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Glucose consumption is generally increased in tumor cells to support tumor growth. Interestingly, we report that glycogen accumulation is a key initiating oncogenic event during liver malignant transformation. We found that glucose-6-phosphatase (G6PC) catalyzing the last step of glycogenolysis is frequently downregulated to augment glucose storage in pre-malignant cells. Accumulated glycogen undergoes liquid-liquid phase separation, which results in the assembly of the Laforin-Mst1/2 complex and consequently sequesters Hippo kinases Mst1/2 in glycogen liquid droplets to relieve their inhibition on Yap. Moreover, G6PC or another glycogenolysis enzyme-liver glycogen phosphorylase (PYGL) deficiency in both human and mice results in glycogen storage disease along with liver enlargement and tumorigenesis in a Yap-dependent manner. Consistently, elimination of glycogen accumulation abrogates liver growth and cancer incidence, whereas increasing glycogen storage accelerates tumorigenesis. Thus, we concluded that cancer-initiating cells adapt a glycogen storing mode, which blocks Hippo signaling through glycogen phase separation to augment tumor incidence.
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Carcinogênese/metabolismo , Carcinogênese/patologia , Glicogênio/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glucose-6-Fosfatase/metabolismo , Glicogênio Fosforilase/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Via de Sinalização Hippo , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Transição de Fase , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina-Treonina Quinase 3/metabolismo , Proteínas de Sinalização YAP/metabolismoRESUMO
In the version of this article initially published, the institution name for affiliation 3 (Maryland Anderson Cancer Center) was incorrect. The correct institution is MD Anderson Cancer Center. The error has been corrected in the HTML and PDF versions of the article.
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This corrects the article DOI: 10.1038/ni.3748.
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An imbalance in the lineages of immunosuppressive regulatory T cells (Treg cells) and the inflammatory TH17 subset of helper T cells leads to the development of autoimmune and/or inflammatory disease. Here we found that TAZ, a coactivator of TEAD transcription factors of Hippo signaling, was expressed under TH17 cell-inducing conditions and was required for TH17 differentiation and TH17 cell-mediated inflammatory diseases. TAZ was a critical co-activator of the TH17-defining transcription factor RORγt. In addition, TAZ attenuated Treg cell development by decreasing acetylation of the Treg cell master regulator Foxp3 mediated by the histone acetyltransferase Tip60, which targeted Foxp3 for proteasomal degradation. In contrast, under Treg cell-skewing conditions, TEAD1 expression and sequestration of TAZ from the transcription factors RORγt and Foxp3 promoted Treg cell differentiation. Furthermore, deficiency in TAZ or overexpression of TEAD1 induced Treg cell differentiation, whereas expression of a transgene encoding TAZ or activation of TAZ directed TH17 cell differentiation. Our results demonstrate a pivotal role for TAZ in regulating the differentiation of Treg cells and TH17 cells.
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Proteínas Adaptadoras de Transdução de Sinal/imunologia , Diferenciação Celular/imunologia , Colite/imunologia , Citocinas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Artrite Reumatoide/imunologia , Estudos de Casos e Controles , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Células HEK293 , Células HeLa , Histona Acetiltransferases/metabolismo , Humanos , Immunoblotting , Lisina Acetiltransferase 5 , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Síndrome de Sjogren/imunologia , Proteínas Smad/imunologia , Proteínas Smad/metabolismo , Fatores de Transcrição de Domínio TEA , Transativadores/metabolismo , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2(D57N) mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.
