RESUMEN
Cocaine abuse leads to neuroinflammation, which, in turn, contributes to the pathogenesis of neurodegeneration associated with advanced HIV-1 infection. Autophagy plays important roles in both innate and adaptive immune responses. However, the possible functional link between cocaine and autophagy has not been explored before. Herein, we demonstrate that cocaine exposure induced autophagy in both BV-2 and primary rat microglial cells as demonstrated by a dose- and time-dependent induction of autophagy-signature proteins such as BECN1/Beclin 1, ATG5, and MAP1LC3B. These findings were validated wherein cocaine treatment of BV-2 cells resulted in increased formation of puncta in cells expressing either endogenous MAP1LC3B or overexpressing GFP-MAP1LC3B. Specificity of cocaine-induced autophagy was confirmed by treating cells with inhibitors of autophagy (3-MA and wortmannin). Intriguingly, cocaine-mediated induction of autophagy involved upstream activation of 2 ER stress pathways (EIF2AK3- and ERN1-dependent), as evidenced by the ability of the ER stress inhibitor salubrinal to ameliorate cocaine-induced autophagy. In vivo validation of these findings demonstrated increased expression of BECN1, ATG5, and MAP1LC3B-II proteins in cocaine-treated mouse brains compared to untreated animals. Increased autophagy contributes to cocaine-mediated activation of microglia since pretreatment of cells with wortmannin resulted in decreased expression and release of inflammatory factors (TNF, IL1B, IL6, and CCL2) in microglial cells. Taken together, our findings suggest that cocaine exposure results in induction of autophagy that is closely linked with neuroinflammation. Targeting autophagic proteins could thus be considered as a therapeutic strategy for the treatment of cocaine-related neuroinflammation diseases.
Asunto(s)
Autofagia/efectos de los fármacos , Cocaína/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Microglía/metabolismo , Transducción de Señal/efectos de los fármacos , Adenina/análogos & derivados , Adenina/farmacología , Androstadienos/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Biomarcadores/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/ultraestructura , Modelos Biológicos , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , WortmaninaRESUMEN
Oogenesis is essential for female gamete production in mammals. The total number of ovarian follicles is determined early in life and production of ovarian oocytes is thought to stop during the lifetime. However, the molecular mechanisms underling oogenesis, particularly autophagy regulation in the ovary, remain largely unknown. Here, we reveal an important MYBL2-VDAC2-BECN1-BCL2L1 pathway linking autophagy suppression in the developing ovary. The transcription factors GATA1 and MYBL2 can bind to and activate the Vdac2 promoter. MYBL2 regulates the spatiotemporal expression of VDAC2 in the developing ovary. Strikingly, in the VDAC2 transgenic pigs (Sus scrofa/Ss), VDAC2 exerts its function by inhibiting autophagy in the ovary. In contrast, Vdac2 knockout promotes autophagy. Moreover, VDAC2-mediated autophagy suppression is dependent on its interactions with both BECN1 and BCL2L1 to stabilize the BECN1 and BCL2L1 complex, suggesting VDAC2 as an autophagy suppressor in the pathway. Our findings provide a functional connection among the VDAC2, MYBL2, the BECN1-BCL2L1 pathway and autophagy suppression in the developing ovary, which is implicated in improving female fecundity.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Mamíferos/metabolismo , Ovario/crecimiento & desarrollo , Transactivadores/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Proteína bcl-X/metabolismo , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Secuencia de Bases , Inmunoprecipitación de Cromatina , Análisis Mutacional de ADN , Ensayo de Cambio de Movilidad Electroforética , Femenino , Factor de Transcripción GATA1/metabolismo , Ratones , Datos de Secuencia Molecular , Ovario/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Sus scrofa , Canal Aniónico 2 Dependiente del Voltaje/deficiencia , Canal Aniónico 2 Dependiente del Voltaje/genéticaRESUMEN
BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a BECN1 knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in BECN1(-/-) cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Lípidos/química , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/metabolismo , Proteínas Reguladoras de la Apoptosis/deficiencia , Secuencia de Bases , Beclina-1 , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/deficiencia , Datos de Secuencia Molecular , Fagosomas/ultraestructuraRESUMEN
By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study selective macroautophagy in mammalian cells.
