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1.
J Cell Sci ; 131(22)2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30333141

RESUMO

Cytokine receptors, such as tumor necrosis factor receptor I (TNFRI, also known as TNFRSF1A) and lymphotoxin ß receptor (LTßR), activate inflammatory nuclear factor (NF)-κB signaling upon stimulation. We have previously demonstrated that depletion of ESCRT components leads to endosomal accumulation of TNFRI and LTßR, and their ligand-independent signaling to NF-κB. Here, we studied whether other perturbations of the endolysosomal system could trigger intracellular accumulation and signaling of ligand-free LTßR. While depletion of the CORVET components had no effect, knockdown of Rab7a or HOPS components, or pharmacological inhibition of lysosomal degradation, caused endosomal accumulation of LTßR and increased its interaction with the TRAF2 and TRAF3 signaling adaptors. However, the NF-κB pathway was not activated under these conditions. We found that knockdown of Rab7a or HOPS components led to sequestration of LTßR in intraluminal vesicles of endosomes, thus precluding NF-κB signaling. This was in contrast to the LTßR localization on the outer endosomal membrane that was seen after ESCRT depletion and was permissive for signaling. We propose that the inflammatory response induced by intracellular accumulation of endocytosed cytokine receptors critically depends on the precise receptor topology within endosomal compartments.


Assuntos
Receptor beta de Linfotoxina/metabolismo , NF-kappa B/metabolismo , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Transporte Proteico , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
2.
Gut ; 67(4): 707-718, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28159836

RESUMO

BACKGROUND AND AIMS: c-Myc is highly expressed in pancreatic multipotent progenitor cells (MPC) and in pancreatic cancer. The transition from MPC to unipotent acinar progenitors is associated with c-Myc downregulation; a role for c-Myc in this process, and its possible relationship to a role in cancer, has not been established. DESIGN: Using coimmunoprecipitation assays, we demonstrate that c-Myc and Ptf1a interact. Using reverse transcriptase qPCR, western blot and immunofluorescence, we show the erosion of the acinar programme. To analyse the genomic distribution of c-Myc and Ptf1a and the global transcriptomic profile, we used ChIP-seq and RNA-seq, respectively; validation was performed with ChIP-qPCR and RT-qPCR. Lineage-tracing experiments were used to follow the effect of c-Myc overexpression in preacinar cells on acinar differentiation. RESULTS: c-Myc binds and represses the transcriptional activity of Ptf1a. c-Myc overexpression in preacinar cells leads to a massive erosion of differentiation. In adult Ela1-Myc mice: (1) c-Myc binds to Ptf1a, and Tcf3 is downregulated; (2) Ptf1a and c-Myc display partially overlapping chromatin occupancy but do not bind the same E-boxes; (3) at the proximal promoter of genes coding for digestive enzymes, we find reduced PTF1 binding and increased levels of repressive chromatin marks and PRC2 complex components. Lineage tracing of committed acinar precursors reveals that c-Myc overexpression does not restore multipotency but allows the persistence of a preacinar-like cell population. In addition, mutant KRas can lead to c-Myc overexpression and acinar dysregulation. CONCLUSIONS: c-Myc repression during development is crucial for the maturation of preacinar cells, and c-Myc overexpression can contribute to pancreatic carcinogenesis through the induction of a dedifferentiated state.


Assuntos
Células Acinares/metabolismo , Regulação para Baixo/genética , Homeostase , Pâncreas/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Diferenciação Celular , Modelos Animais de Doenças , Homeostase/genética , Camundongos , Fatores de Transcrição/genética
3.
Gut ; 64(6): 937-47, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25037190

RESUMO

OBJECTIVE: Pancreatic acinar cell maturation is dependent on the activity of the pancreas transcription factor 1 (PTF1) complex. Induction of pancreatitis leads to MAP kinase activation and transient suppression of the acinar differentiation programme. We investigated the role of MAP kinase-interacting kinase 1 (Mnk1) in mouse exocrine pancreas development and in the response to secretagogue-induced pancreatitis. DESIGN: Mnk1 expression was analysed using immunohistochemistry, RT-qPCR and western blotting. Ptf1a binding to Mnk1 was assessed by chromatin immunoprecipitation and qPCR. Acute pancreatitis was induced in wild type and Mnk1(-/-) mice by 7 h intraperitoneal injections of caerulein. In vitro amylase secretion and trypsinogen activation were assessed using freshly isolated acinar cells. In vivo secretion was quantified by secretin-stimulated MRI. RESULTS: Mnk1 is expressed at the highest levels in pancreatic acinar cells and is a direct PTF1 target. Mnk1 is activated upon induction of pancreatitis and is indispensable for eIF4E phosphorylation. The pancreas of Mnk1(-/-) mice is histologically normal. Digestive enzyme content is significantly increased and c-Myc and Ccnd1 levels are reduced in Mnk1(-/-) mice. Upon induction of acute pancreatitis, Mnk1(-/-) mice show impaired eIF4E phosphorylation, activation of c-Myc and downregulation of zymogen content. Acinar cells show defective relocalisation of digestive enzymes, polarity defects and impaired secretory response in vitro and in vivo. CONCLUSIONS: Mnk1 is a novel pancreatic acinar cell-specific stress response kinase that regulates digestive enzyme abundance and eIF4E phosphorylation. It is required for the physiological secretory response of acinar cells and for the homeostatic response to caerulein administration during acute pancreatitis.