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Fagócitos/imunologia , Fagócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Infecções Bacterianas/etiologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Atividade Bactericida do Sangue/imunologia , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , Fagócitos/microbiologia , Fagossomos/imunologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Proteína Quinase C-alfa/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Sepse/etiologia , Sepse/imunologia , Sepse/metabolismo , Serina-Treonina Quinase 3 , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Receptores Toll-Like/metabolismo , Ubiquitinação , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismoRESUMO
Akt activation is a hallmark of human cancers. Here, we report a critical mechanism for regulation of Akt activity by the splicing kinase SRPK1, a downstream Akt target for transducing growth signals to regulate splicing. Surprisingly, we find that SRPK1 has a tumor suppressor function because ablation of SRPK1 in mouse embryonic fibroblasts induces cell transformation. We link the phenotype to constitutive Akt activation from genome-wide phosphoproteomics analysis and discover that downregulated SRPK1 impairs the recruitment of the Akt phosphatase PHLPP1 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase) to Akt. Interestingly, SRPK1 overexpression is also tumorigenic because excess SRPK1 squelches PHLPP1. Thus, aberrant SRPK1 expression in either direction induces constitutive Akt activation, providing a mechanistic basis for previous observations that SRPK1 is downregulated in some cancer contexts and upregulated in others.
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Carcinogênese/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Adesão Celular , Células Cultivadas , Senescência Celular , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Ativação Enzimática , Feminino , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Nus , Transplante de Neoplasias , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Carga TumoralRESUMO
Drug delivery nanocarriers based on magnetic nanoparticles have attracted increasing attention due to their potential applications in magnetic resonance imaging, photodynamic therapy and targeted drug delivery. Herein, we have fabricated the multifunctional co-loaded magnetic nanocapsules (MNCPs) using a microemulsion process for enhancing targeted magnetic resonance imaging and in vivo photodynamic therapy. MNCPs were synthesized by co-loading Co@Mn magnetic nanoparticles and chlorin e6 into the matrix of an amphiphilic polymer, and further surface covalently coupled with target molecules. This work demonstrates that MNCPs have uniform sizes (dc: ~150â¯nm), favorable biocompatibility, long-term stability, excellent T2 relaxation values, and high drug loading efficiency. These advantages offer MNCPs successfully applied in targeted magnetic resonance imaging, real-time fluorescent labeling, and photodynamic therapy. The research results will contribute to rationally design novel nano-platform and provide a promising approach for further clinical integration of diagnosis and treatment in the near future.
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Materiais Biocompatíveis/farmacologia , Sistemas de Liberação de Medicamentos , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Materiais Biocompatíveis/química , Clorofilídeos , Cobalto/química , Cobalto/farmacologia , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Humanos , Manganês/química , Manganês/farmacologia , Fotoquimioterapia/métodos , Porfirinas/química , Porfirinas/farmacologiaRESUMO
The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. However, the underlying mechanisms are largely unknown. In the present study, we demonstrate that p38b is a major p38 MAPK involved in the regulation of oxidative stress tolerance in addition to p38a and p38c in Drosophila. We further show the importance of MK2 as a p38-activated downstream kinase in resistance to oxidative stresses. Furthermore, we identified the iron-sulfur cluster scaffold protein IscU as a new substrate of MK2 both in Drosophila cells and in mammalian cells. These results imply a new mechanistic connection between the p38 pathway and mitochondria iron-sulfur clusters.
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Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Aconitato Hidratase/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster , Complexo I de Transporte de Elétrons/metabolismo , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Proteínas Ferro-Enxofre/genética , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
The Hippo signaling pathway is an evolutionarily conserved signaling module that plays critical roles in liver size control and tumorigenesis. The Hippo pathway consists of a core kinase cascade in which the mammalian Ste20-like kinases (Mst1/2, orthologs of Drosophila Hippo) and their cofactor Salvador (Sav1) form a complex to phosphorylate and activate the large tumor suppressor (Lats1/2). Lats1/2 kinases in turn phosphorylate and inhibit the transcription co-activators, the Yes-associated protein (YAP) and the transcriptional co-activator with PDZ-binding motif (TAZ), two major downstream effectors of the Hippo pathway. Losses of the Hippo pathway components induce aberrant hepatomegaly and tumorigenesis, in which YAP coordinates regulation of cell proliferation and apoptosis and plays an essential role. This review summarizes the current findings of the regulation of Hippo signaling in liver regeneration and tumorigenesis, focusing on how the loss of tumor suppressor components of the Hippo pathway results in liver cancers and discussing the molecular mechanisms that regulate the expression and activation of its downstream effector YAP in liver tumorigenesis.
