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1.
S Ghavami R H Cunnington S Gupta B Yeganeh K L Filomeno D H Freed S Chen T Klonisch A J Halayko E Ambrose R Singal I M C Dixon 《Cell death & disease》2015,6(3):e1696
Transforming growth factor-β1 (TGF-β1) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β1 may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β1-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β1 to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β1 promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β1 in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β1-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),1 diabetic cardiomyopathy2 and hypertension.3 Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β1.4 TGF-β1 signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.5, 6 Elevated TGF-β1 is observed in post-MI rat heart7 and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.8 The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.9, 10Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.11, 12, 13, 14 Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.15, 16 Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.17, 18, 19, 20 The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.11, 12, 13, 14, 15, 16, 17, 18, 21, 22Recent studies have demonstrated that TGF-β1 may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells15, 23, 24 but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.25 Moreover, it has recently been reported that autophagy can negatively15 and positively25, 26, 27 regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β1-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart. 相似文献
2.
Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod cell loss is the primary pathologic event.Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomes for degradation. Defective autophagy can contribute to the age-dependent accumulation of damaged proteins and organelles leading to altered cellular homeostasis and loss of function.1, 2, 3, 4, 5 The metabolic roles of autophagy can be classified into two types, basal and induced. In nutrient-rich conditions, autophagy is suppressed but still occurs at low levels (basal autophagy); however, when cells are subjected to stress (starvation, injury, hypoxia), autophagy is activated immediately (induced autophagy).6 Induced autophagy maintains the amino acid pool inside cells to adapt to starvation while constitutive autophagy has been shown to function as a cell-repair mechanism that is important for long-lived postmitotic cells.7, 8, 9, 10, 11 Defects in autophagy have been associated with neurodegenerative diseases,12, 13, 14, 15 diabetes,16, 17 lysosomal storage disease18 and the loss of vision.19 In addition to macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) have been described. Although little is known about microautophagy in mammalian cells, macroautophagy (hereafter autophagy) is a major pathway for bulk degradation of cytoplasmic components. CMA is a more selective pathway for degradation of cytosolic proteins that can compensate for the loss of macroautophagy.2, 20, 21, 22Inherited retinal degenerative diseases such as retinitis pigmentosa or Leber''s congenital amaurosis are characterized by premature and progressive death of rod and cone photoreceptor cells.23 These diseases are characterized by the loss of night vision due to the death of rods followed by the loss of cones leading to diminished visual acuity and a reduction in the quality of life for patients. Disease is typically associated with the production of harmful gene products that promote pathology by inhibiting critical pathways resulting in cell death.24, 25, 26 Strategies to prevent photoreceptor death during retinal degenerative disease such as gene replacement therapies or inhibition of cell death pathways have been undertaken with some success;27, 28, 29 however, effective treatments for these blinding disorders are lacking.Another strategy that could be used in conjunction with other therapies might be to enhance survival by stimulating autophagy. Augmenting autophagy would increase the supply of nutrients to stressed cells and accelerate removal of damaged proteins thereby prolonging cell survival beyond what would be possible by only preventing cell death. Although canonical22, 30, 31, 32, 33 and noncanonical autophagic mechanisms34 have been detected in the eye, our knowledge of basic autophagy functions in this organ is still limited. In order to understand how autophagy maintains retinal homeostasis and function, we undertook studies to examine the consequences of deleting the essential autophagy gene Atg5 in rod photoreceptors. 相似文献
3.
J Shi H Wang H Guan S Shi Y Li X Wu N Li C Yang X Bai W Cai F Yang X Wang L Su Z Zheng D Hu 《Cell death & disease》2016,7(3):e2133
Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by excessive hypercellularity and extracellular matrix (ECM) component deposition. Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Previous studies show that IL10 has potential therapeutic benefits in terms of preventing and reducing HS formation. However, no studies have examined IL10-mediated autophagy during the pathological process of HS formation. Here, we examined the effect of IL10 on starvation-induced autophagy and investigated the molecular mechanism underlying IL10-mediated inhibition of autophagy in HS-derived fibroblasts (HSFs) under starvation conditions. Immunostaining and PCR analysis revealed that a specific component of the IL10 receptor, IL10 alpha-chain (IL10Rα), is expressed in HSFs. Transmission electron microscopy and western blot analysis revealed that IL10 inhibited starvation-induced autophagy and induced the expression of p-AKT and p-STAT3 in HSFs in a dose-dependent manner. Blocking IL10R, p-AKT, p-mTOR, and p-STAT3 using specific inhibitors (IL10RB, , rapamycin, and cryptotanshinone, respectively) showed that IL10 inhibited autophagy via IL10Rα-mediated activation of STAT3 (the IL10R-STAT3 pathway) and by directly activating the AKT-mTOR pathway. Notably, these results suggest that IL10-mediated inhibition of autophagy is facilitated by the cross talk between STAT3, AKT, and mTOR; in other words, the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways. Finally, the results also indicate that mTOR-p70S6K is the molecule upon which these two pathways converge to induce IL10-mediated inhibition of autophagy in starved HSFs. In summary, the findings reported herein shed light on the molecular mechanism underlying IL10-mediated inhibition of autophagy and suggest that IL10 is a potential therapeutic agent for the treatment of HS.Autophagy is a degradative process in eukaryotic cells that removes or turns over bulk cytoplasmic constituents through the endosomal and lysosomal fusion system (i.e., autophagosomes). LY2940021, 2 Autophagy is induced by stressful conditions such as starvation and pathogenic invasion.2Hypertrophic scar (HS) is a major skin fibrotic disorder caused by hypercellularity and extracellular matrix (ECM) component deposition.3, 4, 5 HS formation is usually recognized as the consequence of disturbed tissue repair processes and/or disrupted homeostasis in the skin after traumatic injury: HS negatively impacts on patient appearance, skeletal muscle function, and quality of life in general.6, 7, 8, 9 About 40–70% of surgeries and over 91% of burn injuries result in HS.10 A key feature of HS is a metabolic disorder of collagen-based ECM proteins.11, 12, 13 Autophagy has an important role in homeostasis of tissue structure and function.2, 14, 15 Skin autophagic capability is associated with HS and with the pathogenesis of many human diseases.16, 17, 18, 19, 20, 21, 22, 23Existing studies suggest that cytokines are important regulators of the autophagic process in both immune and non-immune cells.24, 25, 26 Interleukin-10 (IL10), expressed by a variety of mammalian cell types, was first described as a cytokine-synthesis-inhibitory factor with immunosuppressive and anti-inflammatory functions.27, 28 IL10 has a pivotal role in wound healing29, 30 and is a promising therapeutic agent for scar improvement in both animal models and human cutaneous wounds.9, 31, 32Fibroblasts are one of the most important effector cells responsible for HS formation.12, 33, 34 Thus, we were prompted to elucidate the mechanisms underlying the interactions among IL10, autophagy, and HS formation, with the aim of providing a molecular foundation for the therapeutic efficacy IL10. We used HS tissue, HS-derived fibroblasts (HSFs), and starvation-induced autophagy in HSFs as our research platform.Here, we report that IL10 inhibited autophagy by interfering with IL10R-mediated activation of IL10R-STAT3, as well as by activating the AKT-mTOR pathway. In addition, cross talk among STAT3, AKT, and mTOR and between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways collaboratively regulated starvation-induced autophagy in HSFs. 相似文献
4.
