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  1. Network analysis reveals crosstalk between autophagy genes and disease genes

    PubMed Central

    Wang, Ji-Ye; Yao, Wei-Xuan; Wang, Yun; Fan, Yi-lei; Wu, Jian-Bing

    2017-01-01

    Autophagy is a protective and life-sustaining process in which cytoplasmic components are packaged into double-membrane vesicles and targeted to lysosomes for degradation. Accumulating evidence supports that autophagy is associated with several pathological conditions. However, research on the functional cross-links between autophagy and disease genes remains in its early stages. In this study, we constructed a disease-autophagy network (DAN) by integrating known disease genes, known autophagy genes and protein-protein interactions (PPI). Dissecting the topological properties of the DAN suggested that nodes that both autophagy and disease genes (inter-genes), are topologically important in the DAN structure. Next, a core network from the DAN was extracted to analyze the functional links between disease and autophagy genes. The genes in the core network were significantly enriched in multiple disease-related pathways, suggesting that autophagy genes may function in various disease processes. Of 17 disease classes, 11 significantly overlapped with autophagy genes, including cancer diseases, metabolic diseases and hematological diseases, a finding that is supported by the literatures. We also found that autophagy genes have a bridging role in the connections between pairs of disease classes. Altogether, our study provides a better understanding of the molecular mechanisms underlying human diseases and the autophagy process. PMID:28295050

  2. Macrophage autophagy protects against liver fibrosis in mice.

    PubMed

    Lodder, Jasper; Denaës, Timothé; Chobert, Marie-Noële; Wan, JingHong; El-Benna, Jamel; Pawlotsky, Jean-Michel; Lotersztajn, Sophie; Teixeira-Clerc, Fatima

    2015-01-01

    Autophagy is a lysosomal degradation pathway of cellular components that displays antiinflammatory properties in macrophages. Macrophages are critically involved in chronic liver injury by releasing mediators that promote hepatocyte apoptosis, contribute to inflammatory cell recruitment and activation of hepatic fibrogenic cells. Here, we investigated whether macrophage autophagy may protect against chronic liver injury. Experiments were performed in mice with mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5(fl/fl) LysM-Cre mice, referred to as atg5(-/-)) and their wild-type (Atg5(fl/fl), referred to as WT) littermates. Liver fibrosis was induced by repeated intraperitoneal injection of carbon tetrachloride. In vitro studies were performed in cultures or co-cultures of peritoneal macrophages with hepatic myofibroblasts. As compared to WT littermates, atg5(-/-) mice exposed to chronic carbon tetrachloride administration displayed higher hepatic levels of IL1A and IL1B and enhanced inflammatory cell recruitment associated with exacerbated liver injury. In addition, atg5(-/-) mice were more susceptible to liver fibrosis, as shown by enhanced matrix and fibrogenic cell accumulation. Macrophages from atg5(-/-) mice secreted higher levels of reactive oxygen species (ROS)-induced IL1A and IL1B. Moreover, hepatic myofibroblasts exposed to the conditioned medium of macrophages from atg5(-/-) mice showed increased profibrogenic gene expression; this effect was blunted when neutralizing IL1A and IL1B in the conditioned medium of atg5(-/-) macrophages. Finally, administration of recombinant IL1RN (interleukin 1 receptor antagonist) to carbon tetrachloride-exposed atg5(-/-) mice blunted liver injury and fibrosis, identifying IL1A/B as central mediators in the deleterious effects of macrophage autophagy invalidation. These results uncover macrophage autophagy as a novel antiinflammatory pathway regulating liver fibrosis.

  3. Autophagy

    PubMed Central

    Hale, Amber N.; Ledbetter, Dan J.; Gawriluk, Thomas R.; Rucker, III, Edmund B.

    2013-01-01

    Autophagy is an evolutionarily conserved cellular process through which long-lived proteins and damaged organelles are recycled to maintain energy homeostasis. These proteins and organelles are sequestered into a double-membrane structure, or autophagosome, which subsequently fuses with a lysosome in order to degrade the cargo. Although originally classified as a type of programmed cell death, autophagy is more widely viewed as a basic cell survival mechanism to combat environmental stressors. Autophagy genes were initially identified in yeast and were found to be necessary to circumvent nutrient stress and starvation. Subsequent elucidation of mammalian gene counterparts has highlighted the importance of this process to normal development. This review provides an overview of autophagy, the types of autophagy, its regulation and its known impact on development gleaned primarily from murine models. PMID:24121596

  4. Autophagy protects intestinal epithelial cells against deoxynivalenol toxicity by alleviating oxidative stress via IKK signaling pathway.

    PubMed

    Tang, Yulong; Li, Jianjun; Li, Fengna; Hu, Chien-An A; Liao, Peng; Tan, Kunrong; Tan, Bie; Xiong, Xia; Liu, Gang; Li, Tiejun; Yin, Yulong

    2015-12-01

    Autophagy is an intracellular process of homeostatic degradation that promotes cell survival under various stressors. Deoxynivalenol (DON), a fungal toxin, often causes diarrhea and disturbs the homeostasis of the intestinal system. To investigate the function of intestinal autophagy in response to DON and associated mechanisms, we firstly knocked out ATG5 (autophagy-related gene 5) in porcine intestinal epithelial cells (IPEC-J2) using CRISPR-Cas9 technology. When treated with DON, autophagy was induced in IPEC-J2 cells but not in IPEC-J2.Atg5ko cells. The deficiency in autophagy increased DON-induced apoptosis in IPEC-J2.atg5ko cells, in part, through the generation of reactive oxygen species (ROS). The cellular stress response can be restored in IPEC-J2.atg5ko cells by overexpressing proteins involved in protein folding. Interestingly, we found that autophagy deficiency downregulated the expression of endoplasmic reticulum folding proteins BiP and PDI when IPEC-J2.atg5ko cells were treated with DON. In addition, we investigated the molecular mechanism of autophagy involved in the IKK, AMPK, and mTOR signaling pathway and found that Bay-117082 and Compound C, specific inhibitors for IKK and AMPK, respectively, inhibited the induction of autophagy. Taken together, our results suggest that autophagy is pivotal for protection against DON in pig intestinal cells.

  5. Protective autophagy is involved in resistance towards MET inhibitors in human gastric adenocarcinoma cells.

    PubMed

    Humbert, Magali; Medová, Michaela; Aebersold, Daniel M; Blaukat, Andree; Bladt, Friedhelm; Fey, Martin F; Zimmer, Yitzhak; Tschan, Mario P

    2013-02-08

    MET, also known as hepatocyte growth factor receptor (HGFR), is a receptor tyrosine kinase with an important role, both in normal cellular function as well as in oncogenesis. In many cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Autophagy, a self-digesting recycling mechanism with cytoprotective functions, is induced by cellular stress. This process is also induced upon cytotoxic drug treatment of cancer cells and partially allows these cells to escape cell death. Thus, since autophagy protects different tumor cells from chemotherapy-induced cell death, current clinical trials aim at combining autophagy inhibitors with different cancer treatments. We found that in a gastric adenocarcinoma cell line GTL-16, where MET activity is deregulated due to receptor overexpression, two different MET inhibitors PHA665752 and EMD1214063 lead to cell death paralleled by the induction of autophagy. A combined treatment of MET inhibitors together with the autophagy inhibitor 3-MA or genetically impairing autophagy by knocking down the key autophagy gene ATG7 further decreased cell viability of gastric cancer cells. In general, we observed the induction of cytoprotective autophagy in MET expressing cells upon MET inhibition and a combination of MET and autophagy inhibition resulted in significantly decreased cell viability in gastric cancer cells.

  6. Overexpression of Annexin II Receptor-Induced Autophagy Protects Against Apoptosis in Uveal Melanoma Cells.

    PubMed

    Zhang, Yuelu; Song, Hongyuan; Guo, Ting; Zhu, Yongzhe; Tang, Hailin; Qi, Zhongtian; Zhao, Ping; Zhao, Shihong

    2016-05-01

    Uveal melanoma is the most common primary malignant intraocular tumor in adults and still lacks effective systemic therapies. Annexin A2 receptor (AXIIR), a receptor for Annexin II, was demonstrated to play an important role in multiple cells, but its role in uveal melanoma cells remains exclusive. Herein, the authors reported that overexpression of AXIIR was able to reduce cell viability and activate apoptosis apparently in the Mum2C uveal melanoma cell line. Meanwhile, overexpression of AXIIR could induce autophagy and increase autophagy flux. After autophagy was inhibited by chloroquine, enhanced apoptosis and cytotoxicity could be detected. In summary, these data highlighted the crucial role of AXIIR in reducing Mum2C cell viability through inducing apoptosis, while autophagy played a protective role in this process. Interference of this gene may be a promising method for uveal melanoma therapy and combination with specific inhibitor of autophagy may serve as a supplementary.

  7. The Cannabinoid Receptor 2 Protects Against Alcoholic Liver Disease Via a Macrophage Autophagy-Dependent Pathway.

    PubMed

    Denaës, Timothé; Lodder, Jasper; Chobert, Marie-Noële; Ruiz, Isaac; Pawlotsky, Jean-Michel; Lotersztajn, Sophie; Teixeira-Clerc, Fatima

    2016-06-27

    Kupffer cells, the resident macrophages of the liver, play a major role in the pathogenesis of alcoholic liver disease. We have previously demonstrated that CB2 receptor protects against alcoholic liver disease by inhibiting alcohol-induced inflammation and steatosis via the regulation of Kupffer cell activation. Here, we explored the mechanism underlying these effects and hypothesized that the anti-inflammatory properties of CB2 receptor in Kupffer cells rely on activation of autophagy. For this purpose, mice invalidated for CB2 receptor (CB2(Mye-/-) mice) or for the autophagy gene ATG5 (ATG5(Mye-/-) mice) in the myeloid lineage, and their littermate wild-type mice were subjected to chronic-plus-binge ethanol feeding. CB2(Mye-/-) mice showed exacerbated alcohol-induced pro-inflammatory gene expression and steatosis. Studies in cultured macrophages demonstrated that CB2 receptor activation by JWH-133 stimulated autophagy via a heme oxygenase-1 dependent pathway. Moreover, JWH-133 reduced the induction of inflammatory genes by lipopolysaccharide in wild-type macrophages, but not in ATG5-deficient cells. The CB2 agonist also protected from alcohol-induced liver inflammation and steatosis in wild-type mice, but not in ATG5(Mye-/-) mice demonstrating that macrophage autophagy mediates the anti-inflammatory and anti-steatogenic effects of CB2 receptor. Altogether these results demonstrate that CB2 receptor activation in macrophages protects from alcohol-induced steatosis by inhibiting hepatic inflammation through an autophagy-dependent pathway.

  8. Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress.

    PubMed

    Kimura, Tomonori; Takahashi, Atsushi; Takabatake, Yoshitsugu; Namba, Tomoko; Yamamoto, Takeshi; Kaimori, Jun-Ya; Matsui, Isao; Kitamura, Harumi; Niimura, Fumio; Matsusaka, Taiji; Soga, Tomoyoshi; Rakugi, Hiromi; Isaka, Yoshitaka

    2013-11-01

    Chronic metabolic stress is related to diseases, whereas autophagy supplies nutrients by recycling the degradative products. Cyclosporin A (CsA), a frequently used immunosuppressant, induces metabolic stress via effects on mitochondrial respiration, and thereby, its chronic usage is often limited. Here we show that autophagy plays a protective role against CsA-induced metabolic stress in kidney proximal tubule epithelial cells. Autophagy deficiency leads to decreased mitochondrial membrane potential, which coincides with metabolic abnormalities as characterized by decreased levels of amino acids, increased tricarboxylic acid (TCA) ratio (the levels of intermediates of the latter part of the TCA cycle, over levels of intermediates in the earlier part), and decreased products of oxidative phosphorylation (ATP). In addition to the altered profile of amino acids, CsA decreased the hyperpolarization of mitochondria with the disturbance of mitochondrial energy metabolism in autophagy-competent cells, i.e., increased TCA ratio and worsening of the NAD(+)/NADH ratio, coupled with decreased energy status, which suggests that adaptation to CsA employs autophagy to supply electron donors from amino acids via intermediates of the latter part of the TCA cycle. The TCA ratio of autophagy-deficient cells was further worsened with decreased levels of amino acids in response to CsA, and, as a result, the deficiency of autophagy failed to adapt to the CsA-induced metabolic stress. Deterioration of the TCA ratio further worsened energy status. The CsA-induced metabolic stress also activated regulatory genes of metabolism and apoptotic signals, whose expressions were accelerated in autophagy-deficient cells. These data provide new perspectives on autophagy in conditions of chronic metabolic stress in disease.

  9. Crosstalk of clock gene expression and autophagy in aging

    PubMed Central

    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

  10. Identification of autophagy genes in Ciona intestinalis: a new experimental model to study autophagy mechanism.

    PubMed

    Godefroy, Nelly; Hoa, Céline; Tsokanos, Foivos; Le Goff, Emilie; Douzery, Emmanuel J P; Baghdiguian, Stephen; Martinand-Mari, Camille

    2009-08-01

    Programmed cell death (PCD) is a mechanism implicated in many physiological and pathological processes. Until recently, apoptosis (self-killing) was the most largely studied mechanism of PCD but a growing number of laboratories are now interested in autophagy (self-eating). In the past few years data showing a tight link between both pathways has accumulated. Until now our laboratory used Ciona intestinalis, a chordate model in which in vivo experiments are possible, to study apoptosis. Recently, we showed that autophagy also occurs in the development of Ciona intestinalis and that the specific markers of both types of death are found in the same tissues and/or in the same cells. These results drove us to postulate that Ciona intestinalis can be a good model to study the link between apoptosis and autophagy. In this article, we conducted an in silico study of autophagy genes. We explored the genomes of Ciona intestinalis, of the second ascidian Ciona savignyi, and those of the classical biological models (Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans and Homo sapiens) to extract and compare autophagy gene sequences. This genomic study was completed by an analysis of: (i) mRNA profile expression during development and (ii) the localization of Beclin protein by immunofluorescent staining in the Ciona intestinalis larvae. Taken together, the results allowed us to conclude that a complex autophagic machinery is present in Ciona intestinalis. Actually, the number of autophagy genes in Ciona intestinalis is comparable to the number of autophagy genes in human.

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

    PubMed Central

    Han, Xiao; Liu, Jian-xun; Li, Xin-zhi

    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 0, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sal B (50 μmol/L) in the presence or absence of chloroquine (3 μmol/L) under GD 3 h, the amount of LC3-II, 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. Moreover, 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-II 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-II, 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. PMID:21113177

  12. Resveratrol protects podocytes against apoptosis via stimulation of autophagy in a mouse model of diabetic nephropathy

    PubMed Central

    Huang, Shan-Shan; Ding, Da-Fa; Chen, Sheng; Dong, Cheng-Long; Ye, Xiao-Long; Yuan, Yang-Gang; Feng, Ya-Min; You, Na; Xu, Jia-Rong; Miao, Heng; You, Qiang; Lu, Xiang; Lu, Yi-Bing

    2017-01-01

    Podocyte apoptosis coincides with albuminuria onset and precedes podocytopenia in diabetic nephropathy. However, there is a lack of effective therapeutic drugs to protect podocytes from apoptosis. Here, we demonstrated that resveratrol relieved a series of indicators of diabetic nephropathy and attenuated apoptosis of podocytes in db/db diabetic model mice. In addition, resveratrol induced autophagy in both db/db mice and human podocytes. Furthermore, inhibition of autophagy by 3-methyladenine (3-MA) and autophagy gene 5 (Atg5) short hairpin RNA (shRNA) reversed the protective effects of resveratrol on podocytes. Finally, we found that resveratrol might regulate autophagy and apoptosis in db/db mice and podocytes through the suppression of microRNA-383-5p (miR-383-5p). Together, our results indicate that resveratrol effectively attenuates high glucose-induced apoptosis via the activation of autophagy in db/db mice and podocytes, which involves miR-383-5p. Thus, this study reveals a new possible strategy to treat diabetic nephropathy. PMID:28374806

  13. Potential protective effects of autophagy activated in MPP+ treated astrocytes

    PubMed Central

    Shen, Cunzhou; Xian, Wenbiao; Zhou, Hongyan; Chen, Ling; Pei, Zhong

    2016-01-01

    Astrocytes, which have various important functions, have previously been associated with Parkinsons disease (PD), particularly in 1-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models of PD. MPP+ is the toxic metabolite of MPTP and is generated by the enzymatic activity of monoamine oxidase B, which is predominantly located in astrocytes. MPP+ acts as a mitochondrial complex I inhibitor. Autophagy is an evolutionarily conserved self-digestion pathway in eukaryotic cells, which occurs in response to various types of stress, including starvation and oxidative stress. Lithium treatment has previously been shown to induce autophagy in astrocytes by inhibiting the enzyme inositol monophosphatase, which may aid in the treatment of neurodegenerative diseases, including Huntington's disease, in which the toxic protein is an autophagy substrate. Therefore, using western blotting and MTT assay, the present study aimed to investigate the protective effects of lithium-induced autophagy against astrocyte injury caused by MPP+ treatment, as well as the potential underlying mechanisms. The results of the present study suggested that lithium was able to induce autophagy in astrocytes treated with MPP+, and this likely occurred via activation of the phosphoinositide 3-kinase/AKT pathway. PMID:27882077

  14. Sirtuin1 and autophagy protect cells from fluoride-induced cell stress.

    PubMed

    Suzuki, Maiko; Bartlett, John D

    2014-02-01

    Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum (ER) stress and oxidative stress. Previously, we reported that fluoride induces ER-stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augment SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50, 100 and 125ppm) in drinking water for 6weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis.

  15. Sirtuin1 and autophagy protect cells from fluoride-induced cell stress

    PubMed Central

    Suzuki, Maiko; Bartlett, John D.

    2014-01-01

    Sirtuin1 (SIRT1) is an (NAD+)-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum stress and oxidative stress. Previously, we reported that fluoride induces endoplasmic reticulum (ER) stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augmented SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50 and 100 ppm) in drinking water for 6 weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis. PMID:24296261

  16. Characterization of a novel autophagy-specific gene, ATG29

    SciTech Connect

    Kawamata, Tomoko; Kamada, Yoshiaki; Suzuki, Kuninori; Kuboshima, Norihiro; Akimatsu, Hiroshi; Ota, Shinichi; Ohsumi, Mariko; Ohsumi, Yoshinori . E-mail: yohsumi@nibb.ac.jp

    2005-12-30

    Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w. atg29{delta} cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins.

  17. Analysis of Autophagy Genes in Microalgae: Chlorella as a Potential Model to Study Mechanism of Autophagy

    PubMed Central

    Jiang, Qiao; Zhao, Li; Dai, Junbiao; Wu, Qingyu

    2012-01-01

    Background Microalgae, with the ability to mitigate CO2 emission and produce carbohydrates and lipids, are considered one of the most promising resources for producing bioenergy. Recently, we discovered that autophagy plays a critical role in the metabolism of photosynthetic system and lipids production. So far, more than 30-autophagy related (ATG) genes in all subtypes of autophagy have been identified. However, compared with yeast and mammals, in silico and experimental research of autophagy pathways in microalgae remained limited and fragmentary. Principal Findings In this article, we performed a genome-wide analysis of ATG genes in 7 microalgae species and explored their distributions, domain structures and evolution. Eighteen “core autophagy machinery” proteins, four mammalian-specific ATG proteins and more than 30 additional proteins (including “receptor-adaptor” complexes) in all subtypes of autophagy were analyzed. Data revealed that receptor proteins in cytoplasm-to-vacuole targeting and mitophagy seem to be absent in microalgae. However, most of the “core autophagy machinery” and mammalian-specific proteins are conserved among microalgae, except for the ATG9-cycling system in Chlamydomonas reinhardtii and the second ubiquitin-like protein conjugation complex in several algal species. The catalytic and binding residues in ATG3, ATG5, ATG7, ATG8, ATG10 and ATG12 are also conserved and the phylogenetic tree of ATG8 coincides well with the phylogenies. Chlorella contains the entire set of the core autophagy machinery. In addition, RT-PCR analysis verified that all crucial ATG genes tested are expressed during autophagy in both Chlorella and Chlamydomonas reinhardtii. Finally, we discovered that addition of 3-Methyladenine (a PI3K specific inhibitor) could suppress the formation of autophagic vacuoles in Chlorella. Conclusions Taken together, Chlorella may represent a potential model organism to investigate autophagy pathways in photosynthetic

  18. MiR224-3p inhibits hypoxia-induced autophagy by targeting autophagy-related genes in human glioblastoma cells

    PubMed Central

    Guo, Xing; Xue, Hao; Guo, Xiaofan; Gao, Xiao; Xu, Shugang; Yan, Shaofeng; Han, Xiao; Li, Tong; Shen, Jie; Li, Gang

    2015-01-01

    Human glioblastoma multiforme (GBM) is a malignant solid tumor characterized by severe hypoxia. Autophagy plays a protective role in cancer cells under hypoxia. However, the microRNA (miRNA)-related molecular mechanisms underlying hypoxia-reduced autophagy remain poorly understood in GBM. In this study, we performed a miRNA microarray analysis on GBM cells and found that numerous miRNAs were differentially expressed under hypoxic conditions. Further research showed that miR224-3p, one of the significantly down-regulated miRNAs, was involved in regulating hypoxia-induced autophagy in GBM cells. Overexpression of miR224-3p abolished hypoxia-induced autophagy, whereas knocking down endogenous miR224-3p increased autophagic activity under normoxia. In addition, we demonstrated that miR224-3p inhibited autophagy by directly suppressing the expression of two autophagy-related genes (ATGs), ATG5 and FAK family-interacting protein of 200 kDa (FIP200). Furthermore, in vitro, miR224-3p attenuated cell proliferation and promoted hypoxia-induced apoptosis, and in vivo, overexpression of miR224-3p inhibited tumorigenesis of GBM cells. Collectively, our study identified a novel hypoxia-down-regulated miRNA, miR224-3p, as a key modulator of autophagy by inhibiting ATGs in GBM cells. PMID:26536662

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

    PubMed Central

    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

  20. Autophagy may protect MC3T3-E1 cells from fluoride-induced apoptosis.

    PubMed

    Wei, Min; Duan, Dongmei; Liu, Yujie; Wang, Zhigang; Li, Zhongli

    2014-06-01

    Fluoride is an essential trace element for all mammalian species; however, excess fluoride intake is known to be toxic to cells in animals and humans. The toxicity of fluoride is mainly exerted via induction of apoptosis. Autophagy is induced by numerous cytotoxic stimuli; however, it is often unclear whether, under specific conditions, autophagy has a pro‑survival or a pro‑apoptotic role. To answer this critical question, the present study assessed autophagy and apoptosis simultaneously in single cells. It was demonstrated that fluoride was able to inhibit cell proliferation and induce apoptosis and autophagy, whereas autophagy appeared to be protective. Further analysis revealed that MAPK/JNK‑dependent autophagy may be protective in fluoride‑induced apoptosis. It is anticipated that the presented single‑cell approach may be a powerful tool for gaining a quantitative understanding of the complex regulation of autophagy, its effect on cell fate and its association with other cellular pathways.

  1. Polymorphisms in Autophagy Genes and Susceptibility to Tuberculosis

    PubMed Central

    Alisjahbana, Bachti; Sahiratmadja, Edhyana; Parwati, Ida; Oosting, Marije; Plantinga, Theo S.; Joosten, Leo A. B.; Netea, Mihai G.; Ottenhoff, Tom H. M.; van de Vosse, Esther; van Crevel, Reinout

    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. 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. PMID:22879892

  2. Dihydromyricetin protects against liver ischemia/reperfusion induced apoptosis via activation of FOXO3a-mediated autophagy

    PubMed Central

    Pi, Huifeng; Qin, Weijia; Chen, Jianwei; Guo, Dengfang; Lin, Jianyu; Chi, Xiaobing; Jiang, Zhelong; Yang, Hejun; Jiang, Yi

    2016-01-01

    Liver ischemia and reperfusion (I/R) injury is characterized by defective liver autophagy accompanied by alterations to the endogenous defense system. Dihydromyricetin (DHM) is a natural flavonoid that demonstrates a wide range of physiological functions, and has been implicated as a regulator of autophagy. This study investigates the protective effects of DHM pretreatment on liver injury caused by ischemia/reperfusion (I/R) and elucidates the potential mechanism of DHM-mediated protection. Mice were subjected to 60 minutes of ischemia followed by 5 hours of reperfusion. DHM (100 mg/kg bw/day) or the vehicle was administered daily by gavage 7 days before ischemia and immediately before reperfusion. In this study, DHM markedly decreased serum aminotransferase activity and inhibited liver I/R -stimulated apoptosis. Moreover, DHM exerted hepatoprotective effects by upregulating mRNA levels of various essential autophagy-related genes including ATG5, ATG12, BECN1, and LC3. Autophagy inhibitor chloroquine or Atg5 knockdown blocked DHM -mediated elevation in liver function. Specifically, DHM significantly increased FOXO3a expression, and enhanced FOXO3a nuclear translocation and Ser588 phosphorylation modification. Importantly, the inhibition of FOXO3a with FOXO3a-siRNA in mice decreased DHM-induced autophagy-related genes and diminished the protective effects of DHM against liver I/R injury. In summary, these findings identify DHM as a novel hepatoprotective small molecule by elevating FOXO3a expression and nuclear translocation, stimulating autophagy-related genes and suppressing liver I/R-induced apoptosis, suggesting FOXO3a may have therapeutic value in liver cell protection in liver I/R injury. PMID:27793014

  3. Protective role of autophagy in methionine-choline deficient diet-induced advanced nonalcoholic steatohepatitis in mice.

    PubMed

    Chen, Rui; Wang, Quanxing; Song, Shaohua; Liu, Fang; He, Bin; Gao, Xiaogang

    2016-01-05

    The methionine choline-deficient (MCD) diet leads to severe liver injury similar to human nonalcoholic steatohepatitis (NASH). Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. The goal of this study was to elucidate the role of autophagy in MCD-induced steatosis, fibrosis, inflammation, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress in mice. Mice were fed with MCD diet and treated with rapamycin (an autophagy enhancer) or chloroquine (an autophagy inhibitor) for 10 weeks. Liver injury was evaluated biochemically and histologically together with hepatic gene expression analysis. Autophagic flux was impaired in livers of mice fed with MCD diet, evidenced by reduced ratio of LC3-II/LC3-I and increased protein expression of p62. It was found that autophagy activation by rapamycin attenuated MCD-induced steatosis, fibrosis, inflammation, mitochondrial dysfunction, and ER stress. By contrast, MCD mice treated with chloroquine developed more liver injury. In conclusions, the autophagic pathway plays an important protective role in MCD-induced advanced NASH. Thus, pharmacological promotion of autophagy may provide a novel therapeutic strategy for treatment of NASH.

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

    PubMed

    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.

  5. MicroRNA-410 regulates autophagy-related gene ATG16L1 expression and enhances chemosensitivity via autophagy inhibition in osteosarcoma

    PubMed Central

    Chen, Ren; Li, Xiaohai; He, Bin; Hu, Wei

    2017-01-01

    Osteosarcoma, which is the most common type of primary bone tumor in adolescents, is characterized by complex genetic alterations and frequent resistance to conventional treatments. MicroRNAs (miRs) have emerged as fundamental regulators in gene expression through their ability to silence gene expression at post-transcriptional and translational levels. The present study investigated the role of miR-410 in the progression of osteosarcoma. The results demonstrated that the expression of miR-410 was markedly downregulated in human osteosarcoma tissues, and U2OS and MG-63 osteosarcoma cell lines. Clinicopathological significance suggested that miR-410 may be a potential biomarker for chemotherapy-resistant osteosarcoma. Furthermore, overexpression of miR-410 exhibited a limited effect on cell viability in U2OS and MG-63 cells. Target prediction algorithms (TargetScan and miRanda) indicated that autophagy related 16-like 1 (ATG16L1) was a potential target gene of miR-410. A luciferase reporter assay demonstrated that miR-410 directly decreased ATG16L1 expression by targeting its 3′-untranslated region. In addition, the results revealed that miR-410 was able to markedly inhibit autophagy. Accordingly, autophagy was activated as a protective mechanism when osteosarcoma cells were exposed to three common anticancer drugs, including rapamycin, doxorubicin and cisplatin. Furthermore, the autophagy inhibitor 3-methyladenine and miR-410 expression were able to improve the therapeutic response of the cells to chemotherapy drugs (rapamycin, doxorubicin and cisplatin), thus indicating that miR-410 enhanced chemosensitivity through autophagy inhibition in osteosarcoma cells. In conclusion, studies regarding the function of miR-410 on autophagy provided insight into the biological function of miR-410 in osteosarcoma and may offer a promising approach for the treatment of osteosarcoma. PMID:28138700

  6. Elevation of protective autophagy as a potential way for preventing developmental neurotoxicity of general anesthetics.

    PubMed

    Li, Guohui; Yu, Buwei

    2014-02-01

    Numerous animal studies have demonstrated that commonly used general anesthetics could cause cognitive impairment in the developing brain. However, the underlying mechanism remains unclear. Recently it is reported that autophagy activation can ameliorate developmental neurotoxicity of ethanol, which is the same GABAA agonist and NMDA antagonist as general anesthetics. We thus intend to propose the possible role of autophagy in the developmental neurotoxicity of general anesthetics. Oxidative stress and neuronal apoptosis can activate autophagy, while autophagy conversely alleviates their levels in the neuron. Crosstalk among neuronal apoptosis, oxidative stress and autophagy resembles the Yin-Yang relationship in Chinese philosophy. Neuronal apoptosis and oxidative stress represent destroyable Yin, while autophagy symbols protective Yang. The destroyable Yin and protective Yang promote and counteract each other. We hypothesize that the destroyable Yin (neuronal apoptosis and oxidative stress injury) prevails over protective Yang (autophagy) when developing brain exposes to general anesthetics. Elevating protective Yang autophagy potentially reverses the neurotoxicity of general anesthetics. Once this hypothesis is proved, it will provide a new perspective to understand the developmental neurotoxicity of general anesthetics and a new way to prevent it.

  7. Autophagy in Aging and Alzheimer’s Disease: Pathologic or Protective?

    PubMed Central

    Barnett, Aaron; Brewer, Gregory J.

    2013-01-01

    Some hypothesize that aging in humans is a cumulative process of macromolecular and mitochondrial damage starting years, even decades before any symptoms arise. Aging may begin when the rate of damage exceeds the rate of continual repair and turnover. Quality control for damaged mitochondria entails cellular digestion by mitophagy, a specialized kind of autophagy. Insufficient protective autophagy could cause damaged cellular components to accumulate over many years until they affect normal function in the cell. Alternatively, aging could be the result of overactive, pathologic autophagy. Current knowledge supports both hypotheses with conflicting data, depending on which stage of autophagy is examined. To distinguish these opposite hypotheses, two criteria need to be observed. First, is there a buildup of undigested waste that can be removed by stimulation of autophagy? Or second, if autophagy is overactive, does inhibition of autophagy rescue cell, organ and organism demise. Both of these are best determined by rate measures rather than measures at a single time point. Here, we review the generalized process of autophagy, with a focus on the limited information available for neuron mitophagy, aging and Alzheimer’s disease. In two mouse models, treatment with rapamycin abolishes the AD pathology and reverses memory deficits. As a working model, we hypothesize that insufficient protective autophagy accelerates both aging and Alzheimer’s disease pathology, possibly caused by defects in autophagosome fusion with lysosomes. PMID:21422527

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

    SciTech Connect

    Cao, Xueming; Chen, Aihua 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.

