RESUMO
Pharmacologic inhibitors of IκB kinase (IKK), especially IKK-ß, have been developed to treat inflammatory diseases. However, their interactions with components of the NF-κB pathways are not fully known in allergic diseases. To examine whether IKK is involved in immediate hypersensitivity reactions and to determine whether counterregulatory mechanisms in the NF-κB activation system were active, we examined the role played by IKK components on mast cell degranulation using a murine ocular immediate hypersensitivity reaction model. Pharmacologic inhibition of IKK in mice caused paradoxical aggravation of the mast cell-mediated immediate hypersensitivity reaction and up-regulation in the expression of inflammatory cytokines. Downstream analyses showed that B-cell deficiency or treatment by IL-1 receptor antagonist corrected the aberrant activation of tissue-resident mast cells, which would indicate contribution by activated B cells. Analyses of co-cultures of tissue-resident mast cells showed the contribution of activated B cells to activation of mast cells and secretion of inflammatory cytokines. Aberrant activation of the NF-κB promoter in isolated B cells was induced exclusively by IKK-ß inhibition and was negated by ablating IKK-α. Aggravated mast cell degranulation by pharmacologic IKK inhibition in the murine immediate hypersensitivity reaction was corrected by B-cell-targeted inhibition of IKK-α. Thus, IKK-ß limits B-cell-mediated mast cell activation and inflammatory cytokine induction in immediate hypersensitivity by counterbalancing the activity of IKK-α.
Assuntos
Linfócitos B/enzimologia , Conjuntivite Alérgica/enzimologia , Quinase I-kappa B/antagonistas & inibidores , Mastócitos/enzimologia , Animais , Antígenos de Plantas/administração & dosagem , Antígenos de Plantas/efeitos adversos , Linfócitos B/efeitos dos fármacos , Biomarcadores/metabolismo , Western Blotting , Conjuntivite Alérgica/etiologia , Conjuntivite Alérgica/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Quinase I-kappa B/metabolismo , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Extratos Vegetais/administração & dosagem , Extratos Vegetais/efeitos adversos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
PURPOSE: The proteasome consists of chymotrypsin-like (CT-L), trypsin-like, and caspase-like subunits that cleave substrates preferentially by amino acid sequence. Proteasomes mediate degradation of regulatory proteins of the p53, Bcl-2, and nuclear factor-κB (NF-κB) families that are aberrantly active in chronic lymphocytic leukemia (CLL). CLL remains an incurable disease, and new treatments are especially needed in the relapsed/refractory setting. We therefore investigated the effects of the proteasome inhibitor carfilzomib (CFZ) in CLL cells. EXPERIMENTAL DESIGN: Tumor cells from CLL patients were assayed in vitro using immunoblotting, real-time polymerase chain reaction, and electrophoretic mobility shift assays. In addition, a p53 dominant-negative construct was generated in a human B-cell line. RESULTS: Unlike bortezomib, CFZ potently induces apoptosis in CLL patient cells in the presence of human serum. CLL cells have significantly lower basal CT-L activity compared to normal B and T cells, although activity is inhibited similarly in T cells versus CLL. Co-culture of CLL cells on stroma protected from CFZ-mediated cytotoxicity; however, PI3K inhibition significantly diminished this stromal protection. CFZ-mediated cytotoxicity in leukemic B cells is caspase-dependent and occurs irrespective of p53 status. In CLL cells, CFZ promotes atypical activation of NF-κB evidenced by loss of cytoplasmic IκBα, phosphorylation of IκBα, and increased p50/p65 DNA binding, without subsequent increases in canonical NF-κB target gene transcription. CONCLUSIONS: Together, these data provide new mechanistic insights into the activity of CFZ in CLL and support phase I investigation of CFZ in this disease.
