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1.
Blood ; 133(4): 331-343, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30429161

RESUMO

Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The Platelet factor 4-Cre recombinase (Pf4-Cre) transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially a result of the Pf4-Cre transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a Gp1ba-Cre transgenic mouse strain in which Cre expression is driven by the endogenous Gp1ba locus. By crossing Gp1ba-Cre and Pf4-Cre mice to the mT/mG dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the Gp1ba-Cre transgene. Broader tissue expression was observed with the Pf4-Cre transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, and dendritic and B and T cells. Direct comparison of phenotypes of Csk, Shp1, or CD148 conditional KO mice generated using either the Gp1ba-Cre or Pf4-Cre strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4-Cre-generated KO mice as a result of nonspecific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the Gp1ba-Cre mouse will advance our understanding of the role of platelets in inflammation and other pathophysiological processes in which platelet-leukocyte interactions are involved.


Assuntos
Plaquetas/metabolismo , Integrases/metabolismo , Leucócitos/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Aglutinação , Animais , Células da Medula Óssea/citologia , Proteína Tirosina Quinase CSK , Linhagem da Célula , Tamanho Celular , Marcação de Genes , Homeostase , Contagem de Linfócitos , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Fenótipo , Agregação Plaquetária , Fator Plaquetário 4/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Recombinação Genética/genética , Baço/citologia , Quinases da Família src/metabolismo
2.
Blood ; 131(10): 1122-1144, 2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29301754

RESUMO

Src family kinases (SFKs) coordinate the initiating and propagating activation signals in platelets, but it remains unclear how they are regulated. Here, we show that ablation of C-terminal Src kinase (Csk) and receptor-like protein tyrosine-phosphatase CD148 in mice results in a dramatic increase in platelet SFK activity, demonstrating that these proteins are essential regulators of platelet reactivity. Paradoxically, Csk/CD148-deficient mice exhibit reduced in vivo and ex vivo thrombus formation and increased bleeding following injury rather than a prothrombotic phenotype. This is a consequence of multiple negative feedback mechanisms, including downregulation of the immunoreceptor tyrosine-based activation motif (ITAM)- and hemi-ITAM-containing receptors glycoprotein VI (GPVI)-Fc receptor (FcR) γ-chain and CLEC-2, respectively and upregulation of the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B and its interaction with the tyrosine phosphatases Shp1 and Shp2. Results from an analog-sensitive Csk mouse model demonstrate the unconventional role of SFKs in activating ITIM signaling. This study establishes Csk and CD148 as critical molecular switches controlling the thrombotic and hemostatic capacity of platelets and reveals cell-intrinsic mechanisms that prevent pathological thrombosis from occurring.


Assuntos
Plaquetas/metabolismo , Homeostase , Trombose/metabolismo , Quinases da Família src/metabolismo , Motivos de Aminoácidos , Animais , Plaquetas/patologia , Proteína Tirosina Quinase CSK , Camundongos , Camundongos Knockout , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Trombose/genética , Quinases da Família src/genética
3.
Proc Natl Acad Sci U S A ; 112(32): E4448-57, 2015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26216992

RESUMO

Aire controls immunologic tolerance by inducing a battery of thymic transcripts encoding proteins characteristic of peripheral tissues. Its unusually broad effect is achieved by releasing RNA polymerase II paused just downstream of transcriptional start sites. We explored Aire's collaboration with the bromodomain-containing protein, Brd4, uncovering an astonishing correspondence between those genes induced by Aire and those inhibited by a small-molecule bromodomain blocker. Aire:Brd4 binding depended on an orchestrated series of posttranslational modifications within Aire's caspase activation and recruitment domain. This interaction attracted P-TEFb, thereby mobilizing downstream transcriptional elongation and splicing machineries. Aire:Brd4 association was critical for tolerance induction, and its disruption could account for certain point mutations that provoke human autoimmune disease. Our findings evoke the possibility of unanticipated immunologic mechanisms subtending the potent antitumor effects of bromodomain blockers.


