RESUMO
The AP-1 Adaptor Complex assists clathrin-coated vesicle assembly in the trans-Golgi network (TGN) of eukaryotic cells. However, the role of AP-1 in the protozoan Trypanosoma cruzi-the Chagas disease parasite-has not been addressed. Here, we studied the function and localization of AP-1 in different T. cruzi life cycle forms, by generating a gene knockout of the large AP-1 subunit gamma adaptin (TcAP1-γ), and raising a monoclonal antibody against TcAP1-γ. Co-localization with a Golgi marker and with the clathrin light chain showed that TcAP1-γ is located in the Golgi, and it may interact with clathrin in vivo, at the TGN. Epimastigote (insect form) parasites lacking TcAP1-γ (TcγKO) have reduced proliferation and differentiation into infective metacyclic trypomastigotes (compared with wild-type parasites). TcγKO parasites have also displayed significantly reduced infectivity towards mammalian cells. Importantly, TcAP1-γ knockout impaired maturation and transport to lysosome-related organelles (reservosomes) of a key cargo-the major cysteine protease cruzipain, which is important for parasite nutrition, differentiation and infection. In conclusion, the defective processing and transport of cruzipain upon AP-1 ablation may underlie the phenotype of TcγKO parasites.
Assuntos
Doença de Chagas/parasitologia , Cisteína Endopeptidases/química , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/fisiologia , Trypanosoma cruzi/genética , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/química , Vesículas Revestidas por Clatrina , Endocitose , Teste de Complementação Genética , Complexo de Golgi/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Organelas , Plasmídeos/metabolismo , Proteínas de Protozoários , Proteínas Recombinantes/química , Rede trans-Golgi/metabolismoRESUMO
The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Produtos do Gene tax/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteínas de Neoplasias/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Divisão Celular , Sequência Consenso , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Produtos do Gene tax/genética , Humanos , Células Jurkat , NF-kappa B/fisiologia , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/fisiologia , Transfecção , Tretinoína/farmacologiaRESUMO
Previously, we have demonstrated that 17α-ethynylestradiol (EE) induces rat multidrug-resistance associated protein 3 (Mrp3, Abcc3) expression transcriptionally through estrogen receptor-α (ER-α) activation. We explored the effect of EE on MRP3 expression of human origin. HepG2 cells were transfected with ER-α and incubated with EE (1-10-50 µM) for 48 h. MRP3 protein and mRNA levels were measured by Western blotting and Real time PCR, respectively. EE up-regulated MRP3 protein and mRNA at 50 µM only in ER-α(+)-HepG2 cells. The in silico analysis of mrp3 promoter region demonstrated absence of estrogen response elements, but showed several Ap-1 binding sites. We further evaluated the potential involvement of the transcription factors c-JUN and c-FOS (members of Ap-1) in MRP3 up-regulation. ER-α(+) HepG2 cells were incubated with EE and c-FOS and c-JUN levels measured by Western blotting in nuclear extracts. EE up-regulated only c-JUN. Experiments of overexpression and knock-down of c-JUN by siRNA further demonstrated that this transcription factor is indeed implicated in MRP3 upregulation by EE. Co-immunoprecipitation assay demonstrated that EE induces c-JUN/ER-α interaction, and chromatin immunoprecipitation assay showed that this complex is recruited to the AP-1 binding consensus element present at the position (-1300/-1078 bp) of human mrp3 promoter. We conclude that EE induces MRP3 expression through ER-α, with recruitment of ER-α in complex with c-JUN to the human mrp3 promoter.
Assuntos
Receptor alfa de Estrogênio/fisiologia , Etinilestradiol/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Fator de Transcrição AP-1/fisiologia , Sequência de Bases , Células Hep G2 , Humanos , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genéticaRESUMO
Previously, we demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and NO release in macrophages by inhibiting NF-kappaB. We now proceeded to elucidate the molecular mechanisms by which BZL exerts its inhibitory action on NF-kappaB. We demonstrated that the inhibitory effect of BZL is not extended to other macrophage responses, since it did not inhibit other typical hallmarks of macrophage activation such as phagocytosis, MHC-II molecules expression or production of reactive oxygen species (ROS) by NADPH oxidase. BZL was able to interfere specifically with the activation of NF-kappaB pathway without affecting AP-1 activation in RAW 264.7 macrophages, not only in LPS-mediated activation, but also for other stimuli, such as pro-inflammatory cytokines (IL-1beta, TNF-alpha), PMA or H(2)O(2). Also, BZL delayed the activation of p38 MAPK, but not that of ERK1/2 and JNK. Finally, treatment with BZL inhibited IkappaBalpha phosporylation and hence its degradation, whereas it did not block IkappaB kinase (IKK) alpha/beta phosphorylation. Collectively, BZL behaves as a broad range specific inhibitor of NF-kappaB activation, independently of the stimuli tested.
