Assuntos
Artrite Reumatoide , Desenvolvimento de Medicamentos , Biomarcadores , Humanos , ProteômicaRESUMO
BACKGROUND: Polarization of macrophages by specific micro-environmental conditions impacts upon their function following subsequent activation. This study aimed to systematically validate robust phenotypic markers for in vitro polarized human macrophages in order to facilitate the study of macrophage subsets in vivo. METHODS: Human peripheral blood monocytes were polarized in vitro with IFN-γ, IL-4, or IL-10. Similar experiments were performed with TNF, IL-13, dexamethasone, M-CSF and GM-CSF as polarizing stimuli. Phenotypic markers were assessed by flow cytometry and qPCR. RESULTS: IFN-γ polarized macrophages (MΦ(IFN-γ)) specifically enhanced membrane expression of CD80 and CD64, IL-4 polarized macrophages (MΦ(IL-4)) mainly upregulated CD200R and CD206, and downregulated CD14 levels, and IL-10 polarized macrophages (MΦ(IL-10)) selectively induced CD163, CD16, and CD32. The expression profiles of the most specific markers were confirmed by qPCR, dose-response experiments, and the use of alternative polarizing factors for each macrophage subset (TNF, IL-13, and dexamethasone, respectively). GM-CSF polarized macrophages (MΦ(GM-CSF)) upregulated CD80 but not CD64 expression, showing a partial phenotypic similarity with MΦ(IFN-γ), and also upregulated the expression of the alternative activation marker CD206. M-CSF polarized macrophages (MΦ(M-CSF)) not only expressed increased levels of CD163 and CD16, resembling MΦ(IL-10,) but also displayed high levels of CD64. The phenotype of MΦ(M-CSF) could be further modulated by additional polarization with IFN-γ, IL-4, or IL-10, whereas MΦ(GM-CSF) showed less phenotypic plasticity. CONCLUSION: This study validated CD80 as the most robust phenotypic marker for human MΦ(IFN-γ), whereas CD200R was upregulated and CD14 was specifically downregulated on MΦ(IL-4). CD163 and CD16 were found to be specific markers for MΦ(IL-10). The GM-CSF/M-CSF differentiation model showed only a partial phenotypic similarity with the IFN-γ/IL-4/IL-10 induced polarization.
Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Diferenciação Celular/fisiologia , Polaridade Celular/fisiologia , Macrófagos/citologia , Macrófagos/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/genética , Polaridade Celular/genética , Dexametasona/farmacologia , Citometria de Fluxo/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interferon gama/metabolismo , Interleucinas/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Monócitos/metabolismo , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To investigate the expression of tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor inducible 14 (Fn14) in the inflamed synovium of patients with arthritis, as TWEAK blockade has been observed to have a beneficial effect in an animal model of rheumatoid arthritis (RA). METHODS: Synovial tissue (ST) biopsies were obtained from 6 early, methotrexate-naive patients with RA as well as 13 patients with RA and 16 patients with psoriatic arthritis (PsA) who were matched for treatment and disease duration. Serial ST samples were obtained from a separate cohort of 13 patients with RA before and after infliximab treatment. TWEAK and Fn14 expression was evaluated by immunohistochemistry and digital image analysis. RESULTS: TWEAK and Fn14 were clearly expressed in ST of patients with RA and PsA. TWEAK expression was significantly higher in RA (sub)lining samples compared to PsA (p = 0.005 and p = 0.014, respectively), but Fn14 expression was comparable. Double immunofluorescence showed TWEAK and Fn14 expression on fibroblast-like synoviocytes and macrophages, but not T cells. Of interest, persistent TWEAK and Fn14 expression was found after anti-TNF therapy. CONCLUSIONS: TWEAK and Fn14 are abundantly expressed in the inflamed synovium of patients with RA and PsA. This raises the possibility that blocking TWEAK/Fn14 signalling could be of therapeutic benefit in inflammatory arthritis.
