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1.
PLoS One ; 7(8): e43805, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22928038

RESUMO

Regulated expression of miRNAs influences development in a wide variety of contexts. We report here that miR290-5p (100049710) and miR292-5p (100049711) are induced at the pre-B stage of murine B cell development and that they influence assembly of the Igκ light chain gene (243469) by contributing to the activation of germline Igκ transcription (κGT). We found that upon forced over-expression of miR290-5p/292-5p in Abelson Murine Leukemia Virus (AMuLV) transformed pro-B cells, two known activators of κGT, E2A (21423) and NF-κB (19697), show increased chromosomal binding to the kappa intronic enhancer. Conversely, knockdown of miR290-5p/292-5p in AMuLV pro-B cells blunts drug-induced activation of κGT. Furthermore, miR290-5p/292-5p knockdown also diminishes κGT activation, but not Rag1/2 (19373, 19374) expression, in an IL-7 dependent primary pro-B cell culture system. In addition, we identified a deficiency in κGT induction in miR290 cluster knockout mice. We hypothesize that increased expression of miR290-5p and miR292-5p contributes to the induction of κGT at the pre-B stage of B cell development through increased binding of NF-κB and E2A to kappa locus regulatory sequences.


Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Loci Gênicos/genética , Cadeias kappa de Imunoglobulina/genética , MicroRNAs/metabolismo , Vírus da Leucemia Murina de Abelson/fisiologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/virologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzamidas , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Transformação Celular Viral/efeitos dos fármacos , Transformação Celular Viral/genética , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Mesilato de Imatinib , Íntrons/genética , Camundongos , MicroRNAs/genética , NF-kappa B/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética
2.
PLoS One ; 6(5): e19854, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21603627

RESUMO

Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34(+) or CD34(+)CD45(+) hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derived-hematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34(+) precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34(+) hematopoietic precursors generated in vitro versus in vivo.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Hematopoéticas/citologia , Antígenos CD34 , Técnicas de Cultura de Células/métodos , Linhagem Celular , Humanos , Antígenos Comuns de Leucócito , Linfócitos T
3.
J Biol Chem ; 285(10): 7556-65, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20068041

RESUMO

Proliferation and apoptosis are diametrically opposite processes. Expression of certain genes like c-Myc, however, can induce both, pointing to a possible linkage between them. Developing CD4(+)CD8(+) thymocytes are intrinsically sensitive to apoptosis, but the molecular basis is not known. We have found that these noncycling cells surprisingly express many cell cycle proteins. We generated transgenic mice expressing a CDK2 kinase-dead (CDK2-DN) protein in the T cell compartment. Analysis of these mice showed that the CDK2-DN protein acts as a dominant negative mutant in mature T cells as expected, but surprisingly, it acts as a dominant active protein in CD4(+)CD8(+) thymocytes. The levels of CDK2 kinase activity, cyclin E, cyclin A, and other cell cycle proteins in transgenic CD4(+)CD8(+) thymocytes are increased. Concurrently, caspase levels are elevated, and apoptosis is significantly enhanced in vitro and in vivo. E2F-1, the unique E2F member capable of inducing apoptosis when overexpressed, is specifically up-regulated in transgenic CD4(+)CD8(+) thymocytes but not in other T cell populations. These results demonstrate that the cell cycle and apoptotic machineries are normally linked, and expression of cell cycle proteins in developing T cells contributes to their inherent 1sensitivity to apoptosis.


Assuntos
Apoptose/fisiologia , Ciclo Celular/fisiologia , Quinase 2 Dependente de Ciclina , Linfócitos T/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Fragmentação do DNA , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Linfócitos T/citologia , Timo/citologia
4.
J Exp Med ; 206(8): 1803-16, 2009 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-19581408

RESUMO

Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens. One such tolerance mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene rearrangement continues in an attempt to edit the autoreactive specificity. Analysis of a heterozygous mutant mouse in which the NF-kappaB-dependent IkappaB alpha gene was replaced with a lacZ (beta-gal) reporter complementary DNA (cDNA; IkappaB alpha(+/lacZ)) suggests a potential role for NF-kappaB in receptor editing. Sorted beta-gal(+) pre-B cells showed increased levels of various markers of receptor editing. In IkappaB alpha(+/lacZ) reporter mice expressing either innocuous or self-specific knocked in BCRs, beta-gal was preferentially expressed in pre-B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IkappaB alpha superrepressor in primary bone marrow cultures resulted in diminished germline kappa and rearranged lambda transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells. Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.