Asunto(s)
Autofagia/efectos de los fármacos , Betaína-Homocisteína S-Metiltransferasa/metabolismo , Glutatión Transferasa/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Endorribonucleasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Leupeptinas/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas/metabolismo , Proteína Sequestosoma-1 , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A1, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Toxinas Shiga/farmacología , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/citología , Escherichia coli , Humanos , Ratones Endogámicos C57BL , Factor de Transcripción CHOPRESUMEN
Bioactive sphingolipids including ceramides are involved in a variety of pathophysiological processes by regulating cell death and survival. The objective of the current study was to examine ceramide metabolism in preeclampsia, a serious disorder of pregnancy characterized by oxidative stress, and increased trophoblast cell death and autophagy. Maternal circulating and placental ceramide levels quantified by tandem mass spectrometry were elevated in pregnancies complicated by preeclampsia. Placental ceramides were elevated due to greater de novo synthesis via high serine palmitoyltransferase activity and reduced lysosomal breakdown via diminished ASAH1 expression caused by TGFB3-induced E2F4 transcriptional repression. SMPD1 activity was reduced; hence, sphingomyelin degradation by SMPD1 did not contribute to elevated ceramide levels in preeclampsia. Oxidative stress triggered similar changes in ceramide levels and acid hydrolase expression in villous explants and trophoblast cells. MALDI-imaging mass spectrometry localized the ceramide increases to the trophophoblast layers and syncytial knots of placentae from pregnancies complicated by preeclampsia. ASAH1 inhibition or ceramide treatment induced autophagy in human trophoblast cells via a shift of the BOK-MCL1 rheostat toward prodeath BOK. Pharmacological inhibition of ASAH1 activity in pregnant mice resulted in increased placental ceramide content, abnormal placentation, reduced fetal growth, and increased autophagy via a similar shift in the BOK-MCL1 system. Our results reveal that oxidative stress-induced reduction of lysosomal hydrolase activities in combination with elevated de novo synthesis leads to ceramide overload, resulting in increased trophoblast cell autophagy, and typifies preeclampsia as a sphingolipid storage disorder.
Asunto(s)
Autofagia/efectos de los fármacos , Ceramidas/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Placenta/metabolismo , Preeclampsia/metabolismo , Esfingolípidos/metabolismo , Animales , Autofagia/fisiología , Células Cultivadas , Femenino , Humanos , Ratones , Preeclampsia/tratamiento farmacológico , EmbarazoRESUMEN
ISG15 (ISG15 ubiquitin-like modifier), a ubiquitin-like protein, is one of the major type I IFN (interferon) effector systems. ISG15 can be conjugated to target proteins (ISGylation) via the stepwise action of E1, E2, and E3 enzymes. Conjugated ISG15 can be removed (deISGylated) from target proteins by USP18 (ubiquitin-specific peptidase 18). Here we investigated the role of deISGylation by USP18 in regulating autophagy and EGFR degradation in cells treated with type I IFNs. We show that type I IFN induced expression of ISG15 leads to ISGylation of BECN1 at Lys117, as well as Lys263, Lys265, and Lys266 which competes with Lys63 ubiquitination of BECN1. We demonstrate that ISGylation of BECN1 at Lys117, as well as Lys263, Lys265, and Lys266 serve an important role in negative regulation of intracellular processes including autophagy and EGFR degradation that are critically dependent upon the activity of class III PtdIns 3-kinase. Our studies provide fundamental new mechanistic insights into the innate immunity response implemented by type I IFNs.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/fisiología , Citocinas/metabolismo , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Ubiquitinas/metabolismo , Beclina-1 , Humanos , Inmunidad Innata , Transducción de Señal/fisiología , Ubiquitina/metabolismo , Ubiquitinación/fisiologíaRESUMEN
