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Sample records for autophagy genes protect

  1. Determination of autophagy gene ATG16L1 polymorphism in human colorectal cancer

    DEFF Research Database (Denmark)

    Nicoli, Elena Raluca; Dumitrescu, Theodor; Uscatu, Constantin Daniel;

    2014-01-01

    Autophagy has emerged not only as an essential repair mechanism to degrade damaged organelles and proteins but also as a major player in protection of tumor cells from multiple stresses. It was shown that autophagy gene polymorphisms are correlated with development of chronic inflammatory lesions...... frequent AA genotype. Such correlation suggests a possible role of autophagy gene polymorphisms in the development of human colorectal cancer....

  2. The protective roles of autophagy in ischemic preconditioning

    Institute of Scientific and Technical Information of China (English)

    Wen-jun YAN; Hai-long DONG; Li-ze XIONG

    2013-01-01

    Autophagy,a process for the degradation of protein aggregates and dysfunctional organelles,is required for cellular homeostasis and cell survival in response to stress and is implicated in endogenous protection.Ischemic preconditioning is a brief and nonlethal episode of ischemia,confers protection against subsequent ischemia-repenfusion through the up-regulation of endogenous protective mechanisms.Emerging evidence shows that autophagy is associated with the protective effect of ischemic preconditioning.This review summarizes recent progress in research on the functions and regulations of the autophagy pathway in preconditioning-induced protection and cellular survival.

  3. Autophagy protects end plate chondrocytes from intermittent cyclic mechanical tension induced calcification.

    Science.gov (United States)

    Xu, Hong-guang; Yu, Yun-fei; Zheng, Quan; Zhang, Wei; Wang, Chuang-dong; Zhao, Xiao-yn; Tong, Wen-xue; Wang, Hong; Liu, Ping; Zhang, Xiao-ling

    2014-09-01

    Calcification of end plate chondrocytes is a major cause of intervertebral disc (IVD) degeneration. However, the underlying molecular mechanism of end plate chondrocyte calcification is still unclear. The aim of this study was to clarify whether autophagy in end plate chondrocytes could protect the calcification of end plate chondrocytes. Previous studies showed that intermittent cyclic mechanical tension (ICMT) contributes to the calcification of end plate chondrocytes in vitro. While autophagy serves as a cell survival mechanism, the relationship of autophagy and induced end plate chondrocyte calcification by mechanical tension in vitro is unknown. Thus, we investigated autophagy, the expression of the autophagy genes, Beclin-1 and LC3, and rat end plate chondrocyte calcification by ICMT. The viability of end plate chondrocytes was examined using the LIVE/DEAD viability/cytotoxicity kit. The reverse transcription-polymerase chain reaction and western blotting were used to detect the expression of Beclin-1; LC3; type I, II and X collagen; aggrecan; and Sox-9 genes. Immunofluorescent and fluorescent microscopy showed decreased autophagy in the 10- and 20-day groups loaded with ICMT. Additionally, Alizarin red and alkaline phosphatase staining detected the palpable calcification of end plate chondrocytes after ICMT treatment. We found that increased autophagy induced by short-term ICMT treatment was accompanied by an insignificant calcification of end plate chondrocytes. To the contrary, the suppressive autophagy inhibited by long-term ICMT was accompanied by a more significant calcification. The process of calcification induced by ICMT was partially resisted by increased autophagy activity induced by rapamycin, implicating that autophagy may prevent end plate chondrocyte calcification.

  4. Crosstalk of clock gene expression and autophagy in aging

    Science.gov (United States)

    Kalfalah, Faiza; Janke, Linda; Schiavi, Alfonso; Tigges, Julia; Ix, Alexander; Ventura, Natascia; Boege, Fritz; Reinke, Hans

    2016-01-01

    Autophagy and the circadian clock counteract tissue degeneration and support longevity in many organisms. Accumulating evidence indicates that aging compromises both the circadian clock and autophagy but the mechanisms involved are unknown. Here we show that the expression levels of transcriptional repressor components of the circadian oscillator, most prominently the human Period homologue PER2, are strongly reduced in primary dermal fibroblasts from aged humans, while raising the expression of PER2 in the same cells partially restores diminished autophagy levels. The link between clock gene expression and autophagy is corroborated by the finding that the circadian clock drives cell-autonomous, rhythmic autophagy levels in immortalized murine fibroblasts, and that siRNA-mediated downregulation of PER2 decreases autophagy levels while leaving core clock oscillations intact. Moreover, the Period homologue lin-42 regulates autophagy and life span in the nematode Caenorhabditis elegans, suggesting an evolutionarily conserved role for Period proteins in autophagy control and aging. Taken together, this study identifies circadian clock proteins as set-point regulators of autophagy and puts forward a model, in which age-related changes of clock gene expression promote declining autophagy levels. PMID:27574892

  5. Protective Effects of Gastrodin Against Autophagy-Mediated Astrocyte Death.

    Science.gov (United States)

    Wang, Xin-shang; Tian, Zhen; Zhang, Nan; Han, Jing; Guo, Hong-liang; Zhao, Ming-gao; Liu, Shui-bing

    2016-03-01

    Gastrodin is an active ingredient derived from the rhizome of Gastrodia elata. This compound is usually used to treat convulsive illness, dizziness, vertigo, and headache. This study aimed to investigate the effect of gastrodin on the autophagy of glial cells exposed to lipopolysaccharides (LPS, 1 µg/mL). Autophagy is a form of programmed cell death, although it also promotes cell survival. In cultured astrocytes, LPS exposure induced excessive autophagy and apoptosis, which were significantly prevented by the pretreatment cells with gastrodin (10 μM). The protective effects of gastrodin via autophagy inhibition were verified by the decreased levels of LC3-II, P62, and Beclin-1, which are classical markers for autophagy. Furthermore, gastrodin protected astrocytes from apoptosis through Bcl-2 and Bax signaling pathway. The treatment of astrocytes with rapamycin (500 nM), wortmannin (100 nM), and LY294002 (10 μM), which are inhibitors of mTOR and PI3K, respectively, eliminated the known effects of gastrodin on the inhibited Beclin-1 expression. Furthermore, gastrodin blocked the down-regulation of glutamine synthetase induced by LPS exposure in astrocytes. Our results suggest that gastrodin can be used as a preventive agent for the excessive autophagy induced by LPS. PMID:26643508

  6. Polymorphisms in autophagy genes and susceptibility to tuberculosis.

    NARCIS (Netherlands)

    Songane, M.; Kleinnijenhuis, J.; Alisjahbana, B.; Sahiratmadja, E.; Parwati, I.; Oosting, M.; Plantinga, T.S.; Joosten, L.A.B.; Netea, M.G.; Ottenhoff, T.H.; Vosse, E. van de; Crevel, R. van

    2012-01-01

    Recent data suggest that autophagy is important for intracellular killing of Mycobacterium tuberculosis, and polymorphisms in the autophagy gene IRGM have been linked with susceptibility to tuberculosis (TB) among African-Americans, and with TB caused by particular M. tuberculosis genotypes in Ghana

  7. Salvianolic acid B inhibits autophagy and protects starving cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    Xiao HAN; Jian-xun LIU; Xin-zhi LI

    2011-01-01

    Aim: To investigate the protective or lethal role of autophagy and the effects of Salvianolic acid B (Sal B) on autophagy in starving myocytes.Methods: Cardiac myocytes were incubated under starvation conditions (GD) for O, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sat B (50 μmol/L) in the presence or absence of chloro-quine (3 μmol/L) under GD 3 h, the amount of LC3-11, the abundance of LC3-positive fluorescent dots in cells, cell viability and cellular ATP levels were determined using immunoblotting, immunofluorescence microscopy, MTT assay and luminometer, respectively. More-over, electron microscopy (EM) and immunofluorescent duel labeling of LC3 and Caspase-8 were used to examine the characteristics of autophagy and apoptosis.Results: Immunoblot analysis showed that the amount of LC3-11 in starving cells increased in a time-dependent manner accompanied by increased LC3-positive fluorescence and decreased cell viability and ATP content. Sal B (50 μmol/L) inhibited the increase in LC3-11, reduced the abundance of LC3 immunofluorescence and intensity of Caspase-8 fluorescence, and enhanced cellular viability and ATP levels in myocytes under GD 3 h, regardless of whether chloroquine was present.Conclusion: Autophagy induced by starvation for 3 h led to cell injury. Sal B protected starving cells by blocking the early stage of autophagic flux and inhibiting apoptosis that occurred during autophagy.

  8. Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus*

    Science.gov (United States)

    Tsuboi, Koichiro; Nishitani, Mayo; Takakura, Atsushi; Imai, Yasuyuki; Komatsu, Masaaki; Kawashima, Hiroto

    2015-01-01

    Genome-wide association studies of inflammatory bowel diseases identified susceptible loci containing an autophagy-related gene. However, the role of autophagy in the colon, a major affected area in inflammatory bowel diseases, is not clear. Here, we show that colonic epithelial cell-specific autophagy-related gene 7 (Atg7) conditional knock-out (cKO) mice showed exacerbation of experimental colitis with more abundant bacterial invasion into the colonic epithelium. Quantitative PCR analysis revealed that cKO mice had abnormal microflora with an increase of some genera. Consistently, expression of antimicrobial or antiparasitic peptides such as angiogenin-4, Relmβ, intelectin-1, and intelectin-2 as well as that of their inducer cytokines was significantly reduced in the cKO mice. Furthermore, secretion of colonic mucins that function as a mucosal barrier against bacterial invasion was also significantly diminished in cKO mice. Taken together, our results indicate that autophagy in colonic epithelial cells protects against colitis by the maintenance of normal gut microflora and secretion of mucus. PMID:26149685

  9. Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus.

    Science.gov (United States)

    Tsuboi, Koichiro; Nishitani, Mayo; Takakura, Atsushi; Imai, Yasuyuki; Komatsu, Masaaki; Kawashima, Hiroto

    2015-08-14

    Genome-wide association studies of inflammatory bowel diseases identified susceptible loci containing an autophagy-related gene. However, the role of autophagy in the colon, a major affected area in inflammatory bowel diseases, is not clear. Here, we show that colonic epithelial cell-specific autophagy-related gene 7 (Atg7) conditional knock-out (cKO) mice showed exacerbation of experimental colitis with more abundant bacterial invasion into the colonic epithelium. Quantitative PCR analysis revealed that cKO mice had abnormal microflora with an increase of some genera. Consistently, expression of antimicrobial or antiparasitic peptides such as angiogenin-4, Relmβ, intelectin-1, and intelectin-2 as well as that of their inducer cytokines was significantly reduced in the cKO mice. Furthermore, secretion of colonic mucins that function as a mucosal barrier against bacterial invasion was also significantly diminished in cKO mice. Taken together, our results indicate that autophagy in colonic epithelial cells protects against colitis by the maintenance of normal gut microflora and secretion of mucus.

  10. Polymorphisms in autophagy genes and susceptibility to tuberculosis.

    Directory of Open Access Journals (Sweden)

    Mario Songane

    Full Text Available Recent data suggest that autophagy is important for intracellular killing of Mycobacterium tuberculosis, and polymorphisms in the autophagy gene IRGM have been linked with susceptibility to tuberculosis (TB among African-Americans, and with TB caused by particular M. tuberculosis genotypes in Ghana. We compared 22 polymorphisms of 14 autophagy genes between 1022 Indonesian TB patients and 952 matched controls, and between patients infected with different M. tuberculosis genotypes, as determined by spoligotyping. The same autophagy polymorphisms were studied in correlation with ex-vivo production of TNF, IL-1β, IL-6, IL-8, IFN-γ and IL-17 in healthy volunteers. No association was found between TB and polymorphisms in the genes ATG10, ATG16L2, ATG2B, ATG5, ATG9B, IRGM, LAMP1, LAMP3, P2RX7, WIPI1, MTOR and ATG4C. Associations were found between polymorphisms in LAMP1 (p = 0.02 and MTOR (p = 0.02 and infection with the successful M. tuberculosis Beijing genotype. The polymorphisms examined were not associated with M. tuberculosis induced cytokines, except for a polymorphism in ATG10, which was linked with IL-8 production (p = 0.04. All associations found lost statistical significance after correction for multiple testing. This first examination of a broad set of polymorphisms in autophagy genes fails to show a clear association with TB, with M. tuberculosis Beijing genotype infection or with ex-vivo pro-inflammatory cytokine production.

  11. Gene expression profiles of autophagy-related genes in multiple sclerosis.

    Science.gov (United States)

    Igci, Mehri; Baysan, Mehmet; Yigiter, Remzi; Ulasli, Mustafa; Geyik, Sirma; Bayraktar, Recep; Bozgeyik, İbrahim; Bozgeyik, Esra; Bayram, Ali; Cakmak, Ecir Ali

    2016-08-15

    Multiple sclerosis (MS) is an imflammatory disease of central nervous system caused by genetic and environmental factors that remain largely unknown. Autophagy is the process of degradation and recycling of damaged cytoplasmic organelles, macromolecular aggregates, and long-lived proteins. Malfunction of autophagy contributes to the pathogenesis of neurological diseases, and autophagy genes may modulate the T cell survival. We aimed to examine the expression levels of autophagy-related genes. The blood samples of 95 unrelated patients (aged 17-65years, 37 male, 58 female) diagnosed as MS and 95 healthy controls were used to extract the RNA samples. After conversion to single stranded cDNA using polyT priming: the targeted genes were pre-amplified, and 96×78 (samples×primers) qRT-PCR reactions were performed for each primer pair on each sample on a 96.96 array of Fluidigm BioMark™. Compared to age- and sex-matched controls, gene expression levels of ATG16L2, ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, HTT were significantly altered (false discovery rate<0.05). Thus, altered expression levels of several autophagy related genes may affect protein levels, which in turn would influence the activity of autophagy, or most probably, those genes might be acting independent of autophagy and contributing to MS pathogenesis as risk factors. The indeterminate genetic causes leading to alterations in gene expressions require further analysis. PMID:27125224

  12. mir-30d Regulates multiple genes in the autophagy pathway and impairs autophagy process in human cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xiaojun [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Department of General Surgery, Gansu Provincial Hospital, Lanzhou, Gansu 710000 (China); Zhong, Xiaomin [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011 (China); Tanyi, Janos L.; Shen, Jianfeng [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States); Xu, Congjian [Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011 (China); Gao, Peng [Department of General Surgery, Gansu Provincial Hospital, Lanzhou, Gansu 710000 (China); Zheng, Tim M. [Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104 (United States); DeMichele, Angela [Division of Hematology and Oncology, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104 (United States); Zhang, Lin, E-mail: linzhang@mail.med.upenn.edu [Ovarian Cancer Research Center and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA 19104 (United States)

    2013-02-15

    Highlights: ► Gene set enrichment analysis indicated mir-30d might regulate the autophagy pathway. ► mir-30d represses the expression of BECN1, BNIP3L, ATG12, ATG5 and ATG2. ► BECN1, BNIP3L, ATG12, ATG5 and ATG2 are direct targets of mir-30d. ► mir-30d inhibits autophagosome formation and LC3B-I conversion to LC3B-II. ► mir-30d regulates the autophagy process. -- Abstract: In human epithelial cancers, the microRNA (miRNA) mir-30d is amplified with high frequency and serves as a critical oncomir by regulating metastasis, apoptosis, proliferation, and differentiation. Autophagy, a degradation pathway for long-lived protein and organelles, regulates the survival and death of many cell types. Increasing evidence suggests that autophagy plays an important function in epithelial tumor initiation and progression. Using a combined bioinformatics approach, gene set enrichment analysis, and miRNA target prediction, we found that mir-30d might regulate multiple genes in the autophagy pathway including BECN1, BNIP3L, ATG12, ATG5, and ATG2. Our further functional experiments demonstrated that the expression of these core proteins in the autophagy pathway was directly suppressed by mir-30d in cancer cells. Finally, we showed that mir-30d regulated the autophagy process by inhibiting autophagosome formation and LC3B-I conversion to LC3B-II. Taken together, our results provide evidence that the oncomir mir-30d impairs the autophagy process by targeting multiple genes in the autophagy pathway. This result will contribute to understanding the molecular mechanism of mir-30d in tumorigenesis and developing novel cancer therapy strategy.

  13. Alpha-lipoic acid protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Xueming; Chen, Aihua, E-mail: aihuachen2012@sina.com; Yang, Pingzhen; Song, Xudong; Liu, Yingfeng; Li, Zhiliang; Wang, Xianbao; Wang, Lizi; Li, Yunpeng

    2013-11-29

    Highlights: •We observed the cell viability and death subjected to H/R in H9c2 cardiomyocytes. •We observed the degree of autophagy subjected to H/R in H9c2 cardiomyocytes. •LA inhibited the degree of autophagy in parallel to the enhanced cell survival. •LA inhibited the autophagy in parallel to the decreased total cell death. •We concluded that LA protected cardiomyocytes against H/R by inhibiting autophagy. -- Abstract: Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Alpha-lipoic acid (LA) plays an important role in the etiology of cardiovascular disease. Autophagy is widely implicated in myocardial I/R injury. We assessed the degree of autophagy by pretreatment with LA exposed to H/R in H9c2 cell based on the expression levels of Beclin-1, LC3II/LC3I, and green fluorescent protein-labeled LC3 fusion proteins. Autophagic vacuoles were confirmed in H9c2 cells exposed to H/R using transmission electron microscopy. Our findings indicated that pretreatment with LA inhibited the degree of autophagy in parallel to the enhanced cell survival and decreased total cell death in H9c2 cells exposed to H/R. We conclude that LA protects cardiomyocytes against H/R injury by inhibiting autophagy.

  14. Gene expression profiles of autophagy-related genes in multiple sclerosis.

    Science.gov (United States)

    Igci, Mehri; Baysan, Mehmet; Yigiter, Remzi; Ulasli, Mustafa; Geyik, Sirma; Bayraktar, Recep; Bozgeyik, İbrahim; Bozgeyik, Esra; Bayram, Ali; Cakmak, Ecir Ali

    2016-08-15

    Multiple sclerosis (MS) is an imflammatory disease of central nervous system caused by genetic and environmental factors that remain largely unknown. Autophagy is the process of degradation and recycling of damaged cytoplasmic organelles, macromolecular aggregates, and long-lived proteins. Malfunction of autophagy contributes to the pathogenesis of neurological diseases, and autophagy genes may modulate the T cell survival. We aimed to examine the expression levels of autophagy-related genes. The blood samples of 95 unrelated patients (aged 17-65years, 37 male, 58 female) diagnosed as MS and 95 healthy controls were used to extract the RNA samples. After conversion to single stranded cDNA using polyT priming: the targeted genes were pre-amplified, and 96×78 (samples×primers) qRT-PCR reactions were performed for each primer pair on each sample on a 96.96 array of Fluidigm BioMark™. Compared to age- and sex-matched controls, gene expression levels of ATG16L2, ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, HTT were significantly altered (false discovery rategenes may affect protein levels, which in turn would influence the activity of autophagy, or most probably, those genes might be acting independent of autophagy and contributing to MS pathogenesis as risk factors. The indeterminate genetic causes leading to alterations in gene expressions require further analysis.

  15. GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes.

    Science.gov (United States)

    Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

    2014-09-01

    Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.

  16. Melatonin Protects N2a against Ischemia/Reperfusion Injury through Autophagy Enhancement

    Institute of Scientific and Technical Information of China (English)

    国艳春; 王剑飞; 王忠强; 杨易; 王西明; 段秋红

    2010-01-01

    Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury.Although melatonin is known as an effective antioxidant,the mechanism of the protection cannot be explained merely by antioxidation.This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy,since autophagy has been frequently suggested to play a crucial role in neuron survival.To find it out,an ischemia/...

  17. Protective autophagy antagonizes oxaliplatin-induced apoptosis in gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Ling Xu; Xiu-Juan Qu; Yun-Peng Liu; Ying-Ying Xu; Jing Liu; Ke-Zuo Hou; Ye Zhang

    2011-01-01

    Oxaliplatin-based chemotherapy is used for treating gastric cancer. Autophagy has been extensively implicated in cancer cells; however, its function is not fully understood. Our study aimed to determine if oxaliplatin induce autophagy in gastric cancer MGC803 cells and to assess the effect of autophagy on apoptosis induced by oxaliplatin. MGC803 cells were cultured with oxaliplatin. Cell proliferation was measured using MTT assay, and apoptosis was determined by flow cytometry. Protein expression was detected by Western blot. Autophagy was observed using fluorescent microscopy. Our results showed that the rate of apoptosis was 9.73% and 16.36% when MGC803 cells were treated with 5 and 20 μg/mL oxaliplatin for 24 h, respectively. In addition, caspase activation and poly ADP-ribose polymerase (PARP)cleavage were detected. Furthermore, when MGC803 cells were treated with oxaliplatin for 24 h, an accumulation of punctate LC3 and an increase of LC3-Ⅱ protein were also detected, indicating the activation of autophagy. Phosphorylation of Akt and mTOR were inhibited by oxaliplatin. Compared to oxaliplatin alone, the combination of autophagy inhibitor chlorochine and oxaliplatin significantly enhanced the inhibition of cell proliferation and the induction of cell apoptosis. In conclusion, oxaliplatin-induced protective autophagy partially prevents apoptosis in gastric cancer MGC803 cells. The combination of autophagy inhibitor and oxaliplatin may be a new therapeutic option for gastric cancer.

  18. Erythropoietin protects epithelial cells from excessive autophagy and apoptosis in experimental neonatal necrotizing enterocolitis.

    Directory of Open Access Journals (Sweden)

    Yueyue Yu

    Full Text Available Neonatal necrotizing enterocolitis (NEC is a devastating gastrointestinal disease of preterm infants. Increased intestinal epithelium permeability is an early event in NEC pathogenesis. Autophagy and apoptosis are induced by multiple stress pathways which may impact the intestinal barrier, and they have been associated with pathogenesis of diverse gastrointestinal diseases including inflammatory bowel disease. Using both in vitro and in vivo models, this study investigates autophagy and apoptosis under experimental NEC stresses. Furthermore this study evaluates the effect of erythropoietin (Epo, a component of breast milk previously shown to decrease the incidence of NEC and to preserve intestinal barrier function, on intestinal autophagy and apoptosis. It was found that autophagy and apoptosis are both rapidly up regulated in NEC in vivo as indicated by increased expression of the autophagy markers Beclin 1 and LC3II, and by evidence of apoptosis by TUNEL and cleaved caspase-3 staining. In the rat NEC experimental model, autophagy preceded the onset of apoptosis in intestine. In vitro studies suggested that Epo supplementation significantly decreased both autophagy and apoptosis via the Akt/mTOR signaling pathway and the MAPK/ERK pathway respectively. These results suggest that Epo protects intestinal epithelium from excessive autophagy and apoptosis in experimental NEC.

  19. Autophagy protects against de novo formation of the [PSI+] prion in yeast.

    Science.gov (United States)

    Speldewinde, Shaun H; Doronina, Victoria A; Grant, Chris M

    2015-12-15

    Prions are self-propagating, infectious proteins that underlie several neurodegenerative diseases. The molecular basis underlying their sporadic formation is poorly understood. We show that autophagy protects against de novo formation of [PSI(+)], which is the prion form of the yeast Sup35 translation termination factor. Autophagy is a cellular degradation system, and preventing autophagy by mutating its core components elevates the frequency of spontaneous [PSI(+)] formation. Conversely, increasing autophagic flux by treating cells with the polyamine spermidine suppresses prion formation in mutants that normally show a high frequency of de novo prion formation. Autophagy also protects against the de novo formation of another prion, namely the Rnq1/[PIN(+)] prion, which is not related in sequence to the Sup35/[PSI(+)] prion. We show that growth under anaerobic conditions in the absence of molecular oxygen abrogates Sup35 protein damage and suppresses the high frequency of [PSI(+)] formation in an autophagy mutant. Autophagy therefore normally functions to remove oxidatively damaged Sup35, which accumulates in cells grown under aerobic conditions, but in the absence of autophagy, damaged/misfolded Sup35 undergoes structural transitions favoring its conversion to the propagatable [PSI(+)] form.

  20. Autophagy Protects against CYP2E1/Chronic Ethanol-Induced Hepatotoxicity

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    Yongke Lu

    2015-10-01

    Full Text Available Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current report was to extend the previous in vitro and acute in vivo experiments to a chronic ethanol model to evaluate whether autophagy is also protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. Wild type (WT, CYP2E1 knockout (KO or CYP2E1 humanized transgenic knockin (KI, mice were fed an ethanol liquid diet or control dextrose diet for four weeks. In the last week, some mice received either saline or 3-methyladenine (3-MA, an inhibitor of autophagy, or rapamycin, which stimulates autophagy. Inhibition of autophagy by 3-MA potentiated the ethanol-induced increases in serum transaminase and triglyceride levels in the WT and KI mice but not KO mice, while rapamycin prevented the ethanol liver injury. Treatment with 3-MA enhanced the ethanol-induced fat accumulation in WT mice and caused necrosis in the KI mice; little or no effect was found in the ethanol-fed KO mice or any of the dextrose-fed mice. 3-MA treatment further lowered the ethanol-decrease in hepatic GSH levels and further increased formation of TBARS in WT and KI mice, whereas rapamycin blunted these effects of ethanol. Neither 3-MA nor rapamycin treatment affected CYP2E1 catalytic activity or content or the induction CYP2E1 by ethanol. The 3-MA treatment decreased levels of Beclin-1 and Atg 7 but increased levels of p62 in the ethanol-fed WT and KI mice whereas rapamycin had the opposite effects, validating inhibition and stimulation of autophagy, respectively. These

  1. Targeting Protective Autophagy Exacerbates UV-Triggered Apoptotic Cell Death

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    Shih-Hwa Chiou

    2012-01-01

    Full Text Available Autophagy is activated by various stresses, including DNA damage, and previous studies of DNA damage-induced autophagy have focused on the response to chemotherapeutic drugs, ionizing radiation, and reactive oxygen species. In this study, we investigated the biological significance of autophagic response to ultraviolet (UV irradiation in A549 and H1299 cells. Our results indicated that UV induces on-rate autophagic flux in these cells. Autophagy inhibition resulting from the knockdown of beclin-1 and Atg5 reduced cell viability and enhanced apoptosis. Moreover, we found that ATR phosphorylation was accompanied by microtubule-associated protein 1 light chain 3B II (LC3B-II expression during the early phases following UV irradiation, which is a well-established inducer of ATR. Knocking down ATR further attenuated the reduction in LC3B-II at early stages in response to UV treatment. Despite the potential role of ATR in autophagic response, reduced ATR expression does not affect autophagy induction during late phases (24 and 48 h after UV treatment. The result is consistent with the reduced ATR phosphorylation at the same time points and suggests that autophagic response at this stage is activated via a distinct pathway. In conclusion, this study demonstrated that autophagy acts as a cytoprotective mechanism against UV-induced apoptosis and that autophagy induction accompanied with apoptosis at late stages is independent of ATR activation.

  2. Sinomenine Hydrochloride Protects against Polymicrobial Sepsis via Autophagy

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    Yu Jiang

    2015-01-01

    Full Text Available Sepsis, a systemic inflammatory response to infection, is the major cause of death in intensive care units (ICUs. The mortality rate of sepsis remains high even though the treatment and understanding of sepsis both continue to improve. Sinomenine (SIN is a natural alkaloid extracted from Chinese medicinal plant Sinomenium acutum, and its hydrochloride salt (Sinomenine hydrochloride, SIN-HCl is widely used to treat rheumatoid arthritis (RA. However, its role in sepsis remains unclear. In the present study, we investigated the role of SIN-HCl in sepsis induced by cecal ligation and puncture (CLP in BALB/c mice and the corresponding mechanism. SIN-HCl treatment improved the survival of BALB/c mice that were subjected to CLP and reduced multiple organ dysfunction and the release of systemic inflammatory mediators. Autophagy activities were examined using Western blotting. The results showed that CLP-induced autophagy was elevated, and SIN-HCl treatment further strengthened the autophagy activity. Autophagy blocker 3-methyladenine (3-MA was used to investigate the mechanism of SIN-HCl in vitro. Autophagy activities were determined by examining the autophagosome formation, which was shown as microtubule-associated protein light chain 3 (LC3 puncta with green immunofluorescence. SIN-HCl reduced lipopolysaccharide (LPS-induced inflammatory cytokine release and increased autophagy in peritoneal macrophages (PM. 3-MA significantly decreased autophagosome formation induced by LPS and SIN-HCl. The decrease of inflammatory cytokines caused by SIN-HCl was partially aggravated by 3-MA treatment. Taken together, our results indicated that SIN-HCl could improve survival, reduce organ damage, and attenuate the release of inflammatory cytokines induced by CLP, at least in part through regulating autophagy activities.

  3. Sinomenine hydrochloride protects against polymicrobial sepsis via autophagy.

    Science.gov (United States)

    Jiang, Yu; Gao, Min; Wang, Wenmei; Lang, Yuejiao; Tong, Zhongyi; Wang, Kangkai; Zhang, Huali; Chen, Guangwen; Liu, Meidong; Yao, Yongming; Xiao, Xianzhong

    2015-01-23

    Sepsis, a systemic inflammatory response to infection, is the major cause of death in intensive care units (ICUs). The mortality rate of sepsis remains high even though the treatment and understanding of sepsis both continue to improve. Sinomenine (SIN) is a natural alkaloid extracted from Chinese medicinal plant Sinomenium acutum, and its hydrochloride salt (Sinomenine hydrochloride, SIN-HCl) is widely used to treat rheumatoid arthritis (RA). However, its role in sepsis remains unclear. In the present study, we investigated the role of SIN-HCl in sepsis induced by cecal ligation and puncture (CLP) in BALB/c mice and the corresponding mechanism. SIN-HCl treatment improved the survival of BALB/c mice that were subjected to CLP and reduced multiple organ dysfunction and the release of systemic inflammatory mediators. Autophagy activities were examined using Western blotting. The results showed that CLP-induced autophagy was elevated, and SIN-HCl treatment further strengthened the autophagy activity. Autophagy blocker 3-methyladenine (3-MA) was used to investigate the mechanism of SIN-HCl in vitro. Autophagy activities were determined by examining the autophagosome formation, which was shown as microtubule-associated protein light chain 3 (LC3) puncta with green immunofluorescence. SIN-HCl reduced lipopolysaccharide (LPS)-induced inflammatory cytokine release and increased autophagy in peritoneal macrophages (PM). 3-MA significantly decreased autophagosome formation induced by LPS and SIN-HCl. The decrease of inflammatory cytokines caused by SIN-HCl was partially aggravated by 3-MA treatment. Taken together, our results indicated that SIN-HCl could improve survival, reduce organ damage, and attenuate the release of inflammatory cytokines induced by CLP, at least in part through regulating autophagy activities.

  4. DRAM1 Protects Neuroblastoma Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Injury via Autophagy

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    Mengqiang Yu

    2014-10-01

    Full Text Available DNA damage-regulated autophagy modulator protein 1 (DRAM1, a multi-pass membrane lysosomal protein, is reportedly a tumor protein p53 (TP53 target gene involved in autophagy. During cerebral ischemia/reperfusion (I/R injury, DRAM1 protein expression is increased, and autophagy is activated. However, the functional significance of DRAM1 and the relationship between DRAM1 and autophagy in brain I/R remains uncertain. The aim of this study is to investigate whether DRAM1 mediates autophagy activation in cerebral I/R injury and to explore its possible effects and mechanisms. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R Neuro-2a cell model to mimic cerebral I/R conditions in vitro, and RNA interference is used to knock down DRAM1 expression in this model. Cell viability assay is performed using the LIVE/DEAD viability/cytotoxicity kit. Cell phenotypic changes are analyzed through Western blot assays. Autophagy flux is monitored through the tandem red fluorescent protein–Green fluorescent protein–microtubule associated protein 1 light chain 3 (RFP–GFP–LC3 construct. The expression levels of DRAM1 and microtubule associated protein 1 light chain 3II/I (LC3II/I are strongly up-regulated in Neuro-2a cells after OGD/R treatment and peaked at the 12 h reperfusion time point. The autophagy-specific inhibitor 3-Methyladenine (3-MA inhibits the expression of DRAM1 and LC3II/I and exacerbates OGD/R-induced cell injury. Furthermore, DRAM1 knockdown aggravates OGD/R-induced cell injury and significantly blocks autophagy through decreasing autophagosome-lysosome fusion. In conclusion, our data demonstrate that DRAM1 knockdown in Neuro-2a cells inhibits autophagy by blocking autophagosome-lysosome fusion and exacerbated OGD/R-induced cell injury. Thus, DRAM1 might constitute a new therapeutic target for I/R diseases.

  5. The influence of autophagy on mouse inflammatory responses caused by Salmonella enterica serovar Typhimurium with spv genes

    Institute of Scientific and Technical Information of China (English)

    LI Yuan-Yuan; WU Shu-Yan; CHU Yuan-Yuan; LIAO LI; LIQiong; HUANG Rui

    2011-01-01

    An investigation into the effects of Salmonella plasmid virulence genes (spv) on autophagy,apoptosis,and inflammation was carried out in mice,using a strain of Salmonella enterica serovar Typhimurium (S.typhimurium) SR-11 carrying spv.Strain BRD509 without spy was used as a control.Results showed that the expression of autophagy protein Beclin-1 in the livers and spleens in the SR-11 group was lower than that in the BRD509 group,while the apoptosis protein,Caspase-3,was higher in the SR-11 group.Inflammatory cytokine levels [interleukin 12 (IL-12) and interferon γ (IFN-γ)] were higher in the SR-11 group compared with those in the BRD509 group since 4 d post-infection.In addition,we found an increase in severe pathological changes and larger viable bacterial amounts in livers and spleens in the SR-11 group.After intervention with autophagy agonist rapamycin (RAPA),Beclin-1 expression increased in both groups,while Caspase-3 expression was different between the two groups: Caspase-3 decreased in the SR-11 group but increased in the BRD509 group.Moreover,RAPA decreased cytokine levels,bacterial quantity and organ-related injury in the SR-11 group whereas RAPA increased cytokine levels and aggravated organ injury in the BRD509 group.Results from these studies suggest that S.typhimurium with spv genes may exacerbate infection by inhibiting autophagy and affecting the production of inflammatory cytokines.RAPA-enhanced autophagy may improve the secretion of cytokines in order to protect the host from damaging by Salmonella infection.Our study suggests that the regulation of cellular autophagy may play a role in the prevention and control of certain infectious diseases.

  6. Induction of cyto-protective autophagy by paramontroseite VO2 nanocrystals

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    Zhou, Wei; Miao, Yanyan; Zhang, Yunjiao; Liu, Liang; Lin, Jun; Yang, James Y.; Xie, Yi; Wen, Longping

    2013-04-01

    A variety of inorganic nanomaterials have been shown to induce autophagy, a cellular degradation process critical for the maintenance of cellular homeostasis. The overwhelming majority of autophagic responses elicited by nanomaterials were detrimental to cell fate and contributed to increased cell death. A widely held view is that the inorganic nanoparticles, when encapsulated and trapped by autophagosomes, may compromise the normal autophagic process due to the inability of the cells to degrade these materials and thus they manifest a detrimental effect on the well-being of a cell. Here we show that, contrary to this notion, nano-sized paramontroseite VO2 nanocrystals (P-VO2) induced cyto-protective, rather than death-promoting, autophagy in cultured HeLa cells. P-VO2 also caused up-regulation of heme oxygenase-1 (HO-1), a cellular protein with a demonstrated role in protecting cells against death under stress situations. The autophagy inhibitor 3-methyladenine significantly inhibited HO-1 up-regulation and increased the rate of cell death in cells treated with P-VO2, while the HO-1 inhibitor protoporphyrin IX zinc (II) (ZnPP) enhanced the occurrence of cell death in the P-VO2-treated cells while having no effect on the autophagic response induced by P-VO2. On the other hand, Y2O3 nanocrystals, a control nanomaterial, induced death-promoting autophagy without affecting the level of expression of HO-1, and the pro-death effect of the autophagy induced by Y2O3. Our results represent the first report on a novel nanomaterial-induced cyto-protective autophagy, probably through up-regulation of HO-1, and may point to new possibilities for exploiting nanomaterial-induced autophagy for therapeutic applications.

  7. Heme oxygenase-1 enhances autophagy in podocytes as a protective mechanism against high glucose-induced apoptosis

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    Dong, Chenglong [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China); Zheng, Haining [Department of Hyperbaric Oxygen, Nanjing General Hospital of Nanjing Military Command, Nanjing (China); Huang, Shanshan; You, Na; Xu, Jiarong; Ye, Xiaolong; Zhu, Qun; Feng, Yamin; You, Qiang; Miao, Heng [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China); Ding, Dafa, E-mail: dingdafa2004@aliyun.com [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China); Lu, Yibing, E-mail: luyibing2004@126.com [Department of Endocrinology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing (China)

    2015-10-01

    Injury and loss of podocytes play vital roles in diabetic nephropathy progression. Emerging evidence suggests autophagy, which is induced by multiple stressors including hyperglycemia, plays a protective role. Meanwhile, heme oxygenase-1 (HO-1) possesses powerful anti-apoptotic properties. Therefore, we investigated the impact of autophagy on podocyte apoptosis under diabetic conditions and its association with HO-1. Mouse podocytes were cultured in vitro; apoptosis was detected by flow cytometry. Transmission electron microscopy and biochemical autophagic flux assays were used to measure the autophagy markers microtubule-associated protein 1 light chain 3-II (LC3-II) and beclin-1. LC3-II and beclin-1 expression peaked 12–24 h after exposing podocytes to high glucose. Inhibition of autophagy with 3-methyladenine or Beclin-1 siRNAs or Atg 5 siRNAs sensitized cells to apoptosis, suggesting autophagy is a survival mechanism. HO-1 inactivation inhibited autophagy, which aggravated podocyte injury in vitro. Hemin-induced autophagy also protected podocytes from hyperglycemia in vitro and was abrogated by HO-1 siRNA. Adenosine monophosphate-activated protein kinase phosphorylation was higher in hemin-treated and lower in HO-1 siRNA-treated podocytes. Suppression of AMPK activity reversed HO-1-mediated Beclin-1 upregulation and autophagy, indicating HO-1-mediated autophagy is AMPK dependent. These findings suggest HO-1 induction and regulation of autophagy are potential therapeutic targets for diabetic nephropathy. - Highlights: • High glucose leads to increased autophagy in podocytes at an early stage. • The early autophagic response protects against high glucose-induced apoptosis. • Heme oxygenase-1 enhances autophagy and decreases high glucose -mediated apoptosis. • Heme oxygenase-1 induces autophagy through the activation of AMPK.

  8. An autophagy gene, HoATG5, is involved in sporulation, cell wall integrity and infection of wounded barley leaves.

    Science.gov (United States)

    Liu, Ning; Ning, Guo-Ao; Liu, Xiao-Hong; Feng, Xiao-Xiao; Lu, Jian-Ping; Mao, Li-Juan; Su, Zhen-Zhu; Wang, Ying; Zhang, Chu-Long; Lin, Fu-Cheng

    2016-11-01

    The endophytic fungus Harpophora oryzae is a beneficial endosymbiont isolated from wild rice. H. oryzae can not only promote rice growth and biomass accumulation but also protect rice roots from invasion by its close relative Magnaporthe oryzae. Autophagy is a highly evolutionary conserved process from lower to higher eukaryotic organisms, and is involved in the maintenance of normal cell differentiation and development. In this study, we isolated a gene (HoATG5) which encodes an essential protein required for autophagy from the beneficial endophyte fungus H. oryzae. Using targeted gene replacement, a ΔHoATG5 mutant was generated and used to investigate the biological functions of autophagy in H. oryzae. We found that the autophagic process was blocked in the HoATG5 deletion mutant. The mutant showed increased vegetative growth and sporulation, and was sensitive to nutrient starvation. The ΔHoATG5 mutant lost its ability to penetrate and infect the wounded barley leaves. These results provide new knowledge to elaborate the molecular machinery of autophagy in endophytic fungi.

  9. Autophagy is a protective response to ethanol neurotoxicity

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    Chen, Gang; Ke, Zunji; Xu, Mei; Liao, Mingjun; Wang, Xin; Qi, Yuanlin; Zhang,Tao; Frank, Jacqueline A.; Bower, Kimberly A.; Shi, Xianglin; Luo, Jia

    2012-01-01

    Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II...

  10. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis

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    Karantza-Wadsworth, Vassiliki; Patel, Shyam; Kravchuk, Olga; Chen, Guanghua; Mathew, Robin; Jin, Shengkan; White, Eileen

    2007-01-01

    Autophagy is a catabolic process involving self-digestion of cellular organelles during starvation as a means of cell survival; however, if it proceeds to completion, autophagy can lead to cell death. Autophagy is also a haploinsufficient tumor suppressor mechanism for mammary tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in breast carcinomas. However, the mechanism by which autophagy suppresses breast cancer remains elusive. Here we show that allelic loss of beclin1 and defective autophagy sensitized mammary epithelial cells to metabolic stress and accelerated lumen formation in mammary acini. Autophagy defects also activated the DNA damage response in vitro and in mammary tumors in vivo, promoted gene amplification, and synergized with defective apoptosis to promote mammary tumorigenesis. Therefore, we propose that autophagy limits metabolic stress to protect the genome, and that defective autophagy increases DNA damage and genomic instability that ultimately facilitate breast cancer progression. PMID:17606641

  11. Differential reliance on autophagy for protection from HSV encephalitis between newborns and adults.

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    Douglas R Wilcox

    2015-01-01

    Full Text Available Newborns are more susceptible to severe disease from infection than adults, with maturation of immune responses implicated as a major factor. The type I interferon response delays mortality and limits viral replication in adult mice in a model of herpes simplex virus (HSV encephalitis. We found that intact type I interferon signaling did not control HSV disease in the neonatal brain. However, the multifunctional HSV protein γ34.5 involved in countering type I interferon responses was important for virulence in the brain in both age groups. To investigate this observation further, we studied a specific function of γ34.5 which contributes to HSV pathogenesis in the adult brain, inhibition of the cellular process of autophagy. Surprisingly, we found that the beclin binding domain of γ34.5 responsible for inhibiting autophagy was dispensable for HSV disease in the neonatal brain, as infection of newborns with the deletion mutant decreased time to mortality compared to the rescue virus. Additionally, a functional beclin binding domain in HSV γ34.5 did not effectively inhibit autophagy in the neonate, unlike in the adult. Type I IFN responses promote autophagy in adult, a finding we confirmed in the adult brain after HSV infection; however, in the newborn brain we observed that autophagy was activated through a type I IFN-independent mechanism. Furthermore, autophagy in the wild-type neonatal mouse was associated with increased apoptosis in infected regions of the brain. Observations in the mouse model were consistent with those in a human case of neonatal HSV encephalitis. Our findings reveal age-dependent differences in autophagy for protection from HSV encephalitis, indicating developmental differences in induction and regulation of this innate defense mechanism after HSV infection in the neonatal brain.

  12. Differential reliance on autophagy for protection from HSV encephalitis between newborns and adults.

    Science.gov (United States)

    Wilcox, Douglas R; Wadhwani, Nitin R; Longnecker, Richard; Muller, William J

    2015-01-01

    Newborns are more susceptible to severe disease from infection than adults, with maturation of immune responses implicated as a major factor. The type I interferon response delays mortality and limits viral replication in adult mice in a model of herpes simplex virus (HSV) encephalitis. We found that intact type I interferon signaling did not control HSV disease in the neonatal brain. However, the multifunctional HSV protein γ34.5 involved in countering type I interferon responses was important for virulence in the brain in both age groups. To investigate this observation further, we studied a specific function of γ34.5 which contributes to HSV pathogenesis in the adult brain, inhibition of the cellular process of autophagy. Surprisingly, we found that the beclin binding domain of γ34.5 responsible for inhibiting autophagy was dispensable for HSV disease in the neonatal brain, as infection of newborns with the deletion mutant decreased time to mortality compared to the rescue virus. Additionally, a functional beclin binding domain in HSV γ34.5 did not effectively inhibit autophagy in the neonate, unlike in the adult. Type I IFN responses promote autophagy in adult, a finding we confirmed in the adult brain after HSV infection; however, in the newborn brain we observed that autophagy was activated through a type I IFN-independent mechanism. Furthermore, autophagy in the wild-type neonatal mouse was associated with increased apoptosis in infected regions of the brain. Observations in the mouse model were consistent with those in a human case of neonatal HSV encephalitis. Our findings reveal age-dependent differences in autophagy for protection from HSV encephalitis, indicating developmental differences in induction and regulation of this innate defense mechanism after HSV infection in the neonatal brain. PMID:25569138

  13. Autophagy induced by p53-reactivating molecules protects pancreatic cancer cells from apoptosis.

    Science.gov (United States)

    Fiorini, Claudia; Menegazzi, Marta; Padroni, Chiara; Dando, Ilaria; Dalla Pozza, Elisa; Gregorelli, Alex; Costanzo, Chiara; Palmieri, Marta; Donadelli, Massimo

    2013-03-01

    TP53 mutations compromising p53 transcriptional function occur in more than 50 % of human cancers, including pancreatic adenocarcinoma, and render cancer cells more resistant to conventional therapy. In the last few years, many efforts have been addressed to identify p53-reactivating molecules able to restore the wild-type transcriptionally competent conformation of the mutated proteins. Here, we show that two of these compounds, CP-31398 and RITA, induce cell growth inhibition, apoptosis, and autophagy by activating p53/DNA binding and p53 phosphorylation (Ser15), without affecting the total p53 amount. These effects occur in both wild-type and mutant p53 pancreatic adenocarcinoma cell lines, whereas they are much less pronounced in normal human primary fibroblasts. Furthermore, CP-31398 and RITA regulate the axis SESN1-2/AMPK/mTOR by inducing AMPK phosphorylation on Thr172, which has a crucial role in the autophagic response. The protective role of autophagy in cell growth inhibition by CP-31398 and RITA is supported by the finding that the AMPK inhibitor compound C or the autophagy inhibitors chloroquine or 3-methyladenine sensitize both pancreatic adenocarcinoma cell lines to the apoptotic response induced by p53-reactivating molecules. Our results demonstrate for the first time a survival role for autophagy induced by p53-reactivating molecules, supporting the development of an anti-cancer therapy based on autophagy inhibition associated to p53 activation.

  14. Autophagy protects cardiomyocytes from the myocardial ischaemia-reperfusion injury through the clearance of CLP36

    Science.gov (United States)

    Li, Shiguo; Liu, Chao; Gu, Lei; Wang, Lina; Shang, Yongliang; Liu, Qiong; Wan, Junyi; Shi, Jian; Wang, Fang; Xu, Zhiliang; Ji, Guangju

    2016-01-01

    Cardiovascular disease (CVD) is the leading cause of the death worldwide. An increasing number of studies have found that autophagy is involved in the progression or prevention of CVD. However, the precise mechanism of autophagy in CVD, especially the myocardial ischaemia-reperfusion injury (MI/R injury), is unclear and controversial. Here, we show that the cardiomyocyte-specific disruption of autophagy by conditional knockout of Atg7 leads to severe contractile dysfunction, myofibrillar disarray and vacuolar cardiomyocytes. A negative cytoskeleton organization regulator, CLP36, was found to be accumulated in Atg7-deficient cardiomyocytes. The cardiomyocyte-specific knockout of Atg7 aggravates the MI/R injury with cardiac hypertrophy, contractile dysfunction, myofibrillar disarray and severe cardiac fibrosis, most probably due to CLP36 accumulation in cardiomyocytes. Altogether, this work reveals autophagy may protect cardiomyocytes from the MI/R injury through the clearance of CLP36, and these findings define a novel relationship between autophagy and the regulation of stress fibre in heart. PMID:27512143

  15. Autophagy Constitutes a Protective Mechanism against Ethanol Toxicity in Mouse Astrocytes and Neurons.

    Science.gov (United States)

    Pla, Antoni; Pascual, María; Guerri, Consuelo

    2016-01-01

    Ethanol induces brain damage and neurodegeneration by triggering inflammatory processes in glial cells through activation of Toll-like receptor 4 (TLR4) signaling. Recent evidence indicates the role of protein degradation pathways in neurodegeneration and alcoholic liver disease, but how these processes affect the brain remains elusive. We have demonstrated that chronic ethanol consumption impairs proteolytic pathways in mouse brain, and the immune response mediated by TLR4 receptors participates in these dysfunctions. We evaluate the in vitro effects of an acute ethanol dose on the autophagy-lysosome pathway (ALP) on WT and TLR4-/- mouse astrocytes and neurons in primary culture, and how these changes affect cell survival. Our results show that ethanol induces overexpression of several autophagy markers (ATG12, LC3-II, CTSB), and increases the number of lysosomes in WT astrocytes, effects accompanied by a basification of lysosomal pH and by lowered phosphorylation levels of autophagy inhibitor mTOR, along with activation of complexes beclin-1 and ULK1. Notably, we found only minor changes between control and ethanol-treated TLR4-/- mouse astroglial cells. Ethanol also triggers the expression of the inflammatory mediators iNOS and COX-2, but induces astroglial death only slightly. Blocking autophagy by using specific inhibitors increases both inflammation and cell death. Conversely, in neurons, ethanol down-regulates the autophagy pathway and triggers cell death, which is partially recovered by using autophagy enhancers. These results support the protective role of the ALP against ethanol-induced astroglial cell damage in a TLR4-dependent manner, and provide new insight into the mechanisms that underlie ethanol-induced brain damage and are neuronal sensitive to the ethanol effects.

  16. No evidence for activated autophagy in left ventricular myocardium at early reperfusion with protection by remote ischemic preconditioning in patients undergoing coronary artery bypass grafting.

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    Nilgün Gedik

    Full Text Available Remote ischemic preconditioning (RIPC by repeated brief limb ischemia/reperfusion reduces myocardial injury in patients undergoing coronary artery bypass grafting (CABG. Activation of signal transducer and activator of transcription 5 (STAT5 in left ventricular (LV myocardium at early reperfusion is associated with such protection. Autophagy, i.e., removal of dysfunctional cellular components through lysosomes, has been proposed as one mechanism of cardioprotection. Therefore, we analyzed whether or not the protection by RIPC is associated with activated autophagy.CABG patients were randomized to undergo RIPC (3×5 min blood pressure cuff inflation/5 min deflation or placebo (cuff deflated before skin incision (n = 10/10. Transmural myocardial biopsies were taken from the LV before cardioplegia (baseline and at early (5-10 min reperfusion. RIPC-induced protection was reflected by decreased serum troponin I concentration area under the curve (194±17 versus 709±129 ng/ml × 72 h, p = 0.002. Western blotting for beclin-1-phosphorylation and protein expression of autophagy-related gene 5-12 (ATG5-12 complex, light chain 3 (LC3, parkin, and p62 was performed. STAT3-, STAT5- and extracellular signal-regulated protein kinase 1/2 (ERK1/2-phosphorylation was used as positive control to confirm signal activation by ischemia/reperfusion.Signals of all analyzed autophagy proteins did not differ between baseline and early reperfusion and not between RIPC and placebo. STAT5-phosphorylation was greater at early reperfusion only with RIPC (2.2-fold, p = 0.02. STAT3- and ERK1/2-phosphorylation were greater at early reperfusion with placebo and RIPC (≥2.7-fold versus baseline, p≤0.05.Protection through RIPC in patients undergoing CABG surgery does not appear to be associated with enhanced autophagy in LV myocardium at early reperfusion.

  17. Autophagy protects type II alveolar epithelial cells from Mycobacterium tuberculosis infection

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    Guo, Xu-Guang [Center for Clinical Laboratory Medicine of PLA, Xijing Hospital, Fourth Military Medical University, Xi' an (China); Department of Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Ji, Tian-Xing [Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Xia, Yong, E-mail: gysyxy@gmail.com [Center for Clinical Laboratory Medicine of PLA, Xijing Hospital, Fourth Military Medical University, Xi' an (China); Ma, Yue-Yun, E-mail: cmbmayy@fmmu.edu.cn [Center for Clinical Laboratory Medicine of PLA, Xijing Hospital, Fourth Military Medical University, Xi' an (China)

    2013-03-08

    Highlights: ► We investigated the protective effect of autophagy pathway against MTB infection. ► MTB-infected A549 cells had higher LDH release. ► Inhibition of autophagy signaling significantly enhanced the MTB-induced necrosis. ► Autophagy prevents apoptosis and promotes cell survival in infected cells. -- Abstract: This study was designed to investigate the protective effect of the autophagy signaling pathway against Mycobacterium tuberculosis infection in type II alveolar epithelial cells. An in vitro M. tuberculosis system was established using human A549 cells. Infection-induced changes in the expression of the autophagic marker LC3 were assessed by reverse transcription-PCR and Western blotting. Morphological changes in autophagosomes were detected by transmission electron microscopy (TEM). The function of the autophagy signaling pathway during infection was assessed by measuring the level of cell death and the amount of lactate dehydrogenase (LDH) released in the presence or absence of the inhibitor 3-methyladenine (3-MA). In addition, effects on LDH release were assessed after the siRNA-mediated knockdown of the essential autophagosomal structural membrane protein Atg5. LC3 mRNA expression was significantly reduced in M.tuberculosis-infected A549 cells (16888.76 ± 1576.34 vs. uninfected: 12744.29 ± 1089.37; P < 0.05). TEM revealed M.tuberculosis bacilli-containing compartments that were surrounded by double membranes characteristic of the autophagic process. M.tuberculosis-infected A549 cells released more LDH (1.45 ± 0.12 vs. uninfected: 0.45 ± 0.04; P < 0.05). The inhibition of autophagy signaling significantly enhanced M.tuberculosis-induced necrosis (3-MA: 75 ± 5% vs. untreated: 15 ± 1%; P < 0.05) and LDH release (3-MA: 2.50 ± 0.24 vs. untreated: 0.45 ± 0.04; Atg5 knockdown: 3.19 ± 0.29 vs. untreated: 1.28 ± 0.11; P < 0.05). Our results indicate that autophagy signaling pathway prevents apoptosis in type II alveolar epithelial cells

  18. Autophagy protects type II alveolar epithelial cells from Mycobacterium tuberculosis infection

    International Nuclear Information System (INIS)

    Highlights: ► We investigated the protective effect of autophagy pathway against MTB infection. ► MTB-infected A549 cells had higher LDH release. ► Inhibition of autophagy signaling significantly enhanced the MTB-induced necrosis. ► Autophagy prevents apoptosis and promotes cell survival in infected cells. -- Abstract: This study was designed to investigate the protective effect of the autophagy signaling pathway against Mycobacterium tuberculosis infection in type II alveolar epithelial cells. An in vitro M. tuberculosis system was established using human A549 cells. Infection-induced changes in the expression of the autophagic marker LC3 were assessed by reverse transcription-PCR and Western blotting. Morphological changes in autophagosomes were detected by transmission electron microscopy (TEM). The function of the autophagy signaling pathway during infection was assessed by measuring the level of cell death and the amount of lactate dehydrogenase (LDH) released in the presence or absence of the inhibitor 3-methyladenine (3-MA). In addition, effects on LDH release were assessed after the siRNA-mediated knockdown of the essential autophagosomal structural membrane protein Atg5. LC3 mRNA expression was significantly reduced in M.tuberculosis-infected A549 cells (16888.76 ± 1576.34 vs. uninfected: 12744.29 ± 1089.37; P < 0.05). TEM revealed M.tuberculosis bacilli-containing compartments that were surrounded by double membranes characteristic of the autophagic process. M.tuberculosis-infected A549 cells released more LDH (1.45 ± 0.12 vs. uninfected: 0.45 ± 0.04; P < 0.05). The inhibition of autophagy signaling significantly enhanced M.tuberculosis-induced necrosis (3-MA: 75 ± 5% vs. untreated: 15 ± 1%; P < 0.05) and LDH release (3-MA: 2.50 ± 0.24 vs. untreated: 0.45 ± 0.04; Atg5 knockdown: 3.19 ± 0.29 vs. untreated: 1.28 ± 0.11; P < 0.05). Our results indicate that autophagy signaling pathway prevents apoptosis in type II alveolar epithelial cells

  19. Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells.

    Directory of Open Access Journals (Sweden)

    Ya C Wu

    Full Text Available Hydrogen sulfide (H(2S is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC and a panel of colon cancer cell lines (HT-29, SW1116, HCT116 were exposed to H(2S at concentrations similar to those found in the human colon. H(2S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2S was accompanied by G(1-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip. Moreover, exposure to H(2S led to features characteristic of autophagy, including increased formation of LC3B(+ autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2S. Further mechanistic investigation revealed that H(2S stimulated the phosphorylation of AMP-activated protein kinase (AMPK and inhibited the phosphorylation of mammalian target of rapamycin (mTOR and S6 kinase. Inhibition of AMPK significantly reversed H(2S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway.

  20. Activation of the MAPK11/12/13/14 (p38 MAPK) pathway regulates the transcription of autophagy genes in response to oxidative stress induced by a novel copper complex in HeLa cells.

    Science.gov (United States)

    Zhong, Wu; Zhu, Haichuan; Sheng, Fugeng; Tian, Yonglu; Zhou, Jun; Chen, Yingyu; Li, Song; Lin, Jian

    2014-07-01

    Transition metal copper (Cu) can exist in oxidized or reduced states in cells, leading to cytotoxicity in cancer cells through oxidative stress. Recently, copper complexes are emerging as a new class of anticancer compounds. Here, we report that a novel anticancer copper complex (HYF127c/Cu) induces oxidative stress-dependent cell death in cancer cells. Further, transcriptional analysis revealed that oxidative stress elicits broad transcriptional changes of genes, in which autophagy-related genes are significantly changed in HYF127c/Cu-treated cells. Consistently, autophagy was induced in HYF127c/Cu-treated cells and inhibitors of autophagy promoted cell death induced by HYF127c/Cu. Further analysis identified that the MAPK11/12/13/14 (formerly known as p38 MAPK) pathway was also activated in HYF127c/Cu-treated cells. Meanwhile, the MAPK11/12/13/14 inhibitor SB203580 downregulated autophagy by inhibiting the transcription of the autophagy genes MAP1LC3B, BAG3, and HSPA1A, and promoted HYF127c/Cu-induced cell death. These data suggest that copper-induced oxidative stress will induce protective autophagy through transcriptional regulation of autophagy genes by activation of the MAPK11/12/13/14 pathway in HeLa cells.

  1. Tissue distribution, gender- and genotype-dependent expression of autophagy-related genes in avian species.

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    Alissa Piekarski

    Full Text Available As a result of the genetic selection of broiler (meat-type breeders chickens for enhanced growth rate and lower feed conversion ratio, it has become necessary to restrict feed intake. When broilers are fed ad libitum, they would become obese and suffer from several health-related problems. A vital adaptation to starvation is autophagy, a self-eating mechanism for recycling cellular constituents. The autophagy pathway has witnessed dramatic growth in the last few years and extensively studied in yeast and mammals however, there is a paucity of information in avian (non-mammalian species. Here we characterized several genes involved in autophagosome initiation and elongation in Red Jungle fowl (Gallus gallus and Japanese quail (coturnix coturnix Japonica. Both complexes are ubiquitously expressed in chicken and quail tissues (liver, leg and breast muscle, brain, gizzard, intestine, heart, lung, kidney, adipose tissue, ovary and testis. Alignment analysis showed high similarity (50.7 to 91.5% between chicken autophagy-related genes and their mammalian orthologs. Phylogenetic analysis demonstrated that the evolutionary relationship between autophagy genes is consistent with the consensus view of vertebrate evolution. Interestingly, the expression of autophagy-related genes is tissue- and gender-dependent. Furthermore, using two experimental male quail lines divergently selected over 40 generations for low (resistant, R or high (sensitive, S stress response, we found that the expression of most studied genes are higher in R compared to S line. Together our results indicate that the autophagy pathway is a key molecular signature exhibited gender specific differences and likely plays an important role in response to stress in avian species.

  2. The autophagy gene Atg16l1 differentially regulates Treg and TH2 cells to control intestinal inflammation.

    Science.gov (United States)

    Kabat, Agnieszka M; Harrison, Oliver J; Riffelmacher, Thomas; Moghaddam, Amin E; Pearson, Claire F; Laing, Adam; Abeler-Dörner, Lucie; Forman, Simon P; Grencis, Richard K; Sattentau, Quentin; Simon, Anna Katharina; Pott, Johanna; Maloy, Kevin J

    2016-01-01

    A polymorphism in the autophagy gene Atg16l1 is associated with susceptibility to inflammatory bowel disease (IBD); however, it remains unclear how autophagy contributes to intestinal immune homeostasis. Here, we demonstrate that autophagy is essential for maintenance of balanced CD4(+) T cell responses in the intestine. Selective deletion of Atg16l1 in T cells in mice resulted in spontaneous intestinal inflammation that was characterized by aberrant type 2 responses to dietary and microbiota antigens, and by a loss of Foxp3(+) Treg cells. Specific ablation of Atg16l1 in Foxp3(+) Treg cells in mice demonstrated that autophagy directly promotes their survival and metabolic adaptation in the intestine. Moreover, we also identify an unexpected role for autophagy in directly limiting mucosal TH2 cell expansion. These findings provide new insights into the reciprocal control of distinct intestinal TH cell responses by autophagy, with important implications for understanding and treatment of chronic inflammatory disorders. PMID:26910010

  3. Downregulation of ATG14 by EGR1-MIR152 sensitizes ovarian cancer cells to cisplatin-induced apoptosis by inhibiting cyto-protective autophagy.

    Science.gov (United States)

    He, Jun; Yu, Jing-Jie; Xu, Qing; Wang, Lin; Zheng, Jenny Z; Liu, Ling-Zhi; Jiang, Bing-Hua

    2015-01-01

    Cisplatin is commonly used in ovarian cancer treatment by inducing apoptosis in cancer cells as a result of lethal DNA damage. However, the intrinsic and acquired resistance to cisplatin in cancer cells remains a big challenge for improving overall survival. The cyto-protective functions of autophagy in cancer cells have been suggested as a potential mechanism for chemoresistance. Here, we reported MIR152 as a new autophagy-regulating miRNA that plays a role in cisplatin-resistance. We showed that MIR152 expression was dramatically downregulated in the cisplatin-resistant cell lines A2780/CP70, SKOV3/DDP compared with their respective parental cells, and in ovarian cancer tissues associated with cisplatin-resistance. Overexpression of MIR152 sensitized cisplatin-resistant ovarian cancer cells by reducing cisplatin-induced autophagy, enhancing cisplatin-induced apoptosis and inhibition of cell proliferation. A mouse subcutaneous xenograft tumor model using A2780/CP70 cells with overexpressing MIR152 was established and displayed decreased tumor growth in response to cisplatin. We also identified that ATG14 is a functional target of MIR152 in regulating autophagy inhibition. Furthermore, we found that EGR1 (early growth response 1) regulated the MIR152 gene at the transcriptional level. Ectopic expression of EGR1 enhanced efficacy of chemotherapy in A2780/CP70 cells. More importantly, these findings were relevant to clinical cases. Both EGR1 and MIR152 expression levels were significantly lower in ovarian cancer tissues with high levels of ERCC1 (excision repair cross-complementation group 1), a marker for cisplatin-resistance. Collectively, these data provide insights into novel mechanisms for acquired cisplatin-resistance. Activation of EGR1 and MIR152 may be a useful therapeutic strategy to overcome cisplatin-resistance by preventing cyto-protective autophagy in ovarian cancer.

  4. Sphingosylphosphorylcholine protects cardiomyocytes against ischemic apoptosis via lipid raft/PTEN/Akt1/mTOR mediated autophagy.

    Science.gov (United States)

    Yue, Hong-Wei; Liu, Jing; Liu, Ping-Ping; Li, Wen-Jing; Chang, Fen; Miao, Jun-Ying; Zhao, Jing

    2015-09-01

    Autophagy, evoked by diverse stresses including myocardial ischemia/reperfusion (I/R), profoundly affects the development of heart failure. However, the specific molecular basis of autophagy remains to be elucidated. Here we report that sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, significantly suppressed apoptosis and induced autophagy in cardiomyocytes. Blocking this SPC evoked autophagy by 3-methyladenine (3MA)-sensitized cardiomyocytes to serum deprivation-induced apoptosis. Subsequent studies revealed that SPC downregulated the phosphorylation of p70S6K and 4EBP1 (two substrates of mTOR) but enhanced that of JNK when inducing autophagy. We identified SPC as a switch for the activity of Akt1, a supposed upstream modulator of both mTOR and JNK. Furthermore, β-cyclodextrin, which destroys membrane cholesterol, abolished the SPC-reduced phosphorylation of both Akt and PTEN, thus inhibiting SPC-induced autophagy. In conclusion, SPC is a novel molecule protecting cardiomyocytes against apoptosis by promoting autophagy. The lipid raft/PTEN/Akt1/mTOR signal pathway is the underlying mechanism and might provide novel targets for cardiac failure therapy.

  5. Viruses, Autophagy Genes, and Crohn’s Disease

    OpenAIRE

    Hubbard, Vanessa M.; Ken Cadwell

    2011-01-01

    The etiology of the intestinal disease Crohn’s disease involves genetic factors as well as ill-defined environmental agents. Several genetic variants linked to this disease are associated with autophagy, a process that is critical for proper responses to viral infections. While a role for viruses in this disease remains speculative, accumulating evidence indicate that this possibility requires serious consideration. In this review, we will examine the three-way relationship between viruses, a...

  6. Perifosine induces protective autophagy and upregulation of ATG5 in human chronic myelogenous leukemia cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yin TONG; Yan-yan LIU; Liang-shun YOU; Wen-bin QIAN

    2012-01-01

    Aim:The efficacy of the Akt inhibitor perifosine against chronic myeloid leukemia (CML)cells and its mechanisms of action are unknown.In this study,the cytotoxic effects of perifosine on CML and acute myeloid leukemia (AML)cell lines were compared to elucidate the mechanisms underlying the differences.Methods:Human AML cell lines Kasumi-1 and HL-60,and the CML cell line K562 were used.Cell viability was quantitated using MTT assay.Apoptosis was determined using Annexin V-FITC/propidium iodide and Hoechst staining,which were followed by flow cytometry and fluorescence microscopy analysis,respectively.Caspase pathway activation and the expression of autophagy-related genes were examined using Western blot.Autophagy was studied using electron microscopy,the acridine orange staining method,and GFP-LC3 was examined with fluorescence microscopy.Results:In contrast to AML cell lines,the CML cell lines K562 and K562/G (an imatinib-insensitive CML cell line)were resistant to perifosine (2.5-20 μmol/L)in respect to inhibiting cell growth and inducing apoptosis.Perifosine (2.5,5,and 10 μmol/L)inhibited Akt and its phosphorylation in AML cells,but not in CML cells.Treatment with perifosine (20 μmol/L)resulted in autophagy in CML cells as shown by the increased formation of acidic vesicular organelles and the accumulation of LC3-II.Treatment of CML cells with perifosine (5,10,and 20 μmol/L)dose-dependently upregulated AGT5,but not Beclin 1 at the protein level.Furthermore,inhibition of autophagyby chloroquine (40 nmol/L)significantly suppressed the cell growth and induced apoptosis in CML cells treated with perifosine (20 μmol/L).Conclusion:Our results show that CML cell lines were resistant to the Akt inhibitor perifosine in vitro,which is due to perifosine-induced protective autophagy and upregulation of ATG5.

  7. Hinokitiol protects primary neuron cells against prion peptide-induced toxicity via autophagy flux regulated by hypoxia inducing factor-1.

    Science.gov (United States)

    Moon, Ji-Hong; Lee, Ju-Hee; Lee, You-Jin; Park, Sang-Youel

    2016-05-24

    Prion diseases are fatal neurodegenerative disorders that are derived from structural changes of the native PrPc. Recent studies indicated that hinokitiol induced autophagy known to major function that keeps cells alive under stressful conditions. We investigated whether hinokitiol induces autophagy and attenuates PrP (106-126)-induced neurotoxicity. We observed increase of LC3-II protein level, GFP-LC3 puncta by hinokitiol in neuronal cells. Addition to, electron microscopy showed that hinokitiol enhanced autophagic vacuoles in neuronal cells. We demonstrated that hinokitiol protects against PrP (106-126)-induced neurotoxicity via autophagy by using autophagy inhibitor, wortmannin and 3MA, and ATG5 small interfering RNA (siRNA). We checked hinokitiol activated the hypoxia-inducible factor-1α (HIF-1α) and identified that hinokitiol-induced HIF-1α regulated autophagy. Taken together, this study is the first report demonstrating that hinokitiol protected against prion protein-induced neurotoxicity via autophagy regulated by HIF-1α. We suggest that hinokitiol is a possible therapeutic strategy in neuronal disorders including prion disease.

