RESUMO
Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.
Assuntos
Bothrops , Venenos de Crotalídeos , Gânglios Espinais , Hiperalgesia , Receptores da Neurocinina-1 , Animais , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Venenos de Crotalídeos/toxicidade , Masculino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Receptores da Neurocinina-1/metabolismo , Minociclina/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Ratos , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Ratos Sprague-DawleyRESUMO
Upon retrieval, an aversive memory can undergo destabilization and reconsolidation. A traumatic-like memory, however, may be resistant to this process. The present study sought to contribute with a strategy to overcome this potential issue by investigating whether generalized fear retrieval is susceptible to destabilization-reconsolidation that can be pharmacologically modified. We hypothesized that exposure to a context that elicits moderate generalization levels would allow a malleable memory state. We developed a fear conditioning protocol in context A (cxt-A) paired with yohimbine administration to promote significant fear to a non-conditioned context B (cxt-B) in rats, mimicking the enhanced noradrenergic activity reported after traumatic events in humans. Next, we attempted to impair the reconsolidation phase by administering clonidine (CLO) immediately after exposure to cxt-A, cxt-B, or a third context C (cxt-C) neither conditioned nor generalized. CLO administered post-cxt-B exposure for two consecutive days subsequently resulted in decreased freezing levels in cxt-A. CLO after cxt-B only once, after cxt-A or cxt-C in two consecutive days, or independently of cxt-B exposures did not affect fear in a later test. A 6-h-delay in CLO treatment post-cxt-B exposures produced no effects, and nimodipine administered pre-cxt-B exposures precluded the CLO action. We then quantified the Egr1/Zif268 protein expression following cxt-B exposures and CLO treatments. We found that these factors interact to modulate this memory destabilization-reconsolidation mechanism in the basolateral amygdala but not the dorsal CA1 hippocampus. Altogether, memory destabilization can accompany generalized fear expression; thus, we may exploit it to potentiate reconsolidation blockers' action.
Assuntos
Medo/psicologia , Generalização Psicológica , Consolidação da Memória/fisiologia , Memória/fisiologia , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Clonidina/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/genética , Extinção Psicológica , Masculino , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/psicologia , Rememoração Mental , Ratos , Ratos Wistar , Simpatolíticos , IoimbinaRESUMO
Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18â¯h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.
Assuntos
Morte Celular , Proteína 1 de Resposta de Crescimento Precoce/genética , Vírus da Encefalite Equina Venezuelana/fisiologia , Encefalomielite Equina Venezuelana/virologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , eIF-2 Quinase/metabolismo , Apoptose , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/virologia , Sobrevivência Celular , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/patologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Transdução de Sinais , Replicação Viral , eIF-2 Quinase/genéticaRESUMO
BACKGROUND: Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma with a poor prognosis owing to its resistance to radiation and chemotherapy. Thus, novel therapeutic strategies for SS are urgently required. Anlotinib, a new oral tyrosine kinase inhibitor, is designed to primarily inhibit multi-targets in vasculogenesis and angiogenesis. This study was designed to characterize its antitumor efficacy and possible mechanism in patients with advanced refractory synovial sarcoma. METHODS: Anlotinib's antitumor effect was evaluated in vivo and vitro. Downstream targets of anlotinib in treating synovial sarcoma were analyzed through microarray assay. Cell proliferation and apoptosis analyses were performed to evaluate the impact of candidate downstream gene depletion in synovial sarcoma cells. Microarray assay were carried out to investigate potential signal network related with candidate downstream gene. RESULTS: Anlotinib significantly suppresses synovial sarcoma proliferation in PDTX model and cell lines. Additionally, GINS1 (also named as PSF1, Partner of SLD Five 1), rather than other conventional gene target, was demonstrated to be a vital target of anlotinib's antitumor effect in synovial sarcoma through microarray assay. Expression of GINS1 was remarkably higher in synovial sarcoma tumor samples and related with poor outcome. Knockdown of GINS1 expression could remarkably inhibit proliferation and promote apoptosis in vitro. Meanwhile, through microarray assay, CITED2, EGR1, SGK1 and SPP1 were identified and further validated by qPCR/WB as downstream targets of GINS1. CONCLUSION: Anlotinib might suppress proliferation of SS through a novel downstream GINS1-regulated network which plays a vital function in SS proliferation and also demonstrated that targeting the GINS1-regulated signal pathway could be a potential strategy for management of SS.