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

    SciTech Connect

    Yang, Xiaojun; Zhong, Xiaomin; Tanyi, Janos L.; Shen, Jianfeng; Xu, Congjian; Gao, Peng; Zheng, Tim M.; DeMichele, Angela; Zhang, Lin

    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.

  10. Stimulation of autophagy by the p53 target gene Sestrin2.

    PubMed

    Maiuri, Maria Chiara; Malik, Shoaib Ahmad; Morselli, Eugenia; Kepp, Oliver; Criollo, Alfredo; Mouchel, Pierre-Luc; Carnuccio, Rosa; Kroemer, Guido

    2009-05-15

    The oncosuppressor protein p53 regulates autophagy in a dual fashion. The pool of cytoplasmic p53 protein represses autophagy in a transcription-independent fashion, while the pool of nuclear p53 stimulates autophagy through the transactivation of specific genes. Here we report the discovery that Sestrin2, a novel p53 target gene, is involved in the induction of autophagy. Depletion of Sestrin2 by RNA interference reduced the level of autophagy in a panel of p53-sufficient human cancer cell lines responding to distinct autophagy inducers. In quantitative terms, Sestrin2 depletion was as efficient in preventing autophagy induction as was the depletion of Dram, another p53 target gene. Knockout of either Sestrin2 or Dram reduced autophagy elicited by nutrient depletion, rapamycin, lithium or thapsigargin. Moreover, autophagy induction by nutrient depletion or pharmacological stimuli led to an increase in Sestrin2 expression levels in p53-proficient cells. In strict contrast, the depletion of Sestrin2 or Dram failed to affect autophagy in p53-deficient cells and did not modulate the inhibition of baseline autophagy by a cytoplasmic p53 mutant that was reintroduced into p53-deficient cells. We conclude that Sestrin2 acts as a positive regulator of autophagy in p53-proficient cells.

  11. Autophagy protects meniscal cells from glucocorticoids-induced apoptosis via inositol trisphosphate receptor signaling.

    PubMed

    Shen, Chao; Gu, Wen; Cai, Gui-Quan; Peng, Jian-Ping; Chen, Xiao-Dong

    2015-09-01

    Intra-articular injection of glucocorticoids (GCs) has been widely used in the management of osteoarthritis and rheumatoid arthritis. Nevertheless, several studies showed that GCs had toxic effects on chondrocytes as well as synovial cells. Previously we reported the protective role of autophagy in the degeneration of meniscal tissues. However, the effects of GCs on autophagy in the meniscal cells have not been fully elucidated. To investigate whether GCs can regulate autophagy in human meniscal cells, the meniscal cells were cultured in vitro and exposed in the presence of dexamethasone. The levels of apoptosis and autophagy were investigated via flow cytometry as well as western blotting analysis. The changes of the aggrecanases were measured using real-time PCR. The role of autophagy in dexamethasone-induced apoptosis was investigated using pharmacological agents and RNA interference technique. An agonist of inositol 1,4,5-trisphosphate receptor (IP3R) was used to investigate the mechanism of dexamethasone-induced autophagy. The results showed that dexamethasone induced autophagy as well as apoptosis in normal human meniscal cells. Using RNA interference technique and pharmacological agents, our results showed that autophagy protected the meniscal cells from dexamethasone-induced apoptosis. Our results also indicated that dexamethasone increased the mRNA levels of aggrecanases. This catabolic effect of dexamethasone was enhanced by 3-MA, the autophagy inhibitor. Furthermore, our results showed that dexamethasone induced autophagy via suppressing the phosphorylation of IP3R. In summary, our results indicated that autophagy protected meniscal cells from GCs-induced apoptosis via inositol trisphosphate receptor signaling.

  12. Protective role of autophagy and autophagy-related protein 5 in early tumorigenesis.

    PubMed

    Liu, He; He, Zhaoyue; Simon, Hans-Uwe

    2015-02-01

    Autophagy, a fundamental cellular catabolic process, is involved in the development of numerous diseases including cancer. Autophagy seems to have an ambivalent impact on tumor development. While increasing evidence indicates a cytoprotective role for autophagy that can contribute to resistance against chemotherapy and even against the adverse, hypoxic environment of established tumors, relatively few publications focus on the role of autophagy in early tumorigenesis. However, the consensus is that autophagy is inhibitory for the genesis of tumors. To understand this apparent contradiction, more detailed information about the roles of the individual participants in autophagy is needed. This review will address this topic with respect to autophagy-related protein 5 (ATG5), which in several lines of investigation has been ascribed special significance in the autophagic pathway. Furthermore, it was recently shown that an ATG5 deficiency in melanocytes interferes with oncogene-induced senescence, thus promoting melanoma tumorigenesis. Similarly, an ATG5 deficiency resulted in tumors of the lung and liver in experimental mouse models. Taken together, these findings indicate that ATG5 and the autophagy to which it contributes are essential gatekeepers restricting early tumorigenesis in multiple tissues.

  13. Autophagy Protects from Raddeanin A-Induced Apoptosis in SGC-7901 Human Gastric Cancer Cells

    PubMed Central

    Liu, Shen-lin; Fang, Liang-hua; Zhou, Jin-yong; Wu, Jian; Xi, Song-yang; Chen, Yan; Zhang, Ying-ying; Xu, Song

    2016-01-01

    Raddeanin A (RA) is an extractive from Anemone raddeana Regel, a traditional Chinese medicine. The aim of this study is to assess the efficacy of RA against human gastric cancer (GC) cells (SGC-7901) and explore its mechanism. MTT assay showed that RA inhibition of proliferation of SGC-7901 cells increased in a dose-dependent manner. Flow cytometry analysis and Hoechst 33258 staining showed that RA induced apoptosis on SGC-7901 cells. Meanwhile, it induced autophagy. Western blotting analysis showed that the RA induces apoptosis and autophagy by activating p38 MAPK pathway and inhibiting mTOR pathway. Further studies showed that autophagy inhibition could protect from RA-induced apoptosis in SGC-7901 cells. In conclusion, RA can induce SGC-7901 cell apoptosis and autophagy by activating p38 MAPK pathway. And autophagy can protect SGC-7901 cells from apoptosis induced by RA. PMID:27974905

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

    PubMed

    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.

  15. Autophagy plays a protective role in cell death of osteoblasts exposure to lead chloride.

    PubMed

    Lv, Xiao-hua; Zhao, Da-hang; Cai, Shi-zhong; Luo, Shi-ying; You, Tingting; Xu, Bi-lian; Chen, Ke

    2015-12-03

    Lead (Pb) is a toxic heavy metal widespreadly used in industrial field. Prior studies showed that Pb exposure had detrimental effects on osteoblasts. The mechanisms underlying Pb-induced damage are complex. Autophagy can protect cells from various cytotoxic stimuli. In the present study, the aim of our research was to investigate whether Pb could activate autophagy to play a protective role against osteoblasts apoptosis. Our results indicated that PbCl2 induced autophagy and autophagic flux in MC3T3-E1 murine osteoblastic cell by RT-PCR, western blot, as well as fluorescence microscopy analysis of GFP-LC3, AO and MDC staining. Pb increased the apoptosis of osteoblasts, evidenced by western blot and Hoechst 33258 staining assessment. In addition, inhibiting autophagy by 3-MA further increased the osteoblasts apoptosis after Pb exposure, showed by flow cytometry and Hoechst 33258 staining. Furthermore, phosphorylation of mTOR and p70S6K was inhibited by Pb exposure, indicating that Pb might induce autophagy in osteoblasts via inhibiting mTOR pathway. Altogether, these evidence suggested that Pb exporsure promoted autophagy flux in osteoblasts. The activation of autophagy by Pb played a protective role in osteoblasts apoptosis, which might be mediated through the mTOR pathway.

  16. Autophagy Protects against Palmitic Acid-Induced Apoptosis in Podocytes in vitro.

    PubMed

    Jiang, Xu-Shun; Chen, Xue-Mei; Wan, Jiang-Min; Gui, Hai-Bo; Ruan, Xiong-Zhong; Du, Xiao-Gang

    2017-02-22

    Autophagy is a highly conserved degradation process that is involved in the clearance of proteins and damaged organelles to maintain intracellular homeostasis and cell integrity. Type 2 diabetes is often accompanied by dyslipidemia with elevated levels of free fatty acids (FFAs). Podocytes, as an important component of the filtration barrier, are susceptible to lipid disorders. The loss of podocytes causes proteinuria, which is involved in the pathogenesis of diabetic nephropathy. In the present study, we demonstrated that palmitic acid (PA) promoted autophagy in podocytes. We further found that PA increased the production of reactive oxygen species (ROS) in podocytes and that NAC (N-acetyl-cysteine), a potent antioxidant, significantly eliminated the excessive ROS and suppressed autophagy, indicating that the increased generation of ROS was associated with the palmitic acid-induced autophagy in podocytes. Moreover, we also found that PA stimulation decreased the mitochondrial membrane potential in podocytes and induced podocyte apoptosis, while the inhibition of autophagy by chloroquine (CQ) enhanced palmitic acid-induced apoptosis accompanied by increased ROS generation, and the stimulation of autophagy by rapamycin (Rap) remarkably suppressed palmitic acid-induced ROS generation and apoptosis. Taken together, these in vitro findings suggest that PA-induced autophagy in podocytes is mediated by ROS production and that autophagy plays a protective role against PA-induced podocyte apoptosis.

  17. Autophagy confers DNA damage repair pathways to protect the hematopoietic system from nuclear radiation injury

    PubMed Central

    Lin, Weiwei; Yuan, Na; Wang, Zhen; Cao, Yan; Fang, Yixuan; Li, Xin; Xu, Fei; Song, Lin; Wang, Jian; Zhang, Han; Yan, Lili; Xu, Li; Zhang, Xiaoying; Zhang, Suping; Wang, Jianrong

    2015-01-01

    Autophagy is essentially a metabolic process, but its in vivo role in nuclear radioprotection remains unexplored. We observed that ex vivo autophagy activation reversed the proliferation inhibition, apoptosis, and DNA damage in irradiated hematopoietic cells. In vivo autophagy activation improved bone marrow cellularity following nuclear radiation exposure. In contrast, defective autophagy in the hematopoietic conditional mouse model worsened the hematopoietic injury, reactive oxygen species (ROS) accumulation and DNA damage caused by nuclear radiation exposure. Strikingly, in vivo defective autophagy caused an absence or reduction in regulatory proteins critical to both homologous recombination (HR) and non-homologous end joining (NHEJ) DNA damage repair pathways, as well as a failure to induce these proteins in response to nuclear radiation. In contrast, in vivo autophagy activation increased most of these proteins in hematopoietic cells. DNA damage assays confirmed the role of in vivo autophagy in the resolution of double-stranded DNA breaks in total bone marrow cells as well as bone marrow stem and progenitor cells upon whole body irradiation. Hence, autophagy protects the hematopoietic system against nuclear radiation injury by conferring and intensifying the HR and NHEJ DNA damage repair pathways and by removing ROS and inhibiting apoptosis. PMID:26197097

  18. Autophagy Protects against Palmitic Acid-Induced Apoptosis in Podocytes in vitro

    PubMed Central

    Jiang, Xu-shun; Chen, Xue-mei; Wan, Jiang-min; Gui, Hai-bo; Ruan, Xiong-zhong; Du, Xiao-gang

    2017-01-01

    Autophagy is a highly conserved degradation process that is involved in the clearance of proteins and damaged organelles to maintain intracellular homeostasis and cell integrity. Type 2 diabetes is often accompanied by dyslipidemia with elevated levels of free fatty acids (FFAs). Podocytes, as an important component of the filtration barrier, are susceptible to lipid disorders. The loss of podocytes causes proteinuria, which is involved in the pathogenesis of diabetic nephropathy. In the present study, we demonstrated that palmitic acid (PA) promoted autophagy in podocytes. We further found that PA increased the production of reactive oxygen species (ROS) in podocytes and that NAC (N-acetyl-cysteine), a potent antioxidant, significantly eliminated the excessive ROS and suppressed autophagy, indicating that the increased generation of ROS was associated with the palmitic acid-induced autophagy in podocytes. Moreover, we also found that PA stimulation decreased the mitochondrial membrane potential in podocytes and induced podocyte apoptosis, while the inhibition of autophagy by chloroquine (CQ) enhanced palmitic acid-induced apoptosis accompanied by increased ROS generation, and the stimulation of autophagy by rapamycin (Rap) remarkably suppressed palmitic acid-induced ROS generation and apoptosis. Taken together, these in vitro findings suggest that PA-induced autophagy in podocytes is mediated by ROS production and that autophagy plays a protective role against PA-induced podocyte apoptosis. PMID:28225005

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

    PubMed Central

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

    2015-01-01

    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. PMID:26490118

  20. Autophagy-related genes from a tick, Haemaphysalis longicornis.

    PubMed

    Umemiya, Rika; Matsuo, Tomohide; Hatta, Takeshi; Sakakibara, Shin-Ichi; Boldbaatar, Damdinsuren; Fujisaki, Kozo

    2008-01-01

    Ticks are gorging-fasting organisms;(1) their life cycle is characterized by alternate off-host (starvation) and on-host (meal) conditions. Their generation time is estimated in several years and many ticks spend more than 95% of their life off the host. They seem to have a unique strategy to endure the off-host state for a long period. Thus, we focused on autophagy, which is induced by starvation and is essential for extension of the lifespan,(2-4) and hypothesized that ticks also have a system of autophagy to overcome the starved condition. Recently, we showed the existence of a homologue of an ATG gene, ATG12, and its expression pattern from nymphal to adult stages in a three-host tick, Haemaphysalis longicornis. The expression level of HlATG12 was downregulated at the beginning of feeding and was highest at 3 months after engorgement. In addition, the HlAtg12 protein was localized to the region around granule-like structures within midgut cells of unfed adults. These results indicate that HlATG12 functions during unfed stages. Here, a potential role of autophagy in unfed ticks is discussed with regard to reports in other animals, such as yeast, mammal, and fruit fly.

  1. Heat shock protein 72 enhances autophagy as a protective mechanism in lipopolysaccharide-induced peritonitis in rats.

    PubMed

    Li, Shu; Zhou, Yi; Fan, Jinjin; Cao, Shirong; Cao, Tao; Huang, Fengxian; Zhuang, Shougang; Wang, Yihan; Yu, Xueqing; Mao, Haiping

    2011-12-01

    Peritoneal dialysis-related peritonitis causes the denudation of mesothelial cells and, ultimately, membrane integrity alterations and peritoneal dysfunction. Because heat shock protein 72 (HSP72) confers protection against apoptosis and because autophagy mediates survival in response to cellular stresses, we examined whether autophagy contributes to HSP72-mediated cytoprotection in lipopolysaccharide (LPS)-induced peritonitis. Exposure of cultured peritoneal mesothelial cells to LPS resulted first in autophagy and later, apoptosis. Inhibition of autophagy by 3-methyladenine or Beclin-1 small-interfering RNA sensitized cells to apoptosis and abolished the antiapoptotic effect of HSP72, suggesting that autophagy activation acts as a prosurvival mechanism. Overexpression of HSP72 augmented autophagy through c-Jun N-terminal kinase (JNK) phosphorylation and Beclin-1 up-regulation. Suppression of JNK activity reversed HSP72-mediated Beclin-1 up-regulation and autophagy, indicating that HSP72-mediated autophagy is JNK dependent. In a rat model of LPS-associated peritonitis, autophagy occurred before apoptosis in peritoneum. Up-regulation of HSP72 by geranylgeranylacetone increased autophagy, inhibited apoptosis, and attenuated peritoneal injury, and these effects were blunted by down-regulation of HSP72 with quercetin. Additionally, blocking autophagy by chloroquine promoted apoptosis and aggravated LPS-associated peritoneal dysfunction. Thus, HSP72 protects peritoneum from LPS-induced mesothelial cells injury, at least in part by enhancing JNK activation-dependent autophagy and inhibiting apoptosis. These findings imply that HSP72 induction might be a potential therapy for peritonitis.

  2. Sinomenine hydrochloride protects against polymicrobial sepsis via autophagy.

    PubMed

    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.

  3. Sinomenine Hydrochloride Protects against Polymicrobial Sepsis via Autophagy

    PubMed Central

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

    2015-01-01

    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. PMID:25625512

  4. Control of autophagy by oncogenes and tumor suppressor genes.

    PubMed

    Maiuri, M C; Tasdemir, E; Criollo, A; Morselli, E; Vicencio, J M; Carnuccio, R; Kroemer, G

    2009-01-01

    Multiple oncogenes (in particular phosphatidylinositol 3-kinase, PI3K; activated Akt1; antiapoptotic proteins from the Bcl-2 family) inhibit autophagy. Similarly, several tumor suppressor proteins (such as BH3-only proteins; death-associated protein kinase-1, DAPK1; the phosphatase that antagonizes PI3K, PTEN; tuberous sclerosic complex 1 and 2, TSC1 and TSC2; as well as LKB1/STK11) induce autophagy, meaning that their loss reduces autophagy. Beclin-1, which is required for autophagy induction acts as a haploinsufficient tumor suppressor protein, and other essential autophagy mediators (such as Atg4c, UVRAG and Bif-1) are bona fide oncosuppressors. One of the central tumor suppressor proteins, p53 exerts an ambiguous function in the regulation of autophagy. Within the nucleus, p53 can act as an autophagy-inducing transcription factor. Within the cytoplasm, p53 exerts a tonic autophagy-inhibitory function, and its degradation is actually required for the induction of autophagy. The role of autophagy in oncogenesis and anticancer therapy is contradictory. Chronic suppression of autophagy may stimulate oncogenesis. However, once a tumor is formed, autophagy inhibition may be a therapeutic goal for radiosensitization and chemosensitization. Altogether, the current state-of-the art suggests a complex relationship between cancer and deregulated autophagy that must be disentangled by further in-depth investigation.

  5. DRAM1 protects neuroblastoma cells from oxygen-glucose deprivation/reperfusion-induced injury via autophagy.

    PubMed

    Yu, Mengqiang; Jiang, Yugang; Feng, Qingliang; Ouyang, Yi'an; Gan, Jie

    2014-10-23

    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.

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

    NASA Astrophysics Data System (ADS)

    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. Protective role of autophagy in AGE-induced early injury of human vascular endothelial cells.

    PubMed

    Xie, Ying; You, Shou-Jiang; Zhang, Yan-Lin; Han, Qiao; Cao, Yong-Jun; Xu, Xing-Shun; Yang, Ya-Ping; Li, Jun; Liu, Chun-Feng

    2011-01-01

    Advanced glycation end-products (AGEs) contribute to the pathogenesis of diabetes mellitus and atherosclerosis by promoting vascular endothelial cell proliferation, migration, damage and death. In this study, we examined the role of autophagy in HUVECs exposed to AGE-modified bovine serum albumin (AGE-BSA). HUVECs incubated with AGE-BSA for 6 h showed an increase in the formation of acidic vesicular organelles and autophagosomes. AGE-BSA-induced upregulation of microtubule associated protein 1 light chain 3-II (LC3-II), a marker of autophagy, was abolished by pretreatment with the autophagy inhibitor 3-methyladenine (3-MA), and was increased by rapamycin, an autophagy inducer. The increase of lactate dehydrogenase (LDH) leakage induced by AGE-BSA was increased by 3-MA, but not rapamycin. An oxidative inhibitor, α-tocopherol, decreased not only the AGE-BSA-induced increase of reactive oxygen species, but also the upregulation of LC3-II protein levels. These results suggest that AGE-BSA increases the level of autophagy, which is protective against HUVEC injury, and that ROS play a role in this activation of autophagy.

  8. Corosolic acid protects hepatocytes against ethanol-induced damage by modulating mitogen-activated protein kinases and activating autophagy.

    PubMed

    Guo, Xiaolan; Cui, Ruibing; Zhao, Jianjian; Mo, Rui; Peng, Lei; Yan, Ming

    2016-11-15

    The reactive oxygen species(ROS)/mitogen-activated protein kinase (MAPK) destroyed autophagy and the reactive oxygen species/mitogen-activated protein kinase (MAPK) pathway are considered closely related to ethanol-induced hepatocellular injury. Previous work indicated that corosolic acid, the natural extracts of leaves of the banaba tree, Lagerstroemia speciosa L., could protect the liver against ethanol-induced damage, but the underlying mechanism is unclear. In the study we found that corosolic acid significantly inhibited ethanol-induced apoptosis, increased level of tumor necrosis factor-α(TNF-α) and reactive oxygen species accumulation in vitro. Corosolic acid inhibited ethanol-activated p38 and c-Jun N-terminal kinase MAPK signaling in BRL-3A and HepG2 cells as well as in experimental rats. Corosolic acid restored the ethanol-suppressed expression of autophagy-related genes, including beclin-1 and the ratio of microtubule-associated protein light chain 3II/I (LC3II/I) via AMP-activated protein kinase (AMPK) activation both in vitro and in vivo. In experimental rats, corosolic acid ameliorated the detrimental histopathological findings. Corosolic acid may protect the liver against ethanol-induced injury by modulation of MAPK signaling and autophagy activation. These findings suggested that corosolic acid might be a promising agent in treatment of alcoholic liver diseases.

  9. Curcumin induces apoptosis and protective autophagy in castration-resistant prostate cancer cells through iron chelation

    PubMed Central

    Yang, Chunguang; Ma, Xueyou; Wang, Zhihua; Zeng, Xing; Hu, Zhiquan; Ye, Zhangqun; Shen, Guanxin

    2017-01-01

    Background Curcumin induces apoptosis and autophagy in different cancer cells. Moreover, chemical and biological experiments have evidenced that curcumin is a biologically active iron chelator and induces cytotoxicity through iron chelation. We thus hypothesized that curcumin may induce apoptosis and autophagy in castration-resistant prostate cancer (CRPC) cells through its iron-chelating properties. Materials and methods CRPC cells were loaded with curcumin alone or in combination with ferric ammonium citrate (FAC). Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was assessed by flow cytometry, terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay and caspase activity. Autophagy status was analyzed by the detection of autophagosomes and light chain 3-II (LC3-II) using transmission electron microscopy and Western blot. Iron-binding activity of curcumin was assessed by spectrophotometry and MTT assay. The expression levels of transferrin receptor 1 (TfR1) and iron regulatory protein 1 (IRP1) were examined by Western blot. Results Curcumin induced apoptosis and autophagy in CRPC cells. Combining curcumin with autophagy inhibitors (3-methyladenine [3-MA]) synergized the apoptotic effect of curcumin. Moreover, curcumin bound to FAC at a ratio of ~1:1, as assessed by spectrophotometry and MTT assay. Apoptosis and autophagy induced by curcumin were counteracted by equal amounts of FAC. At apoptosis- and autophagy-inducing concentrations, curcumin enhanced the expression levels of TfR1 and IRP1, indicative of iron deprivation induced by curcumin. Conclusion Together, our results indicate that curcumin induces apoptosis and protective autophagy in CRPC cells, which are at least partially dependent on its iron-chelating properties. PMID:28243065

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

    SciTech Connect

    Dong, Chenglong; Zheng, Haining; Huang, Shanshan; You, Na; Xu, Jiarong; Ye, Xiaolong; Zhu, Qun; Feng, Yamin; You, Qiang; Miao, Heng; Ding, Dafa; Lu, Yibing

    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.

  11. Nano-TiO2 induces autophagy to protect against cell death through antioxidative mechanism in podocytes.

    PubMed

    Zhang, Xiaochen; Yin, Hongqiang; Li, Zhigui; Zhang, Tao; Yang, Zhuo

    2016-12-01

    Autophagy is a cellular pathway involved in degradation of damaged organelles and proteins in order to keep cellular homeostasis. It plays vital role in podocytes. Titanium dioxide nanoparticles (nano-TiO2) are known to induce autophagy in cells, but little has been reported about the mechanism of this process in podocytes and the role of autophagy in podocyte death. In the present study, we examined how nano-TiO2 induced authophagy. Besides that, whether autophagy could protect podocytes from the damage induced by nano-TiO2 and its mechanism was also investigated. Western blot assay and acridine orange staining presented that nano-TiO2 significantly enhanced autophagy flux in podocytes. In addition, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were involved in such process. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that upregulated level of autophagy induced by rapamycin in high concentration nano-TiO2-treated podocytes could significantly reduce the level of oxidative stress and alleviate podocyte death. Downregulating the level of autophagy with 3-methyladenine had the opposite effects. These findings indicate that nano-TiO2 induces autophagy through activating AMPK to inhibit mTOR in podocytes, and such autophagy plays a protecting role against oxidative stress on the cell proliferation. Changing autophagy level may become a new treatment strategy to relieve the damage induced by nano-TiO2 in podocytes.

  12. Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage.

    PubMed

    Han, Jing; Pan, Xue-Yang; Xu, Yan; Xiao, Yuan; An, Yu; Tie, Lu; Pan, Yan; Li, Xue-Jun

    2012-05-01

    Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H 2O 2-induced viability loss, which specifically evokes an autophagic response. Exposed to H 2O 2, curcumin-treated HUVECs upregulate the level of microtubule-associated protein 1 light chain 3-II (LC3-II), the number of autophagosomes, and the degradation of p62. We show that this compound promotes BECN1 expression and inhibits the phosphatidylinositol 3-kinase (PtdIns3K)-AKT-mechanistic target of rapamycin (MTOR) signaling pathway. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3-II to the expression of RAB7. These results suggest that curcumin induces autophagy, indicating that curcumin has the potential for use as an autophagic-related antioxidant for prevention and treatment of oxidative stress. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress-related cardiovascular diseases.

  13. Melatonin protects N2a against ischemia/reperfusion injury through autophagy enhancement.

    PubMed

    Guo, Yanchun; Wang, Jianfei; Wang, Zhongqiang; Yang, Yi; Wang, Ximing; Duan, Qiuhong

    2010-02-01

    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/reperfusion model in N2a cells was established for examinations. The results showed that autophagy was significantly enhanced in N2a cells treated with melatonin at reperfusion onset following ischemia and greatly promoted cell survival, while autophagy blockage by 3-MA led to the shortened N2a cell survival as assessed by MTT, transmission electron microscopy, and laser confocal scanning microscopy. Besides, the protein levels of LC3II and Beclin1 were remarkably increased in ischemia/reperfusion-injured N2a in the presence of melatonin, whereas the expression of p-PKB, key kinase in PI3K/PKB signaling pathway, showed a decrease when compared with untreated subjects as accessed by immunoblotting. Taken together these data suggest that autophagy is possibly one of the mechanisms underlying neuroprotection of melatonin.

  14. New homoisoflavonoid analogues protect cells by regulating autophagy.

    PubMed

    Gan, Li-She; Zeng, Lin-Wei; Li, Xiang-Rong; Zhou, Chang-Xin; Li, Jie

    2017-03-15

    As a special group of naturally occurring flavonoids, homoisoflavonoids have been discovered as active components of several traditional Chinese medicines for nourishing heart and mind. In this study, twenty homoisoflavonoid analogues, including different substitution groups on rings A and B, as well as heteroaromatic B ring, were synthesized and evaluated for their cardioprotective and neuroprotective activities. In a H2O2-induced H9c2 cardiomyocytes injury assay, nine homoisoflavonoid analogues showed promising activities in the same level as the positive control, diazoxide. Six cardioprotective compounds with representative structure diversities were then evaluated for their neuroprotective effects on MPP+ induced SH-SY5Y cell injury model. Furthermore, autophagy inducing monodansylcadaverine (MDC) fluorescence staining methods and molecular docking studies indicated the action mechanism of these compounds may involve autophagy regulating via class I PI3K signaling pathway.

  15. Genetic Variation in Autophagy-Related Genes Influences the Risk and Phenotype of Buruli Ulcer

    PubMed Central

    Capela, Carlos; Dossou, Ange Dodji; Silva-Gomes, Rita; Sopoh, Ghislain Emmanuel; Makoutode, Michel; Menino, João Filipe; Fraga, Alexandra Gabriel; Cunha, Cristina; Carvalho, Agostinho; Rodrigues, Fernando; Pedrosa, Jorge

    2016-01-01

    Introduction Buruli ulcer (BU) is a severe necrotizing human skin disease caused by Mycobacterium ulcerans. Clinically, presentation is a sum of these diverse pathogenic hits subjected to critical immune-regulatory mechanisms. Among them, autophagy has been demonstrated as a cellular process of critical importance. Since microtubules and dynein are affected by mycolactone, the critical pathogenic exotoxin produced by M. ulcerans, cytoskeleton-related changes might potentially impair the autophagic process and impact the risk and progression of infection. Objective Genetic variants in the autophagy-related genes NOD2, PARK2 and ATG16L1 has been associated with susceptibility to mycobacterial diseases. Here, we investigated their association with BU risk, its severe phenotypes and its progression to an ulcerative form. Methods Genetic variants were genotyped using KASPar chemistry in 208 BU patients (70.2% with an ulcerative form and 28% in severe WHO category 3 phenotype) and 300 healthy endemic controls. Results The rs1333955 SNP in PARK2 was significantly associated with increased susceptibility to BU [odds ratio (OR), 1.43; P = 0.05]. In addition, both the rs9302752 and rs2066842 SNPs in NOD2 gee significantly increased the predisposition of patients to develop category 3 (OR, 2.23; P = 0.02; and OR 12.7; P = 0.03, respectively, whereas the rs2241880 SNP in ATG16L1 was found to significantly protect patients from presenting the ulcer phenotype (OR, 0.35; P = 0.02). Conclusion Our findings indicate that specific genetic variants in autophagy-related genes influence susceptibility to the development of BU and its progression to severe phenotypes. PMID:27128681

  16. Beclin-1-mediated autophagy protects spinal cord neurons against mechanical injury-induced apoptosis.

    PubMed

    Wang, Zhen-Yu; Lin, Jian-Hua; Muharram, Akram; Liu, Wen-Ge

    2014-06-01

    Apoptosis has been widely reported to be involved in the pathogenesis associated with spinal cord injury (SCI). Recently, autophagy has also been implicated in various neuronal damage models. However, the role of autophagy in SCI is still controversial and its interrelationship with apoptosis remains unclear. Here, we used an in vitro SCI model to observe a time-dependent induction of autophagy and apoptosis. Mechanical injury induced autophagy markers such as LC3 lipidation, LC3II/LC3I conversion, and Beclin-1 expression. Injured neurons showed decreased cell viability and increased apoptosis. To elucidate the effect of autophagy on apoptosis, the mechanically-injured neurons were treated with the mTOR inhibitor rapamycin and 3-methyl adenine (3-MA), which are known to regulate autophagy positively and negatively, respectively. Rapamycin-treated neurons showed the highest level of cell viability and lowest level of apoptosis among the injured neurons and those treated with 3-MA showed the reciprocal effect. Notably, rapamycin-treated neurons exhibited slightly reduced Bax expression and significantly increased Bcl-2 expression. Furthermore, by plasmid transfection, we showed that Beclin-1-overexpressing neuronal cells responded to mechanical injury with greater LC3II/LC3I conversion and cell viability, lower levels of apoptosis, higher Bcl-2 expression, and unaltered Bax expression as compared to vector control cells. Beclin-1-knockdown neurons showed almost the opposite effects. Taken together, our results suggest that autophagy may serve as a protection against apoptosis in mechanically-injured spinal cord neurons. Targeting mTOR and/or enhancing Beclin-1 expression might be alternative therapeutic strategies for SCI.