  8. Corresponding erdosteine changes autophagy genes expression in hippocampus on Rhinitis medicamentosa model

    Directory of Open Access Journals (Sweden)

    Dokuyucu Recep

    2015-01-01

    Full Text Available In our study, rats were subjected to Oxymetazoline hydrochloride treatment and Rhinitis medicamentosa (RM was formed and then autophagy gene expression levels were determined after the application of an antioxidant agent erdosteine (ED. The rats were divided into three groups; Group 1 was the control group. Group 2 (RM and group 3 (RM+ED rats received two spray puffs of 0.05% oxymetazoline into the nasal cavities three times daily for eight weeks. After determination of RM in the rats, the RM group were killed. The ED+RM group received 10 mg/kg of an ED suspension. At the end of seven days, these rats were also killed. All groups’ hippocampus tissues were obtained for the measurement of autophagy gene expressions. In rhinitis medicamentosa group Atg5, Atg7 and Atg10 gene expressions in the left hippocampus were reduced as compared to control group (p=0.01, p>0.05, p=0.01, respectively. Also, erdosteine treatments were restored mRNA expression of autophagy genes. In right hippocampus of rhinitis medicamentosa group, Atg5 and Atg10 gene expressions was found to be down-regulated as compared to control group (p>0.05, p<0.05, respectively. Both BECN1 and ULK genes expression were found to be reduced in left hippocampus of rhinitis medicamentosa group. Erdosteine applications was restored the expression of these genes (p=0.03, p=0.03, respectively. Additionally, in right hippocampus, Erdosteine application was restored the expression of ULK gene (p=0.01. This is the first report that evaluated the expression autophagy genes in RM rat models and the changes observed after erdosteine applications.

  9. Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity.

    Science.gov (United States)

    Brigger, D; Schläfli, A M; Garattini, E; Tschan, M P

    2015-01-01

    All-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, is, along with other retinoids, a promising therapeutic agent for the treatment of a variety of solid tumors. On the one hand, preclinical studies have shown promising anticancer effects of ATRA in breast cancer; on the other hand, resistances occurred. Autophagy is a cellular recycling process that allows the degradation of bulk cellular contents. Tumor cells may take advantage of autophagy to cope with stress caused by anticancer drugs. We therefore wondered if autophagy is activated by ATRA in mammary tumor cells and if modulation of autophagy might be a potential novel treatment strategy. Indeed, ATRA induces autophagic flux in ATRA-sensitive but not in ATRA-resistant human breast cancer cells. Moreover, using different RAR agonists as well as RARα-knockdown breast cancer cells, we demonstrate that autophagy is dependent on RARα activation. Interestingly, inhibition of autophagy in breast cancer cells by either genetic or pharmacological approaches resulted in significantly increased apoptosis under ATRA treatment and attenuated epithelial differentiation. In summary, our findings demonstrate that ATRA-induced autophagy is mediated by RARα in breast cancer cells. Furthermore, inhibition of autophagy results in enhanced apoptosis. This points to a potential novel treatment strategy for a selected group of breast cancer patients where ATRA and autophagy inhibitors are applied simultaneously. PMID:26313912

  10. Upregulation of autophagy-related gene-5 (ATG-5 is associated with chemoresistance in human gastric cancer.

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    Jie Ge

    Full Text Available Autophagy-related gene-5 (ATG-5 is one of the key regulators of autophagic cell death. It has been widely regarded as a protective molecular mechanism for tumor cells during the course of chemotherapy. In the present study, we investigated the expression pattern of ATG-5 and multidrug resistance-associated protein-1 (MRP-1 in 135 gastric cancers (GC patients who were treated with epirubicin, cisplatin and 5-FU adjuvant chemotherapy (ECF following surgical resection and explored their potential clinical significance. We found that both ATG-5 (77.78% and MRP-1 (79.26% were highly expressed in GC patients. ATG-5 expression was significantly associated with depth of wall invasion, TNM stages and distant metastasis of GC (P<0.05, whereas MRP-1 expression was significantly linked with tumor size, depth of wall invasion, lymph node metastasis, TNM stages and differentiation status (P<0.05. ATG-5 expression was positively correlated with MRP-1 (rp = 0.616, P<0.01. Increased expression of ATG-5 and MPR-1 was significantly correlated with poor overall survival (OS; P<0.01 and disease free survival (DFS; P<0.01 of our GC cohort. Furthermore, we demonstrated that ATG-5 was involved in drug resistant of GC cells, which was mainly through regulating autophagy. Our data suggest that upregulated expression of ATG-5, an important molecular feature of protective autophagy, is associated with chemoresistance in GC. Expression of ATG-5 and MRP-1 may be independent prognostic markers for GC treatment.

  11. Autophagy attenuates diabetic glomerular damage through protection of hyperglycemia-induced podocyte injury.

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    Li Fang

    Full Text Available Despite the recent attention focused on the important role of autophagy in maintaining podocyte homeostasis, little is known about the changes and mechanisms of autophagy in podocyte dysfunction under diabetic condition. In this study, we investigated the role of autophagy in podocyte biology and its involvement in the pathogenesis of diabetic nephropathy. Podocytes had a high basal level of autophagy. And basal autophagy inhibition either by 3-methyladenenine (3-MA or by Beclin-1 siRNA was detrimental to its architectural structure. However, under diabetic condition in vivo and under high glucose conditions in vitro, high basal level of autophagy in podocytes became defective and defective autophagy facilitated the podocyte injury. Since the dynamics of endoplasmic reticulum(ER seemed to play a vital role in regulating the autophagic flux, the results that Salubrinal/Tauroursodeoxycholic acid (TUDCA could restore defective autophagy further indicated that the evolution of autophagy may be mediated by the changes of cytoprotective output in the ER stress. Finally, we demonstrated in vivo that the autophagy of podocyte was inhibited under diabetic status and TUDCA could improve defective autophagy. Taken together, these data suggested that autophagy might be interrupted due to the failure of ER cytoprotective capacity upon high glucose induced unmitigated stress, and the defective autophagy might accelerate the irreparable progression of diabetic nephropathy.

  12. The autophagy associated gene, ULK1, promotes tolerance to chronic and acute hypoxia

    International Nuclear Information System (INIS)

    Background and purpose: Tumor hypoxia is associated with therapy resistance and malignancy. Previously we demonstrated that activation of autophagy and the unfolded protein response (UPR) promote hypoxia tolerance. Here we explored the importance of ULK1 in hypoxia tolerance, autophagy induction and its prognostic value for recurrence after treatment. Material and methods: Hypoxic regulation of ULK1 mRNA and protein was assessed in vitro and in primary human head and neck squamous cell carcinoma (HNSCC) xenografts. Its importance in autophagy induction, mitochondrial homeostasis and tolerance to chronic and acute hypoxia was evaluated in ULK1 knockdown cells. The prognostic value of ULK1 mRNA expression was assessed in 82 HNSCC patients. Results: ULK1 enrichment was observed in hypoxic tumor regions. High enrichment was associated with a high hypoxic fraction. In line with these findings, high ULK1 expression in HNSCC patients appeared associated with poor local control. Exposure of cells to hypoxia induced ULK1 mRNA in a UPR and HIF1α dependent manner. ULK1 knockdown decreased autophagy activation, increased mitochondrial mass and ROS exposure and sensitized cells to acute and chronic hypoxia. Conclusions: We demonstrate that ULK1 is a hypoxia regulated gene and is associated with hypoxia tolerance and a worse clinical outcome

  13. Corresponding erdosteine changes autophagy genes expression in hippocampus on Rhinitis medicamentosa model

    OpenAIRE

    Dokuyucu Recep; Gogebakan Bulent; Cevik Cengiz

    2015-01-01

    In our study, rats were subjected to Oxymetazoline hydrochloride treatment and Rhinitis medicamentosa (RM) was formed and then autophagy gene expression levels were determined after the application of an antioxidant agent erdosteine (ED). The rats were divided into three groups; Group 1 was the control group. Group 2 (RM) and group 3 (RM+ED) rats received two spray puffs of 0.05% oxymetazoline into the nasal cavities three times daily for eight weeks. After...

  14. Taurine protects against As2O3-induced autophagy in livers of rat offsprings through PPARγ pathway.

    Science.gov (United States)

    Bai, Jie; Yao, Xiaofeng; Jiang, Liping; Zhang, Qiaoting; Guan, Huai; Liu, Shuang; Wu, Wei; Qiu, Tianming; Gao, Ni; Yang, Lei; Yang, Guang; Sun, Xiance

    2016-01-01

    Chronic exposures to arsenic had been associated with metabolism diseases. Peroxisome proliferator-activated receptor gamma (PPARγ) was found in the liver, regulated metabolism. Here, we found that the expression of PPARγ was decreased, the generation of reactive oxygen species (ROS) and autophagy were increased after treatment with As2O3 in offsprings' livers. Taurine (Tau), a sulfur-containing β-amino acid could reverse As2O3-inhibited PPARγ. Tau also inhibit the generation of ROS and autophagy. We also found that As2O3 caused autophagic cell death and ROS accelerated in HepG2 cells. Before incubation with As2O3, the cells were pretreated with PPARγ activator Rosiglitazone (RGS), we found that autophagy and ROS was inhibited in HepG2 cells, suggesting that inhibition of PPARγ contributed to As2O3-induced autophagy and the generation of ROS. After pretreatment with Tau, the level of PPARγ was improved and the autophagy and ROS was inhibited in As2O3-treated cells, suggesting that Tau could protect hepatocytes against As2O3 through modulating PPARγ pathway. PMID:27291853

  15. Variants of autophagy-related gene 5 are associated with neuromyelitis optica in the Southern Han Chinese population.

    Science.gov (United States)

    Cai, Ping-Ping; Wang, Hong-Xia; Zhuang, Jing-Cong; Liu, Qi-Bing; Zhao, Gui-Xian; Li, Zhen-Xin; Wu, Zhi-Ying

    2014-12-01

    Neuromyelitis optica (NMO) and multiple sclerosis (MS) are autoimmune demyelinating diseases of the central nervous system. The discovery of NMO immunoglobulin G (NMO-IgG) antibody has improved the clinical definition of NMO. Recently, the autophagy-related genes (ATGs) have been proved to be associated with several autoimmune and inflammation diseases. Increased T cell expression of ATG5 may be correlated with the pathogenesis of inflammatory demyelination in MS. However, the association of ATG5 variants with MS and NMO patients has not been well studied. In this study, five ATG5 variants were genotyped in 144 MS patients, 109 NMO patients and 288 controls in the Han Chinese population. In the cohort of NMO patients, we observed that the CC genotype of rs548234 increased susceptibility to NMO (p = 0.016), while the allele T of rs548234 (p = 0.003) and the allele A of rs6937876 (p = 0.009) acted as protective factors for NMO-IgG positive NMO patients. However, no association was found between ATG5 variants and MS patients. These results indicated that ATG5 variants are associated with NMO but not MS patients, which may provide a clue for further clarifying the autoimmune mechanisms of autophagy-related pathogenesis in NMO.

  16. Autophagy gene polymorphism is associated with susceptibility to leprosy by affecting inflammatory cytokines.

    Science.gov (United States)

    Yang, Degang; Chen, Jia; Shi, Chao; Jing, Zhichun; Song, Ningjing

    2014-04-01

    Autophagy and inflammation closely interact with each other, and together, they play critical roles in bacterial infection. Leprosy is caused by the infection of Mycobacterium leprae (M. leprae). The objective of the study was to investigate the association between polymorphisms in IRGM, an autophagy gene, and susceptibility to leprosy, and identify possible functions of the polymorphism in the infection of M. leprae. Two polymorphisms in IRGM, rs4958842 and rs13361189, were tested in 412 leprosy cases and 432 healthy controls. Levels of inflammatory cytokines including interleukin 1 beta, IL-4, IL-6, and interferon gamma (INF-γ) were measured after the infection of M. leprae in the peripheral blood mononuclear cell (PBMC) of subjects with different genotypes of rs13361189. Data showed that prevalence of rs13361189TC and CC genotypes were significantly higher in leprosy patients than in healthy controls (odds ratio (OR) = 1.49, 95 % confidence interval (CI) 1.09-2.04, P = 0.012; OR = 2.58, 95 % CI 1.65-4.05, P autophagy gene polymorphism was associated with the increased risk of leprosy by affecting inflammatory cytokines.

  17. Inhibition of glycogen synthase kinase 3β promotes autophagy to protect mice from acute liver failure mediated by peroxisome proliferator-activated receptor α

    OpenAIRE

    Ren, F.; Zhang, L; Zhang, X; Shi, H; T. Wen; Bai, L.; S. Zheng; Y. Chen; Chen, D.; Li, L.; Duan, Z

    2016-01-01

    Our previous studies have demonstrated that inhibition of glycogen synthase kinase 3β (GSK3β) activity protects mice from acute liver failure (ALF), whereas its protective and regulatory mechanism remains elusive. Autophagy is a recently recognized rudimentary cellular response to inflammation and injury. The aim of the present study was to test the hypothesis that inhibition of GSK3β mediates autophagy to inhibit liver inflammation and protect against ALF. In ALF mice model induced by d-gala...

  18. Autophagy protects monocytes from Wolbachia heat shock protein 60-induced apoptosis and senescence.

    Directory of Open Access Journals (Sweden)

    Vijayan Kamalakannan

    2015-04-01

    Full Text Available Monocyte dysfunction by filarial antigens has been a major mechanism underlying immune evasion following hyporesponsiveness during patent lymphatic filariasis. Recent studies have initiated a paradigm shift to comprehend the immunological interactions of Wolbachia and its antigens in inflammation, apoptosis, lymphocyte anergy, etc. Here we showed that recombinant Wolbachia heat shock protein 60 (rWmhsp60 interacts with TLR-4 and induces apoptosis in monocytes of endemic normal but not in chronic patients. Higher levels of reactive oxygen species (ROS induced after TLR-4 stimulation resulted in loss of mitochondrial membrane potential and caspase cascade activation, which are the plausible reason for apoptosis. Furthermore, release in ROS owing to TLR-4 signaling resulted in the activation of NF-κB p65 nuclear translocation which leads to inflammation and apoptosis via TNF receptor pathway following the increase in IL-6 and TNF-α level. Here for the first time, we report that in addition to apoptosis, rWmhsp60 antigen in filarial pathogenesis also induces molecular senescence in monocytes. Targeting TLR-4, therefore, presents a promising candidate for treating rWmhsp60-induced apoptosis and senescence. Strikingly, induction of autophagy by rapamycin detains TLR-4 in late endosomes and subverts TLR-4-rWmhsp60 interaction, thus protecting TLR-4-mediated apoptosis and senescence. Furthermore, rapamycin-induced monocytes were unresponsive to rWmhsp60, and activated lymphocytes following PHA stimulation. This study demonstrates that autophagy mediates the degradation of TLR-4 signaling and protects monocytes from rWmhsp60 induced apoptosis and senescence.

  19. Autophagy protects monocytes from Wolbachia heat shock protein 60-induced apoptosis and senescence.

    Science.gov (United States)

    Kamalakannan, Vijayan; Shiny, Abijit; Babu, Subash; Narayanan, Rangarajan Badri

    2015-04-01

    Monocyte dysfunction by filarial antigens has been a major mechanism underlying immune evasion following hyporesponsiveness during patent lymphatic filariasis. Recent studies have initiated a paradigm shift to comprehend the immunological interactions of Wolbachia and its antigens in inflammation, apoptosis, lymphocyte anergy, etc. Here we showed that recombinant Wolbachia heat shock protein 60 (rWmhsp60) interacts with TLR-4 and induces apoptosis in monocytes of endemic normal but not in chronic patients. Higher levels of reactive oxygen species (ROS) induced after TLR-4 stimulation resulted in loss of mitochondrial membrane potential and caspase cascade activation, which are the plausible reason for apoptosis. Furthermore, release in ROS owing to TLR-4 signaling resulted in the activation of NF-κB p65 nuclear translocation which leads to inflammation and apoptosis via TNF receptor pathway following the increase in IL-6 and TNF-α level. Here for the first time, we report that in addition to apoptosis, rWmhsp60 antigen in filarial pathogenesis also induces molecular senescence in monocytes. Targeting TLR-4, therefore, presents a promising candidate for treating rWmhsp60-induced apoptosis and senescence. Strikingly, induction of autophagy by rapamycin detains TLR-4 in late endosomes and subverts TLR-4-rWmhsp60 interaction, thus protecting TLR-4-mediated apoptosis and senescence. Furthermore, rapamycin-induced monocytes were unresponsive to rWmhsp60, and activated lymphocytes following PHA stimulation. This study demonstrates that autophagy mediates the degradation of TLR-4 signaling and protects monocytes from rWmhsp60 induced apoptosis and senescence. PMID:25849993

  20. A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes

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    Ahnn Joohong

    2010-01-01

    Full Text Available Abstract Background Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines. Results We screened ~15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. Conclusions We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future

  1. Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency.

    Science.gov (United States)

    McAfee, Quentin; Zhang, Zhihui; Samanta, Arabinda; Levi, Samuel M; Ma, Xiao-Hong; Piao, Shengfu; Lynch, John P; Uehara, Takeshi; Sepulveda, Antonia R; Davis, Lisa E; Winkler, Jeffrey D; Amaravadi, Ravi K

    2012-05-22

    Autophagy is a lysosome-dependent degradative process that protects cancer cells from multiple stresses. In preclinical models, autophagy inhibition with chloroquine (CQ) derivatives augments the efficacy of many anticancer therapies, but CQ has limited activity as a single agent. Clinical trials are underway combining anticancer agents with hydroxychloroquine (HCQ), but concentrations of HCQ required to inhibit autophagy are not consistently achievable in the clinic. We report the synthesis and characterization of bisaminoquinoline autophagy inhibitors that potently inhibit autophagy and impair tumor growth in vivo. The structural motifs that are necessary for improved autophagy inhibition compared with CQ include the presence of two aminoquinoline rings and a triamine linker and C-7 chlorine. The lead compound, Lys01, is a 10-fold more potent autophagy inhibitor than HCQ. Compared with HCQ, Lys05, a water-soluble salt of Lys01, more potently accumulates within and deacidifies the lysosome, resulting in impaired autophagy and tumor growth. At the highest dose administered, some mice develop Paneth cell dysfunction that resembles the intestinal phenotype of mice and humans with genetic defects in the autophagy gene ATG16L1, providing in vivo evidence that Lys05 targets autophagy. Unlike HCQ, significant single-agent antitumor activity is observed without toxicity in mice treated with lower doses of Lys05, establishing the therapeutic potential of this compound in cancer. PMID:22566612

  2. Ampelopsin protects endothelial cells from hyperglycemia-induced oxidative damage by inducing autophagy via the AMPK signaling pathway.

    Science.gov (United States)

    Liang, Xinyu; Zhang, Ting; Shi, Linying; Kang, Chao; Wan, Jing; Zhou, Yong; Zhu, Jundong; Mi, Mantian

    2015-01-01

    Diabetic angiopathy is a major diabetes-specific complication that often begins with endothelial dysfunction induced by hyperglycemia; however, the pathological mechanisms of this progression remain unclear. Ampelopsin is a natural flavonol that has strong antioxidant activity, but little information is available regarding its antidiabetic effect. This study focused on the effect of ampelopsin on hyperglycemia-induced oxidative damage and the underlying mechanism of this effect in human umbilical vein endothelial cells (HUVECs). We found that hyperglycemia impaired autophagy in HUVECs through the inhibition of AMP-activated protein kinase (AMPK), which directly led to endothelial cell damage. Ampelopsin significantly attenuated the detrimental effect of hyperglycemia-induced cell dysfunction in a concentration-dependent manner in HUVECs. Ampelopsin significantly upregulated LC3-II, Beclin1, and Atg5 protein levels but downregulated p62 protein levels in HUVECs. Transmission electron microscopy and confocal microscopy indicated that ampelopsin notably induced autophagosomes and LC3-II dots, respectively. Additionally, the autophagy-specific inhibitor 3-MA, as well as Atg5 and Beclin1 siRNA pretreatment, markedly attenuated ampelopsin-induced autophagy, which subsequently abolished the protective effect of ampelopsin against hyperglycemia in HUVECs. Moreover, ampelopsin also increased AMPK activity and inhibited mTOR (mammalian target of rapamycin) complex activation. Ampelopsin-induced autophagy was attenuated by the AMPK antagonist compound C but strengthened by the AMPK agonist AICAR (5-minoimidazole-4-carboxamide ribonucleotide). Furthermore, AMPK siRNA transfection eliminated ampelopsin's alleviation of cell injury induced by hyperglycemia. The protective effect of ampelopsin against hyperglycemia-induced cell damage, which functions by targeting autophagy via AMPK activation, makes it a promising pharmacological treatment for type-2 diabetes.

  3. Characterization of an Autophagy-related Gene MdATG8i from apple

    Directory of Open Access Journals (Sweden)

    Ping eWang

    2016-05-01

    Full Text Available Nutrient deficiencies restrict apple (Malus sp. tree growth and productivity in Northwest China. The process of autophagy, a conserved degradation pathway in eukaryotic cells, has important roles in nutrient-recycling and helps improve plant performance during periods of nutrient-starvation. Little is known about the functioning of autophagy-related genes (ATGs in apple. In this study, one of the ATG8 gene family members MdATG8i was isolated from M. domestica. MdATG8i has conserved putative tubulin binding sites and ATG7 interaction domains. A 1865-bp promoter region cloned from apple genome DNA was predicated to have cis-regulatory elements responsive to light, environmental stresses and hormones. MdATG8i transcriptions were induced in response to leaf senescence, nitrogen depletion, and oxidative stress. At cellular level, MdATG8i protein was expressed in the nucleus and cytoplasm of onion epidermal cells. Yeast two-hybrid tests showed that MdATG8i could interact with MdATG7a and MdATG7b. In Arabidopsis, its heterologous expression was associated with enhanced vegetative growth, leaf senescence, and tolerance to nitrogen- and carbon-starvation. MdATG8i-overexpressing ‘Orin’ apple callus lines also displayed improved tolerance to nutrient-limited conditions. Our results demonstrate that MdATG8i protein could function in autophagy in a conserved way, as a positive regulator in the response to nutrient-starvation.

  4. Autophagy in Inflammatory Diseases

    Directory of Open Access Journals (Sweden)

    Alexander J. S. Choi

    2011-01-01

    Full Text Available Autophagy provides a mechanism for the turnover of cellular organelles and proteins through a lysosome-dependent degradation pathway. During starvation, autophagy exerts a homeostatic function that promotes cell survival by recycling metabolic precursors. Additionally, autophagy can interact with other vital processes such as programmed cell death, inflammation, and adaptive immune mechanisms, and thereby potentially influence disease pathogenesis. Macrophages deficient in autophagic proteins display enhanced caspase-1-dependent proinflammatory cytokine production and the activation of the inflammasome. Autophagy provides a functional role in infectious diseases and sepsis by promoting intracellular bacterial clearance. Mutations in autophagy-related genes, leading to loss of autophagic function, have been implicated in the pathogenesis of Crohn's disease. Furthermore, autophagy-dependent mechanisms have been proposed in the pathogenesis of several pulmonary diseases that involve inflammation, including cystic fibrosis and pulmonary hypertension. Strategies aimed at modulating autophagy may lead to therapeutic interventions for diseases associated with inflammation.

  5. Potential autophagy enhancers protect against fipronil-induced apoptosis in SH-SY5Y cells.

    Science.gov (United States)

    Park, Jae Hyeon; Lee, Jeong Eun; Lee, Soo-Jin; Park, Soo Jin; Park, Kyung Hun; Jeong, Mihye; Koh, Hyun Chul

    2013-10-23

    Oxidative stress created by environmental toxicants activates several signaling pathways. Autophagy is one of the first lines of defense against oxidative stress damage. The autophagy pathway can be induced and up-regulated in response to intracellular reactive oxygen species (ROS). Recently, we reported that fipronil (FPN)-induced mitochondria-dependent apoptosis is mediated through ROS in human neuroblastoma SH-SY5Y cells. In this study, we explored the role of autophagy to prevent FPN neurotoxicity. We investigated the modulation of FPN-induced apoptosis according to autophagy regulation. FPN activated caspase-9 and caspase-3, and induced nuclear fragmentation and condensation, all of which indicate that FPN-induced cell death was due to apoptosis. In addition, we observed FPN-induced autophagic cell death by monitoring the expression of LC3-II and Beclin-1. Exposure to FPN in SH-SY5Y cells led to the production of ROS. Treatment with N-acetyl-cysteine (NAC) effectively blocked both apoptosis and autophagy. Interestingly, pretreatment with rapamycin, an autophagy inducer, significantly enhanced the viability of FPN-exposed cells; the enhancement of cell viability was partially due to alleviation of FPN-induced apoptosis via a decrease in levels of cleaved caspase-3. However, pretreatment with 3-methyladenine (3MA) a specific inhibitor for autophagy, remarkably strengthened FPN toxicity and further induced activation of caspase-3 in these cells. Our studies suggest that FPN-induced cytotoxicity is modified by autophagy regulation and that rapamycin is neuroprotective against FPN-induced apoptosis through enhancing autophagy.

  6. Atorvastatin Protects Vascular Smooth Muscle Cells From TGF-β1-Stimulated Calcification by Inducing Autophagy via Suppression of the β-Catenin Pathway

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    Demin Liu

    2014-01-01

    Full Text Available Background: Arterial calcification is a major event in the progression of atherosclerosis. It is reported that statins exhibit various protective effects against vascular smooth muscle cell (VSMC inflammation and proliferation in cardiovascular remodeling. Although statins counteract atherosclerosis, the molecular mechanisms of statins on the calcium release from VSMCs have not been clearly elucidated. Methods: Calcium content of VSMCs was measured using enzyme-linked immunosorbent assay (ELISA. The expression of proteins involved in cellular transdifferentiation was analyzed by western blot. Cell autophagy was measured by fluorescence microscopic analysis for acridine orange staining and transmission electron microscopy analysis. The autophagic inhibitors (3-MA, chloroquine, NH4Cl and bafilomycin A1 and β-catenin inhibitor JW74 were used to assess the effects of atorvastatin on autophagy and the involvement of β-catenin on cell calcification respectively. Furthermore, cell transfection was performed to overexpress β-catenin. Results: In VSMCs, atorvastatin significantly suppressed transforming growth factor-β1 (TGF-β1-stimulated calcification, accompanied by the induction of autophagy. Downregulation of autophagy with autophagic inhibitors significantly suppressed the inhibitory effect of atorvastatin on cell calcification. Moreover, the beneficial effect of atorvastatin on calcification and autophagy was reversed by β-catenin overexpression. Conversely, JW74 supplement enhanced this effect. Conclusion: These data demonstrated that atorvastatin protect VSMC from TGF-β1-stimulated calcification by inducing autophagy through suppression of the β-catenin pathway, identifying autophagy induction might be a therapeutic strategy for use in vascular calcification.

  7. Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae

    Institute of Scientific and Technical Information of China (English)

    Lei Zhou; Jun Zhao; Wangzhen Guo; Tianzhen Zhang

    2013-01-01

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins,organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi.However,the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood.Here,we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes,VdATG8 and VdATG12,by means of targeted gene replacement and complementadon.Transformation of a cotton-infecting Verticilliun dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 106 conidia.V.dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production.Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants,compared with the wildtype and gene complemented strains.Surprisingly,in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants.These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V.dahliae.

  8. Functional analysis of autophagy genes via Agrobacterium-mediated transformation in the vascular Wilt fungus Verticillium dahliae.

    Science.gov (United States)

    Zhou, Lei; Zhao, Jun; Guo, Wangzhen; Zhang, Tianzhen

    2013-08-20

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 10(6) conidia. V. dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild-type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V. dahliae.

  9. Autophagy Protects Against Senescence and Apoptosis via the RAS-Mitochondria in High-Glucose-Induced Endothelial Cells

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    Fei Chen

    2014-04-01

    Full Text Available Backgrounds: Autophagy is an important process in the pathogenesis of diabetes and plays a critical role in maintaining cellular homeostasis. However, the autophagic response and its mechanism in diabetic vascular endothelium remain unclear. Methods and Results: We studied high-glucose-induced renin-angiotensin system (RAS-mitochondrial damage and its effect on endothelial cells. With regard to therapeutics, we investigated the beneficial effect of angiotensin-converting enzyme inhibitors (ACEIs or angiotensin II type 1 receptor blockers (ARBs against high-glucose-induced endothelial responses. High glucose activated RAS, enhanced mitochondrial damage and increased senescence, apoptosis and autophagic-responses in endothelial cells, and these effects were mimicked by using angiotensin II (Ang. The use of an ACEI or ARB, however, inhibited the negative effects of high glucose. Direct mitochondrial injury caused by carbonyl cyanide 3-chlorophenylhydrazone (CCCP resulted in similar negative effects of high glucose or Ang and abrogated the protective effects of an ACEI or ARB. Additionally, by impairing autophagy, high-glucose-induced senescence and apoptosis were accelerated and the ACEI- or ARB-mediated beneficial effects were abolished. Furthermore, increases in FragEL™ DNA Fragmentation (TUNEL-positive cells, β-galactosidase activation and the expression of autophagic biomarkers were revealed in diabetic patients and rats, and the treatment with an ACEI or ARB decreased these responses. Conclusions: These data suggest that autophagy protects against senescence and apoptosis via RAS-mitochondria in high-glucose-induced endothelial cells.

  10. The V471A polymorphism in autophagy-related gene ATG7 modifies age at onset specifically in Italian Huntington disease patients

    DEFF Research Database (Denmark)

    Metzger, Silke; Walter, Carolin; Riess, Olaf;

    2013-01-01

    , we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second...

  11. N-Acetyl Cysteine Protects against Methamphetamine-Induced Dopaminergic Neurodegeneration via Modulation of Redox Status and Autophagy in Dopaminergic Cells

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    Prashanth Chandramani Shivalingappa

    2012-01-01

    Full Text Available Methamphetamine- (MA- induced neurotoxicity is associated with mitochondrial dysfunction and enhanced oxidative stress. Our previous study demonstrated that MA induces autophagy in a dopaminergic neuronal cell model (N27 cells. The cellular mechanisms underlying MA-induced autophagy and apoptosis remain poorly characterized. In the present study we sought to investigate the importance of GSH redox status in MA-induced neurotoxicity using a thiol antioxidant, N-acetylcysteine (NAC. Morphological and biochemical analysis revealed that MA-induced autophagy in N27 dopaminergic cells was associated with pronounced depletion of GSH levels. Moreover, pretreatment with NAC reduced MA-induced GSH depletion and autophagy, while depletion of GSH using L-buthionine sulfoximine (L-BSO enhanced autophagy. Furthermore, treatment with NAC significantly attenuated MA-induced apoptotic cell death as well as oxidative stress markers, namely, 3-nitrotyrosine (3-NT and 4-hydroxynonenal (4-HNE. Together, these results suggest that NAC exhibits significant protective effects against MA-induced dopaminergic cell death, presumably via modulation of the GSH level and autophagy. Collectively, our data provide mechanistic insights into the role of cellular GSH redox status in MA-induced autophagy and apoptotic cell death, and additional studies are needed to determine the therapeutic effectiveness of cellular redox modifiers in attenuating dopaminergic neurodegeneration in vivo.

  12. Dichloroacetate induces protective autophagy in LoVo cells: involvement of cathepsin D/thioredoxin-like protein 1 and Akt-mTOR-mediated signaling.

    Science.gov (United States)

    Gong, F; Peng, X; Sang, Y; Qiu, M; Luo, C; He, Z; Zhao, X; Tong, A

    2013-11-07

    Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), and recently it has been shown as a promising nontoxic antineoplastic agent. In this study, we demonstrated that DCA could induce autophagy in LoVo cells, which were confirmed by the formation of autophagosomes, appearance of punctate patterns of LC3 immunoreactivity and activation of autophagy associated proteins. Moreover, autophagy inhibition by 3-methyladenine (3-MA) or Atg7 siRNA treatment can significantly enhance DCA-induced apoptosis. To determine the underlying mechanism of DCA-induced autophagy, target identification using drug affinity responsive target stability (DARTS) coupled with ESI-Q-TOF MS/MS analysis were utilized to profile differentially expressed proteins between control and DCA-treated LoVo cells. As a result, Cathepsin D (CTSD) and thioredoxin-like protein 1 (TXNL1) were identified with significant alterations compared with control. Further study indicated that DCA treatment significantly promoted abnormal reactive oxygen species (ROS) production. On the other hand, DCA-triggered autophagy could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we demonstrated that the Akt-mTOR signaling pathway, a major negative regulator of autophagy, was suppressed by DCA treatment. To our knowledge, it was the first study to show that DCA induced protective autophagy in LoVo cells, and the potential mechanisms were involved in ROS imbalance and Akt-mTOR signaling pathway suppression.

  13. Apoptosis-related genes control autophagy and influence DENV-2 infection in the mosquito vector, Aedes aegypti.

    Science.gov (United States)

    Eng, Matthew W; van Zuylen, Madeleine N; Severson, David W

    2016-09-01

    The mosquito Aedes aegypti is the primary urban vector for dengue virus (DENV) worldwide. Insight into interactions occurring between host and pathogen is important in understanding what factors contribute to vector competence. However, many of the molecular mechanisms for vector competence remain unknown. Our previous global transcriptional analysis suggested that differential expression of apoptotic proteins is involved in determining refractoriness vs susceptibility to DENV-2 infection in Ae. aegypti females following a DENV-infected blood meal. To determine whether DENV-refractory Ae. aegypti showed more robust apoptosis upon infection, we compared numbers of apoptotic cells from midguts of refractory and susceptible strains and observed increased numbers of apoptotic cells in only the refractory strain upon DENV-2 infection. Thereafter, we manipulated apoptosis through dsRNA interference of the initiator caspase, Aedronc. Unexpectedly, dsAedronc-treated females showed both decreased frequency of disseminated infection and decreased virus titer in infected individuals. Insect caspases have also previously been identified as regulators of the cellular recycling process known as autophagy. We observed activation of autophagy in midgut and fat body tissues following a blood meal, as well as programmed activation of several apoptosis-related genes, including the effector caspase, Casps7. To determine whether autophagy was affected by caspase knockdown, we silenced Aedronc and Casps7, and observed reduced activation of autophagy upon silencing. Our results provide evidence that apoptosis-related genes are also involved in regulating autophagy, and that Aedronc may play an important role in DENV-2 infection success in Ae. aegypti, possibly through its regulation of autophagy. PMID:27418459

  14. Prohibitin 1 modulates mitochondrial stress-related autophagy in human colonic epithelial cells.

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    Arwa S Kathiria

    Full Text Available INTRODUCTION: Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα, both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB, which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells. METHODS: We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A(1 or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine. RESULTS: TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability. CONCLUSIONS: Decreased PHB levels coupled with dysfunctional

  15. Autophagy protects human brain microvascular endothelial cells against methylglyoxal-induced injuries, reproducible in a cerebral ischemic model in diabetic rats.

    Science.gov (United States)

    Fang, Lili; Li, Xue; Zhong, Yinbo; Yu, Jing; Yu, Lina; Dai, Haibin; Yan, Min

    2015-10-01

    Cerebral microvascular endothelial cells (ECs) are crucial for brain vascular repair and maintenance, but their physiological function may be impaired during ischemic stroke and diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, could exacerbate ischemia-induced EC injury and dysfunction. We investigated the protective effect of autophagy on cultured human brain microvascular endothelial cells (HBMEC) that underwent MGO treatment. A further study was conducted to explore the underlying mechanisms of the protective effect. Autophagic activity was assessed by evaluating protein levels, using western blot. 3-methyladenine (3-MA), bafilomycin A1, ammonium chloride (AC), Beclin 1 siRNA, and chloroquine (CQ) were used to cause autophagy inhibition. Alarmar blue assay and lactate dehydrogenase release assay were used to evaluate cell viability. Streptozotocin was administered to induce type I diabetes in rats and post-permanent middle cerebral artery occlusion was performed to elicit cerebral ischemia. Blood-brain barrier permeability was also assessed. Our study found that MGO reduced HBMEC cell viability in a concentration- and time-dependent manner, and triggered the responsive autophagy activation. Autophagy inhibitors bafilomycin A1, AC, 3-MA, and BECN1 siRNA exacerbated MGO-induced HBMEC injury. FAK phosphorylation inhibitor PF573228 inhibited MGO-triggered autophagy and enhanced lactate dehydrogenase release. Meanwhile, similar autophagy activation in brain vascular ECs was observed during permanent middle cerebral artery occlusion-induced cerebral ischemia in diabetic rats, while chloroquine-induced autophagy inhibition enhanced blood-brain barrier permeability. Taken together, our study indicates that autophagy triggered by MGO defends HBMEC against injuries. PMID:26251121

  16. ISG20L1 is a p53 family target gene that modulates genotoxic stress-induced autophagy

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    Johnson Kimberly N

    2010-04-01

    Full Text Available Abstract Background Autophagy is characterized by the sequestration of cytoplasm and organelles into multimembrane vesicles and subsequent degradation by the cell's lysosomal system. It is linked to many physiological functions in human cells including stress response, protein degradation, organelle turnover, caspase-independent cell death and tumor suppression. Malignant transformation is frequently associated with deregulation of autophagy and several tumor suppressors can modulate autophagic processes. The tumor suppressor p53 can induce autophagy after metabolic or genotoxic stress through transcriptionally-dependent and -independent mechanisms. In this study we expand on the former mechanism by functionally characterizing a p53 family target gene, ISG20L1 under conditions of genotoxic stress. Results We identified a p53 target gene, ISG20L1, and show that transcription of the gene can be regulated by all three p53 family members (p53, p63, and p73. We generated an antibody to ISG20L1 and found that it localizes to the nucleolar and perinucleolar regions of the nucleus and its protein levels increase in a p53- and p73-dependent manner after various forms of genotoxic stress. When ectopically expressed in epithelial cancer-derived cell lines, ISG20L1 expression decreased clonogenic survival without a concomitant elevation in apoptosis and this effect was partially rescued in cells that were ATG5 deficient. Knockdown of ISG20L1 did not alter 5-FU induced apoptosis as assessed by PARP and caspase-3 cleavage, sub-G1 content, and DNA laddering. Thus, we investigated the role of ISG20L1 in autophagy, a process commonly associated with type II cell death, and found that ISG20L1 knockdown decreased levels of autophagic vacuoles and LC3-II after genotoxic stress as assessed by electron microscopy, biochemical, and immunohistochemical measurements of LC3-II. Conclusions Our identification of ISG20L1 as a p53 family target and discovery that modulation

  17. Autophagy in infection.

    Science.gov (United States)

    Deretic, Vojo

    2010-04-01

    Autophagy is a ubiquitous eukaryotic cytoplasmic quality and quantity control pathway. The role of autophagy in cytoplasmic homeostasis seamlessly extends to cell-autonomous defense against intracellular microbes. Recent studies also point to fully integrated, multitiered regulatory and effector connections between autophagy and nearly all facets of innate and adaptive immunity. Autophagy in the immune system as a whole confers measured immune responses; on the flip side, suppression of autophagy can lead to inflammation and tissue damage, as evidenced by Crohn's disease predisposition polymorphisms in autophagy basal apparatus (Atg16L) and regulatory (IRGM) genes. Polymorphisms in the IRGM gene in human populations have also been linked to predisposition to tuberculosis. There are several areas of most recent growth: first, links between autophagy regulators and infectious disease predisposition in human populations; second, demonstration of a role for autophagy in infection control in vivo in animal models; third, the definition of specific antiautophagic defenses in highly evolved pathogens; and fourth, recognition of connections between the ubiquitin system and autophagy of bacteria (and interestingly mitochondria, which are incidentally organelles of bacterial evolutionary origin) via a growing list of modifier and adapter proteins including p62/SQSTM1, NDP52, Atg32, Parkin, and Nix/BNIP3L. PMID:20116986

  18. Autophagy Genes Enhance Murine Gammaherpesvirus 68 Reactivation from Latency by Preventing Virus-Induced Systemic Inflammation.

    Science.gov (United States)

    Park, Sunmin; Buck, Michael D; Desai, Chandni; Zhang, Xin; Loginicheva, Ekaterina; Martinez, Jennifer; Freeman, Michael L; Saitoh, Tatsuya; Akira, Shizuo; Guan, Jun-Lin; He, You-Wen; Blackman, Marcia A; Handley, Scott A; Levine, Beth; Green, Douglas R; Reese, Tiffany A; Artyomov, Maxim N; Virgin, Herbert W

    2016-01-13

    Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine gammaherpesvirus 68 (MHV68) infection is characterized by latency in macrophages, and reactivation is inhibited by interferon-γ (IFN-γ). Using a lysozyme-M-cre (LysMcre) expression system, we show that deletion of autophagy-related (Atg) genes Fip200, beclin 1, Atg14, Atg16l1, Atg7, Atg3, and Atg5, in the myeloid compartment, inhibited MHV68 reactivation in macrophages. Atg5 deficiency did not alter reactivation from B cells, and effects on reactivation from macrophages were not explained by alterations in productive viral replication or the establishment of latency. Rather, chronic MHV68 infection triggered increased systemic inflammation, increased T cell production of IFN-γ, and an IFN-γ-induced transcriptional signature in macrophages from Atg gene-deficient mice. The Atg5-related reactivation defect was partially reversed by neutralization of IFN-γ. Thus Atg genes in myeloid cells dampen virus-induced systemic inflammation, creating an environment that fosters efficient MHV68 reactivation from latency. PMID:26764599

  19. Novel incretin analogues improve autophagy and protect from mitochondrial stress induced by rotenone in SH-SY5Y cells.

    Science.gov (United States)

    Jalewa, Jaishree; Sharma, Mohit Kumar; Hölscher, Christian

    2016-10-01

    Currently, there is no viable treatment available for Parkinson's disease (PD) that stops or reverses disease progression. Interestingly, studies testing the glucagon-like-peptide-1 (GLP-1) mimetic Exendin-4 have shown neuroprotective/neurorestorative properties in pre-clinical tests and in a pilot clinical study of PD. Incretin analogues were originally developed to treat type 2 diabetes and several are currently on the market. In this study, we tested novel incretin analogues on the dopaminergic SH-SY5Y neuroblastoma cells against a toxic mitochondrial complex I inhibitor, Rotenone. Here, we investigate for the first time the effects of six different incretin receptor agonists - Liraglutide, D-Ser2-Oxyntomodulin, a GLP-1/GIP Dual receptor agonist, dAla(2)-GIP-GluPal, Val(8)GLP-1-GluPal and exendin-4. Post-treatment with doses of 1, 10 or 100 nM of incretin analogues for 12 h increased the survival of SH-SY5Y cells treated with 1 μM Rotenone for 12 h. Furthermore, we studied the post-treatment effect of 100 nM incretin analogues against 1 μM Rotenone stress on apoptosis, mitochondrial stress and autophagy markers. We found significant protective effects of the analogues against Rotenone stress on cell survival and on mitochondrial and autophagy-associated markers. The novel GLP-1/GIP Dual receptor agonist was superior and effective at a tenfold lower concentration compared to the other analogues. Using the Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, we further show that the neuroprotective effects are partially PI3K-independent. Our data suggest that the neuroprotective properties exhibited by incretin analogues against Rotenone stress involve enhanced autophagy, increased Akt-mediated cell survival and amelioration of mitochondrial dysfunction. These mechanisms can explain the neuroprotective effects of incretin analogues reported in clinical trials. GLP-1, GIP and dual incretin receptor agonists showed protective effects in SH-SY5Y cells

  20. Cationic poly(amidoamine) dendrimers induced cyto-protective autophagy in hepatocellular carcinoma cells

    Science.gov (United States)

    Li, Yubin; Wang, Shaofei; Wang, Ziyu; Qian, Xiaolu; Fan, Jiajun; Zeng, Xian; Sun, Yun; Song, Ping; Feng, Meiqing; Ju, Dianwen

    2014-09-01

    Poly(amidoamine) (PAMAM) dendrimers are proposed as one of the most promising nanomaterials for biomedical applications because of their unique tree-like structure, monodispersity and tunable properties. In this study, we found that PAMAM dendrimers could induce the formation of autophagosomes and the conversion of microtubule-associated protein 1 light chain 3 (LC3) in hepatocellular carcinoma HepG2 cells, while the inhibition of the Akt/mTOR and activation of the Erk 1/2 signaling pathways were involved in autophagy-induced by PAMAM dendrimers. We also investigated the suppression of autophagy with the obviously enhanced cytotoxicity of PAMAM dendrimers. Moreover, the blockage of a reactive oxygen species (ROS) could enhance the growth inhibition and apoptosis of hepatocellular carcinoma cells, induced by PAMAM dendrimers through reducing autophagic effects. Taken together, these findings explored the role and mechanism of autophagy induced by PAMAM dendrimers in HepG2 cells, provided new insight into the effect of autophagy on drug delivery nanomaterials and tumor cells and contributed to the use of a drug delivery vehicle for hepatocellular carcinoma treatment.

  1. Autophagy and protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 alpha kinase (eIF2α) pathway protect ovarian cancer cells from metformin-induced apoptosis.

    Science.gov (United States)

    Moon, Hee-Sun; Kim, Boyun; Gwak, HyeRan; Suh, Dong Hoon; Song, Yong Sang

    2016-04-01

    Metformin, an oral biguanide for the treatment of type II diabetes, has been shown to have anticancer effects in ovarian cancer. Energy starvation induced by metformin causes endoplasmic reticulum stress-mediated unfolded protein response (UPR) and autophagy. UPR and autophagy act as a survival or death mechanism in cells. In this study, we observed that metformin-induced apoptosis was relieved by autophagy and the PERK/eIF2α pathway in ovarian cancer cells, but not in peripheral blood mononuclear cells (PBMC) or 'normal' ovarian surface epithelial cells (OSE). Increased PARP cleavage and increased LC3B-II with ATG5-ATG12 complex suggested the induction of apoptosis and autophagy, respectively, in metformin-treated ovarian cancer cells. Accumulation of acidic vacuoles in the cytoplasm and downregulation of p62 further supported late-stage autophagy. Interestingly, metformin induced interdependent activation between autophagy and the UPR, especially the PERK/eIF2α pathway. Inhibition of autophagy-induced PERK inhibition, and vice versa, were demonstrated using small molecular inhibitors (PERK inhibitor I, GSK2606414; autophagy inhibitor, 3-MA, and BafA1). Moreover, autophagy and PERK activation protected ovarian cancer cells against metformin-induced apoptosis. Metformin treatment in the presence of inhibitors of PERK and autophagy, however, had no cytotoxic effects on OSE or PBMC. In conclusion, these results suggest that inhibition of autophagy and PERK can enhance the selective anticancer effects of metformin on ovarian cancer cells. © 2015 Wiley Periodicals, Inc.

  2. Autophagy facilitates secretion and protects against degeneration of the Harderian gland.

    Science.gov (United States)

    Koenig, Ulrich; Fobker, Manfred; Lengauer, Barbara; Brandstetter, Marlene; Resch, Guenter P; Gröger, Marion; Plenz, Gabriele; Pammer, Johannes; Barresi, Caterina; Hartmann, Christine; Rossiter, Heidemarie

    2015-01-01

    The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to

  3. Bexarotene targets autophagy and is protective against thromboembolic stroke in aged mice with tauopathy

    Science.gov (United States)

    Huuskonen, Mikko T.; Loppi, Sanna; Dhungana, Hiramani; Keksa-Goldsteine, Velta; Lemarchant, Sighild; Korhonen, Paula; Wojciechowski, Sara; Pollari, Eveliina; Valonen, Piia; Koponen, Juho; Takashima, Akihiko; Landreth, Gary; Goldsteins, Gundars; Malm, Tarja; Koistinaho, Jari; Kanninen, Katja M.

    2016-01-01

    Stroke is a highly debilitating, often fatal disorder for which current therapies are suitable for only a minor fraction of patients. Discovery of novel, effective therapies is hampered by the fact that advanced age, primary age-related tauopathy or comorbidities typical to several types of dementing diseases are usually not taken into account in preclinical studies, which predominantly use young, healthy rodents. Here we investigated for the first time the neuroprotective potential of bexarotene, an FDA-approved agent, in a co-morbidity model of stroke that combines high age and tauopathy with thromboembolic cerebral ischemia. Following thromboembolic stroke bexarotene enhanced autophagy in the ischemic brain concomitantly with a reduction in lesion volume and amelioration of behavioral deficits in aged transgenic mice expressing the human P301L-Tau mutation. In in vitro studies bexarotene increased the expression of autophagy markers and reduced autophagic flux in neuronal cells expressing P301L-Tau. Bexarotene also restored mitochondrial respiration deficits in P301L-Tau neurons. These newly described actions of bexarotene add to the growing amount of compelling data showing that bexarotene is a potent neuroprotective agent, and identify a novel autophagy-modulating effect of bexarotene. PMID:27624652

  4. Bexarotene targets autophagy and is protective against thromboembolic stroke in aged mice with tauopathy.

    Science.gov (United States)

    Huuskonen, Mikko T; Loppi, Sanna; Dhungana, Hiramani; Keksa-Goldsteine, Velta; Lemarchant, Sighild; Korhonen, Paula; Wojciechowski, Sara; Pollari, Eveliina; Valonen, Piia; Koponen, Juho; Takashima, Akihiko; Landreth, Gary; Goldsteins, Gundars; Malm, Tarja; Koistinaho, Jari; Kanninen, Katja M

    2016-01-01

    Stroke is a highly debilitating, often fatal disorder for which current therapies are suitable for only a minor fraction of patients. Discovery of novel, effective therapies is hampered by the fact that advanced age, primary age-related tauopathy or comorbidities typical to several types of dementing diseases are usually not taken into account in preclinical studies, which predominantly use young, healthy rodents. Here we investigated for the first time the neuroprotective potential of bexarotene, an FDA-approved agent, in a co-morbidity model of stroke that combines high age and tauopathy with thromboembolic cerebral ischemia. Following thromboembolic stroke bexarotene enhanced autophagy in the ischemic brain concomitantly with a reduction in lesion volume and amelioration of behavioral deficits in aged transgenic mice expressing the human P301L-Tau mutation. In in vitro studies bexarotene increased the expression of autophagy markers and reduced autophagic flux in neuronal cells expressing P301L-Tau. Bexarotene also restored mitochondrial respiration deficits in P301L-Tau neurons. These newly described actions of bexarotene add to the growing amount of compelling data showing that bexarotene is a potent neuroprotective agent, and identify a novel autophagy-modulating effect of bexarotene. PMID:27624652

  5. The role of autophagy in the intracellular survival of Campylobacter concisus

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    Jose A. Burgos-Portugal

    2014-01-01

    Full Text Available Campylobacter concisus is an emerging pathogen that has been associated with gastrointestinal diseases. Given the importance of autophagy for the elimination of intracellular bacteria and the subversion of this process by pathogenic bacteria, we investigated the role of autophagy in C. concisus intracellular survival. Gentamicin protection assays were employed to assess intracellular levels of C. concisus within Caco-2 cells, following autophagy induction and inhibition. To assess the interaction between C. concisus and autophagosomes, confocal microscopy, scanning electron microscopy, and transmission electron microscopy were employed. Expression levels of 84 genes involved in the autophagy process were measured using qPCR. Autophagy inhibition resulted in two- to four-fold increases in intracellular levels of C. concisus within Caco-2 cells, while autophagy induction resulted in a significant reduction in intracellular levels or bacterial clearance. C. concisus strains with low intracellular survival levels showed a dramatic increase in these levels upon autophagy inhibition. Confocal microscopy showed co-localization of the bacterium with autophagosomes, while transmission electron microscopy identified intracellular bacteria persisting within autophagic vesicles. Further, qPCR showed that following infection, 13 genes involved in the autophagy process were significantly regulated, and a further five showed borderline results, with an overall indication towards a dampening effect exerted by the bacterium on this process. Our data collectively indicates that while autophagy is important for the clearance of C. concisus, some strains may manipulate this process to benefit their intracellular survival.

  6. Autophagy in Hepatic Fibrosis

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    Yang Song

    2014-01-01

    Full Text Available Hepatic fibrosis is a leading cause of morbidity and mortality worldwide. Hepatic fibrosis is usually associated with chronic liver diseases caused by infection, drugs, metabolic disorders, or autoimmune imbalances. Effective clinical therapies are still lacking. Autophagy is a cellular process that degrades damaged organelles or protein aggregation, which participates in many pathological processes including liver diseases. Autophagy participates in hepatic fibrosis by activating hepatic stellate cells and may participate as well through influencing other fibrogenic cells. Besides that, autophagy can induce some liver diseases to develop while it may play a protective role in hepatocellular abnormal aggregates related liver diseases and reduces fibrosis. With a better understanding of the potential effects of autophagy on hepatic fibrosis, targeting autophagy might be a novel therapeutic strategy for hepatic fibrosis in the near future.

  7. Sulforaphane protects against rotenone-induced neurotoxicity in vivo: Involvement of the mTOR, Nrf2, and autophagy pathways

    Science.gov (United States)

    Zhou, Qian; Chen, Bin; Wang, Xindong; Wu, Lixin; Yang, Yang; Cheng, Xiaolan; Hu, Zhengli; Cai, Xueting; Yang, Jie; Sun, Xiaoyan; Lu, Wuguang; Yan, Huaijiang; Chen, Jiao; Ye, Juan; Shen, Jianping; Cao, Peng

    2016-01-01

    Sulforaphane, a naturally occurring compound found in cruciferous vegetables, has been shown to be neuroprotective in several neurological disorders. In this study, we sought to investigate the potential protective effects and associated molecular mechanisms of sulforaphane in an in vivo Parkinson’s disease (PD) model, based on rotenone-mediated neurotoxicity. Our results showed that sulforaphane inhibited rotenone-induced locomotor activity deficiency and dopaminergic neuronal loss. Additionally, sulforaphane treatment inhibited the rotenone-induced reactive oxygen species production, malondialdehyde (MDA) accumulation, and resulted in an increased level of total glutathione and reduced glutathione (GSH): oxidized glutathione (GSSG) in the brain. Western blot analysis illustrated that sulforaphane increased the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase (NQO1), the latter two of which are anti-oxidative enzymes. Moreover, sulforaphane treatment significantly attenuated rotenone-inhibited mTOR-mediated p70S6K and 4E-BP1 signalling pathway, as well as neuronal apoptosis. In addition, sulforaphane rescued rotenone-inhibited autophagy, as detected by LC3-II. Collectively, these findings demonstrated that sulforaphane exert neuroprotective effect involving Nrf2-dependent reductions in oxidative stress, mTOR-dependent inhibition of neuronal apoptosis, and the restoration of normal autophagy. Sulforaphane appears to be a promising compound with neuroprotective properties that may play an important role in preventing PD. PMID:27553905

  8. Autophagy in periodontitis patients and gingival fibroblasts: unraveling the link between chronic diseases and inflammation

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    Bullon Pedro

    2012-10-01

    Full Text Available Abstract Background Periodontitis, the most prevalent chronic inflammatory disease, has been related to cardiovascular diseases. Autophagy provides a mechanism for the turnover of cellular organelles and proteins through a lysosome-dependent degradation pathway. The aim of this research was to study the role of autophagy in peripheral blood mononuclear cells from patients with periodontitis and gingival fibroblasts treated with a lipopolysaccharide of Porphyromonas gingivalis. Autophagy-dependent mechanisms have been proposed in the pathogenesis of inflammatory disorders and in other diseases related to periodontitis, such as cardiovascular disease and diabetes. Thus it is important to study the role of autophagy in the pathophysiology of periodontitis. Methods Peripheral blood mononuclear cells from patients with periodontitis (n = 38 and without periodontitis (n = 20 were used to study autophagy. To investigate the mechanism of autophagy, we evaluated the influence of a lipopolysaccharide from P. gingivalis in human gingival fibroblasts, and autophagy was monitored morphologically and biochemically. Autophagosomes were observed by immunofluorescence and electron microscopy. Results We found increased levels of autophagy gene expression and high levels of mitochondrial reactive oxygen species production in peripheral blood mononuclear cells from patients with periodontitis compared with controls. A significantly positive correlation between both was observed. In human gingival fibroblasts treated with lipopolysaccharide from P. gingivalis, there was an increase of protein and transcript of autophagy-related protein 12 (ATG12 and microtubule-associated protein 1 light chain 3 alpha LC3. A reduction of mitochondrial reactive oxygen species induced a decrease in autophagy whereas inhibition of autophagy in infected cells increased apoptosis, showing the protective role of autophagy. Conclusion Results from the present study suggest that autophagy

  9. Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3.

    Science.gov (United States)

    Duan, Liang; Yi, Min; Chen, Juan; Li, Shengjin; Chen, Weixian

    2016-05-13

    Autophagy plays a crucial role in the progress of Mycobacterium tuberculosis (MTB) infection. Recently, MTB enhanced intracellular survival (EIS) protein was reported to be secreted from MTB cells and linked to the inhibition of autophagy and the intracellular persistence of the pathogen. Here, we investigated the mechanism of EIS-mediated inhibition of autophagy in a human phorbol myristate acetate (PMA)-treated THP-1 cell line as well as in murine macrophages. We confirmed that the presence of EIS led to the inhibition of rapamycin (Rapa)-induced autophagy, while IL-10 gene expression was increased and Akt/mTOR/p70S6K pathway was activated during the process. IL-10 gene silencing led to a significant recovery of EIS-mediated autophagy suppression and decreased activity of the Akt/mTOR/p70S6K pathway. IL-10 promoter activity was unaffected by EIS. Remarkably, EIS increased the acetylation level of histone H3 (Ac-H3), which binds to the SP1 and STAT3 region of the human IL-10 gene promoter sequence. Thus, EIS protein possibly increased IL-10 expression through the regulation of Ac-H3 of its promoter. Our data demonstrated that one possible mechanism of the MTB evasion of autophagy is that the EIS protein up-regulates IL-10 via Ac-H3 and thus activates Akt/mTOR/p70S6K pathway. PMID:27079235

  10. Relationship between autophagy-related genes Beclin-1 and MAP1LC3 expression and biological characteristics of oral cancer

    Institute of Scientific and Technical Information of China (English)

    Xiao-Dong Li; Xiao-Chen Sun; Xin-Mei Li; Jia-Wei Gu

    2016-01-01

    Objective:To study the relationship between autophagy-related genes Beclin-1 and MAP1LC3 expression and biological characteristics of oral cancer. Methods:Oral cancer tissues and precancerous tissues were collected to detect mRNA expression levels of Beclin-1 and MAP1LC3;tongue cancer cell lines CTST-2 and primary epithelial cells of normal buccal mucosa were cultured to detect mRNA expression levels of autophagy marker molecues (Beclin-1 and MAP1LC3), pro-apoptosis genes (P53 and Caspase-3) and anti-apoptosis genes (Survivin, Bcl-2 and Bmi-1). Results:mRNA contents of Beclin-1 and MAP1LC3 in tongue cancer, buccal mucosa cancer, gingival cancer and mouth floor cancer tissues were significantly lower than those in corresponding precancerous tissues; mRNA contents of Beclin-1 and MAP1LC3 in tongue cancer cells CTST-2 were lower than those in normal mucosal cells;mRNA contents of P53 and Caspase-3 in tongue cancer cells CTST-2 were lower than those in normal mucosal cells and positively correlated with mRNA contents of Beclin-1 and MAP1LC3; mRNA contents of survivin, Bcl-2 and Bmi-1 in CTST-2 were higher than those in normal mucosal cells and negatively correlated with mRNA contents of Beclin-1 and MAP1LC3. Conclusion:Expression levels of autophagy-related genes Beclin-1 and MAP1LC3 abnormally reduce in oral cancer and have significant correlation with the expression of pro-apoptosis genes and anti-apoptosis genes of cancer cells.

  11. Animal genes identification and mTOR signaling reactivation in autophagy

    Institute of Scientific and Technical Information of China (English)

    Xuejun Jiang

    2010-01-01

    @@ Autophagy(self-eating)is a self-degradation process essential for survival,differentiation,development,and homeostasis.Conceiving that a process of cellular self-eating could be beneficial may appear bewildering.In its simplest form,however,autophagy is probably a single cell's adjustment to starvation; the cell is forced to break down part of its own reserves to keep alive until circumstances improve(Mizushima and Klionsky,2007).

  12. Autophagy studies in Bombyx mori

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    L Tian

    2015-03-01

    Full Text Available Autophagy, which is well conserved from yeast to mammals, plays essential roles in development and diseases. Using the domesticated silkworm, Bombyx mori, as a model insect, several reports on autophagy have been made recently. Autophagic features are observed in the midgut and fat body during the larval-pupal transition as well as the silk gland and ovarian nurse cells during the pupal stage. There are 14 autophagy related (Atg genes, including at least two transcript variants of Atg1, predicated in Bombyx. Expression of most Atg genes is consistent with the autophagy process in the fat body during the larval-pupal transition, and reduction of Atg1 expression by RNAi blocks this process. The molting hormone, 20-hydroxyecdysone (20E, and starvation induce autophagy in the fat body by upregulating Atg gene expression and blocking the PI3K-TORC1 pathway. Meanwhile, autophagy precedes apoptosis in the midgut and other larval tissues during the larval-pupal transition, while the detailed mechanism is not illustrated yet. We assume that there are at least four future directions about autophagy studies in Bombyx during the next years: (1 physiological functions of autophagy; (2 identification of new components involved in the autophagy process; (3 detailed molecular mechanism of autophagosome formation; (4 functional relationship between autophagy and apoptosis.

  13. A role for autophagy in the extension of lifespan by dietary restriction in C. elegans.

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    Malene Hansen

    2008-02-01

    Full Text Available In many organisms, dietary restriction appears to extend lifespan, at least in part, by down-regulating the nutrient-sensor TOR (Target Of Rapamycin. TOR inhibition elicits autophagy, the large-scale recycling of cytoplasmic macromolecules and organelles. In this study, we asked whether autophagy might contribute to the lifespan extension induced by dietary restriction in C. elegans. We find that dietary restriction and TOR inhibition produce an autophagic phenotype and that inhibiting genes required for autophagy prevents dietary restriction and TOR inhibition from extending lifespan. The longevity response to dietary restriction in C. elegans requires the PHA-4 transcription factor. We find that the autophagic response to dietary restriction also requires PHA-4 activity, indicating that autophagy is a transcriptionally regulated response to food limitation. In spite of the rejuvenating effect that autophagy is predicted to have on cells, our findings suggest that autophagy is not sufficient to extend lifespan. Long-lived daf-2 insulin/IGF-1 receptor mutants require both autophagy and the transcription factor DAF-16/FOXO for their longevity, but we find that autophagy takes place in the absence of DAF-16. Perhaps autophagy is not sufficient for lifespan extension because although it provides raw material for new macromolecular synthesis, DAF-16/FOXO must program the cells to recycle this raw material into cell-protective longevity proteins.

  14. Protective Effect of Astaxanthin on Liver Fibrosis through Modulation of TGF-β1 Expression and Autophagy

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    Miao Shen

    2014-01-01

    Full Text Available Liver fibrosis is a common pathway leading to cirrhosis and a worldwide clinical issue. Astaxanthin is a red carotenoid pigment with antioxidant, anticancer, and anti-inflammatory properties. The aim of this study was to investigate the effect of astaxanthin on liver fibrosis and its potential protective mechanisms. Liver fibrosis was induced in a mouse model using CCL4 (intraperitoneal injection, three times a week for 8 weeks, and astaxanthin was administered everyday at three doses (20, 40, and 80 mg/kg. Pathological results indicated that astaxanthin significantly improved the pathological lesions of liver fibrosis. The levels of alanine aminotransferase aspartate aminotransferase and hydroxyproline were also significantly decreased by astaxanthin. The same results were confirmed in bile duct liagtion, (BDL model. In addition, astaxanthin inhibited hepatic stellate cells (HSCs activation and formation of extracellular matrix (ECM by decreasing the expression of NF-κB and TGF-β1 and maintaining the balance between MMP2 and TIMP1. In addition, astaxanthin reduced energy production in HSCs by downregulating the level of autophagy. These results were simultaneously confirmed in vivo and in vitro. In conclusion, our study showed that 80 mg/kg astaxanthin had a significant protective effect on liver fibrosis by suppressing multiple profibrogenic factors.

  15. Autophagy: for better or for worse

    OpenAIRE

    Wirawan, Ellen; Berghe, Tom Vanden; Lippens, Saskia; Agostinis, Patrizia; Vandenabeele, Peter

    2011-01-01

    Autophagy is a lysosomal degradation pathway that degrades damaged or superfluous cell components into basic biomolecules, which are then recycled back into the cytosol. In this respect, autophagy drives a flow of biomolecules in a continuous degradation-regeneration cycle. Autophagy is generally considered a pro-survival mechanism protecting cells under stress or poor nutrient conditions. Current research clearly shows that autophagy fulfills numerous functions in vital biological processes....

  16. Autophagy During Cardiac Stress: Joys and Frustrations of Autophagy

    Science.gov (United States)

    Gottlieb, Roberta A.; Mentzer, Robert M.

    2013-01-01

    The study of autophagy has been transformed by the cloning of most genes in the pathway and the introduction of GFP-LC3 as a reporter to allow visual assessment of autophagy. The field of cardiac biology is not alone in attempting to understand the implications of autophagy. The purpose of this review is to address some of the controversies and conundrums associated with the evolving studies of autophagy in the heart. Autophagy is a cellular process involving a complex orchestration of regulatory gene products as well as machinery for assembly, selective targeting, and degradation of autophagosomes and their contents. Our understanding of the role of autophagy in human disease is rapidly evolving as investigators examine the process in different tissues and different pathophysiological contexts. In the field of heart disease, autophagy has been examined in the settings of ischemia and reperfusion, preconditioning, cardiac hypertrophy, and heart failure. This review addresses the role of autophagy in cardioprotection, the balance of catabolism and anabolism, the concept of mitochondrial quality control, and the implications of impaired autophagic flux or frustrated autophagy. PMID:20148666

  17. Autophagy in plants and phytopathogens.

    Science.gov (United States)

    Yoshimoto, Kohki; Takano, Yoshitaka; Sakai, Yasuyoshi

    2010-04-01

    Plants and plant-associated microorganisms including phytopathogens have to adapt to drastic changes in environmental conditions. Because of their immobility, plants must cope with various types of environmental stresses such as starvation, oxidative stress, drought stress, and invasion by phytopathogens during their differentiation, development, and aging processes. Here we briefly describe the early studies of plant autophagy, summarize recent studies on the molecular functions of ATG genes, and speculate on the role of autophagy in plants and phytopathogens. Autophagy regulates senescence and pathogen-induced cell death in plants, and autophagy and pexophagy play critical roles in differentiation and the invasion of host cells by phytopathogenic fungi. PMID:20079356

  18. Alcohol Dehydrogenase Protects against Endoplasmic Reticulum Stress-Induced Myocardial Contractile Dysfunction via Attenuation of Oxidative Stress and Autophagy: Role of PTEN-Akt-mTOR Signaling.

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    Jiaojiao Pang

    Full Text Available The endoplasmic reticulum (ER plays an essential role in ensuring proper folding of the newly synthesized proteins. Aberrant ER homeostasis triggers ER stress and development of cardiovascular diseases. ADH is involved in catalyzing ethanol to acetaldehyde although its role in cardiovascular diseases other than ethanol metabolism still remains elusive. This study was designed to examine the impact of ADH on ER stress-induced cardiac anomalies and underlying mechanisms involved using cardiac-specific overexpression of alcohol dehydrogenase (ADH.ADH and wild-type FVB mice were subjected to the ER stress inducer tunicamycin (1 mg/kg, i.p., for 48 hrs. Myocardial mechanical and intracellular Ca(2+ properties, ER stress, autophagy and associated cell signaling molecules were evaluated.ER stress compromised cardiac contractile function (evidenced as reduced fractional shortening, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration and impaired intracellular Ca(2+ homeostasis, oxidative stress and upregulated autophagy (increased LC3B, Atg5, Atg7 and p62, along with dephosphorylation of PTEN, Akt and mTOR, all of which were attenuated by ADH. In vitro study revealed that ER stress-induced cardiomyocyte anomaly was abrogated by ADH overexpression or autophagy inhibition using 3-MA. Interestingly, the beneficial effect of ADH was obliterated by autophagy induction, inhibition of Akt and mTOR. ER stress also promoted phosphorylation of the stress signaling ERK and JNK, the effect of which was unaffected by ADH transgene.Taken together, these findings suggested that ADH protects against ER stress-induced cardiac anomalies possibly via attenuation of oxidative stress and PTEN/Akt/mTOR pathway-regulated autophagy.