An active medicinal component of plant origin with an ability to overcome autophagy by inducing apoptosis should be considered a therapeutically active lead pharmacophore to control malignancies. In this report, we studied the effect of concentration-dependent 3-AWA (3-azido withaferin A) sensitization to androgen-independent prostate cancer (CaP) cells which resulted in a distinct switching of 2 interrelated conserved biological processes, i.e. autophagy and apoptosis. We have observed 3 distinct parameters which are hallmarks of autophagy in our studies. First, a subtoxic concentration of 3-AWA resulted in an autophagic phenotype with an elevation of autophagy markers in prostate cancer cells. This led to a massive accumulation of MAP1LC3B and EGFP-LC3B puncta coupled with gradual degradation of SQSTM1. Second, higher toxic concentrations of 3-AWA stimulated ER stress in CaP cells to turn on apoptosis within 12 h by elevating the expression of the proapoptotic protein PAWR, which in turn suppressed the autophagy-related proteins BCL2 and BECN1. This inhibition of BECN1 in CaP cells, leading to the disruption of the BCL2-BECN1 interaction by overexpressed PAWR has not been reported so far. Third, we provide evidence that pawr-KO MEFs exhibited abundant autophagy signs even at toxic concentrations of 3-AWA underscoring the relevance of PAWR in switching of autophagy to apoptosis. Last but not least, overexpression of EGFP-LC3B and DS-Red-BECN1 revealed a delayed apoptosis turnover at a higher concentration of 3-AWA in CaP cells. In summary, this study provides evidence that 3-AWA is a strong anticancer candidate to abrogate protective autophagy. It also enhanced chemosensitivity by sensitizing prostate cancer cells to apoptosis through induction of PAWR endorsing its therapeutic potential.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Witanólidos/farmacología , Autofagia/fisiología , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.
Asunto(s)
Autofagia , Avibirnavirus/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Membrana Celular/metabolismo , Pollos , Citosol/metabolismo , Células HEK293 , Humanos , Lisosomas/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/metabolismo , Fosforilación , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Protein quality control (proteostasis) depends on constant protein degradation and resynthesis, and is essential for proper homeostasis in systems from single cells to whole organisms. Cells possess several mechanisms and processes to maintain proteostasis. At one end of the spectrum, the heat shock proteins modulate protein folding and repair. At the other end, the proteasome and autophagy as well as other lysosome-dependent systems, function in the degradation of dysfunctional proteins. In this review, we examine how these systems interact to maintain proteostasis. Both the direct cellular data on heat shock control over autophagy and the time course of exercise-associated changes in humans support the model that heat shock response and autophagy are tightly linked. Studying the links between exercise stress and molecular control of proteostasis provides evidence that the heat shock response and autophagy coordinate and undergo sequential activation and downregulation, and that this is essential for proper proteostasis in eukaryotic systems.
Asunto(s)
Autofagia/fisiología , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/fisiología , Lisosomas/metabolismo , Redes y Vías Metabólicas/fisiología , Animales , Humanos , Transducción de Señal/fisiologíaRESUMEN
Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-XL is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-XL may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Compuestos de Bifenilo/química , Proteínas de la Membrana/metabolismo , Nitrofenoles/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/química , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Apoptosis , Beclina-1 , Línea Celular Tumoral , Fibroblastos/metabolismo , Citometría de Flujo , Células HCT116 , Humanos , Ratones , Ratones Noqueados , Microscopía Fluorescente , Fragmentos de Péptidos , Piperazinas/química , Unión Proteica , Proteínas Proto-OncogénicasRESUMEN
P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd(mdx) mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca(2+) influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome--autophagy--and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.
