RESUMO
The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34(+) progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transformação Celular Neoplásica/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Granulócitos/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteína Sequestossoma-1 , Tretinoína/farmacologia , Ubiquitinação , Regulação para CimaRESUMO
BACKGROUND: Plasma and other body fluids contain membranous extracellular vesicles (EVs), which are considered to derive from activated or apoptotic cells. EVs participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. Knowledge on EVs is, however, limited, mainly due to their sub-micrometer size and to intrinsic limitations in methods applied for their characterization. OBJECTIVES: Our aim was to provide a comprehensive description of EVs from plasma of healthy subjects. METHODS: Cryo-transmission electron microscopy combined with receptor-specific gold labeling was used to reveal the morphology, size and phenotype of EVs. An original approach based on sedimentation on electron microscopy grids was developed for enumerating EVs. A correlation was performed between conventional flow cytometry and electron microscopy results. RESULTS: We show that platelet-free plasma samples contain spherical EVs, 30 nm to 1 µm in diameter, tubular EVs, 1-5 µm long, and membrane fragments, 1-8 µm large. We show that only a minority of EVs expose the procoagulant lipid phosphatidylserine, in contrast to the classical theory of EV formation. In addition, the concentrations of the main EV sub-populations are determined after sedimentation on EM grids. Finally, we show that conventional flow cytometry, the main method of EV characterization, detects only about 1% of them. CONCLUSION: This study brings novel insights on EVs from normal plasma and provides a reference for further studies of EVs in disease situations.
Assuntos
Anticorpos Monoclonais/imunologia , Exossomos/química , Plasma/fisiologia , Anticorpos Monoclonais/química , Apoptose , Plaquetas/citologia , Microscopia Crioeletrônica , Citometria de Fluxo , Glicoforinas/metabolismo , Ouro/química , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Nanopartículas Metálicas/química , Fenótipo , Fosfatidilserinas/química , Plasma/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismoRESUMO
AXL receptor tyrosine kinase (RTK) is implicated in proliferation and invasion of many cancers, particularly in pancreatic ductal adenocarcinoma (PDAC), for which new therapeutic options are urgently required. We investigated whether inhibition of AXL activity by specific monoclonal antibodies (mAbs) is efficient in limiting proliferation and migration of pancreatic cancer cells. Expression of AXL was evaluated by immunohistochemistry in 42 PDAC. The AXL role in oncogenesis was studied using the short hairpin RNA approach in a pancreatic carcinoma cell line. We further generated antihuman AXL mAbs and evaluated their inhibitory effects and the AXL downstream signaling pathways first in vitro, in a panel of pancreatic cancer cell lines and then in vivo, using subcutaneous or orthotopic pancreatic tumor xenografts. AXL receptor was found expressed in 76% (32/42) of PDAC and was predominantly present in invasive cells. The AXL-knockdown Panc-1 cells decreased in vitro cell migration, survival and proliferation, and reduced in vivo tumor growth. Two selected anti-AXL mAbs (D9 and E8), which inhibited phosphorylation of AXL and of its downstream target AKT without affecting growth arrest-specific factor 6 (GAS6) binding, induced downexpression of AXL by internalization, leading to an inhibition of proliferation and migration in the four pancreatic cancer cell lines studied. In vivo, treatment by anti-AXL mAbs significantly reduced growth of both subcutaneous and orthotopic pancreatic tumor xenografts independently of their KRAS mutation status. Our in vitro and preclinical in vivo data demonstrate that anti-human AXL mAbs could represent a new approach to the pancreatic cancer immunotherapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Imunoterapia/métodos , Neoplasias Pancreáticas/terapia , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Animais , Carcinoma Ductal Pancreático/metabolismo , Movimento Celular/genética , Sobrevivência Celular/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos Nus , Terapia de Alvo Molecular , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética , Receptor Tirosina Quinase AxlAssuntos
Benzamidas/metabolismo , Piperazinas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Pirimidinas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Transporte Biológico , Humanos , Mesilato de Imatinib , Espaço Intracelular/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Transportador 1 de Cátions Orgânicos/antagonistas & inibidores , Transportador 1 de Cátions Orgânicos/metabolismo , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Resultado do TratamentoRESUMO