Assuntos
Antineoplásicos/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , NF-kappa B/metabolismo , Oligopeptídeos/farmacologia , Inibidores de Proteassoma/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Compostos de Benzil/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Óxidos N-Cíclicos , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidrocarbonetos Fluorados/farmacologia , Indolizinas , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Compostos de Piridínio/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin (Pae) is extracted from the root of paeonia lactiflora which have attracted attention for anti-rheumatic and immune modulating properties. AIM OF THE STUDY: To investigate the role of PI3K/Akt/mTOR signaling mediated by BAFF/BAFF-R in antibodies production and the regulation of Pae on the signaling pathway in rats with collagen-induced arthritis (CIA). MATERIALS AND METHODS: CIA rats were randomly separated into different groups and treated with Pae (25, 100mg/kg) from day 18 to day 38 after immunization. The effects of Pae on B lymphocytes of CIA rats were evaluated by the levels of BAFF, anti-CII antibody, IgA, IgG and IgM, and the expressions of BAFF-R, PI3K, p-Akt and mTOR. RESULTS: In CIA rats, the levels of anti-CII antibody, IgA, IgG and IgM in serum enhanced, BAFF, BAFF-R, PI3K, p-Akt and mTOR were highly expressed. Pae (100mg/kg) obviously decreased arthritis score, relieved ankle and paw swelling, improved spleen histopathology in CIA rats, decreased the levels of IgA, IgM, IgG and anti-CII antibody, and significantly decreased the expressions of BAFF, BAFF-R, PI3K, p-Akt and mTOR. CONCLUSION: PI3K/Akt/mTOR signaling mediated by BAFF/BAFF-R participates in antibodies production by B lymphocytes of CIA rats. Pae had therapeutic effects on rats with CIA. These effects might be relative to regulating PI3K/Akt/mTOR signal mediated by BAFF/BAFF-R, and down regulate the antibodies production further.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Autoanticorpos/sangue , Fator Ativador de Células B/imunologia , Receptor do Fator Ativador de Células B/imunologia , Benzoatos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Glucosídeos/farmacologia , Paeonia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Articulação do Tornozelo/efeitos dos fármacos , Articulação do Tornozelo/enzimologia , Articulação do Tornozelo/imunologia , Articulação do Tornozelo/patologia , Anti-Inflamatórios/isolamento & purificação , Artrite Experimental/enzimologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Linfócitos B/imunologia , Benzoatos/isolamento & purificação , Hidrocarbonetos Aromáticos com Pontes/isolamento & purificação , Glucosídeos/isolamento & purificação , Masculino , Monoterpenos , Paeonia/química , Fosforilação , Fitoterapia , Raízes de Plantas , Plantas Medicinais , Ratos , Ratos Sprague-Dawley , Baço/efeitos dos fármacos , Baço/enzimologia , Baço/imunologia , Baço/patologia , Fatores de TempoRESUMO
Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B-cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X-linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein-protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays, WT BTK, L111P, and E650G variants were expressed in the BTK-deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR-crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK.
Assuntos
Mutação de Sentido Incorreto , Proteínas Tirosina Quinases/genética , Adolescente , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/enzimologia , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Linfócitos B/enzimologia , Linfócitos B/imunologia , Linfócitos B/patologia , Sequência de Bases , Diferenciação Celular , Linhagem Celular , DNA Complementar/genética , Estabilidade Enzimática , Estudos de Associação Genética , Doenças Genéticas Ligadas ao Cromossomo X/enzimologia , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Humanos , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