F Pietrocola S Lachkar D P Enot M Niso-Santano J M Bravo-San Pedro V Sica V Izzo M C Maiuri F Madeo G Mari?o G Kroemer 《Cell death and differentiation》2015,22(3):509-516
Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors.Macroautophagy (herein referred to as ‘autophagy'') consist in the sequestration of cytoplasmic material in autophagosomes, followed by their fusion with lysosomes for the bulk degradation of autophagic cargo by lysosomal hydrolases.1 This phenomenon can be measured by following the redistribution of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) fusion proteins from a diffuse location to autophagosomes (that results in the formation of the so-called GFP-LC3 ‘puncta''), the diminution of the overall abundance of autophagic substrates (such as sequestosome-1, p62/SQSTM1), and the stereotyped activation of proautophagic signals (such as the inhibition of the mammalian target of rapamycin complex 1, mTORC1).2There is growing consensus that the induction of autophagy by nutritional, pharmacological or genetic interventions can reduce age-related pathologies (such as neurodegenerative diseases or type 2 diabetes) and/or extend longevity.3, 4, 5, 6 This applies to caloric restriction or intermediate fasting,7 continuous or intermittent medication of rapamycin,8, 9, 10 administration of the sirtuin 1-activator resveratrol,11, 12 external supply of the polyamine spermidine,13 or genetic ablation of p53.14 In all these cases, inhibition of autophagy by deleting or silencing relevant genes abolishes the extension of health span and/or lifespan.13, 14, 15, 16, 17 Moreover, direct induction of autophagy by transgenic expression of autophagy-relevant genes such as ATG5 in mice is sufficient to increase lifespan.18Recently, acetyltransferases have emerged as a potential target for the pharmaceutical induction of autophagy. Thus, depletion of the sole donor of acetyl groups, acetyl-coenzyme A (acetyl-CoA), is sufficient to reduce the acetylation of cytoplasmic and nuclear proteins coupled to the induction of autophagy.19, 20, 21, 22 Culture of mammalian cells in nutrient-free (NF) conditions or starvation of mice for 24 h reduced the intracellular nucleocytosolic concentrations of acetyl-CoA at the same time as autophagy was induced, and replenishment of acetyl-CoA by external sources (for instance, by providing a membrane-permeant precursor of α-ketoglutarate for anaplerotic reactions or by microinjection of acetyl-CoA) was sufficient to inhibit starvation-induced autophagy.19, 20, 21, 22 Beyond the inhibition of acetyltransferases by acetyl-CoA depletion, direct pharmacological inhibition of acetyltransferases might also contribute to the induction of autophagy. A close correlation between autophagy induction and deacetylation of cytoplasmic proteins was observed in a screen conceived to identify autophagy-stimulating polyphenols23 as well as in in vivo experiments designed to explore the health-improving effects of coffee.24 Spermidine turned out to be an efficient inhibitor of histone acetyltransferases in vitro13 and reduced the global protein acetylation levels in cultured cells.25, 26Driven by these premises, we investigated the hypothesis that several health-related compounds including anacardic acid, curcumin, garcinol and spermidine might induce autophagy by inhibition of acetyltranferases. Here we report results supporting this hypothesis. Moreover, we demonstrate that one particular acetyltransferase, EP300 (E1A-binding protein p300), negatively controls autophagy and that anacardic acid, curcumin, garcinol and spermidine may induce autophagy by directly inhibiting EP300. 相似文献
5.
6.