Imatinib, the anti-Abl tyrosine kinase inhibitor used as first-line therapy in chronic myeloid leukemia (CML), eliminates CML cells mainly by apoptosis and induces autophagy. Analysis of imatinib-treated K562 cells reveals a cell population with cell cycle arrest, p27 increase and senescence-associated beta galactosidase (SA-ß-Gal) staining. Preventing apoptosis by caspase inhibition decreases annexin V-positive cells, caspase-3 cleavage and increases the SA-ß-Gal-positive cell population. In addition, a concomitant increase of the cell cycle inhibitors p21 and p27 is detected emphasizing the senescent phenotype. Inhibition of apoptosis by targeting Bim expression or overexpression of Bcl2 potentiates senescence. The inhibition of autophagy by silencing the expression of the proteins ATG7 or Beclin-1 prevents the increase of SA-ß-Gal staining in response to imatinib plus Z-Vad. In contrast, in apoptotic-deficient cells (Bim expression or overexpression of Bcl2), the inhibition of autophagy did not significantly modify the SA-ß-Gal-positive cell population. Surprisingly, targeting autophagy by inhibiting ATG5 is accompanied by a strong SA-ß-Gal staining, suggesting a specific inhibitory role on senescence. These results demonstrate that in addition to apoptosis and autophagy, imatinib induced senescence in K562 CML cells. Moreover, apoptosis is limiting the senescent response to imatinib, whereas autophagy seems to have an opposite role.
Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Senescência Celular , Piperazinas/toxicidade , Pirimidinas/toxicidade , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 7 Relacionada à Autofagia , Proteína 11 Semelhante a Bcl-2 , Proteína Beclina-1 , Benzamidas , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
Once cleaved by caspases, the Lyn tyrosine kinase (LynDeltaN) is relocalized from the plasma membrane to the cytoplasm of apoptotic cells, but the function of such a cleavage is incompletely understood. We evaluated the effect of LynDeltaN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynDeltaN or its catalytically inactive counterpart LynDeltaNKD. We established that Lyn is cleaved in imatinib-treated parental K562 cells in a caspase-dependent manner. Lyn cleavage also occurred following BCR-ABL silencing by specific short hairpin RNA (sh-RNA). Imatinib-induced apoptosis was abrogated in LynDeltaN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynDeltaN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynDeltaN was suppressed by two inhibitors of Lyn activity. LynDeltaN also inhibits imatinib-mediated caspase-3 activation in the small proportion of nilotinib-resistant K562 cells overexpressing Lyn that can engage an apoptotic program upon imatinib stimulation. Finally, Lyn knockdown by sh-RNA altered neither imatinib-mediated apoptosis nor differentiation. Taken together, our data show that the caspase-cleaved form of Lyn exerts a negative feedback on imatinib-mediated CML cell apoptosis that is entirely dependent on its kinase activity and likely on the BCR-ABL pathway.
Assuntos
Antineoplásicos/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Proteínas de Neoplasias/fisiologia , Piperazinas/antagonistas & inibidores , Inibidores de Proteínas Quinases/antagonistas & inibidores , Pirimidinas/antagonistas & inibidores , Quinases da Família src/fisiologia , Antineoplásicos/farmacologia , Benzamidas , Caspase 9/genética , Inibidores de Caspase , Ativação Enzimática , Eritropoese/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/fisiologia , Humanos , Mesilato de Imatinib , Indóis/farmacologia , Células K562/efeitos dos fármacos , Células K562/enzimologia , Células K562/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/química , Quinases da Família src/genéticaRESUMO
BACKGROUND: Scleroderma skin overexpresses the platelet-derived growth factor receptor beta-subunit (PDGFR-beta) in dermal vessels and PDGFR-beta messenger RNA in cultured fibroblasts. Moreover, increased levels of PDGF and stimulatory autoantibodies to PDGFR have been identified in the serum of scleroderma patients. OBJECTIVE: Imatinib being an inhibitor of tyrosine kinase receptors such as PDGFR, its effect on scleroderma fibroblasts was evaluated in vitro as a preclinical therapeutic step. METHODS: The effect of imatinib on fibroblasts grown from normal or involved/uninvolved scleroderma skin was studied by Western blot and the methyltetrazolium test. The pattern of distribution of PDGFR-beta in scleroderma versus normal skin was studied by immunohistochemistry. RESULTS: In vitro, imatinib inhibited the proliferation of normal dermal and scleroderma fibroblasts at least partly via the inhibition of the phosphorylation of PDGFR. PDGFR-beta was expressed in the epidermis and adnexae in 5 lesional scleroderma biopsies and not in controls. CONCLUSION: This study suggests that imatinib can serve as therapy to limit dermal fibroblast proliferation in scleroderma.