Suckling "F/A2" mice, which overexpress arginase-I in their enterocytes, develop a syndrome (hypoargininemia, reduced hair and muscle growth, impaired B-cell maturation) that resembles IGF1 deficiency. The syndrome may result from an impaired function of the GH-IGF1 axis, activation of the stress-kinase GCN2, and/or blocking of the mTORC1-signaling pathway. Arginine deficiency inhibited GH secretion and decreased liver Igf1 mRNA and plasma IGF1 concentration, but did not change muscle IGF1 concentration. GH supplementation induced Igf1 mRNA synthesis, but did not restore growth, ruling out direct involvement of the GH-IGF1 axis. In C2C12 muscle cells, arginine withdrawal activated GCN2 signaling, without impacting mTORC1 signaling. In F/A2 mice, the reduction of plasma and tissue arginine concentrations to â¼25% of wild-type values activated GCN2 signaling, but mTORC1-mediated signaling remained unaffected. Gcn2-deficient F/A2 mice suffered from hypoglycemia and died shortly after birth. Because common targets of all stress kinases (eIF2α phosphorylation, Chop mRNA expression) were not increased in these mice, the effects of arginine deficiency were solely mediated by GCN2.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Arginase/biossíntese , Arginina/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Erros Inatos do Metabolismo dos Aminoácidos/genética , Animais , Animais Lactentes/metabolismo , Arginase/genética , Arginina/genética , Linfócitos B/enzimologia , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Doenças do Cabelo/enzimologia , Doenças do Cabelo/genética , Hipoglicemia/enzimologia , Hipoglicemia/genética , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Doenças Musculares/enzimologia , Doenças Musculares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas , Síndrome , Serina-Treonina Quinases TORRESUMO
Activation of the B-cell antigen receptor (BCR) signaling pathway contributes to the initiation and maintenance of B-cell malignancies and autoimmune diseases. The Bruton tyrosine kinase (Btk) is specifically required for BCR signaling as demonstrated by human and mouse mutations that disrupt Btk function and prevent B-cell maturation at steps that require a functional BCR pathway. Herein we describe a selective and irreversible Btk inhibitor, PCI-32765, that is currently under clinical development in patients with B-cell non-Hodgkin lymphoma. We have used this inhibitor to investigate the biologic effects of Btk inhibition on mature B-cell function and the progression of B cell-associated diseases in vivo. PCI-32765 blocked BCR signaling in human peripheral B cells at concentrations that did not affect T cell receptor signaling. In mice with collagen-induced arthritis, orally administered PCI-32765 reduced the level of circulating autoantibodies and completely suppressed disease. PCI-32765 also inhibited autoantibody production and the development of kidney disease in the MRL-Fas(lpr) lupus model. Occupancy of the Btk active site by PCI-32765 was monitored in vitro and in vivo using a fluorescent affinity probe for Btk. Active site occupancy of Btk was tightly correlated with the blockade of BCR signaling and in vivo efficacy. Finally, PCI-32765 induced objective clinical responses in dogs with spontaneous B-cell non-Hodgkin lymphoma. These findings support Btk inhibition as a therapeutic approach for the treatment of human diseases associated with activation of the BCR pathway.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Linfócitos B/imunologia , Benzofuranos/farmacologia , Benzofuranos/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Adenina/análogos & derivados , Administração Oral , Tirosina Quinase da Agamaglobulinemia , Animais , Artrite Experimental/tratamento farmacológico , Autoanticorpos/biossíntese , Doenças Autoimunes/enzimologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Benzofuranos/administração & dosagem , Benzofuranos/química , Modelos Animais de Doenças , Cães , Humanos , Linfoma de Células B/enzimologia , Camundongos , Piperidinas , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/administração & dosagem , Pirazóis/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
B-cell receptor signaling contributes to apoptosis resistance in chronic lymphocytic leukemia (CLL), limiting the efficacy of current therapeutic approaches. In this study, we investigated the expression of spleen tyrosine kinase (SYK), a key component of the B-cell receptor signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared with healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCgamma(2), signal transducers and activators of transcription 3, and extracellular signal regulated kinase 1/2 in CLL compared with healthy B cells, suggesting enhanced activation of these mediators in CLL. SYK inhibitors reduced phosphorylation of SYK downstream targets and induced apoptosis in primary CLL cells. With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70(+) cases. Cytotoxic effects of SYK inhibitors also associated with SYK protein expression, potentially predicting response to therapy. Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared with fludarabine therapy alone. We observed no stroma-contact-mediated drug resistance for SYK inhibitors as described for fludarabine treatment. CD40 ligation further enhanced efficacy of SYK inhibition. Our data provide mechanistic insight into the recently observed therapeutic effects of the SYK inhibitor R406 in CLL. Combination of SYK inhibitors with fludarabine might be a novel treatment option particularly for CLL patients with poor prognosis and should be further evaluated in clinical trials.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas Tirosina Quinases/genética , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose , Linfócitos B/enzimologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Curcumina/uso terapêutico , Perfilação da Expressão Gênica , Humanos , Região Variável de Imunoglobulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Mutação , Estadiamento de Neoplasias , Oxazinas/uso terapêutico , Fosforilação , Prognóstico , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/uso terapêutico , Quinase Syk , Vidarabina/análogos & derivados , Vidarabina/uso terapêuticoRESUMO
INTRODUCTION: It was previously shown that lipoxygenase (LO) pathways are important in the rheumatoid arthritis (RA) inflammatory process and that synovial fluid from RA patients contains high amounts of leukotrienes. We therefore aimed to investigate the 5-LO and 15-LO-1 expression pattern in RA and ostheoarthritis (OA) synovial tissue and to study the effect of intraarticular glucocorticoid (GC) therapy on enzyme expression. METHODS: Expression of LOs was evaluated by immunohistochemistry in RA and OA synovial biopsies. Cellular localization of these enzymes was analyzed by double immunofluorescence. In synovial biopsies from 11 RA patients, 5-LO and 15-LO-1 expression was evaluated before and after triamcinolone hexacetonide knee injection and assessed by image analysis to quantify their expression. We also investigated the presence of 15-LO-1 by immunohistochemistry in synovial fluid (SF) cells as well as their ability to form 15-hydroxyeicosatetraenoic acid (15-HETE) following treatment with arachidonic acid (AA). RESULTS: 5-LO and 15-LO-1 are present in RA and OA synovium, with 5-LO being mostly expressed in lining and sublining macrophages, neutrophils and mast cells and 15-LO-1 mainly in lining macrophages, fibroblasts and sublining endothelial cells. Intraarticular GC treatment resulted in a significant suppression of 5-LO expression, but did not influence the 15-LO-1 enzyme significantly. Also, SF cells express a functional 15-LO-1 and produce 15-HETE when challenged with AA. CONCLUSIONS: These data demonstrate that local therapy with GC decreases 5-LO expression in RA synovium and offer an additional possible mechanism for the efficiency of intraarticular adjuvant therapy in RA.
Assuntos
Araquidonato 15-Lipoxigenase/biossíntese , Araquidonato 5-Lipoxigenase/biossíntese , Artrite Reumatoide/enzimologia , Glucocorticoides/administração & dosagem , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Araquidonato 15-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/genética , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Linfócitos B/patologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/uso terapêutico , Humanos , Injeções Intra-Articulares , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Membrana Sinovial/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Linfócitos T/patologiaRESUMO
Tumor cells with different origins have different threshold to apoptosis. Hematopoietic (Jurkat, NCI-H929) cells and non-hematopoietic (A549, MCF-7) cells were received hyperbaric oxygen (HBO(2)) treatment from 2.5 to 3.5 atmosphere absolute (ATA) of 100% oxygen for 6h, and a significant percentage of apoptosis were shown only in hematopoietic Jurkat and NCI-H929 cells by either Annexin V or TUNEL assay. Oxidative stress was illustrated higher in HBO(2)-treated hematopoietic cells by superoxide fluorochrome detectors. HBO(2) treatment leads to caspase-3, caspase-7 activation and further cleavage of PARP within cells. Furthermore, the increased phosphorylation of p38 MAPK was demonstrated in both Jurkat and NCI-H929 cells.
