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1.
Mol Cell ; 57(2): 235-46, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25578880

RESUMO

Uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis and, upon cold exposure, is induced in brown adipose tissue (BAT) and subcutaneous white adipose tissue (iWAT). Here, by high-throughput screening using the UCP1 promoter, we identify Zfp516 as a transcriptional activator of UCP1 as well as PGC1α, thereby promoting a BAT program. Zfp516 itself is induced by cold and sympathetic stimulation through the cAMP-CREB/ATF2 pathway. Zfp516 directly binds to the proximal region of the UCP1 promoter, not to the enhancer region where other transcription factors bind, and interacts with PRDM16 to activate the UCP1 promoter. Although ablation of Zfp516 causes embryonic lethality, knockout embryos still show drastically reduced BAT mass. Overexpression of Zfp516 in adipose tissue promotes browning of iWAT even at room temperature, increasing body temperature and energy expenditure and preventing diet-induced obesity. Zfp516 may represent a future target for obesity therapeutics.


Assuntos
Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/fisiologia , Canais Iônicos/genética , Proteínas Mitocondriais/genética , Transativadores/fisiologia , Adipogenia , Tecido Adiposo Marrom/citologia , Tecido Adiposo Branco/citologia , Animais , Resposta ao Choque Frio , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Canais Iônicos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Desenvolvimento Muscular , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Termogênese , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Proteína Desacopladora 1
2.
Blood ; 123(26): 4089-100, 2014 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-24735967

RESUMO

Cancer develops by a multistep process during which cells acquire characteristics that allow them to evade apoptosis and proliferate unchecked. Sequential acquisition of genetic alterations drives this process but also causes cellular stress, frequently prompting cells to enter a premalignant period during which they mount a defense against transformation. T cell-specific deletion of the tumor suppressor PTEN in mice induces premalignancy in the thymus and development of CD4(+) T-cell lymphomas in the periphery. Here we sought to identify factors mediating the cellular defense against transformation during the premalignant period. We identified several microRNAs upregulated specifically in premalignant thymocytes, including miR-146a, miR-146b, and the miR-183/96/182 cluster. CD4-driven T cell-specific transgenic overexpression of mir-146a and mir-146b significantly delayed PTEN-deficient lymphomagenesis and delayed c-myc oncogene induction, a key driver of transformation in PTEN-deficient T-cell malignancies. We found that miR-146a and miR-146b targeting of Traf6 attenuates TCR signaling in the thymus and inhibits downstream NF-κB-dependent induction of c-myc. Additionally, c-myc repression in mature CD4 T cells by miR-146b impaired TCR-mediated proliferation. Hence, we have identified 2 miRNAs that are upregulated as part of the cellular response against transformation that, when overrepresented, can effectively inhibit progression to malignancy in the context of PTEN deficiency.


Assuntos
Transformação Celular Neoplásica/imunologia , Linfoma de Células T/imunologia , MicroRNAs/imunologia , PTEN Fosfo-Hidrolase/imunologia , RNA Neoplásico/imunologia , Timócitos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Camundongos , Camundongos Knockout , MicroRNAs/biossíntese , MicroRNAs/genética , Família Multigênica/genética , Família Multigênica/imunologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/imunologia , Fator 6 Associado a Receptor de TNF/metabolismo , Timócitos/metabolismo , Timócitos/patologia
3.
Biochem Biophys Res Commun ; 461(4): 630-5, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-25918019