  19. Stress-induced self-cannibalism: on the regulation of autophagy by endoplasmic reticulum stress.

    Science.gov (United States)

    Deegan, Shane; Saveljeva, Svetlana; Gorman, Adrienne M; Samali, Afshin

    2013-07-01

    Macroautophagy (autophagy) is a cellular catabolic process which can be described as a self-cannibalism. It serves as an essential protective response during conditions of endoplasmic reticulum (ER) stress through the bulk removal and degradation of unfolded proteins and damaged organelles; in particular, mitochondria (mitophagy) and ER (reticulophagy). Autophagy is genetically regulated and the autophagic machinery facilitates removal of damaged cell components and proteins; however, if the cell stress is acute or irreversible, cell death ensues. Despite these advances in the field, very little is known about how autophagy is initiated and how the autophagy machinery is transcriptionally regulated in response to ER stress. Some three dozen autophagy genes have been shown to be required for the correct assembly and function of the autophagic machinery; however; very little is known about how these genes are regulated by cellular stress. Here, we will review current knowledge regarding how ER stress and the unfolded protein response (UPR) induce autophagy, including description of the different autophagy-related genes which are regulated by the UPR. PMID:23052213

  20. The autophagy gene Wdr45/Wipi4 regulates learning and memory function and axonal homeostasis.

    Science.gov (United States)

    Zhao, Yan G; Sun, Le; Miao, Guangyan; Ji, Cuicui; Zhao, Hongyu; Sun, Huayu; Miao, Lin; Yoshii, Saori R; Mizushima, Noboru; Wang, Xiaoqun; Zhang, Hong

    2015-01-01

    WDR45/WIPI4, encoding a WD40 repeat-containing PtdIns(3)P binding protein, is essential for the basal autophagy pathway. Mutations in WDR45 cause the neurodegenerative disease β-propeller protein-associated neurodegeneration (BPAN), a subtype of NBIA. We generated CNS-specific Wdr45 knockout mice, which exhibit poor motor coordination, greatly impaired learning and memory, and extensive axon swelling with numerous axon spheroids. Autophagic flux is defective and SQSTM1 (sequestosome-1)/p62 and ubiquitin-positive protein aggregates accumulate in neurons and swollen axons. Nes-Wdr45(fl/Y) mice recapitulate some hallmarks of BPAN, including cognitive impairment and defective axonal homeostasis, providing a model for revealing the disease pathogenesis of BPAN and also for investigating the possible role of autophagy in axon maintenance.

  1. Autophagy blockade sensitizes the anticancer activity of CA-4 via JNK-Bcl-2 pathway

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    Li, Yangling; Luo, Peihua; Wang, Jincheng; Dai, Jiabin; Yang, Xiaochun; Wu, Honghai; Yang, Bo, E-mail: yang924@zju.edu.cn; He, Qiaojun, E-mail: qiaojunhe@zju.edu.cn

    2014-01-15

    Combretastatin A-4 (CA-4) has already entered clinical trials of solid tumors over ten years. However, the limited anticancer activity and dose-dependent toxicity restrict its clinical application. Here, we offered convincing evidence that CA-4 induced autophagy in various cancer cells, which was demonstrated by acridine orange staining of intracellular acidic vesicles, the degradation of p62, the conversion of LC3-I to LC3-II and GFP-LC3 punctate fluorescence. Interestingly, CA-4-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitors (3-methyladenine and bafilomycin A1) or small interfering RNAs against the autophagic genes (Atg5 and Beclin 1). The enhanced anticancer activity of CA-4 and 3-MA was further confirmed in the SGC-7901 xenograft tumor model. These findings suggested that CA-4-elicited autophagic response played a protective role that impeded the eventual cell death while autophagy inhibition was expected to improve chemotherapeutic efficacy of CA-4. Meanwhile, CA-4 treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor or JNK siRNA inhibited autophagy but promoted CA-4-induced apoptosis, indicating a key requirement of JNK-Bcl-2 pathway in the activation of autophagy by CA-4. We also identified that pretreatment of Bcl-2 inhibitor (ABT-737) could significantly enhance anticancer activity of CA-4 due to inhibition of autophagy. Taken together, our data suggested that the JNK-Bcl-2 pathway was considered as the critical regulator of CA-4-induced protective autophagy and a potential drug target for chemotherapeutic combination. - Highlights: • Autophagy inhibition could be a potential for combretastatin A-4 antitumor efficacy. • The JNK-Bcl-2 pathway plays a critical role in CA-4-induced autophagy. • ABT-737 enhances CA-4 anticancer activity due to inhibition of autophagy.

  2. Investigating regulatory signatures of human autophagy related gene 5 (ATG5 through functional in silico analysis

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    Avni Vij

    2016-09-01

    Full Text Available Autophagy is an essential, homeostatic process which removes damaged cellular proteins and organelles for cellular renewal. ATG5, a part of E3 ubiquitin ligase-like complex (Atg12-Atg5/Atg16L1, is a key regulator involved in autophagosome formation - a crucial phase of autophagy. In this study, we used different in silico methods for comprehensive analysis of ATG5 to investigate its less explored regulatory activity. We have predicted various physico-chemical parameters and two possible transmembrane models that helped in exposing its functional regions. Twenty four PTM sites and 44 TFBS were identified which could be targeted to modulate the autophagy pathway. Furthermore, LD analysis identified 3 blocks of genotyped SNPs and 2 deleterious nsSNPs that may have damaging impact on protein function and thus could be employed for carrying genome-wide association studies. In conclusion, the information obtained in this study could be helpful for better understanding of regulatory roles of ATG5 and provides a base for its implication in population-based studies.

  3. Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

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    Wu, Chun-Yan [State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital of Digestive Disease, Fourth Military Medical University, Xi' an 710032 (China); Department of Gastroenterology, Shenyang General Hospital of PLA, 83 Wenhua Road, Shenyang 110016 (China); Yan, Jun; Yang, Yue-Feng; Xiao, Feng-Jun; Li, Qing-Fang; Zhang, Qun-Wei; Wang, Li-Sheng [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Guo, Xiao-Zhong, E-mail: guoxiaozhong1962@163.com [Department of Gastroenterology, Shenyang General Hospital of PLA, 83 Wenhua Road, Shenyang 110016 (China); Wang, Hua, E-mail: wanghua@bmi.ac.cn [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China)

    2011-01-21

    Research highlights: {yields} We first investigate the effects of KAI1 on autophagy in MiaPaCa-2 cells. {yields} Our findings demonstrate that KAI1 induces autophagy, which in turn inhibits KAI1-induced apoptosis. {yields} This study also supplies a possible novel therapeutic method for the treatment of pancreatic cancer using autophagy inhibitors. -- Abstract: KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

  4. Autophagy in sepsis: Degradation into exhaustion?

    Science.gov (United States)

    Ho, Jeffery; Yu, Jun; Wong, Sunny H; Zhang, Lin; Liu, Xiaodong; Wong, Wai T; Leung, Czarina C H; Choi, Gordon; Wang, Maggie H T; Gin, Tony; Chan, Matthew T V; Wu, William K K

    2016-07-01

    Autophagy is one of the innate immune defense mechanisms against microbial challenges. Previous in vitro and in vivo models of sepsis demonstrated that autophagy was activated initially in sepsis, followed by a subsequent phase of impairment. Autophagy modulation appears to be protective against multiple organ injuries in these murine sepsis models. This is achieved in part by preventing apoptosis, maintaining a balance between the productions of pro- and anti-inflammatory cytokines, and preserving mitochondrial functions. This article aims to discuss the role of autophagy in sepsis and the therapeutic potential of autophagy enhancers.

  5. Autophagy and gap junctional intercellular communication inhibition are involved in cadmium-induced apoptosis in rat liver cells

    International Nuclear Information System (INIS)

    Cadmium (Cd) is known to induce hepatotoxicity, yet the underlying mechanism of how this occurs is not fully understood. In this study, Cd-induced apoptosis was demonstrated in rat liver cells (BRL 3A) with apoptotic nuclear morphological changes and a decrease in cell index (CI) in a time- and concentration-dependent manner. The role of gap junctional intercellular communication (GJIC) and autophagy in Cd-induced apoptosis was investigated. Cd significantly induced GJIC inhibition as well as downregulation of connexin 43 (Cx43). The prototypical gap junction blocker carbenoxolone disodium (CBX) exacerbated the Cd-induced decrease in CI. Cd treatment was also found to cause autophagy, with an increase in mRNA expression of autophagy-related genes Atg-5, Atg-7, Beclin-1, and microtubule-associated protein light chain 3 (LC3) conversion from cytosolic LC3-I to membrane-bound LC3-II. The autophagic inducer rapamycin (RAP) prevented the Cd-induced CI decrease, while the autophagic inhibitor chloroquine (CQ) caused a further reduction in CI. In addition, CBX promoted Cd-induced autophagy, as well as changes in expression of Atg-5, Atg-7, Beclin-1 and LC3. CQ was found to block the Cd-induced decrease in Cx43 and GJIC inhibition, whereas RAP had opposite effect. These results demonstrate that autophagy plays a protective role during Cd-induced apoptosis in BRL 3A cells during 6 h of experiment, while autophagy exacerbates Cd-induced GJIC inhibition which has a negative effect on cellular fate. - Highlights: • GJIC and autophagy is crucial for biological processes. • Cd exposure causes GJIC inhibition and autophagy increase in BRL 3A cells. • Autophagy protects Cd induced BRL 3A cells apoptosis at an early stage. • Autophagy exacerbates Cd-induced GJIC inhibition. • GJIC plays an important role in autophagy induced cell death or survival

  6. Autophagy and gap junctional intercellular communication inhibition are involved in cadmium-induced apoptosis in rat liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Hui [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Zhuo, Liling [College of Life Science, Zaozhuang University, Zaozhuang, Shandong, 277160 (China); Han, Tao; Hu, Di; Yang, Xiaokang; Wang, Yi; Yuan, Yan; Gu, Jianhong; Bian, Jianchun; Liu, Xuezhong [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Liu, Zongping, E-mail: liuzongping@yzu.edu.cn [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China)

    2015-04-17

    Cadmium (Cd) is known to induce hepatotoxicity, yet the underlying mechanism of how this occurs is not fully understood. In this study, Cd-induced apoptosis was demonstrated in rat liver cells (BRL 3A) with apoptotic nuclear morphological changes and a decrease in cell index (CI) in a time- and concentration-dependent manner. The role of gap junctional intercellular communication (GJIC) and autophagy in Cd-induced apoptosis was investigated. Cd significantly induced GJIC inhibition as well as downregulation of connexin 43 (Cx43). The prototypical gap junction blocker carbenoxolone disodium (CBX) exacerbated the Cd-induced decrease in CI. Cd treatment was also found to cause autophagy, with an increase in mRNA expression of autophagy-related genes Atg-5, Atg-7, Beclin-1, and microtubule-associated protein light chain 3 (LC3) conversion from cytosolic LC3-I to membrane-bound LC3-II. The autophagic inducer rapamycin (RAP) prevented the Cd-induced CI decrease, while the autophagic inhibitor chloroquine (CQ) caused a further reduction in CI. In addition, CBX promoted Cd-induced autophagy, as well as changes in expression of Atg-5, Atg-7, Beclin-1 and LC3. CQ was found to block the Cd-induced decrease in Cx43 and GJIC inhibition, whereas RAP had opposite effect. These results demonstrate that autophagy plays a protective role during Cd-induced apoptosis in BRL 3A cells during 6 h of experiment, while autophagy exacerbates Cd-induced GJIC inhibition which has a negative effect on cellular fate. - Highlights: • GJIC and autophagy is crucial for biological processes. • Cd exposure causes GJIC inhibition and autophagy increase in BRL 3A cells. • Autophagy protects Cd induced BRL 3A cells apoptosis at an early stage. • Autophagy exacerbates Cd-induced GJIC inhibition. • GJIC plays an important role in autophagy induced cell death or survival.

  7. A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease.

    Directory of Open Access Journals (Sweden)

    Kaisa Kyöstilä

    2015-04-01

    Full Text Available Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136 in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

  8. Suppression of autophagy augments the radiosensitizing effects of STAT3 inhibition on human glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Xiaopeng; Du, Jie; Hua, Song; Zhang, Haowen; Gu, Cheng; Wang, Jie; Yang, Lei; Huang, Jianfeng; Yu, Jiahua, E-mail: yujiahua@suda.edu.cn; Liu, Fenju, E-mail: fangsh@suda.edu.cn

    2015-01-15

    Radiotherapy is an essential component of the standard therapy for newly diagnosed glioblastoma. To increase the radiosensitivity of glioma cells is a feasible solution to improve the therapeutic effects. It has been suggested that inhibition of signal transducer and activator of transcription 3 (STAT3) can radiosensitize glioma cells, probably via the activation of mitochondrial apoptotic pathway. In this study, human malignant glioma cells, U251 and A172, were treated with an STAT3 inhibitor, WP1066, or a short hairpin RNA plasmid targeting STAT3 to suppress the activation of STAT3 signaling. The radiosensitizing effects of STAT3 inhibition were confirmed in glioma cells. Intriguingly, combination of ionizing radiation exposure and STAT3 inhibition triggered a pronounced increase of autophagy flux. To explore the role of autophagy, glioma cells were treated with 3-methyladenine or siRNA for autophagy-related gene 5, and it was demonstrated that inhibition of autophagy further strengthened the radiosensitizing effects of STAT3 inhibition. Accordingly, more apoptotic cells were induced by the dual inhibition of autophagy and STAT3 signaling. In conclusion, our data revealed a protective role of autophagy in the radiosensitizing effects of STAT3 inhibition, and inhibition of both autophagy and STAT3 might be a potential therapeutic strategy to increase the radiosensitivity of glioma cells. - Highlights: • Inactivation of STAT3 signaling radiosensitizes malignant glioma cells. • STAT3 inhibition triggers a significant increase of autophagy flux induced by ionizing radiation in glioma cells. • Suppression of autophagy further strengthens the radiosensitizing effects of STAT3 inhibition in glioma cells. • Dual inhibition of autophagy and STAT3 induce massive apoptotic cells upon exposure to ionizing radiation.

  9. Autophagy in colorectal cancer:An important switch from physiology to pathology

    Institute of Scientific and Technical Information of China (English)

    Florin; Burada; Elena; Raluca; Nicoli; Marius; Eugen; Ciurea; Daniel; Constantin; Uscatu; Mihai; Ioana; Dan; Ionut; Gheonea

    2015-01-01

    Colorectal cancer(CRC) remains a leading cause of cancer death in both men and women worldwide.Among the factors and mechanisms that are involved in the multifactorial etiology of CRC,autophagy is an important transformational switch that occurs when a cell shifts from normal to malignant.In recent years,multiple hypotheses have been considered regarding the autophagy mechanisms that are involved in cancer.The currently accepted hypothesis is that autophagy has dual and contradictory roles in carcinogenesis,but the precise mechanisms leading to autophagy in cancer are not yet fully defined and seem to be context dependent.Autophagy is a surveillance mechanism used by normal cells that protects them from the transformation to malignancy by removing damaged organelles and aggregated proteins and by reducing reactive oxygen species,mitochondrial abnormalities and DNA damage.However,autophagy also supports tumor formation by promoting access to nutrients that are critical to the metabolism and growth of tumor cells and by inhibiting cellular death and increasing drug resistance.Autophagy studies in CRC have focused on several molecules,mainly microtubule-associated protein 1 light chain 3,beclin 1,and autophagy related 5,with conflicting results.Beneficial effects were observed for some agents that modulate autophagy in CRC either alone or,more often,in combination with other agents.More extensive studies are needed in the future to clarify the roles of autophagy-related genes and modulators in colorectal carcinogenesis,and to develop potential beneficial agents for the prognosis and treatment of CRC.

  10. The protective effect of autophagy on mouse spermatocyte derived cells exposure to 1800MHz radiofrequency electromagnetic radiation.

    Science.gov (United States)

    Liu, Kaijun; Zhang, Guowei; Wang, Zhi; Liu, Yong; Dong, Jianyun; Dong, Xiaomei; Liu, Jinyi; Cao, Jia; Ao, Lin; Zhang, Shaoxiang

    2014-08-01

    The increasing exposure to radiofrequency (RF) radiation emitted from mobile phone use has raised public concern regarding the biological effects of RF exposure on the male reproductive system. Autophagy contributes to maintaining intracellular homeostasis under environmental stress. To clarify whether RF exposure could induce autophagy in the spermatocyte, mouse spermatocyte-derived cells (GC-2) were exposed to 1800MHz Global System for Mobile Communication (GSM) signals in GSM-Talk mode at specific absorption rate (SAR) values of 1w/kg, 2w/kg or 4w/kg for 24h, respectively. The results indicated that the expression of LC3-II increased in a dose- and time-dependent manner with RF exposure, and showed a significant change at the SAR value of 4w/kg. The autophagosome formation and the occurrence of autophagy were further confirmed by GFP-LC3 transient transfection assay and transmission electron microscopy (TEM) analysis. Furthermore, the conversion of LC3-I to LC3-II was enhanced by co-treatment with Chloroquine (CQ), indicating autophagic flux could be enhanced by RF exposure. Intracellular ROS levels significantly increased in a dose- and time-dependent manner after cells were exposed to RF. Pretreatment with anti-oxidative NAC obviously decreased the conversion of LC3-I to LC3-II and attenuated the degradation of p62 induced by RF exposure. Meanwhile, phosphorylated extracellular-signal-regulated kinase (ERK) significantly increased after RF exposure at the SAR value of 2w/kg and 4w/kg. Moreover, we observed that RF exposure did not increase the percentage of apoptotic cells, but inhibition of autophagy could increase the percentage of apoptotic cells. These findings suggested that autophagy flux could be enhanced by 1800MHz GSM exposure (4w/kg), which is mediated by ROS generation. Autophagy may play an important role in preventing cells from apoptotic cell death under RF exposure stress.

  11. miR-638 promotes melanoma metastasis and protects melanoma cells from apoptosis and autophagy

    OpenAIRE

    Bhattacharya, Animesh; Schmitz, Ulf; Raatz, Yvonne; Schönherr, Madeleine; Kottek, Tina; Schauer, Marianne; Franz, Sandra; Saalbach, Anja; Anderegg, Ulf; Wolkenhauer, Olaf; Schadendorf, Dirk; Jan C Simon; Magin, Thomas; Vera, Julio; Kunz, Manfred

    2014-01-01

    The present study identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions of melanomas compared with primary melanomas. miR-638 enhanced the tumorigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling identified new candidate genes including TP53INP2 as miR-638 targets, the majority of which are involved in p53 signalling. Overexpression of TP53INP2 severely attenuated proliferative and invasive capacity of ...

  12. MOLECULAR CHARACTERIZATION OF AUTOPHAGY-RELATED GENE 5 FROM Spodoptera exigua AND EXPRESSION ANALYSIS UNDER VARIOUS STRESS CONDITIONS.

    Science.gov (United States)

    Liu, Kai-Yu; Xia, Yu-Qian; Zhou, Jing; Chen, Zu-Wen; Lu, Dandan; Zhang, Ning-Zhao; Liu, Xu-Sheng; Ai, Hui; Zhou, Li-Lin

    2016-08-01

    Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry-SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl-HP cells and partially co-localized with BmATG6-GFP, but it almost has no significant co-localization with GFP-HaATG8. Then, the Western blot analysis demonstrated that GFP-SeATG5 conjugated with ATG12. Moreover, real-time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions. PMID:27226059

  13. Nrf2-Mediated Cardiac Maladaptive Remodeling and Dysfunction in a Setting of Autophagy Insufficiency.

    Science.gov (United States)

    Qin, Qingyun; Qu, Chen; Niu, Ting; Zang, Huimei; Qi, Lei; Lyu, Linmao; Wang, Xuejun; Nagarkatti, Mitzi; Nagarkatti, Prakash; Janicki, Joseph S; Wang, Xing Li; Cui, Taixing

    2016-01-01

    Nuclear factor erythroid-2-related factor 2 (Nrf2) appears to exert either a protective or detrimental effect on the heart; however, the underlying mechanism remains poorly understood. Herein, we uncovered a novel mechanism for turning off the Nrf2-mediated cardioprotection and switching on Nrf2-mediated cardiac dysfunction. In a murine model of pressure overload-induced cardiac remodeling and dysfunction via transverse aortic arch constriction, knockout of Nrf2 enhanced myocardial necrosis and death rate during an initial stage of cardiac adaptation when myocardial autophagy function is intact. However, knockout of Nrf2 turned out to be cardioprotective throughout the later stage of cardiac maladaptive remodeling when myocardial autophagy function became insufficient. Transverse aortic arch constriction -induced activation of Nrf2 was dramatically enhanced in the heart with impaired autophagy, which is induced by cardiomyocyte-specific knockout of autophagy-related gene (Atg)5. Notably, Nrf2 activation coincided with the upregulation of angiotensinogen (Agt) only in the autophagy-impaired heart after transverse aortic arch constriction. Agt5 and Nrf2 gene loss-of-function approaches in combination with Jak2 and Fyn kinase inhibitors revealed that suppression of autophagy inactivated Jak2 and Fyn and nuclear translocation of Fyn, while enhancing nuclear translocation of Nrf2 and Nrf2-driven Agt expression in cardiomyocytes. Taken together, these results indicate that the pathophysiological consequences of Nrf2 activation are closely linked with the functional integrity of myocardial autophagy during cardiac remodeling. When autophagy is intact, Nrf2 is required for cardiac adaptive responses; however, autophagy impairment most likely turns off Fyn-operated Nrf2 nuclear export thus activating Nrf2-driven Agt transcription, which exacerbates cardiac maladaptation leading to dysfunction. PMID:26573705

  14. Molecular mechanism and regulation of autophagy

    Institute of Scientific and Technical Information of China (English)

    Ya-ping YANG; Zhong-qin LIANG; Zhen-lun GU; Zheng-hong QIN

    2005-01-01

    Autophagy is a major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles in eukaryotic cells. A large number of intracellular/extracellular stimuli, including amino acid starvation and invasion of microorganisms, are able to induce the autophagic response in cells. The discovery of the ATG genes in yeast has greatly advanced our understanding of the molecular mechanisms participating in autophagy and the genes involved in regulating the autophagic pathway. Many yeast genes have mammalian homologs,suggesting that the basic machinery for autophagy has been evolutionarily conserved along the eukaryotic phylum. The regulation of autophagy is a very complex process. Many signaling pathways, including target of rapamycin (TOR) or mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase-I (PI3K-I)/PKB, GTPases, calcium and protein synthesis all play important roles in regulating autophagy. The molecular mechanisms and regulation of autophagy are discussed in this review.

  15. Histone deacetylase inhibitors induce apoptosis in myeloid leukemia by suppressing autophagy.

    Science.gov (United States)

    Stankov, M V; El Khatib, M; Kumar Thakur, B; Heitmann, K; Panayotova-Dimitrova, D; Schoening, J; Bourquin, J P; Schweitzer, N; Leverkus, M; Welte, K; Reinhardt, D; Li, Z; Orkin, S H; Behrens, G M N; Klusmann, J H

    2014-03-01

    Histone deacetylase (HDAC) inhibitors (HDACis) are well-characterized anti-cancer agents with promising results in clinical trials. However, mechanistically little is known regarding their selectivity in killing malignant cells while sparing normal cells. Gene expression-based chemical genomics identified HDACis as being particularly potent against Down syndrome-associated myeloid leukemia (DS-AMKL) blasts. Investigating the antileukemic function of HDACis revealed their transcriptional and post-translational regulation of key autophagic proteins, including ATG7. This leads to suppression of autophagy, a lysosomal degradation process that can protect cells against damaged or unnecessary organelles and protein aggregates. DS-AMKL cells exhibit low baseline autophagy due to mammalian target of rapamycin (mTOR) activation. Consequently, HDAC inhibition repressed autophagy below a critical threshold, which resulted in accumulation of mitochondria, production of reactive oxygen species, DNA damage and apoptosis. Those HDACi-mediated effects could be reverted upon autophagy activation or aggravated upon further pharmacological or genetic inhibition. Our findings were further extended to other major acute myeloid leukemia subgroups with low basal level autophagy. The constitutive suppression of autophagy due to mTOR activation represents an inherent difference between cancer and normal cells. Thus, via autophagy suppression, HDACis deprive cells of an essential pro-survival mechanism, which translates into an attractive strategy to specifically target cancer cells. PMID:24080946

  16. Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor.

    Science.gov (United States)

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Lee, Yong Seok; Kang, Sang Sun; Han, Yeon Soo

    2015-07-01

    Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes (ATG) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor (TmATG8). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, TmATG8-silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes. These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor. PMID:25727880

  17. Protective effect of autophagy on human retinal pigment epithelial cells against lipofuscin fluorophore A2E: implications for age-related macular degeneration.

    Science.gov (United States)

    Zhang, J; Bai, Y; Huang, L; Qi, Y; Zhang, Q; Li, S; Wu, Y; Li, X

    2015-11-12

    Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial (RPE) cells is a crucial causative factor responsible for the onset and progression of AMD. A2E, a major component of toxic lipofuscin implicated in AMD, is deposited in RPE cells with age. However, the mechanism whereby A2E may contribute to the pathogenesis of AMD remains unclear. We demonstrated that A2E was a danger signal of RPE cells, which induced autophagy and decreased cell viability in a concentration- and time-dependent manner. Within 15 min after the treatment of RPE with 25 μM A2E, the induction of autophagosome was detected by transmission electron microscopy. After continuous incubating RPE cells with A2E, intense punctate staining of LC3 and increased expression of LC3-II and Beclin-1 were identified. Meanwhile, the levels of intercellular adhesion molecule (ICAM), interleukin (IL)1β, IL2, IL-6, IL-8, IL-17A, IL-22, macrophage cationic peptide (MCP)-1, stromal cell-derived factor (SDF)-1, and vascular endothelial growth factor A (VEGFA) were elevated. The autophagic inhibitor 3-methyladenine (3-MA) and activator rapamycin were also used to verify the effect of autophagy on RPE cells against A2E. Our results revealed that 3-MA decreased the autophagosomes and LC3 puncta induced by A2E, increased inflammation-associated protein expression including ICAM, IL1β, IL2, IL-6, IL-8, IL-17A, IL-22, and SDF-1, and upregulated VEGFA expression. Whereas rapamycin augmented the A2E-mediated autophagy, attenuated protein expression of inflammation-associated and angiogenic factors, and blocked the Akt/mTOR pathway. Taken together, A2E induces autophagy in RPE cells at the early stage of incubation, and this autophagic response can be inhibited by 3-MA or augmented by rapamycin via the mTOR pathway. The enhancement of autophagy has a protective role in RPE cells against the adverse effects of A2E by reducing the

  18. MAP1LC3B overexpression protects against Hermansky-Pudlak syndrome type-1-induced defective autophagy in vitro.

    Science.gov (United States)

    Ahuja, Saket; Knudsen, Lars; Chillappagari, Shashi; Henneke, Ingrid; Ruppert, Clemens; Korfei, Martina; Gochuico, Bernadette R; Bellusci, Saverio; Seeger, Werner; Ochs, Matthias; Guenther, Andreas; Mahavadi, Poornima

    2016-03-15

    Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, and some patients with HPS develop pulmonary fibrosis, known as HPS-associated interstitial pneumonia (HPSIP). We have previously reported that HPSIP is associated with severe surfactant accumulation, lysosomal stress, and alveolar epithelial cell type II (AECII) apoptosis. Here, we hypothesized that defective autophagy might result in excessive lysosomal stress in HPSIP. Key autophagy proteins, including LC3B lipidation and p62, were increased in HPS1/2 mice lungs. Electron microscopy demonstrated a preferable binding of LC3B to the interior of lamellar bodies in the AECII of HPS1/2 mice, whereas in wild-type mice it was present on the limiting membrane in addition to the interior of the lamellar bodies. Similar observations were noted in human HPS1 lung sections. In vitro knockdown of HPS1 revealed increased LC3B lipidation and p62 accumulation, associated with an increase in proapoptotic caspases. Overexpression of LC3B decreased the HPS1 knockdown-induced p62 accumulation, whereas rapamycin treatment did not show the same effect. We conclude that loss of HPS1 protein results in impaired autophagy that is restored by exogenous LC3B and that defective autophagy might therefore play a critical role in the development and progression of HPSIP. PMID:26719147

  19. Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction

    Science.gov (United States)

    Huenchuguala, Sandro; Muñoz, Patricia; Zavala, Patricio; Villa, Mónica; Cuevas, Carlos; Ahumada, Ulises; Graumann, Rebecca; Nore, Beston F; Couve, Eduardo; Mannervik, Bengt; Paris, Irmgard; Segura-Aguilar, Juan

    2014-01-01

    U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit 3H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A1, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A1 pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction. PMID:24434817

  20. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury.

    Science.gov (United States)

    Godar, Rebecca J; Ma, Xiucui; Liu, Haiyan; Murphy, John T; Weinheimer, Carla J; Kovacs, Attila; Crosby, Seth D; Saftig, Paul; Diwan, Abhinav

    2015-01-01

    Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury.

  1. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury.

    Science.gov (United States)

    Godar, Rebecca J; Ma, Xiucui; Liu, Haiyan; Murphy, John T; Weinheimer, Carla J; Kovacs, Attila; Crosby, Seth D; Saftig, Paul; Diwan, Abhinav

    2015-01-01

    Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury. PMID:26103523

  2. Inducing autophagy

    DEFF Research Database (Denmark)

    Harder, Lea M; Bunkenborg, Jakob; Andersen, Jens S.

    2014-01-01

    Autophagy is a lysosomal-mediated catabolic process, which through degradation of different cytoplasmic components aids in maintaining cellular homeostasis and survival during exposure to extra- or intracellular stresses. Ammonia is a potential toxic and stress-inducing byproduct of glutamine...... catabolism, which has recently been found to induce autophagy in an MTOR independent way and support cancer cell survival. In this study, quantitative phosphoproteomics was applied to investigate the initial signaling events linking ammonia to the induction of autophagy. The MTOR inhibitor rapamycin was used...... as a reference treatment to emphasize the differences between an MTOR-dependent and -independent autophagy-induction. By this means 5901 phosphosites were identified of which 626 were treatment-specific regulated and 175 were coregulated. Investigation of the ammonia-specific regulated sites supported that MTOR...

  3. The role of STAT3 in autophagy.

    Science.gov (United States)

    You, Liangkun; Wang, Zhanggui; Li, Hongsen; Shou, Jiawei; Jing, Zhao; Xie, Jiansheng; Sui, Xinbing; Pan, Hongming; Han, Weidong

    2015-01-01

    Autophagy is an evolutionarily conserved process in eukaryotes that eliminates harmful components and maintains cellular homeostasis in response to a series of extracellular insults. However, these insults may trigger the downstream signaling of another prominent stress responsive pathway, the STAT3 signaling pathway, which has been implicated in multiple aspects of the autophagic process. Recent reports further indicate that different subcellular localization patterns of STAT3 affect autophagy in various ways. For example, nuclear STAT3 fine-tunes autophagy via the transcriptional regulation of several autophagy-related genes such as BCL2 family members, BECN1, PIK3C3, CTSB, CTSL, PIK3R1, HIF1A, BNIP3, and microRNAs with targets of autophagy modulators. Cytoplasmic STAT3 constitutively inhibits autophagy by sequestering EIF2AK2 as well as by interacting with other autophagy-related signaling molecules such as FOXO1 and FOXO3. Additionally, the mitochondrial translocation of STAT3 suppresses autophagy induced by oxidative stress and may effectively preserve mitochondria from being degraded by mitophagy. Understanding the role of STAT3 signaling in the regulation of autophagy may provide insight into the classic autophagy model and also into cancer therapy, especially for the emerging targeted therapy, because a series of targeted agents execute antitumor activities via blocking STAT3 signaling, which inevitably affects the autophagy pathway. Here, we review several of the representative studies and the current understanding in this particular field.

  4. Oxidative stress-induced autophagy: Role in pulmonary toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Malaviya, Rama [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States)

    2014-03-01

    Autophagy is an evolutionarily conserved catabolic process important in regulating the turnover of essential proteins and in elimination of damaged organelles and protein aggregates. Autophagy is observed in the lung in response to oxidative stress generated as a consequence of exposure to environmental toxicants. Whether autophagy plays role in promoting cell survival or cytotoxicity is unclear. In this article recent findings on oxidative stress-induced autophagy in the lung are reviewed; potential mechanisms initiating autophagy are also discussed. A better understanding of autophagy and its role in pulmonary toxicity may lead to the development of new strategies to treat lung injury associated with oxidative stress. - Highlights: • Exposure to pulmonary toxicants is associated with oxidative stress. • Oxidative stress is known to induce autophagy. • Autophagy is upregulated in the lung following exposure to pulmonary toxicants. • Autophagy may be protective or pathogenic.

  5. Feedback regulation between autophagy and PKA.

    Science.gov (United States)

    Torres-Quiroz, Francisco; Filteau, Marie; Landry, Christian R

    2015-01-01

    Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA.

  6. Erythropoietin Modulates Autophagy Signaling in the Developing Rat Brain in an In Vivo Model of Oxygen-Toxicity

    Directory of Open Access Journals (Sweden)

    Marco Sifringer

    2012-10-01

    Full Text Available Autophagy is a self-degradative process that involves turnover and recycling of cytoplasmic components in healthy and diseased tissue. Autophagy has been shown to be protective at the early stages of programmed cell death but it can also promote apoptosis under certain conditions. Earlier we demonstrated that oxygen contributes to the pathogenesis of neonatal brain damage, which can be ameliorated by intervention with recombinant human erythropoietin (rhEpo. Extrinsic- and intrinsic apoptotic pathways are involved in oxygen induced neurotoxicity but the role of autophagy in this model is unclear. We analyzed the expression of autophagy activity markers in the immature rodent brain after exposure to elevated oxygen concentrations. We observed a hyperoxia-exposure dependent regulation of autophagy-related gene (Atg proteins Atg3, 5, 12, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3, LC3A-II, and LC3B-II which are all key autophagy activity proteins. Interestingly, a single injection with rhEpo at the onset of hyperoxia counteracted these oxygen-mediated effects. Our results indicate that rhEpo generates its protective effect by modifying the key autophagy activity proteins.

  7. Dysregulation of Autophagy, Mitophagy, and Apoptotic Genes in the Medial Temporal Lobe Cortex in an Ischemic Model of Alzheimer’s Disease

    Science.gov (United States)

    Ułamek-Kozioł, Marzena; Kocki, Janusz; Bogucka-Kocka, Anna; Petniak, Alicja; Gil-Kulik, Paulina; Januszewski, Sławomir; Bogucki, Jacek; Jabłoński, Mirosław; Furmaga-Jabłońska, Wanda; Brzozowska, Judyta; Czuczwar, Stanisław J.; Pluta, Ryszard

    2016-01-01

    Ischemic brain damage is a pathological incident that is often linked with medial temporal lobe cortex injury and finally its atrophy. Post-ischemic brain injury associates with poor prognosis since neurons of selectively vulnerable ischemic brain areas are disappearing by apoptotic program of neuronal death. Autophagy has been considered, after brain ischemia, as a guardian against neurodegeneration. Consequently, we have examined changes in autophagy (BECN 1), mitophagy (BNIP 3), and apoptotic (caspase 3) genes in the medial temporal lobe cortex with the use of quantitative reverse-transcriptase PCR following transient 10-min global brain ischemia in rats with survival 2, 7, and 30 days. The intense significant overexpression of BECN 1 gene was noted on the 2nd day, while on days 7–30 the expression of this gene was still upregulated. BNIP 3 gene was downregulated on the 2nd day, but on days 7–30 post-ischemia, there was a significant reverse tendency. Caspase 3 gene, associated with apoptotic neuronal death, was induced in the same way as BNIP 3 gene after brain ischemia. Thus, the demonstrated changes indicate that the considerable dysregulation of expression of BECN 1, BNIP 3, and caspase 3 genes may be connected with a response of neuronal cells in medial temporal lobe cortex to transient complete brain ischemia. PMID:27472881

  8. Autophagy plays an important role in protecting Pacific oysters from OsHV-1 and Vibrio aestuarianus infections

    OpenAIRE

    Moreau, Pierrick; Moreau, Kevin; Segarra, Am?lie; Tourbiez, Delphine; Travers, Marie-Agn?s; Rubinsztein, David C.; Renault, Tristan

    2015-01-01

    Recent mass mortality outbreaks around the world in Pacific oysters, Crassostrea gigas, have seriously affected the aquaculture economy. Although the causes for these mortality outbreaks appear complex, infectious agents are involved. Two pathogens are associated with mass mortality outbreaks, the virus ostreid herpesvirus 1 (OsHV-1) and the bacterium Vibrio aestuarianus. Here we describe the interactions between these 2 pathogens and autophagy, a conserved intracellular pathway playing a key...

  9. Minocycline protects retinal ganglion cells after optic nerve crush injury in mice by delaying autophagy and upregulating nuclear factor-κB2

    Institute of Scientific and Technical Information of China (English)

    Jiao Xiaoling; Peng Yuan; Yang Liu

    2014-01-01

    Background Currently,no medicine is available that can prevent or treat neural damage associated with optic nerve injury.Minocycline is recently reported to have a neuroprotective function.The aims of this study were to exarmine the neuroprotective effect of minocycline on retinal ganglion cells (RGCs) and determine its underlying mechanisms,using a mouse model of optic nerve crush (ONC).Methods ONC was performed in the left eye of adult male mice,and the mice were randomly divided into minocycline-treated group and saline-treated control group.The mice without receiving ONC injury were used as positive controls.RGC densities were assessed in retinal whole mounts with immunofluorescence labeling of βⅢ-tubulin.Transmission electron microscopy was used to detect RGC morphologies,and Western blotting and real-time PCR were applied to investigate the expression of autophagy markers LC3-Ⅰ,LC3-Ⅱ,and transcriptional factors nuclear factor-κB1 (NF-κB1),NF-κB2.Results In the early stage after ONC (at Days 4 and 7),the density of RGCs in the minocycline-treated group was higher than that of the saline-treated group.Electron micrographs showed that minocycline prevented nuclei and mitochondria injuries at Day 4.Western blotting analysis demonstrated that the conversion of LC3-Ⅰ to LC3-Ⅱ was reduced in the minocycline-treated group at Days 4 and 7,which meant autophagy process was inhibited by minocycline.In addition,the gene expression of NF-κB2 was upregulated by minocycline at Day 4.Conclusion The neuroprotective effect of minocycline is generated in the early stage after ONC in mice,partly through delaying autophagy process and regulating NF-κB2 pathway.

  10. Identification of Barley (Hordeum vulgare L. Autophagy Genes and Their Expression Levels during Leaf Senescence, Chronic Nitrogen Limitation and in Response to Dark Exposure

    Directory of Open Access Journals (Sweden)

    Liliana Avila-Ospina

    2016-02-01

    Full Text Available Barley is a cereal of primary importance for forage and human nutrition, and is a useful model for wheat. Autophagy genes first described in yeast have been subsequently isolated in mammals and Arabidopsis thaliana. In Arabidopsis and maize it was recently shown that autophagy machinery participates in nitrogen remobilization for grain filling. In rice, autophagy is also important for nitrogen recycling at the vegetative stage. In this study, HvATGs, HvNBR1 and HvATI1 sequences were identified from bacterial artificial chromosome (BAC, complementary DNA (cDNA and expressed sequence tag (EST libraries. The gene models were subsequently determined from alignments between genome and transcript sequences. Essential amino acids were identified from the protein sequences in order to estimate their functionality. A total of twenty-four barley HvATG genes, one HvNBR1 gene and one HvATI1 gene were identified. Except for HvATG5, all the genomic sequences found completely matched their cDNA sequences. The HvATG5 gene sequence presents a gap that cannot be sequenced due to its high GC content. The HvATG5 coding DNA sequence (CDS, when over-expressed in the Arabidopsis atg5 mutant, complemented the plant phenotype. The HvATG transcript levels were increased globally by leaf senescence, nitrogen starvation and dark-treatment. The induction of HvATG5 during senescence was mainly observed in the flag leaves, while it remained surprisingly stable in the seedling leaves, irrespective of the leaf age during stress treatment.

  11. Canine hereditary ataxia in old english sheepdogs and gordon setters is associated with a defect in the autophagy gene encoding RAB24.

    Directory of Open Access Journals (Sweden)

    Caryline Agler

    2014-02-01

    Full Text Available Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.

  12. Modulating autophagy: a strategy for cancer therapy

    Institute of Scientific and Technical Information of China (English)

    Jun-Lin Li; Shao-Liang Han; Xia Fan

    2011-01-01

    Autophagy is a process in which long-lived proteins,damaged cell organelles,and other cellular particles are sequestered and degraded.This process is important for maintaining the cellular microenvironment when the cell is under stress.Many studies have shown that autophagy plays a complex role in human diseases,especially in cancer,where it is known to have paradoxical effects.Namely,autophagy provides the energy for metabolism and tumor growth and leads to cell death that promotes tumor suppression.The link between autophagy and cancer is also evident in that some of the genes that regulate carcinogenesis,oncogenes and tumor suppressor genes,participate in or impact the autophagy process.Therefore,modulating autophagy will be a valuable topic for cancer therapy.Many studies have shown that autophagy can inhibit the tumor growth when autophagy modulators are combined with radiotherapy and/or chemotherapy.These findings suggest that autophagy may be a potent target for cancer therapy.

  13. Mechanism of action of the tuberculosis and Crohn disease risk factor IRGM in autophagy

    Science.gov (United States)

    Chauhan, Santosh; Mandell, Michael A.; Deretic, Vojo

    2016-01-01

    ABSTRACT Polymorphisms in the IRGM gene, associated with Crohn disease (CD) and tuberculosis, are among the earliest identified examples documenting the role of autophagy in human disease. Functional studies have shown that IRGM protects against these diseases by modulating autophagy, yet the exact molecular mechanism of IRGM's activity has remained unknown. We have recently elucidated IRGM's mechanism of action. IRGM functions as a platform for assembling, stabilizing, and activating the core autophagic machinery, while at the same time physically coupling it to conventional innate immunity receptors. Exposure to microbial products or bacterial invasion increases IRGM expression, which leads to stabilization of AMPK. Specific protein-protein interactions and post-translational modifications such as ubiquitination of IRGM, lead to a co-assembly with IRGM of the key autophagy regulators ULK1 and BECN1 in their activated forms. IRGM physically interacts with 2 other CD risk factors, ATG16L1 and NOD2, placing these 3 principal players in CD within the same molecular complex. This explains how polymorphisms altering expression or function of any of the 3 factors individually can affect the same process—autophagy. Furthermore, IRGM's interaction with NOD2, and additional pattern recognition receptors such as NOD1, RIG-I, and select TLRs, transduces microbial signals to the core autophagy apparatus. This work solves the long-standing enigma of how IRGM controls autophagy. PMID:26313894

  14. Modulation of Apoptosis Pathways by Oxidative Stress and Autophagy in β Cells

    Directory of Open Access Journals (Sweden)

    Maorong Wang

    2012-01-01

    Full Text Available Human islets isolated for transplantation are exposed to multiple stresses including oxidative stress and hypoxia resulting in significant loss of functional β cell mass. In this study we examined the modulation of apoptosis pathway genes in islets exposed to hydrogen peroxide, peroxynitrite, hypoxia, and cytokines. We observed parallel induction of pro- and antiapoptotic pathways and identified several novel genes including BFAR, CARD8, BNIP3, and CIDE-A. As BNIP3 is an inducer of autophagy, we examined this pathway in MIN6 cells, a mouse beta cell line and in human islets. Culture of MIN6 cells under low serum conditions increased the levels of several proteins in autophagy pathway, including ATG4, Beclin 1, LAMP-2, and UVRAG. Amino acid deprivation led to induction of autophagy in human islets. Preconditioning of islets with inducers of autophagy protected them from hypoxia-induced apoptosis. However, induction of autophagy during hypoxia exacerbated apoptotic cell death. ER stress led to induction of autophagy and apoptosis in β cells. Overexpression of MnSOD, an enzyme that scavenges free radicals, resulted in protection of MIN6 cells from cytokine-induced apoptosis. Ceramide, a mediator of cytokine-induced injury, reduced the active phosphorylated form of Akt and downregulated the promoter activity of the antiapoptotic gene bcl-2. Furthermore, cytokine-stimulated JNK pathway downregulated the bcl-2 promoter activity which was reversed by preincubation with SP600125, a JNK inhibitor. Our findings suggest that β cell apoptosis by multiple stresses in islets isolated for transplantation is the result of orchestrated gene expression in apoptosis pathway.

  15. Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.

    Directory of Open Access Journals (Sweden)

    Zhi-Hua Chen

    Full Text Available BACKGROUND: Chronic obstructive pulmonary disease (COPD is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear. METHODOLOGY AND PRINCIPAL FINDINGS: Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7. Cigarette smoke extract (CSE is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1 and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1(-/- mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema. CONCLUSIONS: We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.

  16. The role and modulation of autophagy in experimental models of myocardial ischemia-reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Carol Chen-Scarabelli; Richard Knight; Pratik R Agrawal; Louis Saravolatz; Cadigia Abuniat; Gabriele Scarabelli; Anastasis Stephanou; Leena Loomba; Jagat Narula; Tiziano M Scarabelli

    2014-01-01

    A physiological sequence called autophagy qualitatively determines cellular viability by removing protein aggregates and damaged cyto-plasmic constituents, and contributes significantly to the degree of myocardial ischemia-reperfusion (I/R) injury. This tightly orchestrated cata-bolic cellular‘housekeeping’ process provides cells with a new source of energy to adapt to stressful conditions. This process was first described as a pro-survival mechanism, but increasing evidence suggests that it can also lead to the demise of the cell. Autophagy has been implicated in the pathogenesis of multiple cardiac conditions including myocardial I/R injury. However, a debate persists as to whether autophagy acts as a protec-tive mechanism or contributes to the injurious effects of I/R injury in the heart. This controversy may stem from several factors including the va-riability in the experimental models and species, and the methodology used to assess autophagy. This review provides updated knowledge on the modulation and role of autophagy in isolated cardiac cells subjected to I/R, and the growing interest towards manipulating autophagy to increase the survival of cardiac myocytes under conditions of stress-most notably being I/R injury. Perturbation of this evolutionarily conserved intracellular cleansing autophagy mechanism, by targeted modulation through, among others, mammalian target of rapamycin (mTOR) inhibitors, adenosine monophosphate-activated protein kinase (AMPK) modulators, calcium lowering agents, resveratrol, longevinex, sirtuin activators, the proapoptotic gene Bnip3, IP3 and lysosome inhibitors, may confer resistance to heart cells against I/R induced cell death. Thus, therapeutic ma-nipulation of autophagy in the challenged myocardium may benefit post-infarction cardiac healing and remodeling.

  17. Autophagy and the nutritional signaling pathway

    Directory of Open Access Journals (Sweden)

    Long HE,Shabnam ESLAMFAM,Xi MA,Defa LI

    2016-09-01

    Full Text Available During their growth and development, animals adapt to tremendous changes in order to survive. These include responses to both environmental and physiological changes and autophagy is one of most important adaptive and regulatory mechanisms. Autophagy is defined as an autolytic process to clear damaged cellular organelles and recycle the nutrients via lysosomic degradation. The process of autophagy responds to special conditions such as nutrient withdrawal. Once autophagy is induced, phagophores form and then elongate and curve to form autophagosomes. Autophagosomes then engulf cargo, fuse with endosomes, and finally fuse with lysosomes for maturation. During the initiation process, the ATG1/ULK1 (unc-51-like kinase 1 and VPS34 (which encodes a class III phosphatidylinositol (PtdIns 3-kinase complexes are critical in recruitment and assembly of other complexes required for autophagy. The process of autophagy is regulated by autophagy related genes (ATGs. Amino acid and energy starvation mediate autophagy by activating mTORC1 (mammalian target of rapamycin and AMP-activated protein kinase (AMPK. AMPK is the energy status sensor, the core nutrient signaling component and the metabolic kinase of cells. This review mainly focuses on the mechanism of autophagy regulated by nutrient signaling especially for the two important complexes, ULK1 and VPS34.

  18. FGF signalling regulates bone growth through autophagy.

    Science.gov (United States)

    Cinque, Laura; Forrester, Alison; Bartolomeo, Rosa; Svelto, Maria; Venditti, Rossella; Montefusco, Sandro; Polishchuk, Elena; Nusco, Edoardo; Rossi, Antonio; Medina, Diego L; Polishchuk, Roman; De Matteis, Maria Antonietta; Settembre, Carmine

    2015-12-10

    Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes. PMID:26595272

  19. The Scope of Gene Patent Protection and the TRIPS Agreement

    DEFF Research Database (Denmark)

    Sommer, Tine

    2007-01-01

    The Scope of Gene Patent Protection and the TRIPS Agreement - An Exclusively Nondiscriminatory Approach?   Gene patenting in Europe has provoked much debate both before and since the adoption of Directive 98/44/EC on the legal protection of biotechnological inventions. Some of the major points...... of discussion have been focused on the scope of protection (e.g. purpose-bound protection) and gene patents being subject to a specific DNA regime on patent rights. The Directive can be interpreted as favouring such a solution, but so far the European Commission has decided neither to support nor reject...

  20. NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Sobhakumari, Arya [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Schickling, Brandon M. [Department of Internal Medicine, The University of Iowa, Iowa City, IA (United States); Love-Homan, Laurie; Raeburn, Ayanna [Department of Pathology, The University of Iowa, Iowa City, IA (United States); Fletcher, Elise V.M. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Case, Adam J. [Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Domann, Frederick E. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics (UIHC), Iowa City, IA (United States); Miller, Francis J. [Department of Internal Medicine, The University of Iowa, Iowa City, IA (United States); Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics (UIHC), Iowa City, IA (United States); and others

    2013-11-01

    Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formation in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect. - Highlights: • Erlotinib increased LC3B-II and autophagosome formation in HNSCC cells. • Inhibition of autophagy sensitized HNSCC cells to erlotinib. • Erlotinib increased NOX4 promoter and 3′UTR luciferase activity. • Manipulating NOX4 decreases or increases autophagy.

  1. MicroRNA-146a Induced by Hypoxia Promotes Chondrocyte Autophagy through Bcl-2

    Directory of Open Access Journals (Sweden)

    Fei Zhang

    2015-10-01

    Full Text Available Background/Aims: There have been many studies on the etiology of osteoarthritis (OA with regard to the function of inflammatory cytokines, the process of cartilage degradation, the function of miR-146a, hypoxia stimulation and autophagy in OA chondrocytes, but there have been no reports on the relationship between miR-146a and autophagy in cartilage, especially under hypoxia. This study aimed to confirm the relationship of miR-146a and autophagy in cartilage under hypoxia. Methods: Chondrocytes were treated by hypoxia gradients, and the main factors including HIF-1α, HIF-2α, miR-146a and Bcl-2 and autophagy markers ULK-1, ATG-5 were detected by quantitative PCR (Q-PCR and western blotting. The autophagy marker LC-3 was detected by immunofluorescence. The reciprocal effects between miR-146a and Bcl-2 were confirmed by several combinations of shRNAs and adenovirus-gene systems followed by Q-PCR and western blot detection. Results: Hypoxia maintained the chondrocytes phenotype and promoted autophagy and miR-146a expression via HIF-1α, but not HIF-2α, while miR-146a did not reversely affect HIF-1α. The autophagy induced by hypoxia through HIF-1α, miR-146a and Bcl-2. Simply, hypoxia induced HIF-1α, and HIF-1α increased miR-146a, but miR-146a suppressed Bcl-2, an autophagy inhibitor. While Bcl-2 affected neither HIF-1α nor miR-146a. The absence of both HIF-1α and miR-146a or Bcl-2 over-expression inhibited hypoxia-induced autophagy. Conclusion: HIF-1α, miR-146a and Bcl-2 play crucial roles during hypoxia-induced autophagy, Hypoxia, HIF-1α and miR-146a promote chondrocytes autophagy via depressing Bcl-2. We conclude that miR-146a may serve as a novel therapeutic target for protecting cartilage from degeneration in OA.

  2. Autophagy in response to photodynamic therapy: cell survival vs. cell death

    Science.gov (United States)

    Oleinick, Nancy L.; Xue, Liang-yan; Chiu, Song-mao; Joseph, Sheeba

    2009-02-01

    Autophagy (or more properly, macroautophagy) is a pathway whereby damaged organelles or other cell components are encased in a double membrane, the autophagosome, which fuses with lysosomes for digestion by lysosomal hydrolases. This process can promote cell survival by removing damaged organelles, but when damage is extensive, it can also be a mechanism of cell death. Similar to the Kessel and Agostinis laboratories, we have reported the vigorous induction of autophagy by PDT; this was found in human breast cancer MCF-7 cells whether or not they were able to efficiently induce apoptosis. One way to evaluate the role of autophagy in PDT-treated cells is to silence one of the essential genes in the pathway. Kessel and Reiners silenced the Atg7 gene of murine leukemia L1210 cells using inhibitory RNA and found sensitization to PDT-induced cell death at a low dose of PDT, implying that autophagy is protective when PDT damage is modest. We have examined the role of autophagy in an epithelium-derived cancer cell by comparing parental and Atg7-silenced MCF-7 cells to varying doses of PDT with the phthalocyanine photosensitizer Pc 4. In contrast to L1210 cells, autophagy-deficient MCF-7 cells were more resistant to the lethal effects of PDT, as judged by clonogenic assays. A possible explanation for the difference in outcome for L1210 vs. MCF-7 cells is the greatly reduced ability of the latter to undergo apoptosis, a deficiency that may convert autophagy into a cell-death process even at low PDT doses. Experiments to investigate the mechanism(s) responsible are in process.

  3. Liver Autophagy in Anorexia Nervosa and Acute Liver Injury

    Directory of Open Access Journals (Sweden)

    Marouane Kheloufi

    2014-01-01

    Full Text Available Autophagy, a lysosomal catabolic pathway for long-lived proteins and damaged organelles, is crucial for cell homeostasis, and survival under stressful conditions. During starvation, autophagy is induced in numerous organisms ranging from yeast to mammals, and promotes survival by supplying nutrients and energy. In the early neonatal period, when transplacental nutrients supply is interrupted, starvation-induced autophagy is crucial for neonates’ survival. In adult animals, autophagy provides amino acids and participates in glucose metabolism following starvation. In patients with anorexia nervosa, autophagy appears initially protective, allowing cells to copes with nutrient deprivation. However, when starvation is critically prolonged and when body mass index reaches 13 kg/m2 or lower, acute liver insufficiency occurs with features of autophagic cell death, which can be observed by electron microscopy analysis of liver biopsy samples. In acetaminophen overdose, a classic cause of severe liver injury, autophagy is induced as a protective mechanism. Pharmacological enhancement of autophagy protects against acetaminophen-induced necrosis. Autophagy is also activated as a rescue mechanism in response to Efavirenz-induced mitochondrial dysfunction. However, Efavirenz overdose blocks autophagy leading to liver cell death. In conclusion, in acute liver injury, autophagy appears as a protective mechanism that can be however blocked or overwhelmed.

  4. Tumor Suppression and Promotion by Autophagy

    Directory of Open Access Journals (Sweden)

    Yenniffer Ávalos

    2014-01-01

    Full Text Available Autophagy is a highly regulated catabolic process that involves lysosomal degradation of proteins and organelles, mostly mitochondria, for the maintenance of cellular homeostasis and reduction of metabolic stress. Problems in the execution of this process are linked to different pathological conditions, such as neurodegeneration, aging, and cancer. Many of the proteins that regulate autophagy are either oncogenes or tumor suppressor proteins. Specifically, tumor suppressor genes that negatively regulate mTOR, such as PTEN, AMPK, LKB1, and TSC1/2 stimulate autophagy while, conversely, oncogenes that activate mTOR, such as class I PI3K, Ras, Rheb, and AKT, inhibit autophagy, suggesting that autophagy is a tumor suppressor mechanism. Consistent with this hypothesis, the inhibition of autophagy promotes oxidative stress, genomic instability, and tumorigenesis. Nevertheless, autophagy also functions as a cytoprotective mechanism under stress conditions, including hypoxia and nutrient starvation, that promotes tumor growth and resistance to chemotherapy in established tumors. Here, in this brief review, we will focus the discussion on this ambiguous role of autophagy in the development and progression of cancer.

  5. Autophagy: A double-edged sword in intervertebral disk degeneration.

    Science.gov (United States)

    Zhang, Shu-Jun; Yang, Wei; Wang, Cheng; He, Wen-Si; Deng, Hai-Yang; Yan, Yi-Guo; Zhang, Jian; Xiang, Yong-Xiao; Wang, Wen-Jun

    2016-06-01

    Autophagy is a homeostatic mechanism through which intracellular damaged organelles and proteins are degraded and recycled in response to increased metabolic demands or stresses. Although primarily cytoprotective, dysfunction of autophagy is often associated with many degenerative diseases, including intervertebral disc (IVD) degeneration (IDD). As a main contributing factor to low back pain, IDD is the pathological basis for various debilitating spinal diseases. Either higher or lower levels of autophagy are observed in degenerative IVD cells. Despite the precise role of autophagy in disc degeneration that is still controversial, with difference from protection to aggravation, targeting autophagy has shown promise for mitigating disc degeneration. In the current review, we summarize the changes of autophagy in degenerative IVD cells and mainly discuss the relationship between autophagy and IDD. With continued efforts, modulation of the autophagic process could be a potential and attractive therapeutic strategy for degenerative disc disease. PMID:27018178

  6. HIV-1 differentially modulates autophagy in neurons and astrocytes.

    Science.gov (United States)

    Mehla, Rajeev; Chauhan, Ashok

    2015-08-15

    Autophagy, a lysosomal degradative pathway that maintains cellular homeostasis, has emerged as an innate immune defense against pathogens. The role of autophagy in the deregulated HIV-infected central nervous system (CNS) is unclear. We have found that HIV-1-induced neuro-glial (neurons and astrocytes) damage involves modulation of the autophagy pathway. Neuro-glial stress induced by HIV-1 led to biochemical and morphological dysfunctions. X4 HIV-1 produced neuro-glial toxicity coupled with suppression of autophagy, while R5 HIV-1-induced toxicity was restricted to neurons. Rapamycin, a specific mTOR inhibitor (autophagy inducer) relieved the blockage of the autophagy pathway caused by HIV-1 and resulted in neuro-glial protection. Further understanding of the regulation of autophagy by cytokines and chemokines or other signaling events may lead to recognition of therapeutic targets for neurodegenerative diseases.

  7. Folate deprivation modulates the expression of autophagy- and circadian-related genes in HT-22 hippocampal neuron cells through GR-mediated pathway.

    Science.gov (United States)

    Sun, Qinwei; Yang, Yang; Li, Xi; He, Bin; Jia, Yimin; Zhang, Nana; Zhao, Ruqian

    2016-08-01

    Folic acid (FA) is an extremely important nutrient for brain formation and development. FA deficiency is highly linked to brain degeneration and age-related diseases, which are also associated with autophagic activities and circadian rhythm in hippocampal neurons. However, little is known how autophagy- and circadian-related genes in hippocampal neurons are regulated under FA deficiency. Here, hippocampal neuroncells (HT-22) were employed to determine the effect of FA deprivation (FD) on the expression of relevant genes and to reveal the potential role of glucocorticoid receptor (GR). FD increased autophagic activities in HT-22 cells, associated with significantly (PChIP assay showed that FD promoted (Pnetwork in response to folate deficiency. PMID:27133904

  8. The metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), inhibits stress-induced autophagy in cancer cells.

    Science.gov (United States)

    Sahni, Sumit; Bae, Dong-Hun; Lane, Darius J R; Kovacevic, Zaklina; Kalinowski, Danuta S; Jansson, Patric J; Richardson, Des R

    2014-04-01

    N-myc downstream regulated gene 1 (NDRG1) is a potent metastasis suppressor with an undefined role in the stress response. Autophagy is a pro-survival pathway and can be regulated via the protein kinase-like endoplasmic reticulum kinase (PERK)/eIF2α-mediated endoplasmic reticulum (ER) stress pathway. Hence, we investigated the role of NDRG1 in stress-induced autophagy as a mechanism of inhibiting metastasis via the induction of apoptosis. As thiosemicarbazone chelators induce stress and up-regulate NDRG1 to inhibit metastasis, we studied their effects on the ER stress response and autophagy. This was important to assess, as little is understood regarding the role of the stress induced by iron depletion and its role in autophagy. We observed that the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which forms redox-active iron and copper complexes, effectively induced ER stress as shown by activation of the PERK/eIF2α pathway. Dp44mT also increased the expression of the autophagic marker, LC3-II, and this was dependent on activation of the PERK/eIF2α axis, as silencing PERK prevented LC3-II accumulation. The effect of Dp44mT on LC3-II expression was at least partially due to iron-depletion, as this effect was also demonstrated with the classical iron chelator, desferrioxamine (DFO), and was not observed for the DFO-iron complex. NDRG1 overexpression also inhibited basal autophagic initiation and the ER stress-mediated autophagic pathway via suppression of the PERK/eIF2α axis. Moreover, NDRG1-mediated suppression of the pro-survival autophagic pathway probably plays a role in its anti-metastatic effects by inducing apoptosis. In fact, multiple pro-apoptotic markers were increased, whereas anti-apoptotic Bcl-2 was decreased upon NDRG1 overexpression. This study demonstrates the role of NDRG1 as an autophagic inhibitor that is important for understanding its mechanism of action. PMID:24532803

  9. Autophagy promotes resistance to photodynamic therapy-induced apoptosis selectively in colorectal cancer stem-like cells.

    Science.gov (United States)

    Wei, Ming-Feng; Chen, Min-Wei; Chen, Ke-Cheng; Lou, Pei-Jen; Lin, Susan Yun-Fan; Hung, Shih-Chieh; Hsiao, Michael; Yao, Cheng-Jung; Shieh, Ming-Jium

    2014-07-01

    Recent studies have indicated that cancer stem-like cells (CSCs) exhibit a high resistance to current therapeutic strategies, including photodynamic therapy (PDT), leading to the recurrence and progression of colorectal cancer (CRC). In cancer, autophagy acts as both a tumor suppressor and a tumor promoter. However, the role of autophagy in the resistance of CSCs to PDT has not been reported. In this study, CSCs were isolated from colorectal cancer cells using PROM1/CD133 (prominin 1) expression, which is a surface marker commonly found on stem cells of various tissues. We demonstrated that PpIX-mediated PDT induced the formation of autophagosomes in PROM1/CD133(+) cells, accompanied by the upregulation of autophagy-related proteins ATG3, ATG5, ATG7, and ATG12. The inhibition of PDT-induced autophagy by pharmacological inhibitors and silencing of the ATG5 gene substantially triggered apoptosis of PROM1/CD133(+) cells and decreased the ability of colonosphere formation in vitro and tumorigenicity in vivo. In conclusion, our results revealed a protective role played by autophagy against PDT in CSCs and indicated that targeting autophagy could be used to elevate the PDT sensitivity of CSCs. These findings would aid in the development of novel therapeutic approaches for CSC treatment.

  10. Aggresome-autophagy involvement in a sarcopenic patient with rigid spine syndrome and a p.C150R mutation in FHL1 gene

    Directory of Open Access Journals (Sweden)

    Patrizia eSabatelli

    2014-08-01

    Full Text Available The four-and-half LIM domain protein 1 (FHL1 is highly expressed in skeletal and cardiac muscle. Mutations of the FHL1 gene have been associated with diverse chronic myopathies including reducing body myopathy (RBM, rigid spine syndrome, and Emery-Dreifuss muscular dystrophy. We investigated a family with a mutation (p.C150R in the second LIM domain of FHL1. In this family, a brother and a sister were affected by rigid spine syndrome, and their mother had a mild lower limbs weakness. The 34-year-old female had an early and progressive rigidity of the cervical spine and severe respiratory insufficiency. Muscle mass evaluated by DXA was markedly reduced, while fat mass was increased to 40%. CT scan showed an almost complete substitution of muscle by fibro-adipose tissue. Muscle biopsy showed accumulation of FHL1 throughout the cytoplasm and around myonuclei into multiproteic aggregates with aggresome/autophagy features as indicated by ubiquitin, p62 and LC3 labeling. DNA deposits, not associated with nuclear lamina components and histones, were also detected into the aggregates, suggesting nuclear degradation. Ultrastructural analysis showed the presence of dysmorphic nuclei, accumulation of tubulofilamentous and granular material and perinuclear accumulation of autophagic vacuoles. These data point to an involvement of the aggresome-autophagy pathway in the pathophysiological mechanism underlying the muscle pathology of FHL1 C150R mutation.

  11. Short-term starvation attenuates liver ischemia-reperfusion injury (IRI) by Sirt1-autophagy signaling in mice

    Science.gov (United States)

    Qin, Jianjie; Zhou, Junjin; Dai, Xinzheng; Zhou, Haoming; Pan, Xiongxiong; Wang, Xuehao; Zhang, Feng; Rao, Jianhua; Lu, Ling

    2016-01-01

    Calorie restriction or starvation (fasting) has some beneficial effects in terms of prolonging life and increasing resistance to stress. It has also been shown that calorie restriction has a protective role during ischemia-reperfusion injury (IRI) in several organs, but the underlying mechanism has not been elucidated. In this study we investigated the effects and molecular mechanisms of short-term starvation (STS) on liver IRI in a mouse liver IRI model. We found that STS significantly attenuated liver IRI in this model, as evidenced by inhibition of serum aminotransferase levels, and decreased pathological damage and hepatocellular apoptosis, especially after 2- or 3-day starvation. Furthermore, we found that 2- or 3-day starvation induced expression of hepatocellular autophagy in vivo and in vitro. Further experiments provided support for the notion that STS-induced autophagy played a key role during starvation-regulated protection against liver IRI via autophagy inhibition with 3-methyladenine. Interestingly, the longevity gene Sirt1 was also significantly up-regulated in liver after STS. Importantly, inhibition of Sirt1 by sirtinol abolished STS-induced autophagy and further abrogated STS-mediated protection against liver IRI. In conclusion, our results indicate that STS attenuates liver IRI via the Sirt1-autophagy pathway. Our findings provide a rationale for a novel therapeutic strategy for managing liver IRI. PMID:27648127

  12. Autophagy-related prognostic signature for breast cancer.

    Science.gov (United States)

    Gu, Yunyan; Li, Pengfei; Peng, Fuduan; Zhang, Mengmeng; Zhang, Yuanyuan; Liang, Haihai; Zhao, Wenyuan; Qi, Lishuang; Wang, Hongwei; Wang, Chenguang; Guo, Zheng

    2016-03-01

    Autophagy is a process that degrades intracellular constituents, such as long-lived or damaged proteins and organelles, to buffer metabolic stress under starvation conditions. Deregulation of autophagy is involved in the progression of cancer. However, the predictive value of autophagy for breast cancer prognosis remains unclear. First, based on gene expression profiling, we found that autophagy genes were implicated in breast cancer. Then, using the Cox proportional hazard regression model, we detected autophagy prognostic signature for breast cancer in a training dataset. We identified a set of eight autophagy genes (BCL2, BIRC5, EIF4EBP1, ERO1L, FOS, GAPDH, ITPR1 and VEGFA) that were significantly associated with overall survival in breast cancer. The eight autophagy genes were assigned as a autophagy-related prognostic signature for breast cancer. Based on the autophagy-related signature, the training dataset GSE21653 could be classified into high-risk and low-risk subgroups with significantly different survival times (HR = 2.72, 95% CI = (1.91, 3.87); P = 1.37 × 10(-5)). Inactivation of autophagy was associated with shortened survival of breast cancer patients. The prognostic value of the autophagy-related signature was confirmed in the testing dataset GSE3494 (HR = 2.12, 95% CI = (1.48, 3.03); P = 1.65 × 10(-3)) and GSE7390 (HR = 1.76, 95% CI = (1.22, 2.54); P = 9.95 × 10(-4)). Further analysis revealed that the prognostic value of the autophagy signature was independent of known clinical prognostic factors, including age, tumor size, grade, estrogen receptor status, progesterone receptor status, ERBB2 status, lymph node status and TP53 mutation status. Finally, we demonstrated that the autophagy signature could also predict distant metastasis-free survival for breast cancer.

  13. The role of autophagy in sensitizing malignant glioma cells to radiation therapy

    Institute of Scientific and Technical Information of China (English)

    Wenzhuo Zhuang; Zhenghong Qin; Zhongqin Liang

    2009-01-01

    Malignant gliomas representthe majority of primary brain tumors.The current standard treatments for malignant gliomas include surgical resection,radiation therapy,and chemotherapy.Radiotherapy,a standard adjuvant therapy,confers some survival advantages,but resistance of the glioma cells to the efficacy of radiation limits the success of the treatment.The mechanisms underlying glioma cell radioresistance have remained elusive.Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm.Recent studies suggest that autophagy may be important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy.Also,autophagy is a novel response of glioma cells to ionizing radiation.Autophagic cell death is considered programmed cell death type Ⅱ,whereas apoptosis is programmed cell death type Ⅰ.These two types of cell death are predominantly distinctive,but many studies demonstrate a cross-talk between them.Whether autophagy in cancer cells causes death or protects cells is controversial.The regulatory pathways of autophagy share several molecules.P13K/Akt/Mtor,DNA-PK,tumor suppressor genes, mitochondrial damage,and lysosome may play important roles in radiation-induced autophagy in glioma cells.Recently,a highly tumorigenic glioma tumor subpopulation,termed cancer stem cell or tumor-initiating cell,has been shown to promote therapeutic resistance.This review summarizes the main mediators associated with radiation-induced autophagy in malignant glioma cells and discusses the implications of the cancer stem cell hypothesis for the development of future therapies for brain tumors.