Assuntos
Quinase I-kappa B/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Nitroimidazóis/farmacologia , Fator de Transcrição AP-1/fisiologia , Animais , Ativação Enzimática/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Inibidor de NF-kappaB alfa , Óxido Nítrico/metabolismo , Fagocitose/efeitos dos fármacos , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The mechanisms by which lymphocytes recognize and interpret mechanical stimuli and translate these into the triggering of select signaling cascades that are critical for lymphocyte function are still not fully understood. In this work, we investigated the association of mechanical stress (MS)-induced changes in membrane physical properties with changes in cytoskeleton dynamics and cell signaling. In Jurkat T cells, MS was associated with the immediate and transient depolymerization of both beta-tubulin and F-actin. The fluidity of the plasma membrane measured in the hydrophobic region of the bilayer, increased 0.5 min post-MS, recovering the initial value in the following 2 min. This effect was accompanied by the rearrangement of lipids in the lateral phase of the plasma membrane, transient lipid rafts' alteration, and membrane hyperpolarization. The consequent increase in cellular [Ca2+] triggered the activation of the transcription factors NFAT, AP-1, and NF-kappaB. Results indicate that the cytoplasmic membrane, through changes in membrane physical properties, senses MS, and transduces an initial physical stimulus into microtubules rearrangements, Ca2+ mobilization, and the subsequent changes in cell signaling.
Assuntos
Membrana Celular/fisiologia , Estresse Mecânico , Actinas/química , Cálcio/metabolismo , Humanos , Células Jurkat , Fluidez de Membrana , NF-kappa B/fisiologia , Fatores de Transcrição NFATC/fisiologia , Proteína Quinase C/fisiologia , Transdução de Sinais , Fator de Transcrição AP-1/fisiologia , Tubulina (Proteína)/químicaRESUMO
Even though RA is involved in differentiation and apoptosis of normal and cancer cells, being sometimes used as adjuvant in chemotherapy, its mechanisms of action involve multiple overlapping pathways that still remain unclear. Recent studies point out that RA exerts rapid and non-genomic effects, which are independent of RAR/RXR-mediated gene transcription. In this work, we reported that RA treatment for 24 h decreases cell viability, induces apoptosis dependent on caspase-3 activation, and activates the transcription factor AP-1 in cultured Sertoli cells. Moreover, RA induced a rapid and non-classical stimulation of ERK1/2. ERK1/2 activation was mediated by MEK1/2, and the protein synthesis inhibitor cycloheximide did not alter the pattern of RA-induced ERK1/2 phosphorylation. Pharmacological inhibition of MEK1/2-ERK1/2 pathway with UO126 blocked caspase-3 activation, decreased AP-1 binding to DNA and inhibited apoptosis. Overall, our data suggest that a rapid and non-genomic effect of RA upon MEK1/2-ERK1/2 pathway leads to caspase-3 activation and caspase-3-dependent apoptosis in cultured Sertoli cells. The non-canonical RA signaling presented in this work evokes new perspectives of RA action, which may play an important role in mediating early biological effects of RA modulating cell death in normal and tumor cells.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células de Sertoli/efeitos dos fármacos , Tretinoína/toxicidade , Vitaminas/toxicidade , Animais , Butadienos/farmacologia , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cicloeximida/farmacologia , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , MAP Quinase Quinase 1/metabolismo , Masculino , Nitrilas/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Células de Sertoli/enzimologia , Células de Sertoli/patologia , Fator de Transcrição AP-1/biossíntese , Fator de Transcrição AP-1/fisiologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Appropriate removal of L: -glutamate from the synaptic cleft is important for prevention of the excitotoxic effects of this neurotransmitter. The Na+-dependent glutamate/aspartate transporter GLAST is regulated in the short term, by a transporter-dependent decrease in uptake activity while in the long term, a receptor's-dependent decrease in GLAST protein levels leads to a severe reduction in glutamate uptake. The promoter region of the mouse glast gene harbors an Activator Protein-1 site (AP-1). To gain insight into the molecular mechanisms triggered by Glu-receptors activation involved in GLAST regulation, we took advantage of the neonatal mouse cerebellar prisms model. We characterized the glutamate uptake activity; the glutamate-dependent effect on GLAST protein levels and over the interaction of nuclear proteins with a mouse glast promoter AP-1 probe. A time and dose dependent decrease in transporter activity matching with a decrease in GLAST levels was recorded upon glutamate treatment. Moreover, a significant increase in glast AP-1 DNA binding was found. Pharmacological experiments established that both effects are mediated through alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, favoring the notion of the critical involvement of glutamate in the regulation of its binding partners: receptors and transporters.