Assuntos
Apoptose , Oxigenoterapia Hiperbárica , Leucemia de Células T/enzimologia , Mieloma Múltiplo/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Anexina A5/metabolismo , Linfócitos B/enzimologia , Linfócitos B/patologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Colágeno Tipo XI/metabolismo , Humanos , Células Jurkat , Leucemia de Células T/patologia , Leucemia de Células T/terapia , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Oxigênio/metabolismo , FosforilaçãoRESUMO
Bruton's tyrosine kinase (Btk) and interleukin-2-inducible T cell kinase (Itk) are members of the TEC family of nonreceptor tyrosine kinases and are expressed primarily in B and T cells, respectively. Both kinases are critically involved in lymphocyte development and signal transduction. In particular, Btk and Itk regulate calcium mobilization subsequent to antigen receptor stimulation. Small molecule antagonists that specifically inhibit either Btk or Itk may allow for selective modulation of B cell or T cell activity and may be useful in treating inflammatory and autoimmune conditions. We have developed a medium-throughput fluorescent imaging plate reader (FLIPR)- based calcium flux assay that can be used to assay potential Btk and Itk inhibitors. This assay takes advantage of Btk-deficient DT40 (DT40-Btk-/-) chicken B cells, which are unable to mobilize calcium in response to cross-linking of their B cell receptor (BCR). Ectopic expression of TEC family kinases can restore antigen receptor signaling in these cells. We have generated stable DT40-Btk-/- lines expressing either wild-type human Btk (huBtk) or a chimeric Btk-Itk kinase (huBtk-Itk) molecule-a Btk protein whose kinase domain has been replaced by the kinase domain of Itk. Expression of either huBtk or huBtk-Itk in DT40-Btk-/- cells restores calcium flux in response to BCR engagement. Using Btk- and Itk-selective inhibitors, we show that inhibition of calcium responses in huBtk-Itk-DT40-Btk-/- cells and huBtk-DT40-Btk-/- cells is dependent on the Itk or Btk kinase domain, respectively. Thus, the FLIPR assay described here can be used to assess, compare, and rank the potency and selectivity of inhibitors of Itk and Btk kinases.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/enzimologia , Cálcio/metabolismo , Galinhas , DNA Complementar/biossíntese , DNA Complementar/genética , Interpretação Estatística de Dados , Avaliação Pré-Clínica de Medicamentos/instrumentação , Corantes Fluorescentes , Fluorometria , Liofilização , Vetores Genéticos/genética , Immunoblotting , Lasers , Organismos Geneticamente Modificados , Regiões Promotoras Genéticas/genética , Engenharia de Proteínas , Proteínas Tirosina Quinases/genéticaRESUMO
The protein tyrosine kinase Syk couples the B-cell receptor (BCR) for antigen to multiple intracellular signaling pathways and also modulates cellular responses to inducers of oxidative stress in a receptor-independent fashion. In B cells, Syk is found in both the nuclear and cytoplasmic compartments but contains no recognizable nuclear localization or export signals. Through the analysis of a series of deletion mutants, we identified the presence of an unconventional shuttling sequence near the junction of the catalytic domain and the linker B region that accounts for Syk's subcellular localization. This localization is altered following prolonged engagement of the BCR, which causes Syk to be excluded from the nucleus. Nuclear exclusion requires the receptor-mediated activation of protein kinase C and new protein synthesis. Both of these processes also potentiate the activation of caspase 3 in cells in response to oxidative stress in a manner that is dependent on the localization of Syk outside of the nucleus. In contrast, restriction of Syk to the nucleus greatly diminishes the stress-induced activation of caspase 3.
Assuntos
Linfócitos B/enzimologia , Núcleo Celular/enzimologia , Citoplasma/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sinais de Localização Nuclear/genética , Proteínas Tirosina Quinases/metabolismo , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Linfócitos B/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Domínio Catalítico , Células Cultivadas , Análise Mutacional de DNA , Ativação Enzimática , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Imunoglobulina M/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/genética , Biossíntese de Proteínas , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Transporte Proteico/genética , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/genética , Deleção de Sequência , Quinase Syk , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
We have taken a knockout approach to interrogate the function of protein kinase D (PKD) serine/threonine kinases in lymphocytes. DT40 B cells express two PKD family members, PKD1 and PKD3, which are both rapidly activated by the B-cell antigen receptor (BCR). DT40 cells with single or dual deletions of PKD1 and/or PKD3 were viable, allowing the role of individual PKD isoforms in BCR signal transduction to be assessed. One proposed downstream target for PKD1 in lymphocytes is the class II histone deacetylases (HDACs). Regulation of chromatin accessibility via class II histone deacetylases is an important mechanism controlling gene expression patterns, but the molecules that control this key process in B cells are not known. Herein, we show that phosphorylation and nuclear export of the class II histone deacetylases HDAC5 and HDAC7 are rapidly induced following ligation of the BCR or after treatment with phorbol esters (a diacylglycerol mimetic). Loss of either PKD1 or PKD3 had no impact on HDAC phosphorylation, but loss of both PKD1 and PKD3 abrogated antigen receptor-induced class II HDAC5/7 phosphorylation and nuclear export. These studies reveal an essential and redundant role for PKD enzymes in controlling class II HDACs in B lymphocytes and suggest that PKD serine kinases are a critical link between the BCR and epigenetic control of chromatin.
