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1.
Nat Commun ; 7: 12207, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27481093

RESUMO

The molecular mechanisms that regulate B-cell development and tolerance remain incompletely understood. In this study, we identify a critical role for the miR-17∼92 microRNA cluster in regulating B-cell central tolerance and demonstrate that these miRNAs control early B-cell development in a cell-intrinsic manner. While the cluster member miR-19 suppresses the expression of Pten and plays a key role in regulating B-cell tolerance, miR-17 controls early B-cell development through other molecular pathways. These findings demonstrate differential control of two closely linked B-cell developmental stages by different members of a single microRNA cluster through distinct molecular pathways.


Assuntos
Linfócitos B/fisiologia , Tolerância Imunológica/genética , Ativação Linfocitária/genética , MicroRNAs/fisiologia , PTEN Fosfo-Hidrolase/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Feminino , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais
2.
EMBO Rep ; 16(5): 638-53, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25770130

RESUMO

The thymus provides a specialized microenvironment in which distinct subsets of thymic epithelial cells (TECs) support T-cell development. Here, we describe the significance of cortical TECs (cTECs) in T-cell development, using a newly established mouse model of cTEC deficiency. The deficiency of mature cTECs caused a massive loss of thymic cellularity and impaired the development of αßT cells and invariant natural killer T cells. Unexpectedly, the differentiation of certain γδT-cell subpopulations-interleukin-17-producing Vγ4 and Vγ6 cells-was strongly dysregulated, resulting in the perturbation of γδT-mediated inflammatory responses in peripheral tissues. These findings show that cTECs contribute to the shaping of the TCR repertoire, not only of "conventional" αßT cells but also of inflammatory "innate" γδT cells.


Assuntos
Epitélio/metabolismo , Interleucina-17/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Timo/metabolismo , Animais , Diferenciação Celular , Sobrevivência Celular/genética , Análise Mutacional de DNA , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/imunologia , Feminino , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Timócitos/citologia , Timócitos/imunologia , Timócitos/metabolismo , Timo/imunologia , Timo/patologia
3.
PLoS One ; 9(2): e89115, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24586531

RESUMO

Themis (also named Gasp) is a newly identified Grb2-binding protein that is essential for thymocyte positive selection. Despite the possible involvement of Themis in TCR-mediated signal transduction, its function remains unresolved and controversial. Themis contains two functionally uncharacterized regions called CABIT (cysteine-containing, all-ß in Themis) domains, a nuclear localization signal (NLS), and a proline-rich sequence (PRS). To elucidate the role of these motifs in Themis's function in vivo, we established a series of mutant Themis transgenic mice on a Themis(-/-) background. Deletion of the highly conserved Core motif of CABIT1 or CABIT2 (Core1 or Core2, respectively), the NLS, or the PRS abolished Grb2-association, as well as TCR-dependent tyrosine-phosphorylation and the ability to induce positive selection in the thymus. The NLS and Core1 motifs were required for the nuclear localization of Themis, whereas Core2 and PRS were not. Furthermore, expression of ΔCore1- but not ΔCore2-Themis conferred dominant negative-type inhibition on T cell development. Collectively, our current results indicate that PRS, NLS, CABIT1, and CABIT2 are all required for positive selection, and that each of the CABIT domains exerts distinct functions during positive selection.


Assuntos
Diferenciação Celular/fisiologia , Proteínas/metabolismo , Linfócitos T/citologia , Timócitos/citologia , Animais , Diferenciação Celular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Transgênicos , Fosforilação , Estrutura Terciária de Proteína , Proteínas/genética , Linfócitos T/metabolismo , Timócitos/metabolismo
4.
EMBO J ; 32(17): 2377-91, 2013 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-23921550

RESUMO

MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR-17~92 expression is frequently found in human cancers, mainly due to gene amplification and Myc-mediated transcriptional upregulation. Here we show that B cell-specific miR-17~92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc-driven lymphomagenesis stringently requires two intact alleles of miR-17~92. We experimentally identified miR-17~92 target genes by PAR-CLIP and validated select target genes in miR-17~92 transgenic mice. These analyses demonstrate that miR-17~92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR-17~92-driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma-bearing mice. These findings establish miR-17~92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation.