  14. Autophagy is an inflammation-related defensive mechanism against disease.

    Science.gov (United States)

    Joven, Jorge; Guirro, Maria; Mariné-Casadó, Roger; Rodríguez-Gallego, Esther; Menéndez, Javier A

    2014-01-01

    The inflammatory response is an energy-intensive process. Consequently, metabolism is closely associated with immune function. The autophagy machinery plays a role in metabolism by providing energy but may also be used to attack invading pathogens (xenophagy). The autophagy machinery may function to protect against not only the threats of infection but also the threats of the host's own response acting on the central immunological tolerance and the negative regulation of innate and inflammatory signaling. The balance between too little and too much autophagy is critical for the survival of immune cells because autophagy is linked to type 2-cell death programmed necrosis and apoptosis. Changes in inflammatory cells are driven by extracellular signals; however, the mechanisms by which cytokines mediate autophagy regulation and govern immune cell function remain unknown. Certain cytokines increase autophagy, whereas others inhibit autophagy. The relationship between autophagy and inflammation is also important in the pathogenesis of metabolic, non-communicable diseases. Inflammation per se is not the cause of obesity-associated diseases, but it is secondary to both the positive energy balance and the specific cellular responses. In metabolic tissues, the suppression of autophagy increases inflammation with the overexpression of cytokines, resulting in an activation of autophagy. The physiological role of these apparently contradictory findings remains uncertain but exemplifies future challenges in the therapeutic modulation of autophagy in the management of disease.

  15. Protective effects of astaxanthin on ConA-induced autoimmune hepatitis by the JNK/p-JNK pathway-mediated inhibition of autophagy and apoptosis.

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    Jingjing Li

    Full Text Available Astaxanthin, a potent antioxidant, exhibits a wide range of biological activities, including antioxidant, atherosclerosis and antitumor activities. However, its effect on concanavalin A (ConA-induced autoimmune hepatitis remains unclear. The aim of this study was to investigate the protective effects of astaxanthin on ConA-induced hepatitis in mice, and to elucidate the mechanisms of regulation.Autoimmune hepatitis was induced in in Balb/C mice using ConA (25 mg/kg, and astaxanthin was orally administered daily at two doses (20 mg/kg and 40 mg/kg for 14 days before ConA injection. Levels of serum liver enzymes and the histopathology of inflammatory cytokines and other maker proteins were determined at three time points (2, 8 and 24 h. Primary hepatocytes were pretreated with astaxanthin (80 μM in vitro 24 h before stimulation with TNF-α (10 ng/ml. The apoptosis rate and related protein expression were determined 24 h after the administration of TNF-α.Astaxanthin attenuated serum liver enzymes and pathological damage by reducing the release of inflammatory factors. It performed anti-apoptotic effects via the descending phosphorylation of Bcl-2 through the down-regulation of the JNK/p-JNK pathway.This research firstly expounded that astaxanthin reduced immune liver injury in ConA-induced autoimmune hepatitis. The mode of action appears to be downregulation of JNK/p-JNK-mediated apoptosis and autophagy.

  16. Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis.

    Science.gov (United States)

    Cabrera, Sandra; Maciel, Mariana; Herrera, Iliana; Nava, Teresa; Vergara, Fabián; Gaxiola, Miguel; López-Otín, Carlos; Selman, Moisés; Pardo, Annie

    2015-04-01

    Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.

  17. SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan); Koya, Daisuke, E-mail: koya0516@kanazawa-med.ac.jp [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer SIRT1 inactivation decreases autophagy in THP-1 cell. Black-Right-Pointing-Pointer Inhibition of autophagy induces inflammation. Black-Right-Pointing-Pointer SIRT1 inactivation induces inflammation through NF-{kappa}B activation. Black-Right-Pointing-Pointer The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-{kappa}B activation. Black-Right-Pointing-Pointer SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD{sup +}-dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-6 through nuclear factor (NF)-{kappa}B signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-{kappa}B activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-{kappa}B activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and

  18. Tyrosinase-Cre-Mediated Deletion of the Autophagy Gene Atg7 Leads to Accumulation of the RPE65 Variant M450 in the Retinal Pigment Epithelium of C57BL/6 Mice

    Science.gov (United States)

    Sukseree, Supawadee; Chen, Ying-Ting; Laggner, Maria; Gruber, Florian; Petit, Valérie; Nagelreiter, Ionela-Mariana; Mlitz, Veronika; Rossiter, Heidemarie; Pollreisz, Andreas; Schmidt-Erfurth, Ursula; Larue, Lionel; Tschachler, Erwin

    2016-01-01

    Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7f/f Tyr-Cre mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete the autophagy gene Atg7. In line with pigment cell-directed blockade of autophagy, the RPE and the melanocytes of the choroid showed strong accumulation of the autophagy adaptor and substrate, sequestosome 1 (Sqstm1)/p62, relative to the levels in control mice. Immunofluorescence and Western blot analysis demonstrated that the RPE, but not the choroid melanocytes, of Atg7f/f Tyr-Cre mice also had strongly increased levels of retinoid isomerohydrolase RPE65, a pivotal enzyme for the maintenance of visual perception. In contrast to Sqstm1, genes involved in retinal regeneration, i.e. Lrat, Rdh5, Rgr, and Rpe65, were expressed at higher mRNA levels. Sequencing of the Rpe65 gene showed that Atg7f/f and Atg7f/f Tyr-Cre mice carry a point mutation (L450M) that is characteristic for the C57BL/6 mouse strain and reportedly causes enhanced degradation of the RPE65 protein by an as-yet unknown mechanism. These results suggest that the increased abundance of RPE65 M450 in the RPE of Atg7f/f Tyr-Cre mice is, at least partly, mediated by upregulation of Rpe65 transcription; however, our data are also compatible with the hypothesis that the RPE65 M450 protein is degraded by Atg7-dependent autophagy in Atg7f/f mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms. PMID:27537685

  19. Autophagy, Metabolism, and Cancer.

    Science.gov (United States)

    White, Eileen; Mehnert, Janice M; Chan, Chang S

    2015-11-15

    Macroautophagy (autophagy hereafter) captures intracellular proteins and organelles and degrades them in lysosomes. The degradation breakdown products are released from lysosomes and recycled into metabolic and biosynthetic pathways. Basal autophagy provides protein and organelle quality control by eliminating damaged cellular components. Starvation-induced autophagy recycles intracellular components into metabolic pathways to sustain mitochondrial metabolic function and energy homeostasis. Recycling by autophagy is essential for yeast and mammals to survive starvation through intracellular nutrient scavenging. Autophagy suppresses degenerative diseases and has a context-dependent role in cancer. In some models, cancer initiation is suppressed by autophagy. By preventing the toxic accumulation of damaged protein and organelles, particularly mitochondria, autophagy limits oxidative stress, chronic tissue damage, and oncogenic signaling, which suppresses cancer initiation. This suggests a role for autophagy stimulation in cancer prevention, although the role of autophagy in the suppression of human cancer is unclear. In contrast, some cancers induce autophagy and are dependent on autophagy for survival. Much in the way that autophagy promotes survival in starvation, cancers can use autophagy-mediated recycling to maintain mitochondrial function and energy homeostasis to meet the elevated metabolic demand of growth and proliferation. Thus, autophagy inhibition may be beneficial for cancer therapy. Moreover, tumors are more autophagy-dependent than normal tissues, suggesting that there is a therapeutic window. Despite these insights, many important unanswered questions remain about the exact mechanisms of autophagy-mediated cancer suppression and promotion, how relevant these observations are to humans, and whether the autophagy pathway can be modulated therapeutically in cancer. See all articles in this CCR Focus section, "Cell Death and Cancer Therapy."

  20. Autophagy: for better or for worse

    Institute of Scientific and Technical Information of China (English)

    Ellen Wirawan; Tom Vanden Berghe; Saskia Lippens; Patrizia Agostinis; Peter Vandenabeele

    2012-01-01

    Autophagy is a lysosomal degradation pathway that degrades damaged or superfluous cell components into basic biomolecules,which are then recycled back into the cytosol.In this respect,autophagy drives a flow of biomolecules in a continuous degradation-regeneration cycle.Autophagy is generally considered a pro-survival mechanism protecting cells under stress or poor nutrient conditions.Current research clearly shows that autophagy fulfills numerous functions in vital biological processes.It is implicated in development,differentiation,innate and adaptive immunity,ageing and cell death.In addition,accumulating evidence demonstrates interesting links between autophagy and several human diseases and tumor development.Therefore,autophagy seems to be an important player in the life and death of cells and organisms.Despite the mounting knowledge about autophagy,the mechanisms through which the autophagic machinery regulates these diverse processes are not entirely understood.In this review,we give a comprehensive overview of the autophagic signaling pathway,its role in general cellular processes and its connection to cell death.In addition,we present a brief overview of the possible contribution of defective autophagic signaling to disease.

  1. WT1 is involved in the Akt-JNK pathway dependent autophagy through directly regulating Gas1 expression in human osteosarcoma cells.

    Science.gov (United States)

    Mo, Hao; He, Juliang; Yuan, Zhenchao; Mo, Ligen; Wu, Zhenjie; Lin, Xiang; Liu, Bin; Guan, Jian

    2016-09-01

    Macroautophagy (herein termed autophagy) works as a protective mechanism in tumorigenesis and development under metabolic stress condition. Multitudes of genes have been found involved in this process during past decades. In the present study, we report that Wilm's tumor suppressor1 (WT1) is involved in autophagy in osteosarcoma (OS) cells. WT1, a transcription factor with multitude of target genes, expresses in a majority of cancer types. Though wide-ranging effect of WT1 is now well documented, the function of WT1 in tumors remains poorly defined. In this chapter, it is found that high expression of WT1 positively correlates with active autophagy in human osteosarcoma cells. And further study on cell signaling pathway illustrates that Akt/JNK pathway acts as a positive regulator of autophagy induced by WT1. Here, we present evidence that WT1 modulates Akt/JNK signaling pathway mediated autophagy by controlling the expression of growth arrest-specific 1 (Gas1). We show that WT1 is required for Gas1 transcription in osteosarcoma cells. And Gas1 is upregulated followed WT1 overexpression in a time-dependent manner. Loss of Gas1 results in a reduction of WT1-induced autophagy. PMID:27453337

  2. Hypoxia, MTOR and autophagy

    OpenAIRE

    Blagosklonny, Mikhail V.

    2013-01-01

    Although hypoxia can cause cell cycle arrest, it may simultaneously suppress a conversion from this arrest to senescence. Furthermore, hypoxia can suppress senescence caused by diverse stimuli, maintaining reversible quiescence instead. Hypoxia activates autophagy and inhibits MTOR, thus also activating autophagy. What is the relationship between autophagy and cellular senescence? Also, can inhibition of MTOR and stimulation of autophagy explain the gerosuppressive effects of hypoxia?

  3. Overexpression of the autophagy-related gene SiATG8a from foxtail millet (Setaria italica L.) confers tolerance to both nitrogen starvation and drought stress in Arabidopsis.

    Science.gov (United States)

    Li, Wei-wei; Chen, Ming; Zhong, Li; Liu, Jia-ming; Xu, Zhao-shi; Li, Lian-cheng; Zhou, Yong-Bin; Guo, Chang-Hong; Ma, You-Zhi

    2015-12-25

    Autophagy is an evolutionarily conserved biological process in all eukaryotes for the degradation of intracellular components for nutrient recycling. Autophagy is known to be involved in responses to low nitrogen stress in Arabidopsis. Foxtail millet has strong abiotic stress resistance to both low nutrient and drought stress. However, to date, there have only been a few genes reported to be related with abiotic stress resistance in foxtail millet. In this study, we identified an autophagy-related gene, SiATG8a, from foxtail millet. SiATG8a is mainly expressed in stems and its expression was dramatically induced by drought stress and nitrogen starvation treatments. SiATG8a was localized in the membrane and cytoplasm of foxtail millet. Overexpression of SiATG8a in Arabidopsis conferred tolerance to both nitrogen starvation and to drought stress. Under nitrogen starvation conditions, the SiATG8a transgenic plants had larger root and leaf areas and accumulated more total nitrogen than wild-type plants. The transgenic plants had lower total protein concentrations than did the WT plants. Under drought stress, the SiATG8a transgenic plants had higher survival rates, chlorophyll content, and proline content, but had lower MDA content than wild type plants. Taken together, our results represent the first identified case where overexpression of autophagy related gene can simultaneously improve plant resistance to low nitrogen and drought stresses. These findings implicate plant autophagy in plant stress responses to low nitrogen and drought and should be helpful in efforts to improve stresses resistance to nitrogen starvation and drought of crops by genetic transformation.

  4. Autophagy in granular corneal dystrophy type 2.

    Science.gov (United States)

    Choi, Seung-Il; Kim, Eung Kweon

    2016-03-01

    Autophagy is a lysosomal degradative process that is essential for cellular homeostasis and metabolic stress adaptation. Defective autophagy is involved in the pathogenesis of many diseases including granular corneal dystrophy type 2 (GCD2). GCD2 is an autosomal dominant disorder caused by substitution of histidine for arginine at codon 124 (R124H) in the transforming growth factor β-induced gene (TGFBI) on chromosome 5q31. Transforming growth factor β-induced protein (TGFBIp) is degraded by autophagy, but mutant-TGFBIp accumulates in autophagosomes and/or lysosomes, despite significant activation of basal autophagy, in GCD2 corneal fibroblasts. Furthermore, inhibition of autophagy induces cell death of GCD2 corneal fibroblasts through active caspase-3. As there is currently no pharmacological treatment for GCD2, development of novel therapies is required. A potential strategy for preventing cytoplasmic accumulation of mutant-TGFBIp in GCD2 corneal fibroblasts is to enhance mutant-TGFBIp degradation. This could be achieved by activation of the autophagic pathway. Here, we will consider the role and the potential therapeutic benefits of autophagy in GCD2, with focus on TGFBIp degradation, in light of the recently established role of autophagy in protein degradation.

  5. Guidelines for monitoring autophagy in Caenorhabditis elegans.

    Science.gov (United States)

    Zhang, Hong; Chang, Jessica T; Guo, Bin; Hansen, Malene; Jia, Kailiang; Kovács, Attila L; Kumsta, Caroline; Lapierre, Louis R; Legouis, Renaud; Lin, Long; Lu, Qun; Meléndez, Alicia; O'Rourke, Eyleen J; Sato, Ken; Sato, Miyuki; Wang, Xiaochen; Wu, Fan

    2015-01-01

    The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.

  6. Neferine Attenuates the Protein Level and Toxicity of Mutant Huntingtin in PC-12 Cells via Induction of Autophagy

    Directory of Open Access Journals (Sweden)

    Vincent Kam Wai Wong

    2015-02-01

    Full Text Available Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington’s disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or a-synuclein. Through the GFP-LC3 autophagy detection platform, we have  identified  neferine,  isolated  from  the  lotus  seed  embryo  of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74 in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

  7. Autophagy in Tuberculosis

    Science.gov (United States)

    Deretic, Vojo

    2014-01-01

    Autophagy as an immune mechanism controls inflammation and acts as a cell-autonomous defense against intracellular microbes including Mycobacterium tuberculosis. An equally significant role of autophagy is its anti-inflammatory and tissue-sparing function. This combination of antimicrobial and anti-inflammatory actions prevents active disease in animal models. In human populations, genetic links between autophagy, inflammatory bowel disease, and susceptibility to tuberculosis provide further support to these combined roles of autophagy. The autophagic control of M. tuberculosis and prevention of progressive disease provide novel insights into physiological and immune control of tuberculosis. It also offers host-based therapeutic opportunities because autophagy can be pharmacologically modulated. PMID:25167980

  8. E50K-OPTN-induced retinal cell death involves the Rab GTPase-activating protein, TBC1D17 mediated block in autophagy.

    Directory of Open Access Journals (Sweden)

    Madhavi Latha Somaraju Chalasani

    Full Text Available The protein optineurin coded by OPTN gene is involved in several functions including regulation of endocytic trafficking, autophagy and signal transduction. Certain missense mutations in the gene OPTN cause normal tension glaucoma. A glaucoma-causing mutant of optineurin, E50K, induces death selectively in retinal cells. This mutant induces defective endocytic recycling of transferrin receptor by causing inactivation of Rab8 mediated by the GTPase-activating protein, TBC1D17. Here, we have explored the mechanism of E50K-induced cell death. E50K-OPTN-induced cell death was inhibited by co-expression of a catalytically inactive mutant of TBC1D17 and also by shRNA mediated knockdown of TBC1D17. Endogenous TBC1D17 colocalized with E50K-OPTN in vesicular structures. Co-expression of transferrin receptor partially protected against E50K-induced cell death. Overexpression of the E50K-OPTN but not WT-OPTN inhibited autophagy flux. Treatment of cells with rapamycin, an inducer of autophagy, reduced E50K-OPTN-induced cell death. An LC3-binding-defective mutant of E50K-OPTN showed reduced cell death, further suggesting the involvement of autophagy. TBC1D17 localized to autophagosomes and inhibited autophagy flux dependent on its catalytic activity. Knockdown of TBC1D17 rescued cells from E50K-mediated inhibition of autophagy flux. Overall, our results suggest that E50K mutant induced death of retinal cells involves impaired autophagy as well as impaired transferrin receptor function. TBC1D17, a GTPase-activating protein for Rab GTPases, plays a crucial role in E50K-induced impaired autophagy and cell death.

  9. Rph1 mediates the nutrient-limitation signaling pathway leading to transcriptional activation of autophagy.

    Science.gov (United States)

    Bernard, Amélie; Klionsky, Daniel J

    2015-04-01

    To maintain proper cellular homeostasis, the magnitude of autophagy activity has to be finely tuned in response to environmental changes. Many aspects of autophagy regulation have been extensively studied: pathways integrating signals through the master regulators TORC1 and PKA lead to multiple post-translational modifications affecting the functions, protein-protein interactions, and localization of Atg proteins. The expression of several ATG genes increases sharply upon autophagy induction conditions, and defects in ATG gene expression are associated with various diseases, pointing to the importance of transcriptional regulation of autophagy. Yet, how changes in ATG gene expression affect the rate of autophagy is not well characterized, and transcriptional regulators of the autophagy pathway remain largely unknown. To identify such regulators, we analyzed the expression of several ATG genes in a library of DNA-binding protein mutants. This led to the identification of Rph1 as a master transcriptional regulator of autophagy.

  10. Role of autophagy in diabetes and endoplasmic reticulum stress of pancreatic β-cells

    OpenAIRE

    Quan, Wenying; Lim, Yu-Mi; Lee, Myung-Shik

    2012-01-01

    Type 2 diabetes mellitus is characterized by insulin resistance and failure of pancreatic β-cells producing insulin. Autophagy plays a crucial role in cellular homeostasis through degradation and recycling of organelles such as mitochondria or endoplasmic reticulum (ER). Here we discussed the role of β-cell autophagy in development of diabetes, based on our own studies using mice with β-cell-specific deletion of Atg7 (autophagy-related 7), an important autophagy gene, and studies by others. β...

  11. Autophagy controls BCG-induced trained immunity and the response to intravesical BCG therapy for bladder cancer.

    Directory of Open Access Journals (Sweden)

    Kathrin Buffen

    2014-10-01

    Full Text Available The anti-tuberculosis-vaccine Bacillus Calmette-Guérin (BCG is the most widely used vaccine in the world. In addition to its effects against tuberculosis, BCG vaccination also induces non-specific beneficial effects against certain forms of malignancy and against infections with unrelated pathogens. It has been recently proposed that the non-specific effects of BCG are mediated through epigenetic reprogramming of monocytes, a process called trained immunity. In the present study we demonstrate that autophagy contributes to trained immunity induced by BCG. Pharmacologic inhibition of autophagy blocked trained immunity induced in vitro by stimuli such as β-glucans or BCG. Single nucleotide polymorphisms (SNPs in the autophagy genes ATG2B (rs3759601 and ATG5 (rs2245214 influenced both the in vitro and in vivo training effect of BCG upon restimulation with unrelated bacterial or fungal stimuli. Furthermore, pharmacologic or genetic inhibition of autophagy blocked epigenetic reprogramming of monocytes at the level of H3K4 trimethylation. Finally, we demonstrate that rs3759601 in ATG2B correlates with progression and recurrence of bladder cancer after BCG intravesical instillation therapy. These findings identify a key role of autophagy for the nonspecific protective effects of BCG.

  12. RUFY4: Immunity piggybacking on autophagy?

    Science.gov (United States)

    Terawaki, Seigo; Camosseto, Voahirana; Pierre, Philippe; Gatti, Evelina

    2016-01-01

    Although autophagy is a highly conserved mechanism among species and cell types, few are the molecules involved with the autophagic process that display cell- or tissue- specific expression. We have unraveled the positive regulatory role on autophagy of RUFY4 (RUN and FYVE domain containing 4), which is expressed in subsets of immune cells, including dendritic cells (DCs). DCs orchestrate the eradication of pathogens by coordinating the action of the different cell types involved in microbe recognition and destruction during the immune response. To fulfill this function, DC display particular regulation of their endocytic and autophagy pathways in response to the immune environment. Autophagy flux is downmodulated in DCs upon microbe sensing, but is remarkably augmented, when cells are differentiated in the presence of the pleiotropic cytokine IL4 (interleukin 4). From gene expression studies aimed at comparing the impact of IL4 on DC differentiation, we identified RUFY4, as a novel regulator that augments autophagy flux and, when overexpressed, induces drastic membrane redistribution and strongly tethers lysosomes. RUFY4 is therefore one of the few known positive regulators of autophagy that is expressed in a cell-specific manner or under specific immunological conditions associated with IL4 expression such as allergic asthma.

  13. Global genomic profiling reveals an extensive p53-regulated autophagy program contributing to key p53 responses

    OpenAIRE

    Kenzelmann Broz, Daniela; Spano Mello, Stephano; Bieging, Kathryn T.; Jiang, Dadi; Rachel L Dusek; Brady, Colleen A.; Sidow, Arend; Attardi, Laura D.

    2013-01-01

    To gain new insights into p53 biology, Kenzelmann Broz et al. used high-throughput sequencing to analyze global p53 transcriptional networks in primary mouse embryo fibroblasts in response to DNA damage. This approach identified autophagy genes as direct p53 target genes. p53-induced autophagy was important for both p53-dependent apoptosis and transformation suppression by p53. These data highlight an intimate connection between p53 and autophagy and suggest that autophagy contributes to p53-...

  14. Application and interpretation of current autophagy inhibitors and activators

    Institute of Scientific and Technical Information of China (English)

    Ya-ping YANG; Li-fang HU; Hui-fen ZHENG; Cheng-jie MAO; Wei-dong HU; Kang-ping XIONG; Fen WANG

    2013-01-01

    Aut ophagy is the major intracellular degradation system,by which cytoplasmic materials are delivered to and degraded in the lysosome.As a quality control mechanism for cytoplasmic proteins and organelles,autophagy plays important roles in a variety of human diseases,including neurodegenerative diseases,cancer,cardiovascular disease,diabetes and infectious and inflammatory diseases.The discovery of ATG genes and the dissection of the signaling pathways involved in regulating autophagy have greatly enriched our knowledge on the occurrence and development of this lysosomal degradation pathway.In addition to its role in degradation,autophagy may also promote a type of programmed cell death that is different from apoptosis,termed type II programmed cell death.Owing to the dual roles of autophagy in cell death and the specificity of diseases,the exact mechanisms of autophagy in various diseases require more investigation.The application of autophagy inhibitors and activators will help us understand the regulation of autophagy in human diseases,and provide insight into the use of autophagy-targeted drugs.In this review,we summarize the latest research on autophagy inhibitors and activators and discuss the possibility of their application in human disease therapy.

  15. Autophagy is essential for cardiac morphogenesis during vertebrate development.

    Science.gov (United States)

    Lee, Eunmyong; Koo, Yeon; Ng, Aylwin; Wei, Yongjie; Luby-Phelps, Kate; Juraszek, Amy; Xavier, Ramnik J; Cleaver, Ondine; Levine, Beth; Amatruda, James F

    2014-04-01

    Genetic analyses indicate that autophagy, an evolutionarily conserved lysosomal degradation pathway, is essential for eukaryotic differentiation and development. However, little is known about whether autophagy contributes to morphogenesis during embryogenesis. To address this question, we examined the role of autophagy in the early development of zebrafish, a model organism for studying vertebrate tissue and organ morphogenesis. Using zebrafish that transgenically express the fluorescent autophagy reporter protein, GFP-LC3, we found that autophagy is active in multiple tissues, including the heart, during the embryonic period. Inhibition of autophagy by morpholino knockdown of essential autophagy genes (including atg5, atg7, and becn1) resulted in defects in morphogenesis, increased numbers of dead cells, abnormal heart structure, and reduced organismal survival. Further analyses of cardiac development in autophagy-deficient zebrafish revealed defects in cardiac looping, abnormal chamber morphology, aberrant valve development, and ectopic expression of critical transcription factors including foxn4, tbx5, and tbx2. Consistent with these results, Atg5-deficient mice displayed abnormal Tbx2 expression and defects in valve development and chamber septation. Thus, autophagy plays an essential, conserved role in cardiac morphogenesis during vertebrate development.

  16. Tomato HsfA1a plays a critical role in plant drought tolerance by activating ATG genes and inducing autophagy

    OpenAIRE

    Wang, Yu; Cai, Shuyu; Yin, Lingling; Kai SHI; Xia, Xiaojian; Zhou, Yanhong; Yu, Jingquan; Zhou, Jie

    2015-01-01

    Autophagy plays critical roles in plant responses to stress. In contrast to the wealth of information concerning the core process of plant autophagosome assembly, our understanding of the regulation of autophagy is limited. In this study, we demonstrated that transcription factor HsfA1a played a critical role in tomato tolerance to drought stress, in part through its positive role in induction of autophagy under drought stress. HsfA1a expression was induced by drought stress. Virus-induced Hs...

  17. Selective Autophagy in Drosophila

    Directory of Open Access Journals (Sweden)

    Ioannis P. Nezis

    2012-01-01

    Full Text Available Autophagy is an evolutionarily conserved process of cellular self-eating and is a major pathway for degradation of cytoplasmic material by the lysosomal machinery. Autophagy functions as a cellular response in nutrient starvation, but it is also associated with the removal of protein aggregates and damaged organelles and therefore plays an important role in the quality control of proteins and organelles. Although it was initially believed that autophagy occurs randomly in the cell, during the last years, there is growing evidence that sequestration and degradation of cytoplasmic material by autophagy can be selective. Given the important role of autophagy and selective autophagy in several disease-related processes such as neurodegeneration, infections, and tumorigenesis, it is important to understand the molecular mechanisms of selective autophagy, especially at the organismal level. Drosophila is an excellent genetically modifiable model organism exhibiting high conservation in the autophagic machinery. However, the regulation and mechanisms of selective autophagy in Drosophila have been largely unexplored. In this paper, I will present an overview of the current knowledge about selective autophagy in Drosophila.

  18. Hsp90 regulates processing of NF-κB2 p100 involving protection of NF-κB-inducing kinase (NIK) from autophagy-mediated degradation

    Institute of Scientific and Technical Information of China (English)

    Guoliang Qing; Pengrong Yan; Zhaoxia Qu; Hudan Liu; Gutian Xiao

    2007-01-01

    NF-κB-inducing kinase (NIK) is required for NF-κB activation based on the processing of NF-κB2 p100. Here we report a novel mechanism of NIK regulation involving the chaperone 90 kDa heat shock protein (Hsp90) and autophagy.Functional inhibition of lisp90 by the anti-tumor agent geldanamycin (GA) efficiently disrupts its interaction with NIK,resulting in NIK degradation and subsequent blockage of p100 processing. Surprisingly, GA-induced NIK degradation is mediated by autophagy, but largely independent of the ubiquitin-proteasome system. Hsp90 seems to be specifically involved in the folding/stabilization of NIK protein, because GA inhibition does not affect NIK mRNA transcription and translation. Furthermore, Hsp90 is not required for NIK-mediated recruitment of the α subunit of IκB kinase to p100, a key step in induction of p100 processing. These findings define an alternative mechanism for Hsp90 client degradation and identify a novel function of autophagy in NF-κB regulation. These findings also suggest a new therapeutic strategy for diseases associated with p100 processing.

  19. Autophagy and cancer

    Institute of Scientific and Technical Information of China (English)

    Si-Zhao; Lu; Duygu; Dee; Harrison-Findik

    2013-01-01

    Autophagy is a homeostatic and evolutionarily conserved mechanism of self-digestion by which the cells degrade and recycle long-lived proteins and excess or damaged organelles.Autophagy is activated in response to both physiological and pathological stimuli including growth factor depletion,energy deficiency or the upregulation of Bcl-2 protein expression.A novel role of autophagy in various cancers has been proposed.Interestingly,evidence that supports both a positive and negative role of autophagy in the pathogenesis of cancer has been reported.As a tumor suppression mechanism,autophagy maintains genome stability,induces senescence and possibly autophagic cell death.On the other hand,autophagy participates in tumor growth and maintenance by supplying metabolic substrate,limiting oxidative stress,and maintaining cancer stem cell population.It has been proposed that the differential roles of autophagy in cancer are disease type and stage specific.In addition,substrate selectivity might be involved in carrying out the specific effect of autophagy in cancer,and represents one of the potential directions for future studies.

  20. Crystal Structure of Oxidative Stress Sensor Keap1 in Complex with Selective Autophagy Substrate p62

    Science.gov (United States)

    Kurokawa, Hirofumi

    Keap1, an adaptor protein of cullin-RING ubiquitin ligase complex, represses cytoprotective transcription factor Nrf2 in an oxidative stress-dependent manner. The accumulation of selective autophagy substrate p62 also activates Nrf2 target genes, but the detailed mechanism has not been elucidated. Crystal structure of Keap1-p62 complex revealed the structural basis for the Nrf2 activation in which Keap1 is inactivated by p62. The accumulation of p62 is observed in hepatocellular carcinoma. The activation of Nrf2 target genes, including detoxifying enzymes and efflux transporters, by p62 may protect the cancer cells from anti-cancer drugs.

  1. Chemical Inhibition of Autophagy

    DEFF Research Database (Denmark)

    Baek, Eric; Lin Kim, Che; Gyeom Kim, Mi;

    2016-01-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical...... autophagy inhibitors on the specific productivity (qp), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine...... significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp. The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical...

  2. A nonapoptotic role for CASP2/caspase 2: modulation of autophagy.

    Science.gov (United States)

    Tiwari, Meenakshi; Sharma, Lokendra K; Vanegas, Difernando; Callaway, Danielle A; Bai, Yidong; Lechleiter, James D; Herman, Brian

    2014-06-01

    CASP2/caspase 2 plays a role in aging, neurodegeneration, and cancer. The contributions of CASP2 have been attributed to its regulatory role in apoptotic and nonapoptotic processes including the cell cycle, DNA repair, lipid biosynthesis, and regulation of oxidant levels in the cells. Previously, our lab demonstrated CASP2-mediated modulation of autophagy during oxidative stress. Here we report the novel finding that CASP2 is an endogenous repressor of autophagy. Knockout or knockdown of CASP2 resulted in upregulation of autophagy in a variety of cell types and tissues. Reinsertion of Caspase-2 gene (Casp2) in mouse embryonic fibroblast (MEFs) lacking Casp2 (casp2(-/-)) suppresses autophagy, suggesting its role as a negative regulator of autophagy. Loss of CASP2-mediated autophagy involved AMP-activated protein kinase, mechanistic target of rapamycin, mitogen-activated protein kinase, and autophagy-related proteins, indicating the involvement of the canonical pathway of autophagy. The present study also demonstrates an important role for loss of CASP2-induced enhanced reactive oxygen species production as an upstream event in autophagy induction. Additionally, in response to a variety of stressors that induce CASP2-mediated apoptosis, casp2(-/-) cells demonstrate a further upregulation of autophagy compared with wild-type MEFs, and upregulated autophagy provides a survival advantage. In conclusion, we document a novel role for CASP2 as a negative regulator of autophagy, which may provide important insight into the role of CASP2 in various processes including aging, neurodegeneration, and cancer.

  3. The yeast chromatin remodeler Rsc1-RSC complex is required for transcriptional activation of autophagy-related genes and inhibition of the TORC1 pathway in response to nitrogen starvation.

    Science.gov (United States)

    Yu, Feifei; Imamura, Yuko; Ueno, Masaru; Suzuki, Sho W; Ohsumi, Yoshinori; Yukawa, Masashi; Tsuchiya, Eiko

    2015-09-01

    The yeast RSC, an ATP-dependent chromatin-remodeling complex, is essential for mitotic and meiotic growth. There are two distinct isoforms of this complex defined by the presence of either Rsc1 or Rsc2; however, the functional differences between these complexes are unclear. Here we show that the RSC complex containing Rsc1, but not Rsc2, functions in autophagy induction. Rsc1 was required not only for full expression of ATG8 mRNA but also for maintenance of Atg8 protein stability. Interestingly, decreased autophagic activity and Atg8 protein stability in rsc1Δ cells, but not the defect in ATG8 mRNA expression, were partially suppressed by deletion of TOR1. In addition, we found that rsc1Δ impaired the binding between the Rho GTPase Rho1 and the TORC1-specific component Kog1, which is required for down-regulation of TORC1 activity. These results suggest that the Rsc1-containing RSC complex plays dual roles in the proper induction of autophagy: 1) the transcriptional activation of autophagy-related genes independent of the TORC1 pathway and 2) the inactivation of TORC1, possibly through enhancement of Rho1-Kog1 binding.

  4. Autophagy and Liver Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Raffaele Cursio

    2015-01-01

    Full Text Available Liver ischemia-reperfusion (I-R injury occurs during liver resection, liver transplantation, and hemorrhagic shock. The main mode of liver cell death after warm and/or cold liver I-R is necrosis, but other modes of cell death, as apoptosis and autophagy, are also involved. Autophagy is an intracellular self-digesting pathway responsible for removal of long-lived proteins, damaged organelles, and malformed proteins during biosynthesis by lysosomes. Autophagy is found in normal and diseased liver. Although depending on the type of ischemia, warm and/or cold, the dynamic process of liver I-R results mainly in adenosine triphosphate depletion and in production of reactive oxygen species (ROS, leads to both, a local ischemic insult and an acute inflammatory-mediated reperfusion injury, and results finally in cell death. This process can induce liver dysfunction and can increase patient morbidity and mortality after liver surgery and hemorrhagic shock. Whether autophagy protects from or promotes liver injury following warm and/or cold I-R remains to be elucidated. The present review aims to summarize the current knowledge in liver I-R injury focusing on both the beneficial and the detrimental effects of liver autophagy following warm and/or cold liver I-R.

  5. Autophagy Is Associated with Pathogenesis of Haemophilus parasuis

    Science.gov (United States)

    Zhang, Yaning; Li, Yufeng; Yuan, Wentao; Xia, Yuting; Shen, Yijuan

    2016-01-01

    Haemophilus parasuis (H. parasuis) is a common commensal Gram-negative extracellular bacterium in the upper respiratory tract of swine, which can cause Glässer's disease in stress conditions. Research on the pathogenicity of H. parasuis has mainly focused on immune evasion and bacterial virulence factors, while few studies have examined the interactions of H. parasuis and its host. Autophagy is associated with the replication and proliferation of many pathogenic bacteria, but whether it plays a role during infection by H. parasuis is unknown. In this study, an adenovirus construct expressing GFP, RFP, and LC3 was used to infect H. parasuis. Western blotting, laser confocal microscopy, and electron microscopy showed that Hps5 infection induced obvious autophagy in PK-15 cells. In cells infected with strains of H. parasuis differing in invasiveness, the levels of autophagy were positively correlated with the presence of alive bacteria in PK-15 cells. In addition, autophagy inhibited the invasion of Hps5 in PK-15 cells. Autophagy related genes Beclin, Atg5 and Atg7 were silenced with RNA interference, the results showed that autophagy induced by H. parasuis infection is a classical pathway. Our observations demonstrate that H. parasuis can induce autophagy and that the levels of autophagy are associated with the presence of alive bacteria in cells, which opened novel avenues to further our understanding of H. parasuis-host interplay and pathogenesis. PMID:27703447

  6. Cellular and Molecular Connections between Autophagy and Inflammation

    Directory of Open Access Journals (Sweden)

    Pierre Lapaquette

    2015-01-01

    Full Text Available Autophagy is an intracellular catabolic pathway essential for the recycling of proteins and larger substrates such as aggregates, apoptotic corpses, or long-lived and superfluous organelles whose accumulation could be toxic for cells. Because of its unique feature to engulf part of cytoplasm in double-membrane cup-shaped structures, which further fuses with lysosomes, autophagy is also involved in the elimination of host cell invaders and takes an active part of the innate and adaptive immune response. Its pivotal role in maintenance of the inflammatory balance makes dysfunctions of the autophagy process having important pathological consequences. Indeed, defects in autophagy are associated with a wide range of human diseases including metabolic disorders (diabetes and obesity, inflammatory bowel disease (IBD, and cancer. In this review, we will focus on interrelations that exist between inflammation and autophagy. We will discuss in particular how mediators of inflammation can regulate autophagy activity and, conversely, how autophagy shapes the inflammatory response. Impact of genetic polymorphisms in autophagy-related gene on inflammatory bowel disease will be also discussed.

  7. Autophagy and Autoimmunity CrossTalks

    Directory of Open Access Journals (Sweden)

    Abhisek eBhattacharya

    2013-04-01

    Full Text Available Autophagy, initially viewed as a conserved bulk-degradation mechanism, has emerged as a central player in a multitude of immune functions. Autophagy is important in host defense against intracellular and extracellular pathogens, metabolic syndromes, immune cell homeostasis, antigen processing and presentation and maintenance of tolerance. The observation that the above processes are implicated in triggering or exacerbating autoimmunity raises the possibility that the autophagy pathway is involved in mediating autoimmune processes, either directly or as a consequence of innate or adaptive functions mediated by the pathway. Genome-wide association studies have shown association between single nucleotide polymorphisms (SNPs in autophagy related gene 5 (Atg5, and Atg16l1 with susceptibility to systemic lupus erythematous (SLE and Crohn’s disease, respectively. Enhanced expression of Atg5 was also reported in blood of mice with experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis (MS, and in T cells isolated from blood or brain tissues from patients with active relapse of MS. This review explores the roles of autophagy pathway in the innate and adaptive immune systems on regulating or mediating the onset, progression or exacerbation of autoimmune processes.

  8. MicroRNA regulation of Autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Lund, Anders H

    2012-01-01

    Macroautophagy (hereafter referred to as autophagy) is a tightly regulated intracellular catabolic pathway involving the lysosomal degradation of cytoplasmic organelles and proteins. Central to this process is the formation of the autophagosome, a double membrane-bound vesicle, which is responsible...... for the delivery of cytoplasmic cargo to the lysosomes. Autophagy levels are constantly changing, allowing adaptation to both immediate and long-term needs of the cell, underlining why tight control of this process is essential in order to prevent the development of pathological disorders. Substantial progress has...... recently contributed to our understanding of the molecular mechanisms of the autophagy machinery, yet several gaps remain in our knowledge of this process. The discovery of microRNAs (miRNAs) established a new paradigm of post-transcriptional gene regulation and during the past decade these small non...

  9. Amino acid metabolism inhibits antibody-driven kidney injury by inducing autophagy.

    Science.gov (United States)

    Chaudhary, Kapil; Shinde, Rahul; Liu, Haiyun; Gnana-Prakasam, Jaya P; Veeranan-Karmegam, Rajalakshmi; Huang, Lei; Ravishankar, Buvana; Bradley, Jillian; Kvirkvelia, Nino; McMenamin, Malgorzata; Xiao, Wei; Kleven, Daniel; Mellor, Andrew L; Madaio, Michael P; McGaha, Tracy L

    2015-06-15

    Inflammatory kidney disease is a major clinical problem that can result in end-stage renal failure. In this article, we show that Ab-mediated inflammatory kidney injury and renal disease in a mouse nephrotoxic serum nephritis model was inhibited by amino acid metabolism and a protective autophagic response. The metabolic signal was driven by IFN-γ-mediated induction of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme activity with subsequent activation of a stress response dependent on the eIF2α kinase general control nonderepressible 2 (GCN2). Activation of GCN2 suppressed proinflammatory cytokine production in glomeruli and reduced macrophage recruitment to the kidney during the incipient stage of Ab-induced glomerular inflammation. Further, inhibition of autophagy or genetic ablation of Ido1 or Gcn2 converted Ab-induced, self-limiting nephritis to fatal end-stage renal disease. Conversely, increasing kidney IDO1 activity or treating mice with a GCN2 agonist induced autophagy and protected mice from nephritic kidney damage. Finally, kidney tissue from patients with Ab-driven nephropathy showed increased IDO1 abundance and stress gene expression. Thus, these findings support the hypothesis that the IDO-GCN2 pathway in glomerular stromal cells is a critical negative feedback mechanism that limits inflammatory renal pathologic changes by inducing autophagy.

  10. Lithium and autophagy.

    Science.gov (United States)

    Motoi, Yumiko; Shimada, Kohei; Ishiguro, Koichi; Hattori, Nobutaka

    2014-06-18

    Lithium, a drug used to treat bipolar disorders, has a variety of neuroprotective mechanisms, including autophagy regulation, in various neuropsychiatric conditions. In neurodegenerative diseases, lithium enhances degradation of aggregate-prone proteins, including mutated huntingtin, phosphorylated tau, and α-synuclein, and causes damaged mitochondria to degrade, while in a mouse model of cerebral ischemia and Alzheimer's disease autophagy downregulation by lithium is observed. The signaling pathway of lithium as an autophagy enhancer might be associated with the mammalian target of rapamycin (mTOR)-independent pathway, which is involved in myo-inositol-1,4,5-trisphosphate (IP3) in Huntington's disease and Parkinson's disease. However, the mTOR-dependent pathway might be involved in inhibiting glycogen synthase kinase-3β (GSK3β) in other diseases. Lithium's autophagy-enhancing property may contribute to the therapeutic benefit of patients with neuropsychiatric disorders. PMID:24738557

  11. Autophagy and cytokines.

    Science.gov (United States)

    Harris, James

    2011-11-01

    Autophagy is a highly conserved homoeostatic mechanism for the lysosomal degradation of cytosolic constituents, including long-lived macromolecules, organelles and intracellular pathogens. Autophagosomes are formed in response to a number of environmental stimuli, including amino acid deprivation, but also by both host- and pathogen-derived molecules, including toll-like receptor ligands and cytokines. In particular, IFN-γ, TNF-α, IL-1, IL-2, IL-6 and TGF-β have been shown to induce autophagy, while IL-4, IL-10 and IL-13 are inhibitory. Moreover, autophagy can itself regulate the production and secretion of cytokines, including IL-1, IL-18, TNF-α, and Type I IFN. This review discusses the potentially pivotal roles of autophagy in the regulation of inflammation and the coordination of innate and adaptive immune responses.

  12. A critical role of autophagy in plant resistance to necrotrophic fungal pathogens.

    Science.gov (United States)

    Lai, Zhibing; Wang, Fei; Zheng, Zuyu; Fan, Baofang; Chen, Zhixiang

    2011-06-01

    Autophagy is a pathway for degradation of cytoplasmic components. In plants, autophagy plays an important role in nutrient recycling during nitrogen or carbon starvation, and in responses to abiotic stress. Autophagy also regulates age- and immunity-related programmed cell death, which is important in plant defense against biotrophic pathogens. Here we show that autophagy plays a critical role in plant resistance to necrotrophic pathogens. ATG18a, a critical autophagy protein in Arabidopsis, interacts with WRKY33, a transcription factor that is required for resistance to necrotrophic pathogens. Expression of autophagy genes and formation of autophagosomes are induced in Arabidopsis by the necrotrophic fungal pathogen Botrytis cinerea. Induction of ATG18a and autophagy by B. cinerea was compromised in the wrky33 mutant, which is highly susceptible to necrotrophic pathogens. Arabidopsis mutants defective in autophagy exhibit enhanced susceptibility to the necrotrophic fungal pathogens B. cinerea and Alternaria brassicicola based on increased pathogen growth in the mutants. The hypersusceptibility of the autophagy mutants was associated with reduced expression of the jasmonate-regulated PFD1.2 gene, accelerated development of senescence-like chlorotic symptoms, and increased protein degradation in infected plant tissues. These results strongly suggest that autophagy cooperates with jasmonate- and WRKY33-mediated signaling pathways in the regulation of plant defense responses to necrotrophic pathogens.

  13. Targeting autophagy in cancer management – strategies and developments

    Directory of Open Access Journals (Sweden)

    Ozpolat B

    2015-09-01

    Full Text Available Bulent Ozpolat,1 Doris M Benbrook2 1Department of Experimental Therapeutics, The University of Texas – Houston, MD Anderson Cancer Center, Houston, TX, 2Department of Obstetrics and Gynecology, University of Oklahoma HSC, Oklahoma City, OK, USA Abstract: Autophagy is a highly regulated catabolic process involving lysosomal degradation of intracellular components, damaged organelles, misfolded proteins, and toxic aggregates, reducing oxidative stress and protecting cells from damage. The process is also induced in response to various conditions, including nutrient deprivation, metabolic stress, hypoxia, anticancer therapeutics, and radiation therapy to adapt cellular conditions for survival. Autophagy can function as a tumor suppressor mechanism in normal cells and dysregulation of this process (ie, monoallelic Beclin-1 deletion may lead to malignant transformation and carcinogenesis. In tumors, autophagy is thought to promote tumor growth and progression by helping cells to adapt and survive in metabolically-challenged and harsh tumor microenvironments (ie, hypoxia and acidity. Recent in vitro and in vivo studies in preclinical models suggested that modulation of autophagy can be used as a therapeutic modality to enhance the efficacy of conventional therapies, including chemo and radiation therapy. Currently, more than 30 clinical trials are investigating the effects of autophagy inhibition in combination with cytotoxic chemotherapies and targeted agents in various cancers. In this review, we will discuss the role, molecular mechanism, and regulation of autophagy, while targeting this process as a novel therapeutic modality, in various cancers. Keywords: autophagy inhibition, chemotherapy, tumor microenvironment

  14. Autophagy research: Lessons from metabolism

    NARCIS (Netherlands)

    A.J. Meijer

    2009-01-01

    Autophagy research continues to expand exponentially. Clearly autophagy and metabolism are intimately connected; however, the rapid expansion of research into this topic inevitably brings the risk that important basic knowledge of metabolism will be overlooked when considering experimental data. Unf

  15. Functional loss of two ceramide synthases elicits autophagy-dependent lifespan extension in C. elegans

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Kruse, Rikke; Harvald, Eva Bang;

    2013-01-01

    of HYL-1 or LAGR-1 does not affect lifespan. We show that loss of HYL-1 and LAGR-1 functions extend lifespan in an autophagy-dependent manner, as knock down of the autophagy-associated gene ATG-12 abolishes hyl-1;lagr-1 longevity. The transcription factors PHA-4/FOXA, DAF-16/FOXO, and SKN-1 are also......-1 result in dietary restriction-induced autophagy and consequently prolonged longevity....

  16. Transcriptional regulation of mammalian autophagy at a glance.

    Science.gov (United States)

    Füllgrabe, Jens; Ghislat, Ghita; Cho, Dong-Hyung; Rubinsztein, David C

    2016-08-15

    Macroautophagy, hereafter referred to as autophagy, is a catabolic process that results in the lysosomal degradation of cytoplasmic contents ranging from abnormal proteins to damaged cell organelles. It is activated  under diverse conditions, including nutrient deprivation and hypoxia. During autophagy, members of the core autophagy-related (ATG) family of proteins mediate membrane rearrangements, which lead to the engulfment and degradation of cytoplasmic cargo. Recently, the nuclear regulation of autophagy, especially by transcription factors and histone modifiers, has gained increased attention. These factors are not only involved in rapid responses to autophagic stimuli, but also regulate the long-term outcome of autophagy. Now there are more than 20 transcription factors that have been shown to be linked to the autophagic process. However, their interplay and timing appear enigmatic as several have been individually shown to act as major regulators of autophagy. This Cell Science at a Glance article and the accompanying poster highlights the main cellular regulators of transcription involved in mammalian autophagy and their target genes. PMID:27528206

  17. The Regulation of Autophagy by Influenza A Virus

    Directory of Open Access Journals (Sweden)

    Rong Zhang

    2014-01-01

    Full Text Available Influenza A virus is a dreadful pathogen of animals and humans, causing widespread infection and severe morbidity and mortality. It is essential to characterize the influenza A virus-host interaction and develop efficient counter measures against the viral infection. Autophagy is known as a catabolic process for the recycling of the cytoplasmic macromolecules. Recently, it has been shown that autophagy is a critical mechanism underlying the interaction between influenza A virus and its host. Autophagy can be induced by the infection with influenza A virus, which is considered as a necessary process for the viral proliferation, including the accumulation of viral elements during the replication of influenza A virus. On the other hand, influenza A virus can inhibit the autophagic formation via interaction with the autophagy-related genes (Atg and signaling pathways. In addition, autophagy is involved in the influenza virus-regulated cell deaths, leading to significant changes in host apoptosis. Interestingly, the high pathogenic strains of influenza A virus, such as H5N1, stimulate autophagic cell death and appear to interplay with the autophagy in distinct ways as compared with low pathogenic strains. This review discusses the regulation of autophagy, an influenza A virus driven process.

  18. Autophagy and Transporter-Based Multi-Drug Resistance

    Directory of Open Access Journals (Sweden)

    Zhe-Sheng Chen

    2012-08-01

    Full Text Available All the therapeutic strategies for treating cancers aim at killing the cancer cells via apoptosis (programmed cell death type I. Defective apoptosis endow tumor cells with survival. The cell can respond to such defects with autophagy. Autophagy is a cellular process by which cytoplasmic material is either degraded to maintain homeostasis or recycled for energy and nutrients in starvation. A plethora of evidence has shown that the role of autophagy in tumors is complex. A lot of effort is needed to underline the functional status of autophagy in tumor progression and treatment, and elucidate how to tweak autophagy to treat cancer. Furthermore, during the treatment of cancer, the limitation for the cure rate and survival is the phenomenon of multi drug resistance (MDR. The development of MDR is an intricate process that could be regulated by drug transporters, enzymes, anti-apoptotic genes or DNA repair mechanisms. Reports have shown that autophagy has a dual role in MDR. Furthermore, it has been reported that activation of a death pathway may overcome MDR, thus pointing the importance of other death pathways to regulate tumor cell progression and growth. Therefore, in this review we will discuss the role of autophagy in MDR tumors and a possible link amongst these phenomena.

  19. Piperlongumine induces autophagy by targeting p38 signaling.

    Science.gov (United States)

    Wang, Y; Wang, J-W; Xiao, X; Shan, Y; Xue, B; Jiang, G; He, Q; Chen, J; Xu, H-G; Zhao, R-X; Werle, K D; Cui, R; Liang, J; Li, Y-L; Xu, Z-X

    2013-01-01

    Piperlongumine (PL), a natural product isolated from the plant species Piper longum L., can selectively induce apoptotic cell death in cancer cells by targeting the stress response to reactive oxygen species (ROS). Here we show that PL induces cell death in the presence of benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD-fmk), a pan-apoptotic inhibitor, and in the presence of necrostatin-1, a necrotic inhibitor. Instead PL-induced cell death can be suppressed by 3-methyladenine, an autophagy inhibitor, and substantially attenuated in cells lacking the autophagy-related 5 (Atg5) gene. We further show that PL enhances autophagy activity without blocking autophagy flux. Application of N-acetyl-cysteine, an antioxidant, markedly reduces PL-induced autophagy and cell death, suggesting an essential role for intracellular ROS in PL-induced autophagy. Furthermore, PL stimulates the activation of p38 protein kinase through ROS-induced stress response and p38 signaling is necessary for the action of PL as SB203580, a p38 inhibitor, or dominant-negative p38 can effectively reduce PL-mediated autophagy. Thus, we have characterized a new mechanism for PL-induced cell death through the ROS-p38 pathway. Our findings support the therapeutic potential of PL by triggering autophagic cell death. PMID:24091667

  20. The Parkinson's disease-associated genes ATP13A2 and SYT11 regulate autophagy via a common pathway

    Science.gov (United States)

    Bento, Carla F.; Ashkenazi, Avraham; Jimenez-Sanchez, Maria; Rubinsztein, David C.

    2016-01-01

    Forms of Parkinson's disease (PD) are associated with lysosomal and autophagic dysfunction. ATP13A2, which is mutated in some types of early-onset Parkinsonism, has been suggested as a regulator of the autophagy–lysosome pathway. However, little is known about the ATP13A2 effectors and how they regulate this pathway. Here we show that ATP13A2 depletion negatively regulates another PD-associated gene (SYT11) at both transcriptional and post-translational levels. Decreased SYT11 transcription is controlled by a mechanism dependent on MYCBP2-induced ubiquitination of TSC2, which leads to mTORC1 activation and decreased TFEB-mediated transcription of SYT11, while increased protein turnover is regulated by SYT11 ubiquitination and degradation. Both mechanisms account for a decrease in the levels of SYT11, which, in turn, induces lysosomal dysfunction and impaired degradation of autophagosomes. Thus, we propose that ATP13A2 and SYT11 form a new functional network in the regulation of the autophagy–lysosome pathway, which is likely to contribute to forms of PD-associated neurodegeneration. PMID:27278822

  1. Autophagy in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Kadija; Abounit; Tiziano; M; Scarabelli; Roy; B; McCauley

    2012-01-01

    Autophagy is a regulated process for the degradation of cellular components that has been well conserved in eukaryotic cells. The discovery of autophagy-regulating proteins in yeast has been important in understanding this process. Although many parallels exist between fungi and mammals in the regulation and execution of autophagy, there are some important differences. The preautophagosomal structure found in yeast has not been identified in mammals, and it seems that there may be multiple origins for autophagosomes, including endoplasmic reticulum, plasma membrane and mitochondrial outer membrane. The maturation of the phagophore is largely dependent on 5’-AMP activated protein kinase and other factors that lead to the dephosphorylation of mammalian target of rapamycin. Once the process is initiated, the mammalian phagophore elongates and matures into an autophagosome by processes that are similar to those in yeast. Cargo selection is dependent on the ubiquitin conjugation of protein aggregates and organelles and recognition of these conjugates by autophagosomal receptors. Lysosomal degradation of cargo produces metabolites that can be recycled during stress. Autophagy is an impor-tant cellular safeguard during starvation in all eukaryotes; however, it may have more complicated, tissue specific roles in mammals. With certain exceptions, autophagy seems to be cytoprotective, and defects in the process have been associated with human disease.

  2. The interplays between autophagy and apoptosis induced by enterovirus 71.

    Directory of Open Access Journals (Sweden)

    Xueyan Xi

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is the causative agent of human diseases with distinct severity, from mild hand, foot and mouth disease to severe neurological syndromes, such as encephalitis and meningitis. The lack of understanding of viral pathogenesis as well as lack of efficient vaccine and drugs against this virus impedes the control of EV71 infection. EV71 virus induces autophagy and apoptosis; however, the relationship between EV71-induced autophagy and apoptosis as well as the influence of autophagy and apoptosis on virus virulence remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it was observed that the Anhui strain of EV71 induced autophagy and apoptosis in human rhabdomyosarcoma (RD-A cells. Additionally, by either applying chemical inhibitors or knocking down single essential autophagic or apoptotic genes, inhibition of EV71 induced autophagy inhibited the apoptosis both at the autophagosome formation stage and autophagy execution stage. However, inhibition of autophagy at the stage of autophagosome and lysosome fusion promoted apoptosis. In reverse, the inhibition of EV71-induced apoptosis contributed to the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I to LC3-II and degradation of sequestosome 1 (SQSTM1/P62. Furthermore, the inhibition of autophagy in the autophagsome formation stage or apoptosis decreased the release of EV71 viral particles. CONCLUSIONS/SIGNIFICANCE: In conclusion, the results of this study not only revealed novel aspect of the interplay between autophagy and apoptosis in EV71 infection, but also provided a new insight to control EV71 infection.

  3. Regulation of autophagy in human skeletal muscle: effects of exercise, exercise training and insulin stimulation

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel; Madsen, Agnete Louise Bjerregaard; Kleinert, Maximilian;

    2016-01-01

    Studies in rodent muscle suggest that autophagy is regulated by acute exercise, exercise training and insulin stimulation. However, little is known about the regulation of autophagy in human skeletal muscle. Here we investigate the autophagic response to acute one-legged exercise, one...... increase the capacity for formation of autophagosomes in muscle. Moreover, AMPK activation during exercise may not be sufficient to regulate autophagy in muscle, while mTORC1 signalling via ULK1 likely mediates the autophagy-inhibiting effect of insulin. This article is protected by copyright. All rights...

  4. Dysregulation of the Autophagy-Endolysosomal System in Amyotrophic Lateral Sclerosis and Related Motor Neuron Diseases

    Directory of Open Access Journals (Sweden)

    Asako Otomo

    2012-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a heterogeneous group of incurable motor neuron diseases (MNDs characterized by a selective loss of upper and lower motor neurons in the brain and spinal cord. Most cases of ALS are sporadic, while approximately 5–10% cases are familial. More than 16 causative genes for ALS/MNDs have been identified and their underlying pathogenesis, including oxidative stress, endoplasmic reticulum stress, excitotoxicity, mitochondrial dysfunction, neural inflammation, protein misfolding and accumulation, dysfunctional intracellular trafficking, abnormal RNA processing, and noncell-autonomous damage, has begun to emerge. It is currently believed that a complex interplay of multiple toxicity pathways is implicated in disease onset and progression. Among such mechanisms, ones that are associated with disturbances of protein homeostasis, the ubiquitin-proteasome system and autophagy, have recently been highlighted. Although it remains to be determined whether disease-associated protein aggregates have a toxic or protective role in the pathogenesis, the formation of them results from the imbalance between generation and degradation of misfolded proteins within neuronal cells. In this paper, we focus on the autophagy-lysosomal and endocytic degradation systems and implication of their dysfunction to the pathogenesis of ALS/MNDs. The autophagy-endolysosomal pathway could be a major target for the development of therapeutic agents for ALS/MNDs.

  5. Inhibition of autophagy attenuates pancreatic cancer growth independent of TP53/TRP53 status.

    Science.gov (United States)

    Yang, Annan; Kimmelman, Alec C

    2014-09-01

    Basal levels of autophagy are elevated in most pancreatic ductal adenocarcinomas (PDAC). Suppressing autophagy pharmacologically using chloroquine (CQ) or genetically with RNAi to essential autophagy genes inhibits human pancreatic cancer growth in vitro and in vivo, which presents possible treatment opportunities for PDAC patients using the CQ-derivative hydroxychloroquine (HCQ). Indeed, such clinical trials are ongoing. However, autophagy is a complex cellular mechanism to maintain cell homeostasis under stress. Based on its biological role, a dual role of autophagy in tumorigenesis has been proposed: at tumor initiation, autophagy helps maintain genomic stability and prevent tumor initiation; while in advanced disease, autophagy degrades and recycles cellular components to meet the metabolic needs for rapid growth. This model was proven to be the case in mouse lung tumor models. However, in contrast to prior work in various PDAC model systems, loss of autophagy in PDAC mouse models with embryonic homozygous Trp53 deletion does not inhibit tumor growth and paradoxically increases progression. This raised concerns whether there may be a genotype-dependent reliance of PDAC on autophagy. In a recent study, our group used a Trp53 heterozygous mouse PDAC model and human PDX xenografts to address the question. Our results demonstrate that autophagy inhibition was effective against PDAC tumors irrespective of TP53/TRP53 status.

  6. Inflammasome-independent modulation of cytokine response by autophagy in human cells.

    Directory of Open Access Journals (Sweden)

    Tania O Crişan

    Full Text Available Autophagy is a cell housekeeping mechanism that has recently received attention in relation to its effects on the immune response. Genetic studies have identified candidate loci for Crohn's disease susceptibility among autophagy genes, while experiments in murine macrophages from ATG16L1 deficient mice have shown that disruption of autophagy increases processing of IL-1β and IL-18 through an inflammasome-dependent manner. Using complementary approaches either inducing or inhibiting autophagy, we describe modulatory effects of autophagy on proinflammatory cytokine production in human cells. Inhibition of basal autophagy in human peripheral blood mononuclear cells (PBMCs significantly enhances IL-1β after stimulation with TLR2 or TLR4 ligands, while at the same time reducing the production of TNFα. In line with this, induction of autophagy by starvation inhibited IL-1β production. These effects of autophagy were not exerted at the processing step, as inflammasome activation was not influenced. In contrast, the effect of autophagy on cytokine production was on transcription level, and possibly involving the inhibition of p38 mitogen activated protein kinase (MAPK phosphorylation. In conclusion, autophagy modulates the secretion of proinflammatory cytokines in human cells through an inflammasome-independent pathway, and this is a novel mechanism that may be targeted in inflammatory diseases.

  7. Receptor Proteins in Selective Autophagy

    Directory of Open Access Journals (Sweden)

    Christian Behrends

    2012-01-01

    Full Text Available Autophagy has long been thought to be an essential but unselective bulk degradation pathway. However, increasing evidence suggests selective autophagosomal turnover of a broad range of substrates. Bifunctional autophagy receptors play a key role in selective autophagy by tethering cargo to the site of autophagosomal engulfment. While the identity of molecular components involved in selective autophagy has been revealed at least to some extent, we are only beginning to understand how selectivity is achieved in this process. Here, we summarize the mechanistic and structural basis of receptor-mediated selective autophagy.

  8. The role of autophagy in cell survival from heavy ion irradiation in the plateau region

    International Nuclear Information System (INIS)

    To study cytotoxic effect of heavy ion irradiation in the plateau region, and investigate whether autophagy induced by heavy ion irradiation is cytoprotective, HeLa cells were irradiated with 350 MeV/u carbon ions beams, and the clonogenic survival was analyzed. The results showed that cell survival decreased with increasing doses. It was also found that G2/M-phase cells increased, and the autophagy-related activity was significantly higher than the control. When autophagy was blocked by 3-methyladenine in carbon-ion irradiated cells, G2/M phase arrest and the percentage of apoptosis cells were further elevated, and cell survival decreased significantly, indicating the induction of cytoprotective autophagy by carbon-ion irradiation. Our results demonstrated that autophagy induced by carbon ion irradiation provided a self-protective mechanism in HeLa cells, short-time inhibition of autophagy before carbon-ion irradiation could enhance radiation cytotoxicity in HeLa cells. (authors)

  9. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

    DEFF Research Database (Denmark)

    Morselli, Eugenia; Mariño, Guillermo; Bennetzen, Martin V;

    2011-01-01

    Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy...... independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation...... and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate...

  10. microRNA-101 is a potent inhibitor of autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Wen, Jiayu; Lees, Michael;

    2011-01-01

    Autophagy is an evolutionarily conserved mechanism of cellular self-digestion in which proteins and organelles are degraded through delivery to lysosomes. Defects in this process are implicated in numerous human diseases including cancer. To further elucidate regulatory mechanisms of autophagy, we...... performed a functional screen in search of microRNAs (miRNAs), which regulate the autophagic flux in breast cancer cells. In this study, we identified the tumour suppressive miRNA, miR-101, as a potent inhibitor of basal, etoposide- and rapamycin-induced autophagy. Through transcriptome profiling, we...... identified three novel miR-101 targets, STMN1, RAB5A and ATG4D. siRNA-mediated depletion of these genes phenocopied the effect of miR-101 overexpression, demonstrating their importance in autophagy regulation. Importantly, overexpression of STMN1 could partially rescue cells from miR-101-mediated inhibition...

  11. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Orfali, Nina [Cork Cancer Research Center, University College Cork, Cork (Ireland); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); McKenna, Sharon L. [Cork Cancer Research Center, University College Cork, Cork (Ireland); Cahill, Mary R. [Department of Hematology, Cork University Hospital, Cork (Ireland); Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu [Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); Mongan, Nigel P., E-mail: nigel.mongan@nottingham.ac.uk [Faculty of Medicine and Health Science, School of Veterinary Medicine and Science, University of Nottingham, LE12 5RD (United Kingdom); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States)

    2014-05-15

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects.

  12. Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells

    International Nuclear Information System (INIS)

    In this study, we confirmed the autophagy induced by porcine reproductive and respiratory syndrome virus (PRRSV) in permissive cells and investigated the role of autophagy in the replication of PRRSV. We first demonstrated that PRRSV infection significantly results in the increased double-membrane vesicles, the accumulation of LC3 fluorescence puncta, and the raised ratio of LC3-II/β-actin, in MARC-145 cells. Then we discovered that induction of autophagy by rapamycin significantly enhances the viral titers of PRRSV, while inhibition of autophagy by 3-MA and silencing of LC3 gene by siRNA reduces the yield of PRRSV. The results showed functional autolysosomes can be formed after PRRSV infection and the autophagosome–lysosome-fusion inhibitor decreases the virus titers. We also examined the induction of autophagy by PRRSV infection in pulmonary alveolar macrophages. These findings indicate that autophagy induced by PRRSV infection plays a role in sustaining the replication of PRRSV in host cells.

  13. Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qinghao; Qin, Yixian; Zhou, Lei; Kou, Qiuwen; Guo, Xin; Ge, Xinna [Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agribiotechnology, China Agricultural University, Beijing (China); Yang, Hanchun, E-mail: yanghanchun1@cau.edu.cn [Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agribiotechnology, China Agricultural University, Beijing (China); Hu, Hongbo, E-mail: hongbo@cau.edu.cn [College of Food Science and Nutritional Engineering, China Agricultural University, Beijing (China)

    2012-08-01

    In this study, we confirmed the autophagy induced by porcine reproductive and respiratory syndrome virus (PRRSV) in permissive cells and investigated the role of autophagy in the replication of PRRSV. We first demonstrated that PRRSV infection significantly results in the increased double-membrane vesicles, the accumulation of LC3 fluorescence puncta, and the raised ratio of LC3-II/{beta}-actin, in MARC-145 cells. Then we discovered that induction of autophagy by rapamycin significantly enhances the viral titers of PRRSV, while inhibition of autophagy by 3-MA and silencing of LC3 gene by siRNA reduces the yield of PRRSV. The results showed functional autolysosomes can be formed after PRRSV infection and the autophagosome-lysosome-fusion inhibitor decreases the virus titers. We also examined the induction of autophagy by PRRSV infection in pulmonary alveolar macrophages. These findings indicate that autophagy induced by PRRSV infection plays a role in sustaining the replication of PRRSV in host cells.

  14. Evolutionary trends and functional anatomy of the human expanded autophagy network.

    Science.gov (United States)

    Till, Andreas; Saito, Rintaro; Merkurjev, Daria; Liu, Jing-Jing; Syed, Gulam Hussain; Kolnik, Martin; Siddiqui, Aleem; Glas, Martin; Scheffler, Björn; Ideker, Trey; Subramani, Suresh

    2015-01-01

    All eukaryotic cells utilize autophagy for protein and organelle turnover, thus assuring subcellular quality control, homeostasis, and survival. In order to address recent advances in identification of human autophagy associated genes, and to describe autophagy on a system-wide level, we established an autophagy-centered gene interaction network by merging various primary data sets and by retrieving respective interaction data. The resulting network ('AXAN') was analyzed with respect to subnetworks, e.g. the prime gene subnetwork (including the core machinery, signaling pathways and autophagy receptors) and the transcription subnetwork. To describe aspects of evolution within this network, we assessed the presence of protein orthologs across 99 eukaryotic model organisms. We visualized evolutionary trends for prime gene categories and evolutionary tracks for selected AXAN genes. This analysis confirms the eukaryotic origin of autophagy core genes while it points to a diverse evolutionary history of autophagy receptors. Next, we used module identification to describe the functional anatomy of the network at the level of pathway modules. In addition to obvious pathways (e.g., lysosomal degradation, insulin signaling) our data unveil the existence of context-related modules such as Rho GTPase signaling. Last, we used a tripartite, image-based RNAi - screen to test candidate genes predicted to play a role in regulation of autophagy. We verified the Rho GTPase, CDC42, as a novel regulator of autophagy-related signaling. This study emphasizes the applicability of system-wide approaches to gain novel insights into a complex biological process and to describe the human autophagy pathway at a hitherto unprecedented level of detail.

  15. Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy.