Assuntos
Fibroblastos/efeitos dos fármacos , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Escleroderma Sistêmico/tratamento farmacológico , Pele/citologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Benzamidas , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Mesilato de Imatinib , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptor beta de Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Pele/efeitos dos fármacos , Pele/metabolismo , Resultado do TratamentoRESUMO
Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 muM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Choque Térmico HSP70/biossíntese , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Regulação para Cima , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismoRESUMO
OBJECTIVE: To detect the redistribution of platelet surface glycoprotein (GP)Ib alpha and cytoskeleton reorganization in the course of thrombin receptor activation, and investigate the mechanism of GPIb alpha re-translocation and the role of thrombin receptors in platelet signal transduction. METHODS: The thrombin receptor activating peptide (PAR1-AP, TRAP) was used for stimulating platelet at different time points (0 - 60 min), then the platelet surface GPIb alpha and P-selectin were examined with flow cytometry, and the alterations of GPIb alpha, actin and myosin were analyzed in cytoskeleton by Western blot and GPIb alpha immunoprecipitation. Cytochalasin D and/or Apyrase VII were used for investigating their inhibitory effect on platelet activation. RESULTS: An increase of P-selectin and reversible internalization of GPIb alpha were observed within platelets upon TRAP activation, and transient changes of actin, myosin and GPIb alpha/myosin, GPIb alpha/actin association were also found in this course. These changes were apparently blocked by cytochalasin D, which inhibited the incorporation of GPIb alpha, actin and myosin into cytoskeleton. Apyrase VII had a weak effect on GPIb alpha internalization, although it accelerated the return of GPIb alpha to platelet surface. In addition, Apyrase VII also quickened the GPIb alpha disappearance in cytoskeleton and the dissociation of GPIb/myosin or GPIb/actin during activation. CONCLUSION: Thrombin receptor activation takes part in platelet signal transduction, inducing a reversible redistribution of GPIb alpha. This process is related to cytoskeleton reorganisation and ADP.
Assuntos
Plaquetas/metabolismo , Citoesqueleto/metabolismo , Ativação Plaquetária/fisiologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Receptores de Trombina/fisiologia , Actinas/metabolismo , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Western Blotting , Células Cultivadas , Humanos , Miosinas/metabolismo , Selectina-P/metabolismo , Fragmentos de Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Receptores de Trombina/metabolismoRESUMO
Thrombin activates human platelets by hydrolyzing the protease-activated receptors PAR-1 and PAR-4, exposing new N-terminal sequences which act as tethered ligands, and binding to glycoprotein (GP) Ib, whose surface accessibility transiently decreases when platelets are stimulated by the enzyme. In an attempt to better understand this latter process, we used the peptides SFLLRNPNDKYEPF (PAR-1-AP or TRAP) and AYPGKF (PAR-4-AP) to study whether hydrolysis of both PAR receptors leads to GPIb redistribution. Both peptides induced surface clearance of GPIb with a maximum at 2 min and 5 min for PAR-1-AP and PAR-4-AP, respectively, followed by a slow return to the surface with levels normalizing between 30 and 60 min. Translocation was associated with the formation of clusters of GPIb as revealed by fluorescence microscopy. This transient redistribution of GPIb was blocked by cytochalasin D and in large part by the membrane permeable Ca2+ chelator, BAPTA. The inhibitor of phosphatidylinositol 3-kinase and myosin light chain kinase, wortmannin, did not significantly modify internalization of GPIb, although its return to the surface was delayed for PAR-1-AP. PAR receptor-mediated association of GPIb to the insoluble cytoskeleton was blocked by cytochalasin D, while BAPTA alone increased and stabilized the presence of GPIb. Globally, immunoprecipitation experiments and analysis of the cytoskeleton confirmed that GPIb translocation is powered by a contractile mechanism involving Ca2+ mobilization, actin polymerization, and myosin incorporation into the cytoskeleton and that both PAR-1 and PAR-4 can activate this process.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Citoesqueleto/metabolismo , Ácido Egtázico/análogos & derivados , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Actinas/metabolismo , Androstadienos/farmacologia , Quelantes/farmacologia , Citocalasina D/farmacologia , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Hidrólise , Ligantes , Microscopia de Fluorescência , Miosinas/metabolismo , Peptídeos/química , Fosfatidilinositol 3-Quinases/metabolismo , Testes de Precipitina , Estrutura Terciária de Proteína , Fatores de Tempo , WortmaninaRESUMO