Assuntos
Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Timo/citologia , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo , Acetilação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/genética , Lisina/metabolismo , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/química , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Elongação da Transcrição Genética/efeitos dos fármacos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcriptoma/genética , Proteína AIRE
4.
J Exp Med ; 212(3): 297-306, 2015 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-25687282

RESUMO

The fidelity of T cell immunity depends greatly on coupling T cell receptor signaling with specific T cell effector functions. Here, we describe a chromatin-based mechanism that enables integration of TCR specificity into definite T cell lineage commitment. Using natural killer T cells (iNKT cell) as a model of a T cell subset that differentiates in response to specific TCR signaling, we identified a key role of histone H3 lysine 27 trimethylation (H3K27me3) in coupling iNKT cell TCR specificity with the generation of iNKT cells. We found that the Zbtb16/PLZF gene promoter that drives iNKT cell differentiation possesses a bivalent chromatin state characterized by the simultaneous presence of negative and positive H3K27me3 and H3K4me3 modifications. Depletion of H3K27me3 at the Zbtb16/PLZF promoter leads to uncoupling of iNKT cell development from TCR specificity and is associated with accumulation of iNKT-like CD4(+) cells that express a non-iNKT cell specific T cell repertoire. In turn, stabilization of H3K27me3 leads to a drastic reduction of the iNKT cell population. Our data suggest that H3K27me3 levels at the bivalent Zbtb16/PLZF gene define a threshold enabling precise coupling of TCR specificity to lineage commitment.


Assuntos
Histonas/metabolismo , Células T Matadoras Naturais/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Animais , Antígenos CD4/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lisina/metabolismo , Metilação , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células T Matadoras Naturais/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores de Antígenos de Linfócitos T/metabolismo
5.
Genes Dev ; 27(16): 1731-8, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23964091

RESUMO

Recent advances in the enzymology of transcription and chromatin regulation have led to the discovery of proteins that play a prominent role in cell differentiation and the maintenance of specialized cell functions. Knowledge about post-synthetic DNA and histone modifications as well as information about the rules that guide the formation of multimolecular chromatin-bound complexes have helped to delineate gene-regulating pathways and describe how these pathways are altered in various pathological conditions. The present review focuses on the emerging area of therapeutic interference with chromatin function for the purpose of cancer treatment and immunomodulation.


Assuntos
Antineoplásicos/uso terapêutico , Cromatina/efeitos dos fármacos , Imunidade , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Humanos , Imunidade/efeitos dos fármacos , Imunidade/genética , Imunomodulação/efeitos dos fármacos , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/metabolismo
6.
Genes Dev ; 26(7): 693-704, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22474261

RESUMO

Argonaute proteins (Ago1-4) are essential components of the microRNA-induced silencing complex and play important roles in both microRNA biogenesis and function. Although Ago2 is the only one with the slicer activity, it is not clear whether the slicer activity is a universally critical determinant for Ago2's function in mammals. Furthermore, functional specificities associated with different Argonautes remain elusive. Here we report that microRNAs are randomly sorted to individual Argonautes in mammals, independent of the slicer activity. When both Ago1 and Ago2, but not either Ago1 or Ago2 alone, are ablated in the skin, the global expression of microRNAs is significantly compromised and it causes severe defects in skin morphogenesis. Surprisingly, Ago3 is able to load microRNAs efficiently in the absence of Ago1 and Ago2, despite a significant loss of global microRNA expression. Quantitative analyses reveal that Ago2 interacts with a majority of microRNAs (60%) in the skin, compared with Ago1 (30%) and Ago3 (<10%). This distribution is highly correlated with the abundance of each Argonaute, as quantified by shotgun proteomics. The quantitative correlation between Argonautes and their associated microRNAs is conserved in human cells. Finally, we measure the absolute expression of Argonaute proteins and determine that their copy number is ~1.4 × 10(5) to 1.7 × 10(5) molecules per cell. Together, our results reveal a quantitative picture for microRNA activity in mammals.