Asunto(s)
Autofagia , Proteínas HSP90 de Choque Térmico/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/farmacología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Canales de Calcio/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Proteínas HSP70 de Choque Térmico/metabolismo , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Músculo Esquelético/efectos de los fármacos , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Mioblastos/patología , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Paclitaxel is recommended as a first-line chemotherapeutic agent against ovarian cancer, but drug resistance becomes a major limitation of its success clinically. The key molecule or mechanism associated with paclitaxel resistance in ovarian cancer still remains unclear. Here, we showed that TXNDC17 screened from 356 differentially expressed proteins by LC-MS/MS label-free quantitative proteomics was more highly expressed in paclitaxel-resistant ovarian cancer cells and tissues, and the high expression of TXNDC17 was associated with poorer prognostic factors and exhibited shortened survival in 157 ovarian cancer patients. Moreover, paclitaxel exposure induced upregulation of TXNDC17 and BECN1 expression, increase of autophagosome formation, and autophagic flux that conferred cytoprotection for ovarian cancer cells from paclitaxel. TXNDC17 inhibition by siRNA or enforced overexpression by a pcDNA3.1(+)-TXNDC17 plasmid correspondingly decreased or increased the autophagy response and paclitaxel resistance. Additionally, the downregulation of BECN1 by siRNA attenuated the activation of autophagy and cytoprotection from paclitaxel induced by TXNDC17 overexpression in ovarian cancer cells. Thus, our findings suggest that TXNDC17, through participation of BECN1, induces autophagy and consequently results in paclitaxel resistance in ovarian cancer. TXNDC17 may be a potential predictor or target in ovarian cancer therapeutics.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Neoplasias Ováricas/metabolismo , Paclitaxel/farmacología , Tiorredoxinas/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/fisiología , Beclina-1 , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Macroautophagy, a catabolic process of cellular self-digestion, is an important tumor cell survival mechanism and a potential target in antineoplastic therapies. Recent discoveries have implicated autophagy in the cellular secretory process, but potential roles of autophagy-mediated secretion in modifying the tumor microenvironment are poorly understood. Furthermore, efforts to inhibit autophagy in clinical trials have been hampered by suboptimal methods to quantitatively measure tumor autophagy levels. Here, we leveraged the autophagy-based involvement in cellular secretion to identify shed proteins associated with autophagy levels in melanoma. The secretome of low-autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu in physiological 3-dimensional cell culture using quantitative proteomics. These comparisons identified candidate autophagy biomarkers IL1B (interleukin 1, ß), CXCL8 (chemokine (C-X-C motif) ligand 8), LIF (leukemia inhibitory factor), FAM3C (family with sequence similarity 3, member C), and DKK3 (dickkopf WNT signaling pathway inhibitor 3) with known roles in inflammation and tumorigenesis, and these proteins were subsequently shown to be elevated in supernatants of an independent panel of high-autophagy melanoma cell lines. Secretion levels of these proteins increased when low-autophagy melanoma cells were treated with the autophagy-inducing tat-BECN1 (Beclin 1) peptide and decreased when ATG7 (autophagy-related 7) was silenced in high-autophagy cells, thereby supporting a mechanistic link between these secreted proteins and autophagy. In addition, serum from metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from patients with low-autophagy tumors. These results suggest that autophagy-related secretion affects the tumor microenvironment and measurement of autophagy-associated secreted proteins in plasma and possibly in tumors can serve as surrogates for intracellular autophagy dynamics in tumor cells.
Asunto(s)
Autofagia , Melanoma/patología , Proteínas de Neoplasias/metabolismo , Proteína 7 Relacionada con la Autofagia , Biomarcadores de Tumor/sangre , Línea Celular Tumoral , Proliferación Celular , Medios de Cultivo , Silenciador del Gen , Humanos , Melanoma/sangre , Melanoma/ultraestructura , Metástasis de la Neoplasia , Proteínas de Neoplasias/sangre , ARN Interferente Pequeño/metabolismo , Esferoides Celulares/patología , Esferoides Celulares/ultraestructura , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.
Asunto(s)
Autofagia , Huesos/metabolismo , Osteoblastos/citología , Animales , Remodelación Ósea , Resorción Ósea , Línea Celular Tumoral , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Homeostasis , Ratones , Ratones Transgénicos , Microscopía Confocal , Subunidad p50 de NF-kappa B/metabolismo , Osteoclastos/metabolismo , Estrés Oxidativo , Ligando RANK/metabolismo , Ratas , Microtomografía por Rayos XRESUMEN
Autophagy has been implicated in the progression and chemoresistance of various cancers. In this study, we have shown that osteosarcoma Saos-2 cells lacking ATG4B, a cysteine proteinase that activates LC3B, are defective in autophagy and fail to form tumors in mouse models. By combining in silico docking with in vitro and cell-based assays, we identified small compounds that suppressed starvation-induced protein degradation, LC3B lipidation, and formation of autophagic vacuoles. NSC185058 effectively inhibited ATG4B activity in vitro and in cells while having no effect on MTOR and PtdIns3K activities. In addition, this ATG4B antagonist had a negative impact on the development of Saos-2 osteosarcoma tumors in vivo. We concluded that tumor suppression was due to a reduction in ATG4B activity, since we found autophagy suppressed within treated tumors and the compound had no effects on oncogenic protein kinases. Our findings demonstrate that ATG4B is a suitable anti-autophagy target and a promising therapeutic target to treat osteosarcoma.