Assuntos
Artrite Psoriásica/metabolismo , Artrite Reumatoide/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Membrana Sinovial/metabolismo , Fatores de Necrose Tumoral/metabolismo , Adulto , Idoso , Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Psoriásica/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Citocina TWEAK , Quimioterapia Combinada , Feminino , Humanos , Infliximab , Masculino , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Receptor de TWEAK , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto JovemRESUMO
OBJECTIVES: All complement pathways lead to the formation of C5a, which is believed to contribute to the influx and activation of C5a-receptor (C5aR) bearing cells into the joints of patients with rheumatoid arthritis (RA). Studies in animal models of RA have suggested therapeutic potential of C5aR blockade. In this study, we examined the effects of the C5aR blockade on synovial inflammation in RA patients. METHODS: We performed a double-blind, placebo-controlled study using an orally administered C5aR-antagonist. Twenty-one patients with active RA were randomized 2:1 to treatment with a C5aR-antagonist AcF- (OpdChaWR) (PMX53) vs placebo for 28 days. Serum concentrations of PMX53 were determined. Synovial tissue was obtained at baseline and after 28 days of treatment for pharmacodynamic analysis using immunohistochemistry and digital image analysis. RESULTS: All patients completed the study. Areas under the curve (AUCs) of PMX53 in patients' blood samples showed a mean of 40.8 nmol h/l. There was neither decrease in cell infiltration, nor changes in key biomarkers associated with clinical efficacy after active treatment. In addition, there was no trend towards clinical improvement in the C5aR-antagonist-treated group compared with placebo nor was there a correlation between the AUC and clinical response. CONCLUSIONS: Treatment with PMX53 did not result in a reduction of synovial inflammation despite reaching serum levels of PMX53 that block C5aR-mediated cell activation in vitro. The data suggest that C5aR blockade does not result in reduced synovial inflammation in RA patients.
Assuntos
Artrite Reumatoide/diagnóstico , Artrite Reumatoide/tratamento farmacológico , Peptídeos Cíclicos/administração & dosagem , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Sinovite/prevenção & controle , Administração Oral , Adolescente , Adulto , Idoso , Artroscopia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Probabilidade , Valores de Referência , Medição de Risco , Índice de Gravidade de Doença , Membrana Sinovial/efeitos dos fármacos , Sinovite/tratamento farmacológico , Resultado do TratamentoRESUMO
OBJECTIVE: Phosphatidylinositol 3-kinase-dependent activation of protein kinase B (PKB) has been observed in rheumatoid arthritis (RA) synovial tissue, and mechanisms that interfere with this process are protective in animal models of arthritis. PKB can regulate cell survival and proliferation via phosphorylation-dependent inactivation of forkhead box class O (FoxO) transcription factors. The present study was undertaken to examine whether FoxO transcription factors are differentially inactivated in RA synovial tissue, and whether this inactivation correlates with laboratory and clinical parameters of disease activity. METHODS: The expression and phosphorylation of FoxO family members were assessed in synovial biopsy tissue from 12 patients with RA and 9 patients with inflammatory osteoarthritis (OA), by immunohistochemistry and quantitative computer-assisted image analysis. Immunoblotting was used to assess the interleukin-1beta (IL-1beta)- and tumor necrosis factor alpha (TNFalpha)-induced phosphorylation of FoxO1 and FoxO4 in cultured fibroblast-like synoviocytes (FLS) and macrophages. RESULTS: FoxO1, FoxO3a, and FoxO4 were expressed and phosphorylated in synovial tissue from both RA patients and OA patients. In RA synovial tissue, phosphorylation of FoxO1 was observed in both FLS and macrophages, FoxO3a in T lymphocytes, and FoxO4 in macrophages alone. Following stimulation with IL-1beta and TNFalpha, FoxO1 and FoxO4 were phosphorylated in both RA and OA FLS and synovial macrophages, respectively. Inactivation of FoxO4 was significantly enhanced in the RA as compared with the OA synovial sublining. There was a strong negative correlation between inactivation of FoxO4 in RA synovial tissue and increased serum C-reactive protein levels and a raised erythrocyte sedimentation rate in RA patients. CONCLUSION: All 3 FoxO family members examined were phosphorylated in both RA and OA synovial tissue; in particular, inactivation of FoxO4 was significantly enhanced in macrophages from RA synovial tissue. Thus, cell-specific inactivation of FoxO family members appears to differentially regulate cell survival and proliferation in the RA synovium.