  17. Orientin protects myocardial cells against hypoxia-reoxygenation injury through induction of autophagy.

    PubMed

    Liu, Liya; Wu, Youxi; Huang, Xiulan

    2016-04-05

    Orientin, a flavonoid exists in Chinese traditional herbal Polygonum orientale L., has been previously demonstrated to protect against myocardial ischemia reperfusion injury (MIRI) through inhibition of apoptosis. However, the underlying mechanisms remain to be elucidated and we therefore in this study investigated the effects of orientin on autophagy during MIRI in rats. The results indicate that orientin, at the concentrations of 10 and 30 μM in the cultures of neonatal rat cardiomyocytes, promoted the induction of autophagy, increasing the formation of autophagosomes and enhancing the expression of LC3 puncta, LC3-II/LC3-I ratio and Beclin 1 after hypoxia/reoxygenation. The induction of autophagy by orientin correlated with enhanced cell viability and decreased apoptosis, which was significantly attenuated by autophagy inhibitor wortmannin, a phosphatidylinositol-3-kinase (PI3K) inhibitor. Moreover, application of orientin increased the activation of AMPK and Akt, downregulated the phosphorylation of mammalian target of rapamycin (mTOR) and the expression of Raptor, and enhanced the interaction between Beclin 1 and Bcl-2 in endoplasmic reticulum due to increased phosphorylation of Beclin 1 and decreased phosphorylation of Bcl-2. Our investigation suggests that the cardioprotective effects of orientin during MIRI may be mediated through the balance of autophagy through regulating AMPK, Akt, mTOR, and Bcl-2 associated signaling pathways.

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

    PubMed Central

    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

  19. Glucosamine protects nucleus pulposus cells and induces autophagy via the mTOR-dependent pathway.

    PubMed

    Jiang, LiBo; Jin, YongLong; Wang, HuiRen; Jiang, YunQi; Dong, Jian

    2014-11-01

    Although glucosamine has been suggested to be effective in the treatment of osteoarthritis, its effect on disc degeneration remains unclear. We sought to explore whether glucosamine can activate autophagy in rat nucleus pulposus (NP) cells and protect cells treated with IL-1β or hydrogen peroxide (H2 O2 ). Autophagy in cells was examined by detecting for LC3, Beclin-1, m-TOR, and p70S6K, as well as by analyzing autophagosomes. To inhibit autophagy, 3-methyladenine (3-MA) was used. In the cells treated with IL-1β, the levels of Adamts-4, Mmp-13, aggrecan, and Col2a1 were analyzed by real-time PCR and immunofluorescence. Apoptosis was analyzed by TUNEL. Cell senescence under H2 O2 was revealed by SA-β-Gal staining. Glucosamine could activate autophagy in a dose-dependent manner within 24 h and inhibit the phosphorylation of m-TOR and p70S6K. Autophagy in IL-1β or H2 O2 -treated cells was increased by glucosamine. Glucosamine attenuated the decrease of aggrecan and prevented the apoptosis of the NP cells induced by IL-1β, whereas 3-MA partly reversed these effects. The percentage of SA-β-Gal-positive cells induced by H2 O2 treatment was decreased by glucosamine, accompanied by the decline of p70S6K phosphorylation. Glucosamine protects NP cells and up-regulates autophagy by inhibiting the m-TOR pathway, which might point a potential therapeutic agent for disc degeneration.

  20. Probiotic Bacillus amyloliquefaciens SC06 Induces Autophagy to Protect against Pathogens in Macrophages

    PubMed Central

    Wu, Yanping; Wang, Yang; Zou, Hai; Wang, Baikui; Sun, Qiming; Fu, Aikun; Wang, Yuanyuan; Wang, Yibing; Xu, Xiaogang; Li, Weifen

    2017-01-01

    Probiotics are increasingly applied in popularity in both humans and animals. Decades of research has revealed their beneficial effects, including the immune modulation in intestinal pathogens inhibition. Autophagy—a cellular process that involves the delivery of cytoplasmic proteins and organelles to the lysosome for degradation and recirculation—is essential to protect cells against bacterial pathogens. However, the mechanism of probiotics-mediated autophagy and its role in the elimination of pathogens are still unknown. Here, we evaluated Bacillus amyloliquefaciens SC06 (Ba)-induced autophagy and its antibacterial activity against Escherichia coli (E. coli) in murine macrophage cell line RAW264.7 cells. Western blotting and confocal laser scanning analysis showed that Ba activated autophagy in a dose- and time-dependent manner. Ba-induced autophagy was found to play a role in the elimination of intracellular bacteria when RAW264.7 cells were challenged with E. coli. Ba induced autophagy by increasing the expression of Beclin1 and Atg5-Atg12-Atg16 complex, but not the AKT/mTOR signaling pathway. Moreover, Ba pretreatment attenuated the activation of JNK in RAW264.7 cells during E. coli infection, further indicating a protective role for probiotics via modulating macrophage immunity. The above findings highlight a novel mechanism underlying the antibacterial activity of probiotics. This study enriches the current knowledge on probiotics-mediated autophagy, and provides a new perspective on the prevention of bacterial infection in intestine, which further the application of probiotics in food products. PMID:28382029

  1. Transcriptional activation of TFEB/ZKSCAN3 target genes underlies enhanced autophagy in spinobulbar muscular atrophy.

    PubMed

    Chua, Jason P; Reddy, Satya L; Merry, Diane E; Adachi, Hiroaki; Katsuno, Masahisa; Sobue, Gen; Robins, Diane M; Lieberman, Andrew P

    2014-03-01

    Spinobulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in exon 1 of the androgen receptor (AR) gene. SBMA demonstrates androgen-dependent toxicity due to unfolding and aggregation of the mutant protein. There are currently no disease-modifying therapies, but of increasing interest for therapeutic targeting is autophagy, a highly conserved cellular process mediating protein quality control. We have previously shown that genetic manipulations inhibiting autophagy diminish skeletal muscle atrophy and extend the lifespan of AR113Q knock-in mice. In contrast, manipulations inducing autophagy worsen muscle atrophy, suggesting that chronic, aberrant upregulation of autophagy contributes to pathogenesis. Since the degree to which autophagy is altered in SBMA and the mechanisms responsible for such alterations are incompletely defined, we sought to delineate autophagic status in SBMA using both cellular and mouse models. Here, we confirm that autophagy is induced in cellular and knock-in mouse models of SBMA and show that the transcription factors transcription factor EB (TFEB) and ZKSCAN3 operate in opposing roles to underlie these changes. We demonstrate upregulation of TFEB target genes in skeletal muscle from AR113Q male mice and SBMA patients. Furthermore, we observe a greater response in AR113Q mice to physiological stimulation of autophagy by both nutrient starvation and exercise. Taken together, our results indicate that transcriptional signaling contributes to autophagic dysregulation and provides a mechanistic framework for the pathologic increase of autophagic responsiveness in SBMA.

  2. Autophagy Protects Advanced Glycation End Product-Induced Apoptosis and Expression of MMP-3 and MMP-13 in Rat Chondrocytes

    PubMed Central

    Wu, Tianlong

    2017-01-01

    Aging is one of the most prominent risk factors for the pathological progression of osteoarthritis (OA). One feature of age-related changes in OA is advanced glycation end products (AGEs) accumulation in articular cartilage. Autophagy plays a cellular housekeeping role by removing dysfunctional cellular organelles and proteins. However, the relationship between autophagy and AGE-associated OA is unknown. The aim of this study is to determine whether autophagy participates in the pathology of AGE-treated chondrocytes and to investigate the exact role of autophagy in AGE-induced cell apoptosis and expression of matrix metalloproteinase- (MMP-) 3 and MMP-13. AGEs induced notable apoptosis that was detected by Annexin V/PI double-staining, and the upregulation of MMP-3 and MMP-13 was confirmed by Western blotting. Autophagy-related proteins were also determined by Western blotting, and chondrocytes were transfected with mCherry-GFP-LC3B-adenovirus to monitor autophagic flux. As a result, autophagy significantly increased in chondrocytes and peaked at 6 h. Furthermore, rapamycin (RA) attenuated AGE-induced apoptosis and expression of MMP-3 and MMP-13 by autophagy activation. In contrast, pretreatment with autophagy inhibitor 3-methyladenine (3-MA) enhanced the abovementioned effects of AGEs. We therefore demonstrated that autophagy is linked with AGE-related pathology in rat chondrocytes and plays a protective role in AGE-induced apoptosis and expression of MMP-3 and MMP-13. PMID:28265573

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

    PubMed

    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.

  4. Data supporting the activation of autophagy genes in the diabetic heart.

    PubMed

    Munasinghe, Pujika Emani; Riu, Federica; Dixit, Parul; Edamatsu, Midori; Saxena, Pankaj; Hamer, Nathan S J; Galvin, Ivor F; Bunton, Richard W; Lequeux, Sharon; Jones, Greg; Lamberts, Regis R; Emanueli, Costanza; Madeddu, Paolo; Katare, Rajesh

    2015-12-01

    This data article contains full list of autophagy related genes that are altered in diabetic heart. This article also shows data from in vitro cultured cardiomyocytes that are exposed the high glucose treatment to simulate hyperglycemic state in vitro. The interpretation of these data and further extensive insights into the regulation of SG biogenesis by AMPK can be found in "Type-2 diabetes increases autophagy in the human heart through promotion of Beclin-1 mediated pathway" (Munasinghe et al., in press) [1].

  5. Autophagy activation protects shock wave induced renal tubular epithelial cell apoptosis may through modulation of Akt/ GSK-3β pathway

    PubMed Central

    Long, Qingzhi; Li, Xiang; He, Hui; He, Dalin

    2016-01-01

    Purpose: Extracorporeal shock wave lithotripsy (ESWL) is well documented to exert destructive effect to renal cells and its mechanism is not clear. Autophagy is one of cell basic response for stressful conditions and it is important to determine cell's fate. The aim of this study is to elucidate the role of autophagy in the process of shock wave-induced renal cells injury. Methods: NRK-52E cell, a rat renal tubular epithelial cell, was exposed to shock wave at the voltage of 14KV. GFP-LC3 puncta was used to monitor Autophagy flux in the process of shock wave injury. Autophagic relative proteins, such as light chain 3 (LC3), beclin-1 and p62, were also examined. Cell variability and apoptosis were detected when inhibition autophagy with 3-methyladenine (3MA) or stimulating its activity with rapamycin during the process of shock wave injury. The role of Akt/ GSK-3β and its connection with autophagy in the process of shock wave injury were also investigated. Results: Shock wave was confirmed to activate autophagy in renal cells, which was manifested in LC3-II turnover, beclin-1 induction and degradation of p62. Inhibition autophagy enhanced cell damage or apoptosis, whereas its stimulating was able to exert protection from shock wave injury. Akt/ GSK-3β, a cell-survival signaling pathway, can also be activated during the process. And its activation could be suppressed by blockade autophagy. Conclusion: Autophagy is a self-protective response for renal cells from shock wave injury. The cyto-protection of autophagy may be connected with modulation Akt/ GSK-3β pathway. PMID:27994511

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

    SciTech Connect

    Guo, Xu-Guang; Ji, Tian-Xing; Xia, Yong; Ma, Yue-Yun

    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

  7. Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis.

    PubMed

    Lenoir, Olivia; Jasiek, Magali; Hénique, Carole; Guyonnet, Léa; Hartleben, Björn; Bork, Tillmann; Chipont, Anna; Flosseau, Kathleen; Bensaada, Imane; Schmitt, Alain; Massé, Jean-Marc; Souyri, Michèle; Huber, Tobias B; Tharaux, Pierre-Louis

    2015-01-01

    The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN.

  8. Heat stroke induces autophagy as a protection mechanism against neurodegeneration in the brain.

    PubMed

    Liu, Tsung-Ta; Hu, Chou-Hui; Tsai, Chu-Dang; Li, Chuan-Wang; Lin, Yuh-Feng; Wang, Jia-Yi

    2010-12-01

    Heat stroke (HS) is defined clinically as a condition when core body temperature rises above 40°C and is accompanied by central nervous system abnormalities. In this study, we established a rat model of HS by exposing anesthetized rats to elevated ambient temperature (40°C) until core temperature reaching 40.5°C (HS onset). The rat was immediately removed from heating chamber, allowed recovery for various time periods, and killed for histological and biochemical studies. Our results indicated neuronal shrinkage and pyknosis of the nucleus and sustained up to 12 h recovery time in cerebral cortex. Elevated expression of autophagy-related proteins, including microtubule associated protein light chain 3 and beclin 1 in cortical tissue at various times (3, 6, 12 h) of recovery was observed. In addition, the number of autophagosomes stained by monodansylcadaverine, a specific autophagosome marker, increased after heat exposure but was reduced by pretreatment with 3-methyladenine, an autophagy inhibitor. Furthermore, heat exposure increased neuronal degeneration in cortical tissue, as evidenced by staining with the fluorescent dye Fluoro-Jade B for degenerating neuron. Pretreatment with 3-methyladenine in HS rats aggravated neurodegeneration. Taken together, these results suggest that HS induces autophagy as a protection mechanism against neurodegeneration. Modulation of autophagy may provide a potential therapeutic approach for HS and await further research.

  9. Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis

    PubMed Central

    Lenoir, Olivia; Jasiek, Magali; Hénique, Carole; Guyonnet, Léa; Hartleben, Björn; Bork, Tillmann; Chipont, Anna; Flosseau, Kathleen; Bensaada, Imane; Schmitt, Alain; Massé, Jean-Marc; Souyri, Michèle; Huber, Tobias B; Tharaux, Pierre-Louis

    2015-01-01

    The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN. PMID:26039325

  10. Autophagy-related gene 7 (ATG7) and reactive oxygen species/extracellular signal-regulated kinase regulate tetrandrine-induced autophagy in human hepatocellular carcinoma.

    PubMed

    Gong, Ke; Chen, Chao; Zhan, Yao; Chen, Yan; Huang, Zebo; Li, Wenhua

    2012-10-12

    Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the broadly used Chinese medicinal herb Stephaniae tetrandrae, exhibits potent antitumor effects and has the potential to be used as a cancer chemotherapeutic agent. We previously reported that high concentrations of tetrandrine induce apoptosis in liver cancer cells. Here, we found that in human hepatocellular carcinoma (HCC) cells, a low dose of tetrandrine (5 μm) induced the expression of LC3-II, resulted in the formation of acidic autophagolysosome vacuoles (AVOs), and caused a punctate fluorescence pattern with the GFP-LC3 protein, which all are markers for cellular autophagy. Tetrandrine induced the production of intracellular reactive oxygen species (ROS), and treatment with ROS scavengers significantly abrogated the tetrandrine-induced autophagy. These results suggest that the generation of ROS plays an important role in promoting tetrandrine-induced autophagy. Tetrandrine-induced mitochondrial dysfunction resulted in ROS accumulation and autophagy. ROS generation activated the ERK MAP kinase, and the ERK signaling pathway at least partially contributed to tetrandrine-induced autophagy in HCC cells. Moreover, we found that tetrandrine transcriptionally regulated the expression of autophagy related gene 7 (ATG7), which promoted tetrandrine-induced autophagy. In addition to in vitro studies, similar results were also observed in vivo, where tetrandrine caused the accumulation of ROS and induced cell autophagy in a tumor xenograft model. Interestingly, tetrandrine treatment also induced autophagy in a ROS-dependent manner in C. elegans muscle cells. Therefore, these findings suggest that tetrandrine is a potent autophagy agonist and may be a promising clinical chemotherapeutic agent.

  11. Triptolide induces protective autophagy through activation of the CaMKKβ-AMPK signaling pathway in prostate cancer cells

    PubMed Central

    Zhang, Zhe; Mao, Lin; Han, Yangyang; Yan, Jun; Lei, Ming

    2016-01-01

    Triptolide, an active compound extracted from the Chinese herb thunder god vine (Tripterygium wilfordii Hook F.), has potent anti-tumor activity. Recently, triptolide was found to induce autophagy in cancer cells. However, the effects of triptolide on autophagy in human prostate cancer (PCa) cells have not yet been clearly elucidated. In this study, we demonstrated that triptolide induces autophagy in three PCa cell lines, PC-3, LNCaP and C4–2. Furthermore, we found that triptolide mediates intracellular accumulation of free calcium by stimulating the endoplasmic reticulum (ER) stress response. This activates the CaMKKβ-AMPK signaling pathway, which in turn inhibits mTOR and activates both ULK1 and Beclin 1, finally resulting in autophagy. Moreover, we found that treatment with autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) enhances triptolide-induced PCa cell death and growth inhibition. Using a PC-3-xenografted mouse model, we showed that blocking autophagy with CQ significantly promoted triptolide-induced tumor growth inhibition in vivo. Overall, our results show that triptolide induces protective autophagy through the CaMKKβ-AMPK pathway in PCa cells, implying that a combination of triptolide with autophagy inhibitors may potentially be an effective therapeutic strategy for PCa. PMID:26734992

  12. Triptolide induces protective autophagy through activation of the CaMKKβ-AMPK signaling pathway in prostate cancer cells.

    PubMed

    Zhao, Fei; Huang, Weiwei; Zhang, Zhe; Mao, Lin; Han, Yangyang; Yan, Jun; Lei, Ming

    2016-02-02

    Triptolide, an active compound extracted from the Chinese herb thunder god vine (Tripterygium wilfordii Hook F.), has potent anti-tumor activity. Recently, triptolide was found to induce autophagy in cancer cells. However, the effects of triptolide on autophagy in human prostate cancer (PCa) cells have not yet been clearly elucidated. In this study, we demonstrated that triptolide induces autophagy in three PCa cell lines, PC-3, LNCaP and C4-2. Furthermore, we found that triptolide mediates intracellular accumulation of free calcium by stimulating the endoplasmic reticulum (ER) stress response. This activates the CaMKKβ-AMPK signaling pathway, which in turn inhibits mTOR and activates both ULK1 and Beclin 1, finally resulting in autophagy. Moreover, we found that treatment with autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) enhances triptolide-induced PCa cell death and growth inhibition. Using a PC-3-xenografted mouse model, we showed that blocking autophagy with CQ significantly promoted triptolide-induced tumor growth inhibition in vivo. Overall, our results show that triptolide induces protective autophagy through the CaMKKβ-AMPK pathway in PCa cells, implying that a combination of triptolide with autophagy inhibitors may potentially be an effective therapeutic strategy for PCa.

  13. Autophagy and formation of tubulovesicular autophagosomes provide a barrier against nonviral gene delivery.

    PubMed

    Roberts, Rebecca; Al-Jamal, Wafa' T; Whelband, Matthew; Thomas, Paul; Jefferson, Matthew; van den Bossche, Jeroen; Powell, Penny P; Kostarelos, Kostas; Wileman, Thomas

    2013-05-01

    Cationic liposome (lipoplex) and polymer (polyplex)-based vectors have been developed for nonviral gene delivery. These vectors bind DNA and enter cells via endosomes, but intracellular transfer of DNA to the nucleus is inefficient. Here we show that lipoplex and polyplex vectors enter cells in endosomes, activate autophagy and generate tubulovesicular autophagosomes. Activation of autophagy was dependent on ATG5, resulting in lipidation of LC3, but did not require the PtdIns 3-kinase activity of PIK3C3/VPS34. The autophagosomes generated by lipoplex fused with each other, and with endosomes, resulting in the delivery of vectors to large tubulovesicular autophagosomes, which accumulated next to the nucleus. The tubulovesicular autophagosomes contained autophagy receptor protein SQSTM1/p62 and ubiquitin, suggesting capture of autophagy cargoes, but fusion with lysosomes was slow. Gene delivery and expression from both lipoplex and polyplex increased 8-fold in atg5 (-/-) cells unable to generate tubulovesicular autophagosomes. Activation of autophagy and capture within tubulovesicular autophagosomes therefore provides a new cellular barrier against efficient gene transfer and should be considered when designing efficient nonviral gene delivery vectors.

  14. Novel functional roles of caspase-related genes in the regulation of apoptosis and autophagy

    PubMed Central

    Shin, Ju-Hyun

    2016-01-01

    Caspases, a family of cysteine proteases, cleave substrates and play significant roles in apoptosis, autophagy, and development. Recently, our group identified 72 genes that interact with Death Caspase-1 (DCP-1) proteins in Drosophila by genetic screening of 15,000 EP lines. However, the cellular functions and molecular mechanisms of the screened genes, such as their involvement in apoptosis and autophagy, are poorly understood in mammalian cells. In order to study the functional characterizations of the genes in human cells, we investigated 16 full-length human genes in mammalian expression vectors and tested their effects on apoptosis and autophagy in human cell lines. Our studies revealed that ALFY, BIRC4, and TAK1 induced autophagy, while SEC61A2, N-PAC, BIRC4, WIPI1, and FALZ increased apoptotic cell death. BIRC4 was involved in both autophagy and apoptosis. Western blot analysis and luciferase reporter activity indicated that ALFY, BIRC4, PDGFA, and TAK1 act in a p53-dependent manner, whereas CPSF1, SEC61A2, N-PAC, and WIPI1 appear to be p53-independent. Overexpression of BIRC4 and TAK1 caused upregulation of p53 and accumulation of its target proteins as well as an increase in p53 mRNA levels, suggesting that these genes are involved in p53 transcription and expression of its target genes followed by p53 protein accumulation. In conclusion, apoptosis and/or autophagy mediated by BIRC4 and TAK1 may be regulated by p53 and caspase activity. These novel findings may provide valuable information that will aid in a better understanding of the roles of caspase-related genes in human cell lines and be useful for the process of drug discovery. PMID:27847434

  15. HK2/hexokinase-II integrates glycolysis and autophagy to confer cellular protection.

    PubMed

    Tan, Valerie P; Miyamoto, Shigeki

    2015-01-01

    Hexokinases (HKs) catalyze the first step of glucose metabolism, phosphorylating glucose to glucose 6-phosphate (G6P). HK2/hexokinase-II is a predominant isoform in insulin-sensitive tissues such as heart, skeletal muscle, and adipose tissues and is also upregulated in many types of tumors associated with enhanced aerobic glycolysis (the Warburg effect). Accumulating evidence indicates that HK2 plays an important role not only in glycolysis but also in cell survival. Although there is increasing recognition that cellular metabolism and cell survival are closely related, the molecular link between metabolism and autophagic pathways has not been fully elucidated. We recently discovered that HK2 facilitates autophagy in response to glucose deprivation (HK substrate deprivation) to protect cardiomyocytes, and suggest that HK2 functions as a molecular switch from glycolysis to autophagy to ensure cellular energy homeostasis under starvation conditions.

  16. Autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell

    PubMed Central

    Li, Wei; Zou, Wei; Yang, Yihong; Chai, Yongping; Chen, Baohui; Cheng, Shiya; Tian, Dong

    2012-01-01

    Apoptotic cell degradation is a fundamental process for organism development, and impaired clearance causes inflammatory or autoimmune disease. Although autophagy genes were reported to be essential for exposing the engulfment signal on apoptotic cells, their roles in phagocytes for apoptotic cell removal are not well understood. In this paper, we develop live-cell imaging techniques to study apoptotic cell clearance in the Caenorhabditis elegans Q neuroblast lineage. We show that the autophagy proteins LGG-1/LC3, ATG-18, and EPG-5 were sequentially recruited to internalized apoptotic Q cells in the phagocyte. In atg-18 or epg-5 mutants, apoptotic Q cells were internalized but not properly degraded; this phenotype was fully rescued by the expression of autophagy genes in the phagocyte. Time-lapse analysis of autophagy mutants revealed that recruitment of the small guanosine triphosphatases RAB-5 and RAB-7 to the phagosome and the formation of phagolysosome were all significantly delayed. Thus, autophagy genes act within the phagocyte to promote apoptotic cell degradation. PMID:22451698

  17. Autophagy in Acute Kidney Injury

    PubMed Central

    Livingston, Man J.; Dong, Zheng

    2014-01-01

    Acute kidney injury is a major kidney disease associated with poor clinical outcomes. The pathogenesis of acute kidney injury is multifactorial and is characterized by tubular cell injury and death. Recent studies have demonstrated autophagy induction in proximal tubular cells during acute kidney injury. The regulatory mechanisms of tubular cell autophagy are poorly understood; however, some recent findings have set up a foundation for further investigation. Although autophagy may promote cell death under certain experimental conditions, pharmacological and autophagy-related gene knockout studies have established a renoprotective role for autophagy in acute kidney injury. The mechanisms by which autophagy protects cells from injury and how, possibly, its pro-survival role switches to pro-death under certain conditions are discussed. Further research is expected to help us understand the regulatory network of tubular cell autophagy, define its precise roles in specific context of acute kidney injury, and identify autophagy-targeting strategies for the prevention and treatment of acute kidney injury. PMID:24485026

  18. Electroacupuncture protects against ischemic stroke by reducing autophagosome formation and inhibiting autophagy through the mTORC1-ULK1 complex-Beclin1 pathway

    PubMed Central

    LIU, WEILIN; SHANG, GUANHAO; YANG, SHANLI; HUANG, JIA; XUE, XIEHUA; LIN, YUNJIAO; ZHENG, YI; WANG, XIAN; WANG, LULU; LIN, RUHUI; TAO, JING; CHEN, LIDIAN

    2016-01-01

    In a previous study by our group, we demonstrated that electroacupuncture (EA) activates the class I phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. There is considerable evidence that the downstream mammalian target of rapamycin complex 1 (mTORC1) plays an important role in autophagy following ischemic stroke. The aim of the present study was to determine whether EA exerts a neuroprotective effect through mTORC1-mediated autophagy following ischemia/reperfusion injury. Our results revealed that EA at the LI11 and ST36 acupoints attenuated motor dysfunction, improved neurological deficit outcomes and decreased the infarct volumes. The number of autophagosomes, autolysosomes and lysosomes was decreased following treatment with EA. Simultaneously, the levels of the autophagosome membrane maker, microtubule-associated protein 1 light chain 3 beta (LC3B)II/I, Unc-51-like kinase 1 (ULK1), autophagy related gene 13 Atg13) and Beclin1 (ser14) were decreased, whereas mTORC1 expression was increased in the peri-infarct cortex. These results suggest that EA protects against ischemic stroke through the inhibition of autophagosome formation and autophagy, which is mediated through the mTORC1-ULK complex-Beclin1 pathway. PMID:26647915

  19. Quercetin induces protective autophagy in gastric cancer cells: involvement of Akt-mTOR- and hypoxia-induced factor 1α-mediated signaling.

    PubMed

    Wang, Kui; Liu, Rui; Li, Jingyi; Mao, Jiali; Lei, Yunlong; Wu, Jinhua; Zeng, Jun; Zhang, Tao; Wu, Hong; Chen, Lijuan; Huang, Canhua; Wei, Yuquan

    2011-09-01

    Quercetin, a dietary antioxidant present in fruits and vegetables, is a promising cancer chemopreventive agent that inhibits tumor promotion by inducing cell cycle arrest and promoting apoptotic cell death. In this study, we examined the biological activities of quercetin against gastric cancer. Our studies demonstrated that exposure of gastric cancer cells AGS and MKN28 to quercetin resulted in pronounced pro-apoptotic effect through activating the mitochondria pathway. Meanwhile, treatment with quercetin induced appearance of autophagic vacuoles, formation of acidic vesicular organelles (AVOs), conversion of LC3-I to LC3-II, recruitment of LC3-II to the autophagosomes as well as activation of autophagy genes, suggesting that quercetin initiates the autophagic progression in gastric cancer cells. Furthermore, either administration of autophagic inhibitor chloroquine or selective ablation of atg5 or beclin 1 using small interfering RNA (siRNA) could augment quercetin-induced apoptotic cell death, suggesting that autophagy plays a protective role against quercetin-induced apoptosis. Moreover, functional studies revealed that quercetin activated autophagy by modulation of Akt-mTOR signaling and hypoxia-induced factor 1α (HIF-1α) signaling. Finally, a xenograft model provided additional evidence for occurrence of quercetin-induced apoptosis and autophagy in vivo. Together, our studies provided new insights regarding the biological and anti-proliferative activities of quercetin against gastric cancer, and may contribute to rational utility and pharmacological study of quercetin in future anti-cancer research.

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

    PubMed

    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.

  1. Bacopa monnieri-Induced Protective Autophagy Inhibits Benzo[a]pyrene-Mediated Apoptosis.

    PubMed

    Das, Durgesh Nandini; Naik, Prajna Paramita; Nayak, Aditi; Panda, Prashanta Kumar; Mukhopadhyay, Subhadip; Sinha, Niharika; Bhutia, Sujit K

    2016-11-01

    Benzo[a]pyrene (B[a]P) is capable of inducing oxidative stress and cellular injuries leading to cell death and associates with a significant risk of cancer development. Prevention of B[a]P-induced cellular toxicity with herbal compound through regulation of mitochondrial oxidative stress might protect cell death and have therapeutic benefit to human health. In this study, we demonstrated the cytoprotective role of Bacopa monnieri (BM) against B[a]P-induced apoptosis through autophagy induction. Pretreatment with BM rescued the reduction in cell viability in B[a]P-treated human keratinocytes (HaCaT) cells indicating the cytoprotective potential of BM against B[a]P. Moreover, BM was found to inhibit B[a]P-mediated reactive oxygen species (ROS)-induced apoptosis activation in HaCaT cells. Furthermore, BM was found to preserve mitochondrial membrane potential and inhibited release of cytochrome c in B[a]P-treated HaCaT cells. Bacopa monnieri induced protective autophagy; we knocked down Beclin-1, and data showed that BM was unable to protect from B[a]P-induced mitochondrial ROS-mediated apoptosis in Beclin-1-deficient HaCaT cells. Moreover, we established that B[a]P-induced damaged mitochondria were found to colocalize and degraded within autolysosomes in order to protect HaCaT cells from mitochondrial injury. In conclusion, B[a]P-induced apoptosis was rescued by BM treatment and provided cytoprotection through Beclin-1-dependent autophagy activation. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Agonist antibodies activating the Met receptor protect cardiomyoblasts from cobalt chloride-induced apoptosis and autophagy.

    PubMed

    Gallo, S; Gatti, S; Sala, V; Albano, R; Costelli, P; Casanova, E; Comoglio, P M; Crepaldi, T

    2014-04-17

    Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), mainly activates prosurvival pathways, including protection from apoptosis. In this work, we investigated the cardioprotective mechanisms of Met activation by agonist monoclonal antibodies (mAbs). Cobalt chloride (CoCl2), a chemical mimetic of hypoxia, was used to induce cardiac damage in H9c2 cardiomyoblasts, which resulted in reduction of cell viability by (i) caspase-dependent apoptosis and (ii) - surprisingly - autophagy. Blocking either apoptosis with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone or autophagosome formation with 3-methyladenine prevented loss of cell viability, which suggests that both processes contribute to cardiomyoblast injury. Concomitant treatment with Met-activating antibodies or HGF prevented apoptosis and autophagy. Pro-autophagic Redd1, Bnip3 and phospho-AMPK proteins, which are known to promote autophagy through inactivation of the mTOR pathway, were induced by CoCl2. Mechanistically, Met agonist antibodies or HGF prevented the inhibition of mTOR and reduced the flux of autophagosome formation. Accordingly, their anti-autophagic function was completely blunted by Temsirolimus, a specific mTOR inhibitor. Targeted Met activation was successful also in the setting of low oxygen conditions, in which Met agonist antibodies or HGF demonstrated anti-apoptotic and anti-autophagic effects. Activation of the Met pathway is thus a promising novel therapeutic tool for ischaemic injury.