Assuntos
Transportador 1 de Aminoácido Excitatório/biossíntese , Regiões Promotoras Genéticas , Receptores de AMPA/fisiologia , Fator de Transcrição AP-1/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Cerebelo/fisiologia , Regulação para Baixo , Transportador 1 de Aminoácido Excitatório/genética , Regulação da Expressão Gênica , Ácido Glutâmico/farmacologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Activator protein-1 (AP1) is a dimeric protein, consisting either of homodimers between c-Jun, JunB, and JunD of by heterodimers with members of the Fos-family by physically interacting via a "leucine zipper" region. AP1 is an important transcription factor initially identified as a DNA binding protein that bound to enhancer sequences of the human metallothionein IIA gene. The protein components of AP1 are encoded by a set of genes known as "immediate-early" genes that can be activated by a variety of growth factors and mitogens through several different signaling pathways. Until recently, AP1 was considered a transcription factor expressed in most tissues to regulate cellular and viral genes now, it is becoming evident that AP1 can be involved in tissue-specific regulation of target genes due to the differential combination of the components of this important transcription factor. AP1 plays a crucial role during human papillomavirus (HPV) early gene expression, in particular of the expression of E6 and E7 oncoproteins. The HPV are a group of DNA viruses consisting of more than 80 different genotypes. Some of these HPV, know as high risk HPV, are important etiologic agents of uterine-cervical cancer (CaCu). Of the different types of cancer, CaCu is one of the most frequent among women worldwide, constituting the second death cause due to neoplasia. During cellular transformation, HPV infect basal cells in stratified epithelium; their DNA integrate into the host genome usually through the E2 gene; as these cells differentiate and migrate into the upper layer of the epithelium, viral oncogene are expressed blocking their differentiation. Mutagenesis in AP1 sites belonging to the HPV promoter region (LCR) completely abolished the HPV promoter activity in different cell lines; these results and biochemistry assays on this AP1 transcription factor, that includes protein-protein interactions between AP1 and another factors as E7 from HPV, and YY-1; the post-translattional modification and, the retinoic acid interaction; suggest a role for this AP1 factor in tissue-specific transcription of the human papillomavirus.
Assuntos
Genes Precoces , Genes Virais , Proteínas Imediatamente Precoces/biossíntese , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Fator de Transcrição AP-1/fisiologia , Proteínas Estruturais Virais/genética , Células Epiteliais/virologia , Feminino , Regulação Viral da Expressão Gênica , Humanos , Modelos Biológicos , Mutagênese , Proteínas Oncogênicas Virais/biossíntese , Especificidade de Órgãos , Papillomaviridae/classificação , Papillomaviridae/fisiologia , Infecções por Papillomavirus/virologia , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Infecções Tumorais por Vírus/virologia , Neoplasias do Colo do Útero/virologia , Neoplasias Uterinas/virologia , Replicação ViralRESUMO
Activator protein-1 (AP1) is a dimeric protein, consisting either of homodimers between c-Jun, JunB, and JunD of by heterodimers with members of the Fos-family by physically interacting via a "leucine zipper" region. AP1 is an important transcription factor initially identified as a DNA binding protein that bound to enhancer sequences of the human metallothionein IIA gene. The protein components of AP1 are encoded by a set of genes known as "immediate-early" genes that can be activated by a variety of growth factors and mitogens through several different signaling pathways. Until recently, AP1 was considered a transcription factor expressed in most tissues to regulate cellular and viral genes now, it is becoming evident that AP1 can be involved in tissue-specific regulation of target genes due to the differential combination of the components of this important transcription factor. AP1 plays a crucial role during human papillomavirus (HPV) early gene expression, in particular of the expression of E6 and E7 oncoproteins. The HPV are a group of DNA viruses consisting of more than 80 different genotypes. Some of these HPV, know as high risk HPV, are important etiologic agents of uterine-cervical cancer (CaCu). Of the different types of cancer, CaCu is one of the most frequent among women worldwide, constituting the second death cause due to neoplasia. During cellular transformation, HPV infect basal cells in stratified epithelium; their DNA integrate into the host genome usually through the E2 gene; as these cells differentiate and migrate into the upper layer of the epithelium, viral oncogene are expressed blocking their differentiation. Mutagenesis in AP1 sites belonging to the HPV promoter region (LCR) completely abolished the HPV promoter activity in different cell lines; these results and biochemistry assays on this AP1 transcription factor, that includes protein-protein interactions between AP1 and another factors as E7 from HPV, and YY-1; the post-translattional modification and, the retinoic acid interaction; suggest a role for this AP1 factor in tissue-specific transcription of the human papillomavirus.