N S Coll A Smidler M Puigvert C Popa M Valls J L Dangl 《Cell death and differentiation》2014,21(9):1399-1408
Autophagy is a major nutrient recycling mechanism in plants. However, its functional connection with programmed cell death (PCD) is a topic of active debate and remains not well understood. Our previous studies established the plant metacaspase AtMC1 as a positive regulator of pathogen-triggered PCD. Here, we explored the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered PCD and aging. We observed that autophagy acts as a positive regulator of pathogen-triggered PCD in a parallel pathway to AtMC1. In addition, we unveiled an additional, pro-survival homeostatic function of AtMC1 in aging plants that acts in parallel to a similar pro-survival function of autophagy. This novel pro-survival role of AtMC1 may be functionally related to its prodomain-mediated aggregate localization and potential clearance, in agreement with recent findings using the single budding yeast metacaspase YCA1. We propose a unifying model whereby autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants, when these functions are not masked by the cumulative stresses of aging, and negatively regulating senescence in older plants.An emerging theme in cell death research is that cellular processes thought to be regulated by linear signaling pathways are, in fact, complex. Autophagy, initially considered merely a nutrient recycling mechanism necessary for cellular homeostasis, was recently shown to regulate cell death, mechanistically interacting with components that control apoptosis. Deficient autophagy can result in apoptosis1, 2, 3 and autophagy hyper-activation can also lead to programmed cell death (PCD).4 In addition, the pro-survival function of autophagy is mediated by apoptosis inhibition and apoptosis mediates autophagy, although this cross-regulation is not fully understood.5In plants, autophagy can also have both pro-survival and pro-death functions. Autophagy-deficient plants exhibit accelerated senescence,6, 7, 8 starvation-induced chlorosis,6, 7, 9 hypersensitivity to oxidative stress10 and endoplasmic reticulum stress.11 Further, autophagy-deficient plants cannot limit the spread of cell death after infection with tissue-destructive microbial infections.12, 13 The plant phytohormone salicylic acid (SA) mediates most of these phenotypes.8 Autophagy has an essential, pro-survival role in situations where there is an increasing load of damaged proteins and organelles that need to be eliminated, that is, during aging or stress. Autophagy has an opposing, pro-death role during developmentally regulated cell death14, 15 or during the pathogen-triggered hypersensitive response PCD (hereafter, HR) that occurs locally at the site of attempted pathogen attack.16, 17 The dual pro-death/pro-survival functions of plant autophagy remain a topic of active debate.Also under scrutiny are possible novel functions of caspases and caspase-like proteins as central regulators of pro-survival processes. Caspases were originally defined as executioners of PCD in animals, but increasing evidence indicates that several caspases have non-apoptotic regulatory roles in cellular differentiation, motility and in the mammalian immune system.18, 19, 20Yeast, protozoa and plants do not have canonical caspases, despite the occurrence of morphologically heterogeneous PCDs.21 More than a decade ago, distant caspase homologs termed metacaspases were identified in these organisms using structural homology searches.22 Metacaspases were classified into type I or type II metacaspases based on the presence or absence of an N-terminal prodomain, reminiscent of the classification in animals into initiator/inflammatory or executioner caspases, respectively. Despite the architectural analogy between caspases and metacaspases, differences in their structure, function, activation and mode of action exist.23, 24, 25Metacaspases mediate PCD in yeast,26, 27, 28, 29, 30, 31 leishmania,32, 33 trypanosoma34 and plants.24 We demonstrated that two type I metacaspases, AtMC1 and AtMC2, antagonistically regulate HR in Arabidopsis thaliana.35 Our work showed that AtMC1 is a positive regulator of HR and that this function is mediated by its catalytic activity and negatively regulated by the AtMC1 N-terminal prodomain. AtMC2 antagonizes AtMC1-mediated HR.Besides AtMC2, new examples of metacaspases with a pro-life/non-PCD role are emerging. Protozoan metacaspases are involved in cell cycle dynamics34, 36, 37, 38 and cell proliferation.39 The yeast metacaspase Yca1 alters cell cycle dynamics40 and interestingly, is required for clearance of insoluble protein aggregates, thus contributing to yeast fitness.41Here, we explore the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered HR and aging. Our data support a model wherein autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants and negatively regulating senescence in older plants. 相似文献
7.
8.
L Wang Y Jiang X Song C Guo F Zhu X Wang Q Wang Y Shi J Wang F Gao W Zhao Y H Chen L Zhang 《Cell death & disease》2016,7(1):e2055
Macrophage foam cells, a major component of the atherosclerotic lesion, have vital roles in the development of atherosclerosis. Lipoautophagy, a type of autophagy characterized by selective delivery of lipid droplet for lysosomal degradation, may impact atherosclerosis by regulating macrophage foam cell formation. Previously, we reported that programmed cell death 4 (PDCD4), a tumor suppressor, negatively regulated autophagy in tumor cells. However, its roles in macrophage lipoautophagy, foam cell formation and atherosclerosis remain to be established. Here we found that Pdcd4 deficiency clearly improved oxidized low-density lipoproteins-impaired autophagy efflux, promoted autophagy-mediated lipid breakdown in murine macrophages and thus prevented macrophage conversion into foam cells. Importantly, Pdcd4 deficiency in mice significantly upregulated macrophage autophagy in local plaques along with attenuated lipid accumulation and atherosclerotic lesions in high-fat-fed Apolipoprotein E knockout mice. Bone marrow transplantation experiment demonstrated that PDCD4-mediated autophagy in hematopoietic cells contributed to the development of atherosclerosis. These results indicate that endogenous PDCD4 promotes for macrophage foam cell formation and atherosclerosis development via inhibiting autophagy and provides new insights into atherogenesis, suggesting that promoting macrophage autophagy through downregulating PDCD4 expression may be beneficial for treating atherosclerosis.Atherosclerosis is a lipid dysfunction-derived chronic inflammatory process in large and medium arterial wall.1 Macrophage foam cell, as a major component in the lesion of atherosclerosis, has vital role in the development of atherosclerosis. In the initial step of atherosclerotic development, circulating monocytes migrate into arterial wall via dysfunctional endothelial cells and differentiate into macrophages.2, 3, 4 The infiltrated macrophages ingest and digest oxidized low-density lipoprotein (ox-LDL), and then transport lipid out of vascular wall.5 However, macrophage with overloaded lipids stored in the form of lipid droplets (LDs) will transform into foam cells. Macrophage foam cell formation could promote the development of atherosclerosis.6 Thus, decreasing the formation of macrophage foam cell would be an attractive strategy to reverse plaque lipid buildup.7The macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved and well-controlled cellular catabolic process. During the process, cytoplasmic components are sequestered in double-membrane vesicles (which is called autophagosome) and degraded by fusion with lysosomal compartments (autophagolysosome) for recycling application.8 The process of autophagy is regulated by several autophagy-related genes (ATGs) encoded proteins, such as ATG5, ATG6 (also known as BECN1), ATG8 (also known as microtubule-associated protein 1 light chain 3, LC3) and ATG12. ATG5 is involved in the early stage of autophagosome formation. ATG5 is conjugated with ATG12 and ATG16L to form ATG12–ATG5–ATG16L complex, which contributes to the elongation and closure of the