  3. PTEN Activation by DNA Damage Induces Protective Autophagy in Response to Cucurbitacin B in Hepatocellular Carcinoma Cells

    PubMed Central

    Niu, Yanan; Sun, Wen; Lu, Jin-Jian; Pei, Lixia

    2016-01-01

    Cucurbitacin B (Cuc B), a natural product, induced both protective autophagy and DNA damage mediated by ROS while the detailed mechanisms remain unclear. This study explored the mechanism of Cuc B-induced DNA damage and autophagy. Cuc B decreased cell viability in concentration- and time-dependent manners. Cuc B caused long comet tails and increased expression of γ-H2AX, phosphorylation of ATM/ATR, and Chk1/Chk2. Cuc B induced autophagy as evidenced by monodansylcadaverine (MDC) staining, increased expression of LC3II, phosphorylated ULK1, and decreased expression of phosphorylated AKT, mTOR. Cuc B induced apoptosis mediated by Bcl-2 family proteins and caspase activation. Furthermore, Cuc B induced ROS formation, which was inhibited by N-acetyl-L-cysteine (NAC). NAC pretreatment dramatically reversed Cuc B-induced DNA damage, autophagy, and apoptosis. Cuc B-induced apoptosis was reversed by NAC but enhanced by 3-methyladenine (3-MA), chloroquine (CQ), and silencing phosphatase and tensin homolog (PTEN). 3-MA and CQ showed no effect on Cuc B-induced DNA damage. In addition, Cuc B increased PTEN phosphorylation and silence PTEN restored Cuc B-induced autophagic protein expressions without affecting DNA damage. In summary, Cuc B induced DNA damage, apoptosis, and protective autophagy mediated by ROS. PTEN activation in response to DNA damage bridged DNA damage and prosurvival autophagy. PMID:28042385

  4. Regulation of autophagy by two products of one gene: TRPM3 and miR-204.

    PubMed

    Cost, Nicolas G; Czyzyk-Krzeska, Maria F

    2015-01-01

    In clear cell renal cell carcinoma (ccRCC), oncogenic autophagy dependent on microtubule-associated protein 1 light chain 3 α and β (LC3A and LC3B) is stimulated by activity of the transient receptor potential melastatin 3 (TRPM3) channel through multiple complementary mechanisms. The Von Hippel-Lindau (VHL) tumor suppressor represses this oncogenic autophagy in a coordinated manner through the activity of miR-204, which is expressed from intron 6 of the gene encoding TRPM3. TRPM3 represents an actionable target for ccRCC treatment.

  5. Induction of the autophagy-associated gene MAP1S via PU.1 supports APL differentiation.

    PubMed

    Haimovici, Aladin; Brigger, Daniel; Torbett, Bruce E; Fey, Martin F; Tschan, Mario P

    2014-09-01

    The PU.1 transcription factor is essential for myeloid development. We investigated if the microtubule-associated protein 1S (MAP1S) is a novel PU.1 target with a link to autophagy, a cellular recycling pathway. Comparable to PU.1, MAP1S expression was significantly repressed in primary AML blasts as compared to mature neutrophils. Accordingly, MAP1S expression was induced during neutrophil differentiation of CD34(+) progenitor and APL cells. Moreover, PU.1 bound to the MAP1S promoter and induced MAP1S expression during APL differentiation. Inhibiting MAP1S resulted in aberrant neutrophil differentiation and autophagy. Taken together, our findings implicate the PU.1-regulated MAP1S gene in neutrophil differentiation and autophagy control.

  6. Cannabidiol protects liver from binge alcohol-induced steatosis by mechanisms including inhibition of oxidative stress and increase in autophagy.

    PubMed

    Yang, Lili; Rozenfeld, Raphael; Wu, Defeng; Devi, Lakshmi A; Zhang, Zhenfeng; Cederbaum, Arthur

    2014-03-01

    Acute alcohol drinking induces steatosis, and effective prevention of steatosis can protect liver from progressive damage caused by alcohol. Increased oxidative stress has been reported as one mechanism underlying alcohol-induced steatosis. We evaluated whether cannabidiol, which has been reported to function as an antioxidant, can protect the liver from alcohol-generated oxidative stress-induced steatosis. Cannabidiol can prevent acute alcohol-induced liver steatosis in mice, possibly by preventing the increase in oxidative stress and the activation of the JNK MAPK pathway. Cannabidiol per se can increase autophagy both in CYP2E1-expressing HepG2 cells and in mouse liver. Importantly, cannabidiol can prevent the decrease in autophagy induced by alcohol. In conclusion, these results show that cannabidiol protects mouse liver from acute alcohol-induced steatosis through multiple mechanisms including attenuation of alcohol-mediated oxidative stress, prevention of JNK MAPK activation, and increasing autophagy.

  7. Lead toxicity induces autophagy to protect against cell death through mTORC1 pathway in cardiofibroblasts.

    PubMed

    Sui, Li; Zhang, Rui-Hong; Zhang, Ping; Yun, Ke-Li; Zhang, Hong-Cai; Liu, Li; Hu, Ming-Xu

    2015-03-31

    Heavy metals, such as lead (Pb(2+)), are usually accumulated in human bodies and impair human's health. Lead is a metal with many recognized adverse health side effects and yet the molecular processes underlying lead toxicity are still poorly understood. In the present study, we proposed to investigate the effects of lead toxicity in cultured cardiofibroblasts. After lead treatment, cultured cardiofibroblasts showed severe endoplasmic reticulum (ER) stress. However, the lead-treated cardiofibroblasts were not dramatically apoptotic. Further, we found that these cells determined to undergo autophagy through inhibiting mammalian target of rapamycin complex 1 (mTORC1) pathway. Moreover, inhibition of autophagy by 3-methyladenine (3-MA) may dramatically enhance lead toxicity in cardiofibroblasts and cause cell death. Our data establish that lead toxicity induces cell stress in cardiofibroblasts and protective autophagy is activated by inhibition of mTORC1 pathway. These findings describe a mechanism by which lead toxicity may promote the autophagy of cardiofibroblasts cells, which protects cells from cell stress. Our findings provide evidence that autophagy may help cells to survive under ER stress conditions in cardiofibroblasts and may set up an effective therapeutic strategy for heavy metal toxicity.

  8. Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

    PubMed Central

    Paris, Irmgard; Muñoz, Patricia; Huenchuguala, Sandro; Couve, Eduardo; Sanders, Laurie H.; Greenamyre, John Timothy; Caviedes, Pablo; Segura-Aguilar, Juan

    2011-01-01

    Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinson’s disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes. PMID:21427056

  9. A comprehensive, genome-wide analysis of autophagy-related genes identified in tobacco suggests a central role of autophagy in plant response to various environmental cues

    PubMed Central

    Zhou, Xue-mei; Zhao, Peng; Wang, Wei; Zou, Jie; Cheng, Tian-he; Peng, Xiong-bo; Sun, Meng-xiang

    2015-01-01

    Autophagy is an evolutionarily conserved mechanism in both animals and plants, which has been shown to be involved in various essential developmental processes in plants. Nicotiana tabacum is considered to be an ideal model plant and has been widely used for the study of the roles of autophagy in the processes of plant development and in the response to various stresses. However, only a few autophagy-related genes (ATGs) have been identified in tobacco up to now. Here, we identified 30 ATGs belonging to 16 different groups in tobacco through a genome-wide survey. Comprehensive expression profile analysis reveals an abroad expression pattern of these ATGs, which could be detected in all tissues tested under normal growth conditions. Our series tests further reveal that majority of ATGs are sensitive and responsive to different stresses including nutrient starvation, plant hormones, heavy metal and other abiotic stresses, suggesting a central role of autophagy, likely as an effector, in plant response to various environmental cues. This work offers a detailed survey of all ATGs in tobacco and also suggests manifold functions of autophagy in both normal plant growth and plant response to environmental stresses. PMID:26205094

  10. Autophagy and gene therapy combine in the treatment of liver disease.

    PubMed

    Thompson, Debra A; Klionsky, Daniel J

    2013-07-01

    Molecular biology holds the promise not only of increasing our understanding of basic cell biology, but also of advancing our ability to design targeted therapeutic methods for treating a range of diseases. One example that seems to hold tremendous potential is gene therapy, the use of exogenous DNA to replace or suppress a mutant gene in the patient's genome, or to boost the activity of a normal gene. A recent report (highlighted in a punctum in this issue of the journal) has brought autophagy into the gene therapy realm.

  11. Heme oxygenase-1-mediated autophagy protects against pulmonary endothelial cell death and development of emphysema in cadmium-treated mice.

    PubMed

    Surolia, Ranu; Karki, Suman; Kim, Hyunki; Yu, Zhihong; Kulkarni, Tejaswini; Mirov, Sergey B; Carter, A Brent; Rowe, Steven M; Matalon, Sadis; Thannickal, Victor J; Agarwal, Anupam; Antony, Veena B

    2015-08-01

    Pulmonary exposure to cadmium, a major component of cigarette smoke, has a dramatic impact on lung function and the development of emphysema. Cigarette smoke exposure induces heme oxygenase-1 (HO-1), a cytoprotective enzyme. In this study, we employed a truncated mouse model of emphysema by intratracheal instillation of cadmium (CdCl2) solution (0.025% per 1 mg/kg body wt) in HO-1(+/+), HO-1(-/-), and overexpressing humanized HO-1 bacterial artificial chromosome (hHO-1BAC) mice. We evaluated the role of HO-1 in cadmium-induced emphysema in mice by analyzing histopathology, micro-computed tomography scans, and lung function tests. CdCl2-exposed HO-1(-/-) mice exhibited more severe emphysema compared with HO-1(+/+) or hHO-1BAC mice. Loss of pulmonary endothelial cells (PECs) from the alveolar capillary membrane is recognized to be a target in emphysema. PECs from HO-1(+/+), HO-1(-/-), and hHO-1BAC were employed to define the underlying molecular mechanism for the protection from emphysema by HO-1. Electron microscopy, expression of autophagic markers (microtubule-associated protein 1B-light chain 3 II, autophagy protein 5, and Beclin1) and apoptotic marker (cleaved caspase 3) suggested induction of autophagy and apoptosis in PECs after CdCl2 treatment. CdCl2-treated HO-1(-/-) PECs exhibited downregulation of autophagic markers and significantly increased cleaved caspase 3 expression and activity (∼4-fold higher). Moreover, hHO-1BAC PECs demonstrated upregulated autophagy and absence of cleaved caspase 3 expression or activity. Pretreatment of HO-1(+/+) PECs with rapamycin induced autophagy and resulted in reduced cell death upon cadmium treatment. Induction of autophagy following CdCl2 treatment was found to be protective from apoptotic cell death. HO-1 induced protective autophagy in PECs and mitigated cadmium-induced emphysema.

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

    PubMed

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

    2016-06-13

    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.

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

    PubMed Central

    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

  14. p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene

    PubMed Central

    He, Z; Liu, H; Agostini, M; Yousefi, S; Perren, A; Tschan, M P; Mak, T W; Melino, G; Simon, H U

    2013-01-01

    p73, a member of the p53 tumor suppressor family, is involved in neurogenesis, sensory pathways, immunity, inflammation, and tumorigenesis. How p73 is able to participate in such a broad spectrum of different biological processes is still largely unknown. Here, we report a novel role of p73 in regulating lipid metabolism by direct transactivation of the promoter of autophagy-related protein 5 (ATG5), a gene whose product is required for autophagosome formation. Following nutrient deprivation, the livers of p73-deficient mice demonstrate a massive accumulation of lipid droplets, together with a low level of autophagy, suggesting that triglyceride hydrolysis into fatty acids is blocked owing to deficient autophagy (macrolipophagy). Compared with wild-type mice, mice functionally deficient in all the p73 isoforms exhibit decreased ATG5 expression and lower levels of autophagy in multiple organs. We further show that the TAp73α is the critical p73 isoform responsible for inducing ATG5 expression in a p53-independent manner and demonstrate that ATG5 gene transfer can correct autophagy and macrolipophagy defects in p73-deficient hepatocytes. These data strongly suggest that the p73–ATG5 axis represents a novel, key pathway for regulating lipid metabolism through autophagy. The identification of p73 as a major regulator of autophagy suggests that it may have an important role in preventing or delaying disease and aging by maintaining a homeostatic control. PMID:23912709

  15. p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene.

    PubMed

    He, Z; Liu, H; Agostini, M; Yousefi, S; Perren, A; Tschan, M P; Mak, T W; Melino, G; Simon, H U

    2013-10-01

    p73, a member of the p53 tumor suppressor family, is involved in neurogenesis, sensory pathways, immunity, inflammation, and tumorigenesis. How p73 is able to participate in such a broad spectrum of different biological processes is still largely unknown. Here, we report a novel role of p73 in regulating lipid metabolism by direct transactivation of the promoter of autophagy-related protein 5 (ATG5), a gene whose product is required for autophagosome formation. Following nutrient deprivation, the livers of p73-deficient mice demonstrate a massive accumulation of lipid droplets, together with a low level of autophagy, suggesting that triglyceride hydrolysis into fatty acids is blocked owing to deficient autophagy (macrolipophagy). Compared with wild-type mice, mice functionally deficient in all the p73 isoforms exhibit decreased ATG5 expression and lower levels of autophagy in multiple organs. We further show that the TAp73α is the critical p73 isoform responsible for inducing ATG5 expression in a p53-independent manner and demonstrate that ATG5 gene transfer can correct autophagy and macrolipophagy defects in p73-deficient hepatocytes. These data strongly suggest that the p73-ATG5 axis represents a novel, key pathway for regulating lipid metabolism through autophagy. The identification of p73 as a major regulator of autophagy suggests that it may have an important role in preventing or delaying disease and aging by maintaining a homeostatic control.

  16. Pyrvinium targets autophagy addiction to promote cancer cell death.

    PubMed

    Deng, Longfei; Lei, Yunlong; Liu, Rui; Li, Jingyi; Yuan, Kefei; Li, Yi; Chen, Yi; Liu, Yi; Lu, You; Edwards, Carl K; Huang, Canhua; Wei, Yuquan

    2013-05-02

    Autophagy is a cellular catabolic process by which long-lived proteins and damaged organelles are degradated by lysosomes. Activation of autophagy is an important survival mechanism that protects cancer cells from various stresses, including anticancer agents. Recent studies indicate that pyrvinium pamoate, an FDA-approved antihelminthic drug, exhibits wide-ranging anticancer activity. Here we demonstrate that pyrvinium inhibits autophagy both in vitro and in vivo. We further demonstrate that the inhibition of autophagy is mammalian target of rapamycin independent but depends on the transcriptional inhibition of autophagy genes. Moreover, the combination of pyrvinium with autophagy stimuli improves its toxicity against cancer cells, and pretreatment of cells with 3-MA or siBeclin1 partially protects cells from pyrvinium-induced cell death under glucose starvation, suggesting that targeted autophagy addiction is involved in pyrvinium-mediated cytotoxicity. Finally, in vivo studies show that the combination therapy of pyrvinium with the anticancer and autophagy stimulus agent, 2-deoxy-D-glucose (2-DG), is significantly more effective in inhibiting tumor growth than pyrvinium or 2-DG alone. This study supports a novel cancer therapeutic strategy based on targeting autophagy addiction and implicates using pyrvinium as an autophagy inhibitor in combination with chemotherapeutic agents to improve their therapeutic efficacy.

  17. Pyrvinium targets autophagy addiction to promote cancer cell death

    PubMed Central

    Deng, Longfei; Lei, Yunlong; Liu, Rui; Li, Jingyi; Yuan, Kefei; Li, Yi; Chen, Yi; Liu, Yi; Lu, You; Edwards III, Carl K; Huang, Canhua; Wei, Yuquan

    2013-01-01

    Autophagy is a cellular catabolic process by which long-lived proteins and damaged organelles are degradated by lysosomes. Activation of autophagy is an important survival mechanism that protects cancer cells from various stresses, including anticancer agents. Recent studies indicate that pyrvinium pamoate, an FDA-approved antihelminthic drug, exhibits wide-ranging anticancer activity. Here we demonstrate that pyrvinium inhibits autophagy both in vitro and in vivo. We further demonstrate that the inhibition of autophagy is mammalian target of rapamycin independent but depends on the transcriptional inhibition of autophagy genes. Moreover, the combination of pyrvinium with autophagy stimuli improves its toxicity against cancer cells, and pretreatment of cells with 3-MA or siBeclin1 partially protects cells from pyrvinium-induced cell death under glucose starvation, suggesting that targeted autophagy addiction is involved in pyrvinium-mediated cytotoxicity. Finally, in vivo studies show that the combination therapy of pyrvinium with the anticancer and autophagy stimulus agent, 2-deoxy-𝒟-glucose (2-DG), is significantly more effective in inhibiting tumor growth than pyrvinium or 2-DG alone. This study supports a novel cancer therapeutic strategy based on targeting autophagy addiction and implicates using pyrvinium as an autophagy inhibitor in combination with chemotherapeutic agents to improve their therapeutic efficacy. PMID:23640456

  18. Metformin Protects Against Cisplatin-Induced Tubular Cell Apoptosis and Acute Kidney Injury via AMPKα-regulated Autophagy Induction.

    PubMed

    Li, Jianzhong; Gui, Yuan; Ren, Jiafa; Liu, Xin; Feng, Ye; Zeng, Zhifeng; He, Weichun; Yang, Junwei; Dai, Chunsun

    2016-04-07

    Metformin, one of the most common prescriptions for patients with type 2 diabetes, is reported to protect the kidney from gentamicin-induced nephrotoxicity. However, the role and mechanisms for metformin in preventing cisplatin-induced nephrotoxicity remains largely unknown. In this study, a single intraperitoneal injection of cisplatin was employed to induce acute kidney injury (AKI) in CD1 mice. The mice exhibited severe kidney dysfunction and histological damage at day 2 after cisplatin injection. Pretreatment of metformin could markedly attenuate cisplatin-induced acute kidney injury, tubular cell apoptosis and inflammatory cell accumulation in the kidneys. Additionally, pretreatment of metformin could enhance both AMPKα phosphorylation and autophagy induction in the kidneys after cisplatin injection. In cultured NRK-52E cells, a rat kidney tubular cell line, metformin could stimulate AMPKα phosphorylation, induce autophagy and inhibit cisplatin-induced cell apoptosis. Blockade of either AMPKα activation or autophagy induction could largely abolish the protective effect of metformin in cisplatin-induced cell death. Together, this study demonstrated that metformin may protect against cisplatin-induced tubular cell apoptosis and AKI through stimulating AMPKα activation and autophagy induction in the tubular cells.

  19. Metformin Protects Against Cisplatin-Induced Tubular Cell Apoptosis and Acute Kidney Injury via AMPKα-regulated Autophagy Induction

    PubMed Central

    Li, Jianzhong; Gui, Yuan; Ren, Jiafa; Liu, Xin; Feng, Ye; Zeng, Zhifeng; He, Weichun; Yang, Junwei; Dai, Chunsun

    2016-01-01

    Metformin, one of the most common prescriptions for patients with type 2 diabetes, is reported to protect the kidney from gentamicin-induced nephrotoxicity. However, the role and mechanisms for metformin in preventing cisplatin-induced nephrotoxicity remains largely unknown. In this study, a single intraperitoneal injection of cisplatin was employed to induce acute kidney injury (AKI) in CD1 mice. The mice exhibited severe kidney dysfunction and histological damage at day 2 after cisplatin injection. Pretreatment of metformin could markedly attenuate cisplatin-induced acute kidney injury, tubular cell apoptosis and inflammatory cell accumulation in the kidneys. Additionally, pretreatment of metformin could enhance both AMPKα phosphorylation and autophagy induction in the kidneys after cisplatin injection. In cultured NRK-52E cells, a rat kidney tubular cell line, metformin could stimulate AMPKα phosphorylation, induce autophagy and inhibit cisplatin-induced cell apoptosis. Blockade of either AMPKα activation or autophagy induction could largely abolish the protective effect of metformin in cisplatin-induced cell death. Together, this study demonstrated that metformin may protect against cisplatin-induced tubular cell apoptosis and AKI through stimulating AMPKα activation and autophagy induction in the tubular cells. PMID:27052588

  20. Autophagy induced by silica nanoparticles protects RAW264.7 macrophages from cell death.

    PubMed

    Marquardt, Clarissa; Fritsch-Decker, Susanne; Al-Rawi, Marco; Diabaté, Silvia; Weiss, Carsten

    2017-03-15

    Although the technological and economic benefits of engineered nanomaterials are obvious, concerns have been raised about adverse effects if such material is inhaled, ingested, applied to the skin or even released into the environment. Here we studied the cytotoxic effects of the most abundant nanomaterial, silica nanoparticles (SiO2-NPs), in murine RAW264.7 macrophages. SiO2-NPs dose-dependently induce membrane leakage and cell death without obvious involvement of reactive oxygen species. Interestingly, at low concentrations SiO2-NPs trigger autophagy, evidenced by morphological and biochemical hallmarks such as autophagolysosomes or increased levels of LC3-II, which serves to protect cells from cytotoxicity. Hence SiO2-NPs initiate an adaptive stress response which dependent on dose serve to balance survival and death and ultimately dictates the cellular fate.

  1. The BAX gene as a candidate for negative autophagy-related genes regulator on mRNA levels in colorectal cancer.

    PubMed

    Gil, Justyna; Ramsey, David; Szmida, Elzbieta; Leszczynski, Przemyslaw; Pawlowski, Pawel; Bebenek, Marek; Sasiadek, Maria M

    2017-02-01

    Autophagy is a catabolic process, which is involved in the maintenance of intracellular homeostasis by degrading redundant molecules and organelles. Autophagy begins with the formation of a double-membrane phagophore, followed by its enclosure, thus leading to the appearance of an autophagosome which fuses with lysosome. This process is highly conserved, precisely orchestrated and regulated by autophagy-related genes. Recently, autophagy has been widely studied in different types of cancers, including colorectal cancer. As it has been revealed, autophagy plays two opposite roles in tumorigenesis, as a tumor suppressor and a tumor enhancer/activator, and therefore is called a double-edge sword. Recently, interaction between autophagy and apoptosis has been found. Therefore, we aimed to study the mRNA levels of genes engaged in autophagy and apoptosis in colorectal cancer tissues. Colorectal cancer and adjacent healthy tissues were obtained from 73 patients diagnosed with primary colorectal cancer. Real-time PCR analysis employing Universal Probe Library was used to assess the expression of the seven following selected genes: BECN1, UVRAG, ULK1, ATG13, Bif-1, BCL2 and BAX. For all but one of the tested genes, a decrease in expression was observed. An increase in expression was observed for BAX. BAX expression decreases consistently from early to more advanced stages. High expression of BAX was strongly associated with negative UVRAG expression. The high expression of the BAX gene seems to be a negative regulator of autophagy in colorectal cancer cells. The relative downregulation of autophagy-related genes was observed in colorectal cancer samples.

  2. AMP-activated protein kinase-dependent autophagy mediated the protective effect of sonic hedgehog pathway on oxygen glucose deprivation-induced injury of cardiomyocytes.

    PubMed

    Xiao, Qing; Yang, Ya; Qin, Yuan; He, Yan-Hua; Chen, Kui-Xiang; Zhu, Jian-Wei; Zhang, Gui-Ping; Luo, Jian-Dong

    2015-02-13

    Sonic hedgehog (Shh) pathway has been reported to protect cardiomyocytes in myocardial infarction (MI), but the underlying mechanism is not clear. Here, we provide evidence that Shh pathway induces cardiomyocytes survival through AMP-activated protein kinase-dependent autophagy. Shh pathway agonist SAG increased the expression of LC3-II, and induced the formation of autophagosomes in cultured H9c2 cardiomyocytes under oxygen glucose deprivation (OGD) 1 h and 4 h. Moreover, SAG induced a profound AMP-activated protein kinase (AMPK) activation, and then directly phosphorylated and activated the downstream autophagy initiator Ulk1, independent of the autophagy suppressor mammalian target of rapamycin (mTOR) complex 1. Taken together, our results have shown that Shh activates AMPK-dependent autophagy in cardiomyocytes under OGD, suggesting a role of autophagy in Shh-induced cellular protection.

  3. Characterization of an Autophagy-Related Gene MdATG8i from Apple

    PubMed Central

    Wang, Ping; Sun, Xun; Jia, Xin; Wang, Na; Gong, Xiaoqing; Ma, Fengwang

    2016-01-01

    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 Malus 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. PMID:27252732

  4. The role of autophagy in liver cancer: molecular mechanisms and potential therapeutic targets.

    PubMed

    Cui, Jianzhou; Gong, Zhiyuan; Shen, Han-Ming

    2013-08-01

    Autophagy is an evolutionarily conserved pathway for degradation of cytoplasmic proteins and organelles via lysosome. Proteins coded by the autophagy-related genes (Atgs) are the core molecular machinery in control of autophagy. Among the various biological functions of autophagy identified so far, the link between autophagy and cancer is probably among the most extensively studied and is often viewed as controversial. Autophagy might exert a dual role in cancer development: autophagy can serve as an anti-tumor mechanism, as defective autophagy (e.g., heterozygous knockdown Beclin 1 and Atg7 in mice) promotes the malignant transformation and spontaneous tumors. On the other hand, autophagy functions as a protective or survival mechanism in cancer cells against cellular stress (e.g., nutrient deprivation, hypoxia and DNA damage) and hence promotes tumorigenesis and causes resistance to therapeutic agents. Liver cancer is one of the common cancers with well-established etiological factors including hepatitis virus infection and environmental carcinogens such as aflatoxin and alcohol exposure. In recent years, the involvement of autophagy in liver cancer has been increasingly studied. Here, we aim to provide a systematic review on the close cross-talks between autophagy and liver cancer, and summarize the current status in development of novel liver cancer therapeutic approaches by targeting autophagy. It is believed that understanding the molecular mechanisms underlying the autophagy modulation and liver cancer development may provoke the translational studies that ultimately lead to new therapeutic strategies for liver cancer.

  5. Liver X receptor activation protects against inflammation and enhances autophagy in myocardium of neonatal mouse challenged by lipopolysaccharides.

    PubMed

    Liu, Peng; He, Siyi; Gao, Junwei; Li, Jingwei; Fan, Xiaotang; Xiao, Ying-Bin

    2014-01-01

    Liver X receptors (LXRs) has been emerged as negative regulators of cardiomyocytic inflammation. The cellular process of autophagy is believed to play a protective role in myocardium during the inflammatory status. In this study, we investigated the role of LXRs agonist TO901317 (TO) on lipopolysaccharides (LPS)-induced myocardial inflammation and autophagy. The results showed that TO pretreatment significantly reduced the LPS-induced infiltration of inflammatory cells, elevation of NF-κB protein, TNF-α, and IL-6 mRNA levels in the myocardium. Moreover, LPS stimulated autophagy in neonatal mice heart, and this effect was further enhanced by TO pretreatment as evidenced by increased LC3-II/GAPDH ratio increment. Furthermore, TUNEL assay revealed LPS stimulation also increased the number of apoptotic cells in the myocardium, and the increment was inhibited by TO pretreatment. Our findings suggested that attenuation of inflammation and apoptosis, and enhancement of autophagy by TO may contribute to the protection of myocardium under inflammatory condition.

  6. Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator

    PubMed Central

    Jung, Jennifer; Nayak, Arnab; Schaeffer, Véronique; Starzetz, Tatjana; Kirsch, Achim K; Müller, Stefan; Dikic, Ivan; Mittelbronn, Michel; Behrends, Christian

    2017-01-01

    Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator. DOI: http://dx.doi.org/10.7554/eLife.23063.001 PMID:28195531

  7. Autophagy protects against dasatinib-induced hepatotoxicity via p38 signaling

    PubMed Central

    Yang, Xiaochun; Wang, Jincheng; Dai, Jiabin; Shao, Jinjin; Ma, Jian; Chen, Chao; Ma, Shenglin; He, Qiaojun; Luo, Peihua; Yang, Bo

    2015-01-01

    Liver dysfunction is a common side effect associated with the treatment of dasatinib and its mechanism is poorly understood. Autophagy has been thought to be a potent survival or death factor for liver dysfunction, which may shed the light on a novel strategy for the intervention of hepatotoxicity caused by dasatinib. In this study, we show for the first time that autophagy is induced, which is consistent with the formation of liver damage. Autophagy inhibition exacerbated dasatinib-induced liver failure, suggesting that autophagy acted as a self-defense mechanism to promote survival. Oxidative stress has been shown to be an important stimulus for autophagy and hepatotoxicity. Interestingly, dasatinib increased the activity of p38, which is a critical modulator of the oxidative stress related to liver injury and autophagy. p38 silencing significantly blocked LC3-II induction and p62 reduction by dasatinib, which was accompanied by increased caspase-3 and PARP cleavage, indicating that autophagy alleviated dasatinib-induced hepatotoxicity via p38 signaling. Finally, the p38 agonist isoproterenol hydrochloride (ISO) alleviated dasatinib-induced liver failure by enhancing autophagy without affecting the anticancer activity of dasatinib. Thus, this study revealed that p38-activated autophagy promoted survival during liver injury, which may provide novel approaches for managing the clinical applications of dasatinib. PMID:25749037

  8. Autophagy: a cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress.

    PubMed

    Bhogal, Ricky H; Weston, Christopher J; Curbishley, Stuart M; Adams, David H; Afford, Simon C

    2012-04-01

    The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.

  9. Tazarotene-Induced Gene 1 Enhanced Cervical Cell Autophagy through Transmembrane Protein 192

    PubMed Central

    Shyu, Rong-Yaun; Wang, Chun-Hua; Wu, Chang-Chieh; Chen, Mao-Liang; Lee, Ming-Cheng; Wang, Lu-Kai; Jiang, Shun-Yuan; Tsai, Fu-Ming

    2016-01-01

    Tazarotene-induced gene 1 (TIG1) is a retinoic acid-inducible protein that is considered a putative tumor suppressor. The expression of TIG1 is decreased in malignant prostate carcinoma or poorly differentiated colorectal adenocarcinoma, but TIG1 is present in benign or well-differentiated tumors. Ectopic TIG1 expression led to suppression of growth in cancer cells. However, the function of TIG1 in cell differentiation is still unknown. Using a yeast two-hybrid system, we found that transmembrane protein 192 (TMEM192) interacted with TIG1. We also found that both TIG1A and TIG1B isoforms interacted and co-localized with TMEM192 in HtTA cervical cancer cells. The expression of TIG1 induced the expression of autophagy-related proteins, including Beclin-1 and LC-3B. The silencing of TMEM192 reduced the TIG1-mediated upregulation of autophagic activity. Furthermore, silencing of either TIG1 or TMEM192 led to alleviation of the upregulation of autophagy induced by all-trans retinoic acid. Our results demonstrate that the expression of TIG1 leads to cell autophagy through TMEM192. Our study also suggests that TIG1 and TMEM192 play an important role in the all-trans retinoic acid-mediated upregulation of autophagic activity. PMID:27989102

  10. Deep hypothermia-enhanced autophagy protects PC12 cells against oxygen glucose deprivation via a mitochondrial pathway.

    PubMed

    Tang, Dang; Wang, Cheng; Gao, Yongjun; Pu, Jun; Long, Jiang; Xu, Wei

    2016-10-06

    Deep hypothermia is known for its organ-preservation properties, which is introduced into surgical operations on the brain and heart, providing both safety in stopping circulation as well as an attractive bloodless operative field. However, the molecular mechanisms have not been clearly identified. This study was undertaken to determine the influence of deep hypothermia on neural apoptosis and the potential mechanism of these effects in PC12 cells following oxygen-glucose deprivation. Deep hypothermia (18°C) was given to PC12 cells while the model of oxygen-glucose deprivation (OGD) induction for 1h. After 24h of reperfusion, the results showed that deep hypothermia decreased the neural apoptosis, and significantly suppressed overexpression of Bax, CytC, Caspase 3, Caspase 9 and cleaved PARP-1, and inhibited the reduction of Bcl-2 expression. While deep hypothermia increased the LC3II/LC3I and Beclin 1, an autophagy marker, which can be inhibited by 3-methyladenine (3-MA), indicating that deep hypothermia-enhanced autophagy ameliorated apoptotic cell death in PC12 cells subjected to OGD. Based on these findings we propose that deep hypothermia protects against neural apoptosis after the induction of OGD by attenuating the mitochondrial apoptosis pathway, moreover, the mechanism of these antiapoptosis effects is related to the enhancement of autophagy, which autophagy might provide a means of neuroprotection against OGD.