RESUMO

Preadipocyte factor-1 (Pref-1) is made as a transmembrane protein containing EGF-repeats at the extracellular domain that can be cleaved to generate a biologically active soluble form. Pref-1 is found in islet ß-cells and its level has been reported to increase in neonatal rat islets upon growth hormone treatment. We found here that Pref-1 can promote growth of pancreatic tumor derived AR42J cells. To examine Pref-1 function in pancreatic islets in vivo, we generated transgenic mouse lines overexpressing the Pref-1/hFc in islet ß-cells using rat insulin II promoter (RIP). These transgenic mice exhibit an increase in islet mass with higher proportion of larger islets in pancreas compared to wild-type littermates. This is in contrast to pancreas from Pref-1 null mice that show higher proportion of smaller islets. Insulin expression and insulin secretion from pancreatic islets from RIP-Pref-1/hFc transgenic mice are increased also. Thus, RIP-Pref-1/hFc transgenic mice show normal glucose levels but with higher plasma insulin levels in both fasting and fed conditions. These mice show improved glucose tolerance. Taken together, we conclude Pref-1 as a positive regulator of islet ß-cells and insulin production.


Assuntos
Proliferação de Células , Hiperinsulinismo/metabolismo , Hiperinsulinismo/patologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Feminino , Resistência à Insulina , Secreção de Insulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regulação para Cima
4.
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
5.
J Exp Med ; 199(1): 69-80, 2004 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-14699085

RESUMO

Survivin is an inhibitor of apoptosis protein that also functions during mitosis. It is expressed in all common tumors and tissues with proliferating cells, including thymus. To examine its role in apoptosis and proliferation, we generated two T cell-specific survivin-deficient mouse lines with deletion occurring at different developmental stages. Analysis of early deleting survivin mice showed arrest at the pre-T cell receptor proliferating checkpoint. Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number. In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis. However, newborn thymocyte homeostatic and mitogen-induced proliferation of survivin-deficient T cells were greatly impaired. These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.


Assuntos
Apoptose/imunologia , Proteínas Associadas aos Microtúbulos/genética , Linfócitos T/imunologia , Animais , Genótipo , Homeostase , Proteínas Inibidoras de Apoptose , Ativação Linfocitária/imunologia , Camundongos , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas de Neoplasias , Mapeamento por Restrição , Survivina
6.
J Exp Med ; 199(6): 825-30, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15007090

RESUMO

Mice deficient for the B cell-restricted transcription factor Pax5 show a defect in the VH to DJH rearrangement step of immunoglobulin heavy chain gene assembly even though the expression of the V(D)J recombinase is not diminished in Pax5-/- pro-B cells. To investigate whether Pax5 is limiting for VH to DJH rearrangement, we generated transgenic mice which express Pax5 in developing thymocytes. We show that enforced expression of Pax5 in thymocytes results in a partial block in T cell development due to defective pre-TCR signaling in beta-selection. Moreover, our results demonstrate that expression of Pax5 in early thymocytes is sufficient to induce VH to DJH rearrangements in CD4+CD8+ T cells and lead us to suggest that Pax5 may play a direct role in the lineage-specific regulation of immunoglobulin heavy chain gene rearrangement.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Rearranjo Gênico do Linfócito B/fisiologia , Genes de Imunoglobulinas/fisiologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Citometria de Fluxo , Camundongos , Camundongos Transgênicos , Fator de Transcrição PAX5 , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Selectinas/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/fisiologia , Timo/citologia , Timo/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , VDJ Recombinases/metabolismo
7.
J Clin Invest ; 111(4): 453-61, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12588883