Assuntos
Artrite Reumatoide/patologia , Fatores de Transcrição Forkhead/antagonistas & inibidores , Membrana Sinovial/patologia , Adulto , Idoso , Animais , Artrite Reumatoide/sangue , Artrite Reumatoide/genética , Biópsia , Sedimentação Sanguínea , Feminino , Fibroblastos/fisiologia , Proteína Forkhead Box O1 , Cabras , Humanos , Imuno-Histoquímica , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Osteoartrite/sangue , Osteoartrite/genética , Osteoartrite/patologia , Fosforilação , CoelhosRESUMO
Although the mechanisms leading to the induction of RA (rheumatoid arthritis) are poorly understood, improper activation, proliferation, survival and retention of neutrophils, macrophages, lymphocytes and other leucocytes contribute to perpetuation of inflammation and eventual joint destruction through activation of stromal fibroblast-like synoviocytes. Fundamental studies in developmental biology, cellular biology and immunology have established critical roles for PI3K (phosphoinositide 3-kinase) signal transduction pathways in cellular chemotactic responses, proliferation, apoptosis and survival. Despite profound alteration of these cellular processes in RA, involvement of PI3K signalling pathways in this chronic inflammatory disease, and their assessment as potential therapeutic targets, has until recently received scant attention. This review highlights recent advances in our understanding of PI3K signalling pathways, in particular regulation of FoxO (forkhead box O) transcription factors, and their relevance to RA.
Assuntos
Artrite Reumatoide/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Divisão Celular , Sobrevivência Celular , Fatores de Transcrição Forkhead/genética , Humanos , Linfócitos/patologia , Linfócitos/fisiologia , Macrófagos/patologia , Macrófagos/fisiologia , Neutrófilos/patologia , Neutrófilos/fisiologia , Transcrição GênicaRESUMO
OBJECTIVE: Oxidative stress contributes to the inflammatory properties of rheumatoid arthritis (RA) synovial T lymphocytes. This study was undertaken to investigate the mechanisms leading to production of reactive oxygen species (ROS) and oxidative stress in RA synovial T lymphocytes. METHODS: ROS production in T lymphocytes from the peripheral blood (PB) of healthy donors and from the PB and synovial fluid (SF) of RA patients was measured by ROS-dependent fluorescence of 6-carboxy-2',7'-dichlorofluorescein. Rap1 GTPase activation was assessed by activation-specific probe precipitation. Proliferation of RA PB and SF T lymphocytes was assayed by 3H-thymidine incorporation. In some experiments, RA PB T cells were preincubated with autologous SF or with PB or SF adherent cells. Experiments were performed in the absence or presence of transwell membranes or CTLA-4Ig fusion proteins. Short- and long-term stimulations of healthy donor PB T lymphocytes were performed with inflammatory cytokines, in the absence or presence of activating anti-CD28 antibodies. RESULTS: T lymphocyte ROS production and Rap1 inactivation were mediated by cell-cell contact with RA synovial adherent cells, and this correlated with T cell mitogenic hyporesponsiveness. CTLA4-Ig blockade of synovial adherent cell signaling to CD28 T cells reversed the inhibition of Rap1 activity and prevented induction of ROS. Introduction of active RapV12 into T cells also prevented induction of ROS production. Coincubation of T cells with stimulating anti-CD28 antibodies and inflammatory cytokines synergistically increased T cell ROS production. CONCLUSION: Cell-cell contact between T cells and RA synovial adherent cells mediates Rap1 inactivation and subsequent ROS production in T lymphocytes following exposure to inflammatory cytokines. This process can be blocked by CTLA4-Ig fusion protein.