  11. p27 Protein Protects Metabolically Stressed Cardiomyocytes from Apoptosis by Promoting Autophagy*

    PubMed Central

    Sun, Xuetao; Momen, Abdul; Wu, Jun; Noyan, Hossein; Li, Renke; von Harsdorf, Rüdiger; Husain, Mansoor

    2014-01-01

    p27Kip1 (p27), a key regulator of cell division, has been implicated in autophagy of cancer cells. However, its role in autophagy, the evolutionarily conserved catabolic process that enables cells to remove unwanted proteins and damaged organelles, had not been examined in the heart. Here we report that ectopic delivery of a p27 fusion protein (TAT-p27) was sufficient to induce autophagy in neonatal rat ventricular cardiomyocytes in vitro, under basal conditions and after glucose deprivation. Conversely, lentivirus-delivered shRNA against p27 successfully reduced p27 levels and suppressed basal and glucose-deprived levels of autophagy in cardiomyocytes in vitro. Glucose deprivation mimics myocardial ischemia and induces apoptosis in cardiomyocytes. During glucose deprivation, TAT-p27 inhibited apoptosis, whereas down-regulation of p27 decreased survival of cardiomyocytes. However, inhibition of autophagy by pharmacological (3-methyladenine, chloroquine, or bafilomycin A1) or genetic approaches (siRNA-mediated knockdown of Atg5) sensitized cardiomyocytes to glucose deprivation-induced apoptosis, even in the presence of TAT-p27. TAT-p27 was also able to provoke greater levels of autophagy in resting and fasting cardiomyocytes in vivo. Further, TAT-p27 enhanced autophagy and repressed cardiomyocytes apoptosis, improved cardiac function, and reduced infarct size following myocardial infarction. Again, these effects were lost when cardiac autophagy in vivo was blocked by chloroquine. Taken together, these data show that p27 positively regulates cardiac autophagy in vitro and in vivo, at rest and after metabolic stress, and that TAT-p27 inhibits apoptosis by promoting autophagy in glucose-deprived cardiomyocytes in vitro and in post-myocardial infarction hearts in vivo. PMID:24794871

  12. Peroxisome proliferator-activated receptor α activation attenuates the inflammatory response to protect the liver from acute failure by promoting the autophagy pathway.

    PubMed

    Jiao, M; Ren, F; Zhou, L; Zhang, X; Zhang, L; Wen, T; Wei, L; Wang, X; Shi, H; Bai, L; Zhang, X; Zheng, S; Zhang, J; Chen, Y; Han, Y; Zhao, C; Duan, Z

    2014-08-28

    Peroxisome proliferator-activated receptor α (PPARα) has been reported to induce a potent anti-inflammatory response. Autophagy is a recently recognized rudimentary cellular response to inflammation and injury. The aim of the present study was to test the hypothesis that PPARα activation mediates autophagy to inhibit liver inflammation and protect against acute liver failure (ALF). PPARα expression during ALF and the impact of PPARα activation by Wy-14 643 on the hepatic immune response were studied in a D-galactosamine/lipopolysaccharide-induced mouse model. Autophagy was inhibited by 3-methyladenine or small interfering RNA (siRNA) against Atg7. In both the mouse model and human ALF subjects, PPARα was significantly downregulated in the injured liver. PPARα activation by pretreatment with Wy-14 643 protected against liver injury in mice. The protective effect of PPARα activation relied on the suppression of inflammatory mechanisms through the induction of autophagy. This hypothesis is supported by the following evidence: first, PPARα activation suppressed proinflammatory responses and inhibited phosphorylated NF-κBp65, phosphorylated JNK and phosphorylated ERK pathways in vivo. Second, protection by PPARα activation was due to the induction of autophagy because inhibition of autophagy by 3-methyladenine or Atg7 siRNA reversed liver protection and inflammation. Third, PPARα activation directly induced autophagy in primary macrophages in vitro, which protected cells from a lipopolysaccharide-induced proinflammatory response. Here, for the first time, we have demonstrated that PPARα-mediated induction of autophagy ameliorated liver injury in cases of ALF by attenuating inflammatory responses, indicating a potential therapeutic application for ALF treatment.

  13. Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication

    PubMed Central

    De Leo, A; Colavita, F; Ciccosanti, F; Fimia, G M; Lieberman, P M; Mattia, E

    2015-01-01

    Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. Latent EBV can periodically reactivate to produce infectious particles that allow the virus to spread and can lead to the death of the infected cell. In this study, we analyzed the relationship between autophagy and EBV reactivation in Burkitt's lymphoma cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation, suggesting that early after activation, the virus is able to suppress autophagy. We report here for the first time that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of Beclin1 gene, highly incremented EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that EBV activation induces the autophagic response, which is soon inhibited by the expression of EBV early lytic products. Moreover, our findings open the possibility that pharmacological inhibitors of autophagy may be used to enhance oncolytic viral therapy of EBV-related lymphomas. PMID:26335716

  14. The Selective Activation of p53 Target Genes Regulated by SMYD2 in BIX-01294 Induced Autophagy-Related Cell Death

    PubMed Central

    Fan, Jia-Dong; Lei, Pin-Ji; Zheng, Jun-Yi; Wang, Xiang; Li, Shangze; Liu, Huan; He, Yi-Lei; Wang, Zhao-Ning; Wei, Gang; Zhang, Xiaodong; Li, Lian-Yun; Wu, Min

    2015-01-01

    Transcription regulation emerged to be one of the key mechanisms in regulating autophagy. Inhibitors of H3K9 methylation activates the expression of LC3B, as well as other autophagy-related genes, and promotes autophagy process. However, the detailed mechanisms of autophagy regulated by nuclear factors remain elusive. In this study, we performed a drug screen of SMYD2-/- cells and discovered that SMYD2 deficiency enhanced the cell death induced by BIX01294, an inhibitor of histone H3K9 methylation. BIX-01294 induces accumulation of LC3 II and autophagy-related cell death, but not caspase-dependent apoptosis. We profiled the global gene expression pattern after treatment with BIX-01294, in comparison with rapamycin. BIX-01294 selectively activates the downstream genes of p53 signaling, such as p21 and DOR, but not PUMA, a typical p53 target gene inducing apoptosis. BIX-01294 also induces other autophagy-related genes, such as ATG4A and ATG9A. SMYD2 is a methyltransferase for p53 and regulates its transcription activity. Its deficiency enhances the BIX-01294-induced autophagy-related cell death through transcriptionally promoting the expression of p53 target genes. Taken together, our data suggest BIX-01294 induces autophagy-related cell death and selectively activates p53 target genes, which is repressed by SMYD2 methyltransferase. PMID:25562686

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

    PubMed Central

    Kabat, Agnieszka M; 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. DOI: http://dx.doi.org/10.7554/eLife.12444.001 PMID:26910010

  16. Simultaneous activation of mitophagy and autophagy by staurosporine protects against dopaminergic neuronal cell death.

    PubMed

    Ha, Ji-Young; Kim, Ji-Soo; Kim, Seo-Eun; Son, Jin H

    2014-02-21

    Abnormal autophagy is frequently observed during dopaminergic neurodegeneration in Parkinson's disease (PD). However, it is not yet firmly established whether active autophagy is beneficial or pathogenic with respect to dopaminergic cell loss. Staurosporine, a common inducer of apoptosis, is often used in mechanistic studies of dopaminergic cell death. Here we report that staurosporine activates both autophagy and mitophagy simultaneously during dopaminergic neuronal cell death, and evaluate the physiological significance of these processes during cell death. First, staurosporine treatment resulted in induction of autophagy in more than 75% of apoptotic cells. Pharmacological inhibition of autophagy by bafilomycin A1 decreased significantly cell viability. In addition, staurosporine treatment resulted in activation of the PINK1-Parkin mitophagy pathway, of which deficit underlies some familial cases of PD, in the dopaminergic neuronal cell line, SN4741. The genetic blockade of this pathway by PINK1 null mutation also dramatically increased staurosporine-induced cell death. Taken together, our data suggest that staurosporine induces both mitophagy and autophagy, and that these pathways exert a significant neuroprotective effect, rather than a contribution to autophagic cell death. This model system may therefore be useful for elucidating the mechanisms underlying crosstalk between autophagy, mitophagy, and cell death in dopaminergic neurons.

  17. Arsenite exposure in human lymphoblastoid cell lines induces autophagy and coordinated induction of lysosomal genes.

    PubMed

    Bolt, Alicia M; Douglas, Randi M; Klimecki, Walter T

    2010-11-30

    Chronic exposure to inorganic arsenic is associated with diverse, complex diseases, making the identification of the mechanism underlying arsenic-induced toxicity a challenge. An increasing body of literature from epidemiological and in vitro studies has demonstrated that arsenic is an immunotoxicant, but the mechanism driving arsenic-induced immunotoxicity is not well established. We have previously demonstrated that in human lymphoblastoid cell lines (LCLs), arsenic-induced cell death is strongly associated with the induction of autophagy. In this study we utilized genome-wide gene expression analysis and functional assays to characterize arsenic-induced effects in seven LCLs that were exposed to an environmentally relevant, minimally cytotoxic, concentration of arsenite (0.75 μM) over an eight-day time course. Arsenic exposure resulted in inhibition of cellular growth and induction of autophagy (measured by expansion of acidic vesicles) over the eight-day exposure duration. Gene expression analysis revealed that arsenic exposure increased global lysosomal gene expression, which was associated with increased functional activity of the lysosome protease, cathepsin D. The arsenic-induced expansion of the lysosomal compartment in LCL represents a novel target that may offer insight into the immunotoxic effects of arsenic.

  18. Evolution of bopA Gene in Burkholderia: A Case of Convergent Evolution as a Mechanism for Bacterial Autophagy Evasion

    PubMed Central

    Yu, Dong; Yin, Zhiqiu; Jin, Yuan; Zhou, Jing; Ren, Hongguang; Hu, Mingda; Li, Beiping; Zhou, Wei

    2016-01-01

    Autophagy is an important defense mechanism targeting intracellular bacteria to restrict their survival and growth. On the other hand, several intracellular pathogens have developed an antiautophagy mechanism to facilitate their own replication or intracellular survival. Up to now, no information about the origin or evolution of the antiautophagic genes in bacteria is available. BopA is an effector protein secreted by Burkholderia pseudomallei via the type three secretion system, and it has been shown to play a pivotal role in their escape from autophagy.  The evolutionary origin of bopA was examined in this work. Sequence similarity searches for BopA showed that no homolog of BopA was detected in eukaryotes. However, eukaryotic linear motifs were detected in BopA. The phylogenetic tree of the BopA proteins in our analysis is congruent with the species phylogeny derived from housekeeping genes. Moreover, there was no obvious difference in GC content values of bopA gene and their respective genomes. Integrated information on the taxonomic distribution, phylogenetic relationships, and GC content of the bopA gene of Burkholderia revealed that this gene was acquired via convergent evolution, not from eukaryotic host through horizontal gene transfer (HGT) event. This work has, for the first time, characterized the evolutionary mechanism of bacterial evasion of autophagy. The results of this study clearly demonstrated the role of convergent evolution in the evolution of how bacteria evade autophagy. PMID:28018913

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

    PubMed

    Moreau, Pierrick; Moreau, Kevin; Segarra, Amélie; Tourbiez, Delphine; Travers, Marie-Agnès; 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 role in innate immunity. We show for the first time that autophagy pathway is present and functional in Pacific oysters and plays an important role to protect animals from infections. This study contributes to better understand the innate immune system of Pacific oysters.

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

    PubMed Central

    Moreau, Pierrick; Moreau, Kevin; Segarra, Amélie; Tourbiez, Delphine; Travers, Marie-Agnès; 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 role in innate immunity. We show for the first time that autophagy pathway is present and functional in Pacific oysters and plays an important role to protect animals from infections. This study contributes to better understand the innate immune system of Pacific oysters. PMID:25714877

  1. Altered Autophagy-Associated Genes Expression in T Cells of Oral Lichen Planus Correlated with Clinical Features

    PubMed Central

    Tan, Ya-Qin; Zhang, Jing; Du, Ge-Fei; Lu, Rui; Chen, Guan-Ying; Zhou, Gang

    2016-01-01

    Oral lichen planus (OLP) is a T cell-mediated inflammatory autoimmune disease. Autophagy has emerged as a fundamental trafficking event in mediating T cell response, which plays crucial roles in innate and adaptive immunity. The present study mainly investigated the mRNA expression of autophagy-associated genes in peripheral blood T cells of OLP patients and evaluated correlations between their expression and the clinical features of OLP. Five differentially expressed autophagy-associated genes were identified by autophagy array. Quantitative real-time RT-PCR results confirmed that IGF1 expression in the peripheral blood T cells of OLP patients was significantly higher than that in controls, especially in female and middle-aged (30–50 years old) OLP patients. In addition, ATG9B mRNA levels were significantly lower in nonerosive OLP patients. However, no significant differences were found in the expression of HGS, ESR1, and SNCA between OLP patients and controls. Taken together, dysregulation of T cell autophagy may be involved in immune response of OLP and may be correlated with clinical patterns. PMID:26980945

  2. L-Ascorbate Protects Against Methamphetamine-Induced Neurotoxicity of Cortical Cells via Inhibiting Oxidative Stress, Autophagy, and Apoptosis.

    PubMed

    Huang, Ya-Ni; Yang, Ling-Yu; Wang, Jing-Ya; Lai, Chien-Cheng; Chiu, Chien-Tsai; Wang, Jia-Yi

    2017-01-01

    Methamphetamine (METH)-induced cell death contributes to the pathogenesis of neurotoxicity; however, the relative roles of oxidative stress, apoptosis, and autophagy remain unclear. L-Ascorbate, also called vitamin (Vit.) C, confers partial protection against METH neurotoxicity via induction of heme oxygenase-1. We further investigated the role of Vit. C in METH-induced oxidative stress, apoptosis, and autophagy in cortical cells. Exposure to lower concentrations (0.1, 0.5, 1 mM) of METH had insignificant effects on ROS production, whereas cells exposed to 5 mM METH exhibited ROS production in a time-dependent manner. We confirmed METH-induced apoptosis (by nuclear morphology revealed by Hoechst 33258 staining and Western blot showing the protein levels of pro-caspase 3 and cleaved caspase 3) and autophagy (by Western blot showing the protein levels of Belin-1 and conversion of microtubule-associated light chain (LC)3-I to LC3-II and autophagosome staining by monodansylcadaverine). The apoptosis as revealed by cleaved caspase-3 expression marked an increase at 18 h after METH exposure while both autophagic markers, Beclin 1 and LC3-II, marked an increase in cells exposed to METH for 6 and 24 h, respectively. Treating cells with Vit. C 30 min before METH exposure time-dependently attenuated the production of ROS. Vitamin C also attenuated METH-induced Beclin 1 and LC3-II expression and METH toxicity. Treatment of cells with Vit. C before METH exposure attenuated the expression of cleaved caspase-3 and reduced the number of METH-induced apoptotic cells. We suggest that the protective effect of Vit. C against METH toxicity might be through attenuation of ROS production, autophagy, and apoptosis.

  3. Pharmacological modulation of autophagy to protect cardiomyocytes according to the time windows of ischaemia/reperfusion

    PubMed Central

    Xu, Qiulin; Li, Xixian; Lu, Yongkang; Shen, Liang; Zhang, Jingwen; Cao, Shiping; Huang, Xiaobo; Bin, Jianping; Liao, Yulin

    2015-01-01

    Background and Purpose Targeted modulation of autophagy induced by myocardial ischaemia/reperfusion has been the subject of intensive investigation, but it is debatable whether autophagy is beneficial or harmful. Hence, we evaluated the effects of pharmacological manipulation of autophagy on the survival of cardiomyocytes in different time windows of ischaemia/reperfusion. Experimental Approach We examined the autophagy and apoptosis in cardiomyocytes subjected to different durations of anoxia/re-oxygenation or ischaemia/reperfusion, and evaluated the effects of the autophagic enhancer rapamycin and inhibitor wortmannin on cell survival. Key Results In neonatal rat cardiomyocytes (NRCs) or murine hearts, autophagy was increased in response to anoxia/reoxygenation or ischaemia/reperfusion in a time-dependent manner. Rapamycin-enhanced autophagy in NRCs led to higher cell viability and less apoptosis when anoxia was sustained for ≦6 h. When anoxia was prolonged to 12 h, rapamycin did not increase cell viability, induced less apoptosis and more autophagic cell death. When anoxia was prolonged to 24 h, rapamycin increased autophagic cell death, while wortmannin reduced autophagic cell death and apoptosis. Similar results were obtained in mice subjected to ischaemia/reperfusion. Rapamycin inhibited the opening of mitochondrial transition pore in NRCs exposed to 6 h anoxia/4 h re-oxygenation but did not exert any effect when anoxia was extended to 24 h. Similarly, rapamycin reduced the myocardial expression of Bax in mice subjected to short-time ischaemia, but this effect disappeared when ischaemia was extended to 24 h. Conclusions and Implications The cardioprotection of autophagy is context-dependent and therapies involving the modification of autophagy should be determined according to the duration of ischaemia/reperfusion. PMID:25660104

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

    PubMed

    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.

  5. Cloning and characterization of the autophagy-related gene 6 from the hard tick, Haemaphysalis longicornis.

    PubMed

    Kawano, Suguru; Umemiya-Shirafuji, Rika; Boldbaatar, Damdinsuren; Matsuoka, Kenji; Tanaka, Tetsuya; Fujisaki, Kozo

    2011-11-01

    Autophagy is the intracellular protein degradation process which is induced by starvation. Ticks have a unique tolerance for starvation, and it is possible that this tolerance is associated with their longevity. Previously, we isolated the homologues of four autophagy-related (ATG) genes in the hard tick, Haemaphysalis longicornis, suggesting that autophagy appeared to play an important role in tolerance for starvation as well as the development of ticks. In this study, the homologue of ATG6 was isolated from H. longicornis (HlATG6). HlATG6 mRNA expression was higher in the egg and unfed larval stages than in other stages and upregulated in ovaries during the blood-feeding period. Moreover, HlATG6-knockdowned ticks laid a few and poorly developed eggs that were white brown in color and not well surface-coated with wax. However, the expression of vitellogenin (Vg)-2, HlVg-2, in the fat body of HlATG6-knockdowned ticks was significantly upregulated. In addition, hemolymph had a deep brown color in HlATG6-knockdowned ticks on day 21 after engorgement and drop-off, indicating that the Vgs synthesized by the fat body and midgut are retained and accumulated in the hemolymph of HlATG6-knockdowned ticks, probably due to the downregulation of the Vg uptake capability of oocytes. Interestingly, HlATG6 knockdown provided non-significant influences on the expression of the Vg receptor (HlVgR) at oocytes, suggesting a non-significant depression of VgR-mediated endocytosis in the oocytes of HlATG6-knockdowned ticks. Therefore, it was interpreted that the repression of Vg uptake in the oocytes of HlATG6-knockdowned ticks may be involved in endocytic processes other than the receptor recognition of Vgs in oocytes.

  6. The metastasis suppressor gene KISS-1 regulates osteosarcoma apoptosis and autophagy processes

    PubMed Central

    Yin, Yiran; Tang, Lian; Shi, Lei

    2017-01-01

    The expression of the metastasis suppressor gene KISS-1 in osteosarcoma cells during apoptosis and autophagy was evaluated. MG-63 osteosarcoma cells were transfected with either KISS-1 overexpression or KISS-1 knockdown expression vector in vitro, and compared with cell lines transfected with empty vector. After 12, 24, 48 and 72 h of cell culture, the cell proliferation was examined. The MTT method was used to detect apoptosis by flow cytometry, and the mRNA levels of apoptosis and autophagy markers caspase-3, Bcl-2, Bax, LC3 and Beclin1 were assessed by RT-PCR. Our results showed that cells in the control and low expression group kept proliferating during the cell culture period of 72 h, while the cells in the overexpression group progressively decreased in number. Also, the proliferation rate of the low expression group was significantly higher than that of the control group. The relative mRNA expression levels of caspase-3 and Bax mRNA in the control and low expression group showed no change (the expression was lowest in the low expression group). Moreover, the mRNA level of Bcl-2 increased in both cell groups. The mRNA expression levels of caspase-3 and Bax in the overexpression group were increased, and the level of Bcl-2 was reduced significantly. At the same time, the relative expression level of LC3 and Beclin1 mRNA in the control and low expression groups remained the same, and that of the overexpression group increased. The mRNA levels of LC3 and Beclin1 in the overexpression group were the highest, and that of the low expression group the lowest. The differences were statistically significant (P<0.05). Based on these results, we showed that KISS-1 inhibited the proliferation of osteosarcoma in vitro, probably by accelerating the processes of apoptosis and autophagy in the cells. PMID:28075440

  7. Time course study of Aβ formation and neurite outgrowth disruption in differentiated human neuroblastoma cells exposed to H2O2: protective role of autophagy.

    PubMed

    Ashabi, Ghorbangol; Ahmadiani, Abolhassan; Abdi, Azadeh; Abraki, Shahnaz Babaei; Khodagholi, Fariba

    2013-09-01

    Here, we tried to elucidate the possible role of autophagy against H2O2 and Amyloid beta (Aβ) induced neurotoxicity using retinoic acid differentiated SH-SY5Y cells. We found that H2O2 disrupted neurite outgrowth concomitant with production of Aβ. Furthermore, we showed that H2O2 could increase the apoptotic factors such as Bax/Bcl-2 ratio, caspase-3 level, and PARP activity in a time course manner. These findings were confirmed by acridine orange/ethidium bromide and Hoechst staining. In addition, we observed that H2O2 led to conversion of LC3 protein from LC3I to LC3II and an increase in autophagy flux. Autophagy factors including LC3B, Atg7, and Atg12 increased and reached their highest level after 2h of insulting and then dropped to a lower level. Our results showed that autophagy could internalize and degrade intra- and extracellular Aβ after 3h treatment with H2O2. However, the remaining amount of Aβ accelerated morphological atrophy and, as a result, increased neuronal death (apoptosis). Inhibition of autophagy influx, using 3-methyl-adenine, increased intra- and extracellular levels of Aβ, providing more proof for a protective role of autophagy against oxidative stress. Further studies can shed light on the important role of autophagy by finding new pathways involved in Aβ degeneration.

  8. Protective effects of crude garlic by reducing iron-mediated oxidative stress, proliferation and autophagy in rats.

    PubMed

    Nahdi, Afef; Hammami, Imen; Kouidhi, Wided; Chargui, Abderrahman; Ben Ammar, Awatef; Hamdaoui, Mohamed Hédi; El May, Ahmed; El May, Michèle

    2010-10-01

    The impact of garlic, known for its antioxidant activities, on iron metabolism has been poorly investigated. The aim of this work was to study the effect of crude garlic pre-treatment on iron-mediated lipid peroxidation, proliferation and autophagy for 5 weeks. Rats were fed distilled water or garlic solution (1 g/kg body weight) by gavage for the first 3 weeks as pre-treatment and received a basal diet supplemented or not with ferrous sulfate (650 mg Fe/kg diet) for the last 2 weeks of treatment. Immunohistochemistry labeling and ultrastuctural observations were used to evaluate the iron deleterious effects in the liver. Iron supplementation induced cell proliferation predominantly in non parenchymal cells comparing to hepatocytes, but not apoptosis. In addition, iron was accumulated within the hepatic lysosomes where it triggers autophagy as evidenced by the formation of autophagic vesicles detected by LC3-II staining. It also induced morphologic alterations of the mitochondrial membranes due to increased lipid peroxidation as shown by elevated iron and malondialdehyde concentrations in serum and tissues. Garlic pre-treatment reduced iron-catalyzed lipid peroxidation by decreasing the malondialdehyde level in the liver and colon and by enhancing the status of antioxidants. In addition, garlic reduced the iron-mediated cell proliferation and autophagy by lowering iron storage in the liver and protected mitochondrial membrane. Based on these results, garlic treatment significantly prevented iron-induced oxidative stress, proliferation and autophagy at both biochemical and histological levels due to its potent free radical scavenging and antioxidant properties.

  9. Thioredoxin-2 protects against oxygen-glucose deprivation/reperfusion injury by inhibiting autophagy and apoptosis in H9c2 cardiomyocytes

    PubMed Central

    Li, Yan-Yan; Xiang, Yin; Zhang, Song; Wang, Yan; Yang, Jie; Liu, Wei; Xue, Feng-Tai

    2017-01-01

    The aim of this study is to examine the role of thioredoxin-2 (Trx2) in autophagy and apoptosis during myocardial ischemia-reperfusion (I/R) injury in vitro. We employed the oxygen-glucose deprivation and reperfusion (OGD/R) model of H9c2 cells and used lentiviral infection to overexpress Trx2. H9c2 cell viability and injury assays were conducted using a Cell Counting Kit-8 (CCK-8) and alactate dehydrogenase (LDH) kit. The effects of Trx2 on autophagy and apoptosis were measured by transmission electron microscopy (TEM), western blot, and flow cytometry. Our results showed that the expression of Trx2 was significantly decreased at reperfusion 6 h after OGD 12 h treatment. Trx2 overexpression inhibited autophagy in H9c2 cells subjected to OGD/R. As the underlying mechanisms, both Akt kinase/the mammalian target of rapamycin (Akt/mTOR) and AMP-activated protein kinase (AMPK)/mTOR signaling pathways were involved in the regulation of Trx2 during autophagy, which was also mediated by reactive oxygen species (ROS). 3-methyladenine (3-MA), an inhibitor of autophagy, not only suppressed OGD/R-induced autophagy but also decreased apoptosis. As a classical autophagy sensitizer, rapamycin (Rapa) augmented autophagy as well as apoptosis. Additionally, we further demonstrated that Trx2 could alleviate OGD/R-induced apoptosis via mitochondrion-mediated intrinsic apoptotic pathway. In summary, our data indicated that Trx2 protects cardiomyocytes under OGD/R by inhibiting autophagy and apoptosis. Trx2 may be a crucial regulatory protein during I/R-induced cardiomyocyte injury and death. PMID:28386372

  10. Taurine protects against As2O3-induced autophagy in pancreas of rat offsprings through Nrf2/Trx pathway.

    PubMed

    Bai, Jie; Yao, Xiaofeng; Jiang, Liping; Qiu, Tianming; Liu, Shuang; Qi, Baoxu; Zheng, Yue; Kong, Yuan; Yang, Guang; Chen, Min; Liu, Xiaofang; Sun, Xiance

    2016-04-01

    Arsenic was increasingly to blame as a risk factor for type 2 diabetes mellitus. In our previous study, we had found iAs stimulated autophagic flux and caused autophagic cell death through ROS pathway in INS-1 cells. Since NF-E2-related factor 2 (Nrf2) and the thioredoxin (Trx) system was a crucial line of defense against ROS, we investigated whether Nrf2/Trx pathway contributed to As2O3-stimulated autophagy and the role of taurine in this study. After treatment with 2 mg/kg BW-8 mg/kg BW As2O3 for 57 d, the expression of Nrf2 protein was decreased significantly in offsprings' pancreas. The expression of Trx gene was decreased significantly in pancreas subsequently. Finally, the generation of reactive oxygen species stimulated autophagy in arsenic-treated pancreas. Taurine could reverse arsenic-inhibited Nrf2 and Trx and inhibit autophagy. In short, inhibition of Nrf2/Trx pathway might play an important role in the pathogenesis of arsenic-related diabetes. Taurine could serve as nutrition supplementation against arsenic-related diabetes in high arsenic exposure area.

  11. Investigating autophagy

    PubMed Central

    Swanlund, Jamie M.; Kregel, Kevin C.; Oberley, Terry D.

    2011-01-01

    Autophagy is a compensatory pathway involving isolation and subsequent degradation of cytosolic material and organelles in eukaryotic cells.1 The autophagic process can provide a “housekeeping” function by removing damaged proteins and organelles in a selective or nonselective fashion in order to exert a protective effect following stress.2 Remarkably, after being discovered to be much more of a targeted process than a random one, the role of autophagy became implicated in many normal cellular and disease processes.3 Several methodologies are routinely employed to monitor the entire autophagic process.4 Microtubule-associated protein light chain 3, a mammalian homolog of yeast Atg8, has been widely used as a specific marker to monitor autophagy in numerous cell types.5 While monitoring autophagic flux is extremely important, it is also beneficial to perform a detailed analysis by electron microscopy (EM) to evaluate changes in various autophagic structures, quantify the areas involved, and determine if any particular organelle(s) or area of the cell cytoplasm is being targeted for degradation.6 The following article describes methods to localize and quantify subcellular areas of autophagy using transmission EM. Also discussed are methods for subcellular localization of specific proteins by employing immunogold EM; this method becomes particularly useful in detecting early changes in cellular homeostasis that may occur before later signs of cellular insult can be observed morphologically. PMID:19923921

  12. Celastrol protects human neuroblastoma SH-SY5Y cells from rotenone-induced injury through induction of autophagy.

    PubMed

    Deng, Yong-Ning; Shi, Jie; Liu, Jie; Qu, Qiu-Min

    2013-07-01

    Celastrol, an active component found in the Chinese herb tripterygium wilfordii has been identified as a neuroprotective agent for neurodegenerative diseases including Parkinson's disease (PD) through unknown mechanism. Celastrol can induce autophagy, which plays a neuroprotective role in PD. We tested the protective effect of celastrol on rotenone-induced injury and investigated the underlying mechanism using human neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were treated with celastrol before rotenone exposure. The cells survival, apoptosis, accumulation of α-synuclein, oxidative stress and mitochondrial function, and autophagy production were analyzed. We found celastrol (500 nM) pre-treatment enhanced cell viability (by 28.99%, P<0.001), decreased cell apoptosis (by 54.38%, P<0.001), increased SOD and GSH (by 120.53% and 90.46%, P<0.01), reduced accumulation of α-synuclein (by 35.93%, P<0.001) and ROS generation (by 33.99%, P<0.001), preserved MMP (33.93±3.62%, vs. 15.10±0.71% of JC-1 monomer, P<0.001) and reduced the level of cytochrome C in cytosol (by 45.57%, P<0.001) in rotenone treated SH-SY5Y cells. Moreover, celastrol increased LC3-II/LC3 I ratio by 60.92% (P<0.001), indicating that celastrol activated autophagic pathways. Inhibiting autophagy by 3-methyladenine (3-MA) abolished the protective effects of celastrol. Our results suggested that celastrol protects SH-SY5Y cells from rotenone induced injuries and autophagic pathway is involved in celastrol neuroprotective effects.

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

    PubMed

    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.

  14. Autophagy and Cancer

    PubMed Central

    Aredia, Francesca; Ortiz, Luis Miguel Guamán; Giansanti, Vincenzo; Scovassi, A. Ivana

    2012-01-01

    Autophagy is a housekeeping survival mechanism with a protective function against stress conditions. However, when stress severity or duration increases, it may promote cell death. Paradoxically, autophagy favors cancer development, since cancer cells could enhance their proliferation potential (thus becoming able to resist anticancer therapy) thanks to the energetic supply provided by organelle degradation typically driven by autophagy following a stepwise pathway. The main actors of the autophagic machinery as well as the features shared with apoptosis will be described. Special attention will be paid to the effects of autophagy manipulation. PMID:24710488

  15. Mesenchymal stem cells protect neurons against hypoxic-ischemic injury via inhibiting parthanatos, necroptosis, and apoptosis, but not autophagy.