Assuntos
Linfócitos B/enzimologia , Histona Desacetilases/metabolismo , Proteína Quinase C/metabolismo , Animais , Linfócitos B/imunologia , Sequência de Bases , Linhagem Celular , Galinhas , DNA Complementar/genética , Epigênese Genética , Deleção de Genes , Histona Desacetilases/classificação , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
Bupleuran 2IIc, a pectic polysaccharide isolated from the roots of bupleurum falcatum L., was previously characterized as a T-cell-independent B cell mitogen. This study focuses on elucidating the mechanism by which bupleuran 2IIc induces cyclin D2 production for inducing mitogenesis in murine B cells. Bupleuran 2IIc was digested with endo-alpha-(1-->4)-D-polygalacturonase and the resulting bupleuran 2IIc/PG-1 ("ramified" region) strongly stimulated cyclin D2 expression. When murine B cells were stimulated with bupleuran 2IIc/PG-1, phosphorylation of tyrosine residues of a number of proteins was observed. Cyclin D2 expression by bupleuran 2IIc/PG-1 was inhibited by the tyrosine kinase inhibitors, genistein and herbimycin A, and the Src family tyrosine kinase inhibitor, PP2, suggesting a possible role for tyrosine kinases. The stimulation by bupleuran 2IIc/PG-1 of cyclin D2 expression was significantly decreased by inhibitors, PI 3-kinase (LY294002 and Wortmannin), PLCgamma (U73122), PKC (H-7), receptor-operated calcium entry inhibitor (SK&F 96365), and calcineurin (FK506). Both PD98059 and U0126, highly selective inhibitors of MEK1 and MEK1/2, respectively, did not strongly suppress the expression of cyclin D2 after stimulation by bupleuran 2IIc/PG-1. The results suggest that (1) bupleuran 2IIc/PG-1 is the active site for induction of cyclin D2 by bupleuran 2IIc, (2) the expression of the cyclin D2 gene by bupleuran 2IIc/PG-1 may be mediated via the activation of PI 3-kinase and PLCgamma followed by activation of PKC and calcium mobilization, and (3) the ERK1/2 cascade is not a central signaling pathway for bupleuran 2IIc/PG-1-induced cyclin D2 expression.
Assuntos
Linfócitos B/efeitos dos fármacos , Ciclinas/biossíntese , Pectinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linfócitos B/enzimologia , Linfócitos B/imunologia , Cálcio/metabolismo , Sequência de Carboidratos , Ciclina D2 , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ativação Linfocitária , Camundongos , Dados de Sequência Molecular , Pectinas/biossíntese , Pectinas/química , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Fosfolipases Tipo C/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4(+) and CD8(+) T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca(2+)) influx, and that either the intracellular Ca(2+) chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca(2+)-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.
Assuntos
Apoptose/fisiologia , Sinalização do Cálcio , Calpaína/fisiologia , Caspase 1/fisiologia , Galectinas/fisiologia , Adjuvantes Imunológicos/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Linfócitos B/citologia , Linfócitos B/enzimologia , Linfócitos B/fisiologia , Cálcio/metabolismo , Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta Imunológica , Células HL-60 , Humanos , Células Jurkat , Monócitos/citologia , Monócitos/enzimologia , Monócitos/fisiologia , Células Mieloides/citologia , Células Mieloides/enzimologia , Células Mieloides/fisiologia , Oligopeptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/citologia , Linfócitos T/enzimologia , Linfócitos T/fisiologia , Células Tumorais CultivadasRESUMO
ES-62 is a phosphorylcholine (PC)-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae which is able to inhibit antigen receptor-stimulated proliferation of B and T lymphocytes in vitro and in vivo. The active component of ES-62 appears to be PC, as the results obtained with ES-62 are broadly mimicked by PC conjugated to bovine serum albumin or PC alone. Such desensitization of lymphocyte responsiveness appears to reflect an uncoupling of the antigen receptors from key intracellular proliferative signaling events, such as the phosphoinositide-3-kinase, protein kinase C and Ras mitogen-activating protein kinase pathways. ES-62 mediates such immunomodulatory effects at concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitized humans and, thus, ES-62 provides a model system for dissecting the mechanisms of immune evasion induced by related PC-containing glycoproteins expressed by human filarial nematodes.