RESUMO

Preadipocyte factor-1 (Pref-1) is a transmembrane protein highly expressed in preadipocytes. Pref-1 expression is, however, completely abolished in adipocytes. The extracellular domain of Pref-1 undergoes two proteolytic cleavage events that generate 50 and 25 kDa soluble products. To understand the function of Pref-1, we generated transgenic mice that express the full ectodomain corresponding to the large cleavage product of Pref-1 fused to human immunoglobulin-gamma constant region. Mice expressing the Pref-1/hFc transgene in adipose tissue, driven by the adipocyte fatty acid-binding protein (aP2, also known as aFABP) promoter, showed a substantial decrease in total fat pad weight. Moreover, adipose tissue from transgenic mice showed reduced expression of adipocyte markers and adipocyte-secreted factors, including leptin and adiponectin, whereas the preadipocyte marker Pref-1 was increased. Pref-1 transgenic mice with a substantial, but not complete, loss of adipose tissue exhibited hypertriglyceridemia, impaired glucose tolerance, and decreased insulin sensitivity. Mice expressing the Pref-1/hFc transgene exclusively in liver under the control of the albumin promoter also showed a decrease in adipose mass and adipocyte marker expression, suggesting an endocrine mode of action of Pref-1. These findings demonstrate the inhibition of adipogenesis by Pref-1 in vivo and the resulting impairment of adipocyte function that leads to the development of metabolic abnormalities.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Intolerância à Glucose/etiologia , Proteínas de Membrana/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sequência de Bases , Osso e Ossos/anormalidades , Osso e Ossos/embriologia , Proteínas de Ligação ao Cálcio , Contagem de Células , Diferenciação Celular , Tamanho Celular , DNA Complementar/genética , Feminino , Expressão Gênica , Intolerância à Glucose/metabolismo , Transtornos do Crescimento/embriologia , Transtornos do Crescimento/genética , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Solubilidade
8.
Mol Cell Biol ; 23(16): 5896-907, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12897158

RESUMO

Upstream regulatory factor (USF) and sterol regulatory element binding protein (SREBP) play key roles in the transcriptional regulation of the fatty acid synthase (FAS) gene by feeding and insulin. Due to the dual binding specificity of SREBP, as well as the presence of multiple consensus sites for these transcription factors in the FAS promoter, their physiologically relevant functional binding sites have been controversial. Here, in order to determine the occupancy of the putative USF and SREBP binding sites, we examined their protein-DNA interactions in living animals by using formaldehyde cross-linking and immunoprecipitation of chromatin and tested the function of these elements by employing mice transgenic for a reporter gene driven by various 5' deletions as well as site-specific mutations of the FAS promoter. We show that the -332 and -65 E-boxes are bound by USF in both fasted and refed mice, while the -150 SRE is bound by SREBP-1 only in refed mice. We also found that mutation of either the -150 SRE or the -65 E-box abolishes the feeding-induced activation of the FAS promoter in transgenic mice. Furthermore, in vivo occupancy of the FAS promoter by SREBP in the fed state can be prevented by mutation not only of the -150 SRE but, unexpectedly, of the -65 E-box as well. We conclude that the FAS promoter is activated during refeeding via the induced binding of SREBP to the -150 SRE and that USF binding to the -65 E-box is also required for SREBP binding and activation of the FAS promoter.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Ligação a DNA/genética , Ácido Graxo Sintases/biossíntese , Ácido Graxo Sintases/genética , Regulação Enzimológica da Expressão Gênica , Fatores de Transcrição , Animais , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Formaldeído/farmacologia , Deleção de Genes , Genes Reporter , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , RNA/metabolismo , Ribonucleases/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1 , Fatores de Tempo , Transcrição Gênica
9.
J Exp Med ; 213(5): 657-65, 2016 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-27045008

RESUMO

NLRs (nucleotide-binding domain [NBD] leucine-rich repeat [LRR]-containing proteins) exhibit diverse functions in innate and adaptive immunity. NAIPs (NLR family, apoptosis inhibitory proteins) are NLRs that appear to function as cytosolic immunoreceptors for specific bacterial proteins, including flagellin and the inner rod and needle proteins of bacterial type III secretion systems (T3SSs). Despite strong biochemical evidence implicating NAIPs in specific detection of bacterial ligands, genetic evidence has been lacking. Here we report the use of CRISPR/Cas9 to generate Naip1(-/-) and Naip2(-/-) mice, as well as Naip1-6(Δ/Δ) mice lacking all functional Naip genes. By challenging Naip1(-/-) or Naip2(-/-) mice with specific bacterial ligands in vivo, we demonstrate that Naip1 is uniquely required to detect T3SS needle protein and Naip2 is uniquely required to detect T3SS inner rod protein, but neither Naip1 nor Naip2 is required for detection of flagellin. Previously generated Naip5(-/-) mice retain some residual responsiveness to flagellin in vivo, whereas Naip1-6(Δ/Δ) mice fail to respond to cytosolic flagellin, consistent with previous biochemical data implicating NAIP6 in flagellin detection. Our results provide genetic evidence that specific NAIP proteins function to detect specific bacterial proteins in vivo.