Assuntos
Artrite Reumatoide/metabolismo , Imunoconjugados/farmacologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Membrana Sinovial/efeitos dos fármacos , Linfócitos T/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Abatacepte , Antirreumáticos/farmacologia , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Antígenos CD28/genética , Antígenos CD28/metabolismo , Comunicação Celular/fisiologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Estresse Oxidativo/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Proteínas rap1 de Ligação ao GTP/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
OBJECTIVE: To investigate the cellular and molecular sources of oxidative stress in patients with rheumatoid arthritis (RA) through analysis of the production of reactive oxygen species (ROS) in synovium. METHODS: Cytochemical procedures based on the 3,3'-diaminobenzidine (DAB)-Mn2+ deposition technique were used on unfixed cryostat sections of synovium from RA patients and rheumatic disease controls. For immunophenotyping, sections were incubated, fixed, and stained with fluorescein isothiocyanate-labeled antibodies. Fluorescence-activated cell sorter analysis of the ROS-reactive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate-di(acetoxymethyl ester) was used to measure intracellular ROS in T lymphocytes from peripheral blood and synovial fluid. To determine which enzymes produced ROS, different inhibitors were tested. RESULTS: Large quantities of DAB precipitated in the majority of RA synovial T lymphocytes, indicative of intracellular ROS production. These ROS-producing T lymphocytes were observed throughout the synovium. Polymerization of DAB was observed to a lesser extent in other forms of chronic arthritis, but was absent in osteoarthritis. DAB staining of cytospin preparations of purified RA synovial fluid T cells confirmed the presence of ROS-producing cells. One of the ROS involved appeared to be H2O2, since catalase suppressed intracellular ROS production. Superoxide dismutase, which uses superoxide as a substrate to form H2O2, diphenyleneiodonium (an inhibitor of NADPH oxidase), N(G)-monomethyl-L-arginine (an inhibitor of nitric oxide synthesis), nordihydroguaiaretic acid (an inhibitor of lipoxygenase), and rotenone (an inhibitor of mitochondrial ROS production) failed to suppress ROS production. CONCLUSION: Our findings show that chronic oxidative stress observed in synovial T lymphocytes is not secondary to exposure to environmental free radicals, but originates from intracellularly produced ROS. Additionally, our data suggest that one of the intracellularly generated ROS is H2O2, although the oxidase(s) involved in its generation remains to be determined.
Assuntos
Artrite Reumatoide/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Membrana Sinovial/metabolismo , Linfócitos T/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Humanos , Osteoartrite/imunologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Linfócitos T/patologiaRESUMO
Ras-like GTPases are ubiquitously expressed, evolutionarily conserved molecular switches that couple extracellular signals to various cellular responses. Rap1, the closest relative of Ras, has attracted much attention because of the possibility that it regulates Ras-mediated signalling. Rap1 is activated by extracellular signals through several regulatory proteins, and it might function in diverse processes, ranging from modulation of growth and differentiation to secretion, integrin-mediated cell adhesion and morphogenesis.
Assuntos
Transdução de Sinais/fisiologia , Proteínas rap1 de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Proteínas rap1 de Ligação ao GTP/químicaRESUMO
The Src homology 2 (SH2) domain adaptor protein Shb has been shown to transmit NGF- and FGF-2-dependent differentiation signals in PC12 cells. To study if this involves signaling through the small GTPase Rap1, Rap1 activity was assessed in Shb-overexpressing PC12 cells. We demonstrate that NGF and EGF induce Rap1 activation in PC12-Shb cells, while FGF-2 fails to do so. However, PC12 cells expressing Shb with an inactivated SH2 domain do not respond to NGF stimulation with Rap1 activation. The CrkII SH2 domain interacts with Shb and a 130- to 135-kDa phosphotyrosine protein present mainly in PC12-Shb cells and these interactions may thus relate to the effect of Shb on Rap1 activation. Transient expression of RalGDS-RBD or Rap1GAP to block the Rap1 pathway reduces the NGF-dependent neurite outgrowth in PC12-Shb cells. These results suggest a role of Shb in NGF-dependent Rap1 signaling and this pathway may be of significance for neurite outgrowth under certain conditions.