    PubMed

    Kong, Deyan; Zhu, Juehua; Liu, Qian; Jiang, Yongjun; Xu, Lily; Luo, Ning; Zhao, Zhenqiang; Zhai, Qijin; Zhang, Hao; Zhu, Mingyue; Liu, Xinfeng

    2017-03-01

    Cellular therapy with mesenchymal stem cells (MSCs) protects cortical neurons against hypoxic-ischemic injury of stroke. Although sorts of efforts have been made to confirm the neuroprotective effect of MSCs on neurons against hypoxic-ischemic injury, the mechanism is until now far away from clear. Here in this study, oxygen-glucose deprivation (OGD)-injured neuron model was applied to mimic the neuronal hypoxic-ischemic injury in vitro. Co-culturing with MSCs in a transwell co-culture system, the OGD injured neurons were rescued by 75.0 %. Further data demonstrated that co-culturing with MSCs protected the cortical neurons from the OGD-induced parthanatos by alleviating apoptosis-inducing factor (AIF) nuclear translocation; attenuated the neuronal necroptosis by down-regulating the expression of the two essential kinases in necroptosis, receptor interacting protein kinase1 (RIP1) and 3 (RIP3); rescued the neurons from apoptosis by deactivating caspase-3; whilst performed no significant influence on OGD-induced neuronal autophagy, according to its failed regulation on Beclin1. In conclusion, MSCs potentially protect the cortical neurons from OGD-injury in vitro, through rescuing neurons from the cell death of parthanatos, necroptosis, and apoptosis, but not autophagy, which could provide some evidence to the mechanism explanation on stem cell treatment for ischemic stroke.

  16. Variants in autophagy-related genes and clinical characteristics in melanoma: a population-based study.

    PubMed

    White, Kirsten A M; Luo, Li; Thompson, Todd A; Torres, Salina; Hu, Chien-An Andy; Thomas, Nancy E; Lilyquist, Jenna; Anton-Culver, Hoda; Gruber, Stephen B; From, Lynn; Busam, Klaus J; Orlow, Irene; Kanetsky, Peter A; Marrett, Loraine D; Gallagher, Richard P; Sacchetto, Lidia; Rosso, Stefano; Dwyer, Terence; Cust, Anne E; Begg, Colin B; Berwick, Marianne

    2016-11-01

    Autophagy has been linked with melanoma risk and survival, but no polymorphisms in autophagy-related (ATG) genes have been investigated in relation to melanoma progression. We examined five single-nucleotide polymorphisms (SNPs) in three ATG genes (ATG5; ATG10; and ATG16L) with known or suspected impact on autophagic flux in an international population-based case-control study of melanoma. DNA from 911 melanoma patients was genotyped. An association was identified between (GG) (rs2241880) and earlier stage at diagnosis (OR 0.47; 95% Confidence Intervals (CI) = 0.27-0.81, P = 0.02) and a decrease in Breslow thickness (P = 0.03). The ATG16L heterozygous genotype (AG) (rs2241880) was associated with younger age at diagnosis (P = 0.02). Two SNPs in ATG5 were found to be associated with increased stage (rs2245214 CG, OR 1.47; 95% CI = 1.11-1.94, P = 0.03; rs510432 CC, OR 1.84; 95% CI = 1.12-3.02, P = 0.05). Finally, we identified inverse associations between ATG5 (GG rs2245214) and melanomas on the scalp or neck (OR 0.20, 95% CI = 0.05-0.86, P = 0.03); ATG10 (CC) (rs1864182) and brisk tumor infiltrating lymphocytes (TILs) (OR 0.42; 95% CI = 0.21-0.88, P = 0.02), and ATG5 (CC) (rs510432) with nonbrisk TILs (OR 0.55; 95% CI = 0.34-0.87, P = 0.01). Our data suggest that ATG SNPs might be differentially associated with specific host and tumor characteristics including age at diagnosis, TILs, and stage. These associations may be critical to understanding the role of autophagy in cancer, and further investigation will help characterize the contribution of these variants to melanoma progression.

  17. Altered gene expression and repressed markers of autophagy in skeletal muscle of insulin resistant patients with type 2 diabetes

    PubMed Central

    Møller, Andreas Buch; Kampmann, Ulla; Hedegaard, Jakob; Thorsen, Kasper; Nordentoft, Iver; Vendelbo, Mikkel Holm; Møller, Niels; Jessen, Niels

    2017-01-01

    This case-control study was designed to investigate the gene expression profile in skeletal muscle from severely insulin resistant patients with long-standing type 2 diabetes (T2D), and to determine associated signaling pathways. Gene expression profiles were examined by whole transcriptome, strand-specific RNA-sequencing and associated signaling was determined by western blot. We identified 117 differentially expressed gene transcripts. Ingenuity Pathway Analysis related these differences to abnormal muscle morphology and mitochondrial dysfunction. Despite a ~5-fold difference in plasma insulin, we did not observe any difference in phosphorylation of AKT or AS160, although other insulin-sensitive cascades, as mTOR/4EBP1, had retained their sensitivity. Autophagy-related gene (ATG14, RB1CC1/FIP200, GABARAPL1, SQSTM1/p62, and WIPI1) and protein (LC3BII, SQSTM1/p62 and ATG5) expression were decreased in skeletal muscle from the patients, and this was associated with a trend to increased phosphorylation of the insulin-sensitive regulatory transcription factor FOXO3a. These data show that gene expression is highly altered and related to mitochondrial dysfunction and abnormal morphology in skeletal muscle from severely insulin resistant patients with T2D, and that this is associated with decreased expression of autophagy-related genes and proteins. We speculate that prolonged treatment with high doses of insulin may suppress autophagy thereby generating a vicious cycle maintaining insulin resistance. PMID:28252104

  18. Reduction of protein translation and activation of autophagy protect against PINK1 pathogenesis in Drosophila melanogaster.

    PubMed

    Liu, Song; Lu, Bingwei

    2010-12-09

    Mutations in PINK1 and Parkin cause familial, early onset Parkinson's disease. In Drosophila melanogaster, PINK1 and Parkin mutants show similar phenotypes, such as swollen and dysfunctional mitochondria, muscle degeneration, energy depletion, and dopaminergic (DA) neuron loss. We previously showed that PINK1 and Parkin genetically interact with the mitochondrial fusion/fission pathway, and PINK1 and Parkin were recently proposed to form a mitochondrial quality control system that involves mitophagy. However, the in vivo relationships among PINK1/Parkin function, mitochondrial fission/fusion, and autophagy remain unclear; and other cellular events critical for PINK1 pathogenesis remain to be identified. Here we show that PINK1 genetically interacted with the protein translation pathway. Enhanced translation through S6K activation significantly exacerbated PINK1 mutant phenotypes, whereas reduction of translation showed suppression. Induction of autophagy by Atg1 overexpression also rescued PINK1 mutant phenotypes, even in the presence of activated S6K. Downregulation of translation and activation of autophagy were already manifested in PINK1 mutant, suggesting that they represent compensatory cellular responses to mitochondrial dysfunction caused by PINK1 inactivation, presumably serving to conserve energy. Interestingly, the enhanced PINK1 mutant phenotype in the presence of activated S6K could be fully rescued by Parkin, apparently in an autophagy-independent manner. Our results reveal complex cellular responses to PINK1 inactivation and suggest novel therapeutic strategies through manipulation of the compensatory responses.

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis.

    PubMed

    Zhang, Yong; Goldman, Scott; Baerga, Rebecca; Zhao, Yun; Komatsu, Masaaki; Jin, Shengkan

    2009-11-24

    White adipocytes have a unique structure in which nearly the entire cell volume is occupied by one large lipid droplet. However, the molecular and cellular processes involved in the cytoplasmic remodeling necessary to create this structure are poorly defined. Autophagy is a membrane trafficking process leading to lysosomal degradation. Here, we investigated the effect of the deletion of an essential autophagy gene, autophagy-related gene 7 (atg7), on adipogenesis. A mouse model with a targeted deletion of atg7 in adipose tissue was generated. The mutant mice were slim and contained only 20% of the mass of white adipose tissue (WAT) found in wild-type mice. Interestingly, approximately 50% of the mutant white adipocytes were multilocular. The mutant white adipocytes were smaller with a larger volume of cytosol and contained more mitochondria. These cells exhibited altered fatty acid metabolism with increased rates of beta-oxidation and reduced rates of hormone-induced lipolysis. Consistently, the mutant mice had lower fed plasma concentrations of fatty acids and the levels decreased at faster rates upon insulin stimuli. These mutant mice exhibited increased insulin sensitivity. The mutant mice also exhibited markedly decreased plasma concentrations of leptin but not adiponectin, lower plasma concentrations of triglyceride and cholesterol, and they had higher levels of basal physical activity. Strikingly, these mutant mice were resistant to high-fat-diet-induced obesity. Taken together, our results indicate that atg7, and by inference autophagy, plays an important role in normal adipogenesis and that inhibition of autophagy by disrupting the atg7 gene has a unique anti-obesity and insulin sensitization effect.

  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.

    PubMed

    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 Genes Enhance Murine Gammaherpesvirus 68 Reactivation From Latency by Preventing Virus-induced Systemic Inflammation

    PubMed Central

    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.

    2016-01-01

    SUMMARY Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine γ-herpesvirus 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

  3. Hsp90 inhibitor 17-AAG sensitizes Bcl-2 inhibitor (-)-gossypol by suppressing ERK-mediated protective autophagy and Mcl-1 accumulation in hepatocellular carcinoma cells.

    PubMed

    Wang, Bin; Chen, Linfeng; Ni, Zhenhong; Dai, Xufang; Qin, Liyan; Wu, Yaran; Li, Xinzhe; Xu, Liang; Lian, Jiqin; He, Fengtian

    2014-11-01

    Natural BH3-memitic (-)-gossypol shows promising antitumor efficacy in several kinds of cancer. However, our previous studies have demonstrated that protective autophagy decreases the drug sensitivities of Bcl-2 inhibitors in hepatocellular carcinoma (HCC) cells. In the present study, we are the first to report that Hsp90 inhibitor 17-AAG enhanced (-)-gossypol-induced apoptosis via suppressing (-)-gossypol-triggered protective autophagy and Mcl-1 accumulation. The suppression effect of 17-AAG on autophagy was mediated by inhibiting ERK-mediated Bcl-2 phosphorylation while was not related to Beclin1 or LC3 protein instability. Meanwhile, 17-AAG downregulated (-)-gossypol-triggered Mcl-1 accumulation by suppressing Mcl-1(Thr163) phosphorylation and promoting protein degradation. Collectively, our study indicates that Hsp90 plays an important role in tumor maintenance and inhibition of Hsp90 may become a new strategy for sensitizing Bcl-2-targeted chemotherapies in HCC cells.

  4. Induction of autophagy by salidroside through the AMPK-mTOR pathway protects vascular endothelial cells from oxidative stress-induced apoptosis.

    PubMed

    Zheng, Xiang-Tao; Wu, Zi-Heng; Wei, Ye; Dai, Ju-Ji; Yu, Guan-Feng; Yuan, FengLai; Ye, Le-Chi

    2017-01-01

    Vascular endothelial cells are highly sensitive to oxidative stress, and this is one of the mechanisms by which widespread endothelial dysfunction is induced in most cardiovascular diseases and disorders. However, how these cells can survive in oxidative stress environments remains unclear. Salidroside, a traditional Chinese medicine, has been shown to confer vascular protective effects. We aimed to understand the role of autophagy and its regulatory mechanisms by treating human umbilical vein endothelial cells (HUVECs) with salidroside under oxidative stress. HUVECs were treated with salidroside and exposed to hydrogen peroxide (H2O2). The results indicated that salidroside exerted cytoprotective effects in an H2O2-induced HUVEC injury model and suppressed H2O2-induced apoptosis of HUVECs. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased oxidative stress-induced HUVEC apoptosis, while the autophagy activator rapamycin induced anti-apoptosis effects in HUVECs. Salidroside increased autophagy and decreased apoptosis of HUVECs in a dose-dependent manner under oxidative stress. Moreover, 3-MA attenuated salidroside-induced HUVEC autophagy and promoted apoptosis, whereas rapamycin had no additional effects compared with salidroside alone. Salidroside upregulated AMPK phosphorylation but downregulated mTOR phosphorylation under oxidative stress; however, administration of compound C, an AMPK inhibitor, abrogated AMPK phosphorylation and increased mTOR phosphorylation and apoptosis compared with salidroside alone. These results suggest that autophagy is a protective mechanism in HUVECs under oxidative stress and that salidroside might promote autophagy through activation of the AMPK pathway and downregulation of mTOR pathway.

  5. Enhancing Autophagy with Drugs or Lung-directed Gene Therapy Reverses the Pathological Effects of Respiratory Epithelial Cell Proteinopathy*

    PubMed Central

    Hidvegi, Tunda; Stolz, Donna B.; Alcorn, John F.; Yousem, Samuel A.; Wang, Jieru; Leme, Adriana S.; Houghton, A. McGarry; Hale, Pamela; Ewing, Michael; Cai, Houming; Garchar, Evelyn Akpadock; Pastore, Nunzia; Annunziata, Patrizia; Kaminski, Naftali; Pilewski, Joseph; Shapiro, Steven D.; Pak, Stephen C.; Silverman, Gary A.; Brunetti-Pierri, Nicola; Perlmutter, David H.

    2015-01-01

    Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy. PMID:26494620

  6. Protective role of cisplatin in ischemic liver injury through induction of autophagy.

    PubMed

    Cardinal, Jon; Pan, Pinhua; Tsung, Allan

    2009-11-01

    High mobility group box 1 (HMGB1) is a nuclear protein released from stressed or damaged cells that activates inflammatory cascades involved in the pathogenesis of liver ischemia reperfusion (I/R) injury. In efforts to develop strategies aimed at preventing its release from ischemic cells following I/R, we studied the use of cisplatin, a member of the platinating chemotherapeutic agents capable of inducing DNA lesions that have high binding affinities for high mobility group proteins inside the nucleus of cells. In addition to demonstrating that cisplatin prevents liver damage associated with liver I/R by sequestering HMGB1 inside the nucleus of ischemic cells, cisplatin also alters cell survival signaling through autophagy. Our results provide a potential approach involving the use of platinating agents and their effects on autophagy in mitigating the deleterious effects of ischemia reperfusion-mediated disease processes.

  7. Autophagy promotes survival in aging submerged cultures of the filamentous fungus Aspergillus niger.

    PubMed

    Nitsche, Benjamin M; Burggraaf-van Welzen, Anne-Marie; Lamers, Gerda; Meyer, Vera; Ram, Arthur F J

    2013-09-01

    Autophagy is a well-conserved catabolic process constitutively active in eukaryotes that is involved in maintaining cellular homeostasis by the targeting of cytoplasmic content and organelles to vacuoles. Autophagy is strongly induced by the limitation of nutrients including carbon, nitrogen, and oxygen and is clearly associated with cell death. It has been demonstrated that the accumulation of empty hyphal compartments and cryptic growth in carbon-starved submerged cultures of the filamentous fungus Aspergillus niger is accompanied by a joint transcriptional induction of autophagy genes. This study examines the role of autophagy by deleting the atg1, atg8, and atg17 orthologs in A. niger and phenotypically analyzing the deletion mutants in surface and submerged cultures. The results indicate that atg1 and atg8 are essential for efficient autophagy, whereas deletion of atg17 has little to no effect on autophagy in A. niger. Depending on the kind of oxidative stress confronted with, autophagy deficiency renders A. niger either more resistant (menadione) or more sensitive (H2O2) to oxidative stress. Fluorescence microscopy showed that mitochondrial turnover upon carbon depletion in submerged cultures is severely blocked in autophagy-impaired A. niger mutants. Furthermore, automated image analysis demonstrated that autophagy promotes survival in maintained carbon-starved cultures of A. niger. Taken together, the results suggest that besides its function in nutrient recycling, autophagy plays important roles in physiological adaptation by organelle turnover and protection against cell death upon carbon depletion in submerged cultures.

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed

    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

  10. FGFR antagonist induces protective autophagy in FGFR1-amplified breast cancer cell.

    PubMed

    Chen, Yi; Xie, Xiaoyan; Li, Xinyi; Wang, Peiqi; Jing, Qian; Yue, Jiaqi; Liu, Yang; Cheng, Zhong; Li, Jingyi; Song, Haixing; Li, Guoyu; Liu, Rui; Wang, Jinhui

    2016-05-20

    Breast cancer, representing approximately 30% of all gynecological cancer cases diagnosed yearly, is a leading cause of cancer-related mortality for women. Amplification of FGFR1 is frequently observed in breast cancers and is associated with poor prognosis. Though FGFRs have long been considered as anti-cancer drug targets, and a cluster of FGFR antagonists are currently under clinical trials, the precise cellular responses under the treatment of FGFR antagonists remains unclear. Here, we show that PD166866, an FGFR1-selective inhibitor, inhibits proliferation and triggers anoikis in FGFR1-amplified breast cancer cell lines. Notably, we demonstrate that PD166866 induces autophagy in FGFR1-amplified breast cancer cell lines, while blockage of autophagy by Atg5 knockdown further enhances the anti-proliferative activities of PD166866. Moreover, mechanistic study reveals that PD166866 induces autophagy through repressing Akt/mTOR signaling pathway. Together, the present study provides new insights into the molecular mechanisms underlying the anti-tumor activities of FGFR antagonists, and may further assist the FGFRs-based drug discovery.

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

    PubMed Central

    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

  12. Protective role of autophagy against Vibrio cholerae cytolysin, a pore-forming toxin from V. cholerae

    PubMed Central

    Gutierrez, Maximiliano Gabriel; Saka, Hector Alex; Chinen, Isabel; Zoppino, Felipe C. M.; Yoshimori, Tamotsu; Bocco, Jose Luis; Colombo, María Isabel

    2007-01-01

    Autophagy is the unique, regulated mechanism for the degradation of organelles. This intracellular process acts as a prosurvival pathway during cell starvation or stress and is also involved in cellular response against specific bacterial infections. Vibrio cholerae is a noninvasive intestinal pathogen that has been studied extensively as the causative agent of the human disease cholera. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. Besides cholera toxin, this bacterium secretes a hemolytic exotoxin termed V. cholerae cytolysin (VCC) that causes extensive vacuolation in epithelial cells. In this work, we explored the relationship between the vacuolation caused by VCC and the autophagic pathway. Treatment of cells with VCC increased the punctate distribution of LC3, a feature indicative of autophagosome formation. Moreover, VCC-induced vacuoles colocalized with LC3 in several cell lines, including human intestinal Caco-2 cells, indicating the interaction of the large vacuoles with autophagic vesicles. Electron microscopy analysis confirmed that the vacuoles caused by VCC presented hallmarks of autophagosomes. Additionally, biochemical evidence demonstrated the degradative nature of the VCC-generated vacuoles. Interestingly, autophagy inhibition resulted in decreased survival of Caco-2 cells upon VCC intoxication. Also, VCC failed to induce vacuolization in Atg5−/− cells, and the survival response of these cells against the toxin was dramatically impaired. These results demonstrate that autophagy acts as a cellular defense pathway against secreted bacterial toxins. PMID:17267617

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

    PubMed Central

    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

  14. Autophagy-related gene 12 (ATG12) is a novel determinant of primary resistance to HER2-targeted therapies: Utility of transcriptome analysis of the autophagy interactome to guide breast cancer treatment

    PubMed Central

    Corominas-Faja, Bruna; Urruticoechea, Ander; Martin-Castillo, Begoña; Menendez, Javier A.

    2012-01-01

    The autophagic process, which can facilitate breast cancer resistance to endocrine, cytotoxic, and molecularly targeted agents, is mainly regulated at the post-translational level. Although recent studies have suggested a possible transcriptome regulation of the autophagic genes, little is known about either the analysis tools that can be applied or the functional importance of putative candidate genes emerging from autophagy-dedicated transcriptome studies. In this context, we evaluated whether the constitutive activation of the autophagy machinery, as revealed by a transcriptome analysis using an autophagy-focused polymerase chain reaction (PCR) array, might allow for the identification of novel autophagy-specific biomarkers for intrinsic (primary) resistance to HER2-targeted therapies. Quantitative real-time PCR (qRT-PCR)-based profiling of 84 genes involved in autophagy revealed that, when compared to trastuzumab-sensitive SKBR3 cells, the positive regulator of autophagic vesicle formation ATG12 (autophagy-related gene 12) was the most differentially up-regulated gene in JIMT1 cells, a model of intrinsic cross-resistance to trastuzumab and other HER1/2-targeting drugs. An analysis of the transcriptional status of ATG12 in > 50 breast cancer cell lines suggested that the ATG12 transcript is commonly upregulated in trastuzumab-unresponsive HER2-overexpressing breast cancer cells. A lentiviral-delivered small hairpin RNA stable knockdown of the ATG12 gene fully suppressed the refractoriness of JIMT1 cells to trastuzumab, erlotinib, gefitinib, and lapatinib in vitro. ATG12 silencing significantly reduced JIMT1 tumor growth induced by subcutaneous injection in nude mice. Remarkably, the outgrowth of trastuzumab-unresponsive tumors was prevented completely when trastuzumab treatment was administered in an ATG12-silenced genetic background. We demonstrate for the first time the usefulness of low-density, autophagy-dedicated qRT-PCR-based platforms for monitoring

  15. SIRT1-mediated FoxOs pathways protect against apoptosis by promoting autophagy in osteoblast-like MC3T3-E1 cells exposed to sodium fluoride

    PubMed Central

    Gu, Xiaolong; Han, Dandan; Chen, Wei; Zhang, Limei; Lin, Qianyun; Gao, Jian; Fanning, Séamus; Han, Bo

    2016-01-01

    Fluorine may result in damage to teeth, bones and other body tissues, and is a serious public health problem. SIRT1 deacetylates FOXOs, which brings about apoptosis and autophagy promotion or suppression. Fluorine may induce cell apoptosis, however, the role of autophagy in apoptosis induced by fluorine is still poorly understood, and the interaction between SIRT1 and FOXOs should be further illustrated. Therefore, this study investigated the mechanisms underlying the NaF- induced apoptosis and autophagy in osteoblast-like MC3T3-E1 cells in vitro through activating or inhibiting SIRT1. Via RT-PCR, western blot, flow cytometry assays, fluorescence and laser confocal microscopy, it was found that NaF induced both cell apoptosis and autophagy. Results also showed that NaF up-regulated SIRT1 expression in a dose-dependent manner. The autophagy of MC3T3-E1 was also up- regulated indirectly whilst apoptosis was significantly attenuated when incubated with the SIRT1 activator SRT1720. When SIRT1 inhibitor Ex-527 was used, the latter effects were reversed. Furthermore, SIRT1 increased deacetylation of FoxO1 and promoted the up-regulation of its target substrate Rab7, as well as increase of Bnip3 which was substrate of FoxO3, and we hypothesize that these pathways may cause an increase in autophagic flux and a reduction in apoptosis. In conclusion, SIRT1-induced autophagy enhancement protects against fluoride-induced apoptosis through autophagy induction in MC3T3-E1 cells, which may be associated with a SIRT1-FoxO1-Rab7 axis and a SIRT1-FoxO3-Binp3 axis. The role of SIRT1 in selecting between cell survival and death provides a potential therapeutic strategy in fluorosis. PMID:27564107

  16. SIRT1-mediated FoxOs pathways protect against apoptosis by promoting autophagy in osteoblast-like MC3T3-E1 cells exposed to sodium fluoride.

    PubMed

    Gu, Xiaolong; Han, Dandan; Chen, Wei; Zhang, Limei; Lin, Qianyun; Gao, Jian; Fanning, Séamus; Han, Bo

    2016-10-04

    Fluorine may result in damage to teeth, bones and other body tissues, and is a serious public health problem. SIRT1 deacetylates FOXOs, which brings about apoptosis and autophagy promotion or suppression. Fluorine may induce cell apoptosis, however, the role of autophagy in apoptosis induced by fluorine is still poorly understood, and the interaction between SIRT1 and FOXOs should be further illustrated. Therefore, this study investigated the mechanisms underlying the NaF- induced apoptosis and autophagy in osteoblast-like MC3T3-E1 cells in vitro through activating or inhibiting SIRT1. Via RT-PCR, western blot, flow cytometry assays, fluorescence and laser confocal microscopy, it was found that NaF induced both cell apoptosis and autophagy. Results also showed that NaF up-regulated SIRT1 expression in a dose-dependent manner. The autophagy of MC3T3-E1 was also up- regulated indirectly whilst apoptosis was significantly attenuated when incubated with the SIRT1 activator SRT1720. When SIRT1 inhibitor Ex-527 was used, the latter effects were reversed. Furthermore, SIRT1 increased deacetylation of FoxO1 and promoted the up-regulation of its target substrate Rab7, as well as increase of Bnip3 which was substrate of FoxO3, and we hypothesize that these pathways may cause an increase in autophagic flux and a reduction in apoptosis. In conclusion, SIRT1-induced autophagy enhancement protects against fluoride-induced apoptosis through autophagy induction in MC3T3-E1 cells, which may be associated with a SIRT1-FoxO1-Rab7 axis and a SIRT1-FoxO3-Binp3 axis. The role of SIRT1 in selecting between cell survival and death provides a potential therapeutic strategy in fluorosis.

  17. Autophagy in hepatic fibrosis.

    PubMed

    Song, Yang; Zhao, Yingying; Wang, Fei; Tao, Lichan; Xiao, Junjie; Yang, Changqing

    2014-01-01

    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.

  18. Autophagy in hypoxia protects cancer cells against apoptosis induced by nutrient deprivation through a Beclin1-dependent way in hepatocellular carcinoma.

    PubMed

    Song, Jianrui; Guo, Xianling; Xie, Xuqin; Zhao, Xue; Li, Ding; Deng, Weijie; Song, Yujiao; Shen, Feng; Wu, Mengchao; Wei, Lixin

    2011-11-01

    Oxygen deficiency and nutrient deprivation widely exists in solid tumors because of the poor blood supply. However, cancer cells can survive this adverse condition and proliferate continuously to develop. To figure out the way to survive, we investigated the role of autophagy in the microenvironment in hepatocellular carcinoma. In order to simulate the tumor microenvironment more veritably, cells were cultured in oxygen-nutrient-deprived condition following a hypoxia preconditioning. As a result, cell death under hypoxia plus nutrient deprivation was much less than that under nutrient deprivation only. And the decreased cell death mainly attributed to the decreased apoptosis. GFP-LC3 and electron microscopy analysis showed that autophagy was significantly activated in the period of hypoxia preconditioning. However, autophagic inhibitor-3-MA significantly abrogated the apoptosis reduction in hypoxia, which implied the involvement of autophagy in protection of hepatocellular carcinoma cells against apoptosis induced by starvation. Furthermore, Beclin 1 was proved to play an important role in this process. siRNA targeting Beclin 1 was transfected into hepatocellular carcinoma cells. And both data from western blot detecting the expression of LC3-II and transmission microscopy observing the accumulation of autophagosomes showed that autophagy was inhibited obviously as a result of Beclin 1 knockdown. Besides, the decreased apoptosis of starved cells under hypoxia was reversed. Taken together, these results suggest that autophagy activated by hypoxia mediates the tolerance of hepatocellular carcinoma cells to nutrient deprivation, and this tolerance is dependent on the activity of Beclin 1.

  19. Inactivation of the Cullin (CUL)-RING E3 ligase by the NEDD8-activating enzyme inhibitor MLN4924 triggers protective autophagy in cancer cells.

    PubMed

    Luo, Zhongguang; Pan, Yongfu; Jeong, Lak Shin; Liu, Jie; Jia, Lijun

    2012-11-01

    The multiunit Cullin (CUL)-RING E3 ligase (CRL) controls diverse biological processes by targeting a mass of substrates for ubiquitination and degradation, whereas its dysfunction causes carcinogenesis. Post-translational neddylation of CUL, a process triggered by the NEDD8-activating enzyme E1 subunit 1 (NAE1), is required for CRL activation. Recently, MLN4924 was discovered via a high-throughput screen as a specific NAE1 inhibitor and first-in-class anticancer drug. By blocking CUL neddylation, MLN4924 inactivates CRL and causes the accumulation of CRL substrates that trigger cell cycle arrest, senescence and/or apoptosis to suppress the growth of cancer cells in vitro and in vivo. Recently, we found that MLN4924 also triggers protective autophagy in response to CRL inactivation. MLN4924-induced autophagy is attributed partially to the inhibition of mechanistic target of rapamycin (also known as mammalian target of rapamycin, MTOR) activity by the accumulation of the MTOR inhibitory protein DEPTOR, as well as reactive oxygen species (ROS)-induced stress. Moreover, the blockage of autophagy response enhances apoptosis in MLN4924-treated cells. Together, our findings not only reveal autophagy as a novel cellular response to CRL inactivation by MLN4924, but also provide a piece of proof-of-concept evidence for the combination of MLN4924 with autophagy inhibitors to enhance therapeutic efficacy.

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

    PubMed Central

    2012-01-01

    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 is an important and shared

  1. Nicotinamide Adenine Dinucleotide Protects against Spinal Cord Ischemia Reperfusion Injury-Induced Apoptosis by Blocking Autophagy

    PubMed Central

    Yu, Sifei; Wang, Zhenfei; Yang, Kai; Liu, Zhuochao

    2017-01-01

    The role of autophagy, neuroprotective mechanisms of nicotinamide adenine dinucleotide (NAD+), and their relationship in spinal cord ischemic reperfusion injury (SCIR) was assessed. Forty-eight Sprague-Dawley rats were divided into four groups: sham, ischemia reperfusion (I/R), 10 mg/kg NAD+, and 75 mg/kg NAD+. Western blotting, immunofluorescence, and immunohistochemistry were used to assess autophagy and apoptosis. Basso, Beattie, and Bresnahan (BBB) scores were used to assess neurological function. Expression levels of Beclin-1, Atg12-Atg5, LC3B-II, cleaved caspase 3, and Bax were upregulated in the I/R group and downregulated in the 75 mg/kg NAD+ group; p-mTOR, p-AKT, p62, and Bcl-2 were downregulated in the I/R group and upregulated in the 75 mg/kg NAD+ group. Numbers of LC3B-positive, caspase 3-positive, Bax-positive, and TUNEL-positive cells were significantly increased in the I/R group and decreased in the 75 mg/kg NAD+ group. The mean integrated option density of Bax increased and that of Nissl decreased in the I/R group, and it decreased and increased, respectively, in the 75 mg/kg NAD+ group. BBB scores significantly increased in the 75 mg/kg NAD+ group relative to the I/R group. No difference was observed between I/R and 10 mg/kg NAD+ groups for these indicators. Therefore, excessive and sustained autophagy aggravates SCIR; administration of NAD+ alleviates injury. PMID:28367271

  2. Sirtuin 3 Protects against Urban Particulate Matter-Induced Autophagy in Human Bronchial Epithelial Cells.

    PubMed

    Chen, I-Chieh; Huang, Hsin-Hsiu; Chen, Pei-Fen; Chiang, Hung-Che

    2016-07-01

    Urban particulate matter (urban PM) is a heterogeneous mixture of various types of particles originating from different sources. Exposure to high concentrations of urban PM leading to adverse health effects is evaluated by using in vitro cultures of human lung epithelial cells. However, the mechanism underlying the correlation between high concentrations of urban PM exposure and adverse health effects has not been fully elucidated; urban PM-induced oxidative stress is considered as an important mechanism of urban PM-mediated cytotoxicity. Sirtuin 3 (SIRT3), a primary mitrochondrial deacetylase, controls cellular reactive oxygen species (ROS) production, and expression of antioxidant enzymes. In this study, we examined the role of SIRT3 in the regulation of urban PM-induced oxidative stress in normal primary human bronchial epithelial cells (HBEpiCs). Cell viability showed a time- and concentration-dependent decrease when exposed to urban PM, which could indicate that the amount of lactate dehydrogenase released from the cell in response to urban PM is related to cell viability in HBEpiC. The effects of urban PM on morphological and biochemical markers of autophagy in HBEpiC were analyzed by electron microscopy and Western blotting. Overexpression of SIRT3 inhibited urban PM-induced ROS generation, while concomitantly increasing the expression of antioxidant enzymes, and decreasing NF-κB activation and release of inflammation factors. Up-regulation of SIRT3 significantly inhibited the expression of autophagy markers and autophagic vacuole formation. Our findings provide a valuable insight into the potential role of the SIRT3 enzyme in regulating urban PM-induced autophagy by mediating urban PM-induced oxidative stress, which may contribute to urban PM-induced impairment of airway epithelial cell function.

