Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Blood ; 125(19): 2995-3005, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25824689

RESUMO

Megakaryocyte maturation and polyploidization are critical for platelet production; abnormalities in these processes are associated with myeloproliferative disorders, including thrombocytopenia. Megakaryocyte maturation signals through cascades that involve p21-activated kinase (Pak) function; however, the specific role for Pak kinases in megakaryocyte biology remains elusive. Here, we identify Pak2 as an essential effector of megakaryocyte maturation, polyploidization, and proplatelet formation. Genetic deletion of Pak2 in murine bone marrow is associated with macrothrombocytopenia, altered megakaryocyte ultrastructure, increased bone marrow megakaryocyte precursors, and an elevation of mature CD41(+) megakaryocytes, as well as an increased number of polyploid cells. In Pak2(-/-) mice, platelet clearance rate was increased, as was production of newly synthesized, reticulated platelets. In vitro, Pak2(-/-) megakaryocytes demonstrate increased polyploidization associated with alterations in ß1-tubulin expression and organization, decreased proplatelet extensions, and reduced phosphorylation of the endomitosis regulators LIM domain kinase 1, cofilin, and Aurora A/B/C. Together, these data establish a novel role for Pak2 as an important regulator of megakaryopoiesis, polyploidization, and cytoskeletal dynamics in developing megakaryocytes.


Assuntos
Plaquetas/metabolismo , Citoesqueleto/patologia , Megacariócitos/metabolismo , Mitose/genética , Fator de Transcrição PAX2/fisiologia , Trombocitopenia/genética , Trombopoese/fisiologia , Animais , Plaquetas/patologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Citoesqueleto/metabolismo , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Microscopia de Fluorescência , Poliploidia , Células-Tronco/metabolismo , Células-Tronco/patologia , Trombocitopenia/patologia
2.
Stem Cells ; 33(5): 1630-41, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25586960

RESUMO

p21-Activated kinase 2 (Pak2), a serine/threonine kinase, has been previously shown to be essential for hematopoietic stem cell (HSC) engraftment. However, Pak2 modulation of long-term hematopoiesis and lineage commitment remain unreported. Using a conditional Pak2 knockout mouse model, we found that disruption of Pak2 in HSCs induced profound leukopenia and a mild macrocytic anemia. Although loss of Pak2 in HSCs leads to less efficient short- and long-term competitive hematopoiesis than wild-type cells, it does not affect HSC self-renewal per se. Pak2 disruption decreased the survival and proliferation of multicytokine stimulated immature progenitors. Loss of Pak2 skewed lineage differentiation toward granulocytopoiesis and monocytopoiesis in mice as evidenced by (a) a three- to sixfold increase in the percentage of peripheral blood granulocytes and a significant increase in the percentage of granulocyte-monocyte progenitors in mice transplanted with Pak2-disrupted bone marrow (BM); (b)Pak2-disrupted BM and c-kit(+) cells yielded higher numbers of more mature subsets of granulocyte-monocyte colonies and polymorphonuclear neutrophils, respectively, when cultured in the presence of granulocyte-macrophage colony-stimulating factor. Pak2 disruption resulted, respectively, in decreased and increased gene expression of transcription factors JunB and c-Myc, which may suggest underlying mechanisms by which Pak2 regulates granulocyte-monocyte lineage commitment. Furthermore, Pak2 disruption led to (a) higher percentage of CD4(+) CD8(+) double positive T cells and lower percentages of CD4(+) CD8(-) or CD4(-) CD8(+) single positive T cells in thymus and (b) decreased numbers of mature B cells and increased numbers of Pre-Pro B cells in BM, suggesting defects in lymphopoiesis.


Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Quinases Ativadas por p21/metabolismo , Anemia Macrocítica/patologia , Animais , Apoptose , Proliferação de Células , Sobrevivência Celular , Deleção de Genes , Regulação da Expressão Gênica , Hematopoese , Leucopenia/patologia , Linfopoese , Camundongos Knockout , Células Mieloides/patologia , Fenótipo , Fatores de Transcrição/metabolismo , Quinases Ativadas por p21/deficiência
3.
EMBO J ; 30(15): 3160-72, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21725281

RESUMO

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ativação Linfocitária , Mastócitos/imunologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Animais , Células Cultivadas , Humanos , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Camundongos , Fosfolipase C gama/metabolismo , Fosforilação , Tirosina/metabolismo
4.
Blood ; 121(13): 2474-82, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23335370

RESUMO

The p21-activated kinases (Paks) are serine/threonine kinases that are major effectors of the Rho guanosine 5'\x{2011}triphosphatase, Rac, and Cdc42. Rac and Cdc42 are known regulators of hematopoietic stem and progenitor cell (HSPC) function, however, a direct role for Paks in HSPCs has yet to be elucidated. Lin(-)Sca1(+)c-kit(+) (LSK) cells from wild-type mice were transduced with retrovirus expressing Pak inhibitory domain (PID), a well-characterized inhibitor of Pak activation. Defects in marrow homing and in vitro cell migration, assembly of the actin cytoskeleton, proliferation, and survival were associated with engraftment failure of PID-LSK. The PID-LSK demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK), whereas constitutive activation of ERK in these cells led to rescue of hematopoietic progenitor cell proliferation in vitro and partial rescue of Pak-deficient HSPC homing and engraftment in vivo. Using conditional knock-out mice, we demonstrate that among group A Paks, Pak2(-/-) HSPC show reduced homing to the bone marrow and altered cell shape similar to PID-LSK cells in vitro and are completely defective in HSPC engraftment. These data demonstrate that Pak proteins are key components of multiple engraftment-associated HSPC functions and play a direct role in activation of ERK in HSPCs, and that Pak2 is specifically essential for HSPC engraftment.


Assuntos
Movimento Celular/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Quinases Ativadas por p21/fisiologia , Animais , Movimento Celular/fisiologia , Proliferação de Células , Sobrevivência Celular/genética , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Quinases Ativadas por p21/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/fisiologia
5.
J Biol Chem ; 288(2): 974-83, 2013 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-23204526

RESUMO

p21-activated kinase-1 (Pak1) is a serine/threonine kinase that plays a key role in mediating antigen-stimulated extracellular calcium influx and degranulation in mast cells. Another isoform in this kinase family, Pak2, is expressed at very high levels in mast cells, but its function is unknown. Here we show that Pak2 loss in murine bone marrow-derived mast cells, unlike loss of Pak1, induces increased antigen-mediated adhesion, degranulation, and cytokine secretion without changes to extracellular calcium influx. This phenotype is associated with an increase in RhoA-GTPase signaling activity to downstream effectors, including myosin light chain and p38(MAPK), and is reversed upon treatment with a Rho-specific inhibitor. Pak2, but not Pak1, negatively regulates RhoA via phosphorylation of the guanine nucleotide exchange factor GEF-H1 at an inhibitory site, leading to increased GEF-H1 microtubule binding and loss of RhoA stimulation. These data suggest that Pak2 plays a unique inhibitory role in mast cell degranulation by down-regulating RhoA via GEF-H1.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Células da Medula Óssea/metabolismo , Cálcio/metabolismo , Adesão Celular , Células Cultivadas , Primers do DNA , Camundongos , Reação em Cadeia da Polimerase , Quinases Ativadas por p21
6.
J Cell Physiol ; 227(6): 2654-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21882191

RESUMO

Understanding the molecular and cellular processes underlying the development, maintenance, and progression of Barrett's esophagus (BE) presents an empirical challenge because there are no simple animal models and standard 2D cell culture can distort cellular processes. Here we describe a three-dimensional (3D) cell culture system to study BE. BE cell lines (CP-A, CP-B, CP-C, and CP-D) and esophageal squamous keratinocytes (EPC2) were cultured on a matrix consisting of esophageal fibroblasts and collagen. Comparison of growth and cytokeratin expression in the presence of all-trans retinoic acid or hydrochloric acid was made by immunohistochemistry and Alcian Blue staining to determine which treatments produced a BE phenotype of columnar cytokeratin expression in 3D culture. All-trans retinoic acid differentially affected the growth of BE cell lines in 3D culture. Notably, the non-dyplastic metaplasia-derived cell line (CP-A) expressed reduced squamous cytokeratins and enhanced columnar cytokeratins upon ATRA treatment. ATRA altered the EPC2 squamous cytokeratin profile towards a more columnar expression pattern. Cell lines derived from patients with high-grade dysplasia already expressed columnar cytokeratins and therefore did not show a systematic shift toward a more columnar phenotype with ATRA treatment. ATRA treatment, however, did reduce the squamoid-like multilayer stratification observed in all cell lines. As the first study to demonstrate long-term 3D growth of BE cell lines, we have determined that BE cells can be cultured for at least 3 weeks on a fibroblast/collagen matrix and that the use of ATRA causes a general reduction in squamous-like multilayered growth and an increase in columnar phenotype with the specific effects cell-line dependent.