Assuntos
Fator de Crescimento Neural/farmacologia , Neuritos/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas rap1 de Ligação ao GTP/metabolismo , Domínios de Homologia de src/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/enzimologia , Neurônios/ultraestrutura , Células PC12 , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-crk , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transfecção , Fator ral de Troca do Nucleotídeo Guanina/genética , Fator ral de Troca do Nucleotídeo Guanina/metabolismo , Proteínas ras/metabolismoRESUMO
The rat pheochromocytoma cell line PC12 is extensively used as a model for studies of neuronal cell differentiation. These cells develop a sympathetic neuron-like phenotype when cultured in the presence of nerve growth factor. The present study was performed in order to assess the role of mouse GTK (previously named BSK/IYK), a cytoplasmic tyrosine kinase belonging to the Src family, for neurite outgrowth in PC12 cells. We report that PC12 cells stably overexpressing GTK exhibit a larger fraction of cells with neurites as compared with control cells, and this response is not accompanied by an increased ERK activity. Treatment of the cells with the MEK inhibitor PD98059 did not reduce the GTK-dependent increased in neurite outgrowth. GTK expression induces a nerve growth factor-independent Rap1 activation, probably through altered CrkII signaling. We observe increased CrkII complex formation with p130(Cas), focal adhesion kinase (FAK), and Shb in PC12-GTK cells. The expression of GTK also correlates with a markedly increased content of FAK, phosphorylation of the adaptor protein Shb, and an association between these two proteins. Transient transfection of GTK-overexpressing cells with RalGDS-RBD or Rap1GAP, inhibitors of the Rap1 pathway, reduces the GTK-dependent neurite outgrowth. These data suggest that GTK participates in a signaling pathway, perhaps involving Shb, FAK and Rap1, that induces neurite outgrowth in PC12 cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Neoplasias , Fator de Crescimento Neural/farmacologia , Neuritos/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Receptor trkA , Tirosina/metabolismo , Proteínas rap1 de Ligação ao GTP/fisiologia , Animais , Proteínas de Transporte/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Proteínas de Membrana/metabolismo , Células PC12 , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-crk , Ratos , Domínios de Homologia de src , Quinases da Família srcRESUMO
The protooncogene product Cbl has emerged as a negative regulator of tyrosine kinases. We have shown previously that Cbl binds to ZAP-70 through its N-terminal tyrosine kinase binding (TKB) domain. In this study, we demonstrate that overexpression of Cbl in Jurkat T cells decreases the TCR-induced phosphorylation of ZAP-70 and other cellular phosphoproteins. Coexpression of Cbl with ZAP-70 in COS cells reproduced the Cbl-induced reduction in the level of phosphorylated ZAP-70. The effect of Cbl was eliminated by the TKB-inactivating G306E mutation in Cbl as well as by a phenylalanine mutation of Tyr292 within the TKB domain binding site on ZAP-70. Notably, the oncogenic Cbl-70Z/3 mutant associated with ZAP-70, but did not reduce the levels of phosphorylated ZAP-70. Overexpression of Cbl, but not Cbl-G306E, in Jurkat T cells led to a decrease in the TCR-induced NF-AT luciferase reporter activity. Overexpression of the TKB domain itself, but not its G306E mutant, functioned in a dominant-negative manner and led to an increase in NF-AT reporter activity. Cbl-70Z/3-overexpressing cells exhibited an increase in both basal and TCR-induced NF-AT luciferase reporter activity, and this trend was reversed by the G306E mutation. Finally, by reconstituting a ZAP-70-deficient Jurkat T cell line, p116, we demonstrate that wild-type ZAP-70 is susceptible to the negative regulatory effect of Cbl, whereas the ZAP-70-Y292F mutant is resistant. Together, our results establish that the linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells.