Asunto(s)
Aminopiridinas/farmacología , Autofagia , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Regulación Neoplásica de la Expresión Génica , Osteosarcoma/metabolismo , Animales , Proteínas Relacionadas con la Autofagia , Dominio Catalítico , Línea Celular Tumoral , Simulación por Computador , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Lípidos/química , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Lipopolysaccharide (LPS)-induced activation of TLR4 (toll-like receptor 4) is followed by a subsequent overwhelming inflammatory response, a hallmark of the first phase of sepsis. Therefore, counteracting excessive innate immunity by autophagy is important to contribute to the termination of inflammation. However, the exact molecular details of this interplay are only poorly understood. Here, we show that PELI3/Pellino3 (pellino E3 ubiquitin protein ligase family member 3), which is an E3 ubiquitin ligase and scaffold protein in TLR4-signaling, is impacted by autophagy in macrophages (MΦ) after LPS stimulation. We noticed an attenuated mRNA expression of proinflammatory Il1b (interleukin 1, ß) in Peli3 knockdown murine MΦ in response to LPS treatment. The autophagy adaptor protein SQSTM1/p62 (sequestosome 1) emerged as a potential PELI3 binding partner in TLR4-signaling. siRNA targeting Sqstm1 and Atg7 (autophagy related 7), pharmacological inhibition of autophagy by wortmannin as well as blocking the lysosomal vacuolar-type H(+)-ATPase by bafilomycin A1 augmented PELI3 protein levels, while inhibition of the proteasome had no effect. Consistently, treatment to induce autophagy by MTOR (mechanistic target of rapamycin (serine/threonine kinase)) inhibition or starvation enhanced PELI3 degradation and reduced proinflammatory Il1b expression. PELI3 was found to be ubiquitinated upon LPS stimulation and point mutation of PELI3-lysine residue 316 (Lys316Arg) attenuated Torin2-dependent degradation of PELI3. Immunofluorescence analysis revealed that PELI3 colocalized with the typical autophagy markers MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ß) and LAMP2 (lysosomal-associated membrane protein 2). Our observations suggest that autophagy causes PELI3 degradation during TLR4-signaling, thereby impairing the hyperinflammatory phase during sepsis.
Asunto(s)
Autofagia , Interleucina-1beta/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Citocinas/metabolismo , Inmunidad Innata , Inflamación , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Ratones , Naftiridinas/metabolismo , Mutación Puntual , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Sepsis/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Ubiquitina/metabolismoRESUMEN
Earlier studies reported allelic deletion of the essential autophagy regulator BECN1 in breast cancers implicating BECN1 loss, and likely defective autophagy, in tumorigenesis. Recent studies have questioned the tumor suppressive role of autophagy, as autophagy-related gene (Atg) defects generally suppress tumorigenesis in well-characterized mouse tumor models. We now report that, while it delays or does not alter mammary tumorigenesis driven by Palb2 loss or ERBB2 and PyMT overexpression, monoallelic Becn1 loss promotes mammary tumor development in 2 specific contexts, namely following parity and in association with wingless-type MMTV integration site family, member 1 (WNT1) activation. Our studies demonstrate that Becn1 heterozygosity, which results in immature mammary epithelial cell expansion and aberrant TNFRSF11A/TNR11/RANK (tumor necrosis factor receptor superfamily, member 11a, NFKB activator) signaling, promotes mammary tumorigenesis in multiparous FVB/N mice and in cooperation with the progenitor cell-transforming WNT1 oncogene. Similar to our Becn1(+/-);MMTV-Wnt1 mouse model, low BECN1 expression and an activated WNT pathway gene signature correlate with the triple-negative subtype, TNFRSF11A axis activation and poor prognosis in human breast cancers. Our results suggest that BECN1 may have nonautophagy-related roles in mammary development, provide insight in the seemingly paradoxical roles of BECN1 in tumorigenesis, and constitute the basis for further studies on the pathophysiology and treatment of clinically aggressive triple negative breast cancers (TNBCs).