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Carcinogênese , Neoplasias Hepáticas/fisiopatologia , Regeneração Hepática/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/fisiologia , Via de Sinalização Hippo , Homeostase/fisiologia , Humanos , Neoplasias Hepáticas/patologia , Modelos Biológicos , Proteínas Nucleares/metabolismo , Tamanho do Órgão/fisiologia , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Ativação Transcricional/fisiologia , Proteínas de Sinalização YAPRESUMO
The signaling network of innate immunity in Drosophila is constructed by multiple evolutionarily conserved pathways, including the Toll- or Imd-regulated NF-κB and JNK pathways. The p38 MAPK pathway is evolutionarily conserved in stress responses, but its role in Drosophila host defense is not fully understood. Here we show that the p38 pathway also participates in Drosophila host defense. In comparison with wild-type flies, the sensitivity to microbial infection was slightly higher in the p38a mutant, significantly higher in the p38b mutant, but unchanged in the p38c mutant. The p38b;p38a double-mutant flies were hypersensitive to septic injury. The immunodeficiency of p38b;p38a mutant flies was also demonstrated by hindgut melanization and larvae stage lethality that were induced by microbes naturally presented in fly food. A canonical MAP3K-MKK cascade was found to mediate p38 activation in response to infection in flies. However, neither Toll nor Imd was required for microbe-induced p38 activation. We found that p38-activated heat-shock factor and suppressed JNK collectively contributed to host defense against infection. Together, our data demonstrate that the p38 pathway-mediated stress response contribute to Drosophila host defense against microbial infection.
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Bactérias/imunologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/imunologia , Fungos/imunologia , Imunidade/imunologia , Sistema de Sinalização das MAP Quinases , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/prevenção & controle , Meios de Cultura , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Ativação Enzimática , Alimentos , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Regulação da Expressão Gênica , Genes de Insetos/genética , Proteínas de Choque Térmico/metabolismo , Imunidade/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Melaninas/metabolismo , Mutação/genética , Sepse/enzimologia , Sepse/prevenção & controleRESUMO
The loss of contact inhibition is a key step during carcinogenesis. The Hippo-Yes-associated protein (Hippo/YAP) pathway is an important regulator of cell growth in a cell density-dependent manner. However, how Hippo signaling senses cell density in this context remains elusive. Here, we report that high cell density induced the phosphorylation of spectrin α chain, nonerythrocytic 1 (SPTAN1), a plasma membrane-stabilizing protein, to recruit NUMB endocytic adaptor protein isoforms 1 and 2 (NUMB1/2), which further sequestered microtubule affinity-regulating kinases (MARKs) in the plasma membrane and rendered them inaccessible for phosphorylation and inhibition of the Hippo kinases sterile 20-like kinases MST1 and MST2 (MST1/2). WW45 interaction with MST1/2 was thereby enhanced, resulting in the activation of Hippo signaling to block YAP activity for cell contact inhibition. Importantly, low cell density led to SPTAN1 dephosphorylation and NUMB cytoplasmic location, along with MST1/2 inhibition and, consequently, YAP activation. Moreover, double KO of NUMB and WW45 in the liver led to appreciable organ enlargement and rapid tumorigenesis. Interestingly, NUMB isoforms 3 and 4, which have a truncated phosphotyrosine-binding (PTB) domain and are thus unable to interact with phosphorylated SPTAN1 and activate MST1/2, were selectively upregulated in liver cancer, which correlated with YAP activation. We have thus revealed a SPTAN1/NUMB1/2 axis that acts as a cell density sensor to restrain cell growth and oncogenesis by coupling external cell-cell contact signals to intracellular Hippo signaling.