Assuntos
Bactérias/imunologia , Proteína Inibidora de Apoptose Neuronal/imunologia , Sistemas de Secreção Tipo III/imunologia , Animais , Bactérias/genética , Flagelina/genética , Flagelina/imunologia , Camundongos , Camundongos Knockout , Proteína Inibidora de Apoptose Neuronal/genética , Sistemas de Secreção Tipo III/genética
10.
Cell Rep ; 8(3): 678-87, 2014 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-25088414

RESUMO

Pref-1 is an EGF-repeat-containing protein that inhibits adipocyte differentiation. To better understand the origin and development of white adipose tissue (WAT), we generated transgenic mouse models for transient or permanent fluorescent labeling of cells using the Pref-1 promoter, facilitating inducible ablation. We show that Pref-1-marked cells retain proliferative capacity and are very early adipose precursors, prior to expression of Zfp423 or PPARγ. In addition, the Pref-1-marked cells establish that adipose precursors are mesenchymal, but not endothelial or pericytal, in origin. During embryogenesis, Pref-1-marked cells first appear in the dorsal mesenteric region as early as embryonic day 10.5 (E10.5). These cells become lipid-laden adipocytes at E17.5 in the subcutaneous region, whereas visceral WAT develops after birth. Finally, ablation of Pref-1-marked cells prevents not only embryonic WAT development but also later adult adipose expansion upon high-fat feeding, demonstrating the requirement of Pref-1 cells for adipogenesis.


Assuntos
Adipogenia , Tecido Adiposo Branco/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/citologia , Adipócitos Brancos/citologia , Adipócitos Brancos/metabolismo , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/embriologia , Animais , Proteínas de Ligação ao Cálcio , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Science ; 339(6124): 1219-24, 2013 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-23471412

RESUMO

Despite considerable interest in the modulation of tumor-associated Foxp3(+) regulatory T cells (T(regs)) for therapeutic benefit, little is known about the developmental origins of these cells and the nature of the antigens that they recognize. We identified an endogenous population of antigen-specific T(regs) (termed MJ23 T(regs)) found recurrently enriched in the tumors of mice with oncogene-driven prostate cancer. MJ23 T(regs) were not reactive to a tumor-specific antigen but instead recognized a prostate-associated antigen that was present in tumor-free mice. MJ23 T(regs) underwent autoimmune regulator (Aire)-dependent thymic development in both male and female mice. Thus, Aire-mediated expression of peripheral tissue antigens drives the thymic development of a subset of organ-specific T(regs), which are likely coopted by tumors developing within the associated organ.


Assuntos
Tolerância Imunológica , Próstata/imunologia , Neoplasias da Próstata/imunologia , Linfócitos T Reguladores/imunologia , Timo/crescimento & desenvolvimento , Timo/imunologia , Fatores de Transcrição/imunologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Autoantígenos/imunologia , Antígenos CD4/análise , Feminino , Fatores de Transcrição Forkhead/análise , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Transgênicos , Antígeno Prostático Específico/imunologia , Fatores de Transcrição/genética , Proteína AIRE
12.
Cell Metab ; 13(6): 739-48, 2011 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-21641555

RESUMO

While fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes, including UCP-1 with lower PPARα binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining a BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis.