  3. A novel protective mechanism for mitochondrial aldehyde dehydrogenase (ALDH2) in type i diabetes-induced cardiac dysfunction: role of AMPK-regulated autophagy.

    PubMed

    Guo, Yuli; Yu, Wenjun; Sun, Dongdong; Wang, Jiaxing; Li, Congye; Zhang, Rongqing; Babcock, Sara A; Li, Yan; Liu, Min; Ma, Meijuan; Shen, Mingzhi; Zeng, Chao; Li, Na; He, Wei; Zou, Qian; Zhang, Yingmei; Wang, Haichang

    2015-02-01

    Mitochondrial aldehyde dehydrogenase (ALDH2) is known to offer myocardial protection against stress conditions including ischemia-reperfusion injury, alcoholism and diabetes mellitus although the precise mechanism is unclear. This study was designed to evaluate the effect of ALDH2 on diabetes-induced myocardial injury with a focus on autophagy. Wild-type FVB and ALDH2 transgenic mice were challenged with streptozotozin (STZ, 200mg/kg, i.p.) for 3months to induce experimental diabetic cardiomyopathy. Diabetes triggered cardiac remodeling and contractile dysfunction as evidenced by cardiac hypertrophy, decreased cell shortening and prolonged relengthening duration, the effects of which were mitigated by ALDH2. Lectin staining displayed that diabetes promoted cardiac hypertrophy, the effect of which was alleviated by ALDH2. Western blot analysis revealed dampened autophagy protein markers including LC3B ratio and Atg7 along with upregulated p62 following experimental diabetes, the effect of which was reconciled by ALDH2. Phosphorylation level of AMPK was decreased and its downstream signaling molecule FOXO3a was upregulated in both diabetic cardiac tissue and in H9C2 cells with high glucose exposure. All these effect were partly abolished by ALDH2 overexpression and ALDH2 agonist Alda1. High glucose challenge dampened autophagy in H9C2 cells as evidenced by enhanced p62 levels and decreased levels of Atg7 and LC3B, the effect of which was alleviated by the ALDH2 activator Alda-1. High glucose-induced cell death and apoptosis were reversed by Alda-1. The autophagy inhibitor 3-MA and the AMPK inhibitor compound C mitigated Alda-1-offered beneficial effect whereas the autophagy inducer rapamycin mimicked or exacerbated high glucose-induced cell injury. Moreover, compound C nullified Alda-1-induced protection against STZ-induced changes in autophagy and function. Our results suggested that ALDH2 protects against diabetes-induced myocardial dysfunction possibly through an

  4. Cloning and characterization of an autophagy-related gene, ATG12, from the three-host tick Haemaphysalis longicornis.

    PubMed

    Umemiya, Rika; Matsuo, Tomohide; Hatta, Takeshi; Sakakibara, Shin-ichi; Boldbaatar, Damdinsuren; Fujisaki, Kozo

    2007-09-01

    Ticks are obligate hematophagous ectoparasites with a life cycle characterized by a period of starvation; many ticks spend more than 95% of their life off the host. Autophagy, which is the process of bulk cytoplasmic degradation in eukaryotic cells, is induced by starvation and is essential for extension of the lifespan. Therefore, we hypothesized that autophagy also occurs in ticks; however, there has been no report on autophagy-related (ATG) genes in ticks. Here, we show the homologue of an ATG gene, ATG12, and its expression pattern from the nymphal to adult stages in the three-host tick Haemaphysalis longicornis. The sequence analysis showed that H. longicornis ATG12 (HlATG12) cDNA is 649bp, has a 411bp ORF coding for a 136-amino acid polypeptide with the carboxy-terminal glycine residue, and has a predicted molecular mass of 15.2kDa. Moreover, RT-PCR revealed that HlATG12 was downregulated at the beginning of feeding, upregulated after engorgement, and downregulated again after molting. The expression level of HlATG12 was highest at 3 months after engorgement. By immuno-electron microscopy, it was demonstrated that HlAtg12 was localized to the region around granule-like structures within midgut cells of unfed adults. In conclusion, HlATG12 might function during unfed and molting stages.

  5. Single-cell RNA sequencing highlights transcription activity of autophagy-related genes during hematopoietic stem cell formation in mouse embryos.

    PubMed

    Hu, Yongfei; Huang, Yan; Yi, Ying; Wang, Hongwei; Liu, Bing; Yu, Jia; Wang, Dong

    2017-04-03

    Accumulating evidence has demonstrated that macroautophagy/autophagy plays an essential role in self-renewal and differentiation in embryonic hematopoiesis. Here, according to the RNA sequencing data sets of 5 population cells related to hematopoietic stem cell (HSC) formation during mouse embryogenesis (endothelial cells, PTPRC/CD45(-) and PTPRC/CD45(+) pre-HSCs in the E11 aorta-gonad-mesonephros (AGM) region, mature HSCs in E12 and E14 fetal liver), we explored the dynamic expression of mouse autophagy-related genes in this course at the single-cell level. Our results revealed that the transcription activity of autophagy-related genes had a substantial increase when endothelial cells (ECs) specified into pre-HSCs, and the upregulation of autophagy-essential genes correlated with reduced NOTCH signaling in pre-HSCs, suggesting the autophagy activity may be greatly enhanced during pre-HSC specification from endothelial precursors. In summary, our results presented strong evidence that autophagy plays a critical role in HSC emergence during mouse midgestation.

  6. The Protective Effect of Aucubin from Eucommia ulmoides Against Status Epilepticus by Inducing Autophagy and Inhibiting Necroptosis.

    PubMed

    Wang, Jin; Li, Ying; Huang, Wei-Hua; Zeng, Xiang-Chang; Li, Xiao-Hui; Li, Jian; Zhou, Jun; Xiao, Jian; Xiao, Bo; Ouyang, Dong-Sheng; Hu, Kai

    2017-04-07

    Eucommia ulmoides Oliv. is a famous traditional Chinese medicine which exhibits anti-oxidative stress ability and neuro-protective effects. Aucubin is the predominant component of Eucommia ulmoides Oliv. Our present study is intended to investigate aucubin's potential protective effects on neurons against epilepsy in the hippocampus by establishing the lithium-pilocarpine induced status epilepticus (SE) rat model in vivo. Aucubin (at a low dose and a high dose of 5[Formula: see text]mg/kg and 10[Formula: see text]mg/kg, respectively) was administered through gavage for two weeks before lithium-pilocarpine injection. Rats were sacrificed at 4, 24 and 72[Formula: see text]h after SE induction. Pretreatment with both low-dose and high-dose aucubin significantly reduced the number of death neurons ([Formula: see text]) and increased the number of surviving neurons ([Formula: see text]) in DG, Hilus, CA1 and CA3 hippocampal regions post SE. Meanwhile, it significantly inhibited necroptosis proteins (MLKL and RIP-1) ([Formula: see text] or [Formula: see text]) and enhanced autophagy protein (Beclin-1 and LC3BII/LC3BI) prevalence in the hippocampus ([Formula: see text] or [Formula: see text]). In conclusion, aucubin appeared to ameliorate damages in lithium-pilocarpine induced SE in hippocampus, reduce the number of apoptotic neurons, and increased the number of survival neurons by inducing autophagy and inhibiting necroptosis. These original findings might provide an important basis for the further investigation of the therapeutic role of aucubin in treatment or prevention of epilepsy-related neuronal damages.

  7. Identification of Autophagy in the Pine Wood Nematode Bursaphelenchus xylophilus and the Molecular Characterization and Functional Analysis of Two Novel Autophagy-Related Genes, BxATG1 and BxATG8

    PubMed Central

    Deng, Li-Na; Wu, Xiao-Qin; Ye, Jian-Ren; Xue, Qi

    2016-01-01

    The pine wood nematode, Bursaphelenchus xylophilus, causes huge economic losses in pine forests, has a complex life cycle, and shows the remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B. xylophilus and no autophagy-related genes have been previously characterized. In this study, transmission electron microscopy was used to confirm that autophagy exists in B. xylophilus. The full-length cDNAs of BxATG1 and BxATG8 were first cloned from B. xylophilus, and BxATG1 and BxATG8 were characterized using bioinformatics methods. The expression pattern of the autophagy marker BxATG8 was investigated using in situ hybridization (ISH). BxATG8 was expressed in esophageal gland and hypodermal seam cells. We tested the effects of RNA interference (RNAi) on BxATG1 and BxATG8. The results revealed that BxATG1 and BxATG8 were likely associated with propagation of nematodes on fungal mats. This study confirmed the molecular characterization and functions of BxATG1 and BxATG8 in B. xylophilus and provided fundamental information between autophagy and B. xylophilus. PMID:26950119

  8. Autophagy activation: a novel mechanism of atorvastatin to protect mesenchymal stem cells from hypoxia and serum deprivation via AMP-activated protein kinase/mammalian target of rapamycin pathway.

    PubMed

    Zhang, Qian; Yang, Yue-Jin; Wang, Hong; Dong, Qiu-Ting; Wang, Tian-Jie; Qian, Hai-Yan; Xu, Hui

    2012-05-20

    Autophagy is a complex "self-eating" process and could be utilized for cell survival under stresses. Statins, which could reduce apoptosis in mesenchymal stem cells (MSCs) during both ischemia and hypoxia/serum deprivation (H/SD), have been proved to induce autophagy in some cell lines. We have previously shown that atorvastatin (ATV) could regulate AMP-activated protein kinase (AMPK), a positive modulator of autophagy, in MSCs. Thus, we hypothesized that autophagy activation through AMPK and its downstream molecule mammalian target of rapamycin (mTOR) may be a novel mechanism of ATV to protect MSCs from apoptosis during H/SD. Here, we demonstrated that H/SD induced autophagy in MSCs significantly as identified by increasing acidic vesicular organelle-positive cells, type II of light chain 3 (LC3-II) expression, and autophagosome formation. The levels of H/SD-induced apoptosis were increased by autophagy inhibitor 3-methyladenine (3-MA) while decreased by rapamycin, an autophagic inducer. ATV further enhanced the autophagic activity observed in MSCs exposed to H/SD. Treatment with 3-MA attenuated ATV-induced autophagy and abrogated the protective effects of ATV on MSC apoptosis, while rapamycin failed to cause additional effects on either autophagy or apoptosis compared with ATV alone. The phosphorylation of AMPK was upregulated whereas the phosphorylation of mTOR was downregulated in ATV-treated MSCs, which were both attenuated by AMPK inhibitor compound C. Further, treatment with compound C reduced the ATV-induced autophagy in MSCs under H/SD. These data suggest that autophagy plays a protective role in H/SD-induced apoptosis of MSCs, and ATV could effectively activate autophagy via AMPK/mTOR pathway to enhance MSC survival during H/SD.

  9. Autophagy deficiency by hepatic FIP200 deletion uncouples steatosis from liver injury in NAFLD.

    PubMed

    Ma, Di; Molusky, Matthew M; Song, Jianrui; Hu, Chun-Rui; Fang, Fang; Rui, Crystal; Mathew, Anna V; Pennathur, Subramaniam; Liu, Fei; Cheng, Ji-Xin; Guan, Jun-Lin; Lin, Jiandie D

    2013-10-01

    Nonalcoholic fatty liver disease is a metabolic disorder commonly associated with obesity. A subset of nonalcoholic fatty liver disease patients further develops nonalcoholic steatohepatitis that is characterized by chronic liver injury, inflammation, and fibrosis. Recent work has implicated the autophagy pathway in the mobilization and oxidation of triglycerides from lipid droplets. However, whether impaired autophagy in hepatocytes drives excess fat accumulation in the liver remains controversial. In addition, the role of autophagy in protecting the liver from gut endotoxin-induced injury has not been elucidated. Here we generated mice with liver-specific autophagy deficiency by the conditional deletion of focal adhesion kinase family kinase-interacting protein of 200 kDa (also called Rb1cc1), a core subunit of the mammalian autophagy related 1 complex. To our surprise, mice lacking FIP200 in hepatocytes were protected from starvation- and high-fat diet-induced fat accumulation in the liver and had decreased expression of genes involved in lipid metabolism. Activation of the de novo lipogenic program by liver X receptor was impaired in FIP200-deficient livers. Furthermore, liver autophagy was stimulated by exposure to low doses of lipopolysaccharides and its deficiency-sensitized mice to endotoxin-induced liver injury. Together these studies demonstrate that hepatocyte-specific autophagy deficiency per se does not exacerbate hepatic steatosis. Instead, autophagy may play a protective role in the liver after exposure to gut-derived endotoxins and its blockade may accelerate nonalcoholic steatohepatitis progression.

  10. Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria.

    PubMed

    Oliveira, C S F; Pereira, H; Alves, S; Castro, L; Baltazar, F; Chaves, S R; Preto, A; Côrte-Real, M

    2015-06-18

    Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. Cat

  11. The role of autophagy in the pathogenesis of glycogen storage disease type II (GSDII).

    PubMed

    Nascimbeni, A C; Fanin, M; Masiero, E; Angelini, C; Sandri, M

    2012-10-01

    Regulated removal of proteins and organelles by autophagy-lysosome system is critical for muscle homeostasis. Excessive activation of autophagy-dependent degradation contributes to muscle atrophy and cachexia. Conversely, inhibition of autophagy causes accumulation of protein aggregates and abnormal organelles, leading to myofiber degeneration and myopathy. Defects in lysosomal function result in severe muscle disorders such as Pompe (glycogen storage disease type II (GSDII)) disease, characterized by an accumulation of autophagosomes. However, whether autophagy is detrimental or not in muscle function of Pompe patients is unclear. We studied infantile and late-onset GSDII patients and correlated impairment of autophagy with muscle wasting. We also monitored autophagy in patients who received recombinant α-glucosidase. Our data show that infantile and late-onset patients have different levels of autophagic flux, accumulation of p62-positive protein aggregates and expression of atrophy-related genes. Although the infantile patients show impaired autophagic function, the late-onset patients display an interesting correlation among autophagy impairment, atrophy and disease progression. Moreover, reactivation of autophagy in vitro contributes to acid α-glucosidase maturation in both healthy and diseased myotubes. Together, our data suggest that autophagy protects myofibers from disease progression and atrophy in late-onset patients.

  12. Inhibition of autophagy enhances Hydroquinone-induced TK6 cell death.

    PubMed

    Xu, Longmei; Liu, Jiaxian; Chen, Yuting; Yun, Lin; Chen, Shaoyun; Zhou, Kairu; Lai, Bei; Song, Li; Yang, Hui; Liang, Hairong; Tang, Huanwen

    2017-03-02

    Hydroquinone (HQ), one of the metabolic products of benzene, is a carcinogen. It can induce apoptosis in lymphoma cells. However, whether HQ can induce autophagy and what roles autophagy plays in TK6 cells exposured to HQ remains unclear. In this study, we found that HQ could induce autophagy through techniques of qRT-PCR, Western blot, immunofluorescent assay of LC3 and transmission electron microscope. Furthermore, inhibiting autophagy using 3-methyladenine (3-MA) or chloroquine (CQ) significantly enhanced HQ-induced cell apoptosis, suggesting that autophagy may be a survival mechanism. Our study also showed that HQ activated PARP-1. Moreover, knockdown of PARP-1 strongly exhibited decreased autophagy related genes expression. In contrast, the absence of SIRT1 increased that. Altogether, our data provided evidence that HQ induced autophagy in TK6 cells and autophagy protected TK6 from HQ attack-induced injury in vitro, and the autophagy was partially mediated via activation of the PARP-1-SIRT1 signaling pathway.

  13. Anti-oxidative cellular protection effect of fasting-induced autophagy as a mechanism for hormesis.

    PubMed

    Moore, Michael N; Shaw, Jennifer P; Ferrar Adams, Dawn R; Viarengo, Aldo

    2015-06-01

    The aim of this investigation was to test the hypothesis that fasting-induced augmented lysosomal autophagic turnover of cellular proteins and organelles will reduce potentially harmful lipofuscin (age-pigment) formation in cells by more effectively removing oxidatively damaged proteins. An animal model (marine snail--common periwinkle, Littorina littorea) was used to experimentally test this hypothesis. Snails were deprived of algal food for 7 days to induce an augmented autophagic response in their hepatopancreatic digestive cells (hepatocyte analogues). This treatment resulted in a 25% reduction in the cellular content of lipofuscin in the digestive cells of the fasting animals in comparison with snails fed ad libitum on green alga (Ulva lactuca). Similar findings have previously been observed in the digestive cells of marine mussels subjected to copper-induced oxidative stress. Additional measurements showed that fasting significantly increased cellular health based on lysosomal membrane stability, and reduced lipid peroxidation and lysosomal/cellular triglyceride. These findings support the hypothesis that fasting-induced augmented autophagic turnover of cellular proteins has an anti-oxidative cytoprotective effect by more effectively removing damaged proteins, resulting in a reduction in the formation of potentially harmful proteinaceous aggregates such as lipofuscin. The inference from this study is that autophagy is important in mediating hormesis. An increase was demonstrated in physiological complexity with fasting, using graph theory in a directed cell physiology network (digraph) model to integrate the various biomarkers. This was commensurate with increased health status, and supportive of the hormesis hypothesis. The potential role of enhanced autophagic lysosomal removal of damaged proteins in the evolutionary acquisition of stress tolerance in intertidal molluscs is discussed and parallels are drawn with the growing evidence for the involvement of

  14. Salmonella plasmid virulence gene spvB enhances bacterial virulence by inhibiting autophagy in a zebrafish infection model.

    PubMed

    Li, Yuan-Yuan; Wang, Ting; Gao, Song; Xu, Guang-Mei; Niu, Hua; Huang, Rui; Wu, Shu-Yan

    2016-02-01

    Salmonella enterica serovar typhimurium (S. typhimurium) is a facultative intracellular pathogen that can cause gastroenteritis and systemic infection in a wide range of hosts. Salmonella plasmid virulence gene spvB is closely related to bacterial virulence in different cells and animal models, and the encoded protein acts as an intracellular toxin required for ADP-ribosyl transferase activity. However, until now there is no report about the pathogenecity of spvB gene on zebrafish. Due to the outstanding advantages of zebrafish in analyzing bacteria-host interactions, a S. typhimurium infected zebrafish model was set up here to study the effect of spvB on autophagy and intestinal pathogenesis in vivo. We found that spvB gene could decrease the LD50 of S. typhimurium, and the strain carrying spvB promoted bacterial proliferation and aggravated the intestinal damage manifested by the narrowed intestines, fallen microvilli, blurred epithelium cell structure and infiltration of inflammatory cells. Results demonstrated the enhanced virulence induced by spvB in zebrafish. In spvB-mutant strain infected zebrafish, the levels of Lc3 turnover and Beclin1 expression increased, and the double-membraned autophagosome structures were observed, suggesting that spvB can inhibit autophagy activity. In summary, our results indicate that S. typhimurium strain containing spvB displays more virulence, triggering an increase in bacterial survival and intestine injuries by suppressing autophagy for the first time. This model provides novel insights into the role of Salmonella plasmid virulence gene in bacterial pathogenesis, and can help to further elucidate the relationship between bacteria and host immune response.

  15. Autophagy Paradox and Ceramide

    PubMed Central

    Jiang, Wenhui; Ogretmen, Besim

    2013-01-01

    Sphingolipid molecules act as bioactive lipid messengers and exert their actions on the regulation of various cellular signaling pathways. Sphingolipids play essential roles in numerous cellular functions, including controlling cell inflammation, proliferation, death, migration, senescence, tumor metastasis and/or autophagy. Dysregulated sphingolipid metabolism has been also implicated in many human cancers. Macroatuophagy (referred to here as autophagy) “self-eating”, is characterized by nonselective sequestering of cytosolic materials by an isolation membrane, which can be either protective or lethal for cells. Ceramide (Cer), a central molecule of sphingolipid metabolism, has been extensively implicated in the control of autophagy. The increasing evidence suggests Cer is highly involved in mediating two opposing autophagic pathways, which regulate either cell survival or death, autophagy paradox. However, the underlying mechanism that regulates the autophagy paradox remains unclear. Therefore, this review focuses on recent studies with regard to the regulation of autophagy by Cer and elucidate the roles and mechanisms of action of Cer in controlling autophagy paradox. PMID:24055889

  16. Autophagy activation attenuates renal ischemia-reperfusion injury in rats

    PubMed Central

    Zhang, Ya-Li; Cui, Li-Yan; Yang, Shuo

    2015-01-01

    Ischemia-reperfusion (I/R) injury is a leading cause of acute kidney injury (AKI), which is a common clinical complication but lacks effective therapies. This study investigated the role of autophagy in renal I/R injury and explored potential mechanisms in an established rat renal I/R injury model. Forty male Wistar rats were randomly divided into four groups: Sham, I/R, I/R pretreated with 3-methyladenine (3-MA, autophagy inhibitor), or I/R pretreated with rapamycin (autophagy activator). All rats were subjected to clamping of the left renal pedicle for 45 min after right nephrectomy, followed by 24 h of reperfusion. The Sham group underwent the surgical procedure without ischemia. 3-MA and rapamycin were injected 15 min before ischemia. Renal function was indicated by blood urea nitrogen and serum creatinine. Tissue samples from the kidneys were scored histopathologically. Autophagy was indicated by light chain 3 (LC3), Beclin-1, and p62 levels and the number of autophagic vacuoles. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and expression of caspase-3. Autophagy was activated after renal I/R injury. Inhibition of autophagy by 3-MA before I/R aggravated renal injury, with worsened renal function, higher renal tissue injury scores, and more tubular apoptosis. In contrast, rapamycin pretreatment ameliorated renal injury, with improved renal function, lower renal tissue injury scores, and inhibited apoptosis based on fewer TUNEL-positive cells and lower caspase-3 expression. Our results demonstrate that autophagy could be activated during I/R injury and play a protective role in renal I/R injury. The mechanisms were involved in the regulation of several autophagy and apoptosis-related genes. Furthermore, autophagy activator may be a promising therapy for I/R injury and AKI in the future. PMID:25898836

  17. Expression of autophagy and UPR genes in the developing brain during ethanol-sensitive and resistant periods.

    PubMed

    Alimov, Alexander; Wang, Haiping; Liu, Mei; Frank, Jacqueline A; Xu, Mei; Ou, Xiaoming; Luo, Jia

    2013-12-01

    Fetal alcohol spectrum disorders (FASD) results from ethanol exposure to the developing fetus and is the leading cause of mental retardation. FASD is associated with a broad range of neurobehavioral deficits which may be mediated by ethanol-induced neurodegeneration in the developing brain. An immature brain is more susceptible to ethanol neurotoxicity. We hypothesize that the enhanced sensitivity of the immature brain to ethanol is due to a limited capacity to alleviate cellular stress. Using a third trimester equivalent mouse model of ethanol exposure, we demonstrated that subcutaneous injection of ethanol induced a wide-spread neuroapoptosis in postnatal day 4 (PD4) C57BL/6 mice, but had little effect on the brain of PD12 mice. We analyzed the expression profile of genes regulating apoptosis, and the pathways of ER stress response (also known as unfolded protein response, UPR) and autophagy during these ethanol-sensitive and resistant periods (PD4 versus PD12) using PCR microarray. The expression of pro-apoptotic genes, such as caspase-3, was much higher on PD4 than PD12; in contrast, the expression of genes that regulate UPR and autophagy, such as atf6, atg4, atg9, atg10, beclin1, bnip3, cebpb, ctsb, ctsd, ctss, grp78, ire1α, lamp, lc3 perk, pik3c3, and sqstm1 was significantly higher on PD12 than PD4. These results suggest that the vulnerability of the immature brain to ethanol could result from high expression of pro-apoptotic proteins and a deficiency in the stress responsive system, such as UPR and autophagy.

  18. Physiological roles of autophagy in plants: does plant autophagy have a pro-death function?

    PubMed

    Yoshimoto, Kohki

    2010-05-01

    Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. Early morphological studies suggested that autophagy occurs in plant cells and predicted that autophagy has a variety of functions in plant growth and development. However, it is only since the identification of autophagy genes that the physiological roles of autophagy in plants have become apparent. Recent reverse genetic studies indicate that autophagy defects in higher plants result in early senescence and excessive immunity-related programmed cell death (PCD), irrespective of nutrient conditions, suggesting that plant autophagy has an important pro-survival function during these types of cell death. Further biochemical and pharmacological studies in combination with double mutant analyses revealed that excessive salicylic acid (SA) signaling is a major factor in autophagy-defective plant-dependent cell death and that the SA signal can induce autophagy. These results demonstrate a novel physiological function for plant autophagy that operates a negative feedback loop to modulate SA signaling.

  19. How and why to study autophagy in Drosophila: it's more than just a garbage chute.

    PubMed

    Nagy, Péter; Varga, Ágnes; Kovács, Attila L; Takáts, Szabolcs; Juhász, Gábor

    2015-03-01

    During the catabolic process of autophagy, cytoplasmic material is transported to the lysosome for degradation and recycling. This way, autophagy contributes to the homeodynamic turnover of proteins, lipids, nucleic acids, glycogen, and even whole organelles. Autophagic activity is increased by adverse conditions such as nutrient limitation, growth factor withdrawal and oxidative stress, and it generally protects cells and organisms to promote their survival. Misregulation of autophagy is likely involved in numerous human pathologies including aging, cancer, infections and neurodegeneration, so its biomedical relevance explains the still growing interest in this field. Here we discuss the different microscopy-based, biochemical and genetic methods currently available to study autophagy in various tissues of the popular model Drosophila. We show examples for results obtained in different assays, explain how to interpret these with regard to autophagic activity, and how to find out which step of autophagy a given gene product is involved in.

  20. Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin.

    PubMed

    Yuk, Jae-Min; Shin, Dong-Min; Lee, Hye-Mi; Yang, Chul-Su; Jin, Hyo Sun; Kim, Kwang-Kyu; Lee, Zee-Won; Lee, Sang-Hee; Kim, Jin-Man; Jo, Eun-Kyeong

    2009-09-17

    Autophagy and vitamin D3-mediated innate immunity have been shown to confer protection against infection with intracellular Mycobacterium tuberculosis. Here, we show that these two antimycobacterial defenses are physiologically linked via a regulatory function of human cathelicidin (hCAP-18/LL-37), a member of the cathelicidin family of antimicrobial proteins. We show that 1,25-dihydroxyvitamin D3 (1,25D3), the active form of vitamin D, induced autophagy in human monocytes via cathelicidin, which activated transcription of the autophagy-related genes Beclin-1 and Atg5. 1,25D3 also induced the colocalization of mycobacterial phagosomes with autophagosomes in human macrophages in a cathelicidin-dependent manner. Furthermore, the antimycobacterial activity in human macrophages mediated by physiological levels of 1,25D3 required autophagy and cathelicidin. These results indicate that human cathelicidin, a protein that has direct antimicrobial activity, also serves as a mediator of vitamin D3-induced autophagy.

  1. 8-p-Hdroxybenzoyl Tovarol Induces Paraptosis Like Cell Death and Protective Autophagy in Human Cervical Cancer HeLa Cells.

    PubMed

    Zhang, Cui; Jiang, Yingnan; Zhang, Jin; Huang, Jian; Wang, Jinhui

    2015-07-02

    8-p-Hdroxybenzoyl tovarol (TAW) is a germacrane-type sesquiterpenoid that can be isolated from the roots of Ferula dissecta (Ledeb.) Ledeb. In this study, the growth inhibitory effects induced by TAW were screened on some types of tumor cells, and the mechanism was investigated on TAW-induced growth inhibition, including paraptosis and autophagy in human cervical cancer HeLa cells. TAW-induced paraptosis involved extensive cytoplasmic vacuolization in the absence of caspase activation. Additionally, TAW evoked cell paraptotic death mediated by endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Autophagy induced by TAW was found to antagonize paraptosis in HeLa cells. This effect was enhanced by rapamycin and suppressed by the autophagy inhibitor, 3-methyladenine (3MA). Loss of beclin 1 (an autophagic regulator) function led to promote ER stress. Taken together, these results suggest that TAW induces paraptosis like cell death and protective autophagy in HeLa cells, which would provide a new clue for exploiting TAW as a promising agent for the treatment of cervical cancer.

