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The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death. 相似文献
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《Autophagy》2013,9(4):457-466
The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death. 相似文献
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Kedi Xu Yi Yang Ming Yan Jianan Zhan Xiao Fu Xiaoxiang Zheng 《Journal of lipid research》2010,51(9):2581-2590
Smooth muscle cells (SMC) make up most of the vascular system. In advanced atherosclerotic plaques, dying SMCs undergo a complex death mode. In the present study, we examined the activation of autophagy in SMCs overloaded with excess free cholesterol (FC) and investigated the possible role which autophagy plays during the FC-induced cell death. After incubation with excess FC, a robust expression of autophagic vacuoles (AV) was detected using both fluorescence microscopy and transmission electron microscopy (TEM). The results revealed that FC induced a time-dependent upregulation of microtubule-associated protein-1 light chain 3-II (LC3-II). Inhibition of autophagy by 3-methyladenine (3-MA) enhanced both cell apoptosis and necrosis, while on the contrary, rapamycin inhibited cell death following cholesterol application. Furthermore, the impact of the colocalization of fragmented mitochondria with AVs was observed after cholesterol treatment. Our results also revealed that the modulation of autophagy directly influenced the cellular organellar stress. In conclusion, our findings demonstrated that excess FC induced the activation of autophagy in SMCs as a cellular defense mechanism, possibly through the degradation of dysfunctional organelles such as mitochondria and endoplasmic reticulum. 相似文献
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Kyohei Takahashi Tomohito Shibata Tatsuya Oba Tetsuya Ishikawa Masahito Yoshikawa Ryosuke Tatsunami Kazuhiko Takahashi Yoshiko Tampo 《Life sciences》2009,84(7-8):211-217
AimsMenadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs).Main methodsBAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody.Key findingsIntracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells.SignificanceWe conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate. 相似文献
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The endoplasmic reticulum (ER) is the primary site for synthesis and folding of secreted and membrane-bound proteins. Proteins are translocated into ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to assist in proper folding. Properly folded proteins traffic from the ER to the Golgi apparatus; misfolded proteins are targeted to degradation. Unfolded protein response (UPR) is a highly regulated intracellular signaling pathway that prevents accumulation of misfolded proteins in the ER lumen. UPR provides an adaptive mechanism by which cells can augment protein folding and processing capacities of the ER. If protein misfolding is not resolved, the UPR triggers apoptotic cascades. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintain cellular homeostasis and determine cell fate under various pathophysiological conditions. Regulated Ca(2+) transfer from the ER to the mitochondria is important in maintaining control of prosurvival/prodeath pathways. We discuss the signaling/communication between the ER and mitochondria and focus on the role of the mitochondrial permeability transition pore in these complex processes. 相似文献
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Sirt3-dependent deacetylation of SOD2 plays a protective role against oxidative stress in oocytes from diabetic mice 总被引:1,自引:0,他引:1
Xiaohui Liu Liang Zhang Pan Wang Xiaoyan Li Danhong Qiu Ling Li 《Cell cycle (Georgetown, Tex.)》2017,16(13):1302-1308
Maternal diabetes has been demonstrated to adversely affect oocyte quality in mouse oocytes. However, the potential molecular mechanisms are poorly understood. Here, we established a type I diabetic mouse model and detected the increased reactive oxygen species (ROS) levels and decreased Sirt3 expression in oocytes from diabetic mice. Furthermore, we found that forced expression of Sirt3 in diabetic oocytes significantly attenuates such an excessive production of ROS. The acetylation status of lysine 68 of superoxide dismutase (SOD2K68) is dependent on Sirt3 in oocytes. In line with this, SOD2K68 acetylation levels were markedly increased in diabetic oocytes, and Sirt3 overexpression could effectively suppress this tendency. Importantly, the deacetylation-mimetic mutant SOD2K68R is capable of partly preventing the oxidative stress in oocytes from diabetic mice. In conclusion, our findings support a model where Sirt3 plays a protective role against oxidative stress in oocytes exposed to maternal diabetes through deacetylating SOD2K68. 相似文献
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Zhou Jing-chao Wang Jing-lin Ren Hao-zhen Shi Xiao-lei 《Journal of physiology and biochemistry》2022,78(1):9-17
Journal of Physiology and Biochemistry - As a highly evolutionarily conserved process, autophagy can be found in all types of eukaryotic cells. Such a constitutive process maintains cellular... 相似文献
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Macroautophagy is a vacuolar, self-digesting mechanism responsible for the removal of long-lived proteins and damaged organelles by the lysosome. The discovery of the ATG genes has provided key information about the formation of the autophagosome, and about the role of macroautophagy in allowing cells to survive during nutrient depletion and/or in the absence of growth factors. Two connected signaling pathways encompassing class-I phosphatidylinositol 3-kinase and (mammalian) target of rapamycin play a central role in controlling macroautophagy in response to starvation. However, a considerable body of literature reports that macroautophagy is also a cell death mechanism that can occur either in the absence of detectable signs of apoptosis (via autophagic cell death) or concomitantly with apoptosis. Macroautophagy is activated by signaling pathways that also control apoptosis. The aim of this review is to discuss the signaling pathways that control macroautophagy during cell survival and cell death. 相似文献
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Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-offunction analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis. 相似文献
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High ER stress in beta-cells stimulates intracellular degradation of misfolded insulin 总被引:5,自引:0,他引:5
Allen JR Nguyen LX Sargent KE Lipson KL Hackett A Urano F 《Biochemical and biophysical research communications》2004,324(1):166-170