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Via de Sinalização Hippo , Proteínas Serina-Treonina Quinases , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Espectrina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Sinalização YAP , Fatores de Transcrição/metabolismo , Carcinogênese/genéticaRESUMO
Intestinal epithelial cells (IECs) compose the first barrier against microorganisms in the gastrointestinal tract. Although the NF-kappaB pathway in IECs was recently shown to be essential for epithelial integrity and intestinal immune homeostasis, the roles of other inflammatory signaling pathways in immune responses in IECs are still largely unknown. Here we show that p38alpha in IECs is critical for chemokine expression, subsequent immune cell recruitment into the intestinal mucosa, and clearance of the infected pathogen. Mice with p38alpha deletion in IECs suffer from a sustained bacterial burden after inoculation with Citrobacter rodentium. These animals are normal in epithelial integrity and immune cell function, but fail to recruit CD4(+) T cells into colonic mucosal lesions. The expression of chemokines in IECs is impaired, which appears to be responsible for the impaired T cell recruitment. Thus, p38alpha in IECs contributes to the host immune responses against enteric bacteria by the recruitment of immune cells.
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Colo/metabolismo , Imunidade nas Mucosas/imunologia , Mucosa Intestinal/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/fisiologia , Linfócitos T/imunologia , Animais , Biomarcadores/metabolismo , Western Blotting , Quimiocinas/metabolismo , Citrobacter rodentium/imunologia , Colo/citologia , Colo/microbiologia , Ensaio de Unidades Formadoras de Colônias , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Técnicas Imunoenzimáticas , Integrases/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL/microbiologia , Camundongos Knockout/microbiologia , NF-kappa B , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/microbiologiaRESUMO
Microtubules play a crucial role during the establishment and maintenance of cell polarity. In fission yeast cells, the microtubule plus-end tracking proteins (+TIPs) (including the CLIP-170 homologue Tip1) regulate microtubule dynamics and also transport polarity factors to the cell cortex. Here, we show that the E3 ubiquitin ligase Dma1 plays an unexpected role in controlling polarized growth through ubiquitinating Tip1. Dma1 colocalizes with Tip1 to cortical sites at cell ends, and is required for ubiquitination of Tip1. Although the absence of dma1+ does not cause apparent polar growth defects in vegetatively growing cells, Dma1-mediated Tip1 ubiquitination is required to restrain polar growth upon DNA replication stress. This mechanism is distinct from the previously recognized calcineurin-dependent inhibition of polarized growth. In this work, we establish a link between Dma1-mediated Tip1 ubiquitination and DNA replication or DNA damage checkpoint-dependent inhibition of polarized growth in fission yeast.
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Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Calcineurina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
TLR4 signaling plays key roles in the innate immune response to microbial infection. Innate immune cells encounter different mechanical cues in both health and disease to adapt their behaviors. However, the impact of mechanical sensing signals on TLR4 signal-mediated innate immune response remains unclear. Here we show that TLR4 signalling augments macrophage bactericidal activity through the mechanical sensor Piezo1. Bacterial infection or LPS stimulation triggers assembly of the complex of Piezo1 and TLR4 to remodel F-actin organization and augment phagocytosis, mitochondrion-phagosomal ROS production and bacterial clearance and genetic deficiency of Piezo1 results in abrogation of these responses. Mechanistically, LPS stimulates TLR4 to induce Piezo1-mediated calcium influx and consequently activates CaMKII-Mst1/2-Rac axis for pathogen ingestion and killing. Inhibition of CaMKII or knockout of either Mst1/2 or Rac1 results in reduced macrophage bactericidal activity, phenocopying the Piezo1 deficiency. Thus, we conclude that TLR4 drives the innate immune response via Piezo1 providing critical insight for understanding macrophage mechanophysiology and the host response.
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Infecções Bacterianas/imunologia , Imunidade Inata , Canais Iônicos/metabolismo , Macrófagos/imunologia , Fagossomos/metabolismo , Receptor 4 Toll-Like/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Infecções por Escherichia coli/imunologia , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Canais Iônicos/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fagocitose/imunologia , Fagossomos/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinase 3 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Mitochondrial adenine nucleotide translocase (ANT) is believed to be a component or a regulatory component of the mitochondrial permeability transition pore (mtPTP), which controls mitochondrial permeability transition during apoptosis. However, the role of ANT in apoptosis is still uncertain, because hepatocytes isolated from ANT knockout and wild-type mice are equally sensitive to TNF- and Fas-induced apoptosis. In a screen for genes required for tumor necrosis factor alpha (TNF-alpha)-induced apoptosis in MCF-7 human breast cancer cells using retrovirus insertion-mediated random mutagenesis, we discovered that the ANT3 gene is involved in TNF-alpha-induced cell death in MCF-7 cells. We further found that ANT3 is selectively required for TNF- and oxidative stress-induced cell death in MCF-7 cells, but it is dispensable for cell death induced by several other inducers. This data supplements previous data obtained from ANT knockout studies, indicating that ANT is involved in some apoptotic processes. We found that the resistance to TNF-alpha-induced apoptosis observed in ANT3 mutant (ANT3(mut)) cells is associated with a deficiency in the regulation of the mitochondrial membrane potential and cytochrome c release. It is not related to intracellular ATP levels or survival pathways, supporting a previous model in which ANT regulates mtPTP. Our study provides genetic evidence supporting a role of ANT in apoptosis and suggests that the involvement of ANT in cell death is cell type- and stimulus-dependent.