Assuntos
Células Acinares/enzimologia , Pâncreas Exócrino/metabolismo , Pancreatite/metabolismo , Pancreatite/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Células Acinares/citologia , Amilases/metabolismo , Animais , Diferenciação Celular , Ceruletídeo , Colangiopancreatografia por Ressonância Magnética , Regulação para Baixo , Ativação Enzimática , Fator de Iniciação 4E em Eucariotos/metabolismo , Marcação de Genes , Resposta ao Choque Térmico/fisiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pancreatite/induzido quimicamente , Fosforilação , Fatores de Transcrição/metabolismo , Tripsinogênio/metabolismo
4.
Gut ; 63(4): 647-55, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23598351

RESUMO

OBJECTIVES: Nr5a2 participates in biliary acid metabolism and is a major regulator of the pancreatic exocrine programme. Single nucleotide polymorphisms in the vicinity of NR5A2 are associated with the risk of pancreatic ductal adenocarcinoma (PDAC). AIMS: To determine the role of Nr5a2 in pancreatic homeostasis, damage-induced regeneration and mutant KRas-driven pancreatic tumourigenesis. DESIGN: Nr5a2+/- and KRas(G12V);Ptf1a-Cre;Nr5a2+/- mice were used to investigate whether a full dose of Nr5a2 is required for normal pancreas development, recovery from caerulein-induced pancreatitis, and protection from tumour development. RESULTS: Adult Nr5a2+/- mice did not display histological abnormalities in the pancreas but showed a more severe acute pancreatitis, increased acino-ductal metaplasia and impaired recovery from damage. This was accompanied by increased myeloid cell infiltration and proinflammatory cytokine gene expression, and hyperactivation of nuclear factor κb and signal transducer and activator of transcription 3 signalling pathways. Induction of multiple episodes of acute pancreatitis was associated with more severe damage and delayed regeneration. Inactivation of one Nr5a2 allele selectively in pancreatic epithelial cells was sufficient to cause impaired recovery from pancreatitis. In comparison with Nr5a2+/+ mice, KRas(G12V);Ptf1a(Cre/+);Nr5a2+/- mice showed a non-statistically significant increase in the area affected by preneoplastic lesions. However, a single episode of acute pancreatitis cooperated with loss of one Nr5a2 allele to accelerate KRas(G12V)-driven development of preneoplastic lesions. CONCLUSIONS: A full Nr5a2 dose is required to restore pancreatic homeostasis upon damage and to suppress the KRas(G12V)-driven mouse pancreatic intraepithelial neoplasia progression, indicating that Nr5a2 is a novel pancreatic tumour suppressor. Nr5a2 could contribute to PDAC through a role in the recovery from pancreatitis-induced damage.


Assuntos
Neoplasias Pancreáticas/genética , Pancreatite/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores Citoplasmáticos e Nucleares/genética , Animais , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/fisiopatologia , Ceruletídeo/farmacologia , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , NF-kappa B/fisiologia , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/fisiopatologia , Pancreatite/induzido quimicamente , Pancreatite/fisiopatologia , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Fator de Transcrição STAT3/fisiologia , Transdução de Sinais/fisiologia
5.
Life Sci Alliance ; 5(7)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35354596