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Proteínas de Ligação a DNA/efeitos dos fármacos , Indóis/uso terapêutico , Proteínas de Neoplasias/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/uso terapêutico , Sarcoma Sinovial/tratamento farmacológico , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/genética , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Proteína 1 de Resposta de Crescimento Precoce/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas Imediatamente Precoces/efeitos dos fármacos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Osteopontina/efeitos dos fármacos , Osteopontina/genética , Osteopontina/metabolismo , Análise Serial de Proteínas , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/análise , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sarcoma Sinovial/genética , Transativadores/efeitos dos fármacos , Transativadores/genética , Transativadores/metabolismoRESUMO
Once viewed as a passive physiological state, sleep is a heterogeneous and complex sequence of brain states with essential effects on synaptic plasticity and neuronal functioning. Rapid-eye-movement (REM) sleep has been shown to promote calcium-dependent plasticity in principal neurons of the cerebral cortex, both during memory consolidation in adults and during post-natal development. This article reviews the plasticity mechanisms triggered by REM sleep, with a focus on the emerging role of kinases and immediate-early genes for the progressive corticalization of hippocampus-dependent memories. The body of evidence suggests that memory corticalization triggered by REM sleep is a systemic phenomenon with cellular and molecular causes.
Assuntos
Consolidação da Memória/fisiologia , Sono REM/fisiologia , Animais , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Hipocampo/metabolismo , Humanos , Plasticidade Neuronal , Receptores de Neurotransmissores/metabolismo , Sinapses/metabolismoRESUMO
Evolution has equipped poxvirus genomes with the coding capacity for several virus-host interaction products which interfere with host cell gene expression and protein function, creating an adequate intracellular environment for a productive infection. We show here that Vaccinia virus (VACV) induces the expression of the cellular transcription factor EGR-1 (early growth response-1) in Mouse Embryonic Fibroblasts (MEFs) through the MEK (mitogen-activated protein kinase (MAPK)/ERK)/ERK (extracellular signal-regulated kinases) pathway, from 3 to 12 h post infection (h.p.i.). By using starved egr-1 knockout (egr-1-/-) MEFs, we demonstrate that VACV replication is reduced by ~1 log in this cell line. Although western blotting and electron microscopy analyses revealed no difference in VACV gene expression or morphogenesis, the specific infectivity of VACV propagated in egr-1-/- MEFs was lower than virus propagated in wild type (WT) cells. This lower infectivity was due to decreased VACV DNA replication during the next cycle of infection. Taken together, these results revealed that EGR-1 appears to facilitate VACV replication in starved fibroblasts by affecting viral particles infectivity.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Vaccinia virus/fisiologia , Vacínia/genética , Vacínia/virologia , Animais , Linhagem Celular , Replicação do DNA , DNA Viral , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Deleção de Genes , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Fosforilação , Vacínia/metabolismo , Replicação ViralRESUMO
Usurpation of the host's signalling pathways is a common strategy employed by viruses to promote their successful replication. Here we show that infection with the orthopoxvirus vaccinia virus (VACV) leads to sustained stimulation of c-Jun activity during the entire infective cycle. This stimulation is temporally regulated through MEK/ERK or MKK/JNK pathways, i.e. during the early/mid phase (1 to 6 hpi) and in the late phase (9 to 24 hpi) of the infective cycle, respectively. As a transcriptional regulator, upon infection with VACV, c-Jun is translocated from the cytoplasm to the nucleus, where it binds to the AP-1 DNA sequence found at the promoter region of its target genes. To investigate the role played by c-Jun during VACV replication cycle, we generated cell lines that stably express a c-Jun-dominant negative (DNc-Jun) mutation. Our data revealed that c-Jun is required during early infection to assist with viral DNA replication, as demonstrated by the decreased amount of viral DNA found in the DNc-Jun cells. We also demonstrated that c-Jun regulates the expression of the early growth response gene (egr-1), a gene previously shown to affect VACV replication mediated by MEK/ERK signalling. VACV-induced stimulation of the MKK/JNK/JUN pathway impacts viral dissemination, as we observed a significant reduction in both viral yield, during late stages of infection, and virus plaque size. Collectively, our data suggest that, by modulating the host's signalling pathways through a common target such as c-Jun, VACV temporally regulates its infective cycle in order to successfully replicate and subsequently spread.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Vaccinia virus/fisiologia , Animais , Linhagem Celular , DNA Viral , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , Fibroblastos/virologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase Quinases/genética , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fosforilação , Proteínas Proto-Oncogênicas c-jun/genética , Replicação ViralRESUMO
Environmental enrichment (EE) is an experimental animal model that enhances an animal's opportunity to interact with sensory, motor, and social stimuli, compared to standard laboratory conditions. A prominent benefit of EE is the reduction of stress-induced anxiety. The relationship between stress and the onset of anxiety-like behavior has been widely investigated in experimental research, showing a clear correlation with structural changes in the hippocampus and basolateral amygdala (BLA). However, the mechanisms by which EE exerts its protective roles in stress and anxiety remain unclear, and it is not known whether EE reduces the effects of acute stress on animal behavior shortly following the cessation of stress. We found that EE can prevent the emergence of anxiety-like symptoms in rats measured immediately after acute restraint stress (1 h) and this effect is not due to changes in systemic release of corticosterone. Rather, we found that stress promotes a rapid increase in the nuclear translocation of glucocorticoid receptor (GR) in the BLA, an effect prevented by previous EE exposure. Furthermore, we observed a reduction of ERK (a MAPK protein) and CREB activity in the BLA promoted by both EE and acute stress. Finally, we found that EE decreases the expression of the immediate-early gene EGR-1 in the BLA, indicating a possible reduction of neuronal activity in this region. Hyperactivity of BLA neurons has been reported to accompany anxiety-like behavior and changes in this process may be one of the mechanism by which EE exerts its protective effects against stress-induced anxiety.
Assuntos
Ansiedade/metabolismo , Complexo Nuclear Basolateral da Amígdala/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Meio Ambiente , Sistema de Sinalização das MAP Quinases/fisiologia , Receptores de Glucocorticoides/fisiologia , Estresse Psicológico/metabolismo , Animais , Ansiedade/genética , Ansiedade/prevenção & controle , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/genética , Genes Precoces/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Distribuição Aleatória , Ratos , Ratos Wistar , Estresse Psicológico/genéticaRESUMO
UNLABELLED: Venezuelan equine encephalitis virus (VEEV) is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing (RNA-Seq) of poly(A) and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response (UPR) was observed. The protein kinase RNA-like endoplasmic reticulum kinase (PERK) arm of the UPR was activated, as the expression of both activating transcription factor 4 (ATF4) and CHOP (DDIT3), critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 (EGR1) was induced in a PERK-dependent manner. EGR1(-/-) mouse embryonic fibroblasts (MEFs) demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE: Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 (EGR1) contributed to VEEV-induced cell death.
Assuntos
Apoptose , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Vírus da Encefalite Equina Venezuelana/fisiologia , Interações Hospedeiro-Patógeno , Resposta a Proteínas não Dobradas , Animais , Linhagem Celular , Proteína 1 de Resposta de Crescimento Precoce/genética , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos KnockoutRESUMO
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.