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Translocador 3 do Nucleotídeo Adenina/metabolismo , Apoptose/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Translocador 3 do Nucleotídeo Adenina/genética , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação/genética , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Fatty acids (FAs) are essential nutrients, but how they are transported into cells remains unclear. Here, we show that FAs trigger caveolae-dependent CD36 internalization, which in turn delivers FAs into adipocytes. During the process, binding of FAs to CD36 activates its downstream kinase LYN, which phosphorylates DHHC5, the palmitoyl acyltransferase of CD36, at Tyr91 and inactivates it. CD36 then gets depalmitoylated by APT1 and recruits another tyrosine kinase SYK to phosphorylate JNK and VAVs to initiate endocytic uptake of FAs. Blocking CD36 internalization by inhibiting APT1, LYN or SYK abolishes CD36-dependent FA uptake. Restricting CD36 at either palmitoylated or depalmitoylated state eliminates its FA uptake activity, indicating an essential role of dynamic palmitoylation of CD36. Furthermore, blocking endocytosis by targeting LYN or SYK inhibits CD36-dependent lipid droplet growth in adipocytes and high-fat-diet induced weight gain in mice. Our study has uncovered a dynamic palmitoylation-regulated endocytic pathway to take up FAs.
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Antígenos CD36/metabolismo , Endocitose/fisiologia , Ácidos Graxos/metabolismo , Lipoilação , Células 3T3-L1 , Aciltransferases/metabolismo , Adipócitos/metabolismo , Animais , Antígenos CD36/deficiência , Antígenos CD36/genética , Cavéolas/metabolismo , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Humanos , Gotículas Lipídicas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Obesidade/tratamento farmacológico , Fosforilação , Transdução de Sinais , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Aumento de Peso/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
The major role of Hippo signaling is to inhibit their downstream effectors YAP/TAZ for organ size control during development and regeneration (Nat Rev Drug Discov 13(1):63-79, 2014; Dev Cell 19(4):491-505, 2010; Cell 163(4):811-828, 2015). We and others have demonstrated that the genetic disruption of kinases Mst1 and Mst2 (Mst1/2), the core components of Hippo signaling, results in YAP activation and sustained liver growth, thereby leading to an eight- to tenfold increase in liver size within 3 months and occurrence of liver cancer within 5 months (Curr Biol 17(23):2054-2060, 2007; Cancer Cell 16(5):425-438, 2009; Cell 130(6):1120-1133, 2007; Cancer Cell 31(5):669-684 e667, 2017; Nat Commun 6:6239, 2015; Cell Rep 3(5):1663-1677, 2013). XMU-MP-1, an Mst1/2 inhibitor, is able to augment mouse liver and intestinal repair and regeneration in both acute and chronic injury mouse models (Sci Transl Med 8:352ra108, 2016).In addition, YAP-deficient mice show an impaired intestinal regenerative response after DSS treatment or gamma irradiation (Proc Natl Acad Sci U S A 108(49):E1312-1320, 2011; Nature 493(7430):106-110, 2013; Genes Dev 24(21):2383-2388, 2010; J Vis Exp (111), 2010). IBS008738, a TAZ activator, facilitates muscle repair after cardiotoxin-induced muscle injury (Mol Cell Biol. 2014;34(9):1607-21). Deletion of Salvador (Sav) in mouse hearts enhances cardiomyocyte regeneration with reduced fibrosis and recovery of pumping function after myocardial infarction (MI) or resection of mouse cardiac apex (Development 140(23):4683-4690, 2013; Sci Signal 8(375):ra41, 2015; Nature 550(7675):260-264, 2017). This chapter provides a detailed description of procedures and important considerations when performing the protocols for the respective assays used to determine the effects of Hippo signaling on tissue repair and regeneration.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Medicina Regenerativa , Transdução de Sinais , Acetaminofen/efeitos adversos , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatectomia , Via de Sinalização Hippo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , RegeneraçãoRESUMO
Reactive oxygen species (ROS) production in phagocytes is a major defense mechanism against pathogens. However, the cellular self-protective mechanism against such potential damage from oxidative stress remains unclear. Here we show that the kinases Mst1 and Mst2 (Mst1/2) sense ROS and maintain cellular redox balance by modulating the stability of antioxidant transcription factor Nrf2. Site-specific ROS release recruits Mst1/2 from the cytosol to the phagosomal or mitochondrial membrane, with ROS subsequently activating Mst1/2 to phosphorylate kelch like ECH associated protein 1 (Keap1) and prevent Keap1 polymerization, thereby blocking Nrf2 ubiquitination and degradation to protect cells against oxidative damage. Treatment with the antioxidant N-acetylcysteine disrupts ROS-induced interaction of Mst1/2 with phagosomes or mitochondria, and thereby diminishes the Mst-Nrf2 signal. Consistently, loss of Mst1/2 results in increased oxidative injury, phagocyte ageing and death. Thus, our results identify the Mst-Nrf2 axis as an important ROS-sensing and antioxidant mechanism during an antimicrobial response.
Assuntos
Macrófagos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Cultivadas , Senescência Celular , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/genética , Proteínas Serina-Treonina Quinases/genética , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinase 3 , Transdução de Sinais/genética , Células THP-1RESUMO
The external factors that modulate Hippo signaling remain elusive. Here, we report that FGF15 activates Hippo signaling to suppress bile acid metabolism, liver overgrowth, and tumorigenesis. FGF15 is induced by FXR in ileal enterocytes in response to increased amounts of intestinal bile. We found that circulating enterohepatic FGF15 stimulates hepatic receptor FGFR4 to recruit and phosphorylate NF2, which relieves the inhibitory effect of Raf on the Hippo kinases Mst1/2, thereby switching FGFR4's role from pro-oncogenic to anti-tumor signaling. The activated Mst1/2 subsequently phosphorylates and stabilizes SHP to downregulate the key bile acid-synthesis enzyme Cyp7a1 expression, thereby limiting bile acid synthesis. In contrast, Mst1/2 deficiency impairs bile acid metabolism and remarkably increases Cyp7a1 expression and bile acid production. Importantly, pharmacological depletion of intestinal bile abrogates Mst1/2-mutant-driven liver overgrowth and oncogenesis. Therefore, FGF15-Hippo signaling along the gut-liver axis acts as a sensor of bile acid availability to restrain liver size and tumorigenesis.
Assuntos
Ácidos e Sais Biliares/metabolismo , Carcinogênese/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Via de Sinalização Hippo , Humanos , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Camundongos Transgênicos , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The Hippo signaling pathway has been established as a key regulator of organ size control, tumor suppression, and tissue regeneration in multiple organisms. Recently, emerging evidence has indicated that Hippo signaling might play an important role in regulating the immune system in both Drosophila and mammals. In particular, patients bearing a loss-of-function mutation of MST1 are reported to have an autosomal recessive primary immunodeficiency syndrome. MST1/2 kinases, the mammalian orthologs of Drosophila Hippo, may activate the non-canonical Hippo signaling pathway via MOB1A/B and/or NDR1/2 or cross-talk with other essential signaling pathways to regulate both innate and adaptive immunity. In this review, we present and discuss recent findings of cellular mechanisms/functions of Hippo signaling in the innate immunity in Drosophila and in mammals, T cell immunity, as well as the implications of Hippo signaling for tumor immunity.