Endoplasmic reticulum (ER) stress, which is caused by the accumulation of misfolded proteins in the ER, elicits an adaptive response, the unfolded protein response (UPR). One component of the UPR, the endoplasmic reticulum-associated protein degradation (ERAD) system, has an important function in the survival of ER stressed cells. Here, we show that HRD1, a component of the ERAD system, is upregulated in pancreatic islets of the Akita diabetes mouse model and enhances intracellular degradation of misfolded insulin. High ER stress in beta-cells stimulated mutant insulin degradation through HRD1 to protect beta-cells from ER stress and ensuing death. If HRD1 serves the same function in humans, it may serve as a target for therapeutic intervention in diabetes. 相似文献
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Oxidative stress is implicated in carcinogenesis, aging, and neurodegenerative diseases. The E3 ligase C terminus of Hsc-70 interacting protein (CHIP) has a protective role against various stresses by targeting damaged proteins for proteasomal degradation, and thus maintains protein quality control. However, the detailed mechanism by which CHIP protects cells from oxidative stress has not been demonstrated. Here, we show that depletion of CHIP led to elevated Endonuclease G (EndoG) levels and enhanced cell death upon oxidative stress. In contrast, CHIP overexpression reduced EndoG levels, and resulted in reduced or no oxidative stress-induced cell death in cancer cells and primary rat cortical neurons. Under normal conditions Hsp70 mediated the interaction between EndoG and CHIP, downregulating EndoG levels in a Hsp70/proteasome-dependent manner. However, under oxidative stress Hsp70 no longer interacted with EndoG, and the stabilized EndoG translocated to the nucleus and degraded chromosomal DNA. Our data suggest that regulation of the level of EndoG by CHIP in normal conditions may determine the sensitivity to cell death upon oxidative stress. Indeed, injection of H2O2 into the rat brain markedly increased cell death in aged mice compared with young mice, which correlated with elevated levels of EndoG and concurrent downregulation of CHIP in aged mice. Taken together, our findings demonstrate a novel protective mechanism of CHIP against oxidative stress through regulation of EndoG, and provide an opportunity to modulate oxidative stress-induced cell death in cancer and aging. 相似文献
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When grown at acidic pH, Escherichia coli cells secrete cadaverine, a polyamine known to inhibit porin-mediated outer membrane permeability. In order to understand the physiological significance of cadaverine excretion and the inhibition of porins, we isolated an OmpC mutant that showed resistance to spermine during growth and polyamine-resistant porin-mediated fluxes. Here, we show that the addition of exogenous cadaverine allows wild-type cells to survive a 30-min exposure to pH 3.6 better than cells expressing the cadaverine-insensitive OmpC porin. Competition experiments between strains expressing either wild-type or mutant OmpC showed that the lack of sensitivity of the porin to cadaverine confers a survival disadvantage to the mutant cells at reduced pH. On the basis of these results, we propose that the inhibition of porins by excreted cadaverine represents a novel mechanism that provides bacterial cells with the ability to survive acid stress. 相似文献
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Adam Prabata Koji Ikeda Elda Putri Rahardini Ken-Ichi Hirata Noriaki Emoto 《The Journal of biological chemistry》2021,297(5)
Obesity is a global health problem that is often related to cardiovascular and metabolic diseases. Chronic low-grade inflammation in white adipose tissue (WAT) is a hallmark of obesity. Previously, during a search for differentially expressed genes in WAT of obese mice, we identified glycoprotein nonmetastatic melanoma protein B (GPNMB), of which expression was robustly induced in pathologically expanded WAT. Here, we investigated the role of GPNMB in obesity-related metabolic disorders utilizing GPNMB-deficient mice. When fed a high-fat diet (HFD), GPNMB-deficient mice showed body weight and adiposity similar to those of wild-type (WT) mice. Nonetheless, insulin and glucose tolerance tests revealed significant obesity-related metabolic disorders in GPNMB-KO mice compared with WT mice fed with HFD. Chronic WAT inflammation was remarkably worsened in HFD-fed GPNMB-KO mice, accompanied by a striking increase in crown-like structures, typical hallmarks for diseased WAT. Macrophages isolated from GPNMB-KO mice were observed to produce more inflammatory cytokines than those of WT mice, a difference abolished by supplementation with recombinant soluble GPNMB extracellular domain. We demonstrated that GPNMB reduced the inflammatory capacity of macrophages by inhibiting NF-κB signaling largely through binding to CD44. Finally, we showed that macrophage depletion by addition of clodronate liposomes abolished the worsened WAT inflammation and abrogated the exacerbation of metabolic disorders in GPNMB-deficient mice fed on HFD. Our data reveal that GPNMB negatively regulates macrophage inflammatory capacities and ameliorates the WAT inflammation in obesity; therefore we conclude that GPNMB is a promising therapeutic target for the treatment of metabolic disorders associated with obesity. 相似文献
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Stover CM Luckett JC Echtenacher B Dupont A Figgitt SE Brown J Männel DN Schwaeble WJ 《Journal of immunology (Baltimore, Md. : 1950)》2008,180(5):3313-3318
Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5alpha. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5alpha. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis. 相似文献
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Liao X Sluimer JC Wang Y Subramanian M Brown K Pattison JS Robbins J Martinez J Tabas I 《Cell metabolism》2012,15(4):545-553
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Codogno P 《Journal de la Société de Biologie》2005,199(3):233-241
Macroautophagy hereafter referred to as autophagy is a major lysosomal catabolic pathway for macromolecules and organelles conserved in eukaryotic cells. The discovery of the molecular basis of autophagy has uncovered its importance during development, life extension and in pathologies such as cancer, certain forms of myopathies and neurodegenerative diseases. Autophagy is a cell survival mechanism during starvation that is controlled by amino acids. Starvation-induced autophagy is an anti-apoptotic mechanism. However autophagy is also an alternative to apoptosis through autophagic cell death. In many situations apoptosis and autophagy can both contribute to cell dismantlement. 相似文献