    Directory of Open Access Journals (Sweden)

    Sung Won Lee

    Full Text Available Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence

  16. Multiple roles of the cytoskeleton in autophagy.

    Science.gov (United States)

    Monastyrska, Iryna; Rieter, Ester; Klionsky, Daniel J; Reggiori, Fulvio

    2009-08-01

    Autophagy is involved in a wide range of physiological processes including cellular remodeling during development, immuno-protection against heterologous invaders and elimination of aberrant or obsolete cellular structures. This conserved degradation pathway also plays a key role in maintaining intracellular nutritional homeostasis and during starvation, for example, it is involved in the recycling of unnecessary cellular components to compensate for the limitation of nutrients. Autophagy is characterized by specific membrane rearrangements that culminate with the formation of large cytosolic double-membrane vesicles called autophagosomes. Autophagosomes sequester cytoplasmic material that is destined for degradation. Once completed, these vesicles dock and fuse with endosomes and/or lysosomes to deliver their contents into the hydrolytically active lumen of the latter organelle where, together with their cargoes, they are broken down into their basic components. Specific structures destined for degradation via autophagy are in many cases selectively targeted and sequestered into autophagosomes. A number of factors required for autophagy have been identified, but numerous questions about the molecular mechanism of this pathway remain unanswered. For instance, it is unclear how membranes are recruited and assembled into autophagosomes. In addition, once completed, these vesicles are transported to cellular locations where endosomes and lysosomes are concentrated. The mechanism employed for this directed movement is not well understood. The cellular cytoskeleton is a large, highly dynamic cellular scaffold that has a crucial role in multiple processes, several of which involve membrane rearrangements and vesicle-mediated events. Relatively little is known about the roles of the cytoskeleton network in autophagy. Nevertheless, some recent studies have revealed the importance of cytoskeletal elements such as actin microfilaments and microtubules in specific aspects of

  17. Inflammasome-independent modulation of cytokine response by autophagy in human cells

    NARCIS (Netherlands)

    Crisan, T.O.; Plantinga, T.S.; Veerdonk, F.L. van de; Farcas, M.F.; Stoffels, M.; Kullberg, B.J.; Meer, J.W. van der; Joosten, L.A.B.; Netea, M.G.

    2011-01-01

    Autophagy is a cell housekeeping mechanism that has recently received attention in relation to its effects on the immune response. Genetic studies have identified candidate loci for Crohn's disease susceptibility among autophagy genes, while experiments in murine macrophages from ATG16L1 deficient m

  18. DNA damage and autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States); Panayiotidis, Mihalis I. [School of Community Health Sciences, University of Nevada, Reno, NV 89557 (United States); Franco, Rodrigo, E-mail: rfrancocruz2@unl.edu [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States)

    2011-06-03

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  19. Dopamine Oxidation and Autophagy

    Directory of Open Access Journals (Sweden)

    Patricia Muñoz

    2012-01-01

    Full Text Available The molecular mechanisms involved in the neurodegenerative process of Parkinson's disease remain unclear. Currently, there is a general agreement that mitochondrial dysfunction, α-synuclein aggregation, oxidative stress, neuroinflammation, and impaired protein degradation are involved in the neurodegeneration of dopaminergic neurons containing neuromelanin in Parkinson's disease. Aminochrome has been proposed to play an essential role in the degeneration of dopaminergic neurons containing neuromelanin by inducing mitochondrial dysfunction, oxidative stress, the formation of neurotoxic α-synuclein protofibrils, and impaired protein degradation. Here, we discuss the relationship between the oxidation of dopamine to aminochrome, the precursor of neuromelanin, autophagy dysfunction in dopaminergic neurons containing neuromelanin, and the role of dopamine oxidation to aminochrome in autophagy dysfunction in dopaminergic neurons. Aminochrome induces the following: (i the formation of α-synuclein protofibrils that inactivate chaperone-mediated autophagy; (ii the formation of adducts with α- and β-tubulin, which induce the aggregation of the microtubules required for the fusion of autophagy vacuoles and lysosomes.

  20. Role of autophagy in prion protein-induced neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    Hao Yao; Deming Zhao; Sher Hayat Khan; Lifeng Yang

    2013-01-01

    Prion diseases,characterized by spongiform degeneration and the accumulation of misfolded and aggregated PrPSc in the central nervous system,are one of fatal neurodegenerative and infectious disorders of humans and animals.In earlier studies,autophagy vacuoles in neurons were frequently observed in neurodegenerative diseases such as Alzheimer's,Parkinson's,and Huntington's diseases as well as prion diseases.Autophagy is a highly conserved homeostatic process by which several cytoplasmic components (proteins or organelles) are sequestered in a doublemembrane-bound vesicle termed 'autophagosome' and degraded upon their fusion with lysosome.The pathway of intercellular self-digestion at basal physiological levels is indispensable for maintaining the healthy status of tissues and organs.In case of prion infection,increasing evidence indicates that autophagy has a crucial ability of eliminating pathological PrPSc accumulated within neurons.In contrast,autophagy dysfunction in affected neurons may contribute to the formation of spongiform changes.In this review,we summarized recent findings about the effect of mammalian autophagy in neurodegenerative disorders,particularly in prion diseases.We also summarized the therapeutic potential of some small molecules (such as lithium,rapamycin,Sirtuin 1 and resveratrol) targets to mitigate such diseases on brain function.Furthermore,we discussed the controversial role of autophagy,whether it mediates neuronal toxicity or serves a protective function in neurodegenerative disorders.

  1. Death and survival of neuronal and astrocytic cells in ischemic brain injury: a role of autophagy

    Institute of Scientific and Technical Information of China (English)

    Min XU; Hui-ling ZHANG

    2011-01-01

    Autophagy is a highly regulated cellular mechanism that leads to degradation of long-lived proteins and dysfunctional organelles. The process has been implicated in a variety of physiological and pathological conditions relevant to neurological diseases. Recent studies show the existence of autophagy in cerebral ischemia, but no consensus has yet been reached regarding the functions of autophagy in this condition. This article highlights the activation of autophagy during cerebral ischemia and/or reperfusion, especially in neurons and astrocytes, as well as the role of autophagy in neuronal or astrocytic cell death and survival. We propose that physiological levels of autophagy, presumably caused by mild to modest hypoxia or ischemia, appear to be protective. However, high levels of autophagy caused by severe hypoxia or ischemia and/or reperfusion may cause self-digestion and eventual neuronal and astrocytic cell death. We also discuss that oxidative and endoplasmic reticulum (ER) stresses in cerebral hypoxia or ischemia and/or reperfusion are potent stimuli of autophagy in neurons and astrocytes. In addition, we review the evidence suggesting a considerable overlap between autophagy on one hand, and apoptosis, necrosis and necroptosis on the other hand, in determining the outcomes and final morphology of damaged neurons and astrocytes.

  2. Nutritional Status and Cardiac Autophagy

    Directory of Open Access Journals (Sweden)

    Jihyun Ahn

    2013-02-01

    Full Text Available Autophagy is necessary for the degradation of long-lasting proteins and nonfunctional organelles, and is activated to promote cellular survival. However, overactivation of autophagy may deplete essential molecules and organelles responsible for cellular survival. Lifelong calorie restriction by 40% has been shown to increase the cardiac expression of autophagic markers, which suggests that it may have a cardioprotective effect by decreasing oxidative damage brought on by aging and cardiovascular diseases. Although cardiac autophagy is critical to regulating protein quality and maintaining cellular function and survival, increased or excessive autophagy may have deleterious effects on the heart under some circumstances, including pressure overload-induced heart failure. The importance of autophagy has been shown in nutrient supply and preservation of energy in times of limitation, such as ischemia. Some studies have suggested that a transition from obesity to metabolic syndrome may involve progressive changes in myocardial inflammation, mitochondrial dysfunction, fibrosis, apoptosis, and myocardial autophagy.

  3. Autophagy resolves early retinal inflammation in Igf1-deficient mice

    Science.gov (United States)

    Rodríguez-de la Rosa, Lourdes; Murillo-Cuesta, Silvia; Vaquero-Villanueva, Laura; Hurlé, Juan M.; Varela-Nieto, Isabel; Valverde, Ángela M.

    2016-01-01

    ABSTRACT Insulin-like growth factor-1 (IGF-1) is a growth factor with differentiating, anti-apoptotic and metabolic functions in the periphery, and anti-inflammatory properties in the nervous system. Mice that have mutations in the Igf1 gene, rendering the gene product inactive (Igf1−/−), present with age-related visual loss accompanied by structural alterations in the first synapses of the retinal pathway. Recent advances have revealed a crucial role of autophagy in immunity and inflammation. Keeping in mind this close relationship, we aimed to decipher these processes in the context of the defects that occur during ageing in the retina of Igf1−/− mice. Tnfa and Il1b mRNAs, and phosphorylation of JNK and p38 MAPK were elevated in the retinas of 6- and 12-month old Igf1−/− mice compared to those in age-matched Igf1+/+ controls. In 6-month-old Igf1−/− retinas, increased mRNA levels of the autophagy mediators Becn1, Atg9, Atg5 and Atg4, decreased p62 (also known as SQSTM1) protein expression together with an increased LC3-II:LC3-I ratio reflected active autophagic flux. However, in retinas from 12-month-old Igf1−/− mice, Nlrp3 mRNA, processing of the IL1β pro-form and immunostaining of active caspase-1 were elevated compared to those in age-matched Igf1+/+ controls, suggesting activation of the inflammasome. This effect concurred with accumulation of autophagosomes and decreased autophagic flux in the retina. Microglia localization and status of activation in the retinas of 12-month-old Igf1+/+ and Igf1−/− mice, analyzed by immunostaining of Cd11b and Iba-1, showed a specific distribution pattern in the outer plexiform layer (OPL), inner plexiform layer (IPL) and inner nuclear layer (INL), and revealed an increased number of activated microglia cells in the retina of 12-month-old blind Igf1−/− mice. Moreover, reactive gliosis was exclusively detected in the retinas from 12-month-old blind Igf1−/− mice. In conclusion, this study

  4. Autophagy and ethanol-induced liver injury

    Institute of Scientific and Technical Information of China (English)

    Terrence M Donohue Jr

    2009-01-01

    The majority of ethanol metabolism occurs in the liver. Consequently, this organ sustains the greatest damage from ethanol abuse. Ethanol consumption disturbs the delicate balance of protein homeostasis in the liver, causing intracellular protein accumulation due to a disruption of hepatic protein catabolism.Evidence indicates that ethanol or its metabolism impairs trafficking events in the liver, including the process of macroautophagy, which is the engulfment and degradation of cytoplasmic constituents by the lysosomal system. Autophagy is an essential, ongoing cellular process that is highly regulated by nutrients,endocrine factors and signaling pathways. A great number of the genes and gene products that govern the autophagic response have been characterized and the major metabolic and signaling pathways that activate or suppress autophagy have been identified. This review describes the process of autophagy, its regulation and the possible mechanisms by which ethanol disrupts the process of autophagic degradation. The implications of autophagic suppression are discussed in relation to the pathogenesis of alcohol-induced liver injury.

  5. Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization.

    Science.gov (United States)

    Liu, Kun; Zhao, Enpeng; Ilyas, Ghulam; Lalazar, Gadi; Lin, Yu; Haseeb, Muhammad; Tanaka, Kathryn E; Czaja, Mark J

    2015-01-01

    Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.

  6. C1q/TNF-Related Protein 9 (CTRP9) attenuates hepatic steatosis via the autophagy-mediated inhibition of endoplasmic reticulum stress.

    Science.gov (United States)

    Jung, Tae Woo; Hong, Ho Cheol; Hwang, Hwan-Jin; Yoo, Hye Jin; Baik, Sei Hyun; Choi, Kyung Mook

    2015-12-01

    C1q/TNF-Related Protein (CTRP) 9, the closest paralog of adiponectin, has been reported to protect against diet-induced obesity and non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanism has not been fully elucidated. We explored the protective effect of CTRP9 against hepatic steatosis and apoptosis, and identified the mechanisms through autophagy and endoplasmic reticulum (ER) stress using in vitro and in vivo experiments. Treating HepG2 cells with human recombinant CTRP9 significantly ameliorated palmitate- or tunicamycin-induced dysregulation of lipid metabolism, caspase 3 activity and chromatin condensation, which lead to reduction of hepatic triglyceride (TG) accumulation. CTRP9 treatment induced autophagy markers including LC3 conversion, P62 degradation, Beclin1 and ATG7 through AMPK phosphorylation in human primary hepatocytes. Furthermore, CTRP9 decreased palmitate- or tunicamycin-induced ER stress markers, such as eIF2α, CHOP and IRE-1, in HepG2 cells. Compound C, an AMPK inhibitor, and 3 methyladenine (3 MA), an autophagy inhibitor, canceled the effects of CTRP9 on ER stress, apoptosis and hepatic steatosis. In the livers of HFD-fed mice, adenovirus-mediated CTRP9 overexpression significantly induced AMPK phosphorylation and autophagy, whereas suppressed ER stress markers. In addition, both SREBP1-mediated lipogenic gene expression and apoptosis were significantly attenuated, which result in improvement in hepatic steatosis by overexpression of CTRP9. These results demonstrate that CTRP9 alleviates hepatic steatosis through relief of ER stress via the AMPK-mediated induction of autophagy. PMID:26419929

  7. Autophagy to Survive

    Directory of Open Access Journals (Sweden)

    Muzeyyen Izmirli

    2014-06-01

    Full Text Available Autophagy is the catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the actions of lysosomes. It helps to keep the cells alive in such cases like oxidative stress, lack of nutrients and growth factors providing recycling of intracellular molecules. However, it works as a part of metabolism regulation, morphogenesis, cell differentiation, senescence, cell death and immune system. As a result of impairment of this mechanism, pathological situations arise including cancer, neurodegenerative and infectious diseases. Consequently, researches about autophagy mechanism are important for the development of novel diagnosis, follow-up and treatment modalities in health problems. [Archives Medical Review Journal 2014; 23(3.000: 411-419

  8. Autophagy in cardiovascular biology

    OpenAIRE

    Lavandero, Sergio; Chiong, Mario; Rothermel, Beverly A.; Hill, Joseph A.

    2015-01-01

    Cardiovascular disease is the leading cause of death worldwide. As such, there is great interest in identifying novel mechanisms that govern the cardiovascular response to disease-related stress. First described in failing hearts, autophagy within the cardiovascular system has been widely characterized in cardiomyocytes, cardiac fibroblasts, endothelial cells, vascular smooth muscle cells, and macrophages. In all cases, a window of optimal autophagic activity appears to be critical to the mai...

  9. AUTOPHAGY IN LUNG CANCER

    OpenAIRE

    Jaboin, Jerry J.; Hwang, Misun; Lu, Bo

    2009-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. The relatively poor cure rate in lung cancer patients has been associated with a resistance to chemotherapy and radiation that is at least in part related to defects in cellular apoptotic machinery. Exploitation of another form of cell death, autophagy, has the capacity to improve the therapeutic gain of current therapies. In an effort to develop novel treatment strategies to enhance the therapeutic ratio for lung cancer, we...

  10. Autophagy in dementias.

    Science.gov (United States)

    Kragh, Christine Lund; Ubhi, Kiren; Wyss-Coray, Tony; Wyss-Corey, Tony; Masliah, Eliezer

    2012-01-01

    Dementias are a varied group of disorders typically associated with memory loss, impaired judgment and/or language and by symptoms affecting other cognitive and social abilities to a degree that interferes with daily functioning. Alzheimer's disease (AD) is the most common cause of a progressive dementia, followed by dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), (VaD) and HIV-associated neurocognitive disorders (HAND). The pathogenesis of this group of disorders has been linked to the abnormal accumulation of proteins in the brains of affected individuals, which in turn has been related to deficits in protein clearance. Autophagy is a key cellular protein clearance pathway with proteolytic cleavage and degradation via the ubiquitin-proteasome pathway representing another important clearance mechanism. Alterations in the levels of autophagy and the proteins associated with the autophagocytic pathway have been reported in various types of dementias. This review will examine recent literature across these disorders and highlight a common theme of altered autophagy across the spectrum of the dementias. PMID:22150925

  11. Novel enabling technologies of gene isolation and plant transformation for improved crop protection

    Energy Technology Data Exchange (ETDEWEB)

    Torok, Tamas

    2013-02-04

    Meeting the needs of agricultural producers requires the continued development of improved transgenic crop protection products. The completed project focused on developing novel enabling technologies of gene discovery and plant transformation to facilitate the generation of such products.

  12. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  13. Autophagy: Regulation by Energy Sensing

    NARCIS (Netherlands)

    A.J. Meijer; P. Codogno

    2011-01-01

    Autophagy is inhibited by the mTOR signaling pathway, which is stimulated by increased amino acid levels. When cellular energy production is compromised, AMP-activated protein kinase is activated, mTOR is inhibited and autophagy is stimulated. Two recent studies have shed light on the molecular mech

  14. Early autophagy activation inhibits podocytes from apoptosis induced by aldosterone

    Institute of Scientific and Technical Information of China (English)

    王文琰

    2013-01-01

    Objective To explore the protection of early autoph-agy activation on podocyte injury induced by aldosterone.Methods In vitro cultured mouse podocyte clones(MPC5) were treated with aldosterone for 6,12,24,48 hrespectively. Apoptosis of podocytes was detected by

  15. Endoplasmic reticulum stress-induced autophagy determines the susceptibility of melanoma cells to dabrafenib

    Science.gov (United States)

    Ji, Chao; Zhang, Ziping; Chen, Lihong; Zhou, Kunli; Li, Dongjun; Wang, Ping; Huang, Shuying; Gong, Ting; Cheng, Bo

    2016-01-01

    Melanoma is one of the deadliest skin cancers and accounts for most skin-related deaths due to strong resistance to chemotherapy drugs. In the present study, we investigated the mechanisms of dabrafenib-induced drug resistance in human melanoma cell lines A375 and MEL624. Our studies support that both endoplasmic reticulum (ER) stress and autophagy were induced in the melanoma cells after the treatment with dabrafenib. In addition, ER stress-induced autophagy protects melanoma cells from the toxicity of dabrafenib. Moreover, inhibition of both ER stress and autophagy promote the sensitivity of melanoma cells to dabrafenib. Taken together, the data suggest that ER stress-induced autophagy determines the sensitivity of melanoma cells to dabrafenib. These results provide us with promising evidence that the inhibition of autophagy and ER stress could serve a therapeutic effect for the conventional dabrafenib chemotherapy. PMID:27536070

  16. Autophagy is required for stem cell mobilization by G-CSF

    DEFF Research Database (Denmark)

    Leveque-El Mouttie, Lucie; Vu, Therese; Lineburg, Katie E.;

    2015-01-01

    Granulocyte colony-stimulating factor (G-CSF) is widely used clinically to prevent neutropenia after cytotoxic chemotherapy and to mobilize hematopoietic stem cells (HSCs) for transplantation. Autophagy, a process of cytoplasmic component recycling, maintains cellular homeostasis and protects...... the cell during periods of metabolic stress or nutrient deprivation. We have observed that G-CSF activates autophagy in neutrophils and HSCs from both mouse and human donors. Furthermore, G-CSF-induced neutrophil and HSC mobilization is impaired in the absence of autophagy. In contrast, autophagy...... is dispensable for direct HSC mobilization in response to the CXCR4 antagonist AMD3100. Altogether, these data demonstrate an important role for G-CSF in invoking autophagy within hematopoietic and myeloid cells and suggest that this pathway is critical for ensuring cell survival in response to clinically...

  17. Live and Let Die: Roles of Autophagy in Cadmium Nephrotoxicity

    Directory of Open Access Journals (Sweden)

    Frank Thévenod

    2015-04-01

    Full Text Available The transition metal ion cadmium (Cd2+ is a significant environmental contaminant. With a biological half-life of ~20 years, Cd2+ accumulates in the kidney cortex, where it particularly damages proximal tubule (PT cells and can result in renal fibrosis, failure, or cancer. Because death represents a powerful means by which cells avoid malignant transformation, it is crucial to clearly identify and understand the pathways that determine cell fate in chronic Cd2+ nephrotoxicity. When cells are subjected to stress, they make a decision to adapt and survive, or—depending on the magnitude and duration of stress—to die by several modes of death (programmed cell death, including autophagic cell death (ACD. Autophagy is part of a larger system of intracellular protein degradation and represents the channel by which organelles and long-lived proteins are delivered to the lysosome for degradation. Basal autophagy levels in all eukaryotic cells serve as a dynamic physiological recycling system, but they can also be induced by intra- or extracellular stress and pathological processes, such as endoplasmic reticulum (ER stress. In a context-dependent manner, autophagy can either be protective and hence contribute to survival, or promote death by non-apoptotic or apoptotic pathways. So far, the role of autophagy in Cd2+-induced nephrotoxicity has remained unsettled due to contradictory results. In this review, we critically survey the current literature on autophagy in Cd2+-induced nephrotoxicity in light of our own ongoing studies. Data obtained in kidney cells illustrate a dual and complex function of autophagy in a stimulus- and time-dependent manner that possibly reflects distinct outcomes in vitro and in vivo. A better understanding of the context-specific regulation of cell fate by autophagy may ultimately contribute to the development of preventive and novel therapeutic strategies for acute and chronic Cd2+ nephrotoxicity.

  18. Crocin-Elicited Autophagy Rescues Myocardial Ischemia/Reperfusion Injury via Paradoxical Mechanisms.

    Science.gov (United States)

    Zeng, Chao; Li, Hu; Fan, Zhiwen; Zhong, Lei; Guo, Zhen; Guo, Yaping; Xi, Yusheng

    2016-01-01

    Crocin, the main effective component of saffron, exerts protective effects against ischemia/reperfusion injury during strokes. However, the effects of crocin in myocardial ischemia/reperfusion injury, and the mechanisms involved, remain unknown. Pretreated with crocin for 7 days, C57BL/6N mice were subjected to 30 min of myocardial ischemia followed by 12[Formula: see text]h of reperfusion (for cardiac function and infarct size, cell apoptosis and necrosis). Neonatal mouse cardiomyocytes were subjected to 2 h of hypoxia followed by 4 h of reoxygenation. NMCM's survival was assessed during hypoxia and reoxygenation in the presence or absence of the autophagy inhibitor 3-methyladenine or the inducer rapamycin. Western blotting was used to evaluate AMPK, Akt, and autophagy-related proteins. Autophagosome was observed using electron microscopy. In the in vivo experiment, crocin pretreatment significantly attenuated infarct size, myocardial apoptosis and necrosis, and improved left ventricular function following ischemia/reperfusion. In vitro data revealed that autophagy was induced during hypoxia, the levels of which were intensely elevated during reoxygenation. Crocin significantly promoted autophagy during ischemia, accompanied with the activation of AMPK. In contrast, crocin overtly inhibited autophagy during reperfusion, accompanied with Akt activation. Induction and inhibition of autophagy mitigated crocin induced protection against NMCMs injury during hypoxia and reoxygenation, respectively. Our data suggest that crocin demonstrated a myocardial protective effect via AMPK/mTOR and Akt/mTOR regulated autophagy against ischemia and reperfusion injury, respectively. PMID:27109157

  19. Neuronal autophagy in cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Feng Xu; Jin-Hua Gu; Zheng-Hong Qin

    2012-01-01

    Autophagy has evolved as a conserved process for the bulk degradation and recycling of cytosolic components,such as long-lived proteins and organelles.In neurons,autophagy is important for homeostasis and protein quality control and is maintained at relatively low levels under normal conditions,while it is upregulated in response to pathophysiological conditions,such as cerebral ischemic injury.However,the role of autophagy is more complex.It depends on age or brain maturity,region,severity of insult,and the stage of ischemia.Whether autophagy plays a beneficial or a detrimental role in cerebral ischemia depends on various pathological conditions.In this review,we elucidate the role of neuronal autophagy in cerebral ischemia.

  20. Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

    Energy Technology Data Exchange (ETDEWEB)

    Sukseree, Supawadee [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok (Thailand); Rossiter, Heidemarie; Mildner, Michael [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Pammer, Johannes [Institute of Clinical Pathology, Medical University of Vienna, Vienna (Austria); Buchberger, Maria; Gruber, Florian [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Watanapokasin, Ramida [Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok (Thailand); Tschachler, Erwin [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Eckhart, Leopold, E-mail: leopold.eckhart@meduniwien.ac.at [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. Black-Right-Pointing-Pointer Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. Black-Right-Pointing-Pointer Autophagy was required for normal terminal differentiation of preputial gland cells. Black-Right-Pointing-Pointer Autophagy-deficient cells of the preputial glands degraded nuclear DNA prematurely. -- Abstract: Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.

  1. Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

    International Nuclear Information System (INIS)

    Highlights: ► We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. ► Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. ► Autophagy was required for normal terminal differentiation of preputial gland cells. ► Autophagy-deficient cells of the preputial glands degraded nuclear DNA prematurely. -- Abstract: Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.

  2. The Autophagy Machinery Controls Cell Death Switching between Apoptosis and Necroptosis.

    Science.gov (United States)

    Goodall, Megan L; Fitzwalter, Brent E; Zahedi, Shadi; Wu, Min; Rodriguez, Diego; Mulcahy-Levy, Jean M; Green, Douglas R; Morgan, Michael; Cramer, Scott D; Thorburn, Andrew

    2016-05-23

    Although autophagy controls cell death and survival, underlying mechanisms are poorly understood, and it is unknown whether autophagy affects only whether or not cells die or also controls other aspects of programmed cell death. MAP3K7 is a tumor suppressor gene associated with poor disease-free survival in prostate cancer. Here, we report that Map3k7 deletion in mouse prostate cells sensitizes to cell death by TRAIL (TNF-related apoptosis-inducing ligand). Surprisingly, this death occurs primarily through necroptosis, not apoptosis, due to assembly of the necrosome in association with the autophagy machinery, mediated by p62/SQSTM1 recruitment of RIPK1. The mechanism of cell death switches to apoptosis if p62-dependent recruitment of the necrosome to the autophagy machinery is blocked. These data show that the autophagy machinery can control the mechanism of programmed cell death by serving as a scaffold rather than by degrading cargo.

  3. [Protection of corneal endothelium from apoptosis by gene and cell therapy].

    Science.gov (United States)

    Fuchsluger, T A

    2016-06-01

    Protection of corneal endothelium from apoptosis using gene and cell therapy is in a translational phase. This approach offers advantages for eye banking and after transplantation. Safe vehicles for gene or cell therapeutic transduction of corneal endothelium with nucleic acids are available. This strategy will be further developed in consultation with the Paul Ehrlich Institute and European regulatory authorities.

  4. Does bilirubin protect against hemochromatosis gene (HFE) related mortality?

    NARCIS (Netherlands)

    Alizadeh, Behrooz Z.; Njajou, Omer T.; Houwing-Duistermaat, Jeanine J.; de Jong, Gerard; Vergeer, Jeannette M.; Hofman, Albert; Pols, Huibert A.P.; van Duijn, Cornelia M.

    2004-01-01

    Serum bilirubin is an important antioxidant that is found at increased levels in hereditary hemochromatosis patients. We hypothesized that increased levels of serum bilirubin may play a protective role against oxidative stress induced by iron overload in carriers of mutations in the hereditary hemoc

  5. Methylprednisolone exerts neuroprotective effects by regulating autophagy and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Wei Gao; Shu-rui Chen; Meng-yao Wu; Kai Gao; Yuan-long Li; Hong-yu Wang; Chen-yuan Li; Hong Li

    2016-01-01

    Methylprednisolone markedly reduces autophagy and apoptosis after secondary spinal cord injury. Here, we investigated whether pretreat-ment of cells with methylprednisolone would protect neuron-like cells from subsequent oxidative damagevia suppression of autophagy and apoptosis. Cultured N2a cells were pretreated with 10 µM methylprednisolone for 30 minutes, then exposed to 100 µM H2O2 for 24 hours. Inverted phase contrast microscope images, MTT assay, lfow cytometry and western blot results showed that, compared to cells ex-posed to 100 µM H2O2 alone, cells pretreated with methylprednisolone had a signiifcantly lower percentage of apoptotic cells, maintained a healthy morphology, and showed downregulation of autophagic protein light chain 3B and Beclin-1 protein expression. These ifndings indicate that methylprednisolone exerted neuroprotective effects against oxidative damage by suppressing autophagy and apoptosis.

  6. Autophagy mediates tolerance to Staphylococcus aureus alpha-toxin.

    Science.gov (United States)

    Maurer, Katie; Reyes-Robles, Tamara; Alonzo, Francis; Durbin, Joan; Torres, Victor J; Cadwell, Ken

    2015-04-01

    Resistance and tolerance are two defense strategies employed by the host against microbial threats. Autophagy-mediated degradation of bacteria has been extensively described as a major resistance mechanism. Here we find that the dominant function of autophagy proteins during infections with the epidemic community-associated methicillin-resistant Staphylococcus aureus USA300 is to mediate tolerance rather than resistance. Atg16L1 hypomorphic mice (Atg16L1(HM)), which have reduced autophagy, were highly susceptible to lethality in both sepsis and pneumonia models of USA300 infection. Autophagy confers protection by limiting the damage caused by α-toxin, particularly to endothelial cells. Remarkably, Atg16L1(HM) mice display enhanced survival rather than susceptibility upon infection with α-toxin-deficient S. aureus. These results identify an essential role for autophagy in tolerance to Staphylococcal disease and highlight how a single virulence factor encoded by a pathogen can determine whether a given host factor promotes tolerance or resistance.

  7. Ammonia Induces Autophagy through Dopamine Receptor D3 and MTOR.

    Science.gov (United States)

    Li, Zhiyuan; Ji, Xinmiao; Wang, Wenchao; Liu, Juanjuan; Liang, Xiaofei; Wu, Hong; Liu, Jing; Eggert, Ulrike S; Liu, Qingsong; Zhang, Xin

    2016-01-01

    Hyperammonemia is frequently seen in tumor microenvironments as well as in liver diseases where it can lead to severe brain damage or death. Ammonia induces autophagy, a mechanism that tumor cells may use to protect themselves from external stresses. However, how cells sense ammonia has been unclear. Here we show that culture medium alone containing Glutamine can generate milimolar of ammonia at 37 degrees in the absence of cells. In addition, we reveal that ammonia acts through the G protein-coupled receptor DRD3 (Dopamine receptor D3) to induce autophagy. At the same time, ammonia induces DRD3 degradation, which involves PIK3C3/VPS34-dependent pathways. Ammonia inhibits MTOR (mechanistic target of Rapamycin) activity and localization in cells, which is mediated by DRD3. Therefore, ammonia has dual roles in autophagy: one to induce autophagy through DRD3 and MTOR, the other to increase autophagosomal pH to inhibit autophagic flux. Our study not only adds a new sensing and output pathway for DRD3 that bridges ammonia sensing and autophagy induction, but also provides potential mechanisms for the clinical consequences of hyperammonemia in brain damage, neurodegenerative diseases and tumors.

  8. Autophagy contributes to regulation of the hypoxia response during submergence in Arabidopsis thaliana.

    Science.gov (United States)

    Chen, Liang; Liao, Bin; Qi, Hua; Xie, Li-Juan; Huang, Li; Tan, Wei-Juan; Zhai, Ning; Yuan, Li-Bing; Zhou, Ying; Yu, Lu-Jun; Chen, Qin-Fang; Shu, Wensheng; Xiao, Shi

    2015-01-01

    Autophagy involves massive degradation of intracellular components and functions as a conserved system that helps cells to adapt to adverse conditions. In mammals, hypoxia rapidly stimulates autophagy as a cell survival response. Here, we examine the function of autophagy in the regulation of the plant response to submergence, an abiotic stress that leads to hypoxia and anaerobic respiration in plant cells. In Arabidopsis thaliana, submergence induces the transcription of autophagy-related (ATG) genes and the formation of autophagosomes. Consistent with this, the autophagy-defective (atg) mutants are hypersensitive to submergence stress and treatment with ethanol, the end product of anaerobic respiration. Upon submergence, the atg mutants have increased levels of transcripts of anaerobic respiration genes (alcohol dehydrogenase 1, ADH1 and pyruvate decarboxylase 1, PDC1), but reduced levels of transcripts of other hypoxia- and ethylene-responsive genes. Both submergence and ethanol treatments induce the accumulation of reactive oxygen species (ROS) in the rosettes of atg mutants more than in the wild type. Moreover, the production of ROS by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases is necessary for plant tolerance to submergence and ethanol, submergence-induced expression of ADH1 and PDC1, and activation of autophagy. The submergence- and ethanol-sensitive phenotypes in the atg mutants depend on a complete salicylic acid (SA) signaling pathway. Together, our findings demonstrate that submergence-induced autophagy functions in the hypoxia response in Arabidopsis by modulating SA-mediated cellular homeostasis.

  9. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Liang [East Hospital, Tongji University School of Medicine, Shanghai (China); Dong, Chuanming [East Hospital, Tongji University School of Medicine, Shanghai (China); Department of Anatomy and Neurobiology, The Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong (China); Sun, Chenxi; Ma, Rongjie; Yang, Danjing [East Hospital, Tongji University School of Medicine, Shanghai (China); Zhu, Hongwen, E-mail: hongwen_zhu@hotmail.com [Tianjin Hospital, Tianjin Academy of Integrative Medicine, Tianjin (China); Xu, Jun, E-mail: xunymc2000@yahoo.com [East Hospital, Tongji University School of Medicine, Shanghai (China)

    2015-08-21

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy.

  10. A role of autophagy in PTP4A3-driven cancer progression.

    Science.gov (United States)

    Huang, Yu-Han; Al-Aidaroos, Abdul Qader O; Yuen, Hiu-Fung; Zhang, Shu-Dong; Shen, Han-Ming; Rozycka, Ewelina; McCrudden, Cian M; Tergaonkar, Vinay; Gupta, Abhishek; Lin, You Bin; Thiery, Jean Paul; Murray, James T; Zeng, Qi

    2014-10-01

    Autophagy, a "self-eating" cellular process, has dual roles in promoting and suppressing tumor growth, depending on cellular context. PTP4A3/PRL-3, a plasma membrane and endosomal phosphatase, promotes multiple oncogenic processes including cell proliferation, invasion, and cancer metastasis. In this study, we demonstrate that PTP4A3 accumulates in autophagosomes upon inhibition of autophagic degradation. Expression of PTP4A3 enhances PIK3C3-BECN1-dependent autophagosome formation and accelerates LC3-I to LC3-II conversion in an ATG5-dependent manner. PTP4A3 overexpression also enhances the degradation of SQSTM1, a key autophagy substrate. These functions of PTP4A3 are dependent on its catalytic activity and prenylation-dependent membrane association. These results suggest that PTP4A3 functions to promote canonical autophagy flux. Unexpectedly, following autophagy activation, PTP4A3 serves as a novel autophagic substrate, thereby establishing a negative feedback-loop that may be required to fine-tune autophagy activity. Functionally, PTP4A3 utilizes the autophagy pathway to promote cell growth, concomitant with the activation of AKT. Clinically, from the largest ovarian cancer data set (GSE 9899, n = 285) available in GEO, high levels of expression of both PTP4A3 and autophagy genes significantly predict poor prognosis of ovarian cancer patients. These studies reveal a critical role of autophagy in PTP4A3-driven cancer progression, suggesting that autophagy could be a potential Achilles heel to block PTP4A3-mediated tumor progression in stratified patients with high expression of both PTP4A3 and autophagy genes.

  11. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

    International Nuclear Information System (INIS)

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy

  12. Regulatory coordination between two major intracellular homeostatic systems: heat shock response and autophagy.

    Science.gov (United States)

    Dokladny, Karol; Zuhl, Micah Nathaniel; Mandell, Michael; Bhattacharya, Dhruva; Schneider, Suzanne; Deretic, Vojo; Moseley, Pope Lloyd

    2013-05-24

    The eukaryotic cell depends on multitiered homeostatic systems ensuring maintenance of proteostasis, organellar integrity, function and turnover, and overall cellular viability. At the two opposite ends of the homeostatic system spectrum are heat shock response and autophagy. Here, we tested whether there are interactions between these homeostatic systems, one universally operational in all prokaryotic and eukaryotic cells, and the other one (autophagy) is limited to eukaryotes. We found that heat shock response regulates autophagy. The interaction between the two systems was demonstrated by testing the role of HSF-1, the central regulator of heat shock gene expression. Knockdown of HSF-1 increased the LC3 lipidation associated with formation of autophagosomal organelles, whereas depletion of HSF-1 potentiated both starvation- and rapamycin-induced autophagy. HSP70 expression but not expression of its ATPase mutant inhibited starvation or rapamycin-induced autophagy. We also show that exercise induces autophagy in humans. As predicted by our in vitro studies, glutamine supplementation as a conditioning stimulus prior to exercise significantly increased HSP70 protein expression and prevented the expected exercise induction of autophagy. Our data demonstrate for the first time that heat shock response, from the top of its regulatory cascade (HSF-1) down to the execution stages delivered by HSP70, controls autophagy thus connecting and coordinating the two extreme ends of the homeostatic systems in the eukaryotic cell. PMID:23576438

  13. 细胞自噬与肿瘤耐药的研究进展%Recent progress of autophagy and drug-resistance

    Institute of Scientific and Technical Information of China (English)

    叶丽霖; 李玉峰

    2013-01-01

    Summary Autophagy is a self degradation process that are responsible for the removal of long lived proteins and damaged organelles, which plays a key role in the elimination of such intracellular waste, reconstruction of structure,growth and development of cells and maintainance of cellular homeostasis. During tumour development, autophagy has paradoxically been reported to have roles in promoting both cell survival and growth. In tumour cells with defects in apoptosis, autophagy allows prolonged survival. Recent evidence suggests that autophagy provides a protective function to limit tumour necrosis and inflammation,and to mitigate genome damage in tumour cells in response to metabolic stress. Many studies show that many cytoxic drugs can induce autophagy in its induction of apoptosis. Activation of autophagy can lead to drug resistance in tumour cells. In this review, we will summarize the relationship between autophagy and drug resistance.

  14. Protecting posted genes: social networking and the limits of GINA.

    Science.gov (United States)

    Soo-Jin Lee, Sandra; Borgelt, Emily

    2014-01-01

    The combination of decreased genotyping costs and prolific social media use is fueling a personal genetic testing industry in which consumers purchase and interact with genetic risk information online. Consumers and their genetic risk profiles are protected in some respects by the 2008 federal Genetic Information Nondiscrimination Act (GINA), which forbids the discriminatory use of genetic information by employers and health insurers; however, practical and technical limitations undermine its enforceability, given the everyday practices of online social networking and its impact on the workplace. In the Web 2.0 era, employers in most states can legally search about job candidates and employees online, probing social networking sites for personal information that might bear on hiring and employment decisions. We examine GINA's protections for online sharing of genetic information as well as its limitations, and propose policy recommendations to address current gaps that leave employees' genetic information vulnerable in a Web-based world. PMID:25325810

  15. Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

    Directory of Open Access Journals (Sweden)

    Hye Rin Lee

    Full Text Available Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ and the aggregated proteins that cause Alzheimer's disease (AD. We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5, and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 μM or resveratrol (20 μM prevented these Aβ1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition. Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion. After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein, cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

  16. Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy.

    Science.gov (United States)

    Sinha, Rohit Anthony; You, Seo-Hee; Zhou, Jin; Siddique, Mobin M; Bay, Boon-Huat; Zhu, Xuguang; Privalsky, Martin L; Cheng, Sheue-Yann; Stevens, Robert D; Summers, Scott A; Newgard, Christopher B; Lazar, Mitchell A; Yen, Paul M

    2012-07-01

    For more than a century, thyroid hormones (THs) have been known to exert powerful catabolic effects, leading to weight loss. Although much has been learned about the molecular mechanisms used by TH receptors (TRs) to regulate gene expression, little is known about the mechanisms by which THs increase oxidative metabolism. Here, we report that TH stimulation of fatty acid β-oxidation is coupled with induction of hepatic autophagy to deliver fatty acids to mitochondria in cell culture and in vivo. Furthermore, blockade of autophagy by autophagy-related 5 (ATG5) siRNA markedly decreased TH-mediated fatty acid β-oxidation in cell culture and in vivo. Consistent with this model, autophagy was altered in livers of mice expressing a mutant TR that causes resistance to the actions of TH as well as in mice with mutant nuclear receptor corepressor (NCoR). These results demonstrate that THs can regulate lipid homeostasis via autophagy and help to explain how THs increase oxidative metabolism.

  17. Autophagy and neurodegenerative disorders

    Institute of Scientific and Technical Information of China (English)

    Evangelia Kesidou; Roza Lagoudaki; Olga Touloumi; Kyriaki-Nefeli Poulatsidou; Constantina Simeonidou

    2013-01-01

    Accumulation of aberrant proteins and inclusion bodies are hallmarks in most neurodegenerative diseases. Consequently, these aggregates within neurons lead to toxic effects, overproduction of reactive oxygen species and oxidative stress. Autophagy is a significant intracel ular mechanism that removes damaged organelles and misfolded proteins in order to maintain cel homeostasis. Excessive or insufficient autophagic activity in neurons leads to altered homeostasis and influences their survival rate, causing neurodegeneration. The review article provides an update of the role of autophagic process in representative chronic and acute neurodegenerative disorders.

  18. A close connection between the PERK and IRE arms of the UPR and the transcriptional regulation of autophagy.

    Science.gov (United States)

    Deegan, Shane; Koryga, Izabela; Glynn, Sharon A; Gupta, Sanjeev; Gorman, Adrienne M; Samali, Afshin

    2015-01-01

    Endoplasmic reticulum (ER) stress is known to lead to activation of both the unfolded protein response (UPR) and autophagy. Although regulatory connections have been identified between the UPR and autophagy, it is still unclear to what extent the UPR regulates the genes involved at the different stages of the autophagy pathway. Here, we carried out a microarray analysis of HCT116 cells subjected to ER stress and observed the transcriptional upregulation of a large cohort of autophagy-related genes. Of particular interest, we identified the transcriptional upregulation of the autophagy receptor genes SQSTM1/p62, NBR1 and BNIP3L/NIX in response to ER stress and show that the inhibition of the UPR transmembrane receptors, PERK and IRE1, abrogates this upregulation. PMID:25475719

  19. Transcriptional and epigenetic regulation of autophagy in aging.

    Science.gov (United States)

    Lapierre, Louis R; Kumsta, Caroline; Sandri, Marco; Ballabio, Andrea; Hansen, Malene

    2015-01-01

    Macroautophagy is a major intracellular degradation process recognized as playing a central role in cell survival and longevity. This multistep process is extensively regulated at several levels, including post-translationally through the action of conserved longevity factors such as the nutrient sensor TOR. More recently, transcriptional regulation of autophagy genes has emerged as an important mechanism for ensuring the somatic maintenance and homeostasis necessary for a long life span. Autophagy is increased in many long-lived model organisms and contributes significantly to their longevity. In turn, conserved transcription factors, particularly the helix-loop-helix transcription factor TFEB and the forkhead transcription factor FOXO, control the expression of many autophagy-related genes and are important for life-span extension. In this review, we discuss recent progress in understanding the contribution of these transcription factors to macroautophagy regulation in the context of aging. We also review current research on epigenetic changes, such as histone modification by the deacetylase SIRT1, that influence autophagy-related gene expression and additionally affect aging. Understanding the molecular regulation of macroautophagy in relation to aging may offer new avenues for the treatment of age-related diseases.

  20. Listeriolysin O is necessary and sufficient to induce autophagy during Listeria monocytogenes infection.

    Directory of Open Access Journals (Sweden)

    Nicole Meyer-Morse

    Full Text Available BACKGROUND: Recent studies have suggested that autophagy is utilized by cells as a protective mechanism against Listeria monocytogenes infection. METHODOLOGY/PRINCIPAL FINDINGS: However we find autophagy has no measurable role in vacuolar escape and intracellular growth in primary cultured bone marrow derived macrophages (BMDMs deficient for autophagy (atg5-/-. Nevertheless, we provide evidence that the pore forming activity of the cholesterol-dependent cytolysin listeriolysin O (LLO can induce autophagy subsequent to infection by L. monocytogenes. Infection of BMDMs with L. monocytogenes induced microtubule-associated protein light chain 3 (LC3 lipidation, consistent with autophagy activation, whereas a mutant lacking LLO did not. Infection of BMDMs that express LC3-GFP demonstrated that wild-type L. monocytogenes was encapsulated by LC3-GFP, consistent with autophagy activation, whereas a mutant lacking LLO was not. Bacillus subtilis expressing either LLO or a related cytolysin, perfringolysin O (PFO, induced LC3 colocalization and LC3 lipidation. Further, LLO-containing liposomes also recruited LC3-GFP, indicating that LLO was sufficient to induce targeted autophagy in the absence of infection. The role of autophagy had variable effects depending on the cell type assayed. In atg5-/- mouse embryonic fibroblasts, L. monocytogenes had a primary vacuole escape defect. However, the bacteria escaped and grew normally in atg5-/- BMDMs. CONCLUSIONS/SIGNIFICANCE: We propose that membrane damage, such as that caused by LLO, triggers bacterial-targeted autophagy, although autophagy does not affect the fate of wild-type intracellular L. monocytogenes in primary BMDMs.

  1. Suppression of autophagy dysregulates the antioxidant response and causes premature senescence of melanocytes.

    Science.gov (United States)

    Zhang, Cheng-Feng; Gruber, Florian; Ni, Chunya; Mildner, Michael; Koenig, Ulrich; Karner, Susanne; Barresi, Caterina; Rossiter, Heidemarie; Narzt, Marie-Sophie; Nagelreiter, Ionela M; Larue, Lionel; Tobin, Desmond J; Eckhart, Leopold; Tschachler, Erwin

    2015-05-01

    Autophagy is the central cellular mechanism for delivering organelles and cytoplasm to lysosomes for degradation and recycling of their molecular components. To determine the contribution of autophagy to melanocyte (MC) biology, we inactivated the essential autophagy gene Atg7 specifically in MCs using the Cre-loxP system. This gene deletion efficiently suppressed a key step in autophagy, lipidation of microtubule-associated protein 1 light chain 3 beta (LC3), in MCs and induced slight hypopigmentation of the epidermis in mice. The melanin content of hair was decreased by 10-15% in mice with autophagy-deficient MC as compared with control animals. When cultured in vitro, MCs from mutant and control mice produced equal amounts of melanin per cell. However, Atg7-deficient MCs entered into premature growth arrest and accumulated reactive oxygen species (ROS) damage, ubiquitinated proteins, and the multi-functional adapter protein SQSTM1/p62. Moreover, nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent expression of NAD(P)H dehydrogenase, quinone 1, and glutathione S-transferase Mu 1 was increased, indicating a contribution of autophagy to redox homeostasis in MCs. In summary, the results of our study suggest that Atg7-dependent autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of MCs. PMID:25290687

  2. Autophagy is required for G₁/G₀ quiescence in response to nitrogen starvation in Saccharomyces cerevisiae.

    Science.gov (United States)

    An, Zhenyi; Tassa, Amina; Thomas, Collin; Zhong, Rui; Xiao, Guanghua; Fotedar, Rati; Tu, Benjamin P; Klionsky, Daniel J; Levine, Beth

    2014-10-01

    In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G₁/G₀ in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G₂/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G₂/M transition and arrest in G₁/G 0₀ autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G₁/G₀ quiescent state.

  3. EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells.

    Science.gov (United States)

    Sugita, Shohei; Ito, Kentaro; Yamashiro, Yutaro; Moriya, Shota; Che, Xiao-Fang; Yokoyama, Tomohisa; Hiramoto, Masaki; Miyazawa, Keisuke

    2015-05-22

    Gefitinib (GEF), an inhibitor for EGFR tyrosine kinase, potently induces autophagy in non-small cell lung cancer (NSCLC) cell lines such as PC-9 cells expressing constitutively activated EGFR kinase by EGFR gene mutation as well as A549 and H226 cells with wild-type EGFR. Unexpectedly, GEF-induced autophagy was also observed in non-NSCLC cells such as murine embryonic fibroblasts (MEF) and leukemia cell lines K562 and HL-60 without EGFR expression. Knockout of EGFR gene in A549 cells by CRISPR/Cas9 system still exhibited autophagy induction after treatment with GEF, indicating that the autophagy induction by GEF is not mediated through inhibiting EGFR kinase activity. Combined treatment with GEF and clarithromycin (CAM), a macrolide antibiotic having the effect of inhibiting autophagy flux, enhances the cytotoxic effect in NSCLC cell lines, although treatment with CAM alone exhibits no cytotoxicity. GEF treatment induced up-regulation of endoplasmic reticulum (ER)-stress related genes such as CHOP/GADD153 and GRP78. Knockdown of CHOP in PC-9 cells and Chop-knockout MEF both exhibited less sensitivity to GEF than controls. Addition of CAM in culture medium resulted in further pronounced GEF-induced ER stress loading, while CAM alone exhibited no effect. These data suggest that GEF-induced autophagy functions as cytoprotective and indicates the potential therapeutic possibility of using CAM for GEF therapy. Furthermore, it is suggested that the intracellular signaling for autophagy initiation in response to GEF can be completely dissociated from EGFR, but unknown target molecule(s) of GEF for autophagy induction might exist.

  4. EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells.

    Science.gov (United States)

    Sugita, Shohei; Ito, Kentaro; Yamashiro, Yutaro; Moriya, Shota; Che, Xiao-Fang; Yokoyama, Tomohisa; Hiramoto, Masaki; Miyazawa, Keisuke

    2015-05-22

    Gefitinib (GEF), an inhibitor for EGFR tyrosine kinase, potently induces autophagy in non-small cell lung cancer (NSCLC) cell lines such as PC-9 cells expressing constitutively activated EGFR kinase by EGFR gene mutation as well as A549 and H226 cells with wild-type EGFR. Unexpectedly, GEF-induced autophagy was also observed in non-NSCLC cells such as murine embryonic fibroblasts (MEF) and leukemia cell lines K562 and HL-60 without EGFR expression. Knockout of EGFR gene in A549 cells by CRISPR/Cas9 system still exhibited autophagy induction after treatment with GEF, indicating that the autophagy induction by GEF is not mediated through inhibiting EGFR kinase activity. Combined treatment with GEF and clarithromycin (CAM), a macrolide antibiotic having the effect of inhibiting autophagy flux, enhances the cytotoxic effect in NSCLC cell lines, although treatment with CAM alone exhibits no cytotoxicity. GEF treatment induced up-regulation of endoplasmic reticulum (ER)-stress related genes such as CHOP/GADD153 and GRP78. Knockdown of CHOP in PC-9 cells and Chop-knockout MEF both exhibited less sensitivity to GEF than controls. Addition of CAM in culture medium resulted in further pronounced GEF-induced ER stress loading, while CAM alone exhibited no effect. These data suggest that GEF-induced autophagy functions as cytoprotective and indicates the potential therapeutic possibility of using CAM for GEF therapy. Furthermore, it is suggested that the intracellular signaling for autophagy initiation in response to GEF can be completely dissociated from EGFR, but unknown target molecule(s) of GEF for autophagy induction might exist. PMID:25858318

  5. Ordered bulk degradation via autophagy

    DEFF Research Database (Denmark)

    Dengjel, Jörn; Kristensen, Anders Riis; Andersen, Jens S

    2008-01-01

    During amino acid starvation, cells undergo macroautophagy which is regarded as an unspecific bulk degradation process. Lately, more and more organelle-specific autophagy subtypes such as reticulophagy, mitophagy and ribophagy have been described and it could be shown, depending on the experimental...... setup, that autophagy specifically can remove certain subcellular components. We used an unbiased quantitative proteomics approach relying on stable isotope labeling by amino acids in cell culture (SILAC) to study global protein dynamics during amino acid starvation-induced autophagy. Looking...... at proteasomal and lysosomal degradation ample cross-talk between the two degradation pathways became evident. Degradation via autophagy appeared to be ordered and regulated at the protein complex/organelle level. This raises several important questions such as: can macroautophagy itself be specific and what...

  6. Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae

    Institute of Scientific and Technical Information of China (English)

    Xiao-hong LIU; Fu-cheng LIN

    2008-01-01

    Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentiation in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.

  7. Autophagy in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Piotr Czarny

    2015-01-01

    Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

  8. Role and Regulation of Autophagy in Heat Stress Responses of Tomato Plants

    Directory of Open Access Journals (Sweden)

    Jie eZhou

    2014-04-01

    Full Text Available As sessile organisms, plants are constantly exposed to a wide spectrum of stress conditions such as high temperature, which causes protein misfolding. Misfolded proteins are highly toxic and must be efficiently removed to reduce cellular proteotoxic stress if restoration of native conformations is unsuccessful. Although selective autophagy is known to function in protein quality control by targeting degradation of misfolded and potentially toxic proteins, its role and regulation in heat stress responses have not been analyzed in crop plants. In the present study, we found that heat stress induced expression of autophagy-related (ATG genes and accumulation of autophagosomes in tomato plants. Virus-induced gene silencing of tomato ATG5 and ATG7 genes resulted in increased sensitivity of tomato plants to heat stress based on both increased development of heat stress symptoms and compromised photosynthetic parameters of heat-stressed leaf tissues. Silencing of tomato homologs for the selective autophagy receptor NBR1, which targets ubiquitinated protein aggregates, also compromised tomato heat tolerance. To better understand the regulation of heat-induced autophagy, we found that silencing of tomato ATG5, ATG7 or NBR1 compromised heat-induced expression of not only the targeted genes but also other autophagy-related genes. Furthermore, we identified two tomato genes encoding proteins highly homologous to Arabidopsis WRKY33 transcription factor, which has been previously shown to interact physically with an autophagy protein. Silencing of tomato WRKY33 genes compromised tomato heat tolerance and reduced heat-induced ATG gene expression and autophagosome accumulation. Based on these results, we propose that heat-induced autophagy in tomato is subject to cooperative regulation by both WRKY33 and ATG proteins and plays a critical role in tomato heat tolerance, mostly likely through selective removal of heat-induced protein aggregates.

  9. Role and regulation of autophagy in heat stress responses of tomato plants.

    Science.gov (United States)

    Zhou, Jie; Wang, Jian; Yu, Jing-Quan; Chen, Zhixiang

    2014-01-01

    As sessile organisms, plants are constantly exposed to a wide spectrum of stress conditions such as high temperature, which causes protein misfolding. Misfolded proteins are highly toxic and must be efficiently removed to reduce cellular proteotoxic stress if restoration of native conformations is unsuccessful. Although selective autophagy is known to function in protein quality control by targeting degradation of misfolded and potentially toxic proteins, its role and regulation in heat stress responses have not been analyzed in crop plants. In the present study, we found that heat stress induced expression of autophagy-related (ATG) genes and accumulation of autophagosomes in tomato plants. Virus-induced gene silencing (VIGS) of tomato ATG5 and ATG7 genes resulted in increased sensitivity of tomato plants to heat stress based on both increased development of heat stress symptoms and compromised photosynthetic parameters of heat-stressed leaf tissues. Silencing of tomato homologs for the selective autophagy receptor NBR1, which targets ubiquitinated protein aggregates, also compromised tomato heat tolerance. To better understand the regulation of heat-induced autophagy, we found that silencing of tomato ATG5, ATG7, or NBR1 compromised heat-induced expression of not only the targeted genes but also other autophagy-related genes. Furthermore, we identified two tomato genes encoding proteins highly homologous to Arabidopsis WRKY33 transcription factor, which has been previously shown to interact physically with an autophagy protein. Silencing of tomato WRKY33 genes compromised tomato heat tolerance and reduced heat-induced ATG gene expression and autophagosome accumulation. Based on these results, we propose that heat-induced autophagy in tomato is subject to cooperative regulation by both WRKY33 and ATG proteins and plays a critical role in tomato heat tolerance, mostly likely through selective removal of heat-induced protein aggregates.

  10. Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus.

    Science.gov (United States)

    Lee, Lucy E; Witter, R L; Reddy, S M; Wu, P; Yanagida, N; Yoshida, S

    2003-01-01

    Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody-positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gl were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.

  11. Macrophage Autophagy in Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Maria Chiara Maiuri

    2013-01-01

    Full Text Available Macrophages play crucial roles in atherosclerotic immune responses. Recent investigation into macrophage autophagy (AP in atherosclerosis has demonstrated a novel pathway through which these cells contribute to vascular inflammation. AP is a cellular catabolic process involving the delivery of cytoplasmic contents to the lysosomal machinery for ultimate degradation and recycling. Basal levels of macrophage AP play an essential role in atheroprotection during early atherosclerosis. However, AP becomes dysfunctional in the more advanced stages of the pathology and its deficiency promotes vascular inflammation, oxidative stress, and plaque necrosis. In this paper, we will discuss the role of macrophages and AP in atherosclerosis and the emerging evidence demonstrating the contribution of macrophage AP to vascular pathology. Finally, we will discuss how AP could be targeted for therapeutic utility.

  12. Autophagy and intestinal homeostasis.

    Science.gov (United States)

    Patel, Khushbu K; Stappenbeck, Thaddeus S

    2013-01-01

    Nutrient absorption is the basic function that drives mammalian intestinal biology. To facilitate nutrient uptake, the host's epithelial barrier is composed of a single layer of cells. This constraint is problematic, as a design of this type can be easily disrupted. The solution during the course of evolution was to add numerous host defense mechanisms that can help prevent local and systemic infection. These mechanisms include specialized epithelial cells that produce a physiochemical barrier overlying the cellular barrier, robust and organized adaptive and innate immune cells, and the ability to mount an inflammatory response that is commensurate with a specific threat level. The autophagy pathway is a critical cellular process that strongly influences all these functions. Therefore, a fundamental understanding of the components of this pathway and their influence on inflammation, immunity, and barrier function will facilitate our understanding of homeostasis in the gastrointestinal tract. PMID:23216414

  13. Cocaine induces astrocytosis through ER stress-mediated activation of autophagy.

    Science.gov (United States)

    Periyasamy, Palsamy; Guo, Ming-Lei; Buch, Shilpa

    2016-08-01

    Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation. PMID:27337297

  14. IL13 activates autophagy to regulate secretion in airway epithelial cells.

    Science.gov (United States)

    Dickinson, John D; Alevy, Yael; Malvin, Nicole P; Patel, Khushbu K; Gunsten, Sean P; Holtzman, Michael J; Stappenbeck, Thaddeus S; Brody, Steven L

    2016-01-01

    Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.

  15. Cross-cancer profiling of molecular alterations within the human autophagy interaction network.

    Science.gov (United States)

    Lebovitz, Chandra B; Robertson, A Gordon; Goya, Rodrigo; Jones, Steven J; Morin, Ryan D; Marra, Marco A; Gorski, Sharon M

    2015-01-01

    Aberrant activation or disruption of autophagy promotes tumorigenesis in various preclinical models of cancer, but whether the autophagy pathway is a target for recurrent molecular alteration in human cancer patient samples is unknown. To address this outstanding question, we surveyed 211 human autophagy-associated genes for tumor-related alterations to DNA sequence and RNA expression levels and examined their association with patient survival outcomes in multiple cancer types with sequence data from The Cancer Genome Atlas consortium. We found 3 (RB1CC1/FIP200, ULK4, WDR45/WIPI4) and one (ATG7) core autophagy genes to be under positive selection for somatic mutations in endometrial carcinoma and clear cell renal carcinoma, respectively, while 29 autophagy regulators and pathway interactors, including previously identified KEAP1, NFE2L2, and MTOR, were significantly mutated in 6 of the 11 cancer types examined. Gene expression analyses revealed that GABARAPL1 and MAP1LC3C/LC3C transcripts were less abundant in breast cancer and non-small cell lung cancers than in matched normal tissue controls; ATG4D transcripts were increased in lung squamous cell carcinoma, as were ATG16L2 transcripts in kidney cancer. Unsupervised clustering of autophagy-associated mRNA levels in tumors stratified patient overall survival in 3 of 9 cancer types (acute myeloid leukemia, clear cell renal carcinoma, and head and neck cancer). These analyses provide the first comprehensive resource of recurrently altered autophagy-associated genes in human tumors, and highlight cancer types and subtypes where perturbed autophagy may be relevant to patient overall survival.

  16. Involvement of autophagy upregulation in 3,4-methylenedioxymethamphetamine ('ecstasy')-induced serotonergic neurotoxicity.

    Science.gov (United States)

    Li, I-Hsun; Ma, Kuo-Hsing; Kao, Tzu-Jen; Lin, Yang-Yi; Weng, Shao-Ju; Yen, Ting-Yin; Chen, Lih-Chi; Huang, Yuahn-Sieh

    2016-01-01

    It has been suggested that autophagy plays pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug that causes long-term serotonergic neurotoxicity in the brain. Apoptosis and necrosis have been implicated in MDMA-induced neurotoxicity, but the role of autophagy in MDMA-elicited serotonergic toxicity has not been investigated. The present study aimed to examine the contribution of autophagy to neurotoxicity in serotonergic neurons in in vitro and in vivo animal models challenged with MDMA. Here, we demonstrated that in cultured rat serotonergic neurons, MDMA exposure induced LC3B-densely stained autophagosome formation, accompanying by a decrease in neurite outgrowth. Autophagy inhibitor 3-methyladenine (3-MA) significantly attenuated MDMA-induced autophagosome accumulation, and ameliorated MDMA-triggered serotonergic neurite damage and neuron death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in serotonergic neurons and aggravated neurite degeneration. In addition, MDMA-induced autophagy activation in cultured serotonergic neurons might be mediated by serotonin transporter (SERT). In an in vivo animal model administered MDMA, neuroimaging showed that 3-MA protected the serotonin system against MDMA-induced downregulation of SERT evaluated by animal-PET with 4-[(18)F]-ADAM, a SERT radioligand. Taken together, our results demonstrated that MDMA triggers upregulation of autophagy in serotonergic neurons, which appears to be detrimental to neuronal growth. PMID:26610922

  17. AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects.

    Science.gov (United States)

    Papp, Diána; Kovács, Tibor; Billes, Viktor; Varga, Máté; Tarnóci, Anna; Hackler, László; Puskás, László G; Liliom, Hanna; Tárnok, Krisztián; Schlett, Katalin; Borsy, Adrienn; Pádár, Zsolt; Kovács, Attila L; Hegedűs, Krisztina; Juhász, Gábor; Komlós, Marcell; Erdős, Attila; Gulyás, Balázs; Vellai, Tibor

    2016-01-01

    Autophagy is a major molecular mechanism that eliminates cellular damage in eukaryotic organisms. Basal levels of autophagy are required for maintaining cellular homeostasis and functioning. Defects in the autophagic process are implicated in the development of various age-dependent pathologies including cancer and neurodegenerative diseases, as well as in accelerated aging. Genetic activation of autophagy has been shown to retard the accumulation of damaged cytoplasmic constituents, delay the incidence of age-dependent diseases, and extend life span in genetic models. This implies that autophagy serves as a therapeutic target in treating such pathologies. Although several autophagy-inducing chemical agents have been identified, the majority of them operate upstream of the core autophagic process, thereby exerting undesired side effects. Here, we screened a small-molecule library for specific inhibitors of MTMR14, a myotubularin-related phosphatase antagonizing the formation of autophagic membrane structures, and isolated AUTEN-67 (autophagy enhancer-67) that significantly increases autophagic flux in cell lines and in vivo models. AUTEN-67 promotes longevity and protects neurons from undergoing stress-induced cell death. It also restores nesting behavior in a murine model of Alzheimer disease, without apparent side effects. Thus, AUTEN-67 is a potent drug candidate for treating autophagy-related diseases. PMID:26312549

  18. Interactions between Autophagy and Inhibitory Cytokines

    Science.gov (United States)

    Wu, Tian-tian; Li, Wei-Min; Yao, Yong-Ming

    2016-01-01

    Autophagy is a degradative pathway that plays an essential role in maintaining cellular homeostasis. Most early studies of autophagy focused on its involvement in age-associated degeneration and nutrient deprivation. However, the immunological functions of autophagy have become more widely studied in recent years. Autophagy has been shown to be an intrinsic cellular defense mechanism in the innate and adaptive immune responses. Cytokines belong to a broad and loose category of proteins and are crucial for innate and adaptive immunity. Inhibitory cytokines have evolved to permit tolerance to self while also contributing to the eradication of invading pathogens. Interactions between inhibitory cytokines and autophagy have recently been reported, revealing a novel mechanism by which autophagy controls the immune response. In this review, we discuss interactions between autophagy and the regulatory cytokines IL-10, transforming growth factor-β, and IL-27. We also mention possible interactions between two newly discovered cytokines, IL-35 and IL-37, and autophagy. PMID:27313501

  19. Autophagy and apoptosis: rivals or mates?

    Directory of Open Access Journals (Sweden)

    Yan Cheng

    2013-03-01

    Full Text Available Autophagy, a cellular process of "self-eating" by which intracellular components are degraded within the lysosome, is an evolutionarily conserved response to various stresses. Autophagy is associated with numerous patho-physiological conditions, and dysregulation of autophagy contributes to the pathogenesis of a variety of human diseases including cancer. Depending on context, activation of autophagy may promote either cell survival or death, two major events that determine pathological process of many illnesses. Importantly, the activity of autophagy is often associated with apoptosis, another critical cellular process determining cellular fate. A better understanding of biology of autophagy and its implication in human health and disorder, as well as the relationship between autophagy and apoptosis, has the potential of facilitating the development of autophagy-based therapeutic interventions for human diseases such as cancer.

  20. Autophagy and apoptosis: rivals or mates?

    Institute of Scientific and Technical Information of China (English)

    Yan Cheng; Jin-Ming Yang

    2013-01-01

    Autophagy,a cellular process of "self-eating" by which intracellular components are degraded within the lysosome,is an evolutionarily conserved response to various stresses.Autophagy is associated with numerous patho-physiological conditions,and dysregulation of autophagy contributes to the pathogenesis of a variety of human diseases including cancer.Depending on context,activation of autophagy may promote either cell survival or death,two major events that determine pathological process of many illnesses.Importantly,the activity of autophagy is often associated with apoptosis,another critical cellular process determining cellular fate.A better understanding of biology of autophagy and its implication in human health and disorder,as well as the relationship between autophagy and apoptosis,has the potential of facilitating the development of autophagy-based therapeutic interventions for human diseases such as cancer.

  1. Fluorescence microscopy: A tool to study autophagy

    Science.gov (United States)

    Rai, Shashank; Manjithaya, Ravi

    2015-08-01

    Autophagy is a cellular recycling process through which a cell degrades old and damaged cellular components such as organelles and proteins and the degradation products are reused to provide energy and building blocks. Dysfunctional autophagy is reported in several pathological situations. Hence, autophagy plays an important role in both cellular homeostasis and diseased conditions. Autophagy can be studied through various techniques including fluorescence based microscopy. With the advancements of newer technologies in fluorescence microscopy, several novel processes of autophagy have been discovered which makes it an essential tool for autophagy research. Moreover, ability to tag fluorescent proteins with sub cellular targets has enabled us to evaluate autophagy processes in real time under fluorescent microscope. In this article, we demonstrate different aspects of autophagy in two different model organisms i.e. yeast and mammalian cells, with the help of fluorescence microscopy.

  2. Interactions between Autophagy and Inhibitory Cytokines.

    Science.gov (United States)

    Wu, Tian-Tian; Li, Wei-Min; Yao, Yong-Ming

    2016-01-01

    Autophagy is a degradative pathway that plays an essential role in maintaining cellular homeostasis. Most early studies of autophagy focused on its involvement in age-associated degeneration and nutrient deprivation. However, the immunological functions of autophagy have become more widely studied in recent years. Autophagy has been shown to be an intrinsic cellular defense mechanism in the innate and adaptive immune responses. Cytokines belong to a broad and loose category of proteins and are crucial for innate and adaptive immunity. Inhibitory cytokines have evolved to permit tolerance to self while also contributing to the eradication of invading pathogens. Interactions between inhibitory cytokines and autophagy have recently been reported, revealing a novel mechanism by which autophagy controls the immune response. In this review, we discuss interactions between autophagy and the regulatory cytokines IL-10, transforming growth factor-β, and IL-27. We also mention possible interactions between two newly discovered cytokines, IL-35 and IL-37, and autophagy.

  3. STAT3-Mediated Autophagy Dependence Identifies Subtypes of Breast Cancer where Autophagy Inhibition can be Efficacious

    OpenAIRE

    Maycotte, Paola; Gearheart, Christy M.; Barnard, Rebecca; Aryal, Suraj; Mulcahy Levy, Jean M.; Fosmire, Susan P.; Hansen, Ryan J.; Morgan, Michael J.; Christopher C Porter; Gustafson, Daniel L.; Thorburn, Andrew

    2014-01-01

    Autophagy is a protein and organelle degradation pathway that is involved in diverse diseases including cancer. Recent evidence suggests that autophagy is a cell survival mechanism in tumor cells and that its inhibition especially in combination with other therapy could be beneficial but it remains unclear if all cancer cells behave the same way when autophagy is inhibited. We inhibited autophagy in a panel of breast cancer cell lines and found that some of them are dependent on autophagy for...

  4. Autophagy and mitophagy in cellular damage control

    Directory of Open Access Journals (Sweden)

    Jianhua Zhang

    2013-01-01

    Full Text Available Autophagy and mitophagy are important cellular processes that are responsible for breaking down cellular contents, preserving energy and safeguarding against accumulation of damaged and aggregated biomolecules. This graphic review gives a broad summary of autophagy and discusses examples where autophagy is important in controlling protein degradation. In addition we highlight how autophagy and mitophagy are involved in the cellular responses to reactive species and mitochondrial dysfunction. The key signaling pathways for mitophagy are described in the context of bioenergetic dysfunction.