Assuntos
Adenilato Quinase/metabolismo , Lipase/genética , Processamento de Proteína Pós-Traducional , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Adiposidade/genética , Animais , Células Cultivadas , Ensaios Enzimáticos , Técnicas de Inativação de Genes , Canais Iônicos/genética , Canais Iônicos/metabolismo , Lipase/metabolismo , Lipólise/genética , Masculino , Camundongos , Camundongos Obesos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação , Termogênese , Proteína Desacopladora 1
13.
Diabetes ; 58(4): 855-66, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19136649

RESUMO

OBJECTIVE: To investigate the role of desnutrin in adipose tissue triacylglycerol (TAG) and fatty acid metabolism. RESEARCH DESIGN AND METHODS: We generated transgenic mice overexpressing desnutrin (also called adipose triglyceride lipase [ATGL]) in adipocytes (aP2-desnutrin) and also performed adenoviral-mediated overexpression of desnutrin in 3T3-L1CARDelta1 adipocytes. RESULTS: aP2-desnutrin mice were leaner with decreased adipose tissue TAG content and smaller adipocyte size. Overexpression of desnutrin increased lipolysis but did not result in increased serum nonesterified fatty acid levels or ectopic TAG storage. We found increased cycling between diacylglycerol (DAG) and TAG and increased fatty acid oxidation in adipocytes from these mice, as well as improved insulin sensitivity. CONCLUSIONS: We show that by increasing lipolysis, desnutrin overexpression causes reduced adipocyte TAG content and attenuation of diet-induced obesity. Desnutrin-mediated lipolysis promotes fatty acid oxidation and re-esterification within adipocytes.


Assuntos
Tecido Adiposo/fisiologia , Tecido Adiposo/fisiopatologia , Hidrolases de Éster Carboxílico/genética , Ingestão de Energia , Obesidade/prevenção & controle , Células 3T3 , Adipócitos/citologia , Adipócitos/fisiologia , Animais , Hidrolases de Éster Carboxílico/fisiologia , Clonagem Molecular , Feminino , Lipase/metabolismo , Lipólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Obesidade/etiologia , Regiões Promotoras Genéticas , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trioleína/metabolismo
14.
Nat Med ; 15(2): 159-68, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19136964

RESUMO

A main function of white adipose tissue is to release fatty acids from stored triacylglycerol for other tissues to use as an energy source. Whereas endocrine regulation of lipolysis has been extensively studied, autocrine and paracrine regulation is not well understood. Here we describe the role of the newly identified major adipocyte phospholipase A(2), AdPLA (encoded by Pla2g16, also called HREV107), in the regulation of lipolysis and adiposity. AdPLA-null mice have a markedly higher rate of lipolysis owing to increased cyclic AMP levels arising from the marked reduction in the amount of adipose prostaglandin E(2) that binds the Galpha(i)-coupled receptor, EP3. AdPLA-null mice have markedly reduced adipose tissue mass and triglyceride content but normal adipogenesis. They also have higher energy expenditure with increased fatty acid oxidation within adipocytes. AdPLA-deficient ob/ob mice remain hyperphagic but lean, with increased energy expenditure, yet have ectopic triglyceride storage and insulin resistance. AdPLA is a major regulator of adipocyte lipolysis and is crucial for the development of obesity.


Assuntos
Adipócitos/enzimologia , Gorduras na Dieta/administração & dosagem , Leptina/deficiência , Lipólise , Obesidade/prevenção & controle , Fosfolipases A2/metabolismo , Adipócitos/metabolismo , Animais , Dinoprostona/metabolismo , Metabolismo Energético , Resistência à Insulina , Camundongos , Camundongos Knockout , Fosfolipases A2/genética
15.
Eur J Immunol ; 38(11): 3200-7, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18991293