Assuntos
Esôfago de Barrett/patologia , Células Epiteliais/patologia , Esôfago/patologia , Fibroblastos/patologia , Queratinócitos/patologia , Linhagem Celular Transformada , Técnicas de Cocultura , Colágeno/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Esôfago/efeitos dos fármacos , Esôfago/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Ácido Clorídrico/farmacologia , Concentração de Íons de Hidrogênio , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinas/metabolismo , Metaplasia , Fenótipo , Telomerase/genética , Telomerase/metabolismo , Fatores de Tempo , Transfecção , Tretinoína/farmacologia
7.
BMC Cancer ; 11: 461, 2011 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-22026449

RESUMO

BACKGROUND: The evolutionary dynamics between interacting heterogeneous cell types are fundamental properties of neoplastic progression but can be difficult to measure and quantify. Cancers are heterogeneous mixtures of mutant clones but the direct effect of interactions between these clones is rarely documented. The implicit goal of most preventive interventions is to bias competition in favor of normal cells over neoplastic cells. However, this is rarely explicitly tested. Here we have developed a cell culture competition model to allow for direct observation of the effect of chemopreventive or therapeutic agents on two interacting cell types. We have examined competition between normal and Barrett's esophagus cell lines, in the hopes of identifying a system that could screen for potential chemopreventive agents. METHODS: One fluorescently-labeled normal squamous esophageal cell line (EPC2-hTERT) was grown in competition with one of four Barrett's esophagus cell lines (CP-A, CP-B, CP-C, CP-D) under varying conditions and the outcome of competition measured over 14 days by flow cytometry. RESULTS: We demonstrate that ascorbic acid (vitamin C) can help squamous cells outcompete Barrett's cells in this system. We are also able to show that ascorbic acid's boost to the relative fitness of squamous cells was increased in most cases by mimicking the pH conditions of gastrointestinal reflux in the lower esophagus. CONCLUSIONS: This model is able to integrate differential fitness effects on various cell types, allowing us to simultaneously capture effects on interacting cell types without having to perform separate experiments. This model system may be used to screen for new classes of cancer prevention agents designed to modulate the competition between normal and neoplastic cells.


Assuntos
Ácido Ascórbico/farmacologia , Esôfago de Barrett/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Esôfago/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Fator de Crescimento Epidérmico/farmacologia , Humanos
8.
Cell Host Microbe ; 20(5): 674-681, 2016 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-27746097

RESUMO

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.


Assuntos
Morte Celular , Glicoproteínas/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A/imunologia , RNA Viral/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Genômica , Glicoproteínas/deficiência , Camundongos , Camundongos Knockout , Mutação , Proteínas Quinases/metabolismo , Proteínas de Ligação a RNA
9.
Cell Host Microbe ; 20(1): 13-24, 2016 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-27321907

RESUMO

Influenza A virus (IAV) is a lytic virus in primary cultures of many cell types and in vivo. We report that the kinase RIPK3 is essential for IAV-induced lysis of mammalian fibroblasts and lung epithelial cells. Replicating IAV drives assembly of a RIPK3-containing complex that includes the kinase RIPK1, the pseudokinase MLKL, and the adaptor protein FADD, and forms independently of signaling by RNA-sensing innate immune receptors (RLRs, TLRs, PKR), or the cytokines type I interferons and TNF-α. Downstream of RIPK3, IAV activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis, with the former reliant on RIPK3 kinase activity and neither on RIPK1 activity. Mice deficient in RIPK3 or doubly deficient in MLKL and FADD, but not MLKL alone, are more susceptible to IAV than their wild-type counterparts, revealing an important role for RIPK3-mediated apoptosis in antiviral immunity. Collectively, these results outline RIPK3-activated cytolytic mechanisms essential for controlling respiratory IAV infection.