Assuntos
Antígenos de Helmintos/metabolismo , Filarioidea/imunologia , Receptores de Antígenos/metabolismo , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Linfócitos B/imunologia , Dipetalonema/imunologia , Filariose/imunologia , Filarioidea/patogenicidade , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Proteínas de Helminto/imunologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/farmacologia , Humanos , Técnicas In Vitro , Ativação Linfocitária/efeitos dos fármacos , Modelos Imunológicos , Proteína Quinase C/metabolismo , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Proteínas ras/metabolismoRESUMO
CD19 is a coreceptor on B cells that enhances the increase in cytoplasmic calcium and ERK2 activation when coligated with the B cell Ag receptor. Constructs containing point mutations and truncations were expressed in Daudi human B lymphoblastoid cells to systematically determine the requirement for individual CD19 cytoplasmic tyrosines in these responses. Evidence for activity was found for Y330, Y360, and Y421 as well as that previously published for Y391. Precipitates formed with phosphopeptides consisting of CD19 sequences flanking these residues were used to screen for cytoplasmic proteins that mediate signaling. Phosphopeptide Y330 precipitated Grb2 and Sos, whereas phosphopeptides Y391 and Y421 both precipitated Vav and phospholipase C-gamma2. These molecules also were found associated with native CD19. In mapping studies with altered constructs, CD19 Y330 and/or Y360 were necessary for binding Grb2 and Sos. Vav associated with CD19 constitutively in unstimulated cells by a tyrosine-independent mechanism requiring the portion of CD19 encoded by exons 9-12. After B cell Ag receptor stimulation, Vav association was tyrosine-dependent, but binding was influenced by multiple residues. However, when maximally phosphorylated by pervanadate, Y391 and, to a lesser extent, Y421 were sufficient. CD19 Y391 was also both necessary and sufficient for binding phospholipase C-gamma2. Thus, different tyrosines along the CD19 cytoplasmic domain provide scaffolding for the formation of complexes of different signaling molecules.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD19/fisiologia , Linfócitos B/imunologia , Proteínas de Ciclo Celular , Ativação Linfocitária/imunologia , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Son Of Sevenless de Drosófila/metabolismo , Fosfolipases Tipo C/metabolismo , Tirosina/fisiologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/fisiologia , Antígenos CD19/genética , Antígenos CD19/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Linfócitos B/metabolismo , Sinalização do Cálcio/imunologia , Citoplasma/imunologia , Citoplasma/metabolismo , Éxons , Proteína Adaptadora GRB2 , Humanos , Isoenzimas/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Peso Molecular , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Fosfolipase C gama , Fosfopeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-vav , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Células Tumorais Cultivadas , Tirosina/genética , Tirosina/metabolismo , Vanadatos/farmacologiaRESUMO
Using a subtractive cloning scheme on cDNA prepared from primary pro-B and pre-B cells, we identified several genes whose products regulate apoptosis. We further characterized one of these genes, encoding protein kinase Ceta (PKCeta). PKCeta transcripts were readily detected in pro-B cells but were absent in pre-B cells. Although both a full-length and a truncated form of PKCeta were detectable in bone marrow pro-B cells, transition to the pre-B-cell stage was associated with increased relative levels of truncated PKCeta. We found that PKCeta is proteolyzed in apoptotic lymphocytes, generating a kinase-active fragment identical to the truncated form which is capable of inducing apoptosis when expressed in a pro-B cell line. Caspase-3 can generate an identical PKCeta cleavage product in vitro, and caspase inhibitors prevent the generation of this product during apoptosis in transfected cell lines. Inducible overexpression of either the full-length or truncated form of PKCeta results in cell cycle arrest at the G(1)/S transition. These results suggest that the expression and proteolytic activation of PKCeta play an important role in the regulation of cell division and cell death during early B-cell development.
