RESUMO
White adipose tissue (WAT) expansion in obesity occurs through enlargement of preexisting adipocytes (hypertrophy) and through formation of new adipocytes (adipogenesis). Adipogenesis results in WAT hyperplasia, smaller adipocytes and a metabolically more favourable form of obesity. How obesogenic WAT hyperplasia is induced remains, however, poorly understood. Here, we show that the mechanosensitive cationic channel Piezo1 mediates diet-induced adipogenesis. Mice lacking Piezo1 in mature adipocytes demonstrated defective differentiation of preadipocyte into mature adipocytes when fed a high fat diet (HFD) resulting in larger adipocytes, increased WAT inflammation and reduced insulin sensitivity. Opening of Piezo1 in mature adipocytes causes the release of the adipogenic fibroblast growth factor 1 (FGF1), which induces adipocyte precursor differentiation through activation of the FGF-receptor-1. These data identify a central feed-back mechanism by which mature adipocytes control adipogenesis during the development of obesity and suggest Piezo1-mediated adipocyte mechano-signalling as a mechanism to modulate obesity and its metabolic consequences.
Assuntos
Adipócitos/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Canais Iônicos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Adipogenia/fisiologia , Tecido Adiposo Branco/metabolismo , Animais , Calorimetria , Células Cultivadas , Feminino , Fator 1 de Crescimento de Fibroblastos/genética , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Insulina/sangue , Interleucina-6/sangue , Canais Iônicos/genética , Masculino , Camundongos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state.
Assuntos
Células-Tronco Embrionárias/citologia , Genes myc , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Blastocisto/metabolismo , Proliferação de Células , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Renal dendritic cells are a major component of the renal mononuclear phagocytic system. In the renal interstitium, these cells are exposed to an osmotic gradient, mainly sodium, whose concentration progressively increases towards inner medulla. Renal allograft rejection affects predominantly the cortex, suggesting a protective role of the renal medullary micromilieu. Whether osmolar variations can modulate the function of renal dendritic cells is currently undefined. Considering the central role of dendritic cells in promoting allorejection, we tested whether the biophysical micromilieu, particularly the interstitial osmotic gradient, influences their alloreactivity. There was a progressive depletion of leukocytes towards the medulla of homeostatic kidney. Only macrophages opposed this tendency. Flow cytometry of homeostatic and post-transplant medullary dendritic cells revealed a switch towards a macrophage-like phenotype. Similarly, bone marrow-derived dendritic cells developed ex vivo in sodium chloride-enriched medium acquired a M2-like signature. Microarray analysis of allotransplant dendritic cells posed a medullary downregulation of genes mainly involved in alloantigen recognition. Gene expression profiles of both medullary dendritic cells and bone marrow-derived dendritic cells matured in hyperosmolar medium had an overlap with the macrophage M2 signature. Thus, the medullary environment inhibits an alloimmune response by modulating the phenotype and function of dendritic cells.
Assuntos
Microambiente Celular , Células Dendríticas/imunologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Fenótipo , Animais , Células da Medula Óssea , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Rejeição de Enxerto/patologia , Homeostase , Imunidade Celular/genética , Imunidade Celular/imunologia , Transplante de Rim , Contagem de Leucócitos , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Concentração Osmolar , Receptores de Superfície Celular/metabolismo , Cloreto de Sódio/farmacologia , TranscriptomaRESUMO
DNA repair and G2-phase cell cycle checkpoint responses are involved in the manifestation of hyper-radiosensitivity (HRS). The low-dose radioresponse of MSH2 isogenic endometrial carcinoma cell lines was examined. Defects in cell cycle checkpoint activation and the DNA damage response in irradiated cells (0.2 Gy) were evaluated. HRS was expressed solely in MSH2+ cells and was associated with efficient activation of the early G2-phase cell cycle checkpoint. Maintenance of the arrest was associated with persistent MRE11, γH2AX, RAD51 foci at 2 h after irradiation. Persistent MRE11 and RAD51 foci were also evident 24 h after 0.2 Gy. MSH2 significantly enhances cell radiosensitivity to low dose IR.