Assuntos
Regulação para Baixo/imunologia , Proteínas Nucleares , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Linfócitos T/enzimologia , Tirosina/metabolismo , Ubiquitina-Proteína Ligases , Animais , Anticorpos Monoclonais/farmacologia , Complexo CD3/imunologia , Células COS , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/imunologia , Genes Reporter , Humanos , Células Jurkat , Luciferases/genética , Luciferases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Fatores de Transcrição NFATC , Fosforilação , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-cbl , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tirosina/fisiologia , Proteína-Tirosina Quinase ZAP-70RESUMO
Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical mitogenic stimuli to regulate leukocyte function remains poorly understood. Here, we show that the cytoplasmic tail of CD31, an important integrin adhesion amplifier, propagates signals that induce T cell adhesion via beta1 (VLA-4) and beta2 (LFA-1) integrins. We identify the small GTPase, Rap1, as a critical mediator of this effect. Importantly, CD31 selectively activated the small Ras-related GTPase, Rap1, but not Ras, R-Ras, or Rap2. An activated Rap1 mutant stimulated T lymphocyte adhesion to intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), as did the Rap1 guanine nucleotide exchange factor C3G and a catalytically inactive mutant of RapGAP. Conversely, negative regulators of Rap1 signaling blocked CD31-dependent adhesion. These findings identify a novel important role for Rap1 in regulating ligand-induced cell adhesion and suggest that Rap1 may play a more general role in coordinating adhesion-dependent signals during leukocyte migration and extravasation. Our findings also suggest an alternative mechanism, distinct from interference with Ras-proximal signaling, by which Rap1 might mediate transformation reversion.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular/fisiologia , Integrinas/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Receptores de Retorno de Linfócitos/fisiologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Antígenos CD/fisiologia , Humanos , Integrina alfa4beta1 , Células Jurkat , Molécula-1 de Adesão Celular Endotelial a Plaquetas/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Linfócitos T/fisiologia , Transfecção , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Members of the Ras superfamily of small molecular weight GTPases play diverse and critical roles in mediating cellular responses to extracellular stimuli, including mitogenesis, cytoskeletal maintenance and rearrangement, and integrin activation. In T lymphocytes, biochemical and genetic evidence demonstrate that Ras plays an essential role in coupling T cell receptor ligation to signaling cascades required for T cell proliferation and development. Recent observations that C3G, a guanine nucleotide exchange factor specific for the Ras-related GTPase Rap1, is recruited into tyrosine-phosphorylated protein signaling complexes in activated T cells have suggested that Rap1 may also play a role in T cell activation. Utilizing a recently developed technique for detection of endogenous, GTP-bound Rap1, we have found that Rap1, but not Rap2, is transiently activated following T cell receptor stimulation of normal human T lymphocytes. Increases in intracellular calcium is both necessary and sufficient to induce Rap1 activation. Remarkably, costimulation of T cells with mitogenic anti-CD28 antibody completely abolished T cell receptor-dependent activation of Rap1. This report demonstrates a potential role for Rap1 in T cell receptor signaling and suggests inactivation of Rap1 as a candidate target of CD28-dependent costimulatory signals required for T cell antigen responsiveness.