Assuntos
Apoptose/genética , Linfócitos B/citologia , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Isoenzimas/fisiologia , Proteína Quinase C/fisiologia , Transcrição Gênica , Animais , Linfócitos B/enzimologia , Caspase 3 , Caspases/fisiologia , Ciclo Celular , Linhagem da Célula , DNA Complementar/genética , Indução Enzimática , Células-Tronco Hematopoéticas/enzimologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Isoenzimas/genética , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Fragmentos de Peptídeos/metabolismo , Proteína Quinase C/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/biossíntese , Inibidores de Serina Proteinase/farmacologia , Técnica de Subtração , TransfecçãoRESUMO
We have identified a novel gene referred to as activation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell.
Assuntos
Linfócitos B/enzimologia , Citidina Desaminase/biossíntese , Centro Germinativo/citologia , Edição de RNA , Desaminase APOBEC-1 , Sequência de Aminoácidos , Animais , Apolipoproteínas B , Cicloeximida/farmacologia , Citidina Desaminase/química , Citidina Desaminase/genética , DNA Complementar/isolamento & purificação , Indução Enzimática/efeitos dos fármacos , Biblioteca Gênica , Centro Germinativo/enzimologia , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
Stimulation of CD4(+) helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-alpha/Ig-beta heterodimers which, second, target antigens to MHC class II-containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-alpha-associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-alpha cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide- MHC class II complexes through antigen targeting by BCR subunits.
Assuntos
Apresentação de Antígeno , Antígenos CD/fisiologia , Proteínas de Ligação a DNA , Precursores Enzimáticos/fisiologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Animais , Antígenos CD/química , Antígenos Virais/metabolismo , Linfócitos B/enzimologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Bacteriófago lambda/imunologia , Antígenos CD79 , Citoplasma/imunologia , Precursores Enzimáticos/metabolismo , Epitopos de Linfócito T/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Ativação Linfocitária , Linfoma de Células B , Camundongos , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptores de Antígenos de Linfócitos B/química , Proteínas Repressoras/imunologia , Quinase Syk , Células Tumorais Cultivadas , Tirosina/fisiologia , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
The TK6 human B lymphoblastoid cell line contains two easily and widely used selectable markers: the X-linked, hemizygous hprt locus, and the heterozygous tk locus on chromosome 17q. In this study, rare APRT heterozygotes were directly isolated from the TK6 population by clonal selection in cell culture medium supplemented with 5 micrograms/ml of 8-azaadenine. One of nine isolated heterozygotes, AZH1, was characterized extensively. APRT- mutants can be recovered from AZH1 at a mutation rate of 1.5 x 10(-7), similar to rates previously determined for the selection of TK- and HPRT- mutants from TK6. A unique sequence alteration was identified in the non-functional aprt allele at position 1930. A G:C to A:T transition at this site alters the canonical AG splice acceptor dinucleotide in exon 3, and also results in the destruction of a Stul recognition sequence. This polymorphism was used to analyze loss of heterozygosity in a set of 32 spontaneous APRT- mutants by restriction analysis following PCR amplification. Analysis of flanking microsatellite dinucleotide polymorphisms demonstrated that LOH occurring in spontaneous APRT- mutants is nearly always a multi-locus event extending at least 7.5 cM along chromosome 16q. This pattern of LOH among APRT- mutants differs from extensive LOH in spontaneous, normal-growth TK- mutants derived from TK6 cells (p < 0.0001), and suggests that cis-acting factors may be equally important in shaping the mutational spectrum as trans-acting factors such as cellular apoptotic capacity.