  5. Matrine-induced autophagy regulated by p53 through AMP-activated protein kinase in human hepatoma cells.

    Science.gov (United States)

    Xie, Shan-Bu; He, Xing-Xing; Yao, Shu-Kun

    2015-08-01

    Matrine, one of the main extract components of Sophora flavescens, has been shown to exhibit inhibitory effects on some tumors through autophagy. However, the mechanism underlying the effect of matrine remains unclear. The cultured human hepatocellular carcinoma cell line HepG2 and SMMC‑7721 were treated with matrine. Signal transduction and gene expression profile were determined. Matrine stimulated autophagy in SMMC‑7721 cells in a mammalian target of rapamycin (mTOR)-dependent manner, but in an mTOR-independent manner in HepG2 cells. Next, in HepG2 cells, autophagy induced by matrine was regulated by p53 inactivation through AMP-activated protein kinase (AMPK) signaling transduction, then AMPK suppression switched autophagy to apoptosis. Furthermore, the interferon (IFN)-inducible genes, including interferon α-inducible protein 27 (IFI27) and interferon induced transmembrane protein 1 (IFITM1), which are downstream effector of p53, might be modulated by matrine-induced autophagy. In addition, we found that the p53 protein isoforms, p53β, p53γ, ∆133p53, and ∆133p53γ, due to alternative splicing of intron 9, might be regulated by the p53-mediated autophagy. These results show that matrine induces autophagy in human hepatoma cells through a novel mechanism, which is p53/AMPK signaling pathway involvement in matrine-promoted autophagy.

  6. Candida albicans autophagy, no longer a bystander: Its role in tolerance to ER stress-related antifungal drugs.

    Science.gov (United States)

    Yu, Qilin; Jia, Chang; Dong, Yijie; Zhang, Bing; Xiao, Chenpeng; Chen, Yulu; Wang, Yuzhou; Li, Xiaoling; Wang, Lei; Zhang, Biao; Li, Mingchun

    2015-08-01

    Autophagy is a degradation process involved in pathogenicity of many pathogenic fungi. However, its roles in Candida albicans, the leading fungal pathogen in human beings, remain to be detailed. Most recently, we found that endoplasmic reticulum (ER) stress-inducing conditions led to transcriptional up-regulation of C. albicans autophagy-related (ATG) genes, implying a possible link between autophagy and ER stress response in this pathogen. Using a series of C. albicans ATG mutants and autophagy reporting systems, we found that both treatment of ER stress-related drugs and loss of the ER calcium pump Spf1 promoted autophagic flux of Atg8 and Lap41 (a homologue of Saccharomyces cerevisiae Ape1), indicating that these conditions induce autophagy. Moreover, deletion of ATG genes in the spf1Δ/Δ mutant rendered cells hypersensitive to these drugs and caused activation of UPR, revealing a role of autophagy in alleviating ER stress. In addition, only treatment of tunicamycin and loss of Spf1 in combination increased autophagic flux of the ER component Sec63, suggesting that most of the ER stress-related conditions cause non-selective autophagy rather than selective ER phagy. This study uncovers the important role of C. albicans autophagy in ER stress response and tolerance to antifungal drugs.

  7. A curated census of autophagy-modulating proteins and small molecules: candidate targets for cancer therapy.

    Science.gov (United States)

    Lorenzi, Philip L; Claerhout, Sofie; Mills, Gordon B; Weinstein, John N

    2014-07-01

    Autophagy, a programmed process in which cell contents are delivered to lysosomes for degradation, appears to have both tumor-suppressive and tumor-promoting functions; both stimulation and inhibition of autophagy have been reported to induce cancer cell death, and particular genes and proteins have been associated both positively and negatively with autophagy. To provide a basis for incisive analysis of those complexities and ambiguities and to guide development of new autophagy-targeted treatments for cancer, we have compiled a comprehensive, curated inventory of autophagy modulators by integrating information from published siRNA screens, multiple pathway analysis algorithms, and extensive, manually curated text-mining of the literature. The resulting inventory includes 739 proteins and 385 chemicals (including drugs, small molecules, and metabolites). Because autophagy is still at an early stage of investigation, we provide extensive analysis of our sources of information and their complex relationships with each other. We conclude with a discussion of novel strategies that could potentially be used to target autophagy for cancer therapy.

  8. Critical role for IL-18 in spontaneous lung inflammation caused by autophagy deficiency.

    Science.gov (United States)

    Abdel Fattah, Elmoataz; Bhattacharya, Abhisek; Herron, Alan; Safdar, Zeenat; Eissa, N Tony

    2015-06-01

    Autophagy is an important component of the immune response. However, the functions of autophagy in human diseases are much less understood. We studied biological consequences of autophagy deficiency in mice lacking the essential autophagy gene Atg7 or Atg5 in myeloid cells. Surprisingly, these mice presented with spontaneous sterile lung inflammation, characterized by marked recruitment of inflammatory cells, submucosal thickening, goblet cell metaplasia, and increased collagen content. Lung inflammation was associated with increase in several proinflammatory cytokines in the bronchoalveolar lavage and in serum. This inflammation was largely driven by IL-18 as a result of constitutive inflammasome activation. Following i.p. LPS injection, autophagy-deficient mice had higher levels of proinflammatory cytokines in lungs and in serum, as well as increased mortality, than control mice. Intranasal bleomycin challenge exacerbated lung inflammation in autophagy-deficient mice and produced more severe fibrotic changes than in control mice. These results uncover a new and important role for autophagy as negative regulator of lung inflammation.

  9. Hepatic autophagy contributes to the metabolic response to dietary protein restriction.

    Science.gov (United States)

    Henagan, Tara M; Laeger, Thomas; Navard, Alexandra M; Albarado, Diana; Noland, Robert C; Stadler, Krisztian; Elks, Carrie M; Burk, David; Morrison, Christopher D

    2016-06-01

    Autophagy is an essential cellular response which acts to release stored cellular substrates during nutrient restriction, and particularly plays a key role in the cellular response to amino acid restriction. However, there has been limited work testing whether the induction of autophagy is required for adaptive metabolic responses to dietary protein restriction in the whole animal. Here, we found that moderate dietary protein restriction led to a series of metabolic changes in rats, including increases in food intake and energy expenditure, the downregulation of hepatic fatty acid synthesis gene expression and reduced markers of hepatic mitochondrial number. Importantly, these effects were also associated with an induction of hepatic autophagy. To determine if the induction of autophagy contributes to these metabolic effects, we tested the metabolic response to dietary protein restriction in BCL2-AAA mice, which bear a genetic mutation that impairs autophagy induction. Interestingly, BCL2-AAA mice exhibit exaggerated responses in terms of both food intake and energy expenditure, whereas the effects of protein restriction on hepatic metabolism were significantly blunted. These data demonstrate that restriction of dietary protein is sufficient to trigger hepatic autophagy, and that disruption of autophagy significantly alters both hepatic and whole animal metabolic response to dietary protein restriction. PMID:27173459

  10. Calpain2调节自噬相关基因ATG7的表达在非酒精性脂肪性肝病中的作用%Role of regulation of autophagy related gene 7 by Calpain 2 in non-alcoholic fatty liver disease in rats

    Institute of Scientific and Technical Information of China (English)

    陈洁; 熊吉; 陈潇迪; 牟歌; 王军; 樊丽琳; 陈东风

    2011-01-01

    Objective To investigate the expression and significance of autophagy related gene 7 ( ATG7 ) and Calpain 2 in nonalcoholic fatty liver disease ( NAFLD ). Methods In vivo model of NAFLD was established in SD rats by high fat diet, while the rats fed with normal food were set as control group. The rats were killed at 4, 8, 12 and 16 weeks after feeding. Blood samples were collected to check serum aspartate transaminase (AST) , alanine aminotransferase (ALT) , and free fatty acid(FFA). Steatosis of liver tissues were observed by HE staining. The expression of Calpain 2 and ATG7 was detected by real-time PCR and Western blotting respectively for mRNA and protein levels. Results HE staining implicated that the degree of hepatic steatosis was increased with the time of high fat diet feeding. Compared with the control group, the serum contents of ALT, AST, and FFA in NAFLD rats were increased with different degree, and significantly increased at 16th week (165.95 ±7. 24 U/L, 249. 52 ±4. 20 U/L, 0. 83 ±0. 05 mmol/L, respectively P < · 0. 01). The relative expression of Calpain 2 at mRNA level was increased after high fat diet feeding and reached its peak at the 16th week, and was 9. 83 ±0. 85 fold higher as compared with the control group (P <0. 01). While the relative expression of ATG7 began to decrease at the 4th week (0. 82 ±0. 02) , and reached its lowest level at the 16th week (0. 20 ±0. 03, P <0. 01) when compared with the control group. As with mRNA level, the protein level of Calpain 2 began to increase at the 4th week (2. 32 ± 0.45 ) , and was 9. 87 ± 1. 20 fold higher as the control group (P <0. 01). While the expression of ATG7 at protein level was decreased with progress of steatosis, and was significantly reduced at 16th week (0.18 ±0.05, P<0.01). Conclusion The up-regulation of Calpain 2 inhibits the expression of ATG7, which further attenuates the cell protection through autophagy, and then induces injury of hepatocyte in NAFLD. Autophagy may

  11. Protection of Mice from Lethal Endotoxemia by Chimeric Human BPI-Fcγ1 Gene Delivery

    Institute of Scientific and Technical Information of China (English)

    Chen Li; Jing Li; Zhe Lv; Xinghua Guo; Qinghua Chen; Qingli Kong; Yunqing An

    2006-01-01

    To evaluate the potentiality of applying gene therapy to endotoxemia in high-risk patients, we investigated the effects of transferring an adeno-associated virus serotype 2 (AAV2)-mediated BPI-Fcγ1 gene on protecting mice from challenge of lethal endotoxin. The chimeric BPI-Fcγ1 gene consists of two parts, one encods functional N-terminus (1 to 199 amino acidic residues) of human BPI, which is a bactericidal/permeability-increasing protein,and the other encodes Fc segment of human immunoglobulin G1 (Fcγ1). Our results indicated that the target protein could be expressed and secreted into the serum of the gene-transferred mice. After lethal endotoxin challenge, the levels of endotoxin and TNF-α in the gene-transferred mice were decreased. The survival rate of the BPI-Fcγ1 gene-transferred mice was markedly increased. Our data suggest that AAV2-mediated chimeric BPI-Fcγ1 gene delivery can potentially be used clinically for the protection and treatment of endotoxemia and endotoxic shock in high-risk individuals.

  12. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

    OpenAIRE

    Godar, Rebecca J.; Ma, Xiucui; Liu, Haiyan; Murphy, John T.; Carla J Weinheimer; Kovacs, Attila; Seth D Crosby; Saftig, Paul; Diwan, Abhinav

    2015-01-01

    Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fe...

  13. p53: The Janus of autophagy?

    OpenAIRE

    Levine, Beth; Abrams, John

    2008-01-01

    The autophagy pathway functions in adaptation to nutrient stress and tumour suppression. The p53 tumour suppressor, previously thought to positively regulate autophagy, may also inhibit it. This dual interplay between p53 and autophagy regulation is enigmatic, but may underlie key aspects of metabolism and cancer biology.

  14. Protective Effect of Gwakhyangjeonggisan Herbal Acupuncture Solution in Glioblastoma Cells: Microarray Analysis of Gene Expression

    Directory of Open Access Journals (Sweden)

    Hong-Seok Lee

    2005-12-01

    Full Text Available Objectives : Neurological disorders have been one of main therapeutic targets of acupuncture. The present study investigated the protective effects of Gwakhyangjeonggisan herbal acupuncture solution (GHAS. Methods : We performed 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay in glioblastoma cells, and did microarray analysis with cells exposed to reactive oxigen species (ROS of hydrogen peroxide by 8.0 k Human cDNA, with cut-off level of 2-fold changes in gene expression. Results : MTT assay showed protective effect of GHAS on the glioblastoma cells exposed to hydrogen peroxide. When glioblastoma cells were exposed to hydrogen peroxide, 24 genes were downregulated. When the cells were pretreated with GHAS before exposure to hydrogen peroxide, 46 genes were downregulated. Many of the genes downregulated by hydrogen peroxide stimulation were decreased in the amount of downregulation or reversed to upregulation. Conclusions : The gene expression changes observed in the present study are supposed to be related to the protective molecular mechanism of GHAS in the glioblastoma cells exposed to ROS stress.

  15. Autophagy, a Conserved Mechanism for Protein Degradation, Responds to Heat, and Other Abiotic Stresses in Capsicum annuum L.

    Science.gov (United States)

    Zhai, Yufei; Guo, Meng; Wang, Hu; Lu, Jinping; Liu, Jinhong; Zhang, Chong; Gong, Zhenhui; Lu, Minghui

    2016-01-01

    Abiotic stresses negatively affect plants growth and development by inducing protein denaturation, and autophagy degrades the damaged proteins to alleviate their toxicity, however, little is known about the involvement of autophagy in pepper (Capsicum annuum L.) tolerances to abiotic stresses. In this study, we identified autophagy-related gene (ATG) members in the whole genome of pepper by HMM method and analyzed their expression profiles in response to heat and other abiotic stresses by quantitative real-time PCR. The results showed that the CaATG contained 15 core ATG members including 29 ATG proteins with their respective conserved functional domains, involving the whole process of autophagy. Under normal environmental condition, the expression of CaATG genes showed tissue- and developmental stage-specific patterns, while under abiotic stresses of salt, drought, heat, cold and carbohydrate starvation, the accumulation of autophagosome punctate increased and the expression level of CaATG genes changed with stress type-dependent pattern, which indicates the linkage of autophagy in pepper response to abiotic stresses. After treated with heat stress, both the number of up-regulated CaATG genes and the increment of autophagosome punctate were higher in pepper thermotolerant line R9 than those in thermosensitive line B6, implying an association of autophagy with heat tolerance. In addition, CaATG6 was predicted to interact with CaHSP90 family members. Our study suggests that autophagy is connected to pepper tolerances to heat and other abiotic stresses. PMID:26904087

  16. Autophagy, a conserved mechanism for protein degradation, responds to heat and other abiotic stresses in Capsicum annuum L.

    Directory of Open Access Journals (Sweden)

    Yufei eZhai

    2016-02-01

    Full Text Available Abiotic stresses negatively affect plants growth and development by inducing protein denaturation, and autophagy degrades the damaged proteins to alleviate their toxicity, however, little is known about the involvement of autophagy in pepper (Capsicum annuum L. tolerances to abiotic stresses. In this study, we identified autophagy-related gene (ATG members in the whole genome of pepper by HMM method and analyzed their expression profiles in response to heat and other abiotic stresses by quantitative real-time PCR. The results showed that the CaATG contained 15 core ATG members including 29 ATG proteins with their respective conserved functional domains, involving the whole process of autophagy. Under normal environmental condition, the expression of CaATG genes showed tissue- and developmental stage-specific patterns, while under abiotic stresses of salt, drought, heat, cold and carbohydrate starvation, the accumulation of autophagosome punctate increased and the expression level of CaATG genes changed with stress type-dependent pattern, which indicates the linkage of autophagy in pepper response to abiotic stresses. After treated with heat stress, both the number of up-regulated CaATG genes and the increment of autophagosome punctate were higher in pepper thermotolerant line R9 than those in thermosensitive line B6, implying an association of autophagy with heat tolerance. In addition, CaATG6 was predicted to interact with CaHSP90 family members. Our study suggests that autophagy is connected to pepper tolerances to heat and other abiotic stresses.

  17. Nicotinate-Curcumin Impedes Foam Cell Formation from THP-1 Cells through Restoring Autophagy Flux.

    Science.gov (United States)

    Gu, Hong-Feng; Li, Hai-Zhe; Tang, Ya-Ling; Tang, Xiao-Qing; Zheng, Xi-Long; Liao, Duan-Fang

    2016-01-01

    Our previous studies have indicated that a novel curcumin derivate nicotinate-curcumin (NC) has beneficial effects on the prevention of atherosclerosis, but the precise mechanisms are not fully understood. Given that autophagy regulates lipid metabolism, the present study was designed to investigate whether NC decreases foam cell formation through restoring autophagy flux in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 cells. Our results showed that ox-LDL (100 μg/ml) was accumulated in THP-1 cells and impaired autophagy flux. Ox-LDL-induced impairment of autophagy was enhanced by treatment with the autophagy inhibitor chloroquine (CQ) and rescued by the autophagy inducer rapamycin. The aggregation of ox-LDL was increased by CQ, but decreased by rapamycin. In addition, colocalization of lipid droplets with LC3-II was remarkably reduced in ox-LDL group. In contrast, NC (10 μM) rescued the impaired autophagy flux by significantly increasing level of LC3-II, the number of autophagolysosomes, and the degradation of p62 in ox-LDL-treated THP-1 cells. Inhibition of the PI3K-Akt-mTOR signaling was required for NC-rescued autophagy flux. Notably, our results showed that NC remarkably promoted the colocalization of lipid droplets with autophagolysosomes, increased efflux of cholesterol, and reduced ox-LDL accumulation in THP-1 cells. However, treatment with 3-methyladenine (3-MA) or CQ reduced the protective effects of NC on lipid accumulation. Collectively, the findings suggest that NC decreases lipid accumulation in THP-1 cells through restoring autophagy flux, and further implicate that NC may be a potential therapeutic reagent to reverse atherosclerosis.

  18. Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Xuchen Cao; Bowen Liu; Wenfeng Cao; Weiran Zhang; Fei Zhang; Hongmeng Zhao; Ran Meng

    2013-01-01

    Apigenin (4',5,7-trihydroxyflavone) is a member of the flavone subclass of flavonoids present in fruits and vegetables.The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated.Cell proliferation and viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays.Flow cytometry,fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy,and the role of autophagy was assessed using autophagy inhibitors.Apigenin dose-and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines.The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3,PARP cleavage and Bax/Bcl-2 ratios.The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis.In addition,the apigenin-treated cells exhibited autophagy,as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles (AVOs)by flow cytometry.Furthermore,the results of the Western blot analysis revealed that the level of LC3-Ⅱ,the processed form of LC3-Ⅰ,was increased.Treatment with the autophagy inhibitor,3-methyladenine (3-MA),significantly enhanced the apoptosis induced by apigenin,which was accompanied by an increase in the level of PARP cleavage.Similar results were also confirmed by flow cytometry and fluorescence microscopy.These results indicate that apigenin has apoptosis-and autophagy-inducing effects in breast cancer cells.Autophagy plays a cyto-protective role in apigenin-induced apoptosis,and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control.

  19. Nicotinate-Curcumin Impedes Foam Cell Formation from THP-1 Cells through Restoring Autophagy Flux.

    Directory of Open Access Journals (Sweden)

    Hong-Feng Gu

    Full Text Available Our previous studies have indicated that a novel curcumin derivate nicotinate-curcumin (NC has beneficial effects on the prevention of atherosclerosis, but the precise mechanisms are not fully understood. Given that autophagy regulates lipid metabolism, the present study was designed to investigate whether NC decreases foam cell formation through restoring autophagy flux in oxidized low-density lipoprotein (ox-LDL-treated THP-1 cells. Our results showed that ox-LDL (100 μg/ml was accumulated in THP-1 cells and impaired autophagy flux. Ox-LDL-induced impairment of autophagy was enhanced by treatment with the autophagy inhibitor chloroquine (CQ and rescued by the autophagy inducer rapamycin. The aggregation of ox-LDL was increased by CQ, but decreased by rapamycin. In addition, colocalization of lipid droplets with LC3-II was remarkably reduced in ox-LDL group. In contrast, NC (10 μM rescued the impaired autophagy flux by significantly increasing level of LC3-II, the number of autophagolysosomes, and the degradation of p62 in ox-LDL-treated THP-1 cells. Inhibition of the PI3K-Akt-mTOR signaling was required for NC-rescued autophagy flux. Notably, our results showed that NC remarkably promoted the colocalization of lipid droplets with autophagolysosomes, increased efflux of cholesterol, and reduced ox-LDL accumulation in THP-1 cells. However, treatment with 3-methyladenine (3-MA or CQ reduced the protective effects of NC on lipid accumulation. Collectively, the findings suggest that NC decreases lipid accumulation in THP-1 cells through restoring autophagy flux, and further implicate that NC may be a potential therapeutic reagent to reverse atherosclerosis.

  20. Effect of autophagy induced by dexamethasone on senescence in chondrocytes

    Science.gov (United States)

    Xue, Enxing; Zhang, Yu; Song, Bing; Xiao, Jun; Shi, Zhanjun

    2016-01-01

    The aim of the current study was to explore the effects of dexamethasone (DXM) on autophagy and senescence in chondrocytes. Collagen II and aggrecan were examined in normal chondrocytes isolated from Sprague-Dawley rats. Following stimulation with DXM, LysoTracker Red staining, monodansylcadaverine (MDC) staining, green fluorescent protein-red fluorescent protein-light chain 3 (LC3) and western blotting were used to detect autophagy levels in the chondrocytes. Mechanistic target of rapamycin (mTOR) pathway-associated molecules were investigated by western blotting. Cell senescence was analyzed by senescence-associated (SA)-β-galactosidase (β-gal) staining. A dose-dependent increase in the number of autophagic vacuoles was observed in the DXM-treated chondrocytes, as demonstrated by LysoTracker Red and MDC staining. A dose-dependent increase in autophagosome formation was observed in the DXM-treated chondrocytes. Expression of LC3-II and beclin-1 was increased by DXM, in particular in the cells treated with DXM for 4 days. However, P62 expression was reduced as a result of treatment. SA-β-gal staining indicated that DXM increased cell senescence. Notably, DXM-induced cell senescence was exacerbated by the autophagic inhibitor 3-MA. Autophagy induced by DXM protected chondrocytes from senescence, and it is suggested that the mTOR pathway may be involved in the activation of DXM-induced autophagy. PMID:27572674

  1. Activation of autophagy in photoreceptor necroptosis after experimental retinal detachment

    Institute of Scientific and Technical Information of China (English)

    Kai; Dong; Zi-Cheng; Zhu; Feng-Hua; Wang; Gen-Jie; Ke; Zhang; Yu; Xun; Xu

    2014-01-01

    AIM:To investigate whether photoreceptor necroptosis induced by z-VAD-FMK(pan caspase inhibitor) was involved the activation of autophagy and whether Necrostatin-1, a specific necroptosis inhibitor, could inhibit this induction of autophagy after experimental retinal detachment.METHODS:Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate and subretinal injections of z-VAD-FMK, vehicle or z-VAD-FMK plus Necrostatin-1.Three days after retinal detachment, morphologic changes were observed by transmission electron microscopy. In other animals, retinas were subjected to immunoprecipitation and Western Blotting, then probed with anti-RIP1, phosphoserine, LC-3II or caspase 8antibody.RESULTS:It was proved by immunoprecipitation and western blotting, that photoreceptor necroptosis was mediated by caspase-8 inhibition and receptor interacting protein kinase(RIP1) phosphorylation activation. Transmission electron microscope and western blotting results indicated that photoreceptornecroptosis was involved the LC-3II and autophagosomes induction. We also discovered Necrostatin-1 could inhibit RIP1 phosphorylation and LC-3II induction.CONCLUSION:These data firstly indicate photoreceptor necroptosis is associated with the activation of autophagy. Necrostatin-1 protects photoreceptors from necroptosis and autophagy by down-regulation of RIP1 phosphorylation and LC-3II.

  2. Chaperone-mediated autophagy: roles in neuroprotection.

    Science.gov (United States)

    Cai, Zhibiao; Zeng, Weijun; Tao, Kai; E, Zhen; Wang, Bao; Yang, Qian

    2015-08-01

    Chaperone-mediated autophagy (CMA), one of the main pathways of lysosomal proteolysis, is characterized by the selective targeting and direct translocation into the lysosomal lumen of substrate proteins containing a targeting motif biochemically related to the pentapeptide KFERQ. Along with the other two lysosomal pathways, macro- and micro-autophagy, CMA is essential for maintaining cellular homeostasis and survival by selectively degrading misfolded, oxidized, or damaged cytosolic proteins. CMA plays an important role in pathologies such as cancer, kidney disorders, and neurodegenerative diseases. Neurons are post-mitotic and highly susceptible to dysfunction of cellular quality-control systems. Maintaining a balance between protein synthesis and degradation is critical for neuronal functions and homeostasis. Recent studies have revealed several new mechanisms by which CMA protects neurons through regulating factors critical for their viability and homeostasis. In the current review, we summarize recent advances in the understanding of the regulation and physiology of CMA with a specific focus on its possible roles in neuroprotection. PMID:26206599

  3. ER stress, autophagy, and RNA viruses

    Directory of Open Access Journals (Sweden)

    Jia-Rong eJheng

    2014-08-01

    Full Text Available Endoplasmic reticulum (ER stress is a general term for representing the pathway by which various stimuli affect ER functions. ER stress induces the evolutionarily conserved signaling pathways, called the unfolded protein response (UPR, which compromises the stimulus and then determines whether the cell survives or dies. In recent years, ongoing research has suggested that these pathways may be linked to the autophagic response, which plays a key role in the cell’s response to various stressors. Autophagy performs a self-digestion function, and its activation protects cells against certain pathogens. However, the link between the UPR and autophagy may be more complicated. These two systems may act dependently, or the induction of one system may interfere with the other. Experimental studies have found that different viruses modulate these mechanisms to allow them to escape the host immune response or, worse, to exploit the host’s defense to their advantage; thus, this topic is a critical area in antiviral research. In this review, we summarize the current knowledge about how RNA viruses, including influenza virus, poliovirus, coxsackievirus, enterovirus 71, Japanese encephalitis virus, hepatitis C virus, and dengue virus, regulate these processes. We also discuss recent discoveries and how these will produce novel strategies for antiviral treatment.

  4. Precision autophagy: Will the next wave of selective autophagy markers and specific autophagy inhibitors feed clinical pipelines?

    Science.gov (United States)

    Lebovitz, Chandra B; DeVorkin, Lindsay; Bosc, Damien; Rothe, Katharina; Singh, Jagbir; Bally, Marcel; Jiang, Xiaoyan; Young, Robert N; Lum, Julian J; Gorski, Sharon M

    2015-01-01

    Research presented at the Vancouver Autophagy Symposium (VAS) 2014 suggests that autophagy's influence on health and disease depends on tight regulation and precision targeting of substrates. Discussions recognized a pressing need for robust biomarkers that accurately assess the clinical utility of modulating autophagy in disease contexts. Biomarker discovery could flow from investigations of context-dependent triggers, sensors, and adaptors that tailor the autophagy machinery to achieve target specificity. In his keynote address, Dr. Vojo Deretic (University of New Mexico) described the discovery of a cargo receptor family that utilizes peptide motif-based cargo recognition, a mechanism that may be more precise than generic substrate tagging. The keynote by Dr. Alec Kimmelman (Harvard Medical School) emphasized that unbiased screens for novel selective autophagy factors may accelerate the development of autophagy-based therapies. Using a quantitative proteomics screen for de novo identification of autophagosome substrates in pancreatic cancer, Kimmelman's group discovered a new type of selective autophagy that regulates bioavailable iron. Additional presentations revealed novel autophagy regulators and receptors in metabolic diseases, proteinopathies, and cancer, and outlined the development of specific autophagy inhibitors and treatment regimens that combine autophagy modulation with anticancer therapies. VAS 2014 stimulated interdisciplinary discussions focused on the development of biomarkers, drugs, and preclinical models to facilitate clinical translation of key autophagy discoveries.

  5. Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression.

    Science.gov (United States)

    Leventhal, Jeremy S; Ni, Jie; Osmond, Morgan; Lee, Kyung; Gusella, G Luca; Salem, Fadi; Ross, Michael J

    2016-01-01

    Sepsis related acute kidney injury (AKI) is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC) from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS), a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO). Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3) and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling.

  6. Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression.

    Directory of Open Access Journals (Sweden)

    Jeremy S Leventhal

    Full Text Available Sepsis related acute kidney injury (AKI is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS, a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO. Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3 and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling.

  7. Autophagy in 5-Fluorouracil Therapy in Gastrointestinal Cancer: Trends and Challenges

    Institute of Scientific and Technical Information of China (English)

    Jia-Cheng Tang; Yi-Li Feng; Xiao Liang; Xiu-Jun Cai

    2016-01-01

    Objective: 5-Fluorouracil (5-FU)-based combination therapies are standard treatments for gastrointestinal cancer, where the modulation of autophagy is becoming increasingly important in offering effective treatment for patients in clinical practice.This review focuses on the role of autophagy in 5-FU-induced tumor suppression and cancer therapy in the digestive system.Data Sources: All articles published in English from 1996 to date those assess the synergistic effect ofautophagy and 5-FU in gastrointestinal cancer therapy were identified through a systematic online search by use of PubMed.The search terms were "autophagy" and "5-FU" and ("colorectal cancer" or"hepatocellular carcinoma" or"pancreatic adenocarcinoma" or"esophageal cancer" or"gallbladder carcinoma" or "gastric cancer").Study Selection: Critical reviews on relevant aspects and original articles reporting in vitro and/or in vivo results regarding the efficiency ofautophagy and 5-FU in gastrointestinal cancer therapy were reviewed, analyzed, and summarized.The exclusion criteria for the articles were as follows: (1) new materials (e.g., nanomaterial)-induced autophagy;(2) clinical and experimental studies on diagnostic and/or prognostic biomarkers in digestive system cancers;and (3) immunogenic cell death for anticancer chemotherapy.Results: Most cell and animal experiments showed inhibition ofautophagy by either pharmacological approaches or via genetic silencing of autophagy regulatory gene, resulting in a promotion of 5-FU-induced cancer cells death.Meanwhile, autophagy also plays a pro-death role and may mediate cell death in certain cancer cells where apoptosis is defective or difficult to induce.The dual role of autophagy complicates the use of autophagy inhibitor or inducer in cancer chemotherapy and generates inconsistency to an extent in clinic trials.Conclusion: Autophagy might be a therapeutic target that sensitizes the 5-FU treatment in gastrointestinal cancer.

  8. p53 status determines the role of autophagy in pancreatic tumour development

    Science.gov (United States)

    Rosenfeldt, Mathias T.; O'Prey, Jim; Morton, Jennifer P.; Nixon, Colin; Mackay, Gillian; Mrowinska, Agata; Au, Amy; Rai, Taranjit Singh; Zheng, Liang; Ridgway, Rachel; Adams, Peter D.; Anderson, Kurt I.; Gottlieb, Eyal; Sansom, Owen J.; Ryan, Kevin M.

    2013-12-01

    Macroautophagy (hereafter referred to as autophagy) is a process in which organelles termed autophagosomes deliver cytoplasmic constituents to lysosomes for degradation. Autophagy has a major role in cellular homeostasis and has been implicated in various forms of human disease. The role of autophagy in cancer seems to be complex, with reports indicating both pro-tumorigenic and tumour-suppressive roles. Here we show, in a humanized genetically-modified mouse model of pancreatic ductal adenocarcinoma (PDAC), that autophagy's role in tumour development is intrinsically connected to the status of the tumour suppressor p53. Mice with pancreases containing an activated oncogenic allele of Kras (also called Ki-Ras)--the most common mutational event in PDAC--develop a small number of pre-cancerous lesions that stochastically develop into PDAC over time. However, mice also lacking the essential autophagy genes Atg5 or Atg7 accumulate low-grade, pre-malignant pancreatic intraepithelial neoplasia lesions, but progression to high-grade pancreatic intraepithelial neoplasias and PDAC is blocked. In marked contrast, in mice containing oncogenic Kras and lacking p53, loss of autophagy no longer blocks tumour progression, but actually accelerates tumour onset, with metabolic analysis revealing enhanced glucose uptake and enrichment of anabolic pathways, which can fuel tumour growth. These findings provide considerable insight into the role of autophagy in cancer and have important implications for autophagy inhibition in cancer therapy. In this regard, we also show that treatment of mice with the autophagy inhibitor hydroxychloroquine, which is currently being used in several clinical trials, significantly accelerates tumour formation in mice containing oncogenic Kras but lacking p53.

  9. Study on the Mechanism of mTOR-Mediated Autophagy during Electroacupuncture Pretreatment against Cerebral Ischemic Injury

    Science.gov (United States)

    Wu, Zhou-Quan; Cui, Su-yang; Zhu, Liang

    2016-01-01

    This study is aimed at investigating the association between the electroacupuncture (EA) pretreatment-induced protective effect against early cerebral ischemic injury and autophagy. EA pretreatment can protect cerebral ischemic and reperfusion injuries, but whether the attenuation of early cerebral ischemic injury by EA pretreatment was associated with autophagy is not yet clear. This study used the middle cerebral artery occlusion model to monitor the process of ischemic injury. For rats in the EA pretreatment group, EA pretreatment was conducted at Baihui acupoint before ischemia for 30 min for 5 consecutive days. The results suggested that EA pretreatment significantly increased the expression of autophagy in the cerebral cortical area on the ischemic side of rats. But the EA pretreatment-induced protective effects on the brain could be reversed by the specific inhibitor 3-methyladenine of autophagy. Additionally, the Pearson correlation analysis indicated that the impact of EA pretreatment on p-mTOR (2481) was negatively correlated with its impact on autophagy. In conclusion, the mechanism of EA pretreatment at Baihui acupoint against cerebral ischemic injury is mainly associated with the upregulation of autophagy expression, and its regulation of autophagy may depend on mTOR-mediated signaling pathways. PMID:27547233

  10. Host-induced silencing of Fusarium culmorum genes protects wheat from infection.

    Science.gov (United States)

    Chen, Wanxin; Kastner, Christine; Nowara, Daniela; Oliveira-Garcia, Ely; Rutten, Twan; Zhao, Yusheng; Deising, Holger B; Kumlehn, Jochen; Schweizer, Patrick

    2016-09-01

    Plants producing antisense or double-stranded RNA molecules that target specific genes of eukaryotic pests or pathogens can become protected from their attack. This beneficial effect was also reported for plant-fungus interactions and is believed to reflect uptake of the RNAs by the fungus via an as yet unknown mechanism, followed by target gene silencing. Here we report that wheat plants pre-infected with Barley stripe mosaic virus (BSMV) strains containing antisense sequences against target genes of the Fusarium head blight (FHB) fungus F. culmorum caused a reduction of corresponding transcript levels in the pathogen and reduced disease symptoms. Stable transgenic wheat plants carrying an RNAi hairpin construct against the β-1, 3-glucan synthase gene FcGls1 of F. culmorum or a triple combination of FcGls1 with two additional, pre-tested target genes also showed enhanced FHB resistance in leaf and spike inoculation assays under greenhouse and near-field conditions, respectively. Microscopic evaluation of F. culmorum development in plants transiently or stably expressing FcGls1 silencing constructs revealed aberrant, swollen fungal hyphae, indicating severe hyphal cell wall defects. The results lead us to propose host-induced gene silencing (HIGS) as a plant protection approach that may also be applicable to highly FHB-susceptible wheat genotypes. PMID:27540093

  11. Establishing a novel C.elegans model to investigate the role of autophagy in amyotrophic lateral sclerosis

    Institute of Scientific and Technical Information of China (English)

    Jia LI; Kai-xing HUANG; Wei-dong LE

    2013-01-01

    Aim:To develop a C.elegans model of amyotrophic lateral sclerosis (ALS) and to evaluate the role of autophagy in the disease.Methods:Stable transgenic worms expressing the G93A mutant form of Cu,Zn-superoxide dismutase (SOD1) in GABAergic motor neurons were generated.Axon guidance and protein aggregation in the motor neurons were observed with fluorescence microscopy.A paralysis assay was performed to evaluate the motor function of the transgenic worms.The expression of autophagic genes in daf2(e1370) mutants was analyzed using real-time PCR.The reporter GFP::LGG-1 was used to verify whether autophagy was induced in motor neurons.Results:Expression of G93A SOD1 in motor neurons caused age-dependent motor defects accompanied by significant SOD1 aggregation and axon guidance failure.After 12 d,over 80% of the G93A worms became paralyzed,whereas less than 10% of the controls showed a paralytic phenotype.In the daf-2(e1370) mutants of C.elegans,the levels of autophagic genes bec-1,atg-7,Igg-1,and atg-18 were upregulated by approximately 1.5-fold,the level of unc-51 increased by approximately fourfold,and autophagosomes in motor neurons was markedly increased.Crossing the daf-2(e1370) mutation into the G93A SOD1 mutant worms significantly ameliorated the motor defects,SOD1 aggregation,and axon guidance failure.Conclusion:G93A SOD1 expression in motor neurons of C.elegans results in characteristic alterations of ALS.Increased autophagy protects C.elegans motor neurons against the toxicity of mutant SOD1.

  12. Autophagy and obesity%自噬和肥胖

    Institute of Scientific and Technical Information of China (English)

    张林; 胡茂清

    2015-01-01

    [Summary] Autophagy is an intracellular degradation process by which the damaged organelles and macromolecules are lysosomal dependently degraded by auto‐phagocyte under the control of autophagy‐related genes. The autophagy level in hypothalamus and adipose tissue changes in obese individuals.Autophagy participates in the regulation of food intake and energy balance ,and associates with adipocyte differentiation and adipogenesis. More and more attention has been paid to the relationship between autophagy and obesity w hich may guide the new research direction of pathogenesis study and therapy of obesity.%自噬是在相关基因的调控下,自噬细胞溶酶体依赖性的降解细胞内受损的细胞器及大分子物质的过程。肥胖个体下丘脑、脂肪组织自噬水平改变,自噬参与了下丘脑控制进食及能量平衡调节,并且与脂肪细胞的分化、脂肪形成有关。自噬与肥胖发生的关系日益受到重视,为肥胖的发病机制研究及治疗提供了新的方向。

  13. Telemetric control of peripheral lipophagy by hypothalamic autophagy.

    Science.gov (United States)

    Martinez-Lopez, Nuria; Singh, Rajat

    2016-08-01

    Autophagy maintains cellular quality control by degrading organelles, and cytosolic proteins and their aggregates in lysosomes. Autophagy also degrades lipid droplets (LD) through a process termed lipophagy. During lipophagy, LD are sequestered within autophagosomes and degraded by lysosomal acid lipases to generate free fatty acids that are β-oxidized for energy. Lipophagy was discovered in hepatocytes, and since then has been shown to function in diverse cell types. Whether lipophagy degrades LD in the major fat storing cell-the adipocyte-remained unclear. We have found that blocking autophagy in brown adipose tissues (BAT) by deleting the autophagy gene Atg7 in BAT MYF5 (myogenic factor 5)-positive progenitors increases basal lipid content in BAT and decreases lipid utilization during cold exposure-indicating that lipophagy contributes to lipohomeostasis in the adipose tissue. Surprisingly, knocking out Atg7 in hypothalamic proopiomelanocortin (POMC) neurons also blocks lipophagy in BAT and liver suggesting that specific neurons within the central nervous system (CNS) exert telemetric control over lipophagy in BAT and liver. PMID:27341145

  14. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia.

    LENUS (Irish Health Repository)

    Orfali, Nina

    2014-05-15

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies.

  15. Anti-tumor immunity, autophagy and chemotherapy

    Institute of Scientific and Technical Information of China (English)

    Gy(o)rgyi Müzes; Ferenc Sipos

    2012-01-01

    Autophagy or self-digestion of cells is activated upon various stressful stimuli and has been found to be a survival and drug resistance pathway in cancer.However,genetic studies support that autophagy can act as a tumor suppressor.Furthermore,defective autophagy is implicated in tumorigenesis,as well.The precise impact of autophagy on malignant transformation has not yet been clarified,but recent data suggest that this complex process is mainly directed by cell types,phases,genetic background and microenvironment.Relation of autophagy to anticancer immune responses may indicate a novel aspect in cancer chemotherapy.

  16. The cellular decision between apoptosis and autophagy

    Institute of Scientific and Technical Information of China (English)

    Yong-Jun Fan; Wei-Xing Zong

    2013-01-01

    Apoptosis and autophagy are important molecular processes that maintain organismal and cellular homeostasis,respectively.While apoptosis fulfills its role through dismantling damaged or unwanted cells,autophagy maintains cellular homeostasis through recycling selective intracellular organelles and molecules.Yet in some conditions,autophagy can lead to cell death.Apoptosis and autophagy can be stimulated by the same stresses.Emerging evidence indicates an interplay between the core proteins in both pathways,which underlies the molecular mechanism of the crosstalk between apoptosis and autophagy.This review summarizes recent literature on molecules that regulate both the apoptotic and autophagic processes.

  17. AUTOPHAGY AND IL-1 FAMILY CYTOKINES

    Directory of Open Access Journals (Sweden)

    James Harris

    2013-01-01

    Full Text Available Autophagy is an important intracellular homeostatic mechanism for the targeting of cytosolic constituents, including organelles, for lysosomal degradation. Autophagy plays roles in numerous physiological processes, include immune cell responses to endogenous and exogenous pathogenic stimuli. Moreover, autophagy has a potentially pivotal role in the regulation of inflammatory responses. In particular, autophagy regulates endogenous inflammasome activators, as well as inflammasome components and pro-IL-1β. This review focuses specifically on the role autophagy plays in regulating the production, processing and secretion of IL-1 family cytokines.

  18. Intestinal Epithelium and Autophagy: Partners in Gut Homeostasis

    OpenAIRE

    Randall-Demllo, Sarron; Chieppa, Marcello; Eri, Rajaraman

    2013-01-01

    One of the most significant challenges of cell biology is to understand how each type of cell copes with its specific workload without suffering damage. Among the most intriguing questions concerns intestinal epithelial cells in mammals; these cells act as a barrier between the internally protected region and the external environment that is exposed constantly to food and microbes. A major process involved in the processing of microbes is autophagy. In the intestine, through multiple, complex...

  19. NAC1 and HMGB1 enter a partnership for manipulating autophagy.

    Science.gov (United States)

    Zhang, Yi; Yang, Jay W; Ren, Xingcong; Yang, Jin-Ming

    2011-12-01

    Our recent study revealed a new role of nucleus accumbens-1 (NAC1), a transcription factor belonging to the BTB/POZ gene family, in regulating autophagy. Moreover, we found that the high-mobility group box 1 (HMGB1), a chromatin-associated nuclear protein acting as an extracellular damage associated molecular pattern molecule (DAMP), is the downstream executor of NAC1 in modulating autophagy. In response to stress such as therapeutic insults, NAC1 increases the expression, cytosolic translocation and release of HMGB1; elevated level of the cytoplasmic HMGB1 leads to activation of autophagy. The NAC1-HMGB1 partnership may represent a previously unrecognized pathway that regulates autophagy in response to various stresses such as chemotherapy.

  20. Transcription factors and cognate signalling cascades in the regulation of autophagy.

    Science.gov (United States)

    Chandra, Vemika; Bhagyaraj, Ella; Parkesh, Raman; Gupta, Pawan

    2016-05-01

    Autophagy is a process that maintains the equilibrium between biosynthesis and the recycling of cellular constituents; it is critical for avoiding the pathophysiology that results from imbalance in cellular homeostasis. Recent reports indicate the need for the design of high-throughput screening assays to identify targets and small molecules for autophagy modulation. For such screening, however, a better understanding of the regulation of autophagy is essential. In addition to regulation by various signalling cascades, regulation of gene expression by transcription factors is also critical. This review focuses on the various transcription factors as well as the corresponding signalling molecules that act together to translate the stimuli to effector molecules that up- or downregulate autophagy. This review rationalizes the importance of these transcription factors functioning in tandem with cognate signalling molecules and their interfaces as possible therapeutic targets for more specific pharmacological interventions. PMID:25651938

  1. Ribose 5-phosphate isomerase inhibits LC3 processing and basal autophagy.

    Science.gov (United States)

    Heintze, Jacob; Costa, Joana R; Weber, Melanie; Ketteler, Robin

    2016-09-01

    Autophagy and cellular metabolism are tightly linked processes, but how individual metabolic enzymes regulate the process of autophagy is not well understood. This study implicates ribose-5-phosphate isomerase (RPIA), a key regulator of the pentose phosphate pathway, in the control of autophagy. We used a dual gene deletion strategy, combining shRNA-mediated knockdown studies with CRISPR/Cas9 genome editing. Knockdown of RPIA by shRNA or genomic deletion by CRISPR/Cas9 genome editing, results in an increase of ATG4B-mediated LC3 processing and in the appearance of LC3-positive autophagosomes in cells. Increased LC3 processing upon knockdown of RPIA can be reversed by treatment with the antioxidant N-acetyl cysteine. The results are consistent with a model in which RPIA suppresses autophagy and LC3 processing by modulation of redox signaling. PMID:27328773

  2. Stable Expression of Hantavirus H8205 Strain G1/IL-2 Gene and Immune Protection of the Fusion Gene

    Institute of Scientific and Technical Information of China (English)

    XIONG Ying; YUAN Yuan; JIA Min; YU Bing; HUANG Hanju

    2007-01-01

    To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE electrophoresis were used to determine the stable transfection and expression of recombinant protein.Each mouse was inoculated with plasmids intramuscularly (i.m.) three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies (NAb) were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1:20-1:80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

  3. Autophagy in immune cell regulation and dysregulation.

    Science.gov (United States)

    Chaturvedi, Akanksha; Pierce, Susan K

    2009-09-01

    Autophagy is an ancient pathway required for cell and tissue homeostasis and differentiation. Initially thought to be a process leading to cell death, autophagy is currently viewed as a beneficial catabolic process that promotes cell survival under starvation conditions by sequestering components of the cytoplasm, including misfolded proteins, protein aggregates, and damaged organelles, and targeting them for lysosome-mediated degradation. In this way, autophagy plays a role in maintaining a balance between degradation and recycling of cellular material. The importance of autophagy is underscored by the fact that malfunctioning of this pathway results in neurodegeneration, cancer, susceptibility to microbial infection, and premature aging. Autophagy occurs in almost all cell types, including immune cells. Recent advances in the field suggest that autophagy plays a central role in regulating the immune system at multiple levels. In this review, we focus on recent developments in the area of autophagy-mediated modulation of immune responses. PMID:19671376

  4. The symphony of autophagy and calcium signaling.

    Science.gov (United States)

    Yao, Zhiyuan; Klionsky, Daniel J

    2015-01-01

    Posttranslational regulation of macroautophagy (hereafter autophagy), including phosphorylating and dephosphorylating components of the autophagy-related (Atg) core machinery and the corresponding upstream transcriptional factors, is important for the precise modulation of autophagy levels. Several kinases that are involved in phosphorylating autophagy-related proteins have been identified in both yeast and mammalian cells. However, there has been much less research published with regard to the identification of the complementary phosphatases that function in autophagy. A recent study identified PPP3/calcineurin, a calcium-dependent phosphatase, as a regulator of autophagy, and demonstrated that one of the key targets of PPP3/calcineurin is TFEB, a master transcriptional factor that controls autophagy and lysosomal function in mammalian cells.

  5. Modulation of pathogen recognition by autophagy

    Directory of Open Access Journals (Sweden)

    Ji Eun eOh

    2012-03-01

    Full Text Available Autophagy is an ancient biological process for maintaining cellular homeostasis by degradation of long-lived cytosolic proteins and organelles. Recent studies demonstrated that autophagy is availed by immune cells to regulate innate immunity. On the one hand, cells exert direct effector function by degrading intracellular pathogens; on the other hand, autophagy modulates pathogen recognition and downstream signaling for innate immune responses. Pathogen recognition via pattern recognition receptors induces autophagy. The function of phagocytic cells is enhanced by recruitment of autophagy-related proteins. Moreover, autophagy acts as a delivery system for viral replication complexes to migrate to the endosomal compartments where virus sensing occurs. In another case, key molecules of the autophagic pathway have been found to negatively regulate immune signaling, thus preventing aberrant activation of cytokine production and consequent immune responses. In this review, we focus on the recent advances in the role of autophagy in pathogen recognition and modulation of innate immune responses.

  6. Liraglutide prevents high glucose level induced insulinoma cells apoptosis by targeting autophagy

    Institute of Scientific and Technical Information of China (English)

    CHEN Ze-fang; LI Yan-bo; HAN Jun-yong; YIN Jia-jing; WANG Yang; ZHU Li-bo; XIE Guang-ying

    2013-01-01

    Background The pathophysiology of type 2 diabetes is progressive pancreatic beta cell failure with consequential reduced insulin secretion.Glucotoxicity results in the reduction of beta cell mass in type 2 diabetes by inducing apoptosis.Autophagy is essential for the maintenance of normal islet architecture and plays a crucial role in maintaining the intracellular insulin content by accelerating the insulin degradation rate in beta cells.Recently more attention has been paid to the effect of autophagy in type 2 diabetes.The regulatory pathway of autophagy in controlling pancreatic beta cells is still not clear.The aim of our study was to evaluate whether liraglutide can inhibit apoptosis and modulate autophagy in vitro in insulinoma cells (INS-1 cells).Methods INS-1 cells were incubated for 24 hours in the presence or absence of high levels of glucose,liraglutide (a long-acting human glucagon-like peptide-1 analogue),or 3-methyadenine (3-MA).Cell viability was measured using the Cell Counting Kit-8 (CCK8) viability assay.Autophagy of INS-1 cells was tested by monodansylcadaverine (MDC)staining,an autophagy fluorescent compound used for the labeling of autophagic vacuoles,and by Western blotting of microtubule-associated protein I light chain 3 (LC3),a biochemical markers of autophagic initiation.Results The viability of INS-1 cells was reduced after treatment with high levels of glucose.The viability of INS-1 cells was reduced and apoptosis was increased when autophagy was inhibited.The viability of INS-1 cells was significantly increased by adding liraglutide to supplement high glucose level medium compared with the cells treated with high glucose levels alone.Conclusions Apoptosis and autophagy were increased in rat INS-1 cells when treated with high level of glucose,and the viability of INS-1 cells was significantly reduced by inhibiting autophagy.Liraglutide protected INS-1 cells from high glucose level-induced apoptosis that is accompanied by a significant

  7. Historical landmarks of autophagy research

    OpenAIRE

    Ohsumi, Yoshinori

    2013-01-01

    The year of 2013 marked the 50th anniversary of C de Duve's coining of the term “autophagy” for the degradation process of cytoplasmic constituents in the lysosome/vacuole. This year we regretfully lost this great scientist, who contributed much during the early years of research to the field of autophagy. Soon after the discovery of lysosomes by de Duve, electron microscopy revealed autophagy as a means of delivering intracellular components to the lysosome. For a long time after the discove...

  8. The Pseudomonas aeruginosa pfpI gene plays an antimutator role and provides general stress protection.

    Science.gov (United States)

    Rodríguez-Rojas, Alexandro; Blázquez, Jesús

    2009-02-01

    Hypermutator Pseudomonas aeruginosa strains, characterized by an increased spontaneous-mutation rate, are found at high frequencies in chronic lung infections. Hypermutability is associated with the loss of antimutator genes related to DNA repair or damage avoidance systems. Only a few antimutator genes have been described in P. aeruginosa, although there is some evidence that additional genes may be involved in naturally occurring hypermutability. In order to find new P. aeruginosa antimutator genes, we constructed and screened a library of random insertions in the PA14 strain. Some previously described P. aeruginosa and/or Escherichia coli antimutator genes, such as mutS, mutL, uvrD, mutT, ung, and mutY, were detected, indicating a good coverage of our insertional library. One additional mutant contained an insertion in the P. aeruginosa PA14-04650 (pfpI) gene, putatively encoding a member of the DJ-1/ThiJ/PfpI superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1a. The pfpI-defective mutants in both PAO1 and PA14 showed higher spontaneous mutation rates than the wild-type strains, suggesting that PfpI plays a key role in DNA protection under nonstress conditions. Moreover, the inactivation of pfpI resulted in a dramatic increase in the H(2)O(2)-induced mutant frequency. Global transcription studies showed the induction of bacteriophage Pf1 genes and the repression of genes related to iron metabolism, suggesting that the increased spontaneous-mutant frequency may be due to reduced protection against the basal level of reactive oxygen species. Finally, pfpI mutants are more sensitive to different types of stress and are affected in biofilm formation. PMID:19028889

  9. Autophagy attenuates the catabolic effect during inflammatory conditions in nucleus pulposus cells, as sustained by NF-κB and JNK inhibition.

    Science.gov (United States)

    Xu, Kang; Chen, Weijian; Wang, Xiaofei; Peng, Yan; Liang, Anjing; Huang, Dongsheng; Li, Chunhai; Ye, Wei

    2015-09-01

    Proteoglycan degradation contributing to the pathogenesis of intervertebral disc (IVD) degeneration is induced by inflammatory cytokines, such as tumor necrosis factor‑α (TNF‑α) and interleukin‑1β (IL‑1β). Cell autophagy exists in degenerative diseases, including osteoarthritis and intervertebral disc degeneration. However, the autophagy induced by TNF‑α and IL‑1β and the corresponding molecular mechanism appear to be cell‑type dependent. The effect and mechanism of autophagy regulated by TNF‑α and IL‑1β in IVDs remains unclear. Additionally, the impact of autophagy on the catabolic effect in inflammatory conditions also remains elusive. In the present study, autophagy activator and inhibitor were used to demonstrate the impact of autophagy on the catabolic effect induced by TNF‑α. A critical role of autophagy was identified in rat nucleus pulposus (NP) cells: Inhibition of autophagy suppresses, while activation of autophagy enhances, the catabolic effect of cytokines. Subsequently, the autophagy‑related gene expression in rat NP cells following TNF‑α and IL‑1β treatment was observed using immunofluorescence, quantitative polymerase chain reaction and western blot analysis; however, no association was present. In addition, nuclear factor κB (NF‑κB), c‑Jun N‑terminal kinase (JNK), extracellular signal‑regulated kinases and p38 mitogen‑activated protein kinase inhibitors and TNF‑α were used to determine the molecular mechanism of autophagy during the inflammatory conditions, and only the NF‑κB and JNK inhibitor were found to enhance the autophagy of rat NP cells. Finally, IKKβ knockdown was used to further confirm the effect of the NF‑κB signal on human NP cells autophagy, and the data showed that IKKβ knockdown upregulated the autophagy of NP cells during inflammatory conditions.

  10. Congenital disorders of autophagy: an emerging novel class of inborn errors of neuro-metabolism.

    Science.gov (United States)

    Ebrahimi-Fakhari, Darius; Saffari, Afshin; Wahlster, Lara; Lu, Jenny; Byrne, Susan; Hoffmann, Georg F; Jungbluth, Heinz; Sahin, Mustafa

    2016-02-01

    Single gene disorders of the autophagy pathway are an emerging, novel and diverse group of multisystem diseases in children. Clinically, these disorders prominently affect the central nervous system at various stages of development, leading to brain malformations, developmental delay, intellectual disability, epilepsy, movement disorders, and neurodegeneration, among others. Frequent early and severe involvement of the central nervous system puts the paediatric neurologist, neurogeneticist, and neurometabolic specialist at the forefront of recognizing and treating these rare conditions. On a molecular level, mutations in key autophagy genes map to different stages of this highly conserved pathway and thus lead to impairment in isolation membrane (or phagophore) and autophagosome formation, maturation, or autophagosome-lysosome fusion. Here we discuss 'congenital disorders of autophagy' as an emerging subclass of inborn errors of metabolism by using the examples of six recently identified monogenic diseases: EPG5-related Vici syndrome, beta-propeller protein-associated neurodegeneration due to mutations in WDR45, SNX14-associated autosomal-recessive cerebellar ataxia and intellectual disability syndrome, and three forms of hereditary spastic paraplegia, SPG11, SPG15 and SPG49 caused by SPG11, ZFYVE26 and TECPR2 mutations, respectively. We also highlight associations between defective autophagy and other inborn errors of metabolism such as lysosomal storage diseases and neurodevelopmental diseases associated with the mTOR pathway, which may be included in the wider spectrum of autophagy-related diseases from a pathobiological point of view. By exploring these emerging themes in disease pathogenesis and underlying pathophysiological mechanisms, we discuss how congenital disorders of autophagy inform our understanding of the importance of this fascinating cellular pathway for central nervous system biology and disease. Finally, we review the concept of modulating

  11. FLCN, a novel autophagy component, interacts with GABARAP and is regulated by ULK1 phosphorylation.

    Science.gov (United States)

    Dunlop, Elaine A; Seifan, Sara; Claessens, Tijs; Behrends, Christian; Kamps, Miriam Af; Rozycka, Ewelina; Kemp, Alain J; Nookala, Ravi K; Blenis, John; Coull, Barry J; Murray, James T; van Steensel, Maurice Am; Wilkinson, Simon; Tee, Andrew R

    2014-10-01

    Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.

  12. Deletion of autophagy inducer RB1CC1 results in degeneration of the retinal pigment epithelium.

    Science.gov (United States)

    Yao, Jingyu; Jia, Lin; Khan, Naheed; Lin, Chengmao; Mitter, Sayak K; Boulton, Michael E; Dunaief, Joshua L; Klionsky, Daniel J; Guan, Jun-Lin; Thompson, Debra A; Zacks, David N

    2015-01-01

    Autophagy regulates cellular homeostasis and response to environmental stress. Within the retinal pigment epithelium (RPE) of the eye, the level of autophagy can change with both age and disease. The purpose of this study is to determine the relationship between reduced autophagy and age-related degeneration of the RPE. The gene encoding RB1CC1/FIP200 (RB1-inducible coiled-coil 1), a protein essential for induction of autophagy, was selectively knocked out in the RPE by crossing Best1-Cre mice with mice in which the Rb1cc1 gene was flanked with Lox-P sites (Rb1cc1(flox/flox)). Ex vivo and in vivo analyses, including western blot, immunohistochemistry, transmission electron microscopy, fundus photography, optical coherence tomography, fluorescein angiography, and electroretinography were performed to assess the structure and function of the retina as a function of age. Deletion of Rb1cc1 resulted in multiple autophagy defects within the RPE including decreased conversion of LC3-I to LC3-II, accumulation of autophagy-targeted precursors, and increased numbers of mitochondria. Age-dependent degeneration of the RPE occurred, with formation of atrophic patches, subretinal migration of activated microglial cells, subRPE deposition of inflammatory and oxidatively damaged proteins, subretinal drusenoid deposits, and occasional foci of choroidal neovascularization. There was secondary loss of photoreceptors overlying the degenerated RPE and reduction in the electroretinogram. These observations are consistent with a critical role of autophagy in the maintenance of normal homeostasis in the aging RPE, and indicate that disruption of autophagy leads to retinal phenotypes associated with age-related degeneration.

  13. Induction of autophagy by Imatinib sequesters Bcr-Abl in autophagosomes and down-regulates Bcr-Abl protein.

    LENUS (Irish Health Repository)

    Elzinga, Baukje M

    2013-06-01

    Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1\\/ATG7). Pharmacological inhibition of autophagy also reduced Bcr-Abl\\/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib.

  14. miR-224-3p inhibits autophagy in cervical cancer cells by targeting FIP200

    Science.gov (United States)

    Fang, Wang; Shu, Shan; Yongmei, Li; Endong, Zhu; Lirong, Yin; Bei, Sun

    2016-01-01

    Cervical cancer (CC) is a malignant solid tumor, which is one of the main causes of morbidity and mortality in women. Persistent High-risk human papillomavirus (hrHPV) infection is closely related to cervical cancer and autophagy has been suggested to inhibit viral infections. miRNAs have been reported to regulate autophagy in many solid tumors with many studies implicating miR-224-3p in the regulation of autophagy. In this study, we performed a miRNA microarray analysis on CC tissues and found that a large number of miRNAs with differential expressions in hrHPV-infected tissues. We identified miR-224-3p as a candidate miRNA selectively up regulated in HPV-infected tissues and cell lines. Further analysis revealed that miR-224-3p regulates autophagy in cervical cancer tissues and cell lines. While the overexpression of miR-224-3p inhibits autophagy in HPV-infected cells, knocking down endogenous miR-224-3p increases autophagy activity in the same cells. In addition, we found that miR-224-3p directly inhibits the expression of autophagy related gene, FAK family-interacting protein of 200 kDa (FIP200). In summary, we found that miR-224-3p regulates autophagy in hrHPV-induced cervical cancer cells through targeting FIP200 expression. PMID:27615604

  15. miR-224-3p inhibits autophagy in cervical cancer cells by targeting FIP200.

    Science.gov (United States)

    Fang, Wang; Shu, Shan; Yongmei, Li; Endong, Zhu; Lirong, Yin; Bei, Sun

    2016-01-01

    Cervical cancer (CC) is a malignant solid tumor, which is one of the main causes of morbidity and mortality in women. Persistent High-risk human papillomavirus (hrHPV) infection is closely related to cervical cancer and autophagy has been suggested to inhibit viral infections. miRNAs have been reported to regulate autophagy in many solid tumors with many studies implicating miR-224-3p in the regulation of autophagy. In this study, we performed a miRNA microarray analysis on CC tissues and found that a large number of miRNAs with differential expressions in hrHPV-infected tissues. We identified miR-224-3p as a candidate miRNA selectively up regulated in HPV-infected tissues and cell lines. Further analysis revealed that miR-224-3p regulates autophagy in cervical cancer tissues and cell lines. While the overexpression of miR-224-3p inhibits autophagy in HPV-infected cells, knocking down endogenous miR-224-3p increases autophagy activity in the same cells. In addition, we found that miR-224-3p directly inhibits the expression of autophagy related gene, FAK family-interacting protein of 200 kDa (FIP200). In summary, we found that miR-224-3p regulates autophagy in hrHPV-induced cervical cancer cells through targeting FIP200 expression. PMID:27615604

  16. Posttranslational modification of autophagy-related proteins in macroautophagy.

    Science.gov (United States)

    Xie, Yangchun; Kang, Rui; Sun, Xiaofang; Zhong, Meizuo; Huang, Jin; Klionsky, Daniel J; Tang, Daolin

    2015-01-01

    Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases.

  17. Neem oil limonoids induces p53-independent apoptosis and autophagy.

    Science.gov (United States)

    Srivastava, Pragya; Yadav, Neelu; Lella, Ravi; Schneider, Andrea; Jones, Anthony; Marlowe, Timothy; Lovett, Gabrielle; O'Loughlin, Kieran; Minderman, Hans; Gogada, Raghu; Chandra, Dhyan

    2012-11-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells. PMID:22915764

  18. Neem oil limonoids induces p53-independent apoptosis and autophagy.

    Science.gov (United States)

    Srivastava, Pragya; Yadav, Neelu; Lella, Ravi; Schneider, Andrea; Jones, Anthony; Marlowe, Timothy; Lovett, Gabrielle; O'Loughlin, Kieran; Minderman, Hans; Gogada, Raghu; Chandra, Dhyan

    2012-11-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.

  19. Age-regulated function of autophagy in the mouse inner ear.

    Science.gov (United States)

    de Iriarte Rodríguez, Rocío; Pulido, Sara; Rodríguez-de la Rosa, Lourdes; Magariños, Marta; Varela-Nieto, Isabel

    2015-12-01

    Autophagy is a highly conserved catabolic process essential for embryonic development and adult homeostasis. The autophagic machinery supplies energy by recycling intracellular components and facilitates the removal of apoptotic cells. In the inner ear, autophagy has been reported to play roles during early development in the chicken embryo and in the response to otic injury in the adult mouse. However, there are no studies on the expression of the autophagy machinery in the postnatal and adult inner ear. Insulin-like growth factor 1 (IGF-1) is one of the factors that regulate both otic development and cochlear postnatal maturation and function. Here, we hypothesised that autophagy could be one of the processes involved in the cochlear development and functional maturation. We report that autophagy-related genes (ATG) Becn1, Atg4g and Atg5 are expressed in the mouse cochlea, vestibular system and brainstem cochlear nuclei from late developmental stages to adulthood. Atg9 was studied in the mouse cochlea and showed a similar pattern. The presence of autophagic flux was confirmed by decreased sequestosome 1 (SQSTM1/p62) and increased relative levels of microtubule-associated protein light chain 3-II (LC3-II). Inner ear autophagy flux is developmentally regulated and is lower at perinatal stages than in the adult mouse, where an expression plateau is reached at the age of two-months, coinciding with the age at which full functional activity is reached. Expression is maintained in adult mice and declines after the age of twelve months. LC3B labelling showed that autophagy was primarily associated with spiral ganglion neurons. Over time, Igf1 wild type mice showed lower expression of genes coding for IGF-1 high affinity receptor and the family factor IGF-2 than null mice. Parallel analysis of autophagy machinery gene expression showed no significant differences between the genotypes over the lifespan of the null mice. Taken together, these results show that the

  20. The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection.

    Science.gov (United States)

    Wise, Andrew K; Tu, Tian; Atkinson, Patrick J; Flynn, Brianna O; Sgro, Beatrice E; Hume, Cliff; O'Leary, Stephen J; Shepherd, Robert K; Richardson, Rachael T

    2011-08-01

    A cochlear implant can restore hearing function by electrically exciting spiral ganglion neurons (SGNs) in the deaf cochlea. However, following deafness SGNs undergo progressive degeneration ultimately leading to their death. One significant cause of SGN degeneration is the loss of neurotrophic support that is normally provided by cells within the organ of Corti (OC). The administration of exogenous neurotrophins (NTs) can protect SGNs from degeneration but the effects are short-lived once the source of NTs has been exhausted. NT gene therapy, whereby cells within the cochlea are transfected with genes enabling them to produce NTs, is one strategy for providing a cellular source of NTs that may provide long-term support for SGNs. As the SGNs normally innervate sensory cells within the OC, targeting residual OC cells for gene therapy in the deaf cochlea may provide a source of NTs for SGN protection and targeted regrowth of their peripheral fibers. However, the continual degeneration of the OC over extended periods of deafness may deplete the cellular targets for NT gene therapy and hence limit the effectiveness of this method in preventing SGN loss. This study examined the effects of deafness duration on the efficacy of NT gene therapy in preventing SGN loss in guinea pigs that were systemically deafened with aminoglycosides. Adenoviral vectors containing green fluorescent protein (GFP) with or without genes for Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT3) were injected into the scala media (SM) compartment of cochleae that had been deafened for one, four or eight weeks prior to the viral injection. The results showed that viral transfection of cells within the SM was still possible even after severe degeneration of the OC. Supporting cells (pillar and Deiters' cells), cells within the stria vascularis, the spiral ligament, endosteal cells lining the scala compartments and interdental cells in the spiral limbus were transfected. However, the

  1. HSF-1 is involved in attenuating the release of inflammatory cytokines induced by LPS through regulating autophagy.

    Science.gov (United States)

    Tong, Zhongyi; Jiang, Bimei; Zhang, Lingli; Liu, Yanjuan; Gao, Min; Jiang, Yu; Li, Yuanbin; Lu, Qinglan; Yao, Yongming; Xiao, Xianzhong

    2014-05-01

    Autophagy plays a protective role in endotoxemic mice. Heat shock factor 1 (HSF-1) also plays a crucial protective role in endotoxemic mice by decreasing inflammatory cytokines. The purpose of this study was to determine whether HSF-1 is involved in attenuating the release of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated mice and peritoneal macrophages (PMs) through regulating autophagy activity. Autophagosome formation in HSF-1(+/+) and HSF-1(-/-) mice and PMs stimulated by LPS was examined by Western blotting and immunofluorescence. Lipopolysaccharide-induced autophagy and inflammatory cytokines were examined in HSF-1(+/+) and HSF-1(-/-) PMs treated with 3-methyladenine (3-MA) or rapamycin. Results showed that LPS-induced autophagy was elevated transiently at 12 h but declined at 24 h in the livers and lungs of mice. Higher levels of inflammatory cytokines and lower autophagy activity were detected in HSF-1(-/-) mice and PMs compared with HSF-1(+/+) mice and PMs. Interestingly, LPS-induced release of inflammatory cytokines did not further increase in HSF-1(-/-) PMs treated with 3-MA but aggravated in HSF-1(+/+) PMs. Lipopolysaccharide-induced autophagy did not decrease in HSF-1(-/-) PMs treated with 3-MA but decreased in HSF-1 PMs(+/+). Taken together, our results suggested that HSF-1 attenuated the release of inflammatory cytokines induced by LPS by regulating autophagy activity.

  2. BRAF associated autophagy exploitation: BRAF and autophagy inhibitors synergise to efficiently overcome resistance of BRAF mutant colorectal cancer cells.

    Science.gov (United States)

    Goulielmaki, Maria; Koustas, Evangelos; Moysidou, Eirini; Vlassi, Margarita; Sasazuki, Takehiko; Shirasawa, Senji; Zografos, George; Oikonomou, Eftychia; Pintzas, Alexander

    2016-02-23

    Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components. Autophagy has a controversial role in cancer--both in protecting against tumor progression by isolation of damaged organelles, or by potentially contributing to cancer growth. The impact of autophagy in RAS induced transformation still remains to be further analyzed based on the differential effect of RAS isoforms and tumor cell context. In the present study, the effect of KRAS/BRAF/PIK3CA oncogenic pathways on the autophagic cell properties and on main components of the autophagic machinery like p62 (SQSTM1), Beclin-1 (BECN1) and MAP1LC3 (LC3) in colon cancer cells was investigated. This study provides evidence that BRAF oncogene induces the expression of key autophagic markers, like LC3 and BECN1 in colorectal tumor cells. Herein, PI3K/AKT/MTOR inhibitors induce autophagic tumor properties, whereas RAF/MEK/ERK signalling inhibitors reduce expression of autophagic markers. Based on the ineffectiveness of BRAFV600E inhibitors in BRAFV600E bearing colorectal tumors, the BRAF related autophagic properties in colorectal cancer cells are further exploited, by novel combinatorial anti-cancer protocols. Strong evidence is provided here that pre-treatment of autophagy inhibitor 3-MA followed by its combination with BRAFV600E targeting drug PLX4720 can synergistically sensitize resistant colorectal tumors. Notably, colorectal cancer cells are very sensitive to mono-treatments of another autophagy inhibitor, Bafilomycin A1. The findings of this study are expected to provide novel efficient protocols for treatment of otherwise resistant colorectal tumors bearing BRAFV600E, by exploiting the autophagic properties induced by BRAF oncogene.