RESUMO

Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of proteins residing in endosomal membranes. Here, we show that mammalian ESCRT-I restricts the size of lysosomes and promotes degradation of proteins from lysosomal membranes, including MCOLN1, a Ca2+ channel protein. The altered lysosome morphology upon ESCRT-I depletion coincided with elevated expression of genes annotated to biogenesis of lysosomes due to prolonged activation of TFEB/TFE3 transcription factors. Lack of ESCRT-I also induced transcription of cholesterol biosynthesis genes, in response to inefficient delivery of cholesterol from endolysosomal compartments. Among factors that could possibly activate TFEB/TFE3 signaling upon ESCRT-I deficiency, we excluded lysosomal cholesterol accumulation and Ca2+-mediated dephosphorylation of TFEB/TFE3. However, we discovered that this activation occurs due to the inhibition of Rag GTPase-dependent mTORC1 pathway that specifically reduced phosphorylation of TFEB at S112. Constitutive activation of the Rag GTPase complex in cells lacking ESCRT-I restored S112 phosphorylation and prevented TFEB/TFE3 activation. Our results indicate that ESCRT-I deficiency evokes a homeostatic response to counteract lysosomal nutrient starvation, that is, improper supply of nutrients derived from lysosomal degradation.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Complexos Endossomais de Distribuição Requeridos para Transporte , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Lisossomos/metabolismo , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais
6.
Autophagy ; 16(12): 2303-2304, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33025853

RESUMO

BMP2K (BMP2 inducible kinase) is a serine-threonine kinase with high amino acid homology to a known endocytic regulator, AAK1, and thus has been suspected to act in endocytosis. In our recent study, we report that BMP2K kinase regulates erythroid maturation in a manner that could not be explained by its involvement in endocytosis. Instead, we discovered that in erythroid cells, its splicing variants (BMP2K-L and BMP2K-S) act in opposing ways to regulate autophagic degradation, an important event in erythroid maturation. We also found that both isoforms could interact with a mammalian counterpart of yeast Sec16, SEC16A, a regulator of COPII vesicle-dependent secretory trafficking. BMP2K-L and -S differentially affect SEC16A levels and distribution, as well as abundance of SEC31A at COPII assemblies (SEC31A load). The regulation of SEC31A load by BMP2K variants concerned assemblies positive for SEC24B, a SEC16A interactor implicated in macroautophagy/autophagy. Hence, we found an unusual mechanism of two splicing variants of a kinase playing opposing roles in autophagy, potentially via differential regulation of SEC16A-dependent COPII assembly. Thereby they constitute a regulatory system, that we call the BMP2K-L/S system, fine-tuning autophagy and modulating erythroid maturation.


Assuntos
Retículo Endoplasmático , Proteínas de Transporte Vesicular , Animais , Autofagia/genética , Células Eritroides , Complexo de Golgi
7.
Elife ; 92020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32795391

RESUMO

Intracellular transport undergoes remodeling upon cell differentiation, which involves cell type-specific regulators. Bone morphogenetic protein 2-inducible kinase (BMP2K) has been potentially implicated in endocytosis and cell differentiation but its molecular functions remained unknown. We discovered that its longer (L) and shorter (S) splicing variants regulate erythroid differentiation in a manner unexplainable by their involvement in AP-2 adaptor phosphorylation and endocytosis. However, both variants interact with SEC16A and could localize to the juxtanuclear secretory compartment. Variant-specific depletion approach showed that BMP2K isoforms constitute a BMP2K-L/S regulatory system that controls the distribution of SEC16A and SEC24B as well as SEC31A abundance at COPII assemblies. Finally, we found L to promote and S to restrict autophagic degradation and erythroid differentiation. Hence, we propose that BMP2K-L and BMP2K-S differentially regulate abundance and distribution of COPII assemblies as well as autophagy, possibly thereby fine-tuning erythroid differentiation.


Assuntos
Processamento Alternativo/genética , Autofagia/fisiologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Proteínas Serina-Treonina Quinases/genética , Animais , Diferenciação Celular/genética , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo
9.
Cytokine Growth Factor Rev ; 32: 63-73, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27461871

RESUMO

Signaling of plasma membrane receptors can be regulated by endocytosis at different levels, including receptor internalization, endocytic sorting towards degradation or recycling, and using endosomes as mobile signaling platforms. Increasing number of reports underscore the importance of endocytic mechanisms for signaling of cytokine receptors. In this short review we present both consistent and conflicting data regarding endocytosis and its role in signaling of receptors from the tumor necrosis factor receptor superfamily (TNFRSF) and those for interleukins (ILRs) and interferons (IFNRs). These receptors can be internalized through various endocytic routes and most of them are able to activate downstream pathways from endosomal compartments. Moreover, some of the cytokine receptors clearly require endocytosis for proper signal transduction. Still, the data describing internalization mechanisms and fate of cytokine receptors are often fragmentary and barely address the relation between their endocytosis and signaling. In the light of growing knowledge regarding different mechanisms of endocytosis, extending it to the regulation of cytokine receptor signaling may improve our understanding of the complex and pleiotropic functions of these molecules.


Assuntos
Subunidades Proteicas/metabolismo , Receptores de Citocinas/metabolismo , Endocitose , Humanos , Transdução de Sinais
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