autophagosomes in the generation of lipidated forms of LC3 family proteins.9 Lipoautophagy, a type of autophagy that selectively delivers LDs for lysosomal degradation,10 regulates lipid metabolism and is involved in the process of atherosclerosis.11, 12, 13, 14 In advanced atherosclerosis, macrophage autophagy becomes dysfunctional. However, the basic autophagy deficiency in macrophage by specific Atg5 knockout accelerates atherosclerotic plaques in high-fat-fed ldlr−/− mice via promoting oxidative stress, plaque necrosis12 or inflammasome hyperactivation.13 More interestingly, autophagy can enhance brokendown of lipid in LD, cholesterol efflux from macrophage foam cells and further inhibit atherogenisis.14 Stent-based delivery of everolimus (mTOR inhibitor) in atherosclerotic plaques of cholesterol-fed rabbits leads to a marked reduction of macrophages via autophagic cell death.15 Therefore, regulating the level of macrophage autophagy and macrophage conversion into foam cells would be a potential target for preventing the atherosclerotic plaques formation.16Programmed cell death 4 (PDCD4), an inhibitor of protein translation, inhibits translation initiation via binding to the translation initiation factor eIF4A or translation elongation by direct or indirectly binding to the coding region of specific RNAs.17, 18 Accumulated evidence has demonstrated PDCD4 as a tumor suppressor.19 PDCD4 can inhibit promotion and progression of tumors, such as lung cancer,20 hepatocellular carcinoma cells,21 colon cancer,22 ovarian cancer23 and glioma.24 In addition, it has been reported that PDCD4 is also involved in the development of inflammatory diseases.25, 26, 27, 28, 29, 30 For example, Pdcd4-deficient mice are resistant to experimental allergic encephalitis,25 LPS-induced endotoxin shock26 and type-1 diabetes.27 In addition, Pdcd4-deficient mice are sensitive to LPS/D-galactosamine-induced acute liver injury.28 Recently, we reported that Pdcd4 deficiency attenuated adipocyte foam cells, diet-induced obesity, obesity-associated inflammation and insulin resistance,29 and increased IL-10 expression by macrophages that partly involved in atherosclerosis in hyperlipidemic mice,30 suggesting that PDCD4 may be involved in the metabolism-related diseases. Furthermore, we found that PDCD4 negatively regulated autophagy by inhibiting ATG5 expression in tumor cells.31 However, its role in macrophage lipoautophagy and foam formation, and association with atherosclerosis remain to be investigated.In the present study, we found that Pdcd4 deficiency improved ox-LDL-impaired autophagy efflux in murine macrophage and subsequently attenuated macrophage conversion into foam cells in an autophagy-dependent manner and further attenuated the formation of atherosclerotic lesions in hyperlipidemia mice. These results indicate that PDCD4 is critical for macrophage foam cell formation in atherosclerosis development and provides new insights into atherogenesis, and potential therapeutic avenues to treat atherosclerosis-associated diseases. 相似文献
9.
J Liu X Bi T Chen Q Zhang S-X Wang J-J Chiu G-S Liu Y Zhang P Bu F Jiang 《Cell death & disease》2015,6(7):e1827
Disturbed cell autophagy is found in various cardiovascular disease conditions. Biomechanical stimuli induced by laminar blood flow have important protective actions against the development of various vascular diseases. However, the impacts and underlying mechanisms of shear stress on the autophagic process in vascular endothelial cells (ECs) are not entirely understood. Here we investigated the impacts of shear stress on autophagy in human vascular ECs. We found that shear stress induced by laminar flow, but not that by oscillatory or low-magnitude flow, promoted autophagy. Time-course analysis and flow cessation experiments confirmed that this effect was not a transient adaptive stress response but appeared to be a sustained physiological action. Flow had no effect on the mammalian target of rapamycin-ULK pathway, whereas it significantly upregulated Sirt1 expression. Inhibition of Sirt1 blunted shear stress-induced autophagy. Overexpression of wild-type Sirt1, but not the deacetylase-dead mutant, was sufficient to induce autophagy in ECs. Using both of gain- and loss-of-function experiments, we showed that Sirt1-dependent activation of FoxO1 was critical in mediating shear stress-induced autophagy. Shear stress also induced deacetylation of Atg5 and Atg7. Moreover, shear stress-induced Sirt1 expression and autophagy were redox dependent, whereas Sirt1 might act as a redox-sensitive transducer mediating reactive oxygen species-elicited autophagy. Functionally, we demonstrated that flow-conditioned cells are more resistant to oxidant-induced cell injury, and this cytoprotective effect was abolished after inhibition of autophagy. In summary, these results suggest that Sirt1-mediated autophagy in ECs may be a novel mechanism by which laminar flow produces its vascular-protective actions.Vascular endothelial cells (ECs) are fundamentally important in maintaining structural and functional homeostasis of blood vessels. Normal biological functions of ECs are highly sensitive to the biomechanical stimuli induced by blood flow, of which shear stress acting on the surface of EC has been recognized to be one of the most important vasoactive factors in EC.1, 2 A relatively high level of laminar shear stress is cytoprotective, whereas abnormal (low-magnitude or oscillatory) shear stress is a detrimental cellular stress to ECs.1 Transduction of the mechanical signals involves multiple messenger molecules and signaling proteins, which collectively regulate important endothelial functions, such as gene expression, proliferation, migration, morphogenesis, permeability, thrombogenicity, and inflammation.2Autophagy (also known as macroautophagy) is an evolutionarily conserved cellular stress response.3, 4 Autophagy is a cellular self-digestion process, which is responsible for degradation of misfolded proteins and damaged organelles. Autophagic process is mainly mediated by the formation of autophagosome, a double-membrane vacuole structure containing engulfed cellular components. This process requires expression of a group of key genes involved in autophagy, including LC3A, beclin-1, Atg5, Atg7, and Atg12, for example.3, 5 Autophagosomes fuse with lysosomes, forming autolysosomes, where the cellular components are degraded by various hydrolases in an acidified environment.4, 5 In ECs, an autophagic response can be initiated by different stress stimuli.6, 7, 8 It is noted that the cellular outcome following autophagy induction in ECs varies depending on the nature of stimuli and specific experimental settings.6, 7, 9, 10 Moreover, there is evidence showing that autophagy may also be involved in modulating other EC functions such as angiogenesis and cellular senescence.11, 12 Therefore, understanding the regulatory mechanisms of autophagy in ECs will be important for discovery of strategies to protect normal endothelial functions. Recently, Guo et al. provided some evidence indicating that the autophagic process in EC might be affected by shear stress.13 This argument, however, was only based on observations of changed expression levels of LC3 and beclin-1; further experimental evidence is needed to confirm such an effect of shear stress on autophagy. More importantly, the mechanisms underlying this phenomenon are not understood. Different signaling pathways may be involved in modulating autophagy in ECs.14, 15, 16 For example, inhibition of the mTOR (mammalian target of rapamycin) pathway by rapamycin-induced endothelial autophagy and prevented energy stress-triggered cell damage.16 There is also evidence indicating a potential role of Sirt1.14 Moreover, accumulating evidence has suggested that reactive oxygen species (ROS) are closely implicated in modulating autophagic responses via complex interactions with other autophagy-related factors.15 Despite of these results, the signaling mechanisms of shear stress-regulated autophagy in EC remain to be defined. Hence, here we aim to delineate the impacts and underlying mechanisms of shear stress on autophagy in human vascular ECs. 相似文献
10.