  3. BRAF associated autophagy exploitation: BRAF and autophagy inhibitors synergise to efficiently overcome resistance of BRAF mutant colorectal cancer cells

    Science.gov (United States)

    Goulielmaki, Maria; Koustas, Evangelos; Moysidou, Eirini; Vlassi, Margarita; Sasazuki, Takehiko; Shirasawa, Senji; Zografos, George; Oikonomou, Eftychia; Pintzas, Alexander

    2016-01-01

    Autophagy is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components. Autophagy has a controversial role in cancer – both in protecting against tumor progression by isolation of damaged organelles, or by potentially contributing to cancer growth. The impact of autophagy in RAS induced transformation still remains to be further analyzed based on the differential effect of RAS isoforms and tumor cell context. In the present study, the effect of KRAS/BRAF/PIK3CA oncogenic pathways on the autophagic cell properties and on main components of the autophagic machinery like p62 (SQSTM1), Beclin-1 (BECN1) and MAP1LC3 (LC3) in colon cancer cells was investigated. This study provides evidence that BRAF oncogene induces the expression of key autophagic markers, like LC3 and BECN1 in colorectal tumor cells. Herein, PI3K/AKT/MTOR inhibitors induce autophagic tumor properties, whereas RAF/MEK/ERK signalling inhibitors reduce expression of autophagic markers. Based on the ineffectiveness of BRAFV600E inhibitors in BRAFV600E bearing colorectal tumors, the BRAF related autophagic properties in colorectal cancer cells are further exploited, by novel combinatorial anti-cancer protocols. Strong evidence is provided here that pre-treatment of autophagy inhibitor 3-MA followed by its combination with BRAFV600E targeting drug PLX4720 can synergistically sensitize resistant colorectal tumors. Notably, colorectal cancer cells are very sensitive to mono-treatments of another autophagy inhibitor, Bafilomycin A1. The findings of this study are expected to provide novel efficient protocols for treatment of otherwise resistant colorectal tumors bearing BRAFV600E, by exploiting the autophagic properties induced by BRAF oncogene. PMID:26802026

  4. Sucrose induces vesicle accumulation and autophagy.

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    Higuchi, Takahiro; Nishikawa, Jun; Inoue, Hiroko

    2015-04-01

    It has been shown that the treatment of mammalian cells with sucrose leads to vacuole accumulation associated with lysosomes and upregulation of lysosomal enzyme expression and activity. Autophagy is an evolutionarily conserved homeostatic process by which cells deliver cytoplasmic material for degradation into lysosomes, thus it is probable that sucrose affects the autophagic activity. The role of sucrose in autophagy is unknown; however, another disaccharide, trehalose has been shown to induce autophagy. In the current study, we used mouse embryonic fibroblasts to investigate whether sucrose induces autophagy and whether vesicle formation is associated with autophagy. The results showed that sucrose induces autophagy while being accumulated within the endosomes/lysosomes. These vesicles were swollen and packed within the cytoplasm. Furthermore, trehalose and the trisaccharide raffinose, which are not hydrolyzed in mammalian cells, increased the rate of vesicles accumulation and LC3-II level (a protein marker of autophagy). However, fructose and maltose did not show the same effects. The correlation between the two processes, vesicle accumulation and autophagy induction, was confirmed by treatment of cells with sucrose plus invertase, or maltose plus acarbose-the α-glucosidase inhibitor-and by sucrose deprivation. Results also showed that vesicle accumulation was not affected by autophagy inhibition. Therefore, the data suggest that sucrose-induced autophagy through accumulation of sucrose-containing vesicles is caused by the absence of hydrolysis enzymes.

  5. Sucrose induces vesicle accumulation and autophagy.

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    Higuchi, Takahiro; Nishikawa, Jun; Inoue, Hiroko

    2015-04-01

    It has been shown that the treatment of mammalian cells with sucrose leads to vacuole accumulation associated with lysosomes and upregulation of lysosomal enzyme expression and activity. Autophagy is an evolutionarily conserved homeostatic process by which cells deliver cytoplasmic material for degradation into lysosomes, thus it is probable that sucrose affects the autophagic activity. The role of sucrose in autophagy is unknown; however, another disaccharide, trehalose has been shown to induce autophagy. In the current study, we used mouse embryonic fibroblasts to investigate whether sucrose induces autophagy and whether vesicle formation is associated with autophagy. The results showed that sucrose induces autophagy while being accumulated within the endosomes/lysosomes. These vesicles were swollen and packed within the cytoplasm. Furthermore, trehalose and the trisaccharide raffinose, which are not hydrolyzed in mammalian cells, increased the rate of vesicles accumulation and LC3-II level (a protein marker of autophagy). However, fructose and maltose did not show the same effects. The correlation between the two processes, vesicle accumulation and autophagy induction, was confirmed by treatment of cells with sucrose plus invertase, or maltose plus acarbose-the α-glucosidase inhibitor-and by sucrose deprivation. Results also showed that vesicle accumulation was not affected by autophagy inhibition. Therefore, the data suggest that sucrose-induced autophagy through accumulation of sucrose-containing vesicles is caused by the absence of hydrolysis enzymes. PMID:25389129

  6. Inhibition of autophagy ameliorates pulmonary microvascular dilation and PMVECs excessive proliferation in rat experimental hepatopulmonary syndrome

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    Xu, Duo; Chen, Bing; Gu, Jianteng; Chen, Lin; Belguise, Karine; Wang, Xiaobo; Yi, Bin; Lu, Kaizhi

    2016-01-01

    Hepatopulmonary syndrome (HPS) is a defective liver-induced pulmonary vascular disorder with massive pulmonary microvascular dilation and excessive proliferation of pulmonary microvascular endothelial cells (PMVECs). Growing evidence suggests that autophagy is involved in pulmonary diseases, protectively or detrimentally. Thus, it is interesting and important to explore whether autophagy might be involved in and critical in HPS. In the present study, we report that autophagy was activated in common bile duct ligation (CBDL) rats and cultured pulmonary PMVECs induced by CBDL rat serum, two accepted in vivo and in vitro experimental models of HPS. Furthermore, pharmacological inhibition of autophagy with 3-methyladenine (3-MA) significantly alleviated pathological alterations and typical symptom of HPS in CBDL rats in vivo, and consistently 3-MA significantly attenuated the CBDL rat serum-induced excessive proliferation of PMVECs in vitro. All these changes mediated by 3-MA might explain the observed prominent improvement of pulmonary appearance, edema, microvascular dilatation and arterial oxygenation in vivo. Collectively, these results suggest that autophagy activation may play a critical role in the pathogenesis of HPS, and autophagy inhibition may have a therapeutic potential for this disease. PMID:27480323

  7. Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes

    Institute of Scientific and Technical Information of China (English)

    Lu-yang YU; Bo LIN; Zhen-lin ZHANG; Li-he GUO

    2004-01-01

    AIM: To investigate the efficiency of transfer of A20 gene into pancreas against STZ-induced diabetes. METHODS:PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2 d before STZ injection. The uptake of plasmid pcDNA3-LacZ or pcDNA3-A20 was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A20 expression in the pancreas of pcDNA3-A20 transgenic mice was measured by RT-PCR and Westem blots. Urine amylase, NO generation, and histological examination were examined. RESULTS:Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 36 h later. On d 33 LacZ expression could be found in spleen,duodenum, and islets. The development of diabetes was prevented by direct A20 gene transferring into the pancreas and A20-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A20-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A20 gene into the pancreas protected mice from STZ-induced diabetes.

  8. Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection

    Institute of Scientific and Technical Information of China (English)

    Jiang-Li DONG; Bo ZHOU; Gang SHENG; Tao WANG

    2005-01-01

    Elevated expression of the rotavirus VP6 antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized (sVP6). The VP6 and sVp6genes were transformed into tobacco (Nicotiana tabacum L.) plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8-34-fold higher than that of controls containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein (TSP). Then, BALB/c female mice that had been gavaged weekly with 10 mg TSP containing 34 μg VP6 protein, in which VP6-specific serum IgG and mucosal IgA antibodies were investigated. The severity and duration of diarrhea caused by simian rotavirus SA-11 challenge were reduced significantly in passively immunized pups, which indicates that anti-VP6 antibodies generated in orally immunized female mice can be passed onto pups and provide heterotypic protection. An edible vaccine based on the VP6 of human rotavirus group A could provide a means to protect children and young animals from severe acute diarrhea.

  9. Protection of rat islet viability following heme oxygenase-1 gene transfection via adenoviral vector in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiaobo Chen; Yongxiang Li; Weiping Dong; Yang Jiao; Jianming Tan

    2007-01-01

    Objective: To investigate the effect of Heme oxygenase-1 (HO-1) gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods:Recombinant adenovirus vector containing human HO-1 gene(Ad-HO-1 ) or enhanced green fluorescent protein gene(Ad-EGFP) was generated by using AdEasy system respectively.The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index (SI) was calculated. Glucose-stimulated insulin release was usedto assess islet viability. Results:Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index(SI) of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets(P < 0.05).Conclusion: Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.

  10. RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy.

    Science.gov (United States)

    Luan, Qi; Jin, Lei; Jiang, Chen Chen; Tay, Kwang Hong; Lai, Fritz; Liu, Xiao Ying; Liu, Yi Lun; Guo, Su Tang; Li, Chun Ying; Yan, Xu Guang; Tseng, Hsin-Yi; Zhang, Xu Dong

    2015-01-01

    Although RIPK1 (receptor [TNFRSF]-interacting protein kinase 1) is emerging as a critical determinant of cell fate in response to cellular stress resulting from activation of death receptors and DNA damage, its potential role in cell response to endoplasmic reticulum (ER) stress remains undefined. Here we report that RIPK1 functions as an important prosurvival mechanism in melanoma cells undergoing pharmacological ER stress induced by tunicamycin (TM) or thapsigargin (TG) through activation of autophagy. While treatment with TM or TG upregulated RIPK1 and triggered autophagy in melanoma cells, knockdown of RIPK1 inhibited autophagy and rendered the cells sensitive to killing by TM or TG, recapitulating the effect of inhibition of autophagy. Consistently, overexpression of RIPK1 enhanced induction of autophagy and conferred resistance of melanoma cells to TM- or TG-induced cell death. Activation of MAPK8/JNK1 or MAPK9/JNK2, which phosphorylated BCL2L11/BIM leading to its dissociation from BECN1/Beclin 1, was involved in TM- or TG-induced, RIPK1-mediated activation of autophagy; whereas, activation of the transcription factor HSF1 (heat shock factor protein 1) downstream of the ERN1/IRE1-XBP1 axis of the unfolded protein response was responsible for the increase in RIPK1 in melanoma cells undergoing pharmacological ER stress. Collectively, these results identify upregulation of RIPK1 as an important resistance mechanism of melanoma cells to TM- or TG-induced ER stress by protecting against cell death through activation of autophagy, and suggest that targeting the autophagy-activating mechanism of RIPK1 may be a useful strategy to enhance sensitivity of melanoma cells to therapeutic agents that induce ER stress.

  11. Does the KIR2DS5 gene protect from some human diseases?

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    Izabela Nowak

    Full Text Available BACKGROUND: KIR2DS5 gene encodes an activating natural killer cell receptor whose ligand is not known. It was recently reported to affect the outcome of hematopoietic stem cell transplantation. METHODOLOGY/PRINCIPAL FINDINGS: In our studies on KIR2DS5 gene associations with human diseases, we compared the frequencies of this gene in patients and relevant controls. Typing for KIR2DS5 gene was performed by either individual or multiplex polymerase chain reactions which, when compared in the same samples, gave concordant results. We noted an apparently protective effect of KIR2DS5 gene presence in several clinical conditions, but not in others. Namely, this effect was observed in ankylosing spondylitis (p=0.003, odds ratio [OR]=0.47, confidence interval [CI]=0.28-0.79, endometriosis (p=0.03, OR=0.25, CI = 0.07-0.82 and acute rejection of kidney graft (p=0.0056, OR=0.44, CI=0.24-0.80, but not in non-small-cell lung carcinoma, rheumatoid arthritis, spontaneous abortion, or leukemia (all p>0.05. In addition, the simultaneous presence of KIR2DS5 gene and HLA-C C1 allotype exhibited an even stronger protective effect on ankylosing spondylitis (p=0.0003, OR=0.35, CI=0.19-0.65, whereas a lack of KIR2DS5 and the presence of the HLA-C C2 allotype was associated with ankylosing spondylitis (p=0.0017, OR=1.92, CI=1.28-2.89, whereas a lack of KIR2DS5 and presence of C1 allotype was associated with rheumatoid arthritis (p=0.005, OR=1.47, CI=1.13-1.92. The presence of both KIR2DS5 and C1 seemed to protect from acute kidney graft rejection (p=0.017, OR=0.47, CI=0.25-0.89, whereas lack of KIR2DS5 and presence of C2 seemed to favor rejection (p=0.0015, OR=2.13, CI=1.34-3.37. CONCLUSIONS/SIGNIFICANCE: Our results suggest that KIR2DS5 may protect from endometriosis, ankylosing spondylitis, and acute rejection of kidney graft.

  12. Autophagy is induced in the skeletal muscle of cachectic cancer patients

    Science.gov (United States)

    Aversa, Zaira; Pin, Fabrizio; Lucia, Simone; Penna, Fabio; Verzaro, Roberto; Fazi, Maurizio; Colasante, Giuseppina; Tirone, Andrea; Fanelli, Filippo Rossi; Ramaccini, Cesarina; Costelli, Paola; Muscaritoli, Maurizio

    2016-01-01

    Basal rates of autophagy can be markedly accelerated by environmental stresses. Recently, autophagy has been involved in cancer-induced muscle wasting. Aim of this study has been to evaluate if autophagy is induced in the skeletal muscle of cancer patients. The expression (mRNA and protein) of autophagic markers has been evaluated in intraoperative muscle biopsies. Beclin-1 protein levels were increased in cachectic cancer patients, suggesting autophagy induction. LC3B-I protein levels were not significantly modified. LC3B-II protein levels were significantly increased in cachectic cancer patients suggesting either increased autophagosome formation or reduced autophagosome turnover. Conversely, p62 protein levels were increased in cachectic and non-cachectic cancer patients, suggesting impaired autophagosome clearance. As for mitophagy, both Bnip3 and Nix/Bnip3L show a trend to increase in cachectic patients. In the same patients, Parkin levels significantly increased, while PINK1 was unchanged. At gene level, Beclin-1, p-62, BNIP3, NIX/BNIP3L and TFEB mRNAs were not significantly modulated, while LC3B and PINK1 mRNA levels were increased and decreased, respectively, in cachectic cancer patients. Autophagy is induced in the skeletal muscle of cachectic cancer patients, although autophagosome clearance appears to be impaired. Further studies should evaluate whether modulation of autophagy could represent a relevant therapeutic strategy in cancer cachexia. PMID:27459917

  13. Autophagy-preferential degradation of MIR224 participates in hepatocellular carcinoma tumorigenesis.

    Science.gov (United States)

    Lan, Sheng-Hui; Wu, Shan-Ying; Zuchini, Roberto; Lin, Xi-Zhang; Su, Ih-Jen; Tsai, Ting-Fen; Lin, Yen-Ju; Wu, Cheng-Tao; Liu, Hsiao-Sheng

    2014-09-01

    Autophagy and microRNA (miRNA) are important regulators during cancer cell tumorigenesis. Impaired autophagy and high expression of the oncogenic microRNA MIR224 are prevalent in hepatocellular carcinoma (HCC); however, the relationship between the 2 phenomena remains elusive. In this study, we are the first to reveal that autophagy selectively regulates MIR224 expression through an autophagosome-mediated degradation system. Based on this finding, we further demonstrated that in hepatitis B virus (HBV)-related HCC, aberrant autophagy (low autophagic activity) results in accumulation of MIR224 and decreased expression of the target gene Smad4, which leads to increased cell migration and tumor formation. Preferential recruitment of MIR224 into the autophagosome was clearly demonstrated by a) miRNA in situ hybridization under confocal microscopy, and b) immunogold labeling of MIR224 under electron microscopy compared with a ubiquitously expressed microRNA MIRlet7e/let-7. Furthermore, we found that off-label use of amiodarone, an antiarrhythmic agent, effectively suppressed HCC tumorigenesis through autophagy-mediated MIR224 degradation both in vitro and in vivo. In summary, we identified amiodarone as a new autophagy inducer, which may provide an alternative approach in HCC therapy through a novel tumor suppression mechanism.

  14. Blocking tumor growth by targeting autophagy and SQSTM1 in vivo.

    Science.gov (United States)

    Wei, Huijun; Guan, Jun-Lin

    2015-01-01

    Autophagy is a highly conserved cellular process for degradation of bulk cytoplasmic materials in response to starvation and maintenance of cellular homeostasis. Dysfunction of autophagy is implicated in a variety of diseases including cancer. In a recent study, we devised a system for inducible deletion of an essential autophagy gene Rb1cc1/Fip200 in established tumor cells in vivo and showed that Rb1cc1 is required for maintaining tumor growth. We further investigated the role of the accumulated SQSTM1 in Rb1cc1-null autophagy-deficient tumor cells. To our surprise, the increased SQSTM1 was not responsible for the inhibition of tumor growth, but rather supported the residual growth of tumors (i.e., partially compensated for the defective growth caused by Rb1cc1 deletion). Further analysis indicated that SQSTM1 promoted tumor growth in autophagy-deficient cells at least partially through its activation of the NFKB signaling pathway. A working model is proposed to account for our findings, which suggest that targeting both autophagy and the consequently increased SQSTM1 may be exploited for developing more effective cancer therapies.

  15. Autophagy is essential for ultrafine particle-induced inflammation and mucus hyperproduction in airway epithelium.

    Science.gov (United States)

    Chen, Zhi-Hua; Wu, Yin-Fang; Wang, Ping-Li; Wu, Yan-Ping; Li, Zhou-Yang; Zhao, Yun; Zhou, Jie-Sen; Zhu, Chen; Cao, Chao; Mao, Yuan-Yuan; Xu, Feng; Wang, Bei-Bei; Cormier, Stephania A; Ying, Song-Min; Li, Wen; Shen, Hua-Hao

    2016-01-01

    Environmental ultrafine particulate matter (PM) is capable of inducing airway injury, while the detailed molecular mechanisms remain largely unclear. Here, we demonstrate pivotal roles of autophagy in regulation of inflammation and mucus hyperproduction induced by PM containing environmentally persistent free radicals in human bronchial epithelial (HBE) cells and in mouse airways. PM was endocytosed by HBE cells and simultaneously triggered autophagosomes, which then engulfed the invading particles to form amphisomes and subsequent autolysosomes. Genetic blockage of autophagy markedly reduced PM-induced expression of inflammatory cytokines, e.g. IL8 and IL6, and MUC5AC in HBE cells. Mice with impaired autophagy due to knockdown of autophagy-related gene Becn1 or Lc3b displayed significantly reduced airway inflammation and mucus hyperproduction in response to PM exposure in vivo. Interference of the autophagic flux by lysosomal inhibition resulted in accumulated autophagosomes/amphisomes, and intriguingly, this process significantly aggravated the IL8 production through NFKB1, and markedly attenuated MUC5AC expression via activator protein 1. These data indicate that autophagy is required for PM-induced airway epithelial injury, and that inhibition of autophagy exerts therapeutic benefits for PM-induced airway inflammation and mucus hyperproduction, although they are differentially orchestrated by the autophagic flux.

  16. Autophagy is Required for the Maintenance of Liver Progenitor Cell Functionality

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    Yiji Cheng

    2015-06-01

    Full Text Available Background: Liver progenitor cells (LPCs are bipotent stem cells existing in the adult liver, which could be activated upon massive liver injury and contribute to liver regeneration. However, mechanisms of maintenance of LPC functionality remain poorly understood. Previous studies found that autophagy was required for the self-renewal and differentiation of several tissue stem cells. Methods: The study compared the level of autophagic activity in LPCs and differentiated hepatocytes. Then, autophagic activity was inhibited in LPCs by lentivirus-mediated autophagy-related gene 5 or Beclin 1 knockdown. Clonogenic assay, cell viability assays, hepatic differentiation assay, and senescence analysis were conducted to assess the role of autophagy in regulating self-renewal, hepatic differentiation and senescence of LPCs. Results: We observed high autophagic activity in LPCs compared with differentiated hepatocytes. We found that inhibition of autophagy impaired the self-renewal, proliferation, and hepatic differentiation capability of LPCs under normal cultural condition, but had little impact on cell viability. Interestingly, while wild-type LPCs remained rarely affected by the toxin, etoposide, inhibition of autophagy induced the senescent phenotype of LPCs. Overexpression of Beclin 1 in Beclin 1-knockdown LPCs restored the functionality of stem cells. Conclusion: Our findings indicate that autophagy may function as a critical regulator of LPC functionality under both physiological and pathological condition.

  17. T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy

    Science.gov (United States)

    Alessandri, Cristiano; Barbati, Cristiana; Vacirca, Davide; Piscopo, Paola; Confaloni, Annamaria; Sanchez, Massimo; Maselli, Angela; Colasanti, Tania; Conti, Fabrizio; Truglia, Simona; Perl, Andras; Valesini, Guido; Malorni, Walter; Ortona, Elena; Pierdominici, Marina

    2012-01-01

    Autophagy, the cytoprotection mechanism that takes place under metabolic impairment, has been implicated in the pathogenesis of autoimmunity. Here, we investigated the spontaneous and induced autophagic behavior of T lymphocytes from patients with systemic lupus erythematosus (SLE) compared with that of T lymphocytes from healthy donors by measuring the autophagy marker microtubule-associated protein 1 light chain 3 (LC3)-II. No significant differences in spontaneous autophagy were found between T lymphocytes from patients with SLE and from healthy donors, apart from CD4+ naive T cells from patients with SLE in which constitutively higher levels of autophagy (P<0.001) were detected. At variance, whereas treatment of T lymphocytes from healthy donors with serum IgG from patients with SLE resulted in a 2-fold increase in LC3-II levels (P<0.001), T lymphocytes from SLE patients were resistant to autophagic induction and also displayed an up-regulation of genes negatively regulating autophagy, e.g., α-synuclein. These findings could open new perspectives in the search for pathogenetic determinants of SLE progression and in the development of therapeutic strategies aimed to recover T-cell compartment homeostasis by restoring autophagic susceptibility.—Alessandri, C., Barbati, C., Vacirca, D., Piscopo, P., Confaloni, A., Sanchez, M., Maselli, A., Colasanti, T., Conti, F., Truglia, S., Perl, A., Valesini, G., Malorni, W., Ortona, E., Pierdominici, M. T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy. PMID:22835828

  18. EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy.

    Science.gov (United States)

    Li, Mengtao; Lu, Guang; Hu, Jia; Shen, Xue; Ju, Jiabao; Gao, Yuanxu; Qu, Liujing; Xia, Yan; Chen, Yingyu; Bai, Yun

    2016-03-01

    Self-renewal and differentiation of neural stem cells is essential for embryonic neurogenesis, which is associated with cell autophagy. However, the mechanism by which autophagy regulates neurogenesis remains undefined. Here, we show that Eva1a/Tmem166, an autophagy-related gene, regulates neural stem cell self-renewal and differentiation. Eva1a depletion impaired the generation of newborn neurons, both in vivo and in vitro. Conversely, overexpression of EVA1A enhanced newborn neuron generation and maturation. Moreover, Eva1a depletion activated the PIK3CA-AKT axis, leading to the activation of the mammalian target of rapamycin and the subsequent inhibition of autophagy. Furthermore, addition of methylpyruvate to the culture during neural stem cell differentiation rescued the defective embryonic neurogenesis induced by Eva1a depletion, suggesting that energy availability is a significant factor in embryonic neurogenesis. Collectively, these data demonstrated that EVA1A regulates embryonic neurogenesis by modulating autophagy. Our results have potential implications for understanding the pathogenesis of neurodevelopmental disorders caused by autophagy dysregulation. PMID:26905199

  19. EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy

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    Mengtao Li

    2016-03-01

    Full Text Available Self-renewal and differentiation of neural stem cells is essential for embryonic neurogenesis, which is associated with cell autophagy. However, the mechanism by which autophagy regulates neurogenesis remains undefined. Here, we show that Eva1a/Tmem166, an autophagy-related gene, regulates neural stem cell self-renewal and differentiation. Eva1a depletion impaired the generation of newborn neurons, both in vivo and in vitro. Conversely, overexpression of EVA1A enhanced newborn neuron generation and maturation. Moreover, Eva1a depletion activated the PIK3CA-AKT axis, leading to the activation of the mammalian target of rapamycin and the subsequent inhibition of autophagy. Furthermore, addition of methylpyruvate to the culture during neural stem cell differentiation rescued the defective embryonic neurogenesis induced by Eva1a depletion, suggesting that energy availability is a significant factor in embryonic neurogenesis. Collectively, these data demonstrated that EVA1A regulates embryonic neurogenesis by modulating autophagy. Our results have potential implications for understanding the pathogenesis of neurodevelopmental disorders caused by autophagy dysregulation.

  20. Development of potent autophagy inhibitors that sensitize oncogenic BRAF V600E mutant melanoma tumor cells to vemurafenib.

    Science.gov (United States)

    Goodall, Megan L; Wang, Tong; Martin, Katie R; Kortus, Matthew G; Kauffman, Audra L; Trent, Jeffrey M; Gately, Stephen; MacKeigan, Jeffrey P

    2014-06-01

    Autophagy is a dynamic cell survival mechanism by which a double-membrane vesicle, or autophagosome, sequesters portions of the cytosol for delivery to the lysosome for recycling. This process can be inhibited using the antimalarial agent chloroquine (CQ), which impairs lysosomal function and prevents autophagosome turnover. Despite its activity, CQ is a relatively inadequate inhibitor that requires high concentrations to disrupt autophagy, highlighting the need for improved small molecules. To address this, we screened a panel of antimalarial agents for autophagy inhibition and chemically synthesized a novel series of acridine and tetrahydroacridine derivatives. Structure-activity relationship studies of the acridine ring led to the discovery of VATG-027 as a potent autophagy inhibitor with a high cytotoxicity profile. In contrast, the tetrahydroacridine VATG-032 showed remarkably little cytotoxicity while still maintaining autophagy inhibition activity, suggesting that both compounds act as autophagy inhibitors with differential effects on cell viability. Further, knockdown of autophagy-related genes showed no effect on cell viability, demonstrating that the ability to inhibit autophagy is separate from the compound cytotoxicity profiles. Next, we determined that both inhibitors function through lysosomal deacidification mechanisms and ultimately disrupt autophagosome turnover. To evaluate the genetic context in which these lysosomotropic inhibitors may be effective, they were tested in patient-derived melanoma cell lines driven by oncogenic BRAF (v-raf murine sarcoma viral oncogene homolog B). We discovered that both inhibitors sensitized melanoma cells to the BRAF V600E inhibitor vemurafenib. Overall, these autophagy inhibitors provide a means to effectively block autophagy and have the potential to sensitize mutant BRAF melanomas to first-line therapies.

  1. Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human macrophages.

    Science.gov (United States)

    Rekha, Rokeya Sultana; Rao Muvva, S S V Jagadeeswara; Wan, Min; Raqib, Rubhana; Bergman, Peter; Brighenti, Susanna; Gudmundsson, Gudmundur H; Agerberth, Birgitta

    2015-01-01

    LL-37 is a human antimicrobial peptide (AMP) of the cathelicidin family with multiple activities including a mediator of vitamin D-induced autophagy in human macrophages, resulting in intracellular killing of Mycobacterium tuberculosis (Mtb). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent Mtb-killing can be further enhanced by 4-phenylbutyrate (PBA), also an inducer of LL-37 expression. Here, we explore a potential mechanism(s) behind PBA and LL-37-induced autophagy and intracellular killing of Mtb. Mtb infection of macrophages downregulated the expression of both the CAMP transcript and LL-37 peptide as well as certain autophagy-related genes (BECN1 and ATG5) at both the mRNA and protein levels. In addition, activation of LC3-II in primary macrophages and THP-1 cells was not detected. PBA and the active form of vitamin D3 (1,25[OH]2D3), separately or particularly in combination, were able to overcome Mtb-induced suppression of LL-37 expression. Notably, reactivation of autophagy occurred by stimulation of macrophages with PBA and promoted colocalization of LL-37 and LC3-II in autophagosomes. Importantly, PBA treatment failed to induce autophagy in Mtb-infected THP-1 cells, when the expression of LL-37 was silenced. However, PBA-induced autophagy was restored when the LL-37 knockdown cells were supplemented with synthetic LL-37. Interestingly, we have found that LL-37-induced autophagy was mediated via P2RX7 receptor followed by enhanced cytosolic free Ca(2+), and activation of AMPK and PtdIns3K pathways. Altogether, these results suggest a novel activity for PBA as an inducer of autophagy, which is LL-37-dependent and promotes intracellular killing of Mtb in human macrophages.

  2. Autophagy and IL-1 family cytokines

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    James eHarris

    2013-04-01

    Full Text Available Autophagy is an important intracellular homeostatic mechanism for the targeting of cytosolic constituents, including organelles, for lysosomal degradation. Autophagy plays roles in numerous physiological processes, including immune cell responses to endogenous and exogenous pathogenic stimuli. Moreover, autophagy has a potentially pivotal role to play in the regulation of inflammatory responses. In particular, autophagy regulates endogenous inflammasome activators, as well as inflammasome components and pro-IL-1β. As a result, autophagy acts a key modulator of IL-1β and IL-18, as well as IL-1α, release. This review focuses specifically on the role autophagy plays in regulating the production, processing and secretion of IL-1 and IL-18 and the consequences of this important function.

  3. Autophagy and IL-1 Family Cytokines.

    Science.gov (United States)

    Harris, James

    2013-01-01

    Autophagy is an important intracellular homeostatic mechanism for the targeting of cytosolic constituents, including organelles, for lysosomal degradation. Autophagy plays roles in numerous physiological processes, including immune cell responses to endogenous and exogenous pathogenic stimuli. Moreover, autophagy has a potentially pivotal role to play in the regulation of inflammatory responses. In particular, autophagy regulates endogenous inflammasome activators, as well as inflammasome components and pro-IL-1β. As a result, autophagy acts a key modulator of IL-1β and IL-18, as well as IL-1α, release. This review focuses specifically on the role autophagy plays in regulating the production, processing, and secretion of IL-1 and IL-18 and the consequences of this important function.

  4. Protective Effect of CRHR1 Gene Variants on the Development of Adult Depression Following Childhood Maltreatment

    Science.gov (United States)

    Polanczyk, Guilherme; Caspi, Avshalom; Williams, Benjamin; Price, Thomas S.; Danese, Andrea; Sugden, Karen; Uher, Rudolf; Poulton, Richie; Moffitt, Terrie E.

    2013-01-01

    Context A previous study reported a gene × environment interaction in which a haplotype in the corticotropin-releasing hormone receptor 1 gene (CRHR1) was associated with protection against adult depressive symptoms in individuals who were maltreated as children (as assessed by the Childhood Trauma Questionnaire [CTQ]). Objective To replicate the interaction between childhood maltreatment and a TAT haplotype formed by rs7209436, rs110402, and rs242924 in CRHR1, predicting adult depression. Design Two prospective longitudinal cohort studies. Setting England and New Zealand. Participants Participants in the first sample were women in the E-Risk Study (N= 1116), followed up to age 40 years with 96% retention. Participants in the second sample were men and women in the Dunedin Study (N= 1037), followed up to age 32 years with 96% retention. Main Outcome Measure Research diagnoses of pastyear and recurrent major depressive disorder. Results In the E-Risk Study, the TAT haplotype was associated with a significant protective effect. In this effect, women who reported childhood maltreatment on the CTQ were protected against depression. In the Dunedin Study, which used a different type of measure of maltreatment, this finding was not replicated. Conclusions A haplotype in CRHR1 has been suggested to exert a protective effect against adult depression among research participants who reported maltreatment on the CTQ, a measure that elicits emotional memories. This suggests the hypothesis that CRHR1’s protective effect may relate to its function in the consolidation of memories of emotionally arousing experiences. PMID:19736354

  5. Autophagy in muscle of glucose-infusion hyperglycemia rats and streptozotocin-induced hyperglycemia rats via selective activation of m-TOR or FoxO3.

    Directory of Open Access Journals (Sweden)

    Pengfei Lv

    Full Text Available Autophagy is a conserved process in eukaryotes required for metabolism and is involved in diverse diseases. To investigate autophagy in skeletal muscle under hyperglycemia status, we established two hyperglycemia-rat models that differ in their circulating insulin levels, by glucose infusion and singe high-dose streptozotocin injection. We then detected expression of autophagy related genes with real-time PCR and western blot. We found that under hyperglycemia status induced by glucose-infusion, autophagy was inhibited in rat skeletal muscle, whereas under streptozotocin-induced hyperglycemia status autophagy was enhanced. Meanwhile, hyperglycemic gastrocnemius muscle was more prone to autophagy than soleus muscle. Furthermore, inhibition of autophagy in skeletal muscle in glucose-infusion hyperglycemia rats was mediated by the m-TOR pathway while m-TOR and FoxO3 both contributed to enhancement of autophagy in gastrocnemius muscle in streptozotocin-induced hyperglycemia rats. These data shows that insulin plays a relatively more important role than hyperglycemia in regulating autophagy in hyperglycemia rat muscle through selectively activating the m-TOR or FoxO3 pathway in a fiber-selective manner.

  6. Cytoprotective role of autophagy against BH3 mimetic gossypol in ATG5 knockout cells generated by CRISPR-Cas9 endonuclease.

    Science.gov (United States)

    Kim, Na-Yeon; Han, Byeal-I; Lee, Michael

    2016-01-01

    Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas-mediated genome editing. The MTT assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5.

  7. Oxidative Stress and Autophagy in Cardiovascular Homeostasis

    OpenAIRE

    Morales, Cyndi R.; Pedrozo, Zully; Lavandero, Sergio; Hill, Joseph A.

    2014-01-01

    Significance: Autophagy is an evolutionarily ancient process of intracellular protein and organelle recycling required to maintain cellular homeostasis in the face of a wide variety of stresses. Dysregulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) leads to oxidative damage. Both autophagy and ROS/RNS serve pathological or adaptive roles within cardiomyocytes, depending on the context. Recent Advances: ROS/RNS and autophagy communicate with each other via both tra...

  8. Autophagy and oxidative stress in cardiovascular diseases

    OpenAIRE

    Mei, Yu; Thompson, Melissa D.; Cohen, Richard A.; Tong, XiaoYong

    2014-01-01

    Autophagy is a highly conserved degradation process by which intracellular components, including soluble macromolecules (e.g. nucleic acids, proteins, carbohydrates, and lipids) and dysfunctional organelles (e.g. mitochondria, ribosomes, peroxisomes, and endoplasmic reticulum) are degraded by the lysosome. Autophagy is orchestrated by the autophagy related protein (Atg) composed protein complexes to form autophagosomes, which fuse with lysosomes to generate autolysosomes where the contents ar...

  9. The role of autophagy in Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    Lei Zhang; Yaru Dong; Xiaoheng Xu; Zhong Xu

    2012-01-01

    Although Parkinson's disease is the most common neurodegenerative movement disorder, the mechanisms of pathogenesis remain poorly understood. Recent findings have shown that deregulation of the autophagy-lysosome pathway is involved in the pathogenesis of Parkinson's disease. This review summarizes the most recent findings and discusses the unique role of the autophagy-lysosome pathway in Parkinson's disease to highlight the possibility of Parkinson's disease treatment strategies that incorporate autophagy-lysosome pathway modulation.

  10. Autophagy gets in on the regulatory act

    Institute of Scientific and Technical Information of China (English)

    Steven K. Backues; Daniel J. Klionsky

    2011-01-01

    Autophagy down-regulates the Wnt signal transduction pathway via targeted degradation of a key signaling protein. This may provide an explanation for autophagy's role in tumor suppression.%@@ The eukaryotic cell has at its disposal two primary methods for getting rid of unwanted proteins: the proteasome and autophagy.The proteasome is a large protein complex comprising regulatory and proteolytic subunits whose core function is the degradation of damaged or misfolded proteins.

  11. Icariin Attenuates OGD/R-Induced Autophagy via Bcl-2-Dependent Cross Talk between Apoptosis and Autophagy in PC12 Cells

    Science.gov (United States)

    2016-01-01

    Icariin (ICA), an active component of Epimedium brevicornum Maxim, exerts a variety of neuroprotective effects such as antiapoptosis. However, the mechanisms underlying antiapoptosis of ICA in neurons exposed to oxygen-glucose deprivation and reperfusion (OGD/R) are unclear. The B-cell lymphoma-2 (Bcl-2) protein family plays an important role in the regulation of apoptosis and autophagy through Bcl-2-dependent cross talk. Bcl-2 suppresses apoptosis by binding to Bax and inhibits autophagy by binding to Beclin-1 which is an autophagy related protein. In the present study, MTT result showed that ICA increased cell viability significantly in OGD/R treated PC12 cells (P < 0.01). Results of western blotting analysis showed that ICA increased Bcl-2 expression significantly and decreased expressions of Bax, cleaved Caspase-3, Beclin-1, and LC3-II significantly in OGD/R treated PC12 cells (P < 0.01). These results suggest that ICA protects PC12 cells from OGD/R induced autophagy via Bcl-2-dependent cross talk between apoptosis and autophagy. PMID:27610184

  12. The Role of the Antiviral APOBEC3 Gene Family in Protecting Chimpanzees against Lentiviruses from Monkeys

    Science.gov (United States)

    Etienne, Lucie; Bibollet-Ruche, Frederic; Sudmant, Peter H.; Wu, Lily I.; Hahn, Beatrice H.; Emerman, Michael

    2015-01-01

    Cross-species transmissions of viruses from animals to humans are at the origin of major human pathogenic viruses. While the role of ecological and epidemiological factors in the emergence of new pathogens is well documented, the importance of host factors is often unknown. Chimpanzees are the closest relatives of humans and the animal reservoir at the origin of the human AIDS pandemic. However, despite being regularly exposed to monkey lentiviruses through hunting, chimpanzees are naturally infected by only a single simian immunodeficiency virus, SIVcpz. Here, we asked why chimpanzees appear to be protected against the successful emergence of other SIVs. In particular, we investigated the role of the chimpanzee APOBEC3 genes in providing a barrier to infection by most monkey lentiviruses. We found that most SIV Vifs, including Vif from SIVwrc infecting western-red colobus, the chimpanzee’s main monkey prey in West Africa, could not antagonize chimpanzee APOBEC3G. Moreover, chimpanzee APOBEC3D, as well as APOBEC3F and APOBEC3H, provided additional protection against SIV Vif antagonism. Consequently, lentiviral replication in primary chimpanzee CD4+ T cells was dependent on the presence of a lentiviral vif gene that could antagonize chimpanzee APOBEC3s. Finally, by identifying and functionally characterizing several APOBEC3 gene polymorphisms in both common chimpanzees and bonobos, we found that these ape populations encode APOBEC3 proteins that are uniformly resistant to antagonism by monkey lentiviruses. PMID:26394054

  13. The Role of the Antiviral APOBEC3 Gene Family in Protecting Chimpanzees against Lentiviruses from Monkeys.

    Directory of Open Access Journals (Sweden)

    Lucie Etienne

    2015-09-01

    Full Text Available Cross-species transmissions of viruses from animals to humans are at the origin of major human pathogenic viruses. While the role of ecological and epidemiological factors in the emergence of new pathogens is well documented, the importance of host factors is often unknown. Chimpanzees are the closest relatives of humans and the animal reservoir at the origin of the human AIDS pandemic. However, despite being regularly exposed to monkey lentiviruses through hunting, chimpanzees are naturally infected by only a single simian immunodeficiency virus, SIVcpz. Here, we asked why chimpanzees appear to be protected against the successful emergence of other SIVs. In particular, we investigated the role of the chimpanzee APOBEC3 genes in providing a barrier to infection by most monkey lentiviruses. We found that most SIV Vifs, including Vif from SIVwrc infecting western-red colobus, the chimpanzee's main monkey prey in West Africa, could not antagonize chimpanzee APOBEC3G. Moreover, chimpanzee APOBEC3D, as well as APOBEC3F and APOBEC3H, provided additional protection against SIV Vif antagonism. Consequently, lentiviral replication in primary chimpanzee CD4(+ T cells was dependent on the presence of a lentiviral vif gene that could antagonize chimpanzee APOBEC3s. Finally, by identifying and functionally characterizing several APOBEC3 gene polymorphisms in both common chimpanzees and bonobos, we found that these ape populations encode APOBEC3 proteins that are uniformly resistant to antagonism by monkey lentiviruses.

  14. Autophagy in the thymic epithelium is dispensable for the development of self-tolerance in a novel mouse model.

    Directory of Open Access Journals (Sweden)

    Supawadee Sukseree

    Full Text Available The thymic epithelium plays critical roles in the positive and negative selection of T cells. Recently, it was proposed that autophagy in thymic epithelial cells is essential for the induction of T cell tolerance to self antigens and thus for the prevention of autoimmune diseases. Here we have tested this hypothesis using mouse models in which autophagy was blocked specifically in epithelial cells expressing keratin 14 (K14, including the precursor of thymic epithelial cells. While the thymic epithelial cells of mice carrying the floxed Atg7 gene (ATG7 f/f showed a high level of autophagy, as determined by LC3 Western blot analysis and fluorescence detection of the recombinant green fluorescent protein (GFP-LC3 reporter protein on autophagosomes, autophagy in the thymic epithelium was efficiently suppressed by deletion of the Atg7 gene using the Cre-loxP system (ATG7 f/f K14-Cre. Suppression of autophagy led to the massive accumulation of p62/sequestosome 1 (SQSTM1 in thymic epithelial cells. However, the structure of the thymic epithelium as well as the organization and the size of the thymus were not altered in mutant mice. The ratio of CD4 to CD8-positive T cells, as well as the frequency of activated (CD69+ CD4 T cells in lymphoid organs, did not differ between mice with autophagy-competent and autophagy-deficient thymic epithelium. Inflammatory infiltrating cells, potentially indicative of autoimmune reactions, were present in the liver, lung, and colon of a similar fraction of ATG7 f/f and ATG7 f/f K14-Cre mice. In contrast to previously reported mice, that had received an autophagy-deficient thymus transplant, ATG7 f/f K14-Cre mice did not suffer from autoimmunity-induced weight loss. In summary, the results of this study suggest that autophagy in the thymic epithelium is dispensable for negative selection of autoreactive T cells.

  15. Effect of Exendin-4 on Autophagy Clearance in Beta Cell of Rats with Tacrolimus-induced Diabetes Mellitus

    Science.gov (United States)

    Lim, Sun Woo; Jin, Long; Jin, Jian; Yang, Chul Woo

    2016-01-01

    Growing evidence suggests that GLP-1 protects beta cells against various cellular injuries by modulating autophagy. In this study, we examined whether exendin-4 (Ex-4), a GLP-1 analog, had preventive effects on tacrolimus (Tac)-induced beta cell injury by improving autophagy clearance. Rats with Tac-induced diabetes mellitus exhibited increased autophagy-associated protein expression, light chain 3B levels, and autophagic vacuole numbers in pancreatic beta cells. Additionally, Tac increased autophagy in a dose- and time-dependent manner in vitro, and inhibition of autophagosome using 3-methyladenine reduced Tac-induced islet cell injury by decreasing reactive oxygen species production and apoptosis. Ex-4 treatment decreased Tac-induced hyperglycaemia, oxidative stress, and apoptosis, accompanied by decreased autophagy-associated protein expression and autophagosome numbers. In vivo and in vitro studies showed that Tac treatment impaired lysosomal function and autophagosome-lysosome fusion; these processes were improve by Ex-4 treatment. Moreover, addition of bafilomycin A1, an inhibitor of lysosomal function, abolished the protective effects of Ex-4. Our findings reveal that Tac-induced diabetes mellitus was a state of excessive burden of autophagosomes and impairment of autophagy clearance and that Ex-4 protected against Tac-induced pancreatic islet injury by reducing the burden of autophagosomes via activation of autophagosome clearance. Thus, Ex-4 had therapeutic effects on Tac-induced pancreatic beta cell injury. PMID:27436514

  16. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

    OpenAIRE

    Hou, Ying-Chen Claire; Chittaranjan, Suganthi; Barbosa, Sharon González; McCall, Kimberly; Gorski, Sharon M.

    2008-01-01

    A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a r...

  17. LAPping up dead cells to prevent lupus nephritis: a novel role for noncanonical autophagy in autoimmunity.

    Science.gov (United States)

    Leventhal, Jeremy S; Ross, Michael J

    2016-08-01

    The mechanisms underlying the development of systemic lupus erythematosus and lupus nephritis remain poorly understood. A recent study demonstrates that deficiencies in the immune system's ability to degrade scavenged dead cells via noncanonical autophagy is sufficient to break immune tolerance and produce features commonly seen in lupus, including circulating autoantibodies, inflammatory cytokines, and nephritis. This work provides a possible mechanism for the association of polymorphisms in autophagy genes with the risk of lupus. PMID:27418084

  18. LAPping up dead cells to prevent lupus nephritis: a novel role for noncanonical autophagy in autoimmunity.

    Science.gov (United States)

    Leventhal, Jeremy S; Ross, Michael J

    2016-08-01

    The mechanisms underlying the development of systemic lupus erythematosus and lupus nephritis remain poorly understood. A recent study demonstrates that deficiencies in the immune system's ability to degrade scavenged dead cells via noncanonical autophagy is sufficient to break immune tolerance and produce features commonly seen in lupus, including circulating autoantibodies, inflammatory cytokines, and nephritis. This work provides a possible mechanism for the association of polymorphisms in autophagy genes with the risk of lupus.

  19. The C/EBPbeta isoform, liver-inhibitory protein (LIP), induces autophagy in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Abreu, Maria M. [Department of Cancer Biology, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States); Sealy, Linda, E-mail: Linda.sealy@vanderbilt.edu [Department of Cancer Biology, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States); Department of Molecular Physiology and Biophysics, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States)

    2010-11-15

    Autophagy is a process involving the bulk degradation of cellular components in the cytoplasm via the lysosomal degradation pathway. Autophagy manifests a protective role in stressful conditions such as nutrient or growth factor depletion; however, extensive degradation of regulatory molecules or organelles essential for survival can lead to the demise of the cell, or autophagy-mediated cell death. The role of autophagy in cancer is complex with roles in both tumor suppression and tumor promotion proposed. Here we report that an isoform of the C/EBPbeta transcription factor, liver-enriched inhibitory protein (LIP), induces cell death in human breast cancer cells and stimulates autophagy. Overexpression of LIP is incompatible with cell growth and when cell cycle analysis was performed, a DNA profile of cells undergoing apoptosis was not observed. Instead, LIP expressing cells appeared to have large autophagic vesicles when examined via electron microscopy. Autophagy was further assessed in LIP expressing cells by monitoring the development of acidic vesicular organelles and conversion of LC3 from the cytoplasmic form to the membrane-bound form. Our work shows that C/EBPbeta isoform, LIP, is another member of the group of transcription factors, including E2F1 and p53, which are capable of playing a role in autophagy.

  20. Involvement of Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Advanced Glycation End Products-Induced Glomerular Mesangial Cell Injury

    Science.gov (United States)

    Chiang, Chih-Kang; Wang, Ching-Chia; Lu, Tien-Fong; Huang, Kuo-How; Sheu, Meei-Ling; Liu, Shing-Hwa; Hung, Kuan-Yu

    2016-01-01

    Advanced glycation end-products (AGEs)-induced mesangial cell death is one of major causes of glomerulus dysfunction in diabetic nephropathy. Both endoplasmic reticulum (ER) stress and autophagy are adaptive responses in cells under environmental stress and participate in the renal diseases. The role of ER stress and autophagy in AGEs-induced mesangial cell death is still unclear. Here, we investigated the effect and mechanism of AGEs on glomerular mesangial cells. AGEs dose-dependently decreased mesangial cell viability and induced cell apoptosis. AGEs also induced ER stress signals in a time- and dose-dependent manner. Inhibition of ER stress with 4-phenylbutyric acid effectively inhibited the activation of eIF2α and CHOP signals and reversed AGEs-induced cell apoptosis. AGEs also activated LC-3 cleavage, increased Atg5 expression, and decreased p62 expression, which indicated the autophagy induction in mesangial cells. Inhibition of autophagy by Atg5 siRNAs transfection aggravated AGEs-induced mesangial cell apoptosis. Moreover, ER stress inhibition by 4-phenylbutyric acid significantly reversed AGEs-induced autophagy, but autophagy inhibition did not influence the AGEs-induced ER stress-related signals activation. These results suggest that AGEs induce mesangial cell apoptosis via an ER stress-triggered signaling pathway. Atg5-dependent autophagy plays a protective role. These findings may offer a new strategy against AGEs toxicity in the kidney. PMID:27665710

  1. Salmonella enterica serovar Typhimurium lacking hfq gene confers protective immunity against murine typhoid.

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    Uday Shankar Allam

    Full Text Available Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Δhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4(+ T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate.

  2. Autophagy-deficiency in hepatic progenitor cells leads to the defects of stemness and enhances susceptibility to neoplastic transformation.

    Science.gov (United States)

    Xue, Feng; Hu, Lei; Ge, Ruiliang; Yang, Lixue; Liu, Kai; Li, Yunyun; Sun, Yanfu; Wang, Kui

    2016-02-01

    Autophagy is a highly conserved and lysosome-dependent degradation process which assists in cell survival and tissue homeostasis. Although previous reports have shown that deletion of the essential autophagy gene disturbs stem cell maintenance in some cell types such as hematopoietic and neural cells, it remains unclear how autophagy-deficiency influences hepatic progenitor cells (HPCs). Here we report that Atg5-deficiency in HPCs delays HPC-mediated rat liver regeneration in vivo. In vitro researches further demonstrate that loss of autophagy decreases the abilities of colony and spheroid formations, and disrupts the induction of hepatic differentiation in HPCs. Meanwhile, autophagy-deficiency increases the accumulations of damaged mitochondria and mitochondrial reactive oxygen species (mtROS) and suppresses homologous recombination (HR) pathway of DNA damage repair in HPCs. Moreover, in both diethylnitrosamine (DEN) and CCl4 models, autophagy-deficiency accelerates neoplastic transformation of HPCs. In conclusion, these findings demonstrate that autophagy contributes to stemness maintenance and reduces susceptibility to neoplastic transformation in HPCs.

  3. Coffee induces autophagy in vivo.

    Science.gov (United States)

    Pietrocola, Federico; Malik, Shoaib Ahmad; Mariño, Guillermo; Vacchelli, Erika; Senovilla, Laura; Chaba, Kariman; Niso-Santano, Mireia; Maiuri, Maria Chiara; Madeo, Frank; Kroemer, Guido

    2014-01-01

    Epidemiological studies and clinical trials revealed that chronic consumption coffee is associated with the inhibition of several metabolic diseases as well as reduction in overall and cause-specific mortality. We show that both natural and decaffeinated brands of coffee similarly rapidly trigger autophagy in mice. One to 4 h after coffee consumption, we observed an increase in autophagic flux in all investigated organs (liver, muscle, heart) in vivo, as indicated by the increased lipidation of LC3B and the reduction of the abundance of the autophagic substrate sequestosome 1 (p62/SQSTM1). These changes were accompanied by the inhibition of the enzymatic activity of mammalian target of rapamycin complex 1 (mTORC1), leading to the reduced phosphorylation of p70(S6K), as well as by the global deacetylation of cellular proteins detectable by immunoblot. Immunohistochemical analyses of transgenic mice expressing a GFP-LC3B fusion protein confirmed the coffee-induced relocation of LC3B to autophagosomes, as well as general protein deacetylation. Altogether, these results indicate that coffee triggers 2 phenomena that are also induced by nutrient depletion, namely a reduction of protein acetylation coupled to an increase in autophagy. We speculate that polyphenols contained in coffee promote health by stimulating autophagy.

  4. Autophagy selectivity through receptor clustering

    Science.gov (United States)

    Rutenberg, Andrew; Brown, Aidan

    Substrate selectivity in autophagy requires an all-or-none cellular response. We focus on peroxisomes, for which autophagy receptor proteins NBR1 and p62 are well characterized. Using computational models, we explore the hypothesis that physical clustering of autophagy receptor proteins on the peroxisome surface provides an appropriate all-or-none response. We find that larger peroxisomes nucleate NBR1 clusters first, and lose them due to competitive coarsening last, resulting in significant size-selectivity. We then consider a secondary hypothesis that p62 inhibits NBR1 cluster formation. We find that p62 inhibition enhances size-selectivity enough that, even if there is no change of the pexophagy rate, the volume of remaining peroxisomes can significantly decrease. We find that enhanced ubiquitin levels suppress size-selectivity, and that this effect is more pronounced for individual peroxisomes. Sufficient ubiquitin allows receptor clusters to form on even the smallest peroxisomes. We conclude that NBR1 cluster formation provides a viable physical mechanism for all-or-none substrate selectivity in pexophagy. We predict that cluster formation is associated with significant size-selectivity. Now at Simon Fraser University.

  5. Coffee induces autophagy in vivo

    Science.gov (United States)

    Pietrocola, Federico; Malik, Shoaib Ahmad; Mariño, Guillermo; Vacchelli, Erika; Senovilla, Laura; Chaba, Kariman; Niso-Santano, Mireia; Maiuri, Maria Chiara; Madeo, Frank; Kroemer, Guido

    2014-01-01

    Epidemiological studies and clinical trials revealed that chronic consumption coffee is associated with the inhibition of several metabolic diseases as well as reduction in overall and cause-specific mortality. We show that both natural and decaffeinated brands of coffee similarly rapidly trigger autophagy in mice. One to 4 h after coffee consumption, we observed an increase in autophagic flux in all investigated organs (liver, muscle, heart) in vivo, as indicated by the increased lipidation of LC3B and the reduction of the abundance of the autophagic substrate sequestosome 1 (p62/SQSTM1). These changes were accompanied by the inhibition of the enzymatic activity of mammalian target of rapamycin complex 1 (mTORC1), leading to the reduced phosphorylation of p70S6K, as well as by the global deacetylation of cellular proteins detectable by immunoblot. Immunohistochemical analyses of transgenic mice expressing a GFP–LC3B fusion protein confirmed the coffee-induced relocation of LC3B to autophagosomes, as well as general protein deacetylation. Altogether, these results indicate that coffee triggers 2 phenomena that are also induced by nutrient depletion, namely a reduction of protein acetylation coupled to an increase in autophagy. We speculate that polyphenols contained in coffee promote health by stimulating autophagy. PMID:24769862

  6. Autophagy pathway is required for IL-6 induced neuroendocrine differentiation and chemoresistance of prostate cancer LNCaP cells.

    Directory of Open Access Journals (Sweden)

    Pei-Ching Chang

    Full Text Available Prostate cancer (PCa cells undergoing neuroendocrine differentiation (NED are clinically relevant to the development of relapsed castration-resistant PCa. Increasing evidences show that autophagy involves in the development of neuroendocrine (NE tumors, including PCa. To clarify the effect of autophagy on NED, androgen-sensitive PCa LNCaP cells were examined. Treatment of LNCaP cells with IL-6 resulted in an induction of autophagy. In the absence of androgen, IL-6 caused an even stronger activation of autophagy. Similar result was identified in NED induction. Inhibition of autophagy with chloroquine (CQ markedly decreased NED. This observation was confirmed by beclin1 and Atg5 silencing experiments. Further supporting the role of autophagy in NED, we found that LC3 was up-regulated in PCa tissue that had relapsed after androgen-deprivation therapy when compared with their primary tumor counterpart. LC3 staining in relapsed PCa tissue showed punctate pattern similar to the staining of chromogranin A (CgA, a marker for NED cells. Moreover, autophagy inhibition induced the apoptosis of IL-6 induced NE differentiated PCa cells. Consistently, inhibition of autophagy by knockdown of beclin1 or Atg5 sensitized NE differentiated LNCaP cells to etoposide, a chemotherapy drug. To identify the mechanisms, phosphorylation of IL-6 downstream targets was analyzed. An increase in phospho-AMPK and a decrease in phospho-mTOR were found, which implies that IL-6 regulates autophagy through the AMPK/mTOR pathway. Most important to this study is the discovery of REST, a neuronal gene-specific transcriptional repressor that is involved in autophagy activation. REST was down-regulated in IL-6 treatment. Knockdown experiments suggest that REST is critical to NED and autophagy activation by IL-6. Together, our studies imply that autophagy is involved in PCa progression and plays a cytoprotective role when NED is induced in PCa cells by IL-6 treatment. These results

  7. Autophagy Pathway Is Required for IL-6 Induced Neuroendocrine Differentiation and Chemoresistance of Prostate Cancer LNCaP Cells

    Science.gov (United States)

    Chang, Yi-Ting; Chu, Cheng-Ying; Lee, Chin-Ling; Hsu, Hung-Wei; Zhou, Tyng-An; Wu, Zhaoju; Kim, Randie H.; Desai, Sonal J.; Liu, Shangqin; Kung, Hsing-Jien

    2014-01-01

    Prostate cancer (PCa) cells undergoing neuroendocrine differentiation (NED) are clinically relevant to the development of relapsed castration-resistant PCa. Increasing evidences show that autophagy involves in the development of neuroendocrine (NE) tumors, including PCa. To clarify the effect of autophagy on NED, androgen-sensitive PCa LNCaP cells were examined. Treatment of LNCaP cells with IL-6 resulted in an induction of autophagy. In the absence of androgen, IL-6 caused an even stronger activation of autophagy. Similar result was identified in NED induction. Inhibition of autophagy with chloroquine (CQ) markedly decreased NED. This observation was confirmed by beclin1 and Atg5 silencing experiments. Further supporting the role of autophagy in NED, we found that LC3 was up-regulated in PCa tissue that had relapsed after androgen-deprivation therapy when compared with their primary tumor counterpart. LC3 staining in relapsed PCa tissue showed punctate pattern similar to the staining of chromogranin A (CgA), a marker for NED cells. Moreover, autophagy inhibition induced the apoptosis of IL-6 induced NE differentiated PCa cells. Consistently, inhibition of autophagy by knockdown of beclin1 or Atg5 sensitized NE differentiated LNCaP cells to etoposide, a chemotherapy drug. To identify the mechanisms, phosphorylation of IL-6 downstream targets was analyzed. An increase in phospho-AMPK and a decrease in phospho-mTOR were found, which implies that IL-6 regulates autophagy through the AMPK/mTOR pathway. Most important to this study is the discovery of REST, a neuronal gene-specific transcriptional repressor that is involved in autophagy activation. REST was down-regulated in IL-6 treatment. Knockdown experiments suggest that REST is critical to NED and autophagy activation by IL-6. Together, our studies imply that autophagy is involved in PCa progression and plays a cytoprotective role when NED is induced in PCa cells by IL-6 treatment. These results reveal the

  8. Detecting Autophagy in Caenorhabditis elegans Embryos Using Markers of P Granule Degradation.

    Science.gov (United States)

    Palmisano, Nicholas J; Meléndez, Alicia

    2016-01-01

    Autophagy plays an active role during the early stages of embryogenesis in the nematode Caenorhabditis elegans. Although their exact function is unknown, P granules are ribonucleoprotein particles that play a role in germ cell specification. The localization of P granules is restricted to the germline precursor cells in wild-type embryos, as a result of their degradation in the somatic cell lineage. Autophagy is known to be required for the degradation of P granules, as mutations in various autophagy genes, including those encoding the adaptor SEPA-1 and the p62-like adaptor SQST-1, result in the accumulation of the P granule components PGL-1 and PGL-3 (termed PGL granules) in the somatic cells of C. elegans embryos. In this protocol, we present a methodology for using fusion reporters of SEPA-1, SQST-1, and PGL-1 that have aided in the identification of new genes for normal autophagy activity by screening for mutant animals that lack the degradation of these autophagy substrates. PMID:26729906

  9. Characterization of the autophagy marker protein Atg8 reveals atypical features of autophagy in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Rahul Navale

    Full Text Available Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8 employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64 and aspartic (pepstatin protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine, indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in

  10. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jae Hyeon [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Shin, In Chul [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Koh, Hyun Chul, E-mail: hckoh@hanyang.ac.kr [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)

    2013-04-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  11. Autophagy- An emerging target for melanoma therapy

    Science.gov (United States)

    Ndoye, Abibatou; Weeraratna, Ashani T.

    2016-01-01

    Melanoma accounts for only 5% of all cancers but is the leading cause of skin cancer death due to its high metastatic potential. Patients with metastatic melanoma have a 10-year survival rate of less than 10%. While the clinical landscape for melanoma is evolving rapidly, lack of response to therapies, as well as resistance to therapy remain critical obstacles for treatment of this disease. In recent years, a myriad of therapy resistance mechanisms have been unravelled, one of which is autophagy, the focus of this review. In advanced stages of malignancy, melanoma cells hijack the autophagy machinery in order to alleviate drug-induced and metabolic stress in the tumor microenvironment, thereby promoting resistance to multiple therapies, tumor cell survival, and progression.  Autophagy is an essential cellular process that maintains cellular homeostasis through the recycling of intracellular constituents. Early studies on the role of autophagy in cancer generated controversy as to whether autophagy was pro- or anti-tumorigenic. Currently, there is a consensus that autophagy is tumor-suppressive in the early stages of cancer and tumor-promoting in established tumors.  This review aims to highlight current understandings on the role of autophagy in melanoma malignancy, and specifically therapy resistance; as well as to evaluate recent strategies for therapeutic autophagy modulation. PMID:27583134

  12. Autophagy in allografts rejection: A new direction?

    Science.gov (United States)

    Sun, Hukui; Cheng, Dayan; Ma, Yuanyuan; Wang, Huaiquan; Liang, Ting; Hou, Guihua

    2016-03-18

    Despite the introduction of new and effective immunosuppressive drugs, acute cellular graft rejection is still a major risk for graft survival. Modulating the dosage of immunosuppressive drugs is not a good choice for all patients, new rejection mechanisms discovery are crucial to limit the inflammatory process and preserve the function of the transplant. Autophagy, a fundamental cellular process, can be detected in all subsets of lymphocytes and freshly isolated naive T lymphocytes. It is required for the homeostasis and function of T lymphocytes, which lead to cell survival or cell death depending on the context. T cell receptor (TCR) stimulation and costimulator signals induce strong autophagy, and autophagy deficient T cells leads to rampant apoptosis upon TCR stimulation. Autophagy has been proved to be activated during ischemia-reperfusion (I/R) injury and associated with grafts dysfunction. Furthermore, Autophagy has also emerged as a key mechanism in orchestrating innate and adaptive immune response to self-antigens, which relates with negative selection and Foxp3(+) Treg induction. Although, the role of autophagy in allograft rejection is unknown, current data suggest that autophagy indeed sweeps across both in the graft organs and recipients lymphocytes after transplantation. This review presents the rationale for the hypothesis that targeting the autophagy pathway could be beneficial in promoting graft survival after transplantation.

  13. Keeping autophagy in cheCK1

    Science.gov (United States)

    Cheong, Jit Kong; Virshup, David M.

    2016-01-01

    Abstract Mutant RAS-driven cancer cells cope with proliferative stress by increasing basal autophagy to maintain protein/organelle and energy homeostasis. We recently demonstrated that casein kinase 1 alpha (CK1α), a therapeutically tractable enzyme, is critical for fine-tuning the transcriptional regulation of mutant RAS-induced autophagy and the development of mutant RAS-driven cancers. PMID:27314070

  14. Autophagy- An emerging target for melanoma therapy.

    Science.gov (United States)

    Ndoye, Abibatou; Weeraratna, Ashani T

    2016-01-01

    Melanoma accounts for only 5% of all cancers but is the leading cause of skin cancer death due to its high metastatic potential. Patients with metastatic melanoma have a 10-year survival rate of less than 10%. While the clinical landscape for melanoma is evolving rapidly, lack of response to therapies, as well as resistance to therapy remain critical obstacles for treatment of this disease. In recent years, a myriad of therapy resistance mechanisms have been unravelled, one of which is autophagy, the focus of this review. In advanced stages of malignancy, melanoma cells hijack the autophagy machinery in order to alleviate drug-induced and metabolic stress in the tumor microenvironment, thereby promoting resistance to multiple therapies, tumor cell survival, and progression.  Autophagy is an essential cellular process that maintains cellular homeostasis through the recycling of intracellular constituents. Early studies on the role of autophagy in cancer generated controversy as to whether autophagy was pro- or anti-tumorigenic. Currently, there is a consensus that autophagy is tumor-suppressive in the early stages of cancer and tumor-promoting in established tumors.  This review aims to highlight current understandings on the role of autophagy in melanoma malignancy, and specifically therapy resistance; as well as to evaluate recent strategies for therapeutic autophagy modulation. PMID:27583134

  15. Lacritin and other autophagy associated proteins in ocular surface health.

    Science.gov (United States)

    Karnati, Roy; Talla, Venu; Peterson, Katherine; Laurie, Gordon W

    2016-03-01

    Advantage may be taken of macroautophagy ('autophagy') to promote ocular health. Autophagy continually captures aged or damaged cellular material for lysosomal degradation and recyling. When autophagic flux is chronically elevated, or alternatively deficient, health suffers. Chronic elevation of flux and stress are the consequence of inflammatory cytokines or of dry eye tears but not normal tears invitro. Exogenous tear protein lacritin transiently accelerates flux to restore homeostasis invitro and corneal health invivo, and yet the monomeric active form of lacritin appears to be selectively deficient in dry eye. Tissue transglutaminase-dependent cross-linking of monomer decreases monomer quantity and monomer affinity for coreceptor syndecan-1 thereby abrogating activity. Tissue transglutaminase is elevated in dry eye. Mutation of arylsulfatase A, arylsulfatase B, ceroid-lipofuscinosis neuronal 3, mucolipin, or Niemann-Pick disease type C1 respectively underlie several diseases of apparently insufficient autophagic flux that affect the eye, including: metachromatic leukodystrophy, mucopolysaccharidosis type VI, juvenile-onset Batten disease, mucolipidosis IV, and Niemann-Pick type C associated with myelin sheath destruction of corneal sensory and ciliary nerves and of the optic nerve; corneal clouding, ocular hypertension, glaucoma and optic nerve atrophy; accumulation of 'ceroid-lipofuscin' in surface conjunctival cells, and in ganglion and neuronal cells; decreased visual acuity and retinal dystrophy; and neurodegeneration. For some, enzyme or gene replacement, or substrate reduction, therapy is proving to be successful. Here we discuss examples of restoring ocular surface homeostasis through alteration of autophagy, with particular attention to lacritin. PMID:26318608

  16. Lacritin and other autophagy associated proteins in ocular surface health.

    Science.gov (United States)

    Karnati, Roy; Talla, Venu; Peterson, Katherine; Laurie, Gordon W

    2016-03-01

    Advantage may be taken of macroautophagy ('autophagy') to promote ocular health. Autophagy continually captures aged or damaged cellular material for lysosomal degradation and recyling. When autophagic flux is chronically elevated, or alternatively deficient, health suffers. Chronic elevation of flux and stress are the consequence of inflammatory cytokines or of dry eye tears but not normal tears invitro. Exogenous tear protein lacritin transiently accelerates flux to restore homeostasis invitro and corneal health invivo, and yet the monomeric active form of lacritin appears to be selectively deficient in dry eye. Tissue transglutaminase-dependent cross-linking of monomer decreases monomer quantity and monomer affinity for coreceptor syndecan-1 thereby abrogating activity. Tissue transglutaminase is elevated in dry eye. Mutation of arylsulfatase A, arylsulfatase B, ceroid-lipofuscinosis neuronal 3, mucolipin, or Niemann-Pick disease type C1 respectively underlie several diseases of apparently insufficient autophagic flux that affect the eye, including: metachromatic leukodystrophy, mucopolysaccharidosis type VI, juvenile-onset Batten disease, mucolipidosis IV, and Niemann-Pick type C associated with myelin sheath destruction of corneal sensory and ciliary nerves and of the optic nerve; corneal clouding, ocular hypertension, glaucoma and optic nerve atrophy; accumulation of 'ceroid-lipofuscin' in surface conjunctival cells, and in ganglion and neuronal cells; decreased visual acuity and retinal dystrophy; and neurodegeneration. For some, enzyme or gene replacement, or substrate reduction, therapy is proving to be successful. Here we discuss examples of restoring ocular surface homeostasis through alteration of autophagy, with particular attention to lacritin.