Activation of platelets by collagen is mediated by the complex glycoprotein VI (GPVI)/Fc receptor gamma (FcR gamma chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling has been examined using murine platelets deficient in one or both kinases. In the fyn(-/-) platelets, tyrosine phosphorylation of FcR gamma chain, phopholipase C (PLC) activity, aggregation, and secretion are reduced, though the time of onset of response is unchanged. In the lyn(-/-) platelets, there is a delay of up to 30 seconds in the onset of tyrosine phosphorylation and functional responses, followed by recovery of phosphorylation and potentiation of aggregation and alpha-granule secretion. Tyrosine phosphorylation and aggregation in response to stimulation by collagen-related peptide is further attenuated and delayed in fyn(-/-)lyn(-/-) double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune receptor tyrosine-based activation motif phosphorylation and PLC gamma 2 activation after the ligation of GPVI. Lyn plays an additional role in inhibiting platelet activation through an uncharacterized inhibitory pathway. (Blood. 2000;96:4246-4253)
Assuntos
Plaquetas/metabolismo , Proteínas de Transporte , Glicoproteínas da Membrana de Plaquetas/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores de IgG/metabolismo , Quinases da Família src/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Colágeno/farmacologia , Retroalimentação , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fosfolipase C gama , Fosforilação/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas/farmacologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas c-fyn , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/metabolismo , Quinases da Família src/deficiência , Quinases da Família src/fisiologiaRESUMO
The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P(3)) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P(3) was increased in platelets from mice deficient in the SH2 domain-containing inositol 5-phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cgamma2 (PLCgamma2) were unaltered in SHIP(-/-) platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca(2+), maximal expression of P-selectin, and potentiation of Ca(2+) and aminophospholipid exposure to CRP in SHIP(-/-) platelets in the presence of Ca(2+) (1 mM). Microinjection of PI3,4, 5P(3) into megakaryocytes caused a 3-fold increase in Ca(2+) in response to CRP, which was absent in X-linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca(2+) in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca(2+) entry that involves PI3,4,5P(3) and Btk, and which is independent of increased PLC activity.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Megacariócitos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fosfolipases Tipo C/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Transporte Biológico , Coagulantes/análise , Ligação Genética , Humanos , Síndromes de Imunodeficiência/genética , Camundongos , Selectina-P , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Glicoproteínas da Membrana de Plaquetas/agonistas , Precursores de Proteínas/análise , Imunodeficiência Combinada Severa , Cromossomo X/genéticaRESUMO
The Src homology (SH)2 domain-containing protein-tyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosine-phosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50-55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP- but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosine-phosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.
Assuntos
Integrinas/fisiologia , Ativação Plaquetária , Proteínas Tirosina Fosfatases/fisiologia , Cálcio/fisiologia , Colágeno/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Receptores de Colágeno , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Tirosina/metabolismo , Domínios de Homologia de src , Quinases da Família src/fisiologiaRESUMO
Collagen activates platelets through a tyrosine kinase-dependent pathway, involving phospholipase Cgamma2. Functional responses such as aggregation and secretion induced by collagen are potentiated by preincubation with thrombopoietin (TPO). In this study, we show that collagen and thrombopoietin activate the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and that this contributes to their respective actions. The structurally distinct inhibitors of PI 3-kinase, wortmannin, and LY294002, completely inhibit formation of phosphatidylinositol 3,4,5-trisphosphate by collagen. This leads to a substantial reduction in the formation of inositol phosphates and phosphatidic acid, 2 indices of PLC activity, and the consequent inhibition of intracellular Ca(++) [Ca(++)](i), aggregation and secretion. Potentiation of the collagen response by TPO is prevented in the presence of wortmannin and LY294002. However, when the 2 PI 3-kinase inhibitors are given after the addition of TPO but before the collagen, recovery of potentiation is observed. This suggests that potentiation is mediated through activation of PI 3-kinase. TPO stimulates aggregation of platelets from a low percentage of donors and this is also blocked by wortmannin. These results suggest that the PI 3-kinase pathway plays an important role in signaling by collagen and in the priming action of TPO.