S Saez-Atienzar L Bonet-Ponce J R Blesa F J Romero M P Murphy J Jordan M F Galindo 《Cell death & disease》2014,5(8):e1368
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been associated with Parkinson''s disease, and its inhibition opens potential new therapeutic options. Among the drug inhibitors of both wild-type and mutant LRRK2 forms is the 2-arylmethyloxy-5-subtitutent-N-arylbenzamide GSK257815A. Using the well-established dopaminergic cell culture model SH-SY5Y, we have investigated the effects of GSK2578215A on crucial neurodegenerative features such as mitochondrial dynamics and autophagy. GSK2578215A induces mitochondrial fragmentation of an early step preceding autophagy. This increase in autophagosome results from inhibition of fusion rather than increases in synthesis. The observed effects were shared with LRRK2-IN-1, a well-described, structurally distinct kinase inhibitor compound or when knocking down LRRK2 expression using siRNA. Studies using the drug mitochondrial division inhibitor 1 indicated that translocation of the dynamin-related protein-1 has a relevant role in this process. In addition, autophagic inhibitors revealed the participation of autophagy as a cytoprotective response by removing damaged mitochondria. GSK2578215A induced oxidative stress as evidenced by the accumulation of 4-hydroxy-2-nonenal in SH-SY5Y cells. The mitochondrial-targeted reactive oxygen species scavenger MitoQ positioned these species as second messengers between mitochondrial morphologic alterations and autophagy. Altogether, our results demonstrated the relevance of LRRK2 in mitochondrial-activated pathways mediating in autophagy and cell fate, crucial features in neurodegenerative diseases.Nowadays, Parkinson''s disease (PD) constitutes the main motor disorder and the second neurodegenerative disease after Alzheimer''s disease. Etiology of PD remains unknown, but both environmental and genetic factors have been implicated. Among the genes associated with PD is the leucine-rich repeat kinase 2 (LRRK2, PARK8, OMIM 607060) encoding gene encoded by PARK8. Indeed, LRRK2 mutations have been described in a substantial number of idiopathic late-onset PD patients without a known family history of the disease.1, 2, 3The physiologic function remains unknown. It localizes in the cytosol as well as in specific membrane subdomains, including mitochondria, autophagosomes and autolysosomes,4 and interacts with a whole array of proteins, including both α- and β-tubulin,5, 6 tau,7
α-synuclein8 and F-actin.9 LRRK2 gene mutations, including the most common G2019S,3 are associated with increases in toxic putative kinase activity.1, 10 LRRK2 overexpression is toxic to cultured cells,11, 12 and LRRK2 loss did not cause neurodegenerative changes (for a review see Tong and Shen13). However, LRRK2 transgenic mice lack obvious PD-like behavioral phenotypes.14 LRRK2-associated PD patients show degeneration of dopaminergic neurons in the substantia nigra.15 Data from our own group and others have associated mitochondrial apoptotical pathways with PD,16, 17, 18 and, in this context, LRRK2 mutant-mediated toxicity could be due to mitochondria-dependent apoptosis.19 There is considerable evidence for impaired mitochondrial function and morphology in both early-onset, autosomal recessive inherited PD and late-onset sporadic PD.Mitochondrial dynamics include several mechanisms, such as fission, fusion and mitophagy.20, 21 Altered fission/fusion dynamics might be a common pathogenic pathway of neurodegenerative diseases. It is well documented that mitochondrial dynamics constitute a relevant issue in some experimental neurodegenerative models.20, 22, 23, 24, 25 Mitochondrial dynamics is tightly regulated by cellular pathways including those participated by the dynamin-related protein-1 (Drp1). Drp1 mostly locates in the cytoplasm, but is stimulated after fission stimuli to migrate to the mitochondria. Once there, Drp1 forms ring-like structures, which wrap around the scission site to constrict the mitochondrial membrane resulting in mitochondrial fission.26, 27 Interestingly, a functional interaction between PD-associated LRRK2 and members of the dynamin GTPase superfamily has been described.28Macroautophagy (hereafter referred to as autophagy) is an active cellular response, which functions in the intracellular degradation system of cellular debris such as damaged organelles. Whether autophagy promotes cell death or enhances survival is still controversial.29, 30 It requires the formation of autophagosomes where cellular content is to be degraded by the action of lysosomal enzymatic content. Autophagosome formation is regulated by an orderly action of >30 autophagy-related (Atg) proteins. Among them is the microtubule-associated protein 1A/1B-light chain 3 (LC3), a homolog of Apg8p, which is essential for autophagy in yeast and is associated with autophagosome membranes.31 Interestingly, these vesicles are mostly highly mobile in the cytoplasm.32 Wild-type and mutant LRRK2 expression has been related to autophagy.4, 33, 34, 35, 36 Reactive oxygen species (ROS) function as relevant second messengers after several stimuli, including mitochondrial disruption. Exacerbated ROS increases might result in overactivation of antioxidant systems and yield harmful oxidative stress. Among oxidative stress hallmarks is the accumulation of α,β-unsaturated hydroxyalkenal 4-hydroxy-2-nonenal (4-HNE), whose accumulation has been reported in PD post-mortem patient brains,37, 38 thus giving a significant relevance to ROS in the pathogenesis of PD.All these results indicate LRRK2 as a promising pharmacologic target in PD treatment.39 Several LRRK2 inhibitor drugs have been synthetized, such as the potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide (GSK2578215A). GSK2578215A exhibits biochemical IC50s of 10.9 nM against wild-type LRRK2, and possesses a high ratio of brain to plasma distribution.40 This study provides key insights into the mechanisms downstream of LRRK2 inhibition, and spreads light onto an underexplored, yet potentially tractable therapeutic target for treating LRRK2-associated PD. We demonstrate how inhibition of this kinase results in the activation of cellular death pathways such as the mitochondrial fission machinery, and how cells reply by activating a protective autophagic response. Our results show the presence of oxidative stress hallmarks, thus pointing to a key function for ROS, placed downstream of mitochondrial fission. 相似文献
11.