  17. Role of autophagy in the pathogenesis of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Lee, Jae Keun; Shin, Jin Hee; Lee, Ji Eun; Choi, Eui-Ju

    2015-11-01

    Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease characterized by the selective degeneration of upper and lower motor neurons associated with the abnormal aggregation of ubiquitinated proteins. The molecular mechanisms underlying the pathogenesis of ALS remain unclear, however. Autophagy is a major pathway for the elimination of protein aggregates and damaged organelles and therefore contributes to cellular homeostasis. This catabolic process begins with the formation of the double membrane-bound autophagosome that engulfs portions of the cytoplasm and subsequently fuses with a lysosome to form an autolysosome, in which lysosomal enzymes digest autophagic substrates. Defects at various stages of autophagy have been associated with pathological mutations of several ALS-linked genes including SOD1, p62, TDP-43, and optineurin, suggesting that such defects may play a causative role in the pathogenesis of this condition. In this review, we summarize the dysregulation of autophagy associated with ALS as well as potential therapeutic strategies based on modulation of the autophagic process. PMID:26264610

  18. Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways

    Science.gov (United States)

    Buckingham, Erin M.; Jarosinski, Keith W.; Jackson, Wallen; Carpenter, John E.

    2016-01-01

    intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells. PMID:27440906

  19. 细胞自噬的研究方法%Methods for Autophagy Detection

    Institute of Scientific and Technical Information of China (English)

    马泰; 孙国平; 李家斌

    2012-01-01

    Autophagy extensively participate in physiological and pathological processes, and has been focused by contemporary biomedicine scientists in recent years. Transmission electron microscopy, immunofluorescence and immunoblotting techniques were common used in detection of autophagy. Deeper research needs more accurate detection of autophagy. Dysfunction of autophagy involves formation and degeneration of autophosome, accordingly, accurate and comprehensive evaluation of autophagy includes autophosome detection, as well as the fluency of autophagic degeneration, I.e. Autophagic flux assay. Additionally, artificial up- or down-regulation of autophagy by drugs or gene interferences in in vitro or in vivo models has also been considered as important part of autophagy analysis. Any method currently used alone may not been as evidence of autophagy. More careful attention should be paid on results of any assays of autophagy, especially DO NOT interpret "increase or decrease of autophosome" (also "up- or down-expression of autophagy-related proteins") as "enhancement or attenuation of autophagic function".%细胞自噬的研究是目前生物医学领域热点之一,广泛参与各种生理和病理过程.目前普遍采用的自噬检测方法包括电镜、免疫荧光、蛋白质印迹等方法检测自噬体及其标志蛋白.研究的深入对自噬的检测方法也提出了更高的要求,自噬功能障碍包括自噬体形成和降解障碍,因此,准确全面地评估自噬不仅包括自噬体的检测,还包括动态观察整个自噬性降解的过程是否顺畅(即自噬潮分析).另外,通过药物或基因干预技术来人为地调控自噬以观察其在体内体外模型中的作用也是自噬分析的重要内容.需要注意的是,任何一种方法单独应用均不能作为自噬的依据,对任何方法得到的结果进行解释时必须慎重,特别是不能将自噬体的增多减少或自噬相关蛋白表达的高低等同于自噬的增强或减弱.

  20. Autophagy, inflammation and innate immunity in inflammatory myopathies.

    Directory of Open Access Journals (Sweden)

    Cristina Cappelletti

    Full Text Available Autophagy has a large range of physiological functions and its dysregulation contributes to several human disorders, including autoinflammatory/autoimmune diseases such as inflammatory myopathies (IIMs. In order to better understand the pathogenetic mechanisms of these muscular disorders, we sought to define the role of autophagic processes and their relation with the innate immune system in the three main subtypes of IIM, specifically sporadic inclusion body myositis (sIBM, polymyositis (PM, dermatomyositis (DM and juvenile dermatomyositis (JDM. We found that although the mRNA transcript levels of the autophagy-related genes BECN1, ATG5 and FBXO32 were similar in IIM and controls, autophagy activation in all IIM subgroups was suggested by immunoblotting results and confirmed by immunofluorescence. TLR4 and TLR3, two potent inducers of autophagy, were highly increased in IIM, with TLR4 transcripts significantly more expressed in PM and DM than in JDM, sIBM and controls, and TLR3 transcripts highly up-regulated in all IIM subgroups compared to controls. Co-localization between autophagic marker, LC3, and TLR4 and TLR3 was observed not only in sIBM but also in PM, DM and JDM muscle tissues. Furthermore, a highly association with the autophagic processes was observed in all IIM subgroups also for some TLR4 ligands, endogenous and bacterial HSP60, other than the high-mobility group box 1 (HMGB1. These findings indicate that autophagic processes are active not only in sIBM but also in PM, DM and JDM, probably in response to an exogenous or endogenous 'danger signal'. However, autophagic activation and regulation, and also interaction with the innate immune system, differ in each type of IIM. Better understanding of these differences may lead to new therapies for the different IIM types.

  1. Emerging connections between RNA and autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Lubas, Michal; Lund, Anders H

    2016-01-01

    Macroautophagy/autophagy is a key catabolic process, essential for maintaining cellular homeostasis and survival through the removal and recycling of unwanted cellular material. Emerging evidence has revealed intricate connections between the RNA and autophagy research fields. While a majority...... of studies have focused on protein, lipid and carbohydrate catabolism via autophagy, accumulating data supports the view that several types of RNA and associated ribonucleoprotein complexes are specifically recruited to phagophores (precursors to autophagosomes) and subsequently degraded in the lysosome....../vacuole. Moreover, recent studies have revealed a substantial number of novel autophagy regulators with RNA-related functions, indicating roles for RNA and associated proteins not only as cargo, but also as regulators of this process. In this review, we discuss widespread evidence of RNA catabolism via autophagy...

  2. Stress management by autophagy: Implications for chemoresistance.

    Science.gov (United States)

    Huang, Zhao; Zhou, Li; Chen, Zhibin; Nice, Edouard C; Huang, Canhua

    2016-07-01

    Development of chemoresistance, which limits the efficiency of anticancer agents, has long been a major problem in cancer therapy and urgently needs to be solved to improve clinical outcomes. Factors contributing to chemoresistance are various, but a key factor is the cell's capability for stress management. Autophagy, a favored survival strategy that organisms employ to get over many kinds of stress, is emerging as a crucial player in drug resistance. It has been shown that autophagy facilitates the resistance of tumor cells to anticancer agents, and abrogation of autophagy could be therapeutically beneficial in some cases, suggesting autophagy could be a promising target for cancer treatments. Thus, defining the roles of autophagy in chemoresistance, and the mechanisms involved, will be critical to enhance the efficiency of chemotherapy and develop novel anticancer strategy interventions.

  3. Inhibition of mTOR-dependent autophagy sensitizes leukemic cells to cytarabine-induced apoptotic death.

    Directory of Open Access Journals (Sweden)

    Mihajlo Bosnjak

    Full Text Available The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4 and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR, and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.

  4. Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis.

    Science.gov (United States)

    Lee, Hae-Youn; Kim, Jinyoung; Quan, Wenying; Lee, June-Chul; Kim, Min-Soo; Kim, Seok-Hyung; Bae, Jin-Woo; Hur, Kyu Yeon; Lee, Myung-Shik

    2016-08-01

    Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mφs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 β release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD(+):NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mφs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mφs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mφs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mφs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis. PMID:27337687

  5. THE RISK OF GENE TRANSFERRING IN THE INSURANCE PROTECTION OF AGRICULTERE

    Directory of Open Access Journals (Sweden)

    M. Malik

    2016-05-01

    Full Text Available The paper justified essence of genetic engineering as the object of insurance services. Defines the concept of risk gene transferring. The character features of this specific risk. The influence and consequences for agricultural producers. The description of the possible creation of the concept of insurance services that cover risk of gene transferring. The study reveals of the use of GMOs in agriculture, due to issues of economic security of a particular region or country as a whole. To determined the impact of risks and control for developing and developed countries that are important aspects of farming. Changes in weather, climate, productivity, price values, public policy, the situation on global markets can cause large fluctuations in agricultural production, and consequently affecting the income of agricultural producers. Risk management includes a range of strategies that reduce the social and financial implications of possible changes affecting the production and income of farmers. There is a need for an in-depth study of the theoretical and practical aspects of the impact of the risk of gene transferring in the context of insurance protection.

  6. THE RISK OF GENE TRANSFERRING IN THE INSURANCE PROTECTION OF AGRICULTERE

    Directory of Open Access Journals (Sweden)

    M. Malik

    2016-03-01

    Full Text Available The paper justified essence of genetic engineering as the object of insurance services. Defines the concept of risk gene transferring. The character features of this specific risk. The influence and consequences for agricultural producers. The description of the possible creation of the concept of insurance services that cover risk of gene transferring. The study reveals of the use of GMOs in agriculture, due to issues of economic security of a particular region or country as a whole. To determined the impact of risks and control for developing and developed countries that are important aspects of farming. Changes in weather, climate, productivity, price values, public policy, the situation on global markets can cause large fluctuations in agricultural production, and consequently affecting the income of agricultural producers. Risk management includes a range of strategies that reduce the social and financial implications of possible changes affecting the production and income of farmers. There is a need for an in-depth study of the theoretical and practical aspects of the impact of the risk of gene transferring in the context of insurance protection.

  7. Inhibition of autophagy enhances heat-induced apoptosis in human non-small cell lung cancer cells through ER stress pathways.

    Science.gov (United States)

    Xie, Wen-Yue; Zhou, Xiang-Dong; Yang, Juan; Chen, Ling-Xiu; Ran, Dan-Hua

    2016-10-01

    The occurrence and mechanisms of autophagy induced by heat stress are not well known in lung cancer cells. Here, we have demonstrated that heat stress induces autophagy in A549 and NCI-H460 cells through morphological and biochemical analyses. The inhibition of autophagy by chloroquine, 3-methyladenine and Beclin 1 siRNA enhanced heat-induced apoptosis. Moreover, the combination of chloroquine and heat stress inhibited tumor growth and enhanced apoptosis in vivo experiments. In addition, heat-induced autophagy involved the ER stress pathway (PERK- or IRE1-dependent). Further, heat treatment led to the increased phosphorylation of AMPK and the decreased phosphorylation of mTOR in vitro and in vivo. Knockdown of GRP78 inhibited the AMPK-mTOR pathway, and the AMPK inhibitor compound C decreased heat-induced autophagy, suggesting that activation of ER stress was involved in autophagy induction and promotion of the AMPK-mTOR pathway. In conclusion, our data suggested that the heat treatment of lung cancer cells triggered protective autophagy, as mediated by ER stress. Thus, inhibition of autophagy can be a promising strategy to enhance hyperthermia in the treatment of lung cancer patients.

  8. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis.

    Science.gov (United States)

    Hou, Ying-Chen Claire; Chittaranjan, Suganthi; Barbosa, Sharon González; McCall, Kimberly; Gorski, Sharon M

    2008-09-22

    A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death-related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes--death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53-as well as Ras-Raf-mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo. PMID:18794330

  9. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis.

    Science.gov (United States)

    Hou, Ying-Chen Claire; Chittaranjan, Suganthi; Barbosa, Sharon González; McCall, Kimberly; Gorski, Sharon M

    2008-09-22

    A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death-related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes--death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53-as well as Ras-Raf-mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.

  10. A Pichia pastoris VPS15 homologue is required in selective peroxisome autophagy

    NARCIS (Netherlands)

    Stasyk, OV; van der Klei, IJ; Bellu, AR; Shen, SG; Kiel, JAKW; Cregg, JM; Veenhuis, M; Stasyk, Oleh V.; Shen, Shigang; Cregg, James M.

    1999-01-01

    Methylotrophic yeasts contain large peroxisomes during growth on methanol. Upon exposure to excess glucose or ethanol these organelles are selectively degraded by autophagy, Here we describe the cloning of a Pichia pastoris gene (PpVPS15) involved ill peroxisome degradation, which is homologous to S

  11. Autophagy-associated dengue vesicles promote viral transmission avoiding antibody neutralization.

    Science.gov (United States)

    Wu, Yan-Wei; Mettling, Clément; Wu, Shang-Rung; Yu, Chia-Yi; Perng, Guey-Chuen; Lin, Yee-Shin; Lin, Yea-Lih

    2016-01-01

    One of the major defense mechanisms against virus spread in vivo is the blocking of viral infectibility by neutralizing antibodies. We describe here the identification of infectious autophagy-associated dengue vesicles released from infected cells. These vesicles contain viral proteins E, NS1, prM/M, and viral RNA, as well as host lipid droplets and LC3-II, an autophagy marker. The viral RNA can be protected within the autophagic organelles since anti-dengue neutralizing antibodies do not have an effect on the vesicle-mediated transmission that is able to initiate a new round of infection in target cells. Importantly, such infectious vesicles were also detected in a patient serum. Our study suggests that autophagy machinery plays a new role in dengue virus transmission. This discovery explains the inefficiency of neutralizing antibody upon dengue infection as a potential immune evasion mechanism in vivo. PMID:27558165

  12. Autophagy-associated dengue vesicles promote viral transmission avoiding antibody neutralization

    Science.gov (United States)

    Wu, Yan-Wei; Mettling, Clément; Wu, Shang-Rung; Yu, Chia-Yi; Perng, Guey-Chuen; Lin, Yee-Shin; Lin, Yea-Lih

    2016-01-01

    One of the major defense mechanisms against virus spread in vivo is the blocking of viral infectibility by neutralizing antibodies. We describe here the identification of infectious autophagy-associated dengue vesicles released from infected cells. These vesicles contain viral proteins E, NS1, prM/M, and viral RNA, as well as host lipid droplets and LC3-II, an autophagy marker. The viral RNA can be protected within the autophagic organelles since anti-dengue neutralizing antibodies do not have an effect on the vesicle-mediated transmission that is able to initiate a new round of infection in target cells. Importantly, such infectious vesicles were also detected in a patient serum. Our study suggests that autophagy machinery plays a new role in dengue virus transmission. This discovery explains the inefficiency of neutralizing antibody upon dengue infection as a potential immune evasion mechanism in vivo. PMID:27558165

  13. Autophagy induced by snakehead fish vesiculovirus inhibited its replication in SSN-1 cell line.

    Science.gov (United States)

    Wang, Yao; Chen, Nan; Hegazy, Abeer M; Liu, Xiaodan; Wu, Zhixin; Liu, Xueqin; Zhao, Lijuan; Qin, Qiwei; Lan, Jiangfeng; Lin, Li

    2016-08-01

    Autophagy plays an important role in host protection against pathogen infection through activating innate and adaptive immunity. In the present study, we observed that the infection of snakehead fish vesiculovirus (SHVV) could induce apparent autophagy in striped snakehead fish cell line (SSN-1), including clear double-membrane vesicles, fluorescent punctate pattern of microtubule-associated protein 1 light chain 3B (SSN-LC3B) and the conversion of SSN-LC3B-Ⅰ to SSN-LC3B-Ⅱ. Furthermore, we verified that autophagy inhibited the replication of SHVV by assessing mRNA and protein level of nucleoprotein as well as virus titer in the supernatant. These results will shed a new light on the prevention of the infection of SHVV.

  14. Inhibition of caspase-9 aggravates acute liver injury through suppression of cytoprotective autophagy

    Science.gov (United States)

    Guo, Rui; Lin, Bin; Pan, Jing Fei; Liong, Emily C.; Xu, Ai Min; Youdim, Moussa; Fung, Man Lung; So, Kwok Fai; Tipoe, George L.

    2016-01-01

    Acute liver disease is characterized by inflammation, oxidative stress and necrosis, which can greatly influence the long term clinical outcome and lead to liver failure or cancer. Here, we initially demonstrated the beneficial role of caspase-9-dependent autophagy in acute liver injury. Treatment with caspase-9 inhibitor z-LEHD-FMK in HepG2 cells, AML12 cells and C57BL/b6N mice exacerbated CCl4-induced acute hepatocellular damage, and also down-regulated autophagy markers expression levels, indicating that caspase-9 inhibition may aggravate acute liver damage by suppressing cytoprotective autophagy. CCl4 was used as an acute liver injury inducer which caused oxidative stress and apoptosis through up-regulation of HIF-1α, as well as triggered hepatic inflammation and necroptosis via TLR4/NF-κB pathway. Caspase-9 Thr125 site was firstly phosphorylated by ERK1/2 which subsequently activated the cytoprotective autophagy process to attenuate acute CCl4 injury. Caspase-9 inhibition further aggravated hepatic necroptosis through NF-κB expression, leading to increased pro-inflammatory mediators levels, suggesting a protective role of caspase-9-dependent autophagy in the inflammatory process as well as its possibility being a new therapeutic target for the treatment of acute liver injury. PMID:27580936

  15. Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids.

    Science.gov (United States)

    Kuramoto, Kenta; Wang, Nan; Fan, Yuying; Zhang, Weiran; Schoenen, Frank J; Frankowski, Kevin J; Marugan, Juan; Zhou, Yifa; Huang, Sui; He, Congcong

    2016-09-01

    Cannabinoids and related drugs generate profound behavioral effects (such as analgesic effects) through activating CNR1 (cannabinoid receptor 1 [brain]). However, repeated cannabinoid administration triggers lysosomal degradation of the receptor and rapid development of drug tolerance, limiting the medical use of marijuana in chronic diseases. The pathogenic mechanisms of cannabinoid tolerance are not fully understood, and little is known about its prevention. Here we show that a protein involved in macroautophagy/autophagy (a conserved lysosomal degradation pathway), BECN2 (beclin 2), mediates cannabinoid tolerance by preventing CNR1 recycling and resensitization after prolonged agonist exposure, and deletion of Becn2 rescues CNR1 activity in mouse brain and conveys resistance to analgesic tolerance to chronic cannabinoids. To target BECN2 therapeutically, we established a competitive recruitment model of BECN2 and identified novel synthetic, natural or physiological stimuli of autophagy that sequester BECN2 from its binding with GPRASP1, a receptor protein for CNR1 degradation. Co-administration of these autophagy inducers effectively restores the level and signaling of brain CNR1 and protects mice from developing tolerance to repeated cannabinoid usage. Overall, our findings demonstrate the functional link among autophagy, receptor signaling and animal behavior regulated by psychoactive drugs, and develop a new strategy to prevent tolerance and improve medical efficacy of cannabinoids by modulating the BECN2 interactome and autophagy activity. PMID:27305347

  16. Suppression of autophagy and antigen presentation by Mycobacterium tuberculosis PE_PGRS47.

    Science.gov (United States)

    Saini, Neeraj K; Baena, Andres; Ng, Tony W; Venkataswamy, Manjunatha M; Kennedy, Steven C; Kunnath-Velayudhan, Shajo; Carreño, Leandro J; Xu, Jiayong; Chan, John; Larsen, Michelle H; Jacobs, William R; Porcelli, Steven A

    2016-01-01

    Suppression of major histocompatibility complex (MHC) class II antigen presentation is believed to be among the major mechanisms used by Mycobacterium tuberculosis to escape protective host immune responses. Through a genome-wide screen for the genetic loci of M. tuberculosis that inhibit MHC class II-restricted antigen presentation by mycobacteria-infected dendritic cells, we identified the PE_PGRS47 protein as one of the responsible factors. Targeted disruption of the PE_PGRS47 (Rv2741) gene led to attenuated growth of M. tuberculosis in vitro and in vivo, and a PE_PGRS47 mutant showed enhanced MHC class II-restricted antigen presentation during in vivo infection of mice. Analysis of the effects of deletion or over-expression of PE_PGRS47 implicated this protein in the inhibition of autophagy in infected host phagocytes. Our findings identify PE_PGRS47 as a functionally relevant, non-redundant bacterial factor in the modulation of innate and adaptive immunity by M. tuberculosis, suggesting strategies for improving antigen presentation and the generation of protective immunity during vaccination or infection. PMID:27562263

  17. Suppression of autophagy and antigen presentation by Mycobacterium tuberculosis PE_PGRS47.

    Science.gov (United States)

    Saini, Neeraj K; Baena, Andres; Ng, Tony W; Venkataswamy, Manjunatha M; Kennedy, Steven C; Kunnath-Velayudhan, Shajo; Carreño, Leandro J; Xu, Jiayong; Chan, John; Larsen, Michelle H; Jacobs, William R; Porcelli, Steven A

    2016-08-15

    Suppression of major histocompatibility complex (MHC) class II antigen presentation is believed to be among the major mechanisms used by Mycobacterium tuberculosis to escape protective host immune responses. Through a genome-wide screen for the genetic loci of M. tuberculosis that inhibit MHC class II-restricted antigen presentation by mycobacteria-infected dendritic cells, we identified the PE_PGRS47 protein as one of the responsible factors. Targeted disruption of the PE_PGRS47 (Rv2741) gene led to attenuated growth of M. tuberculosis in vitro and in vivo, and a PE_PGRS47 mutant showed enhanced MHC class II-restricted antigen presentation during in vivo infection of mice. Analysis of the effects of deletion or over-expression of PE_PGRS47 implicated this protein in the inhibition of autophagy in infected host phagocytes. Our findings identify PE_PGRS47 as a functionally relevant, non-redundant bacterial factor in the modulation of innate and adaptive immunity by M. tuberculosis, suggesting strategies for improving antigen presentation and the generation of protective immunity during vaccination or infection.

  18. Baicalein Induces Apoptosis and Autophagy via Endoplasmic Reticulum Stress in Hepatocellular Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Zhongxia Wang

    2014-01-01

    Full Text Available Background. Hepatocellular carcinoma (HCC remains a disastrous disease and the treatment for HCC is rather limited. Separation and identification of active compounds from traditionally used herbs in HCC treatment may shed light on novel therapeutic drugs for HCC. Methods. Cell viability and colony forming assay were conducted to determine anti-HCC activity. Morphology of cells and activity of caspases were analyzed. Antiapoptotic Bcl-2 family proteins and JNK were also examined. Levels of unfolded protein response (UPR markers were determined and intracellular calcium was assayed. Small interfering RNAs (siRNAs were used to investigate the role of UPR and autophagy in baicalein-induced cell death. Results. Among four studied flavonoids, only baicalein exhibited satisfactory inhibition of viability and colony formation of HCC cells within water-soluble concentration. Baicalein induced apoptosis via endoplasmic reticulum (ER stress, possibly by downregulating prosurvival Bcl-2 family, increasing intracellular calcium, and activating JNK. CHOP was the executor of cell death during baicalein-induced ER stress while eIF2α and IRE1α played protective roles. Protective autophagy was also triggered by baicalein in HCC cells. Conclusion. Baicalein exhibits prominent anti-HCC activity. This flavonoid induces apoptosis and protective autophagy via ER stress. Combination of baicalein and autophagy inhibitors may represent a promising therapy against HCC.

  19. Beclin1-induced autophagy abrogates radioresistance of lung cancer cells by suppressing osteopontin

    International Nuclear Information System (INIS)

    Osteopontin (OPN) serves as an indicator of resistance to radiotherapy. However, the role of OPN in the development of acquired radioresistance in human lung cancer cells has not yet been fully elucidated. Therefore, the potential importance of OPN as a marker of lung cancer with a potential significant role in the development of radioresistance against repeated radiotherapy has prompted us to define the pathways by which OPN regulates lung cancer cell growth. In addition, autophagy has been reported to play a key role in the radiosensitization of cancer cells. Here, we report that increased OPN expression through induction of nuclear p53 following irradiation was inhibited by exogenous beclin-1 (BECN1). Our results clearly show that BECN1 gene expression led to induction of autophagy and inhibition of cancer cell growth and angiogenesis. Our results suggest that the induction of autophagy abrogated the radioresistance of the cancer cells. Interestingly, we showed that knockdown of OPN by lentivirus-mediated shRNA induced the autophagy of human lung cancer cell. Taken together, these results suggest that OPN and BECN1 can be molecular targets for overcoming radioresistance by controlling autophagy. (author)

  20. Shared developmental roles and transcriptional control of autophagy and apoptosis in Caenorhabditis elegans.

    Science.gov (United States)

    Erdélyi, Péter; Borsos, Eva; Takács-Vellai, Krisztina; Kovács, Tibor; Kovács, Attila L; Sigmond, Tímea; Hargitai, Balázs; Pásztor, Líz; Sengupta, Tanima; Dengg, Marlene; Pécsi, Ildikó; Tóth, Judit; Nilsen, Hilde; Vértessy, Beáta G; Vellai, Tibor

    2011-05-01

    Autophagy is a lysosome-mediated self-degradation process of eukaryotic cells that, depending on the cellular milieu, can either promote survival or act as an alternative mechanism of programmed cell death (PCD) in terminally differentiated cells. Despite the important developmental and medical implications of autophagy and the main form of PCD, apoptosis, orchestration of their regulation remains poorly understood. Here, we show in the nematode Caenorhabditis elegans, that various genetic and pharmacological interventions causing embryonic lethality trigger a massive cell death response that has both autophagic and apoptotic features. The two degradation processes are also redundantly required for normal development and viability in this organism. Furthermore, the CES-2-like basic region leucine-zipper (bZip) transcription factor ATF-2, an upstream modulator of the core apoptotic cell death pathway, is able to directly regulate the expression of at least two key autophagy-related genes, bec-1/ATG6 and lgg-1/ATG8. Thus, the two cell death mechanisms share a common method of transcriptional regulation. Together, these results imply that under certain physiological and pathological conditions, autophagy and apoptosis are co-regulated to ensure the proper morphogenesis and survival of the developing organism. The identification of apoptosis and autophagy as compensatory cellular pathways in C. elegans might help us to understand how dysregulated PCD in humans can lead to diverse pathologies, including cancer, neurodegeneration and diabetes. PMID:21502138

  1. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy.

    Science.gov (United States)

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Bedia, Carmen; Daniels, Craig; Abraham, Gilu; Stogios, Peter J; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Ong, Sze Ying; Hartland, Elizabeth L; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-02-16

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis. PMID:26831115

  2. Relationship between autophagy and aging%细胞自噬与衰老

    Institute of Scientific and Technical Information of China (English)

    席兴字

    2011-01-01

    Aging is an intrinsic property of organism, which is characterized by the accumulation of damaged protein aggregates in cells and tissues. Clearance of protein aggregates mainly relies on ubiquitin-proteasome system and lysosome-dependent autophagy pathway.In recent years, some autophagy genes have been reported to play an important role in aging in models organisms, and these findings motivate interests in developing anti-aging drugs. This paper summarized the mechanisms of autophagy, and discussed the connections between aging and autophagy.%衰老是生物体内在的性质,细胞中损伤蛋白质积累是生物体衰老的一个重要特征.蛋门质聚集体的清除主要依赖于泛素-蛋白酶体途径和依赖溶酶体的细胞自噬(autophagy)途径.近年来,研究人员在许多模式生物中发现一些细胞自噬基因在衰老中起重要作用,这些研究激发人们利用细胞自噬开发抗衰老药物的兴趣.本文就细胞自噬的机理、衰老与自噬之间的联系等进行综述.

  3. The expression and significance of autophagy related gene in rats with non-alcoholic fatty liver disease%自噬相关基因在大鼠非酒精性脂肪性肝病中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    罗燕; 王燕; 庄振杰; 柳银兰; 施军平; 娄国强

    2015-01-01

    Objective To investigate the expression and significance of autophagy related gene in rats with non-alcoholic fatty liver disease (NAFLD).Methods In vivo model of NAFLD was established in SD rats by high fat diet (model group,n =24),while the rats with normal food were set as control group (n =18).The rats of each group were sacrificed at the end of 4,8,12 weeks respectively.Body weight,liver wet weight,liver function,liver lipid metabolism and other indicators were measured.Hepatic histology was evaluated by hematoxylin-eosin (HE) staining and oil red O staining.The identification of autophagy were morphologically visualized by transmission electron microscopy (TEM).The expression of autophagy related gene ATG7,beclin1,microtubule-associated protein 1 lightchain3 (LC3) were detected by real-time quantitative RT-PCR (qRT-PCR) for mRNA levels.Result ① Compared with the control group at the corresponding time point,the weights of the mice and their livers.②Hepatic oil red O staining areas and density in high fat diet group all increased.HE staining revealed different degrees of macrosteatosis accompanied with intralobular inflammatary foci.With a remarkable histological change of NASH (typical hepatocellular ballooning and perisinusoidal fibrosis) about 80% of mice from high fat diet group had NASH [NAFLD activity score (NAS) ≥5] at the end of 12 weeks.③ TEM pictures showed that autophagy bubble appeared since high fat diet feeding for 4 weeks.④ The relative expression of ATG7,Beclin1,LC3-Ⅰ,LC3-Ⅱ at mRNA level were up-regulated after high fat diet,and reached their peak at the end of 8 weeks when compared with the control group.The differences can be applied to statistical significance (P < 0.05).The expression of these autophagy related genes in rats of high fat diet group were down-regulated at the end of 12 weeks,which had no difference the control group.Conclusion ①High fat diet feeding is able to induce a rat model of NAFLD.②The expression

  4. Induction of Autophagy in the Striatum and Hypothalamus of Mice after 835 MHz Radiofrequency Exposure.

    Science.gov (United States)

    Kim, Ju Hwan; Huh, Yang Hoon; Kim, Hak Rim

    2016-01-01

    The extensive use of wireless mobile phones and associated communication devices has led to increasing public concern about potential biological health-related effects of the exposure to electromagnetic fields (EMFs). EMFs emitted by a mobile phone have been suggested to influence neuronal functions in the brain and affect behavior. However, the affects and phenotype of EMFs exposure are unclear. We applied radiofrequency (RF) of 835 MHz at a specific absorption rate (SAR) of 4.0 W/kg for 5 hours/day for 4 and 12 weeks to clarify the biological effects on mouse brain. Interestingly, microarray data indicated that a variety of autophagic related genes showed fold-change within small range after 835 MHz RF exposure. qRT-PCR revealed significant up-regulation of the autophagic genes Atg5, LC3A and LC3B in the striatum and hypothalamus after a 12-week RF. In parallel, protein expression of LC3B-II was also increased in both brain regions. Autophagosomes were observed in the striatum and hypothalamus of RF-exposed mice, based on neuronal transmission electron microscopy. Taken together, the results indicate that RF exposure of the brain can induce autophagy in neuronal tissues, providing insight into the protective mechanism or adaptation to RF stress. PMID:27073885

  5. Coordination of autophagy with other cellular activities

    Institute of Scientific and Technical Information of China (English)

    Yan WANG; Zheng-hong QIN

    2013-01-01

    The cell biological phenomenon of autophagy has attracted increasing attention in recent years,partly as a consequence of the discovery of key components of its cellular machinery.Autophagy plays a crucial role in a myriad of cellular functions.Autophagy has its own regulatory mechanisms,but this process is not isolated.Autophagy is coordinated with other cellular activities to maintain cell homeostasis.Autophagy is critical for a range of human physiological processes.The multifunctional roles of autophagy are explained by its ability to interact with several key components of various cell pathways.In this review,we focus on the coordination between autophagy and other physiological processes,including the ubiquitin-proteasome system (UPS),energy homeostasis,aging,programmed cell death,the immune responses,microbial invasion and inflammation.The insights gained from investigating autophagic networks should increase our understanding of their roles in human diseases and their potential as targets for therapeutic intervention.

  6. Autophagy in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Thien Tra

    Full Text Available Autophagy (macroautophagy is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

  7. Autophagy in Mycobacterium tuberculosis and HIV infections

    Directory of Open Access Journals (Sweden)

    Lucile eEspert

    2015-06-01

    Full Text Available Human Immunodeficiency Virus (HIV and Mycobacterium tuberculosis (M.tb are among the most lethal human pathogens worldwide, each being responsible for around 1.5 million deaths annually. Moreover, synergy between acquired immune deficiency syndrome (AIDS and tuberculosis (TB has turned HIV/M.tb co-infection into a major public health threat in developing countries. In the past decade, autophagy, a lysosomal catabolic process, has emerged as a major host immune defense mechanism against infectious agents like M.tb and HIV. Nevertheless, in some instances, autophagy machinery appears to be instrumental for HIV infection. Finally, there is mounting evidence that both pathogens deploy various countermeasures to thwart autophagy. This mini-review proposes an overview of the roles and regulations of autophagy in HIV and M.tb infections with an emphasis on microbial factors. We also discuss the role of autophagy manipulation in the context of HIV/M.tb co-infection. In future, a comprehensive understanding of autophagy interaction with these pathogens will be critical for development of autophagy-based prophylactic and therapeutic interventions for AIDS and TB.

  8. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  9. Effect of Autophagy Over Liver Diseases

    Institute of Scientific and Technical Information of China (English)

    Dong-qian Yi; Xue-feng Yang; Duan-fang Liao; Qing Wu; Nian Fu; Yang Hu; Ting Cao

    2016-01-01

    Abstract In recent years, increasingly evidences show that autophagy plays an important role in the pathogenesis and development of liver diseases, and the relationship between them has increasingly become a focus of concern. Autophagy refers to the process through which the impaired organelles, misfolded protein, and intruding microorganisms is degraded by lysosomes to maintain stability inside cells. This article states the effect of autophagy on liver diseases (hepatic fibrosis, fatty liver, viral hepatitis, and liver cancer), which aims to provide a new direction for the treatment of liver diseases.

  10. RAP1 Protects from Obesity through Its Extratelomeric Role Regulating Gene Expression

    Directory of Open Access Journals (Sweden)

    Paula Martínez

    2013-06-01

    Full Text Available RAP1 is part of shelterin, the protective complex at telomeres. RAP1 also binds along chromosome arms, where it is proposed to regulate gene expression. To investigate the nontelomeric roles of RAP1 in vivo, we generated a RAP1 whole-body knockout mouse. These mice show early onset of obesity, which is more severe in females than in males. Rap1-deficient mice show accumulation of abdominal fat, hepatic steatosis, and high-fasting plasma levels of insulin, glucose, cholesterol, and alanine aminotransferase. Gene expression analyses of liver and visceral white fat from Rap1-deficient mice before the onset of obesity show deregulation of metabolic programs, including fatty acid, glucose metabolism, and PPARα signaling. We identify Pparα and Pgc1α as key factors affected by Rap1 deletion in the liver. We show that RAP1 binds to Pparα and Pgc1α loci and modulates their transcription. These findings reveal a role for a telomere-binding protein in the regulation of metabolism.

  11. Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo

    OpenAIRE

    Motoki, Atsuko; Merkel, Matthias J.; Packwood, William H.; Cao, Zhiping; Liu, Lijuan; Iliff, Jeffrey; Alkayed, Nabil J.; Van Winkle, Donna M.

    2008-01-01

    Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ...

  12. 自噬调节与肿瘤治疗%Autophagy modulation in cancer therapy

    Institute of Scientific and Technical Information of China (English)

    杨晓亮

    2011-01-01

    自噬是一个细胞质成分被双层膜的囊泡包裹并与溶酶体融合降解的过程.肿瘤细胞利用自噬过程存活于代谢应激下,因此推测自噬抑制剂在肿瘤治疗中具有临床应用价值.与此相矛盾的是在人类乳腺肿瘤、卵巢肿瘤、前列腺肿瘤中,重要的自噬基因beclinl等位性丢失发生很频繁,表明自噬的受损促使肿瘤的发生.与自噬缺陷和对代谢应激损伤耐受有关的肿瘤,其发生的可能原因包括增加炎症反应导致的细胞死亡及细胞因子的产生,染色体组的损伤.这表明自噬诱导剂通过限制细胞基因组损伤、细胞死亡及炎症来预防肿瘤可能具有价值.因为自噬是细胞应激反应的重要组成部分,所以通过调节自噬过程进行肿瘤的预防及治疗将是一个很有前景的新领域.%Autophagy is a process by which cytoplasmic components are sequestered in double membrane vesicles and degraded upon fusion with lysosomal compartments. Autophagy is utilized by tumor cells to survive meta bolic stress and as such autophagy inhibitors are predicted to have clinical value in the setting of cancer therapy. Paradoxically, allelic loss of the essential autophagy gene beclinl is found with high frequency in human breast, ovarian and prostate cancers, suggesting that impaired autophagy promotes cancer. Probable causes of cancer associated with defects in autophagy and impaired tolerance to stress include enhanced cell death resulting in an inflammatory response and cytokine production, and also genome damage. This suggests that autophagy inducers would also be valuable in the setting of cancer prevention to limit cellular genome damage, cell death and inflammation. As autophagy is a major component of the cellular stress response, modulating the autophagy pathway is a promising new area in cancer prevention and therapy.

  13. Autophagy supports color vision.

    Science.gov (United States)

    Zhou, Zhenqing; Vinberg, Frans; Schottler, Frank; Doggett, Teresa A; Kefalov, Vladimir J; Ferguson, Thomas A

    2015-01-01

    Cones comprise only a small portion of the photoreceptors in mammalian retinas. However, cones are vital for color vision and visual perception, and their loss severely diminishes the quality of life for patients with retinal degenerative diseases. Cones function in bright light and have higher demand for energy than rods; yet, the mechanisms that support the energy requirements of cones are poorly understood. One such pathway that potentially could sustain cones under basal and stress conditions is macroautophagy. We addressed the role of macroautophagy in cones by examining how the genetic block of this pathway affects the structural integrity, survival, and function of these neurons. We found that macroautophagy was not detectable in cones under normal conditions but was readily observed following 24 h of fasting. Consistent with this, starvation induced phosphorylation of AMPK specifically in cones indicating cellular starvation. Inhibiting macroautophagy in cones by deleting the essential macroautophagy gene Atg5 led to reduced cone function following starvation suggesting that cones are sensitive to systemic changes in nutrients and activate macroautophagy to maintain their function. ATG5-deficiency rendered cones susceptible to light-induced damage and caused accumulation of damaged mitochondria in the inner segments, shortening of the outer segments, and degeneration of all cone types, revealing the importance of mitophagy in supporting cone metabolic needs. Our results demonstrate that macroautophagy supports the function and long-term survival of cones providing for their unique metabolic requirements and resistance to stress. Targeting macroautophagy has the potential to preserve cone-mediated vision during retinal degenerative diseases.

  14. Effect of P39 Gene Deletion in Live Brucella Vaccine Strains on Residual Virulence and Protective Activity in Mice

    OpenAIRE

    Tibor, Anne; Jacques, Isabelle; Guilloteau, Laurence; Verger, Jean-Michel; Grayon, Maggy; Wansard, Valerie; Letesson, Jean-Jacques

    1998-01-01

    The 39-kilodalton protein (P39) has previously been shown to be an immunodominant protein in Brucella infections. P39 gene deletion mutants of vaccine strains Brucella abortus S19 and Brucella melitensis Rev.1 were constructed by gene replacement. This deletion did not significantly modify the residual virulence of both vaccine strains in CD-1 mice. CD-1 mice vaccinated with the parent or mutant strains were protected against a virulent challenge. Mutant vaccine strains devoid of P39 could pr...

  15. Absolute Protection and Relative Protection:Comparative Thinking of Gene Patent Protection Principle%绝对保护与相对保护:基因专利保护原则的选择

    Institute of Scientific and Technical Information of China (English)

    高莉

    2014-01-01

    Although most countries agree,to a certain extent,that gene has patent qualification,issues such as the scope of protection of gene patent,patent assessment are still the focus of the argument in recent years. Absolute protection and relative protection are now mainly taken by all countries on the patent protection of gene two principles,which define the scope of protection of gene patent. Each has its own advantages and disadvantages. No matter what kind of protection prin-ciple is used alone,it is biased. At the same time,although the relative protectionism,also known as "functional limit"principle is proposed based on the criticism of the absolute protection,its adoption of patent qualification has caused new influence and brought new challenges.%尽管大多数国家都在一定程度上承认基因具有可专利性资格,然而,基因专利保护范围的界定、可专利性审查等问题仍然是近年来争论的焦点。绝对保护与相对保护是目前各国主要采用的有关基因专利保护的两种原则,这两种原则对于基因专利保护范围的界定各有利弊,如单独适用会有失偏颇。同时,虽然相对保护主义,是在对绝对保护主义批判的基础上提出的,但是它的采用可能影响可专利性资格的确定。因此,两种原则应因情况,融合适用。

  16. Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly.

    Science.gov (United States)

    Lapaquette, Pierre; Glasser, Anne-Lise; Huett, Alan; Xavier, Ramnik J; Darfeuille-Michaud, Arlette

    2010-01-01

    Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells. Recent genome-wide association studies have highlighted the autophagy pathway as being associated with CD risk. In the present study we investigated whether defects in autophagy enhance replication of commensal and pathogenic Escherichia coli and CD-associated AIEC. We show that functional autophagy limits intracellular AIEC replication and that a subpopulation of the intracellular bacteria is located within LC3-positive autophagosomes. In IRGM and ATG16L1 deficient cells intracellular AIEC LF82 bacteria have enhanced replication. Surprisingly autophagy deficiency did not interfere with the ability of intracellular bacteria to survive and/or replicate for any other E. coli strains tested, including non-pathogenic, environmental, commensal, or pathogenic strains involved in gastro enteritis. Together these findings demonstrate a central role for autophagy restraining Adherent-Invasive E. coli strains associated with ileal CD. AIEC infection in patients with polymorphisms in autophagy genes may have a significant impact on the outcome of intestinal inflammation. PMID:19747213

  17. Autophagy-dependent regulatory T cells are critical for the control of graft-versus-host disease

    Science.gov (United States)

    Le Texier, Laëtitia; Lineburg, Katie E.; Leveque-El Mouttie, Lucie; Nicholls, Jemma; Melino, Michelle; Nalkurthi, Blessy C.; Alexander, Kylie A.; Teal, Bianca; Blake, Stephen J.; Souza-Fonseca-Guimaraes, Fernando; Engwerda, Christian R.; Kuns, Rachel D.; Lane, Steven W.; Teh, Charis; Gray, Daniel; Clouston, Andrew D.; Nilsson, Susan K.; Blazar, Bruce R.; Hill, Geoffrey R.; MacDonald, Kelli P.A.

    2016-01-01

    Regulatory T cells (Tregs) play a crucial role in the maintenance of peripheral tolerance. Quantitative and/or qualitative defects in Tregs result in diseases such as autoimmunity, allergy, malignancy, and graft-versus-host disease (GVHD), a serious complication of allogeneic stem cell transplantation (SCT). We recently reported increased expression of autophagy-related genes (Atg) in association with enhanced survival of Tregs after SCT. Autophagy is a self-degradative process for cytosolic components that promotes cell homeostasis and survival. Here, we demonstrate that the disruption of autophagy within FoxP3+ Tregs (B6.Atg7fl/fl-FoxP3cre+) resulted in a profound loss of Tregs, particularly within the bone marrow (BM). This resulted in dysregulated effector T cell activation and expansion, and the development of enterocolitis and scleroderma in aged mice. We show that the BM compartment is highly enriched in TIGIT+ Tregs and that this subset is differentially depleted in the absence of autophagy. Moreover, following allogeneic SCT, recipients of grafts from B6.Atg7fl/fl-FoxP3cre+ donors exhibited reduced Treg reconstitution, exacerbated GVHD, and reduced survival compared with recipients of B6.WT-FoxP3cre+ grafts. Collectively, these data indicate that autophagy-dependent Tregs are critical for the maintenance of tolerance after SCT and that the promotion of autophagy represents an attractive immune-restorative therapeutic strategy after allogeneic SCT. PMID:27699243

  18. Hypercapnia Inhibits Autophagy and Bacterial Killing in Human Macrophages by Increasing Expression of Bcl-2 and Bcl-xL

    Science.gov (United States)

    Casalino-Matsuda, S. Marina; Nair, Aisha; Beitel, Greg J.; Gates, Khalilah L.; Sporn, Peter H. S.

    2015-01-01

    Hypercapnia, the elevation of CO2 in blood and tissue, commonly develops in patients with advanced lung disease and severe pulmonary infections, and is associated with high mortality. We previously reported that hypercapnia alters expression of host defense genes, inhibits phagocytosis, and increases the mortality of Pseudomonas pneumonia in mice. However, the effect of hypercapnia on autophagy, a conserved process by which cells sequester and degrade proteins and damaged organelles that also plays a key role in antimicrobial host defense and pathogen clearance, has not previously been examined. In the present study we show that hypercapnia inhibits autophagy induced by starvation, rapamycin, LPS, heat-killed and live bacteria in the human macrophage. Inhibition of autophagy by elevated CO2 was not attributable to acidosis. Hypercapnia also reduced macrophage killing of Pseudomonas aeruginosa. Moreover, elevated CO2 induced the expression of Bcl-2 and Bcl-xL, anti-apoptotic factors that negatively regulate autophagy by blocking Beclin 1, an essential component of the autophagy initiation complex. Furthermore, siRNA targeting Bcl-2 and Bcl-xL and the small molecule Z36, which blocks Bcl-2 and Bcl-xL binding to Beclin 1, prevented hypercapnic inhibition of autophagy and bacterial killing. These results suggest that targeting the Bcl-2/Bcl-xL-Beclin 1 interaction may hold promise for ameliorating hypercapnia-induced immunosuppression and improving resistance to infection in patients with advanced lung disease and hypercapnia. PMID:25895534

  19. Nanomaterial-modulated autophagy: underlying mechanisms and functional consequences.

    Science.gov (United States)

    Zheng, Wei; Wei, Min; Li, Song; Le, Weidong

    2016-06-01

    Autophagy is an essential lysosome-dependent process that controls the quality of the cytoplasm and maintains cellular homeostasis, and dysfunction of this protein degradation system is correlated with various disorders. A growing body of evidence suggests that nanomaterials (NMs) have autophagy-modulating effects, thus predicting a valuable and promising application potential of NMs in the diagnosis and treatment of autophagy-related diseases. NMs exhibit unique physical, chemical and biofunctional properties, which may endow NMs with capabilities to modulate autophagy via various mechanisms. The present review highlights the impacts of various NMs on autophagy and their functional consequences. The possible underlying mechanisms for NM-modulated autophagy are also discussed.

  20. Autophagy inhibition plays the synergetic killing roles with radiation in the multi-drug resistant SKVCR ovarian cancer cells

    International Nuclear Information System (INIS)

    Autophagy has attracted attentions as a novel mechanism for tumor development. In this study Human ovarian carcinoma cell line SKOV3 and multidrug-resistant phenotype SKVCR cells were used and the roles of autophagy in radiation-induced cell death were analyzed. Cell viability was examined by colony formation and cell counting kit-8 (CCK-8) assay, 3MA and ZVAD were used to block autophagy and apoptosis, respectively. Quantitative real-time PCR was used to detect mRNA level and Western blot was used to detect protein expression, monodansylcadaverine (MDC) staining and flow cytometery were used for autophagy, apoptosis and cell cycle dynamics, respectively. (1) The radiosensitivity exhibited differently in SKOV3 and SKVCR cells (SKOV3: D0=3.37, SKVCR: D0= 4.18); compared with SKOV3 the constitutive expression of MAPLC3 in SKVCR was higher, but no change of Caspase-3 and cleaved Caspase-3. (2) The ionizing radiation (IR)- induced apoptosis and autophagy were significant in both cells (P<0.05); inhibition of apoptosis with ZVAD showed no impact on survival of SKOV3 and SKVCR cells after radiation, while inhibition of autophagy significantly decreased viability in SKVCR cells, for SKVO3 cells only low level of radiation (2 Gy and 4 Gy) could decrease the viability(P<0.05). (3) ZVAD inhibited apoptosis and autophagy in both cells, 3MA inhibit apoptosis in SKOV3, and promote apoptosis in SKVCR, together with inhibition of autophagy. (4) G2/M arrest was induced by radiation in both cells; the accumulation of G2/M was more significant in SKOV3, 3MA attenuated the radiation-induced S phase delay in SKVCR. IR-induced autophagy provides a self-protective mechanism against radiotherapy in SKVCR cells, the use of autophagy inhibitor, 3MA, increases the killing effects of radiation by inhibiting autophagy and radiation- induced S phase delay, also by the increase of apoptosis, which suggests a better therapeutic strategy in drug- resistant SKVCR ovarian cancer cells

  1. The role of autophagy in Parkinson's disease☆

    OpenAIRE

    Zhang, Lei; Dong, Yaru; Xu, Xiaoheng; Xu, Zhong

    2012-01-01

    Although Parkinson's disease is the most common neurodegenerative movement disorder, the mechanisms of pathogenesis remain poorly understood. Recent findings have shown that deregulation of the autophagy-lysosome pathway is involved in the pathogenesis of Parkinson's disease. This review summarizes the most recent findings and discusses the unique role of the autophagy-lysosome pathway in Parkinson's disease to highlight the possibility of Parkinson's disease treatment strategies that incorpo...

  2. Skeletal Muscle Autophagy: A New Metabolic Regulator

    OpenAIRE

    Neel, Brian A.; Lin, Yuxi; Pessin, Jeffrey E.

    2013-01-01

    Autophagy classically functions as a physiological process to degrade cytoplasmic components, protein aggregates, and/or organelles, as a mechanism for nutrient breakdown, and as a regulator of cellular architecture. Proper autophagic flux is vital for both functional skeletal muscle, which controls support and movement of the skeleton, and muscle metabolism. The role of autophagy as a metabolic regulator in muscle has been previously studied; however, the underlying molecular mechanisms that...

  3. Mechanisms of mitochondria and autophagy crosstalk

    OpenAIRE

    Rambold, Angelika S.; Lippincott-Schwartz, Jennifer

    2011-01-01

    Autophagy is a cellular survival pathway that recycles intracellular components to compensate for nutrient depletion and ensures the appropriate degradation of organelles. Mitochondrial number and health are regulated by mitophagy, a process by which excessive or damaged mitochondria are subjected to autophagic degradation. Autophagy is thus a key determinant for mitochondrial health and proper cell function. Mitophagic malfunction has been recently proposed to contribute to progressive neuro...

  4. Autophagy in lung disease pathogenesis and therapeutics

    OpenAIRE

    Ryter, Stefan W.; Augustine M K Choi

    2015-01-01

    Autophagy, a cellular pathway for the degradation of damaged organelles and proteins, has gained increasing importance in human pulmonary diseases, both as a modulator of pathogenesis and as a potential therapeutic target. In this pathway, cytosolic cargos are sequestered into autophagosomes, which are delivered to the lysosomes where they are enzymatically degraded and then recycled as metabolic precursors. Autophagy exerts an important effector function in the regulation of inflammation, an...

  5. Autophagy and its neuroprotection in neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    Ping Gu; Avaneesh Jakkoju; Mingwei Wang; Weidong Le

    2011-01-01

    It has been suggested that protein misfolding and aggregation contribute significantly to the development of neurodegenerative diseases. Misfolded and aggregated proteins are cleared by ubiquitin proteasomal system (UPS) and by both Micro and Macro autophagy lysosomal pathway (ALP). Autophagosomal dysfunction has been implicated in an increasing number of diseases including neurodegenerative diseases. Autophagy is a cellular self-eating process that plays an important role in neuroprotection as well as neuronal injury and death. While a decrease in autophagic activity interferes with protein degradation and possibly organelle turnover, increased autophagy has been shown to facilitate the clearance of aggregation-prone proteins and promote neuronal survival in a number of disease models. On the other hand, too much autophagic activity can be detrimental, suggesting the regulation of autophagy is critical in dictating cell fate. In this review paper, we will discuss various aspects of ALP biology and its dual functions in neuronal cell death and survival. We will also evaluate the role of autophagy in neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis. Finally, we will explore the therapeutic potential of autophagy modifiers in several neurodegenerative diseases.

  6. Autophagy in the control of food intake.

    Science.gov (United States)

    Singh, Rajat

    2012-04-01

    The cellular nutrient sensing apparatus detects nutritional depletion and transmits this information to downstream effectors that generate energy from alternate sources. Autophagy is a crucial catabolic pathway that turns over redundant cytoplasmic components in lysosomes to provide energy to the starved cell. Recent studies have described a role for hypothalamic autophagy in the control of food intake and energy balance. Activated autophagy in hypothalamic neurons during starvation mobilized neuron-intrinsic lipids to generate free fatty acids that increased AgRP levels. AgRP neuron-specific inhibition of autophagy decreased fasting-induced increases in AgRP levels and food intake. Deletion of autophagy in AgRP neurons led to constitutive increases in levels of proopiomelanocortin and its active processed product, α-melanocyte stimulating hormone that contributed to reduced adiposity in these rodents. The current manuscript discusses these new findings and raises additional questions that may help understand how hypothalamic autophagy controls food intake and energy balance. These studies may have implications for designing new therapies against obesity and insulin resistance. PMID:23700515

  7. Autophagy in stem and progenitor cells.

    Science.gov (United States)

    Rodolfo, Carlo; Di Bartolomeo, Sabrina; Cecconi, Francesco

    2016-02-01

    Autophagy is a highly conserved cellular process, responsible for the degradation and recycling of damaged and/or outlived proteins and organelles. This is the major cellular pathway, acting throughout the formation of cytosolic vesicles, called autophagosomes, for the delivering to lysosome. Recycling of cellular components through autophagy is a crucial step for cell homeostasis as well as for tissue remodelling during development. Impairment of this process has been related to the pathogenesis of various diseases, such as cancer and neurodegeneration, to the response to bacterial and viral infections, and to ageing. The ability of stem cells to self-renew and differentiate into the mature cells of the body renders this unique type of cell highly crucial to development and tissue renewal, not least in various diseases. During the last two decades, extensive knowledge about autophagy roles and regulation in somatic cells has been acquired; however, the picture about the role and the regulation of autophagy in the different types of stem cells is still largely unknown. Autophagy is a major player in the quality control and maintenance of cellular homeostasis, both crucial factors for stem cells during an organism's life. In this review, we have highlighted the most significant advances in the comprehension of autophagy regulation in embryonic and tissue stem cells, as well as in cancer stem cells and induced pluripotent cells.

  8. Effect of rumen-protected choline supplementation on liver and adipose gene expression during the transition period in dairy cattle

    NARCIS (Netherlands)

    Goselink, R.M.A.; Baal, van J.; Widjaja, H.C.A.; Dekker, R.A.; Zom, R.L.G.; Veth, M.J.; Vuuren, van A.M.

    2013-01-01

    We previously reported that supplementation of rumen-protected choline (RPC) reduces the hepatic triacylglycerol concentration in periparturient dairy cows during early lactation. Here, we investigated the effect of RPC on the transcript levels of lipid metabolism-related genes in liver and adipose

  9. Autophagy provides nutrients for nonassimilating fungal structures and is necessary for plant colonization but not for infection in the necrotrophic plant pathogen Fusarium graminearum

    DEFF Research Database (Denmark)

    Josefsen, Lone; Droce, Aida; Søndergaard, Teis;

    2012-01-01

    The role of autophagy in necrotrophic fungal physiology and infection biology is poorly understood. We have studied autophagy in the necrotrophic plant pathogen Fusarium graminearum in relation to development of nonassimilating structures and infection. We identified an ATG8 homolog F. graminearum...... starvation is severely inhibited in the Delta Fgatg8 strain demonstrating autophagy-dependent lipid utilization, lipophagy, in fungi. Radial growth rate of the Delta Fgatg8 strain is reduced compared with the wild type and the mutant does not grow over inert plastic surfaces in contrast to the wild type....... The ability to infect barley and wheat is normal but the mutant is unable to spread from spikelet to spikelet in wheat. Complementation by inserting the F. graminearum atg8 gene into a region adjacent to the actin gene in Delta Fgatg8 fully restores the WT phenotype. The results showed that autophagy plays...

  10. 6-Gingerol Protects against Nutritional Steatohepatitis by Regulating Key Genes Related to Inflammation and Lipid Metabolism

    Directory of Open Access Journals (Sweden)

    Thing-Fong Tzeng

    2015-02-01

    Full Text Available Non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis (NASH, appears to be increasingly common worldwide. The aim of the study was to investigate the effects of 6-gingerol ((S-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl-3-decanone, a bioactive ingredient of plants belonging to the Zingiberaceae family, on experimental models of NASH. In HepG2 cells, 6-gingerol (100 μmol/L treatment inhibited free fatty acids mixture (0.33 mmol/L palmitate and 0.66 mmol/L oleate-induced triglyceride and inflammatory marker accumulations. Male C57BL/6 mice were fed with a methionine and choline-deficient (MCD diet to induce steatohepatitis. After four weeks of MCD diet feeding, the mice were dosed orally with 6-gingerol (25, 50 or 100 mg/kg/day once daily for another four weeks. 6-Gingerol (100 mg/kg/day attenuated liver steatosis and necro-inflammation in MCD diet-fed mice. The expressions of inflammatory cytokine genes, including those for monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6, and nuclear transcription factor (NF-κB, which were increased in the livers of MCD diet-fed mice, were attenuated by 6-gingerol. 6-Gingerol possesses a repressive property on hepatic steatosis, which is associated with induction of peroxisome proliferator-activated receptor α. Our study demonstrated the protective role of 6-gingerol in ameliorating nutritional steatohepatitis. The effect was mediated through regulating key genes related to lipid metabolism and inflammation.

  11. Anti tumor and autophagy%细胞自噬与抗肿瘤

    Institute of Scientific and Technical Information of China (English)

    史海涛; 王攀

    2009-01-01

    Autophagy is a catabolic process that widely presents in eukaryotic cells. It is involved in many physiological and pathological processes. The regulation of autophagy is complex and signaling pathways play important roles in this process. Abnormal autophagy is related with occurrence and development of tumor. And it has multi-faceted impact on the process of tumor. The research of autophagy not only reveals complexity and diversity of organism's own regulation and control, but also provides new ideas for tumor gene therapy and for overcoming tumor drug resistance research. Here, this article reviews the latest research progress on the occurrence, regulation, functions, molecular mechanism of autophagy as well as the relationship between autophagy and tumors, to provide clues to anti-cancer research.%细胞的自体吞噬现象普遍存在于真核细胞,其参与细胞诸多生理和病理过程,井受复杂的信号级联网络调控.自噬异常与肿瘤的发生、发展有关,可从多个层面影响肿瘤发生的进程.研究细胞自噬不仅能揭示生物自身调控的复杂性和多样性,同时为肿瘤基因治疗及克服肿瘤耐药性的研究提供了新的思路.本文拟通过对自噬的发生、功能、分子机制及与肿瘤的关系的最新研究进展综述,为抗肿瘤研究提供线索.

  12. Elastase induces lung epithelial cell autophagy through placental growth factor: a new insight of emphysema pathogenesis.

    Science.gov (United States)

    Hou, Hsin-Han; Cheng, Shih-Lung; Chung, Kuei-Pin; Kuo, Mark Yen-Ping; Yeh, Cheng-Chang; Chang, Bei-En; Lu, Hsuan-Hsuan; Wang, Hao-Chien; Yu, Chong-Jen

    2014-09-01

    Chronic obstructive pulmonary disease (COPD) is a devastating disease, which is associated with increasing mortality and morbidity. Therefore, there is a need to clearly define the COPD pathogenic mechanism and to explore effective therapies. Previous studies indicated that cigarette smoke (CS) induces autophagy and apoptosis in lung epithelial (LE) cells. Excessive ELANE/HNE (elastase, neutrophil elastase), a factor involved in protease-antiprotease imbalance and the pathogenesis of COPD, causes LE cell apoptosis and upregulates the expression of several stimulus-responsive genes. However, whether or not elastase induces autophagy in LE cell remains unknown. The level of PGF (placental growth factor) is higher in COPD patients than non-COPD controls. We hypothesize that elastase induces PGF expression and causes autophagy in LE cells. In this study, we demonstrated that porcine pancreatic elastase (PPE) induced PGF expression and secretion in LE cells in vitro and in vivo. The activation of MAPK8/JNK1 (mitogen-activated protein kinase 8) and MAPK14/p38alpha MAPK signaling pathways was involved in the PGF mediated regulation of the TSC (tuberous sclerosis complex) pathway and autophagy in LE cells. Notably, PGF-induced MAPK8 and MAPK14 signaling pathways mediated the inactivation of MTOR (mechanistic target of rapamycin), the upregulation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) and the increase of autophagosome formation in mice. Furthermore, the PPE-induced autophagy promotes further apoptosis in vitro and in vivo. In summary, elastase-induced autophagy promotes LE cell apoptosis and pulmonary emphysema through the upregulation of PGF. PGF and its downstream MAPK8 and MAPK14 signaling pathways are potential therapeutic targets for the treatment of emphysema and COPD.

  13. Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis.

    Science.gov (United States)

    Floyd, Brice E; Morriss, Stephanie C; MacIntosh, Gustavo C; Bassham, Diane C

    2015-01-01

    Ribosomes account for a majority of the cell's RNA and much of its protein and represent a significant investment of cellular resources. The turnover and degradation of ribosomes has been proposed to play a role in homeostasis and during stress conditions. Mechanisms for the turnover of rRNA and ribosomal proteins have not been fully elucidated. We show here that the RNS2 ribonuclease and autophagy participate in RNA turnover in Arabidopsis thaliana under normal growth conditions. An increase in autophagosome formation was seen in an rns2-2 mutant, and this increase was dependent on the core autophagy genes ATG9 and ATG5. Autophagosomes and autophagic bodies in rns2-2 mutants contain RNA and ribosomes, suggesting that autophagy is activated as an attempt to compensate for loss of rRNA degradation. Total RNA accumulates in rns2-2, atg9-4, atg5-1, rns2-2 atg9-4, and rns2-2 atg5-1 mutants, suggesting a parallel role for autophagy and RNS2 in RNA turnover. rRNA accumulates in the vacuole in rns2-2 mutants. Vacuolar accumulation of rRNA was blocked by disrupting autophagy via an rns2-2 atg5-1 double mutant but not by an rns2-2 atg9-4 double mutant, indicating that ATG5 and ATG9 function differently in this process. Our results suggest that autophagy and RNS2 are both involved in homeostatic degradation of rRNA in the vacuole.

  14. Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling.

    Directory of Open Access Journals (Sweden)

    Dong-Min Shin

    Full Text Available The "enhanced intracellular survival" (eis gene of Mycobacterium tuberculosis (Mtb is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.

  15. The Retinoblastoma Tumor Suppressor Protein (pRb)/E2 Promoter Binding Factor 1 (E2F1) Pathway as a Novel Mediator of TGFβ-induced Autophagy.

    Science.gov (United States)

    Korah, Juliana; Canaff, Lucie; Lebrun, Jean-Jacques

    2016-01-29

    TGFβ is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Although autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFβ signaling and autophagy is context-dependent and remains unclear. TGFβ-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFβ in hepatocarcinoma cells. In the present study, we define a novel process of TGFβ-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFβ is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFβ tumor suppressive effects. We further determined that TGFβ induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFβ induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes and further highlight the central relevance of the pRb/E2F1 pathway downstream of TGFβ signaling in tumor suppression.

  16. Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

    Science.gov (United States)

    Ejaz, Asim; Mitterberger, Maria C; Lu, Zhen; Mattesich, Monika; Zwierzina, Marit E; Hörl, Susanne; Kaiser, Andreas; Viertler, Hans-Peter; Rostek, Ursula; Meryk, Andreas; Khalid, Sana; Pierer, Gerhard; Bast, Robert C; Zwerschke, Werner

    2016-04-01

    Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells. PMID:27211557

  17. p62/SQSTM1 differentially removes the toxic mutant androgen receptor via autophagy and inclusion formation in a spinal and bulbar muscular atrophy mouse model.

    Science.gov (United States)

    Doi, Hideki; Adachi, Hiroaki; Katsuno, Masahisa; Minamiyama, Makoto; Matsumoto, Shinjiro; Kondo, Naohide; Miyazaki, Yu; Iida, Madoka; Tohnai, Genki; Qiang, Qiang; Tanaka, Fumiaki; Yanagawa, Toru; Warabi, Eiji; Ishii, Tetsuro; Sobue, Gen

    2013-05-01

    Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders that are caused by the expansion of trinucleotide CAG repeats in the causative genes. Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease that is caused by the expansion of a polyQ tract within the androgen receptor (AR). p62 is a ubiquitin- and light-chain 3-binding protein that is known to regulate the degradation of targeted proteins via autophagy and inclusion formation. In this study, we examined the effects of p62 depletion and overexpression on cultured cells and in a transgenic mouse model that overexpressed the mutant AR. Here, we demonstrate that depletion of p62 significantly exacerbated motor phenotypes and the neuropathological outcome, whereas overexpression of p62 protected against mutant AR toxicity in SBMA mice. Depletion of p62 significantly increased the levels of monomeric mutant AR and mutant AR protein complexes in an SBMA mouse model via the impairment of autophagic degradation. In addition, p62 overexpression improved SBMA mouse phenotypes by inducing cytoprotective inclusion formation. Our results demonstrate that p62 provides two different therapeutic targets in SBMA pathogenesis: (1) autophagy-dependent degradation and (2) benevolent inclusion formation of the mutant AR.

  18. Autophagy in acute brain injury: feast, famine, or folly?

    Science.gov (United States)

    Smith, Craig M; Chen, Yaming; Sullivan, Mara L; Kochanek, Patrick M; Clark, Robert S B

    2011-07-01

    In the central nervous system, increased autophagy has now been reported after traumatic brain and spinal cord injury, cerebral ischemia, intracerebral hemorrhage, and seizures. This increase in autophagy could be physiologic, converting damaged or dysfunctional proteins, lipids, and/or organelles to their amino acid and fatty acid components for recycling. On the other hand, this increase in autophagy could be supraphysiologic, perhaps consuming and eliminating functional proteins, lipids, and/or organelles as well. Whether an increase in autophagy is beneficial (feast) or detrimental (famine) in brain likely depends on both the burden of intracellular substrate targeted for autophagy and the capacity of the cell's autophagic machinery. Of course, increased autophagy observed after brain injury could also simply be an epiphenomenon (folly). These divergent possibilities have clear ramifications for designing therapeutic strategies targeting autophagy after acute brain injury and are the subject of this review. This article is part of a Special Issue entitled "Autophagy and protein degradation in neurological diseases."

  19. Modulation of inflammation by autophagy: consequences for Crohn's disease.

    NARCIS (Netherlands)

    Plantinga, T.S.; Joosten, L.A.B.; Meer, J.W.M. van der; Netea, M.G.

    2012-01-01

    Autophagy, the cellular machinery for targeting intracellular components for lysosomal degradation, is critically involved in the host defence to pathogenic microorganisms. Recent studies have unveiled several aspects of the immune response that are regulated by autophagy, including antigen presenta

  20. Risperidone and NAP protect cognition and normalize gene expression in a schizophrenia mouse model.

    Science.gov (United States)

    Vaisburd, Sinaya; Shemer, Zeev; Yeheskel, Adva; Giladi, Eliezer; Gozes, Illana

    2015-11-10

    Mutated disrupted in schizophrenia 1 (DISC1), a microtubule regulating protein, leads to schizophrenia and other psychiatric illnesses. It is hypothesized that microtubule stabilization may provide neuroprotection in schizophrenia. The NAP (NAPVSIPQ) sequence of activity-dependent neuroprotective protein (ADNP) contains the SxIP motif, microtubule end binding (EB) protein target, which is critical for microtubule dynamics leading to synaptic plasticity and neuroprotection. Bioinformatics prediction for FDA approved drugs mimicking SxIP-like motif which displace NAP-EB binding identified Risperidone. Risperidone or NAP effectively ameliorated object recognition deficits in the mutated DISC1 mouse model. NAP but not Risperidone, reduced anxiety in the mutated mice. Doxycycline, which blocked the expression of the mutated DISC1, did not reverse the phenotype. Transcripts of Forkhead-BOX P2 (Foxp2), a gene regulating DISC1 and associated with human ability to acquire a spoken language, were increased in the hippocampus of the DISC1 mutated mice and were significantly lowered after treatment with NAP, Risperidone, or the combination of both. Thus, the combination of NAP and standard of care Risperidone in humans may protect against language disturbances associated with negative and cognitive impairments in schizophrenia.

  1. Dual role of autophagy in HIV-1 replication and pathogenesis

    OpenAIRE

    Killian M

    2012-01-01

    Abstract Autophagy, the major mechanism for degrading long-lived intracellular proteins and organelles, is essential for eukaryotic cell homeostasis. Autophagy also defends the cell against invasion by microorganisms and has important roles in innate and adaptive immunity. Increasingly evident is that HIV-1 replication is dependent on select components of autophagy. Fittingly, HIV-1 proteins are able to modulate autophagy to maximize virus production. At the same time, HIV-1 proteins appear t...

  2. Bone Cell Autophagy Is Regulated by Environmental Factors

    OpenAIRE

    Zahm, Adam M.; Bohensky, Jolene; Adams, Christopher S.; Shapiro, Irving M.; Srinivas, Vickram

    2011-01-01

    The goal of this investigation was to ascertain whether bone cells undergo autophagy and to determine if this process is regulated by environmental factors. We showed that osteocytes in both murine and human cortical bone display a punctuate distribution of microtubule-associated protein light chain 3, indicative of autophagy. In addition, we noted a basal level of autophagy in preosteocyte-like murine long bone-derived osteocytic (MLO)-A5 cells. Autophagy was upregulated following nutrient d...

  3. Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells

    Directory of Open Access Journals (Sweden)

    Chan Shih-Hung

    2012-07-01

    Full Text Available Abstract Background Insulin receptor substrate (IRS-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels. Methods and results In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expre