Assuntos
Proteínas Argonautas/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Proteínas Argonautas/deficiência , Proteínas Argonautas/genética , Proliferação de Células , Fatores de Iniciação em Eucariotos/deficiência , Fatores de Iniciação em Eucariotos/genética , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Pele/citologia , Pele/metabolismo
7.
Cell Cycle ; 10(22): 3834-40, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22071625

RESUMO

MET, a receptor protein tyrosine kinase activated by hepatocyte growth factor (HGF), is a crucial determinant of metastatic progression. Recently, we have identified p53 as an important regulator of MET-dependent cell motility and invasion. This regulation occurs via feedforward loop suppressing MET expression by miR-34-dependent and -independent mechanisms. Here, by using Dicer conditional knockout, we provide further evidence for microRNA-independent MET regulation by p53. Furthermore, we show that while MET levels increase immediately after p53 inactivation, mutant cells do not contain active phosphorylated MET and remain non-invasive for a long latency period at contrary to cell culture observations. Evaluation of mouse models of ovarian and prostate carcinogenesis indicates that formation of desmoplastic stroma, associated production of HGF by stromal cells and coinciding MET phosphorylation precede cancer invasion. Thus, initiation mutation of p53 is sufficient for preprogramming motile and invasive properties of epithelial cells, but the stromal reaction may represent a critical step for their manifestation during cancer progression.


Assuntos
Fator de Crescimento de Hepatócito/fisiologia , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Animais , Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica , Masculino , Camundongos , MicroRNAs/fisiologia , Invasividade Neoplásica/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/fisiologia , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
8.
Nat Immunol ; 12(1): 29-36, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21131967

RESUMO

Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.


Assuntos
Artrite Reumatoide/imunologia , NF-kappa B/metabolismo , Proteínas Metiltransferases/metabolismo , Fator de Transcrição RelA/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Montagem e Desmontagem da Cromatina/genética , Metilação de DNA , Células HEK293 , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Inflamação , Lisina/metabolismo , NF-kappa B/genética , NF-kappa B/imunologia , Ligação Proteica/genética , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/imunologia , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia
9.
Cancer Cell ; 18(6): 568-79, 2010 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-21156281

RESUMO

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease composed of at least two distinct subtypes: germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. These phenotypic subtypes segregate with largely unique genetic lesions, suggesting the involvement of different pathogenetic mechanisms. In this report we show that the BLIMP1/PRDM1 gene is inactivated by multiple mechanisms, including homozygous deletions, truncating or missense mutations, and transcriptional repression by constitutively active BCL6, in ∼53% of ABC-DLBCL. In vivo, conditional deletion of Blimp1 in mouse B cells promotes the development of lymphoproliferative disorders recapitulating critical features of the human ABC-DLBCL. These results demonstrate that BLIMP1 is a bona fide tumor-suppressor gene whose loss contributes to lymphomagenesis by blocking plasma cell differentiation.


Assuntos
Genes Supressores de Tumor , Linfoma Difuso de Grandes Células B/genética , Fatores de Transcrição/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Humanos , Linfoma Difuso de Grandes Células B/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-bcl-6
10.
Cancer Cell ; 18(6): 580-9, 2010 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-21156282

RESUMO

Diffuse large B cell lymphoma (DLBCL) comprises disease entities with distinct genetic profiles, including germinal center B cell (GCB)-like and activated B cell (ABC)-like DLBCLs. Major differences between these two subtypes include genetic aberrations leading to constitutive NF-κB activation and interference with terminal B cell differentiation through BLIMP1 inactivation, observed in ABC- but not GCB-DLBCL. Using conditional gain-of-function and/or loss-of-function mutagenesis in the mouse, we show that constitutive activation of the canonical NF-κB pathway cooperates with disruption of BLIMP1 in the development of a lymphoma that resembles human ABC-DLBCL. Our work suggests that both NF-κB signaling, as an oncogenic event, and BLIMP1, as a tumor suppressor, play causal roles in the pathogenesis of ABC-DLBCL.


Assuntos
Linfoma Difuso de Grandes Células B/etiologia , NF-kappa B/metabolismo , Fatores de Transcrição/fisiologia , Animais , Proliferação de Células , Centro Germinativo/fisiologia , Quinase I-kappa B/genética , Camundongos , Mutação , Plasmócitos/fisiologia , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fatores de Transcrição/genética
11.
Nature ; 468(7327): 1119-23, 2010 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-21068722

RESUMO

Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by 'mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.