Y Liu L Zhao Y Ju W Li M Zhang Y Jiao J Zhang S Wang Y Wang M Zhao B Zhang Y Zhao 《Cell death & disease》2014,5(8):e1361
Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.Osteosarcoma, a common primary bone tumor in children and adolescents, is prone to early metastasis through blood.1 Treatment with a combination of surgery and aggressive adjuvant chemotherapy has improved the survival rate of osteosarcoma patients. The 5-year-survival rates of non-metastatic patients have reached a plateau of approximately 70%.2, 3 However, patients with poor responses to chemotherapeutics will undergo local recurrence and metastasis, which reduce the 5-year-survival rates to only 20% despite additional doses or drugs.4, 5 Drug resistance is responsible for the poor prognosis. Attenuating chemoresistance facilitates better treatment of osteosarcoma.6, 7 Novel treatment strategies that combine anticancer drugs with adjuvant agents could improve the antitumor effects.8, 9In the 1960s, macrophage migration inhibitory factor (MIF) was identified as a pluripotent protein that modulates inflammation.10 Increasing evidence suggests that inflammation is closely related to tumorigenesis.11 MIF plays a bridging role between inflammation and tumorigenesis.12, 13, 14 MIF triggers the activation of the MAPK and PI3K pathways by binding its membrane receptor CD74, resulting in the inhibition of cell apoptosis.15 Recently, MIF was demonstrated to be involved in cell proliferation, differentiation, angiogenesis and tumorigenesis.16, 17, 18 Some evidence has indicated that MIF is abundantly expressed in various cancers and is significantly associated with tumor invasion and metastasis.19, 20, 21 MIF has been well established to be involved in the development of glioblastoma,22 breast cancer,23 bladder cancer24 and colon cancer.20, 25 MIF was also upregulated in osteosarcoma.26, 27 The knockdown of MIF blocked osteosarcoma cell proliferation and invasion.26 However, the effect of MIF on drug resistance in osteosarcoma has not yet been investigated. Wu et al.
23 have revealed that MIF knockdown promoted chemosensitivity by inducing autophagy in breast cancer. In contrast, autophagy reportedly contributed to chemoresistance in osteosarcoma.6 These controversial results prompted us to confirm the role of MIF in drug resistance in osteosarcoma.In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. Both N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced reactive oxygen species (ROS)-mediated autophagy to rescue cell death. Furthermore, MIF inhibition by DSTD enhances chemosensitivity by downregulating HMGB1 in osteosarcoma cells. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma. 相似文献
12.
13.
A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.7-10,15,17,19,21,25-28,31,33,34,37,38,42 A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.7-10,15,17,19,21,25-28,31,33,34,37,38,42 The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.7-10,15,17,19,21,25-28,31,33,34,37,38,42 Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge. 相似文献
14.
Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, exhibits diverse beneficial properties, including antiviral activity. Autophagy is a cellular process that is involved in the degradation of long-lived proteins and damaged organelles. Recent evidence indicates that modulation of autophagy is a potential therapeutic strategy for various viral diseases. In the present study, we investigated the effect of EGCG on hepatitis B virus (HBV) replication and the possible involvement of autophagy in this process. Our results showed that HBV induced autophagosome formation, which was required for replication of itself. However, although EGCG efficiently inhibited HBV replication, it enhanced, but not inhibited, autophagosome formation in hepatoma cells. Further study showed that HBV induced an incomplete autophagy, while EGCG, similar to starvation, was able to induce a complete autophagic process, which appeared to be unfavorable for HBV replication. Furthermore, it was found that HBV induced an incomplete autophagy by impairing lysosomal acidification, while it lost this ability in the presence of EGCG. Taken together, these data demonstrated that EGCG treatment opposed HBV-induced incomplete autophagy via enhancing lysosomal acidification, which was unfavorable for HBV replication.Macroautophagy (hereafter autophagy) is a conserved cellular process through which cytoplasmic materials are sequestered into double-membrane vacuole called autophagosomes and destined for degradation through fusion with lysosomes.1, 2, 3 Accumulating evidence indicates that autophagy is involved in diverse pathophysiological processes, including cancer, neurodegenerative disorders, and cardiovascular diseases.4, 5, 6, 7 Recent studies show that autophagy has an important role in regulating the replication of many viruses, including dengue virus, coxsackievirus B3 virus (CVB3), hepatitis C virus (HCV), and influenza virus A.8, 9, 10, 11, 12 Several investigations also indicate that autophagy has an important role in hepatitis B virus (HBV) replication: autophagy is induced by HBV and is required for HBV replication; however, the underlying mechanisms remains still unclear.13, 14, 15, 16Green tea is the most commonly consumed beverage worldwide. In traditional Chinese medicine, green tea is considered to have beneficial properties for human health, including antitumorigenic, antioxidant, and anti-inflammatory activities.17, 18, 19 Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea and appears to be the primary active ingredient accounting for the latter''s biological effects. In recent years, EGCG is revealed to display inhibitory effect on diverse viruses, such as human immunodeficiency virus type-1, Epstein–Barr virus (EBV), and HCV.20, 21, 22, 23, 24, 25 Of interest, EGCG is also found to regulate autophagy formation, although it seems to be cell-type specific.26, 27, 28, 29, 30 Given the potential therapeutic effect of EGCG on viral infection and its role in autophagy regulation, we investigated the effect of EGCG on HBV replication and the possible involvement of autophagy in this process.Here we showed that HBV induced an incomplete autophagy that was required for HBV replication; however, a complete autophagic process induced by EGCG appeared to be unfavorable for HBV replication. Further study showed that HBV hampered the autophagic flux by impairing lysosomal acidification, which could be opposed by the treatment of EGCG. 相似文献
15.
16.
17.