RESUMO

The PI3K-AKT pathway can mediate diverse biological responses and is crucial for optimal immune responses and lymphocyte development. Deletion of PI3K subunits or AKT leads to blockage of T-cell development at the TCR-beta checkpoint. Studies with over-expression of constitutively activated AKT have implicated this pathway in anti-apoptosis of developing thymocytes and in development of regulatory T cells. However, the role of endogenous PI3K-AKT in T-cell development beyond the TCR-beta checkpoint remains unclear. Here, we inhibited the endogenous PI3K-AKT pathway in thymocytes after double negative stages by expressing the negative regulator, PTEN. These mice exhibit normal early T-cell development, but the transition from intermediate single positive to double positive (DP) thymocytes is inhibited, leading to a significantly decreased number of DP, single positive thymocytes and peripheral T cells. Proliferation of peripheral T cells is reduced but apoptosis of DP cells and subsequent T-cell maturation, including regulatory T cells, are normal. AKT phosphorylation can be readily observed in most WT T-cell compartments but not DP thymocytes in response to TCR activation. Thus, the PI3K-AKT pathway is crucial for the transition of intermediate single positive to DP thymocytes but is dispensable for apoptosis and maturation of developing thymocytes.


Assuntos
Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/fisiologia , Linfócitos T/fisiologia , Animais , Apoptose , Camundongos , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia
16.
J Exp Med ; 205(4): 929-38, 2008 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-18378791

RESUMO

The production of distinct sets of T cell receptor (TCR) gammadelta(+) T cells occurs in an ordered fashion in thymic development. The Vgamma3 and Vgamma4 genes, located downstream in the TCRgamma Cgamma1 gene cluster, are expressed by the earliest waves of developing TCRgammadelta(+) T cells in the fetal thymus, destined for intraepithelial locations. Upstream Vgamma2 and Vgamma5 genes are expressed in later waves in the adult and constitute most TCRgammadelta(+) T cells in secondary lymphoid tissue. This developmental pattern is caused in part by a preference for rearrangements of the downstream Vgamma3 and Vgamma4 genes in the early fetal stage, which switches to a preference for rearrangements of the upstream Vgamma2 and Vgamma5 gene rearrangements in the adult. Our gene targeting studies show that the downstream Vgamma genes rearrange preferentially in the early fetal thymus because of their downstream location, independent of promoter or recombination signal sequences and unrelated to the extent of germline transcription. Remarkably, gene deletion studies show that the downstream Vgamma genes competitively inhibit upstream Vgamma rearrangements at the fetal stage. These data provide a mechanism for specialization of the fetal thymus for the production of T cells expressing specific Vgamma genes.


Assuntos
Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T/genética , Animais , Enzimas de Restrição do DNA/metabolismo , Feto/imunologia , Deleção de Genes , Marcação de Genes , Células Germinativas , Camundongos , Timo/citologia , Timo/imunologia , Transcrição Gênica
17.
Science ; 319(5860): 215-20, 2008 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-18187659

RESUMO

Substantial evidence exists that many tumors can be specifically recognized by CD8+ T lymphocytes. The definition of antigens targeted by these cells is paramount for the development of effective immunotherapeutic strategies for treating human cancers. In a screen for endogenous tumor-associated T cell responses in a primary mouse model of prostatic adenocarcinoma, we identified a naturally arising CD8+ T cell response that is reactive to a peptide derived from histone H4. Despite the ubiquitous nature of histones, T cell recognition of histone H4 peptide was specifically associated with the presence of prostate cancer in these mice. Thus, the repertoire of antigens recognized by tumor-infiltrating T cells is broader than previously thought and includes peptides derived from ubiquitous self antigens that are normally sequestered from immune detection.


Assuntos
Adenocarcinoma/imunologia , Antígenos de Neoplasias/imunologia , Autoantígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Histonas/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias da Próstata/imunologia , Transferência Adotiva , Animais , Apresentação de Antígeno , Linhagem Celular , Epitopos de Linfócito T/imunologia , Hibridomas , Masculino , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia
18.
J Immunol ; 174(11): 6732-41, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15905513

RESUMO

Although Notch plays a crucial role in T cell development, regulation of Notch signaling in the thymus is not well understood. Kuzbanian, an ADAM protease, has been implicated in the cleavage of both Notch receptors and the Notch ligand, Delta. In this study we show that the expression of a dominant-negative form of Kuzbanian (dnKuz) leads to reduced TCRbeta expression in double-negative thymocytes and to a partial block between the double-negative to double-positive stages of development. These defects were rescued by overexpression of Delta-1 on thymocytes. Mixed chimeras showed a cell-autonomous block by dnKuz, but non-cell-autonomous rescue by Delta-1. This suggests that dnKuz impairs Notch signaling in receiving cells, and increasing Delta-1 on sending cells overcomes this defect. Interestingly, the expression of an activated form of Notch-1 rescued some, but not all, the defects in dnKuz Tg mice. Our data suggest that multiple Notch-dependent steps in early thymocyte development require Kuzbanian, but differ in the involvement of other Notch signaling components.