Assuntos
Anti-Inflamatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Inflamação , Macrófagos/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Benzodiazepinas , Células Cultivadas , Epigenômica , Estudo de Associação Genômica Ampla , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Inflamação/tratamento farmacológico , Inflamação/prevenção & controle , Estimativa de Kaplan-Meier , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/imunologia , Infecções por Salmonella/fisiopatologia , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium , Sepse/tratamento farmacológico , Sepse/prevenção & controle , Choque Séptico/tratamento farmacológico , Choque Séptico/prevenção & controle
12.
Science ; 327(5962): 213-6, 2010 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-20056891

RESUMO

Cocaine-induced alterations in gene expression cause changes in neuronal morphology and behavior that may underlie cocaine addiction. In mice, we identified an essential role for histone 3 lysine 9 (H3K9) dimethylation and the lysine dimethyltransferase G9a in cocaine-induced structural and behavioral plasticity. Repeated cocaine administration reduced global levels of H3K9 dimethylation in the nucleus accumbens. This reduction in histone methylation was mediated through the repression of G9a in this brain region, which was regulated by the cocaine-induced transcription factor DeltaFosB. Using conditional mutagenesis and viral-mediated gene transfer, we found that G9a down-regulation increased the dendritic spine plasticity of nucleus accumbens neurons and enhanced the preference for cocaine, thereby establishing a crucial role for histone methylation in the long-term actions of cocaine.


Assuntos
Comportamento Animal/efeitos dos fármacos , Cocaína/administração & dosagem , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Núcleo Accumbens/metabolismo , Animais , Cocaína/farmacologia , Transtornos Relacionados ao Uso de Cocaína/etiologia , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Espinhas Dendríticas/fisiologia , Regulação para Baixo , Repressão Enzimática , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Lisina/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Núcleo Accumbens/citologia , Núcleo Accumbens/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Recompensa , Autoadministração , Transcrição Gênica
13.
Proc Natl Acad Sci U S A ; 107(1): 187-92, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-19966290

RESUMO

Chromosome translocations between Ig (Ig) and non-Ig genes are frequently associated with B-cell lymphomas in humans and mice. The best characterized of these is c-myc/IgH translocation, which is associated with Burkitt's lymphoma. These translocations are caused by activation-induced cytidine deaminase (AID), which produces double-strand DNA breaks in both genes. c-myc/IgH translocations are rare events, in part because ATM, p53, and p19 actively suppress them. To further define the mechanism of protection against the accumulation of cells that bear c-myc/IgH translocation, we assayed B cells from mice that carry mutations in cell-cycle and apoptosis regulator proteins that act downstream of p53. We find that PUMA, Bim, and PKCdelta are required for protection against c-myc/IgH translocation, whereas Bcl-XL and BAFF enhance c-myc/IgH translocation. Whether these effects are general or specific to c-myc/IgH translocation and whether AID produces dsDNA breaks in genes other than c-myc and Ig is not known. To examine these questions, we developed an assay for translocation between IgH and Igbeta, both of which are somatically mutated by AID. Igbeta/IgH, like c-myc/IgH translocations, are AID-dependent, and AID is responsible for lesions on IgH and the non-IgH translocation partners. However, ATM, p53, and p19 do not protect against Igbeta/IgH translocations. Instead, B cells are protected against Igbeta/IgH translocations by a BAFF- and PKCdelta-dependent pathway. We conclude that AID-induced double-strand breaks in non-Ig genes other than c-myc lead to their translocation, and that at least two nonoverlapping pathways protect against translocations in primary B cells.


Assuntos
Citidina Desaminase/metabolismo , Translocação Genética , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/imunologia , Proteína 11 Semelhante a Bcl-2 , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p19/genética , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Citidina Desaminase/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes de Imunoglobulinas , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Hipermutação Somática de Imunoglobulina , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
14.
Genes Dev ; 23(8): 975-85, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19390090

RESUMO

Proliferation of pancreatic islet beta cells is an important mechanism for self-renewal and for adaptive islet expansion. Increased expression of the Ink4a/Arf locus, which encodes the cyclin-dependent kinase inhibitor p16(INK4a) and tumor suppressor p19(Arf), limits beta-cell regeneration in aging mice, but the basis of beta-cell Ink4a/Arf regulation is poorly understood. Here we show that Enhancer of zeste homolog 2 (Ezh2), a histone methyltransferase and component of a Polycomb group (PcG) protein complex, represses Ink4a/Arf in islet beta cells. Ezh2 levels decline in aging islet beta cells, and this attrition coincides with reduced histone H3 trimethylation at Ink4a/Arf, and increased levels of p16(INK4a) and p19(Arf). Conditional deletion of beta-cell Ezh2 in juvenile mice also reduced H3 trimethylation at the Ink4a/Arf locus, leading to precocious increases of p16(INK4a) and p19(Arf). These mutant mice had reduced beta-cell proliferation and mass, hypoinsulinemia, and mild diabetes, phenotypes rescued by germline deletion of Ink4a/Arf. beta-Cell destruction with streptozotocin in controls led to increased Ezh2 expression that accompanied adaptive beta-cell proliferation and re-establishment of beta-cell mass; in contrast, mutant mice treated similarly failed to regenerate beta cells, resulting in lethal diabetes. Our discovery of Ezh2-dependent beta-cell proliferation revealed unique epigenetic mechanisms underlying normal beta-cell expansion and beta-cell regenerative failure in diabetes pathogenesis.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Diabetes Mellitus/metabolismo , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Células Secretoras de Insulina/metabolismo , Envelhecimento/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexo Repressor Polycomb 2 , Estreptozocina/farmacologia
15.
Cell ; 136(6): 1122-35, 2009 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-19303854

RESUMO

Although in vitro studies of embryonic stem cells have identified polycomb repressor complexes (PRCs) as key regulators of differentiation, it remains unclear as to how PRC-mediated mechanisms control fates of multipotent progenitors in developing tissues. Here, we show that an essential PRC component, Ezh2, is expressed in epidermal progenitors but diminishes concomitant with embryonic differentiation and with postnatal decline in proliferative activity. We show that Ezh2 controls proliferative potential of basal progenitors by repressing the Ink4A-Ink4B locus and tempers the developmental rate of differentiation by preventing premature recruitment of AP1 transcriptional activator to the structural genes that are required for epidermal differentiation. Together, our studies reveal that PRCs control epigenetic modifications temporally and spatially in tissue-restricted stem cells. They maintain their proliferative potential and globally repressing undesirable differentiation programs while selectively establishing a specific terminal differentiation program in a stepwise fashion.


Assuntos
Diferenciação Celular , Células Epidérmicas , Epiderme/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco/metabolismo , Animais , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Histonas/metabolismo , Humanos , Metilação , Camundongos , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Proteínas Repressoras/metabolismo
16.
Proc Natl Acad Sci U S A ; 105(34): 12435-8, 2008 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-18713867

RESUMO

B cell activating factor (BAFF) signals through BAFF-R to promote mature B cell survival. Recent analyses of BAFF-induced signaling revealed direct association between augmented B cell metabolic fitness and activation of Akt, one of the key regulators of cell survival. The strongest and most reproducible induction of Akt occurs with significant delay (24 h) after BAFF treatment, where it precedes activation of anabolism. It was also recently shown that BAFF induces sustained Erk activation and increased turnover of the proapoptotic molecule Bim. Here we show that these BAFF-induced signaling pathways are mediated by BAFF-R and represent previously unknown arms of I kappa B kinase (IKK)1-dependent signaling. In combination with the known role of IKK1 in regulating transcription of prosurvival genes, our data underscore the central role of IKK1 in coordinating multiple BAFF-R-mediated signaling pathways controlling mature B cell homeostasis.


Assuntos
Fator Ativador de Células B/metabolismo , Linfócitos B/metabolismo , Quinase I-kappa B/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Receptor do Fator Ativador de Células B/metabolismo , Linfócitos B/citologia , Tamanho Celular , Células Cultivadas , Homeostase , Camundongos , Fosforilação
17.
Mol Cell ; 30(4): 426-36, 2008 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-18498747

RESUMO

The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis. Cbp-deficient cells are more susceptible to v-Src transformation than their parental cells. Upon phosphorylation, Cbp specifically binds to activated c-Src and sequesters it in lipid rafts, resulting in an efficient suppression of c-Src function independent of Csk. In some human cancer cells and tumors, Cbp is downregulated and the introduction of Cbp significantly suppresses tumorigenesis. These findings indicate a potential role for Cbp as a suppressor of c-Src-mediated tumor progression.