Assuntos
Plaquetas/fisiologia , Colágeno/farmacologia , Fosfatos de Inositol/sangue , Integrinas/sangue , Fosfatidilinositol 3-Quinases/sangue , Transdução de Sinais , Trombopoetina/farmacologia , Androstadienos/farmacologia , Plaquetas/efeitos dos fármacos , Cálcio/sangue , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Cinética , Ácidos Fosfatídicos/sangue , Fosfatidilinositóis/sangue , Agregação Plaquetária , Receptores de Colágeno , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trombopoetina/fisiologia , WortmaninaRESUMO
In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Plaquetas/fisiologia , Proteínas de Transporte/metabolismo , Integrinas/metabolismo , Isoenzimas/metabolismo , Proteínas de Membrana , Fosfoproteínas/metabolismo , Ativação Plaquetária/fisiologia , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismo , Animais , Plaquetas/metabolismo , Ativação Enzimática , Humanos , Camundongos , Fosfolipase C gama , Fosforilação , Receptores de ColágenoRESUMO
The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)(10)GCP*G](n); P*=hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 3,4-bisphosphate [PI(3, 4)P(2)] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca(2+) ([Ca(2+)](i)) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca(2+) by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4, 5)P(3), but not the R28C (Arg(28)-->Cys) mutant which binds to PI(3, 4,5)P(3) with low affinity, also inhibits elevation of [Ca(2+)](i) in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCgamma2. These results demonstrate that PI 3-kinase is required for full activation of PLCgamma2 by GPVI in platelets and megakaryocytes.
Assuntos
Plaquetas/enzimologia , Proteínas de Transporte , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Tirosina Quinase da Agamaglobulinemia , Androstadienos/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/fisiologia , Cálcio/metabolismo , Cromonas/farmacologia , Humanos , Integrinas/metabolismo , Megacariócitos/efeitos dos fármacos , Megacariócitos/enzimologia , Megacariócitos/metabolismo , Camundongos , Morfolinas/farmacologia , Ácidos Fosfatídicos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfolipase C gama , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteínas/antagonistas & inibidores , Receptores de Colágeno , WortmaninaRESUMO
Phosphatidylserine exposure and microvesicle release give rise to procoagulant activity during platelet activation. We have previously shown that whereas the Ca2+ ionophore A23187 and 2,5-di-(t-butyl)-1, 4-benzohydroquinone, a Ca2+-ATPase inhibitor, induce phosphatidylserine exposure, only the former triggers microvesicle release. We now report that microvesicle formation with ionophore A23187 is specifically associated with mu-calpain activation, increased protein tyrosine phosphatase (PTP) activity and decreased tyrosine phosphorylation. The degree to which calpain and individual PTPs were activated in response to A23187 depended on the extent of bivalent cation chelation in the external medium. EGTA (2 mM) blocked or severely retarded their activation, and addition of extracellular Ca2+ in excess (2 mM) resulted in virtually immediate tyrosine dephosphorylation. Dephosphorylation was correlated with an increase in total PTP activity in platelet lysates. In platelets stimulated by a combination of thrombin and collagen, only the subpopulation undergoing microvesicle release and isolated by their binding to annexin-V-coated magnetic beads exhibited protein tyrosine dephosphorylation. Detection of PTP activity in an 'in-gel' assay showed the Ca2+-dependent appearance of active low-molecular-mass bands at 38, 36 and 27 kDa. Individual PTPs varied in their protease sensitivity to changes in intracellular Ca2+ levels. For example, PTP1B was a more sensitive substrate than SH2-domain-containing tyrosine phosphatase-1 for mu-calpain cleavage. Incubation of platelets with the PTP inhibitors, phenylarsine oxide and benzylphosphonic acid acetoxymethyl ester, led to increased tyrosine phosphorylation and the surface expression of aminophospholipids but little microvesicle formation. Furthermore, microvesicle release in response to ionophore A23187 was inhibited. We conclude that platelet microvesicle formation is associated with extensive protein tyrosine dephosphorylation.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Calcimicina/farmacologia , Colágeno/farmacologia , Hemostáticos/farmacologia , Ionóforos/farmacologia , Fosfatidilserinas/sangue , Trombina/farmacologia , Tirosina/metabolismo , Plaquetas/enzimologia , Cálcio/sangue , Calpaína/metabolismo , Ativação Enzimática , Humanos , Hidrólise , Cinética , Fosforilação , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Estimulação QuímicaRESUMO