Assuntos
NF-kappa B/metabolismo , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/metabolismo , Edição de RNA , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Primers do DNA/genética , DNA Complementar/genética , Rearranjo Gênico de Cadeia Leve de Linfócito B , Proteínas I-kappa B/genética , Fatores Reguladores de Interferon/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Inibidor de NF-kappaB alfa , Células Precursoras de Linfócitos B/citologia , Receptores de Antígenos de Linfócitos B/genética , Tolerância a Antígenos Próprios/genética
5.
Proc Natl Acad Sci U S A ; 106(2): 522-7, 2009 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-19116268

RESUMO

Allelic exclusion of Ig gene expression is necessary to limit the number of functional receptors to one per B cell. The mechanism underlying allelic exclusion is unknown. Because germline transcription of Ig and TCR loci is tightly correlated with rearrangement, we created two novel knock-in mice that report transcriptional activity of the Jkappa germline promoters in the Igkappa locus. Analysis of these mice revealed that germline transcription is biallelic and occurs in all pre-B cells. Moreover, we found that the two germline promoters in this region are not equivalent but that the distal promoter accounts for the vast majority of observed germline transcript in pre-B cells while the activity of the proximal promoter increases later in development. Allelic exclusion of the Igkappa locus thus occurs at the level of rearrangement, but not germline transcription.


Assuntos
Alelos , Linfócitos B/citologia , Cadeias kappa de Imunoglobulina/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Linfócitos B/imunologia , Técnicas de Introdução de Genes , Rearranjo Gênico , Humanos , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/imunologia
6.
Science ; 320(5881): 1349-52, 2008 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-18535245

RESUMO

Gene regulatory networks direct the progressive determination of cell fate during embryogenesis, but how they control cell behavior during morphogenesis remains largely elusive. Cell sorting, microarrays, and targeted molecular manipulations were used to analyze cardiac cell migration in the ascidian Ciona intestinalis. The heart network regulates genes involved in most cellular activities required for migration, including adhesion, cell polarity, and membrane protrusions. We demonstrated that fibroblast growth factor signaling and the forkhead transcription factor FoxF directly upregulate the small guanosine triphosphatase RhoDF, which synergizes with Cdc42 to contribute to the protrusive activity of migrating cells. Moreover, RhoDF induces membrane protrusions independently of other cellular activities required for migration. We propose that transcription regulation of specific effector genes determines the coordinated deployment of discrete cellular modules underlying migration.


Assuntos
Movimento Celular , Ciona intestinalis/embriologia , Ciona intestinalis/genética , Redes Reguladoras de Genes , Coração/embriologia , Transcrição Gênica , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Linhagem da Célula , Extensões da Superfície Celular/ultraestrutura , Ciona intestinalis/citologia , Ciona intestinalis/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Modelos Animais , Morfogênese , Células Musculares/citologia , Miocárdio/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso , Transdução de Sinais , Regulação para Cima , Proteína cdc42 de Ligação ao GTP/metabolismo
7.
Proc Natl Acad Sci U S A ; 105(6): 2022-7, 2008 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-18250301

RESUMO

PTEN is a tumor suppressor gene but whether cancer can develop in all PTEN-deficient cells is not known. In T cell-specific PTEN-deficient (tPTEN-/-) mice, which suffer from mature T cell lymphomas, we found that premalignancy, as defined by elevated AKT and senescence pathways, starts in immature T cell precursors and surprisingly not in mature T cells. Premalignancy only starts in 6-week-old mice and becomes much stronger in 9-week-old mice although PTEN is lost since birth. tPTEN-/- immature T cells do not become tumors, and senescence has no role in this model because these cells exist in a novel cell cycle state, expressing proliferating proteins but not proliferating to any significant degree. Instead, the levels of p27(kip1), which is lower in tPTEN-/- immature T cells and almost nonexistent in tPTEN-/- mature T cells, correlate with the proliferation capability of these cells. Interestingly, transient reduction of these cancer precursor cells in adult tPTEN-/- mice within a crucial time window significantly delayed lymphomas and mouse lethality. Thus, loss of PTEN alone is not sufficient for cells to become cancerous, therefore other developmental events are necessary for tumor formation.