A Trocoli P Bensadoun E Richard G Labrunie F Merhi A M Schl?fli D Brigger S Souquere G Pierron J-M Pasquet P Soubeyran J Reiffers E Ségal-Bendirdjian M P Tschan M Djavaheri-Mergny 《Cell death and differentiation》2014,21(12):1852-1861
The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34+ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.p62 or sequestosome 1 (p62/SQSTM1) is a scaffold protein, implicated in a variety of biological processes including those that control cell death, inflammation, and metabolism.1, 2 Through its multi-domain structure, p62/SQSTM1 interacts specifically with key signaling proteins, including atypical PKC family members, NF-κB, and mTOR to control cellular responses.3, 4, 5, 6, 7 p62/SQSTM1 functions also as a key mediator of autophagy. Through its interaction with LC3, an essential protein involved in autophagy, p62/SQSTM1 selectively directs ubiquitinated substrates to autophagosomes leading to their subsequent degradation in lysosomes.8, 9 At the molecular level, p62/SQSTM1 acts as a pro-tumoral molecule by ensuring efficient and selective activation of cell signaling axes involved in cell survival, proliferation, and metabolism (i.e., NF-κB, mTOR, and Nrf-2 pathways).3, 5, 6, 7, 10, 11, 12, 13 p62/SQSTM1 can also signal anti-tumoral responses either by inactivating the pro-oncogenic signaling through BCR-ABL14 and Wnt pathways15, 16 or by inducing the activation of caspase 8, a pro-death protein.17, 18 Interestingly, in response to stress, autophagy promotes the degradation of p62, thus limits the activation of p62-regulatory pathways that control tumorigenesis.10 In addition, p62/SQSTM1 controls pathways that modulate differentiation of normal and cancerous cells. For example, p62/SQSTM1 has been shown to antagonize basal ERK activity and adipocyte differentiation.19 In contrast, p62/SQSTM1 favors differentiation of osteoclasts,20 osteoblasts,21 neurons,22 megakaryocytes23 and macrophages.24 The role and regulation of p62/SQSTM1 during leukemia cell differentiation has been poorly documented.Acute myeloid leukemia (AML) is a hematological disease characterized by multiple deregulated pathways resulting in a blockade of myeloid precursors at different stages of maturation.25, 26 Acute promyelocyte leukemia (APL) is the M3 type of AML characterized by an arrest of the terminal differentiation of promyelocytes into granulocytes and frequently associated with the expression of the oncogenic PML-RAR alpha fusion gene.27, 28 All-trans retinoic acid (ATRA), a potent activator of cellular growth arrest, differentiation, and death of APL cells, has been shown to effectively promote complete clinical remission of APL when combined with chemotherapy.29, 30, 31 Despite the success of this treatment, some APL patients are refractory to ATRA treatment or relapse owing to the development of resistance to ATRA in leukemia cells.32, 33, 34Our previous results revealed that autophagy flux is activated during granulocyte differentiation of myeloid leukemia cell lines induced by ATRA.35 In the present study, we observed that p62/SQSTM1, an autophagic substrate, is markedly upregulated at both mRNA and protein levels during the granulocytic differentiation process. Here, we investigated the regulation and the function of p62/SQSTM1 during AML cells differentiation into neutrophils/granulocytes. 相似文献
18.
M Casanova I J Gonzalez C Sprissler H Zalila M Dacher L Basmaciyan G F Sp?th N Azas N Fasel 《Cell death & disease》2015,6(10):e1933
Metacaspases (MCAs) are cysteine peptidases expressed in plants, fungi and protozoa, with a caspase-like histidine–cysteine catalytic dyad, but differing from caspases, for example, in their substrate specificity. The role of MCAs is subject to debate: roles in cell cycle control, in cell death or even in cell survival have been suggested. In this study, using a Leishmania major MCA-deficient strain, we showed that L. major MCA (LmjMCA) not only had a role similar to caspases in cell death but also in autophagy and this through different domains. Upon cell death induction by miltefosine or H2O2, LmjMCA is processed, releasing the catalytic domain, which activated substrates via its catalytic dyad His/Cys and a proline-rich C-terminal domain. The C-terminal domain interacted with proteins, notably proteins involved in stress regulation, such as the MAP kinase LmaMPK7 or programmed cell death like the calpain-like cysteine peptidase. We also showed a new role of LmjMCA in autophagy, acting on or upstream of ATG8, involving Lmjmca gene overexpression and interaction of the C-terminal domain of LmjMCA with itself and other proteins. These results allowed us to propose two models, showing the role of LmjMCA in the cell death and also in the autophagy pathway, implicating different protein domains.Apoptosis is, in most cases, associated with and depends on the activation of cys-dependent peptidases, named caspases.1, 2 Once activated, initiator caspases induce a proteolytic cascade via the activation of effector caspases that ultimately cleave numerous substrates, thereby causing the typical morphological features of apoptosis.3, 4 Despite their essential role in apoptosis, caspases are also involved in non-apoptotic events, including inflammation, cell proliferation, cell differentiation5 and the cell survival process autophagy, a major catabolic process in eukaryotic cells that allows cells to survive nutrient starvation due to engulfment of a portion of the cytoplasm by a specific membrane, delivery to lysosomes or vacuoles and digestion by hydrolytic enzymes.6, 7, 8, 9, 10 Plants, fungi and protozoa are devoid of caspases but express metacaspases (MCAs).11MCAs are cysteine peptidases of the clan CD, family 14, with a caspase-like histidine–cysteine catalytic dyad.12, 13 However, besides their distant similarity to caspases,14 MCAs prefer arginine/lysine in the P1 position, whereas caspases prefer aspartic residues.15, 16 The role of MCAs in cell death is still enigmatic. For example, in the yeast Saccharomyces cerevisiae, YCA1 has a role in cell death,17, 18 whereas, although only partly dependent on its conserved catalytic cysteine, it also facilitates the removal of unfolded proteins, prolonging cellular life span.19 Similarly, some metacaspases have roles, outside of death, in stress acclimation pathways, as in Aspergillus fumigatus20 or in the unicellular planctonic organisms diatoms.21, 22 In Arabidopsis thaliana, AtMC1 is a positive regulator of cell death and a survival factor for aging plants,23 whereas AtMC2 negatively regulates cell death.24
Trypanosoma brucei TbMCA2, TbMCA3 and TbMCA5 and Leishmania major MCA are involved in cell cycle regulation.25, 26Leishmania are parasitic protozoa responsible for the neglected tropical disease leishmaniasis, transmitted to humans by the bite of the sand fly. In the insect, parasites proliferate as free-living flagellated forms called procyclic promastigotes within the midgut before differentiating into virulent metacyclic promastigotes and migrating to the proboscis.27, 28 In the mammalian host, promastigotes are taken up by macrophages and transform into amastigotes. Under a variety of stress stimuli, apoptosis-like morphological and biochemical features have been described in Leishmania, among which are cell shrinkage, chromatin condensation, DNA fragmentation or mitochondrial depolarization.29, 30, 31, 32, 33, 34, 35, 36, 37, 38 Despite the evidence of morphological and biochemical markers of cell death in dying Leishmania, very little is known about the cell death pathway and the implicated executioner proteins. Indeed, essential proteins involved in mammalian apoptosis, death receptors, small pro- and anti-apoptotic molecules and caspases, are apparently not encoded in the genome of Leishmania39 and the role of Leishmania MCA in cell death is still controversial, certain authors suggesting a role as a negative regulator of intracellular amastigote proliferation, instead of having a caspase-like role in the execution of cell death.40LmjMCA contains different domains: an N-terminal domain with a Mitochondrion Localization Signal (MLS),41 a caspase-like catalytic domain and a C-terminal proline-rich domain.41 On the basis of this domain structure, LmjMCA can be classified among the type I metacaspases,16 a subclass more generally defined in higher plants and characterized by the presence of an N-terminal prodomain and a short linker between the large and small subunits, as initiator caspases in metazoans.11 Upon induction of cell death by heat shock, H2O2 or drugs like miltefosine or curcumin, LmjMCA is processed and the catalytic domain is released,41 liberating the C-terminal domain. It was therefore interesting to investigate the functional roles of the different domains.In this report, we studied the role of L. major MCA (LmjMCA), using an MCA-deficient strain and overexpressing independently the catalytic and the C-terminal domains. The results confirmed that MCA was not essential to L. major survival. In contrast, LmjMCA processing, releasing its catalytic and C-terminal domains, induced cell death in L. major, whereas the overexpression of Lmjmca gene triggered autophagy after interaction of the C-terminal domain with itself and with other proteins, acting on or upstream of the autophagic protein ATG8. 相似文献
19.