Assuntos
Apoptose , Proteína de Domínio de Morte Associada a Fas/metabolismo , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/imunologia , Necrose , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/fisiologia , Células Epiteliais/virologia , Proteína de Domínio de Morte Associada a Fas/genética , Fibroblastos/fisiologia , Fibroblastos/virologia , Humanos , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/patologia , Proteínas Quinases/genética , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
10.
Nat Rev Cancer ; 14(1): 13-25, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24505617

RESUMO

p21-Activated kinases (PAKs) are positioned at the nexus of several oncogenic signalling pathways. Overexpression or mutational activation of PAK isoforms frequently occurs in various human tumours, and recent data suggest that excessive PAK activity drives many of the cellular processes that are the hallmarks of cancer. In this Review, we discuss the mechanisms of PAK activation in cancer, the key substrates that mediate the developmental and oncogenic effects of this family of kinases, and how small-molecule inhibitors of these enzymes might be best developed and deployed for the treatment of cancer.


Assuntos
Neoplasias/enzimologia , Quinases Ativadas por p21/metabolismo , Animais , Antineoplásicos/farmacologia , Progressão da Doença , Ativação Enzimática , Amplificação de Genes , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Neovascularização Patológica/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genética
11.
Elife ; 3: e02270, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24843022

RESUMO

The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR ß-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation.DOI: http://dx.doi.org/10.7554/eLife.02270.001.


Assuntos
Citoesqueleto de Actina/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Timócitos/citologia , Quinases Ativadas por p21/fisiologia , Animais , Camundongos , Camundongos Knockout , Quinases Ativadas por p21/genética
12.
F1000 Biol Rep ; 22010 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-20948805

RESUMO

Knowledge of spatial patterning of GTPases is critical to understanding protein function since subcellular localization is essential for normal protein function. In this paper, we discuss how Hahn's group has created a new type of genetically encoded, light-activated enzyme that allows precise temporal and spatial control of the small GTPase, Rac1. This reagent enables this group to analyze the roles of effector proteins in Rac1 function and may be applicable to other signaling proteins.

13.
Cancer Res ; 70(5): 1941-50, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20160040

RESUMO

Curcumin, a plant polyphenol, is a widely studied chemopreventive agent with demonstrated antitumor activities in preclinical studies and low toxicity profiles in multiple clinical trials against human malignancies. We previously showed that curcumin radiosensitizes cervical tumor cells without increasing the cytotoxic effects of radiation on normal human fibroblasts. Here we report that an inhibitory activity of curcumin on the antioxidant enzyme thioredoxin reductase-1 (TxnRd1) is required for curcumin-mediated radiosensitization of squamous carcinoma cells. Stable knockdown of TxnRd1 in both HeLa and FaDu cells nearly abolished curcumin-mediated radiosensitization. TxnRd1 knockdown cells showed decreased radiation-induced reactive oxygen species and sustained extracellular signal-regulated kinase 1/2 activation, which we previously showed was required for curcumin-mediated radiosensitization. Conversely, overexpressing catalytically active TxnRd1 in HEK293 cells, with low basal levels of TxnRd1, increased their sensitivity to curcumin alone and to the combination of curcumin and ionizing radiation. These results show the critical role of TxnRd1 in curcumin-mediated radiosensitization and suggest that TxnRd1 levels in tumors could have clinical value as a predictor of response to curcumin and radiotherapy.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/radioterapia , Curcumina/farmacologia , Radiossensibilizantes/farmacologia , Tiorredoxina Redutase 1/biossíntese , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/enzimologia , Transformação Celular Neoplásica/metabolismo , Ativação Enzimática , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Redutase 1/deficiência , Tiorredoxina Redutase 1/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA