RESUMO
Asymmetric cell division is a key step in cellular differentiation in multicellular organisms. In plants, asymmetric zygotic division produces the apical and basal cells. The mitogen-activated protein kinase (MPK) cascade in Arabidopsis acts in asymmetric divisions such as zygotic division and stomatal development, but whether the effect on cellular differentiation of this cascade is direct or indirect following asymmetric division is not clear. Here, we report the analysis of a rice mutant, globular embryo 4 (gle4). In two- and four-cell-stage embryos, asymmetric zygotic division and subsequent cell division patterns were indistinguishable between the wild type and gle4 mutants. However, marker gene expression and transcriptome analyses showed that specification of the basal region was compromised in gle4 We found that GLE4 encodes MPK6 and that GLE4/MPK6 is essential in cellular differentiation rather than in asymmetric zygotic division. Our findings provide a new insight into the role of MPK in plant development. We propose that the regulation of asymmetric zygotic division is separate from the regulation of cellular differentiation that leads to apical-basal polarity.
Assuntos
Divisão Celular Assimétrica/genética , Proteína Quinase 6 Ativada por Mitógeno/fisiologia , Oryza , Zigoto/citologia , Divisão Celular/genética , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteína Quinase 6 Ativada por Mitógeno/genética , Oryza/embriologia , Oryza/enzimologia , Oryza/genética , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/metabolismoRESUMO
BACKGROUND: Arsenic trioxide (ATO) is commonly used in the treatment of acute promyelocytic leukemia (APL), but does not benefit patients with solid tumors. When combined with other agents or radiation, ATO showed treatment benefits with manageable toxicity. Previously, we reported that metformin amplified the inhibitory effect of ATO on intrahepatic cholangiocarcinoma (ICC) cells more significantly than other agents. Here, we investigated the chemotherapeutic sensitization effect of metformin in ATO-based treatment in ICC in vitro and in vivo and explored the underlying mechanisms. METHODS: ICC cell lines (CCLP-1, RBE, and HCCC-9810) were treated with metformin and/or ATO; the anti-proliferation effect was evaluated by cell viability, cell apoptosis, cell cycle, and intracellular-reactive oxygen species (ROS) assays. The in vivo efficacy was determined in nude mice with CCLP-1 xenografts. The active status of AMPK/p38 MAPK and mTORC1 pathways was detected by western blot. In addition, an antibody array was used screening more than 200 molecules clustered in 12 cancer-related pathways in CCLP-1 cells treated with metformin and/or ATO. Methods of genetic modulation and pharmacology were further used to demonstrate the relationship of the molecule. Seventy-three tumor samples from ICC patients were used to detect the expression of ERK3 by immunohistochemistry. The correlation between ERK3 and the clinical information of ICC patients were further analyzed. RESULTS: Metformin and ATO synergistically inhibited proliferation of ICC cells by promoting cell apoptosis, inducing G0/G1 cell cycle arrest, and increasing intracellular ROS. Combined treatment with metformin and ATO efficiently reduced ICC growth in an ICC xenograft model. Mechanistically, the antibody array revealed that ERK3 exhibited the highest variation in CCLP-1 cells after treatment with metformin and ATO. Results of western blot confirm that metformin and ATO cooperated to inhibit mTORC1, activate AMP-activated protein kinase (AMPK), and upregulate ERK3. Metformin abrogated the activation of p38 MAPK induced by ATO, and this activity was partially dependent on AMPK activation. Inactivation of p38 MAPK by SB203580 or specific short interfering RNA (siRNA) promoted the inactivation of mTORC1 in ICC cells treated with metformin and ATO. Activation of p38 MAPK may be responsible for resistance to ATO in ICC. The relationship between p38 MAPK and ERK3 was not defined by our findings. Finally, AMPK is a newfound positive regulator of ERK3. Overexpression of EKR3 in ICC cells inhibited cell proliferation through inactivation of mTORC1. ERK3 expression is associated with a better prognosis in ICC patients. CONCLUSIONS: Metformin sensitizes arsenic trioxide to suppress intrahepatic cholangiocarcinoma via the regulation of AMPK/p38 MAPK-ERK3/mTORC1 pathways. ERK3 is a newfound potential prognostic predictor and a tumor suppressor in ICC.
Assuntos
Arsenicais/farmacologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Sinergismo Farmacológico , Metformina/farmacologia , Óxidos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/fisiologia , Animais , Trióxido de Arsênio , Arsenicais/uso terapêutico , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Camundongos , Camundongos Nus , Proteína Quinase 6 Ativada por Mitógeno/genética , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Proteína Quinase 6 Ativada por Mitógeno/fisiologia , Óxidos/uso terapêutico , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Plants can successfully improve their resistance to previously lethal salinity stress by a short exposure to low levels of salt stress, a process known as salt acclimation (SA). In spite of its fundamental significance in theoretical study and agricultural practice, the molecular mechanisms underlying plant SA remain elusive. In this study, we found that salt acclimated Arabidopsis young seedlings can survive subsequent 200 mM NaCl stress. RNA-seq was performed to analyze the genome-wide transcriptional response under SA conditions. Among 518 differentially expressed genes (DEGs) under SA, 366 up-regulated genes were enriched for cell wall biosynthesis, osmoregulation, oxidative stress, or transcription factors. Seven DEGs participate in the synthesis of lignin and 24 DEGs encode plant cell wall proteins, suggesting the importance of cell wall remodeling under SA. Furthermore, in comparison to non-acclimated salt stress, 228 of 245 DEGs were repressed by acclimated salt stress, including many genes related to ethylene biosynthesis and signaling pathway. In addition, MAPK6, a major component of the ethylene signaling pathway, was found to play a crucial role in SA. Our transcriptomic analysis has provided important insight on the roles of transcription factors, cell wall remodeling, and the ethylene biosynthesis and signaling pathways during SA in Arabidopsis.
Assuntos
Aclimatação/genética , Arabidopsis/genética , Parede Celular/metabolismo , Etilenos/metabolismo , Cloreto de Sódio/metabolismo , Transcriptoma , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Vias Biossintéticas/genética , Parede Celular/ultraestrutura , Perfilação da Expressão Gênica , Proteína Quinase 6 Ativada por Mitógeno/genética , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Proteína Quinase 6 Ativada por Mitógeno/fisiologia , Pressão Osmótica , Estresse Oxidativo , Tolerância ao Sal/genética , Transdução de SinaisRESUMO
Previous studies suggest that hippocampal CA1, CA3, and DG regions may have distinct roles in alcohol dependence. Extracellular signal-regulated kinases (ERKs) and Ca(2+)/calmodulin dependent protein kinase II (CaMKII) have been shown to contribute to the molecular mechanism underlying drug dependence and relapse, and there may be an interaction between the activation of ERKs and CaMKII. However, little is known regarding the mechanisms underlying the effects of alcohol exposure, withdrawal, and relapse, particularly with regard to the interaction between CaMKII and ERK1/2 signaling in hippocampal subregions. In the present study, rats were provided water containing 6% alcohol as their only drinking source. We found that alcohol exerted locomotor stimulant and anxiolytic effects on rats in open field behaviors. Following chronic alcohol exposure, phospho-ERK1/2 was significantly decreased in the DG. Alcohol withdrawal was associated with an increase of phospho-ERK1/2 in the CA1 and DG, while alcohol re-exposure induced a decrease of phospho-ERK1/2 in the CA1, CA3, and DG. The activation of CaMKII (Thr286) correlated with the effects of alcohol on phospho-ERK1/2. Our results indicate that region-specific activation CaMKII-ERK1/2 signaling in the hippocampal CA1 and DG may play an important role in alcohol dependence.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Região CA1 Hipocampal/enzimologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Etanol/administração & dosagem , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteína Quinase 6 Ativada por Mitógeno/fisiologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