Assuntos
Transformação Celular Neoplásica , PTEN Fosfo-Hidrolase/fisiologia , Linfócitos T/citologia , Animais , Sequência de Bases , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Primers do DNA , Feminino , Citometria de Fluxo , Masculino , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo
8.
J Exp Med ; 204(13): 3247-56, 2007 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-18056289

RESUMO

Complete IgHC gene rearrangement occurs only in B cells in a stage-specific and ordered manner. We used gene targeting to reposition a distal V(H) gene segment to a region just 5' of the D(H) gene cluster and found its activation to be highly dependent on the chromosomal domain within which it resides. The targeted V(H) gene segment rearranged at a higher frequency than its endogenous counterpart, its rearrangement was no longer ordered, and its ability to be silenced by allelic exclusion was lost. Additionally, the targeted V(H) gene segment lost lineage specificity, as VDJ(H) rearrangement was observed in thymocytes. These data suggest that locus contraction, mimicked by proximal targeting, can override any regulation imposed by DNA sequences immediately surrounding V(H) gene segments.


Assuntos
Rearranjo Gênico , Genes de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , VDJ Recombinases/genética , Alelos , Animais , Linfócitos B/metabolismo , Linhagem da Célula , Mapeamento Cromossômico , Cromossomos , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Transdução de Sinais , Timo/metabolismo
9.
Nat Immunol ; 8(1): 101-8, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17128277

RESUMO

Immunosurveillance by cytotoxic T cells requires that cells generate a diverse spectrum of peptides for presentation by major histocompatibility complex (MHC) class I molecules. Those peptides are generated by proteolysis, which begins in the cytoplasm and continues in the endoplasmic reticulum by the unique aminopeptidase ERAAP. The overall extent to which trimming by ERAAP modifies the peptide pool and the immunological consequences of ERAAP deficiency are unknown. Here we show that the peptide-MHC repertoire of ERAAP-deficient mice was missing many peptides. Furthermore, ERAAP-deficient cells presented many unstable and structurally unique peptide-MHC complexes, which elicited potent CD8+ T cell and B cell responses. Thus, ERAAP is a 'quintessential editor' of the peptide-MHC repertoire and, paradoxically, its absence enhances immunogenicity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Leucil Aminopeptidase/deficiência , Peptídeos/imunologia , Animais , Apresentação de Antígeno/imunologia , Células Cultivadas , Feminino , Leucil Aminopeptidase/genética , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
10.
J Immunol ; 177(4): 2356-64, 2006 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16887997

RESUMO

We report in this study that B7h, the ligand for the ICOS costimulatory receptor, is rapidly shed from mouse B cells following either ICOS binding or BCR engagement. Shedding occurs through proteolytic cleavage that releases the extracellular ICOS-binding region of B7h. Prior exposure of B7h-expressing APCs to ICOS-expressing cells inhibits their subsequent ability to costimulate IFN-gamma and IL-4 production from CD4+ T cells. Shedding is regulated as TLR7/8 and TLR9 ligands inhibit B7h shedding. A shedding-resistant B7h mutant elicits greater costimulation of IFN-gamma production from CD4+ T cells than does wild-type B7h. These data define shedding of B7h as a novel mechanism for controlling costimulatory signaling by B7-CD28 family members that is regulated on B cells by TLR signaling.


Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Linfócitos B/metabolismo , Glicoproteínas de Membrana/fisiologia , Proteínas/metabolismo , Receptor 7 Toll-Like/fisiologia , Receptor 8 Toll-Like/fisiologia , Receptor Toll-Like 9/fisiologia , Animais , Autoanticorpos/biossíntese , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células CHO , Células Cultivadas , Técnicas de Cocultura , Cricetinae , Cricetulus , Regulação para Baixo/imunologia , Ligante Coestimulador de Linfócitos T Induzíveis , Proteína Coestimuladora de Linfócitos T Induzíveis , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas/antagonistas & inibidores , Transdução de Sinais/imunologia
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