Assuntos
Antígenos CD28/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Complexo CD3/metabolismo , Cálcio/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Jurkat/metabolismo , Transdução de Sinais , Linfócitos T Citotóxicos/metabolismo , Proteínas rap de Ligação ao GTPRESUMO
Shb is a recently described Src homology 2 (SH2) domain-containing adaptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains. As a result, a number of proteins detected in anti-Shb and anti-Grb2 immunoprecipitates are shared, including phosphoproteins of 22, 36/38, 55/57 and 70 kDa. Shb-association with p22, which represents the T cell receptor associated zeta chain, occurs through the Shb SH2 domain. The central region of Shb binds p36/38. Since this interaction was inhibited by phosphotyrosine, this region of Shb is likely to contain a non-SH2 PTB (phosphotyrosine binding) domain. The Shb PTB domain was found to preferentially bind the sequence Asp-Asp-X-pTyr when incubated with a phosphopeptide library. A peptide corresponding to a phosphorylation site in 34 kDa Lnk inhibited association between Shb and p36/38. Overexpression of Shb in Jurkat cells led to increased basal phosphorylation of Shb-associated p36/38 and p70 proteins. Inactivation of the Shb SH2 domain by an R522K mutation resulted in a reduced stimulation of tyrosine phosphorylation of several proteins in response to CD3 crosslinking when expressed in Jurkat cells. Together, our results show three distinct domains of Shb all participate in the formulation of multimeric signaling complexes in activated T cells. These results indicate that the Shb protein functions in T cell receptor signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Sítios de Ligação , Complexo CD3/metabolismo , Proteínas de Transporte/metabolismo , Proteína Adaptadora GRB2 , Expressão Gênica , Humanos , Fosfotirosina/metabolismo , Ligação Proteica , Proteínas/fisiologia , Proteínas Recombinantes , Transdução de Sinais , Transfecção , Células Tumorais CultivadasRESUMO
Transformation of hematopoietic cells by the Bcr-abl oncoprotein leads to constitutive tyrosine phosphorylation of a number of cellular polypeptides that function in normal growth factor-dependent cell proliferation. Recent studies have shown that the CrkL adaptor protein and the Cbl protooncoprotein are constitutively tyrosine phosphorylated and form a preformed complex in cells expressing Bcr-abl. In the current study, we have examined cytokine-dependent tyrosine phosphorylation of Cbl and its association with Crk proteins. Erythropoietin (EPO) and interleukin-3 induced a dose and time-dependent tyrosine phosphorylation of Cbl in both EPO-dependent Ba/F3 and DA-3 transfectants, and the erythroid cell line HCD-57. Furthermore, once phosphorylated, Cbl associated with Crk adaptor proteins. Of the three Crk isoforms expressed in hematopoietic cells (CrkL, CrkII, and CrkI), tyrosine phosphorylated Cbl binds preferentially to CrkL and CrkII. The amount of Cbl associated with CrkL and CrkII exceeded the fraction of Cbl associated with Grb2 indicating that unlike other receptor systems, the Cbl-Crk association represents the dominant complex of Cbl in growth factor-stimulated hematopoietic cells. In factor-dependent hematopoietic cell lines, CrkL constitutively associated with the guanine nucleotide release factor, C3G, which is known to interact via Crk src-homology 3 (SH3) domains. Our data suggest that the inducible Cbl-Crk association is a proximal component of a signaling pathway downstream of multiple cytokine receptors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Eritropoetina/farmacologia , Interleucina-3/farmacologia , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases , Linhagem Celular , Humanos , Cinética , Modelos Biológicos , Proteínas Nucleares/isolamento & purificação , Fosfoproteínas/isolamento & purificação , Fosforilação , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-cbl , Receptores da Eritropoetina/isolamento & purificação , Receptores da Eritropoetina/fisiologia , Transdução de Sinais , Domínios de Homologia de srcRESUMO
The protooncogene product Cbl has emerged as a novel signal transduction protein downstream of a number of cell surface receptors coupled to tyrosine kinases. Recently, we and others have reported the activation-dependent association of Cbl with the Syk and ZAP-70 tyrosine kinases through presently undefined mechanisms. Potential Src homology 2 and 3 domain binding sites within the C-terminal half of Cbl mediate in vivo interactions with several signaling proteins; however, the N-terminal transforming region (Cbl-N) lacks recognizable catalytic or protein interaction motifs. Here, we show that in vitro Cbl-N (amino acids 1-357) but not Cbl-C (amino acids 358-906) binds to ZAP-70 in a T cell-activation-dependent manner. A point mutation in Cbl-N, G306E, corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue, SLI-1, severely compromised Cbl-N/ZAP-70 binding. Cbl-N/ZAP-70 binding was direct and phosphotyrosine-dependent, thus identifying a phosphotyrosine-binding domain within the transforming region of Cbl. In vivo, Cbl-N expressed in T cells selectively associated with the ZAP-70/zeta complex. These results identify a novel mechanism for the direct participation of the N-terminal region of Cbl in ZAP-70 signal transduction, and suggest a biochemical mechanism for the leukemogenicity of the oncogene v-cbl through potential interaction with proliferation-related phosphotyrosyl proteins.