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Mamarias Animales/metabolismo , Proteína Wnt1/metabolismo , Alelos , Animales , Apoptosis , Autofagia , Beclina-1 , Neoplasias de la Mama/metabolismo , Proliferación Celular , Células Epiteliales/citología , Femenino , Regulación Neoplásica de la Expresión Génica , Heterocigoto , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Células Madre/citología , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
The hypoxia inducible transcription factor HIF1 activates autophagy, a general catabolic pathway involved in the maintenance of cellular homeostasis. Dysfunction in both autophagy and HIF1 has been implicated in an increasing number of human diseases, including inflammatory bowel disease (IBD), such as Crohn disease (CD). Adherent invasive E. coli (AIEC) colonize ileal mucosa of CD patients and strongly promote gastrointestinal inflammatory disorders by activation of HIF-dependent responses. Here, we aim to characterize the contribution of HIF1 in xenophagy, a specialized form of autophagy involved in the degradation of intracellular bacteria. Our results showed that endogenous HIF1A knockdown increased AIEC survival in intestinal epithelial cells. We demonstrate that the increase in survival rate correlates with a dramatic impairment of the autophagic flux at the autolysosomal maturation step. Furthermore, we show that AIEC remained within single-membrane LC3-II-positive vesicles and that they were unable to induce the phosphorylation of ULK1. These results suggested that, in the absence of HIF1A, AIEC were found within LC3-associated phagosomes. Using blocking antibodies against TLR5 and CEACAM6, the 2 well-known AIEC-bound receptors, we showed that downstream receptor signaling was necessary to mediate ULK1 phosphorylation. Finally, we provide evidence that HIF1 mediates CEACAM6 expression and that CEACAM6 is necessary to recruit ULK1 in a bacteria-containing signaling hub. Collectively, these results identify a new function for HIF1 in AIEC-dedicated xenophagy, and suggest that coactivation of autophagy and HIF1A expression may be a potential new therapy to resolve AIEC infection in CD patients.
Asunto(s)
Autofagia/fisiología , Células Epiteliales/microbiología , Infecciones por Escherichia coli/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Línea Celular , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/metabolismoRESUMEN
Impaired autophagy function and enhanced ARG2 (arginase 2)-MTOR (mechanistic target of rapamycin) crosstalk are implicated in vascular aging and atherosclerosis. We are interested in the role of ARG2 and the potential underlying mechanism(s) in modulation of endothelial autophagy. Using human nonsenescent "young" and replicative senescent endothelial cells as well as Apolipoprotein E-deficient (apoe(-/-)Arg2(+/+)) and Arg2-deficient apoe(-/-) (apoe(-/-)arg2(-/-)) mice fed a high-fat diet for 10 wk as the atherosclerotic animal model, we show here that overexpression of ARG2 in the young cells suppresses endothelial autophagy with concomitant enhanced expression of RICTOR, the essential component of the MTORC2 complex, leading to activation of the AKT-MTORC1-RPS6KB1/S6K1 (ribosomal protein S6 kinase, 70kDa, polypeptide 1) cascade and inhibition of PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit). Expression of an inactive ARG2 mutant (H160F) had the same effect. Moreover, silencing RPS6KB1 or expression of a constitutively active PRKAA prevented autophagy suppression by ARG2 or H160F. In senescent cells, enhanced ARG2-RICTOR-AKT-MTORC1-RPS6KB1 and decreased PRKAA signaling and autophagy were observed, which was reversed by silencing ARG2 but not by arginase inhibitors. In line with the above observations, genetic ablation of Arg2 in apoe(-/-) mice reduced RPS6KB1, enhanced PRKAA signaling and endothelial autophagy in aortas, which was associated with reduced atherosclerosis lesion formation. Taken together, the results demonstrate that ARG2 impairs endothelial autophagy independently of the L-arginine ureahydrolase activity through activation of RPS6KB1 and inhibition of PRKAA, which is implicated in atherogenesis.