressed in epithelial and vascular tissues. In vascular tissues, expression of the gene is induced by mechanical stretch, and overexpression of the gene prevents injury-induced vascular smooth muscle hypertrophy and neointimal hyperplasia. We now show that deletion of the gly96/IEX-1 gene in mice is associated with development of elevated blood pressure, cardiac hypertrophy, and diminished fractional shortening of the left ventricle. Systolic blood pressure in conscious male gly96/IEX-1-/- mice is 20-25 mmHg higher than in gly96/IEX-1+/+ mice. Serum and/or urine concentrations of sodium, potassium, creatinine, angiotensin II, corticosterone, aldosterone, epinephrine, norepinephrine, prostaglandin E2, thromboxane B2, prostaglandin-6-keto-1alpha, nitrites and nitrates, cAMP, and cGMP are normal in gly96/IEX-1-/- mice. Alterations in dietary sodium intake do not alter blood pressure in gly96/IEX-1-/- mice. Aortic mRNAs for endothelial nitric oxide synthase, guanylate cyclase-alpha, and cGMP kinase-1 are increased in gly96/IEX-1-/- mice. Treatment with Nomega-nitro-L-arginine methyl ester or L-arginine does not alter blood pressure in gly96/IEX-1-/- mice. Gly96/IEX-1-/- mice respond to infused sodium nitroprusside with decrements in blood pressure similar to those seen in wild-type littermate mice. In contrast to gly96/IEX-1 transgenic mice that have abnormalities in immune function, gly96/IEX-1-/- mice have normal lymphoid tissue architecture and a normal complement of T and B cells in lymphoid tissues. Ablation of the gly96/IEX-1 gene results in hypertension and cardiac hypertrophy, suggesting a novel role for this gene in cardiovascular physiology.
Assuntos
Cardiomegalia/genética , Hipertensão/genética , Proteínas Imediatamente Precoces/genética , Animais , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/farmacologia , Aorta/enzimologia , Pressão Sanguínea , Cardiomegalia/enzimologia , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Hipertensão/enzimologia , Hipertensão/prevenção & controle , Proteínas Imediatamente Precoces/deficiência , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III , Nitroprussiato/administração & dosagem , Nitroprussiato/farmacologia , RNA Mensageiro/metabolismo , Disfunção Ventricular Esquerda/enzimologia , Disfunção Ventricular Esquerda/genéticaRESUMO
Macromolecules are transported in and out of the nucleus through nuclear pores. It is poorly understood how these megadalton conduits support nucleocytoplasmic traffic during genetic reprogramming associated with cell commitment to a specific lineage. Murine embryonic stem cells were differentiated into cardiomyocytes within embryoid bodies, and contracting cells expressing myocardial-specific proteins were isolated from the mesodermal layer. Compared with postmitotic cardiac cells from heart muscle, these proliferative and differentiating stem cell-derived cardiomyocytes demonstrated a significantly lower density of nuclear pores. At nanoscale resolution, the pore channel was commonly unoccupied in heart muscle-isolated cardiac cells, yet a dense material, presumably the central transporter, protruded toward the cytosolic face of the nuclear pore complex in stem cell-derived cardiomyocytes. Stem cell-derived cardiac cells distributed the nuclear transport factor Ran in the nucleus, decreased the number of spare nuclear pore complexes from the cytosolic annulate lamellae reservoir, and expressed a set of nucleoporins, NUP214, NUP358, NUP153, and p62, involved in nuclear transport. Stem cell-derived cardiomyocytes secured transport of nuclear constitutive proteins, cardiogenic transcription factors, and cell cycle regulators, including the prototypic histone H1, myocyte enhancer binding factor 2, and p53. Thus, differentiating stem cell-derived cardiomyocytes undergo structural adaptation and mobilize nuclear transport regulators in support of nucleocytoplasmic communication during commitment to mature cardiac lineage.
Assuntos
Miócitos Cardíacos/fisiologia , Poro Nuclear/fisiologia , Células-Tronco/citologia , Animais , Transporte Biológico , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Linhagem da Célula , Camundongos , Microfibrilas/metabolismo , Microfibrilas/ultraestrutura , Microscopia Eletrônica , Microscopia de Contraste de Fase , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , Células-Tronco/ultraestrutura , Fatores de Tempo , Proteína ran de Ligação ao GTP/metabolismoRESUMO
B7-H3 is a cell surface molecule in the immunoglobulin superfamily that is frequently upregulated in response to autoantigens and pathogens during host T cell immune responses. However, B7-H3's role in the differential regulation of T cell subsets remains largely unknown. Therefore, we constructed a new B7-H3 deficient mouse strain (B7-H3 KO) and evaluated the functions of B7-H3 in the regulation of Th1, Th2, and Th17 subsets in experimental autoimmune encephalomyelitis (EAE), experimental asthma, and collagen-induced arthritis (CIA); these mouse models were used to predict human immune responses in multiple sclerosis, asthma, and rheumatoid arthritis, respectively. Here, we demonstrate that B7-H3 KO mice have significantly less inflammation, decreased pathogenesis, and limited disease progression in both EAE and CIA mouse models when compared with littermates; these results were accompanied by a decrease in IFN-γ and IL-17 production. In sharp contrast, B7-H3 KO mice developed severe ovalbumin (OVA)-induced asthma with characteristic infiltrations of eosinophils in the lung, increased IL-5 and IL-13 in lavage fluid, and elevated IgE anti-OVA antibodies in the blood. Our results suggest B7-H3 has a costimulatory function on Th1/Th17 but a coinhibitory function on Th2 responses. Our studies reveal that B7-H3 could affect different T cell subsets which have important implications for regulating pathogenesis and disease progression in human autoimmune disease.
Assuntos
Artrite Experimental/patologia , Asma/patologia , Antígenos B7/fisiologia , Encefalomielite Autoimune Experimental/patologia , Inflamação/patologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia , Animais , Apoptose , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Asma/induzido quimicamente , Asma/imunologia , Western Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Inflamação/induzido quimicamente , Inflamação/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CREB-binding protein (CBP) and its para-log p300 are transcriptional coactivators that physically or functionally interact with over 320 mammalian and viral proteins, including 36 that are essential for B cells in mice. CBP and p300 are generally considered limiting for transcription, yet their roles in adult cell lineages are largely unknown since homozygous null mutations in either gene or compound heterozygosity cause early embryonic lethality in mice. We tested the hypotheses that CBP and p300 are limiting and that each has unique properties in B cells, by using mice with Cre/LoxP conditional knockout alleles for CBP (CBP(flox)) and p300 (p300(flox)), which carry CD19(Cre) that initiates floxed gene recombination at the pro-B-cell stage. CD19(Cre)-mediated loss of CBP or p300 led to surprisingly modest deficits in B-cell numbers, whereas inactivation of both genes was not tolerated by peripheral B cells. There was a moderate decrease in B-cell receptor (BCR)-responsive gene expression in CBP or p300 homozygous null B cells, suggesting that CBP and p300 are essential for this signaling pathway that is crucial for B-cell homeostasis. These results indicate that individually CBP and p300 are partially limiting beyond the pro-B-cell stage and that other coactivators in B cells cannot replace their combined loss.
Assuntos
Linfócitos B/citologia , Proteína de Ligação a CREB/fisiologia , Fatores de Transcrição de p300-CBP/fisiologia , Alelos , Animais , Antígenos CD19 , Homeostase , Integrases , Contagem de Linfócitos , CamundongosRESUMO
Forkhead transcription factors FOXO1 (FKHR), FOXO3a (FKHRL1), and FOXO4 (AFX) play a pivotal role in tumor suppression by inducing growth arrest and apoptosis. Loss of function of these factors due to phosphorylation and proteasomal degradation has been implicated in cell transformation and malignancy. However, the ubiquitin ligase necessary for the ubiquitination of the FOXO factors and the relevance of this regulation to tumorigenesis have not been characterized. Here we demonstrate that Skp2, an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex, interacts with, ubiquitinates, and promotes the degradation of FOXO1. This effect of Skp2 requires Akt-specific phosphorylation of FOXO1 at Ser-256. Moreover, expression of Skp2 inhibits transactivation of FOXO1 and abolishes the inhibitory effect of FOXO1 on cell proliferation and survival. Furthermore, expression of the FOXO1 protein is lost in a mouse lymphoma model, where Skp2 is overexpressed. These data suggest that the Skp2-promoted proteolysis of FOXO1 plays a key role in tumorigenesis.