Assuntos
Transformação Celular Neoplásica , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/metabolismo , Ubiquitina-Proteína Ligases , Linhagem Celular , Humanos , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-cbl , Transdução de Sinais , Relação Estrutura-Atividade , Proteína-Tirosina Quinase ZAP-70RESUMO
We and others have recently identified Cbl, the protein product of the c-cbl protooncogene, as an early tyrosine kinase substrate upon T cell activation and have shown that Cbl forms in vivo complexes with Src family tyrosine kinases, Grb2 adaptor protein, and the p85 subunit of PI-3 kinase. Here we show that Cbl associates with all three forms of the human Crk protein, predominantly CrkL, following T cell receptor activation of Jurkat T cells. Association between Cbl and Crk proteins was confirmed in normal human peripheral blood-derived T cells. In vitro, Cbl was able to interact with the Crk SH2 domain but not the SH3 domain. A phosphopeptide corresponding to a potential Crk SH2 domain-binding motif in Cbl (pYDVP) specifically inhibited binding between Cbl and Crk SH2 domain. Anti-Cbl antibody completely immunodepleted the CrkL-associated 120kDa phosphotyrosyl polypeptide, suggesting that the recently described p130cas-related Crk-associated p116 of T cells may be Cbl. Consistent with this possibility, the 4F4 antibody used to characterize the p116 polypeptide cross-reacted with Cbl protein when it was resolved on one- or two-dimensional gels. CrkL was constitutively associated with a substantial amount of the guanine nucleotide exchange protein C3G, and a fraction of the C3G protein was coimmunoprecipitated with Cbl in activated Jurkat T cells. These results suggest the possibility that Cbl may participate in a signaling pathway that regulates guanine nucleotide exchange on small G-proteins in T cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Proteína Substrato Associada a Crk , Fatores de Troca do Nucleotídeo Guanina , Humanos , Zíper de Leucina , Ativação Linfocitária , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-cbl , Proteínas Recombinantes , Proteína p130 Retinoblastoma-Like , Transdução de Sinais , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Domínios de Homologia de srcRESUMO
Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as zeta and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-gamma 1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85 alpha cDNA into Jurkat cells followed by anti-hemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI-3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Isoenzimas/metabolismo , Fosfoproteínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Proteína Adaptadora GRB2 , Humanos , Cinética , Ativação Linfocitária , Camundongos , Dados de Sequência Molecular , Peso Molecular , Fosfatidilinositol 3-Quinases , Fosfolipase C gama , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotirosina , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Previously, we have identified p120 as a Fyn/Lck SH3 and SH2 domain-binding protein that is tyrosine phosphorylated rapidly after T cell receptor triggering. Here, we used direct protein purification, amino acid sequence analysis, reactivity with antibodies, and two-dimensional gel analyses to identify p120 as the human c-cbl protooncogene product. We demonstrate in vivo complexes of p120cbl with Fyn tyrosine kinase, the adaptor protein Grb2, and the p85 subunit of phosphatidylinositol (PI) 3-kinase. The association of p120cbl with Fyn and the p85 subunit of PI 3-kinase (together with PI 3-kinase activity) was markedly increased by T cell activation, consistent with in vitro binding of p120cbl to their SH2 as well as SH3 domains. In contrast, a large fraction of p120cbl was associated with Grb2 prior to activation, and this association did not change upon T cell activation. In vitro, p120cbl interacted with Grb2 exclusively through its SH3 domains. These results demonstrate a novel Grb2-p120cbl signaling complex in T cells, distinct from the previously analyzed Grb2-Sos complex. The association of p120cbl with ubiquitous signaling proteins strongly suggests a general signal transducing function for this enigmatic protooncogene with established leukemogenic potential but unknown physiological function.