Autophagy is a catabolic mechanism facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner. Autophagy flux is necessary for normal neuronal homeostasis and its dysfunction contributes to neuronal cell death in several neurodegenerative diseases. Elevated autophagy has been reported after spinal cord injury (SCI); however, its mechanism, cell type specificity and relationship to cell death are unknown. Using a rat model of contusive SCI, we observed accumulation of LC3-II-positive autophagosomes starting at posttrauma day 1. This was accompanied by a pronounced accumulation of autophagy substrate protein p62, indicating that early elevation of autophagy markers reflected disrupted autophagosome degradation. Levels of lysosomal protease cathepsin D and numbers of cathepsin-D-positive lysosomes were also decreased at this time, suggesting that lysosomal damage may contribute to the observed defect in autophagy flux. Normalization of p62 levels started by day 7 after SCI, and was associated with increased cathepsin D levels. At day 1 after SCI, accumulation of autophagosomes was pronounced in ventral horn motor neurons and dorsal column oligodendrocytes and microglia. In motor neurons, disruption of autophagy strongly correlated with evidence of endoplasmic reticulum (ER) stress. As autophagy is thought to protect against ER stress, its disruption after SCI could contribute to ER-stress-induced neuronal apoptosis. Consistently, motor neurons showing disrupted autophagy co-expressed ER-stress-associated initiator caspase 12 and cleaved executioner caspase 3. Together, these findings indicate that SCI causes lysosomal dysfunction that contributes to autophagy disruption and associated ER-stress-induced neuronal apoptosis.In the United States, spinal cord injury (SCI) has an annual incidence of 11 000 and prevalence of nearly 500 000. Neuronal cell death is an important contributor to SCI-induced neurological deficits. Many of the affected neurons do not die because of direct mechanical damage but rather show delayed cell death as a result of injury-induced biochemical changes (secondary injury).1, 2, 3, 4 Thus, blocking or attenuating secondary neuronal death may serve to limit posttraumatic disabilities.Macroautophagy (hereafter called autophagy) is a lysosome-dependent catabolic pathway degrading cytoplasmic proteins, protein aggregates and organelles.5, 6, 7 Autophagy is initiated by the formation of autophagosomes, double membrane vesicles containing cytoplasmic components that include potentially toxic protein aggregates and damaged organelles. Autophagosomes then fuse with lysosomes to allow degradation of their contents by lysosomal hydrolases.8, 9, 10, 11 This progress of cargo, from sequestration in autophagosomes, to their delivery and degradation in lysosomes, is termed autophagy flux. Autophagy flux is important for homeostasis in all cells but appears especially critical in terminally differentiated cells such as neurons.12, 13 It is also upregulated, and often plays a protective function, in response to cell injury.14, 15 For example, autophagy is activated in response to and can limit effects of homeostasis perturbation in the endoplasmic reticulum (ER stress).16, 17 Thus, autophagy plays an important neuroprotective function, while impaired autophagy flux has been implicated in neurodegenerative disorders such as Parkinson''s and Alzheimer''s diseases.18, 19, 20, 21Upregulation of autophagy markers has been observed after SCI,22, 23 but its mechanisms and function remain controversial, with both beneficial and detrimental roles proposed. Under certain circumstances, pathologically increased autophagy can contribute to cell death,21, 24 particularly when autophagy flux is blocked, for example, because of lysosomal defects. Defects in autophagy flux can also exacerbate ER stress and potentiate ER-stress-induced apoptosis.16, 17 ER stress has long been implicated as part of the secondary injury after central nervous system trauma,25, 26 but its mechanisms remain unknown.In the current study, we characterized the temporal distribution and cell-type specificity of autophagy following contusive SCI in a rat model. Our data demonstrate that autophagosome accumulation after SCI is not due to increased initiation of autophagy, but rather due to inhibition of autophagy flux. This likely reflects the disruption of lysosomal function after SCI. Pathological accumulation of autophagosomes is prominent in ventral horn (VH) motor neurons, where it is associated with signs of ER stress and related apoptosis. Together, our findings suggest that autophagy is disrupted after SCI and may exacerbate ER stress and neuronal cell death. 相似文献
20.
O Maximyuk V Khmyz C-J Lindskog V Vukojevi? T Ivanova I Bazov K F Hauser G Bakalkin O Krishtal 《Cell death & disease》2015,6(3):e1683
Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.1, 2, 3, 4 Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.16, 17, 22, 25 Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations30, 31 and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.17, 22 Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.22, 32 Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.25Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.27, 33 Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.27, 34 Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36 Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.37, 38 The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.39 The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury. 相似文献