Assuntos
Diferenciação Celular/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Metaloendopeptidases/genética , Receptores de Superfície Celular/metabolismo , Linfócitos T/metabolismo , Timo/metabolismo , Fatores de Transcrição/metabolismo , Proteínas ADAM , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Animais , Diferenciação Celular/genética , Regulação da Expressão Gênica/imunologia , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Metaloendopeptidases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Quimera por Radiação , Receptor Notch1 , Receptores de Antígenos de Linfócitos T alfa-beta/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/enzimologia , Linfócitos T/patologia , Timo/enzimologia , Timo/patologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
19.
Immunity ; 16(3): 453-63, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11911829

RESUMO

Many mammalian genes, including those encoding antigen receptors, contain more than one enhancer element. Deleting one element often does not prevent expression, but functional redundancy has never been directly demonstrated by gene targeting of multiple elements. We demonstrate that simultaneous deletion of two enhancer/LCR-like elements in the TCR Cgamma1 cluster, HsA and 3'E(Cgamma1), severely diminishes TCRgamma transcription, selectively impairs development of gammadelta thymocyte subsets, but only modestly reduces TCRgamma gene rearrangement, while deletion of each element separately has little effect. In contrast to these results in thymocytes, deletion of HsA alone reduces transcription of one Vgamma gene specifically in peripheral gammadelta T cells. Thus, the two elements exhibit functional redundancy in thymocytes but also have unique functions in other settings.


Assuntos
Elementos Facilitadores Genéticos/genética , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/imunologia , Animais , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Recombinação Genética
20.
Proc Natl Acad Sci U S A ; 101(17): 6780-5, 2004 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-15090646

RESUMO

Adipocyte-specific secretory factor (ADSF)/resistin is a small cysteine-rich protein secreted from adipose tissue that belongs to a gene family found in inflammatory zone (FIZZ) or found in resistin-like molecule (RELM). ADSF has been implicated in modulating adipogenesis and insulin resistance. To examine the long-term function of ADSF in adipogenesis and glucose homeostasis, we constructed an expression vector for a dominant inhibitory form of ADSF by fusing it to the human IgGgamma constant region (hFc). ADSF-hFc not only homodimerizes but heterooligomerizes with ADSF/resistin and prevents ADSF/resistin inhibition of adipocyte differentiation of 3T3-L1 cells in a dominant negative manner. Transgenic mice overexpressing ADSF-hFc in adipose tissue show increased adiposity with elevated expression of adipocyte markers as well as enlarged adipocyte size. This finding clearly demonstrates in vivo the inhibitory role of ADSF in adipogenesis. ADSF-hFc transgenic mice with impaired ADSF function exhibit improved glucose tolerance and insulin sensitivity either on chow or high-fat diets. Because of the enhanced adipocyte differentiation, the ADSF-hFc transgenic mice show increased expression of leptin and adiponectin in adipose tissue. The elevated circulating levels for these adipocyte-derived hormones with decreased plasma triglyceride and free fatty acid levels may account for the improved glucose and insulin tolerance in these transgenic mice.


Assuntos
Adipócitos/citologia , Divisão Celular/fisiologia , Hormônios Ectópicos/fisiologia , Células 3T3-L1 , Animais , Northern Blotting , Western Blotting , Diferenciação Celular/fisiologia , Teste de Tolerância a Glucose , Resistência à Insulina , Camundongos , Camundongos Transgênicos , Resistina , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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