Assuntos
Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Proteína Tirosina Quinase CSK , Fracionamento Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/fisiologia , Técnicas de Transferência de Genes , Humanos , Microdomínios da Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , Neoplasias/metabolismo , Fosfoproteínas/genética , Proteínas Tirosina Quinases/genética , Quinases da Família src
18.
Carcinogenesis ; 28(10): 2074-81, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17494055

RESUMO

The Src family kinases (SFKs) are believed to play critical roles in malignant transformation, as well as in growth, invasion and dissemination of neoplastic tissue. Inhibition of SFK-mediated signal transduction and activation of downstream targets inhibits tumor progression. To determine whether constitutive activity of SFK per se is sufficient to induce tumorigenesis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of the SFK-negative regulator csk (Csk-K5 mice). Even though expression levels of SFKs were lower in C-terminal Src kinase (Csk)-null keratinocytes, activity levels were higher than in control keratinocytes. At the age of 3 months, all Csk-K5 mice displayed signs of chronic inflammation in dermis and epidermal hyperplasia. About 19% of Csk-K5 mice (7 out of 36) developed papillomatous lesions. However, these lesions did not show any signs of neoplastic transformation over the next 8 months. Epidermal hyperplasia and hyperkeratosis in Csk-K5 mice were associated with an increased number of stem cells in the interfollicular epidermis, an increased proliferation of basal keratinocytes and a delayed terminal differentiation of the suprabasal keratinocytes. Our results clearly demonstrate that even though SFK-mediated signaling promotes tumor progression, elevated activity of SFKs in vivo alone is not sufficient to induce neoplastic transformation.


Assuntos
Epiderme/patologia , Queratinócitos/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Papiloma/patologia , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Animais , Animais Recém-Nascidos , Deleção de Genes , Hiperplasia , Queratinócitos/patologia , Camundongos , Papiloma/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Ferimentos e Lesões/patologia , Ferimentos e Lesões/fisiopatologia
19.
J Exp Med ; 203(11): 2551-62, 2006 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-17060474

RESUMO

B cell life depends critically on the cytokine B cell-activating factor of the tumor necrosis factor family (BAFF). Lack of BAFF signaling leads to B cell death and immunodeficiency. Excessive BAFF signaling promotes lupus-like autoimmunity. Despite the great importance of BAFF to B cell biology, its signaling mechanism is not well characterized. We show that BAFF initiates signaling and transcriptional programs, which support B cell survival, metabolic fitness, and readiness for antigen-induced proliferation. We further identify a BAFF-specific protein kinase C beta-Akt signaling axis, which provides a connection between BAFF and generic growth factor-induced cellular responses.


Assuntos
Fator Ativador de Células B/fisiologia , Linfócitos B/metabolismo , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Linfócitos B/enzimologia , Proliferação de Células , Sobrevivência Celular/imunologia , Células Cultivadas , Camundongos , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Proteína Quinase C beta , Transdução de Sinais/imunologia
20.
Cell ; 126(3): 597-609, 2006 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-16901790

RESUMO

Epidermal lineage commitment occurs when multipotent stem cells are specified to three lineages: the epidermis, the hair follicle, and the sebaceous gland (SG). How and when a lineage becomes specified remains unknown. Here, we report the existence of a population of unipotent progenitor cells that reside in the SG and express the transcriptional repressor Blimp1. Using cell-culture studies and genetic lineage tracing, we demonstrate that Blimp1-expressing cells are upstream from other cells of the SG lineage. Blimp1 appears to govern cellular input into the gland since its loss leads to elevated c-myc expression, augmented cell proliferation, and SG hyperplasia. Finally, BrdU labeling experiments demonstrate that the SG defects associated with loss of Blimp1 lead to enhanced bulge stem cell activity, suggesting that when normal SG homeostasis is perturbed, multipotent stem cells in the bulge can be mobilized to correct this imbalance.


Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Células Epiteliais/metabolismo , Proteínas Repressoras/metabolismo , Glândulas Sebáceas/embriologia , Glândulas Sebáceas/crescimento & desenvolvimento , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Bromodesoxiuridina , Contagem de Células , Movimento Celular/genética , Proliferação de Células , Células Cultivadas , Células Epiteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Folículo Piloso/citologia , Folículo Piloso/embriologia , Folículo Piloso/crescimento & desenvolvimento , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/fisiopatologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/genética , Glândulas Sebáceas/citologia , Células-Tronco/citologia , Fatores de Transcrição/genética
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