Assuntos
Regulação Neoplásica da Expressão Gênica , Linfoma/genética , MicroRNAs/fisiologia , Animais , Linfócitos B/patologia , Linfócitos B/fisiologia , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Proliferação de Células , Sobrevivência Celular/genética , Proteínas de Homeodomínio/genética , Humanos , Imidazóis/farmacologia , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfolinas/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/administração & dosagem , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Quinoxalinas/farmacologia , RNA Longo não Codificante , Reprodutibilidade dos Testes
5.
Immunol Lett ; 151(1-2): 1-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23499578

RESUMO

RhoH is a new member of the atypical G proteins exclusively expressed in hematopoietic lineage cells. It has been shown to act as an adaptor for ZAP70, Syk, Lck and Csk kinases in signal transduction, and is required for positive selection of thymocytes as well as activation of peripheral T cells and mast cells. In the present study, we showed that RhoH is required not only for positive selection but also for negative selection of thymocytes. Regarding development of unconventional T cell subsets, development of NKT and regulatory T cells was also inhibited, whereas development of TCRαß CD8αα intestinal intraepithelial lymphocytes (IEL) was not affected by the absence of RhoH. TCR-dependent in vitro activation of TCRαß CD8αα IEL required RhoH, suggesting that overall development of IEL does not critically depend on TCR signaling but more on cytokine-dependent expansion and survival in the periphery. Our current results indicate differential requirements for RhoH in the development of TCRαß CD8αα IELs compared to other subsets of T cells including agonist selected T cells.


Assuntos
Antígenos CD8/metabolismo , Diferenciação Celular , Mucosa Intestinal/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Feminino , Interferon gama/biossíntese , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células T Matadoras Naturais/metabolismo , Baço/citologia , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/metabolismo , Timo/citologia , Timo/imunologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genética
6.
J Immunol ; 182(2): 957-62, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19124738

RESUMO

RhoH is an atypical small G protein with defective GTPase activity that is specifically expressed in hematopoietic lineage cells. RhoH has been implicated in regulation of several physiological processes including hematopoiesis, integrin activation, and T cell differentiation and activation. In the present study, we investigated the role of RhoH in mast cells by generating RhoH knockout mice. Despite observing normal development of mast cells in vivo, passive systemic anaphylaxis and histamine release were impaired in these mice. We also observed defective degranulation and cytokine production upon FcepsilonRI ligation in RhoH-deficient bone marrow-derived mast cells. Furthermore, FcepsilonRI-dependent activation of Syk and phosphorylation of its downstream targets, including LAT, SLP76, PLCgamma1, and PLCgamma2 were impaired, however phosphorylation of the gamma-subunit of FcepsilonRI remained intact. We also found RhoH-Syk association that was greatly enhanced by active Fyn. Our results indicate that RhoH regulates FcepsilonRI signaling in mast cells by facilitating Syk activation, possibly as an adaptor molecule for Syk.


Assuntos
Mastócitos/enzimologia , Mastócitos/imunologia , Receptores de IgE/fisiologia , Transdução de Sinais/imunologia , Fatores de Transcrição/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Degranulação Celular/genética , Degranulação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Ativação Enzimática/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Anafilaxia Cutânea Passiva/genética , Anafilaxia Cutânea Passiva/imunologia , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteínas rho de Ligação ao GTP/biossíntese , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genética
7.
J Leukoc Biol ; 81(2): 500-8, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17090688

RESUMO

Rac1, one of the Rho family small guanosine triphosphatases, has been shown to work as a "molecular switch" in various signal transduction pathways. To assess the function of Rac1 in the differentiation process of CD4 single-positive (CD4-SP) T cells from CD4CD8 double-positive (DP) cells, we used a DP cell line DPK, which can differentiate into CD4-SP cells upon TCR stimulation in vitro. DPK expressing dominant-negative (dn)Rac1 underwent massive apoptosis upon TCR stimulation and resulted in defective differentiation of CD4-SP cells. Conversely, overexpression of dnRac2 did not affect differentiation. TCR-dependent actin polymerization was inhibited, whereas early ERK activation was unaltered in dnRac1-expressing DPK. We found that TCR-dependent induction of Bcl-2 was suppressed greatly in dnRac1-expressing DPK, and this suppression was independent of actin rearrangement. Furthermore, introduction of exogenous Bcl-2 inhibited TCR-dependent induction of apoptosis and restored CD4-SP generation in dnRac1-expressing DPK without restoring TCR-induced actin polymerization. Collectively, these data indicate that Rac1 is critical in differentiation of CD4-SP from the DP cell line by preventing TCR-induced apoptosis via Bcl-2 up-regulation.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Timo/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Apoptose/imunologia , Diferenciação Celular/imunologia , Linhagem Celular , Humanos , Timo/citologia
8.
J Biol Chem ; 281(26): 17736-42, 2006 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-16636057

RESUMO

Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85alpha regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-gamma stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.


Assuntos
Macrófagos/enzimologia , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Biopterinas/metabolismo , Células Cultivadas , Infecções por Chlamydophila/imunologia , Infecções por Chlamydophila/metabolismo , Chlamydophila pneumoniae/crescimento & desenvolvimento , Dimerização , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , GTP Cicloidrolase/metabolismo , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Óxido Nítrico/biossíntese , Fosfatidilinositol 3-Quinases/genética , Especificidade por Substrato
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