Methyl jasmonate (MeJA) is an important signalling molecule that has been reported to be able to promote plant senescence. The cell death suppressor Bax inhibitor-1 (BI1) has been found to suppress stress factor-mediated cell death in yeast and Arabidopsis. However, the effect and the genetic mechanism of Arabidopsis thaliana BI1 (AtBI1) on leaf senescence remain unclear. It was found here that the AtBI1 mutant, atbi1-2 (a gene knock-out), showed accelerated progression of MeJA-induced leaf senescence, while the AtBI1 complementation lines displayed similar symptoms as the WT during the senescence process. In addition, over-expression of the AtBI1 gene delayed the onset of MeJA-induced leaf senescence. Further analyses showed that during the process of MeJA-induced senescence, the activity of MPK6, a mitogen-activated protein kinase (MAPK), increased in WT plants, whereas it was significantly suppressed in AtBI1-overexpressing plants. Under the MeJA treatment, cytosolic calcium ([Ca(2+)](cyt)) functioned upstream of MPK6 activation and the elevation of [Ca(2+)](cyt) was reduced in AtBI1-overexpressing leaves. These results suggested a role of AtBI1 over-expression in delaying MeJA-induced leaf senescence by suppressing the [Ca(2+)](cyt)-dependent activation of MPK6, thus providing a new insight into the function and mechanism of AtBI1 in plant senescence.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/genética , Proteína Quinase 6 Ativada por Mitógeno/genética , Folhas de Planta/fisiologia , Acetatos/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Cálcio/análise , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Morte Celular , Senescência Celular , Clorofila/metabolismo , Ciclopentanos/farmacologia , Ativação Enzimática/fisiologia , Técnicas de Inativação de Genes , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Proteína Quinase 6 Ativada por Mitógeno/fisiologia , Modelos Biológicos , Oxilipinas/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de TempoRESUMO
Cdc14 is a member of the dual-specificity phosphatase family, which is essential for faithful cell cycle progression in eukaryotic cells of different origin. The function of human Cdc14A (hCdc14A), however, has not been fully elucidated as only few physiological substrates have been identified. To gain insight into the biological role of Cdc14A, we performed a yeast two-hybrid screen designed to isolate substrates of this human phosphatase. Using this genetic approach, we here report the identification of Erk3, an atypical mitogen-activated protein kinase (MAPK), as a specific binding partner of hCdc14A. GST pull-down assays show that Erk3 interacts directly with hCdc14A in vitro via its unique C-terminal domain. Furthermore, biochemical analysis reveals that hCdc14A can remove cyclin-dependent kinase (Cdk)-mediated phosphorylation of Erk3 in vitro raising the possibility that Erk3 may be a potential substrate for hCdc14A in vivo. Consistent with a physiologically relevant cross-talk in vivo, we find that Cdc14A forms a stable complex with Erk3 in human cells independent of its intrinsic phosphatase activity but mediated by its regulatory C-terminal domain. We show that hCdc14A impacts the emerging signaling pathway between Erk3 and MK5, a MAPK-activated protein kinase. We document that hCdc14A upregulation leads to redistribution of the Erk3 substrate MK5 from the nucleus to the cytoplasm. In addition, we find that hCdc14A stabilizes complex formation between Erk3 and its binding partner cyclin D3, a D-type cyclin implicated in both cellular proliferation and differentiation. Collectively, our findings suggest an intimate functional relationship between the Cdc14A phosphatase and the Erk3 kinase in signaling pathways that regulate key cell-fate decisions in human cells.
Assuntos
Proteína Quinase 6 Ativada por Mitógeno/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 6 Ativada por Mitógeno/genética , Monoéster Fosfórico Hidrolases/genética , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases , Técnicas do Sistema de Duplo-HíbridoRESUMO
Signal transduction pathways in eukaryotic cells integrate diverse extracellular signals, and regulate complex biological responses such as growth, differentiation and death. One group of proline-directed Ser/Thr protein kinases, the mitogen-activated protein kinases (MAPKs), plays a central role in these signalling pathways. Much attention has focused in recent years on three subfamilies of MAPKs, the extracellular signal regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the p38 MAPKs. However, the ERK family is broader than the ERK1 and ERK2 proteins that have been the subject of most studies in this area. Here we overview the work on ERKs 3 to 8, emphasising where possible their biological activities as well as distinctive biochemical properties. It is clear from these studies that these additional ERKs show similarities to ERK1 and ERK2, but with some interesting differences that challenge the paradigm of the archetypical ERK1/2 MAPK pathway.