We have used flow cytometry to compare the temporal relationship between cytoplasmic Ca(2+)-fluxes and micro-vesiculation during platelet activation. Changes in fluorescence of the Ca(2+)-dye, fluo-3, and in forward light scatter as a measure of the decrease in platelet size that accompanies micro-vesiculation, were assessed simultaneously. In other experiments, changes in Ca(2+) levels and aminophospholipid exposure were assessed using fura-red, which is a long wavelength range indicator, and FITC-annexin V. Results obtained using the ionophore A23 187 and the ATPase inhibitor, thapsigargin, showed that micro-vesiculation is a relatively late event compared with intracellular Ca(2+) elevation. The relatively slow binding kinetics of annexin V prevented the establishment of a temporal relationship between increases in intracellular Ca(2+) and aminophospholipid exposure. Nevertheless, the combined use of fura-red and annexin V highlighted the heterogeneous response seen on some occasions with thapsigargin and always with a thrombin plus collagen mixture, and confirmed that individual platelets that bound annexin V were also those with elevated intracellular Ca(2+) levels.
RESUMO
The Scott syndrome is a rare inherited haemorrhagic disorder characterized by the inability of blood cells to expose aminophospholipids and to shed microparticles. We have had the opportunity to study a recently reported French patient with this syndrome and have confirmed by means of a fluorescence assay for transbilayer lipid movement a reduced aminophospholipid exposure when platelets were stimulated with the calcium-ionophore ionomycin, in spite of a normal elevation of intracellular Ca2+. Secretion and calpain activation were also shown to be normal. Significantly, the level of phosphotyrosine-labelled proteins in platelets treated with thrombin or a thrombin + collagen mixture and in particular the phosphorylation of a 40 kD band were severely reduced. Furthermore, inhibition of thiol-containing enzymes. including tyrosine-phosphatases, by N-ethyl maleimide did not lead to aminophospholipid exposure in the patient's platelets, in spite of increased tyrosine protein phosphorylation. In contrast, amphiphilic membrane drugs such as tetracaine and propranolol induced both surface aminophospholipid exposure in Scott platelets and the shedding of microparticles, thereby showing that membrane perturbation can lead to loss of phospholipid asymmetry in this syndrome. Our results provide the first insight that the lack of expression of procoagulant phospholipids and microparticle formation in Scott syndrome platelets is associated with a defect of intracellular signalling.
Assuntos
Transtornos Plaquetários/sangue , Plaquetas/enzimologia , Hemorragia/sangue , Proteínas Tirosina Fosfatases/metabolismo , Idoso , Transtornos Plaquetários/enzimologia , Western Blotting , Feminino , Hemorragia/enzimologia , Humanos , Microesferas , Fosforilação , Síndrome , Tetracaína/farmacologia , Trombina/farmacologiaRESUMO
Platelet activation by agents such as the Ca2+-ionophore A23187 or Ca2+-ATPase inhibitors leads to the generation of a procoagulant surface and the formation of microparticles. These responses are late events of platelet activation and readily detected by flow cytometry using annexin V-FITC as an aminophospholipid probe. One Ca2+-ATPase inhibitor, 2,5-di-(tertbutyl)-1,4-benzohydroquinone induced aminophospholipid exposure without microparticle formation. Previous work has shown that microparticle formation is strictly linked to the activation of calpain, a thiol-protease that modifies the platelet cytoskeleton and some signal transduction enzymes. We now report how the detection of platelet tyrosine phosphorylation by western-blotting clearly shows that when platelet activation and aminophospholipid exposure are accompanied by microparticle formation there is a decrease in the tyrosine phosphorylation of proteins.