Assuntos
Carcinoma/metabolismo , Sobrevivência Celular/efeitos da radiação , Reparo de Erro de Pareamento de DNA , Neoplasias do Endométrio/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Expressão Gênica , Histonas/metabolismo , Humanos , Proteína Homóloga a MRE11 , Proteína 2 Homóloga a MutS/genética , Tolerância a Radiação , Reparo de DNA por RecombinaçãoRESUMO
Cytomegalovirus (CMV) coinfection is associated with infant HIV-1 disease progression and mortality. In a cohort of Kenyan HIV-infected infants, the frequencies of activated (CD38(+) HLA-DR(+)) and apoptosis-vulnerable (CD95(+) Bcl-2(-)) CD4(+) and CD8(+) T cells increased substantially during acute CMV infection. The frequency of activated CD4(+) T cells was strongly associated with both concurrent CMV coinfection (P = 0.001) and HIV-1 viral load (P = 0.05). The frequency of apoptosis-vulnerable cells was also associated with CMV coinfection in the CD4 (P = 0.02) and CD8 (P < 0.001) T cell subsets. Similar observations were made in HIV-exposed uninfected infants. CMV-induced increases in T cell activation and apoptosis may contribute to the rapid disease progression in coinfected infants.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/imunologia , Infecções por HIV/complicações , Infecções por HIV/imunologia , HIV-1 , Ativação Linfocitária , ADP-Ribosil Ciclase 1/análise , Apoptose , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Coinfecção , Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Progressão da Doença , Infecções por HIV/virologia , HIV-1/imunologia , Antígenos HLA-DR/análise , Humanos , Lactente , Quênia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Carga Viral , Receptor fas/biossínteseRESUMO
CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/imunologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Mitógenos/farmacologia , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/metabolismo , Neoplasias/imunologia , Fenótipo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologiaRESUMO
Although CD8(+) T cells play an important role in the containment of adult HIV-1 replication, their role in infant HIV-1 infection is not as well understood. Impaired HIV-specific CD8(+) T cell responses may underlie the persistently high viral loads observed in infants. We examined the frequency and phenotype of infant HIV-specific CD8(+) T cells in 7 HIV-infected antiretroviral therapy-naïve infants during the first 2 years of life, using class I HLA tetramers and IFN-γ-ELISPOT. The frequency (0.088-3.9% of CD3(+)CD8(+) cells) and phenotype (CD27(+)CD28(-), CD45RA(+/-), CD57(+/-), HLA-DR(+), CD95(+)) of infant HIV-specific CD8(+) T cells were similar to reports in adults undergoing early infection. Unlike adults, at 23-24 months post-infection a high frequency of HIV-specific CD8(+) T cells expressed HLA-DR (mean 80%, range 68-85%) and CD95 (mean 88%, range 79-96%), suggesting sustained activation and vulnerability to apoptosis. Despite comparable expansion of HIV-specific CD8(+) T cells of a similar phenotype to adults during early infection, infant T cells failed to contain HIV-1 replication, and remained persistently activated and vulnerable to apoptosis during chronic infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/patogenicidade , Antígenos CD28/metabolismo , Antígenos CD57/metabolismo , HIV-1/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Lactente , Recém-Nascido , Antígenos Comuns de Leucócito/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Receptor fas/metabolismoRESUMO
Megakaryopoiesis is a complex process that involves major cellular and nuclear changes and relies on controlled coordination of cellular proliferation and differentiation. These mechanisms are orchestrated in part by transcriptional regulators. The key hematopoietic transcription factor stem cell leukemia (SCL)/TAL1 is required in early hematopoietic progenitors for specification of the megakaryocytic lineage. These early functions have, so far, prevented full investigation of its role in megakaryocyte development in loss-of-function studies. Here, we report that SCL critically controls terminal megakaryocyte maturation. In vivo deletion of Scl specifically in the megakaryocytic lineage affects all key attributes of megakaryocyte progenitors (MkPs), namely, proliferation, ploidization, cytoplasmic maturation, and platelet release. Genome-wide expression analysis reveals increased expression of the cell-cycle regulator p21 in Scl-deleted MkPs. Importantly, p21 knockdown-mediated rescue of Scl-mutant MkPs shows full restoration of cell-cycle progression and partial rescue of the nuclear and cytoplasmic maturation defects. Therefore, SCL-mediated transcriptional control of p21 is essential for terminal maturation of MkPs. Our study provides a mechanistic link between a major hematopoietic transcriptional regulator, cell-cycle progression, and megakaryocytic differentiation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Megacariócitos/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Trombocitopenia/fisiopatologia , Trombopoese/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células da Medula Óssea/fisiologia , Células da Medula Óssea/ultraestrutura , Divisão Celular/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Citoplasma/fisiologia , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/ultraestrutura , Megacariócitos/ultraestrutura , Camundongos , Microscopia Eletrônica , Poliploidia , Proteínas Proto-Oncogênicas/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Trombocitopenia/patologiaRESUMO
The relationships between normal and leukemic stem/progenitor cells are unclear. We show that in â¼80% of primary human CD34+ acute myeloid leukemia (AML), two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. Both populations have leukemic stem cell (LSC) activity and are hierarchically ordered; one LSC population gives rise to the other. Global gene expression profiling shows the LSC populations are molecularly distinct and resemble normal progenitors but not stem cells. The more mature LSC population most closely mirrors normal granulocyte-macrophage progenitors (GMP) and the immature LSC population a previously uncharacterized progenitor functionally similar to lymphoid-primed multipotential progenitors (LMPPs). This suggests that in most cases primary CD34+ AML is a progenitor disease where LSCs acquire abnormal self-renewal potential.
Assuntos
Células Progenitoras de Granulócitos e Macrófagos/citologia , Leucemia Mieloide Aguda/patologia , Células Progenitoras Linfoides/citologia , Células-Tronco Neoplásicas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Perfilação da Expressão Gênica , Sobrevivência de Enxerto , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Células Progenitoras Linfoides/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/transplante , Transplante Heterólogo/patologia , Adulto JovemRESUMO
Precise spatiotemporal control of Gata1 expression is required in both early hematopoietic progenitors to determine erythroid/megakaryocyte versus granulocyte/monocyte lineage output and in the subsequent differentiation of erythroid cells and megakaryocytes. An enhancer element upstream of the mouse Gata1 IE (1st exon erythroid) promoter, mHS-3.5, can direct both erythroid and megakaryocytic expression. However, loss of this element ablates only megakaryocytes, implying that an additional element has erythroid specificity. Here, we identify a double DNaseI hypersensitive site, mHS-25/6, as having erythroid but not megakaryocytic activity in primary cells. It binds an activating transcription factor complex in erythroid cells where it also makes physical contact with the Gata1 promoter. Deletion of mHS-25/6 or mHS-3.5 in embryonic stem cells has only a modest effect on in vitro erythroid differentiation, whereas loss of both elements ablates both primitive and definitive erythropoiesis with an almost complete loss of Gata1 expression. Surprisingly, Gata2 expression was also concomitantly low, suggesting a more complex interaction between these 2 factors than currently envisaged. Thus, whereas mHS-3.5 alone is sufficient for megakaryocytic development, mHS-3.5 and mHS-25/6 collectively regulate erythroid Gata1 expression, demonstrating lineage-specific differences in Gata1 cis-element use important for development of these 2 cell types.
Assuntos
Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Células Eritroides/metabolismo , Eritropoese/fisiologia , Fator de Transcrição GATA1/biossíntese , Regulação da Expressão Gênica/fisiologia , Megacariócitos/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Células Eritroides/citologia , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Megacariócitos/citologia , Camundongos , Regiões Promotoras Genéticas/fisiologia , Deleção de SequênciaRESUMO
Understanding cancer pathogenesis requires knowledge of not only the specific contributory genetic mutations but also the cellular framework in which they arise and function. Here we explore the clonal evolution of a form of childhood precursor-B cell acute lymphoblastic leukemia that is characterized by a chromosomal translocation generating a TEL-AML1 fusion gene. We identify a cell compartment in leukemic children that can propagate leukemia when transplanted in mice. By studying a monochorionic twin pair, one preleukemic and one with frank leukemia, we establish the lineal relationship between these "cancer-propagating" cells and the preleukemic cell in which the TEL-AML1 fusion first arises or has functional impact. Analysis of TEL-AML1-transduced cord blood cells suggests that TEL-AML1 functions as a first-hit mutation by endowing this preleukemic cell with altered self-renewal and survival properties.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Doenças em Gêmeos , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Pré-Leucemia/patologia , ADP-Ribosil Ciclase 1/análise , Doença Aguda , Animais , Antígenos CD19/análise , Antígenos CD34/análise , Apoptose , Transplante de Medula Óssea , Pré-Escolar , Subunidade alfa 2 de Fator de Ligação ao Core/análise , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Doenças em Gêmeos/genética , Doenças em Gêmeos/metabolismo , Doenças em Gêmeos/patologia , Feminino , Sangue Fetal/transplante , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Células Precursoras de Linfócitos B/química , Células Precursoras de Linfócitos B/fisiologia , Pré-Leucemia/genética , Pré-Leucemia/metabolismo , Recombinação Genética , Transplante Heterólogo , Gêmeos MonozigóticosRESUMO
Human NK cells are divided into CD56(bright)CD16(-) cells and CD56(dim)CD16(+) cells. We tested the hypothesis that CD56(bright) NK cells can differentiate into CD56(dim) cells by prospectively isolating and culturing each NK subset in vitro and in vivo. Our results show that CD56(bright) cells can differentiate into CD56(dim) both in vitro, in the presence of synovial fibroblasts, and in vivo, upon transfer into NOD-SCID mice. In vitro, this differentiation was inhibited by fibroblast growth factor receptor-1 Ab, demonstrating a role of the CD56 and fibroblast growth factor receptor-1 interaction in this process. Differentiated CD56(dim) cells had reduced IFN-gamma production but increased perforin expression and cytolysis of cell line K562 targets. Flow cytometric fluorescent in situ hybridization demonstrated that CD56(bright) NK cells had longer telomere length compared with CD56(dim) NK cells, implying the former are less mature. Our data support a linear differentiation model of human NK development in which immature CD56(bright) NK cells can differentiate into CD56(dim) cells.
Assuntos
Antígeno CD56/biossíntese , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Fibroblastos/imunologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Adulto , Anticorpos Monoclonais/fisiologia , Antígeno CD56/metabolismo , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica , Fibroblastos/citologia , Inibidores do Crescimento/fisiologia , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/metabolismo , Modelos Imunológicos , Estudos Prospectivos , Receptores de Fatores de Crescimento de Fibroblastos/imunologia , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismoRESUMO
Cell migration within a natural context is tightly controlled, often by specific transcription factors. However, the switch from stationary to migratory behavior is poorly understood. Border cells perform a spatially and temporally controlled invasive migration during Drosophila oogenesis. Slbo, a C/EBP family transcriptional activator, is required for them to become migratory. We purified wild-type and slbo mutant border cells as well as nonmigratory follicle cells and performed comparative whole-genome expression profiling, followed by functional tests of the contributions of identified targets to migration. About 300 genes were significantly upregulated in border cells, many dependent on Slbo. Among these, the microtubule regulator Stathmin was strongly upregulated and was required for normal migration. Actin cytoskeleton regulators were also induced, including, surprisingly, a large cluster of "muscle-specific" genes. We conclude that Slbo induces multiple cytoskeletal effectors, and that each contributes to the behavioral changes in border cells.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Movimento Celular/fisiologia , Proteínas de Drosophila/fisiologia , Perfilação da Expressão Gênica , Oogênese/fisiologia , Ovário/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas do Citoesqueleto/fisiologia , Drosophila , Proteínas de Drosophila/genética , Feminino , Oogênese/genética , Ovário/citologia , Ovário/metabolismo , Estatmina/fisiologia , Fatores de Transcrição/genética , Regulação para CimaRESUMO
The DNA-binding hemopoietic zinc finger transcription factor GATA1 promotes terminal megakaryocyte differentiation and restrains abnormal immature megakaryocyte expansion. How GATA1 coordinates these fundamental processes is unclear. Previous studies of synthetic and naturally occurring mutant GATA1 molecules demonstrate that DNA-binding and interaction with the essential GATA1 cofactor FOG-1 (via the N-terminal finger) are required for gene expression in terminally differentiating megakaryocytes and for platelet production. Moreover, acquired mutations deleting the N-terminal 84 amino acids are specifically detected in megakaryocytic leukemia in human Down syndrome patients. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by ectopically expressing modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte progenitors. In addition to DNA binding, distinct N-terminal regions, including residues in the first 84 amino acids, promote platelet release and restrict megakaryocyte growth. In contrast, abrogation of GATA1-FOG-1 interaction leads to loss of differentiation, but growth of blocked immature megakaryocytes is controlled. Thus, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth, and these processes can be uncoupled.
Assuntos
Fator de Transcrição GATA1/fisiologia , Megacariócitos/citologia , Sequência de Aminoácidos , Animais , Antígenos CD/biossíntese , Plaquetas/metabolismo , Western Blotting , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Separação Celular , Galinhas , Clonagem Molecular , DNA/química , Citometria de Fluxo , Fator de Transcrição GATA1/química , Fator de Transcrição GATA1/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Megacariócitos/metabolismo , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Fenótipo , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Ligação Proteica , Estrutura Terciária de Proteína , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Células-Tronco/citologia , Tetraspanina 29 , Dedos de ZincoRESUMO
Plasmacytoid dendritic cells (PDCs) are a unique leukocyte population capable of secreting high levels of type I interferon (IFN) in response to viruses and bacterial stimuli. In vitro experiments have shown that upon maturation, human and murine PDCs develop into potent immunostimulatory cells; however, their ability to prime an immune response in vivo remains to be addressed. We report that CpG-matured murine PDCs are capable of eliciting in naive mice antigen-specific CTLs against endogenous antigens as well as exogenous peptides, but not against an exogenous antigen. Type I IFN is not required for priming, as injection of CpG-matured PDCs into type I IFN receptor-deficient mice elicits functional CTL responses. Mature PDCs prime CTLs that secrete IFN-gamma and protect mice from a tumor challenge. In contrast, immature PDCs are unable to prime antigen-specific CTLs. However, mice injected with immature PDCs are fully responsive to secondary antigenic challenges, suggesting that PDCs have not induced long-lasting tolerance via anergic or regulatory T cells. Our results underline the heterogeneity and plasticity of different antigen-presenting cells, and reveal an important role of mature PDCs in priming CD8 responses to endogenous antigens, in addition to their previously reported ability to modulate antiviral responses via type I IFN.