Assuntos
Linfoma/prevenção & controle , Proteínas Quinases Associadas a Fase S/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Ubiquitina/metabolismo , Células 3T3 , Animais , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Chlorocebus aethiops , Inibidor de Quinase Dependente de Ciclina p27 , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Humanos , Linfoma/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Linfócitos T/fisiologia , Fatores de Transcrição/genética , Transfecção , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Upon systemic activation by antigens, CD8(+), but not CD4(+), T cells selectively accumulate and undergo apoptosis in the liver, a mechanism associated with the induction of hepatic tolerance and chronic infection. The molecular basis for CD8(+) T cell preference in this process is unknown. We prepared B7-H1-deficient mice by gene targeting and found spontaneous accumulation of CD8(+) T cells in the liver while CD4(+) T cell levels remained normal. Moreover, antigen-driven CD8(+) T cells proliferated normally while apoptotic levels during the contraction phase was selectively impaired in the liver, leading to accelerated hepatocyte damage in experimental autoimmune hepatitis. Therefore, B7-H1 is a key protein selectively regulating the accumulation and deletion of intrahepatic CD8(+) T cells and may also contribute to inflammation, autoimmune diseases, and tolerance in the liver.
Assuntos
Apoptose , Antígeno B7-1/fisiologia , Proteínas Sanguíneas , Linfócitos T CD8-Positivos/imunologia , Fígado/imunologia , Peptídeos , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Antígeno B7-1/genética , Antígeno B7-H1 , Linfócitos T CD8-Positivos/patologia , Movimento Celular , Hepatite Animal/imunologia , Hepatite Animal/patologia , Cinética , Fígado/citologia , Fígado/patologia , Ativação Linfocitária , Glicoproteínas de Membrana , Camundongos , Camundongos KnockoutRESUMO
Interactions of LIGHT and its receptors, herpesvirus entry mediator on T cells and lymphotoxin beta receptor on stromal cells, are implicated in the regulation of lymphoid organogenesis, costimulation of T cells, and activation of dendritic cells. In this work we report that LIGHT-deficient mice had normal lymphoid organs with T cells and APCs that normally responded to Ag stimulation and normally stimulated T cells. Although the number of Vbeta8(+) T cells in naive LIGHT(+/+) and LIGHT(-/-) mice was identical, Vbeta8(+)CD8(+) T cell proliferation in response to staphylococcal enterotoxin B was significantly lower in LIGHT(-/-) mice. Consistently, induction and cytokine secretion of CD8(+) CTL to MHC class I-restricted peptide was also reduced in LIGHT(-/-) mice. However, the proliferative response of Vbeta8(+)CD4(+) T cells to staphylococcal enterotoxin B was comparable in LIGHT(-/-) and LIGHT(+/+) mice. Our results suggest that LIGHT is required for activation of normal CD8(+) T cells but not CD4(+) T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética , Animais , Formação de Anticorpos/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Epitopos de Linfócito T/imunologia , Feminino , Marcação de Genes/métodos , Haptenos , Hemocianinas/administração & dosagem , Hemocianinas/imunologia , Ativação Linfocitária/genética , Tecido Linfoide/imunologia , Tecido Linfoide/patologia , Receptor beta de Linfotoxina , Linfotoxina-alfa/genética , Linfotoxina-beta , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Picratos/imunologia , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Acrosome biogenesis involves the transport and fusion of Golgi-derived proacrosomal vesicles along the acroplaxome, an F-actin/keratin 5-containing cytoskeletal plate anchored to the spermatid nucleus. A significant issue is whether the acroplaxome develops in acrosomeless mutant mice. Male mice with a Hrb null mutation are infertile and both spermatids and sperm are round-headed and lack an acrosome. Hrb, a protein that contains several NPF motifs (Asn-Pro-Phe) and interacts with proteins with Eps15 homology domains, is regarded as critical for the docking and/or fusion of Golgi-derived proacrosomal vesicles. Here we report that the lack of an acrosome in Hrb mutant spermatids does not prevent the development of the acroplaxome. Yet the acroplaxome in the mutant contains F-actin but is deficient in keratin 5. We also show that the actin-based motor protein myosin Va and its receptor, Rab27a/b, known to be involved in vesicle transport, are present in the Golgi and Golgi-derived proacrosomal vesicles in wild-type and Hrb mutant mouse spermatids. In the Hrb mutant, myosin-Va-bound proacrosome vesicles tether to the acroplaxome, where they flatten and form a flat sac, designated pseudoacrosome. As spermiogenesis advances, round-shaped spermatid nuclei of the mutant display several nuclear protrusions, designated nucleopodes. Nucleopodes are consistently found at the acroplaxome- pseudoacrosome site. Our findings support the interpretation that the acroplaxome provides a focal point for myosin-Va/ Rab27a/b-driven proacrosomal vesicles to accumulate, coalesce, and form an acrosome in wild-type spermatids and a pseudoacrosome in Hrb mutant spermatids. We suggest that nucleopodes develop at a site where a keratin 5-deficient acroplaxome may not withstand tension forces operating during spermatid nuclear shaping.