  2. Role of FOXO1 in aldosterone-induced autophagy: A compensatory protective mechanism related to podocyte injury

    PubMed Central

    Wang, Bin; Ding, Wei; Zhang, Minmin; Li, Hongmei; Guo, Honglei; Lin, Lilu; Chen, Jing; Gu, Yong

    2016-01-01

    This study was undertaken to elucidate whether and how autophagy was regulated in aldosterone (Aldo)-induced podocyte injury and to examine its role in this model both in vitro and in vivo. In cultured podocytes, Aldo increased autophagy flux as indicated by the enhanced expression of LC3-II/LC3-I and the reduction of p62. Autophagy induction with rapamycin (RP) provided a cytoprotective effect, and inhibition of autophagy with Atg7-specific siRNA, chloroquine (CQ) or 3-methyladenine (3-MA) worsened Aldo-induced podocyte injury by attenuating endoplasmic reticulum (ER) stress. Aldo inhibited Akt phosphorylation but increased the mammalian target of rapamycin (mTOR) signaling pathway; however, Aldo up-regulated the expression of FOXO1 and its downstream effector Rab7. Either knockdown of FOXO1 or Rab7 inhibited Aldo-induced autophagy. Additionally, an elevated level of P300-regulated acetylation of FOXO1 and the interaction of acetylated FOXO1 and Atg7 were also confirmed to be involved in regulating autophagy in Aldo-induced podocytes. Similar results were further confirmed in vivo. We propose that autophagy enhancement through enhancing of the FOXO1/Rab7 axis and post-translational modification of FOXO1 may represent a potential therapeutic strategy against podocyte injury by promoting autophagy. PMID:27244896

  3. Protective effects of ethyl pyruvate on lipopolysaccharide-induced acute lung injury through inhibition of autophagy in neutrophils

    PubMed Central

    Zhu, Qingteng; Wang, Hui; Wang, Hairong; Luo, Yong; Yu, Yang; Du, Qirong; Fei, Aihua; Pan, Shuming

    2017-01-01

    Among a number of clinical factors, bacterial infection is one of the most common causes of acute lung injury (ALI), a serious complication that carries a high risk of mortality (~40%). During the process of ALI, intense local and systemic inflammation is elicited, which exacerbates the injury. Neutrophil infiltration into airspace is observed in early stage of ALI, and is required for the full development of ALI through an array of mechanisms, including the release of granule contents and the production of pro-inflammatory cytokines, due to the overactivation of complement and cytokines. The present study noted that ethyl pyruvate alleviated ALI in lipopolysaccharide (LPS)-induced ALI mice. Increased autophagy in neutrophils from ALI mice was observed, while ethyl pyruvate diminished autophagy in neutrophils and constrained granule release, and therefore myeloperoxidase (MPO) in bronchoalveolar lavage fluid and the production of proinflammatory cytokines. Using neutrophil cells, it was identified that autophagy was required for neutrophil activation and granule release, and that ethyl pyruvate caused neutrophil autophagy, leading to the restriction of granule release, and thus contributing to the mitigation of ALI. If autophagy was obviated through knockdown of key regulator of autophagy Atg5, the effects of ethyl pyruvate on granule release by neutrophils disappeared. Taken together, the results demonstrated that ethyl pyruvate alleviates ALI through inhibition of autophagy-induced granule release by neutrophils, and this mechanism suggested a novel potential therapeutic target in autophagy regulation for ALI. PMID:28098908

  4. Insulin Protects Hepatic Lipotoxicity by Regulating ER Stress through the PI3K/Akt/p53 Involved Pathway Independently of Autophagy Inhibition

    PubMed Central

    Ning, Hua; Sun, Zongxiang; Liu, Yunyun; Liu, Lei; Hao, Liuyi; Ye, Yaxin; Feng, Rennan; Li, Jie; Li, Ying; Chu, Xia; Li, Songtao; Sun, Changhao

    2016-01-01

    The detrimental role of hepatic lipotoxicity has been well-implicated in the pathogenesis of NAFLD. Previously, we reported that inhibiting autophagy aggravated saturated fatty acid (SFA)-induced hepatotoxicity. Insulin, a physiological inhibitor of autophagy, is commonly increased within NAFLD mainly caused by insulin resistance. We therefore hypothesized that insulin augments the sensitivity of hepatocyte to SFA-induced lipotoxicity. The present study was conducted via employing human and mouse hepatocytes, which were exposed to SFAs, insulin, or their combination. Unexpectedly, our results indicated that insulin protected hepatocytes against SFA-induced lipotoxicity, based on the LDH, MTT, and nuclear morphological measurements, and the detection from cleaved-Parp-1 and -caspase-3 expressions. We subsequently clarified that insulin led to a rapid and short-period inhibition of autophagy, which was gradually recovered after 1 h incubation in hepatocytes, and such extent of inhibition was insufficient to aggravate SFA-induced lipotoxicity. The mechanistic study revealed that insulin-induced alleviation of ER stress contributed to its hepatoprotective role. Pre-treating hepatocytes with insulin significantly stimulated phosphorylated-Akt and reversed SFA-induced up-regulation of p53. Chemical inhibition of p53 by pifithrin-α robustly prevented palmitate-induced cell death. The PI3K/Akt pathway blockade by its special antagonist abolished the protective role of insulin against SFA-induced lipotoxicity and p53 up-regulation. Furthermore, we observed that insulin promoted intracellular TG deposits in hepatocytes in the present of palmitate. However, blocking TG accumulation via genetically silencing DGAT-2 did not prevent insulin-protected lipotoxicity. Our study demonstrated that insulin strongly protected against SFA-induced lipotoxicity in hepatocytes mechanistically through alleviating ER stress via a PI3K/Akt/p53 involved pathway but independently from autophagy

  5. In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver

    SciTech Connect

    Donati, Alessio; Ventruti, Annamaria; Cavallini, Gabriella; Masini, Matilde; Vittorini, Simona; Chantret, Isabelle; Codogno, Patrice; Bergamini, Ettore

    2008-02-15

    Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5'-dimethylpyrazole, DMP, 12 mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis.

  6. In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver.

    PubMed

    Donati, Alessio; Ventruti, Annamaria; Cavallini, Gabriella; Masini, Matilde; Vittorini, Simona; Chantret, Isabelle; Codogno, Patrice; Bergamini, Ettore

    2008-02-15

    Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5'-dimethylpyrazole, DMP, 12mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis.

  7. p53-regulated autophagy is controlled by glycolysis and determines cell fate.

    PubMed

    Duan, Lei; Perez, Ricardo E; Davaadelger, Batzaya; Dedkova, Elena N; Blatter, Lothar A; Maki, Carl G

    2015-09-15

    The tumor suppressor p53 regulates downstream targets that determine cell fate. Canonical p53 functions include inducing apoptosis, growth arrest, and senescence. Non-canonical p53 functions include its ability to promote or inhibit autophagy and its ability to regulate metabolism. The extent to which autophagy and/or metabolic regulation determines cell fate by p53 is unclear. To address this, we compared cells resistant or sensitive to apoptosis by the p53 activator Nutlin-3a. In resistant cells, glycolysis was maintained upon Nutlin-3a treatment, and activated p53 promoted prosurvival autophagy. In contrast, in apoptosis sensitive cells activated p53 increased superoxide levels and inhibited glycolysis through repression of glycolytic pathway genes. Glycolysis inhibition and increased superoxide inhibited autophagy by repressing ATG genes essential for autophagic vesicle maturation. Inhibiting glycolysis increased superoxide and blocked autophagy in apoptosis-resistant cells, causing p62-dependent caspase-8 activation. Finally, treatment with 2-DG or the autophagy inhibitors chloroquine or bafilomycin A1 sensitized resistant cells to Nutlin-3a-induced apoptosis. Together, these findings reveal novel links between glycolysis and autophagy that determine apoptosis-sensitivity in response to p53. Specifically, the findings indicate 1) that glycolysis plays an essential role in autophagy by limiting superoxide levels and maintaining expression of ATG genes required for autophagic vesicle maturation, 2) that p53 can promote or inhibit autophagy depending on the status of glycolysis, and 3) that inhibiting protective autophagy can expand the breadth of cells susceptible to Nutlin-3a induced apoptosis.

  8. Quercetin induces protective autophagy and apoptosis through ER stress via the p-STAT3/Bcl-2 axis in ovarian cancer.

    PubMed

    Liu, Y; Gong, W; Yang, Z Y; Zhou, X S; Gong, C; Zhang, T R; Wei, X; Ma, D; Ye, F; Gao, Q L

    2017-04-01

    Quercetin (3,3',4',5,7-pentahydroxyflavone, Qu) is a promising cancer chemo-preventive agent for various cancers because it inhibits disease progression and promotes apoptotic cell death. In our previous study, we demonstrated that Qu could evoke ER stress to enhance drug cytotoxicity in ovarian cancer (OC). However, Qu-induced ER stress in OC is still poorly understood. Here, we demonstrated that Qu evoked ER stress to involve in mitochondria apoptosis pathway via the p-STAT3/Bcl-2 axis in OC cell lines and in primary OC cells. Unexpectedly, inhibition of ER stress did not reverse Qu-induced cell death. Further functional studies revealed that Qu-induced ER stress could activate protective autophagy concomitantly by activating the p-STAT3/Bcl-2 axis in this process. Moreover, the autophagy scavenger 3-MA was shown to enhance Qu's anticancer effects in an ovarian cancer mice xenograft model. These findings revealed a novel role of ER stress as a "double edge sword" participating in Qu-induced apoptosis of OC and might provide a new angle to consider in clinical studies of biological modifiers that may circumvent drug resistance in patients by targeting protective autophagy pathways.

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

    SciTech Connect

    Li, Yangling; Luo, Peihua; Wang, Jincheng; Dai, Jiabin; Yang, Xiaochun; Wu, Honghai; Yang, Bo He, Qiaojun

    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.

  10. Detection of Autophagy in Caenorhabditis elegans

    PubMed Central

    Palmisano, Nicholas J.; Meléndez, Alicia

    2017-01-01

    Autophagy is a dynamic and catabolic process that results in the breakdown and recycling of cellular components through the autophagosomal-lysosomal pathway. Many autophagy genes identified in yeast and mammals have orthologs in C. elegans. In recent years, gene inactivation, by RNAi and/or chromosomal mutations, has been useful to probe the functions of autophagy in C. elegans, and a role for autophagy has been shown in multiple processes such as, the adaptation to stress, longevity, cell death, cell growth control, clearance of aggregate prone proteins, degradation of P granules during embryogenesis, and apoptotic cell clearance. Here we discuss some of these roles and describe methods that can be used to study autophagy in C. elegans. Specifically, we summarize how to visualize autophagy in embryos, larva, or adults, how to detect the lipidation of LGG-1 by western blot, and how to inactivate autophagy genes by RNAi. PMID:26729905

  11. Detection of Autophagy in Caenorhabditis elegans.

    PubMed

    Palmisano, Nicholas J; Meléndez, Alicia

    2016-02-01

    Autophagy is a dynamic and catabolic process that results in the breakdown and recycling of cellular components through the autophagosomal-lysosomal pathway. Many autophagy genes identified in yeasts and mammals have orthologs in the nematode Caenorhabditis elegans. In recent years, gene inactivation by RNA interference (RNAi) and chromosomal mutations has been useful to probe the functions of autophagy in C. elegans, and a role for autophagy has been shown to contribute to multiple processes, such as the adaptation to stress, longevity, cell death, cell growth control, clearance of aggregation-prone proteins, degradation of P granules during embryogenesis, and apoptotic cell clearance. Here, we discuss some of these roles and describe methods that can be used to study autophagy in C. elegans. Specifically, we summarize how to visualize autophagy in embryos, larva, or adults, how to detect the lipidation of the ubiquitin-like modifier LGG-1 by western blot, and how to inactivate autophagy genes by RNAi.

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

    PubMed

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

    2016-06-09

    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.

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

    SciTech Connect

    Wu, Chun-Yan; Yan, Jun; Yang, Yue-Feng; Xiao, Feng-Jun; Li, Qing-Fang; Zhang, Qun-Wei; Wang, Li-Sheng; Guo, Xiao-Zhong; Wang, Hua

    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.

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

    SciTech Connect

    Zou, Hui; Zhuo, Liling; Han, Tao; Hu, Di; Yang, Xiaokang; Wang, Yi; Yuan, Yan; Gu, Jianhong; Bian, Jianchun; Liu, Xuezhong; Liu, Zongping

    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.

  15. Cerebral ischemic post-conditioning induces autophagy inhibition and a HMGB1 secretion attenuation feedback loop to protect against ischemia reperfusion injury in an oxygen glucose deprivation cellular model

    PubMed Central

    Wang, Jue; Han, Dong; Sun, Miao; Feng, Juan

    2016-01-01

    Cerebral ischemic postconditioning (IPOC) has been demonstrated to be neuroprotective against cerebral ischemia reperfusion injury. The present study aimed to determine whether IPOC could inhibit autophagy and high mobility group box 1 (HMGB1) release in a PC12 cell oxygen glucose deprivation/reperfusion (OGD/R) model. An 8 h OGD and 24 h reperfusion cellular model was developed to mimic cerebral ischemia reperfusion injury, with 3 cycles of 10 min OGD/5 min reperfusion treatment to imitate IPOC. Cell viability was determined to demonstrate the efficiency of OGD/R, IPOC and autophagy activator, rapamycin (RAP), treatment. Transmission electron microscopy was performed to observe the formation of autophagosomes, and immunofluorescence, western blot and co-immunoprecipitation were used to examine the expression of autophagy-associated proteins and HMGB1. Enzyme-linked immunosorbent assay analysis was conducted to examine the level of HMGB1 in cell supernatants. Additionally, PC12 cells were treated with RAP to examine the effect of autophagy on HMGB1 release, and the effect of recombinant human HMGB1 and Beclin1 small interfering RNA on autophagy was investigated. The present study confirmed that IPOC inhibited autophagy and HMGB1 secretion, autophagy inhibition induced a decrease in HMGB1 secretion, and HMGB1 secretion attenuation caused autophagy inhibition in return, as demonstrated by immunofluorescence and western blot analyses. Autophagy inhibition and HMGB1 secretion attenuation were, therefore, demonstrated to form a feedback loop under IPOC. These mechanisms illustrated the protective effects of IPOC and may accelerate the clinical use of IPOC. PMID:27666823

  16. Autophagy is involved in TGF-β1-induced protective mechanisms and formation of cancer-associated fibroblasts phenotype in tumor microenvironment

    PubMed Central

    Liu, Fang-Lan; Mo, En-Pan; Yang, Liu; Du, Jun; Wang, Hong-Sheng; Zhang, Huan; Kurihara, Hiroshi; Xu, Jun; Cai, Shao-Hui

    2016-01-01

    Transforming growth factor-β1 (TGF-β1) present in tumor microenvironment acts in a coordinated fashion to either suppress or promote tumor development. However, the molecular mechanisms underlying the effects of TGF-β1 on tumor microenvironment are not well understood. Our clinical data showed a positive association between TGF-β1 expression and cancer-associated fibroblasts (CAFs) in tumor microenvironment of breast cancer patients. Thus we employed starved NIH3T3 fibroblasts in vitro and 4T1 cells mixed with NIH3T3 fibroblasts xenograft model in vivo to simulate nutritional deprivation of tumor microenvironment to explore the effects of TGF-β1. We demonstrated that TGF-β1 protected NIH3T3 fibroblasts from Star-induced growth inhibition, mitochondrial damage and cell apoptosis. Interestingly, TGF-β1 induced the formation of CAFs phenotype in starvation (Star)-treated NIH3T3 fibroblasts and xenografted Balb/c mice, which promoted breast cancer tumor growth. In both models, autophagy agonist rapamycin increased TGF-β1-induced protective effects and formation of CAFs phenotypes, while autophagy inhibitor 3-methyladenine, Atg5 knockdown or TGF-β type I receptor kinase inhibitor LY-2157299 blocked TGF-β1 induced these effects. Taken together, our results indicated that TGF-β/Smad autophagy was involved in TGF-β1-induced protective effects and formation of CAFs phenotype in tumor microenvironment, which may be used as therapy targets in breast cancer. PMID:26716641

  17. Autophagy in cancer: good, bad, or both?

    PubMed

    Hippert, Melanie M; O'Toole, Patrick S; Thorburn, Andrew

    2006-10-01

    Autophagy has been recognized as an important cellular process for at least 50 years; however, it is only with the recent identification of key regulators of autophagy (Atg genes) that we have begun a mechanistic exploration of its importance in cancer. 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. However, the role of autophagy in these processes is complicated and may, depending on the circumstances, have diametrically opposite consequences for the tumor. In this article, we discuss recent discoveries regarding autophagy in cancer.

  18. Comparative gene identification-58 (CGI-58) promotes autophagy as a putative lysophosphatidylglycerol acyltransferase.

    PubMed

    Zhang, Jun; Xu, Dan; Nie, Jia; Han, Ruili; Zhai, Yonggong; Shi, Yuguang

    2014-11-21

    CGI-58 is a lipid droplet-associated protein that, when mutated, causes Chanarin-Dorfman syndrome in humans, which is characterized by excessive storage of triglyceride in various tissues. However, the molecular mechanisms underlying the defect remain elusive. CGI-58 was previously reported to catalyze the resynthesis of phosphatidic acid as a lysophosphatidic acid acyltransferase. In addition to triglyceride, phosphatidic acid is also used a substrate for the synthesis of various mitochondrial phospholipids. In this report, we investigated the propensity of CGI-58 in the remodeling of various phospholipids. We found that the recombinant CGI-58 overexpressed in mammalian cells or purified from Sf9 insect cells catalyzed efficiently the reacylation of lysophosphatidylglycerol to phosphatidylglycerol (PG), which requires acyl-CoA as the acyl donor. In contrast, the recombinant CGI-58 was devoid of acyltransferase activity toward other lysophospholipids. Accordingly, overexpression and knockdown of CGI-58 adversely affected the endogenous PG level in C2C12 cells. PG is a substrate for the synthesis of cardiolipin, which is required for mitochondrial oxidative phosphorylation and mitophagy. Consequently, overexpression and knockdown of CGI-58 adversely affected autophagy and mitophagy in C2C12 cells. In support for a key role of CGI-58 in mitophagy, overexpression of CGI-58 significantly stimulated mitochondrial fission and translocation of PINK1 to mitochondria, key steps involved in mitophagy. Furthermore, overexpression of CGI-58 promoted mitophagic initiation through activation of 5'-AMP-activated protein kinase and inhibition of mTORC1 mammalian target of rapamycin complex 1 signaling, the positive and negative regulators of autophagy, respectively. Together, these findings identified novel molecular mechanisms by which CGI-58 regulates lipid homeostasis, because defective autophagy is implicated in dyslipidemia and fatty liver diseases.

  19. Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency.

    PubMed

    Pastore, Nunzia; Blomenkamp, Keith; Annunziata, Fabio; Piccolo, Pasquale; Mithbaokar, Pratibha; Maria Sepe, Rosa; Vetrini, Francesco; Palmer, Donna; Ng, Philip; Polishchuk, Elena; Iacobacci, Simona; Polishchuk, Roman; Teckman, Jeffrey; Ballabio, Andrea; Brunetti-Pierri, Nicola

    2013-03-01

    Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins.

  20. Identification of Autophagy-Related Genes and Their Regulatory miRNAs Associated with Celiac Disease in Children.

    PubMed

    Comincini, Sergio; Manai, Federico; Meazza, Cristina; Pagani, Sara; Martinelli, Carolina; Pasqua, Noemi; Pelizzo, Gloria; Biggiogera, Marco; Bozzola, Mauro

    2017-02-12

    Celiac disease (CD) is a severe genetic autoimmune disorder, affecting about one in 100 people, where the ingestion of gluten leads to damage in the small intestine. Diagnosing CD is quite complex and requires blood tests and intestinal biopsy examinations. Controversy exists regarding making the diagnosis without biopsy, due to the large spectrum of manifesting symptoms; furthermore, small-intestinal gastroscopy examinations have a relatively complex management in the pediatric population. To identify novel molecular markers useful to increase the sensitivity and specificity in the diagnosis of pediatric CD patients, the expression levels of two key autophagy executor genes (ATG7 and BECN1) and their regulatory validated miRNAs (miR-17 and miR-30a, respectively) were analyzed by relative quantitative real-time-PCR on a cohort of confirmed CD patients compared to age-related controls. Among the investigated targets, the non-parametric Mann-Whitney U test and ROC analysis indicated the highest significant association of BECN1 with CD status in the blood, while in intestinal biopsies, all of the investigated sequences were positively associated with CD diagnosis. Nomogram-based analysis showed nearly opposite expression trends in blood compared to intestine tissue, while hierarchical clustering dendrograms enabled identifying CD and control subgroups based on specific genes and miRNA expression signatures. Next, using an established in vitro approach, through digested gliadin administration in Caco-2 cells, we also highlighted that the modulation of miR-17 endogenous levels using enriched exosomes increased the intracellular autophagosome content, thereby altering the autophagic status. Altogether, these results highlighted novel molecular markers that might be useful to increase the accuracy in CD diagnosis and in molecular-based stratification of the patients, further reinforcing the functional involvement of the regulation of the autophagy process within a

  1. Identification of Autophagy-Related Genes and Their Regulatory miRNAs Associated with Celiac Disease in Children

    PubMed Central

    Comincini, Sergio; Manai, Federico; Meazza, Cristina; Pagani, Sara; Martinelli, Carolina; Pasqua, Noemi; Pelizzo, Gloria; Biggiogera, Marco; Bozzola, Mauro

    2017-01-01

    Celiac disease (CD) is a severe genetic autoimmune disorder, affecting about one in 100 people, where the ingestion of gluten leads to damage in the small intestine. Diagnosing CD is quite complex and requires blood tests and intestinal biopsy examinations. Controversy exists regarding making the diagnosis without biopsy, due to the large spectrum of manifesting symptoms; furthermore, small-intestinal gastroscopy examinations have a relatively complex management in the pediatric population. To identify novel molecular markers useful to increase the sensitivity and specificity in the diagnosis of pediatric CD patients, the expression levels of two key autophagy executor genes (ATG7 and BECN1) and their regulatory validated miRNAs (miR-17 and miR-30a, respectively) were analyzed by relative quantitative real-time-PCR on a cohort of confirmed CD patients compared to age-related controls. Among the investigated targets, the non-parametric Mann–Whitney U test and ROC analysis indicated the highest significant association of BECN1 with CD status in the blood, while in intestinal biopsies, all of the investigated sequences were positively associated with CD diagnosis. Nomogram-based analysis showed nearly opposite expression trends in blood compared to intestine tissue, while hierarchical clustering dendrograms enabled identifying CD and control subgroups based on specific genes and miRNA expression signatures. Next, using an established in vitro approach, through digested gliadin administration in Caco-2 cells, we also highlighted that the modulation of miR-17 endogenous levels using enriched exosomes increased the intracellular autophagosome content, thereby altering the autophagic status. Altogether, these results highlighted novel molecular markers that might be useful to increase the accuracy in CD diagnosis and in molecular-based stratification of the patients, further reinforcing the functional involvement of the regulation of the autophagy process within a

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

    PubMed

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

    2014-08-04

    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.

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

    SciTech Connect

    Yuan, Xiaopeng; Du, Jie; Hua, Song; Zhang, Haowen; Gu, Cheng; Wang, Jie; Yang, Lei; Huang, Jianfeng; Yu, Jiahua Liu, Fenju

    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.

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

    PubMed

    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 (P<0.05) enhanced GR activation indicated by higher ratio of GR phosphorylation. Out of 17 autophagy-related genes determined, 8 was significantly (P<0.05) up-regulated in FD group, which includes ATG2b, ATG3, ATG4c, ATG5, ATG10, ATG12, ATG13 and ATG14. Meanwhile, 4 out of 7 circadian-related genes detected, Clock, Cry1, Cry2 and Per2, were significantly (P<0.05) up-regulated. The protein content of autophagy markers, LC3A and LC3B, was also increased significantly (P<0.05). ChIP assay showed that FD promoted (P<0.05) GR binding to the promoter sequence of ATG3 and Per2. Moreover, MeDIP analysis demonstrated significant (P<0.05) hypomethylation in the promoter sequence of ATG12, ATG13 and Per2 genes. Together, we speculate that FD increases the transcription of autophagy- and circadian-related genes through, at least partly, GR-mediated pathway. Our results provide a basis for future investigations into the intracellular regulatory network in response to folate deficiency.

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

    PubMed

    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

    2014-04-01

    U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit (3)H-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 A 1, 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 A 1 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.

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

    PubMed Central

    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

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

    PubMed Central

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

    2015-01-01

    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

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

    PubMed

    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.

  9. Protective autophagy induced by RBX1/ROC1 knockdown or CRL inactivation via modulating the DEPTOR-MTOR axis.

    PubMed

    Yang, Dongqin; Zhao, Yongchao; Liu, Jie; Sun, Yi; Jia, Lijun

    2012-12-01

    RBX1/ROC1 is an essential subunit of the largest multiunit Cullin-RING E3 ligase (CRL), which controls the degradation of diverse substrates, thereby regulating numerous cellular processes. Recently, we reported that RBX1 is overexpressed in hepatocellular carcinomas (HCC) and its expression is negatively correlated with patient survival. Moreover, siRNA silencing of RBX1 inhibits the proliferation of liver cancer cells both in vitro and in vivo by inducing CDKN1A/p21-dependent cell senescence. Interestingly, independent of senescence, RBX1 knockdown also triggers an autophagy response, due, at least in part, to the accumulation of the MTOR-inhibitory protein DEPTOR, a recently identified CRL substrate. Biologically, blockage of autophagy significantly enhances the growth-suppressive effect of RBX1 knockdown by triggering massive apoptosis, indicating that the autophagy response upon RBX1 knockdown serves as a survival signal in liver cells. Similar observations were also made in many types of human cancer cells upon inhibition of CRL by MLN4924. These findings suggest that RBX1-CRL is a promising anti-cancer drug target and provide proof-of-concept evidence for a novel drug combination of RBX1-CRL inhibitor and autophagy inhibitor for effective treatment of human cancer.

  10. Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway.

    PubMed

    Vucicevic, Ljubica; Misirkic, Maja; Janjetovic, Kristina; Vilimanovich, Urosh; Sudar, Emina; Isenovic, Esma; Prica, Marko; Harhaji-Trajkovic, Ljubica; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir; Trajkovic, Vladimir

    2011-01-01

    In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential

  11. Autophagy inhibition uncovers the neurotoxic action of the antipsychotic drug olanzapine.

    PubMed

    Vucicevic, Ljubica; Misirkic-Marjanovic, Maja; Paunovic, Verica; Kravic-Stevovic, Tamara; Martinovic, Tamara; Ciric, Darko; Maric, Nadja; Petricevic, Sasa; Harhaji-Trajkovic, Ljubica; Bumbasirevic, Vladimir; Trajkovic, Vladimir

    2014-01-01

    We investigated the role of autophagy, a controlled cellular self-digestion process, in regulating survival of neurons exposed to atypical antipsychotic olanzapine. Olanzapine induced autophagy in human SH-SY5Y neuronal cell line, as confirmed by the increase in autophagic flux and presence of autophagic vesicles, fusion of autophagosomes with lysosomes, and increase in the expression of autophagy-related (ATG) genes ATG4B, ATG5, and ATG7. The production of reactive oxygen species, but not modulation of the main autophagy repressor MTOR or its upstream regulators AMP-activated protein kinase and AKT1, was responsible for olanzapine-triggered autophagy. Olanzapine-mediated oxidative stress also induced mitochondrial depolarization and damage, and the autophagic clearance of dysfunctional mitochondria was confirmed by electron microscopy, colocalization of autophagosome-associated MAP1LC3B (LC3B henceforth) and mitochondria, and mitochondrial association with the autophagic cargo receptor SQSTM1/p62. While olanzapine-triggered mitochondrial damage was not overtly toxic to SH-SY5Y cells, their death was readily initiated upon the inhibition of autophagy with pharmacological inhibitors, RNA interference knockdown of BECN1 and LC3B, or biological free radical nitric oxide. The treatment of mice with olanzapine for 14 d increased the brain levels of autophagosome-associated LC3B-II and mRNA encoding Atg4b, Atg5, Atg7, Atg12, Gabarap, and Becn1. The administration of the autophagy inhibitor chloroquine significantly increased the expression of proapoptotic genes (Trp53, Bax, Bak1, Pmaip1, Bcl2l11, Cdkn1a, and Cdkn1b) and DNA fragmentation in the frontal brain region of olanzapine-exposed animals. These data indicate that olanzapine-triggered autophagy protects neurons from otherwise fatal mitochondrial damage, and that inhibition of autophagy might unmask the neurotoxic action of the drug.

  12. The putative role of autophagy in the pathogenesis of abdominal aortic aneurysms.

    PubMed

    Ramadan, Azza; Al-Omran, Mohammed; Verma, Subodh

    2017-02-01

    Abdominal aortic aneurysms (AAA) are a significant cause of worldwide mortality and morbidity. While the histopathological characteristics of AAA are well documented, the cellular and molecular mechanisms involved in the pathogenesis of AAA are not entirely understood. Autophagy is a highly conserved basal cellular process in eukaryotic cells that involves the turnover of organelles and proteins. It is also activated as an adaptive response to stressful conditions to promote cell survival. While autophagy typically promotes pro-survival processes, it can sometimes lead to cellular demise. Preclinical studies have revealed autophagy to be a protective mechanism in certain vascular diseases with several autophagy-related genes reported to be markedly upregulated in human aneurysmal tissue. The role autophagy plays in the pathogenesis of AAA, however, remains poorly defined. In this review, we discuss the putative role of autophagy in AAA by reviewing several in vitro and in vivo studies that address the functional significance of autophagy in cells that are involved in the pathophysiology of AAA, amongst which are macrophages, smooth muscle and endothelial cells.

  13. 2-Methoxyestradiol protects against ischemia/reperfusion injury in alcoholic fatty liver by enhancing sirtuin 1-mediated autophagy.

    PubMed

    Cho, Hong-Ik; Seo, Min-Jong; Lee, Sun-Mee

    2017-02-16

    Alcoholic fatty liver (AFL) is susceptible to ischemia/reperfusion (I/R) injury, responding with inflammation and extensive hepatocellular damage. Autophagy maintains cellular homeostasis and regulates inflammation and lipid metabolism. 2-Methoxyestradiol (2-ME2), an endogenous metabolite of estradiol, exhibits antioxidant and anti-inflammatory properties. This study examined the cytoprotective mechanisms of 2-ME2 on hepatic I/R in AFL, focusing on autophagy signaling. C57BL/6 mice were fed an ethanol diet (ED) to induce AFL, or a control diet (CD) for 6weeks, and then subjected to 60min of ischemia and 5h of reperfusion. 2-ME2 (15mg/kg, i.p.) was administered 12h before ischemia and 10min before reperfusion, and sirtinol (sirtuin 1 (SIRT1) inhibitor, 10mg/kg, i.p.) was administered 30min before reperfusion. After reperfusion, ED animals showed higher serum aminotransferase activities and proinflammatory cytokine levels, and more severe histological changes compared with CD animals. These alterations were attenuated by 2-ME2. In the ED I/R group, autophagy and mitophagy were significantly impaired, as indicated by decreased hepatic levels of microtubule-associated protein 1 light chain 3 II and parkin protein expression, and increased p62 protein expression, which were attenuated by 2-ME2. The hepatic levels of Atg12-5 complex, Atg3, Atg7, lysosomal-associated membrane protein 2 and Rab7 protein expression significantly decreased in ED I/R animals, which were attenuated by 2-ME2. In the ED I/R group, the level of SIRT1 protein expression and its catalytic activity significantly decreased, which were attenuated by 2-ME2. Sirtinol reversed the stimulatory effect of 2-ME2 on autophagy. Our findings suggest that 2-ME2 ameliorates I/R-induced hepatocellular damage in AFL through activating SIRT1-mediated autophagy signaling.

  14. Cerebral ischemic post‑conditioning induces autophagy inhibition and a HMGB1 secretion attenuation feedback loop to protect against ischemia reperfusion injury in an oxygen glucose deprivation cellular model.

    PubMed

    Wang, Jue; Han, Dong; Sun, Miao; Feng, Juan

    2016-11-01

    Cerebral ischemic postconditioning (IPOC) has been demonstrated to be neuroprotective against cerebral ischemia reperfusion injury. The present study aimed to determine whether IPOC could inhibit autophagy and high mobility group box 1 (HMGB1) release in a PC12 cell oxygen glucose deprivation/reperfusion (OGD/R) model. An 8 h OGD and 24 h reperfusion cellular model was developed to mimic cerebral ischemia reperfusion injury, with 3 cycles of 10 min OGD/5 min reperfusion treatment to imitate IPOC. Cell viability was determined to demonstrate the efficiency of OGD/R, IPOC and autophagy activator, rapamycin (RAP), treatment. Transmission electron microscopy was performed to observe the formation of autophagosomes, and immunofluorescence, western blot and co‑immunoprecipitation were used to examine the expression of autophagy‑associated proteins and HMGB1. Enzyme‑linked immunosorbent assay analysis was conducted to examine the level of HMGB1 in cell supernatants. Additionally, PC12 cells were treated with RAP to examine the effect of autophagy on HMGB1 release, and the effect of recombinant human HMGB1 and Beclin1 small interfering RNA on autophagy was investigated. The present study confirmed that IPOC inhibited autophagy and HMGB1 secretion, autophagy inhibition induced a decrease in HMGB1 secretion, and HMGB1 secretion attenuation caused autophagy inhibition in return, as demonstrated by immunofluorescence and western blot analyses. Autophagy inhibition and HMGB1 secretion attenuation were, therefore, demonstrated to form a feedback loop under IPOC. These mechanisms illustrated the protective effects of IPOC and may accelerate the clinical use of IPOC.

  15. Lipopolysaccharide induction of autophagy is associated with enhanced bactericidal activity in Dictyostelium discoideum

    PubMed Central

    Pflaum, Katherine; Gerdes, Kimberly; Yovo, Kossi; Callahan, Jennifer; Snyder, Michelle L.D.

    2012-01-01

    Innate immune cells respond to microbial invaders using pattern recognition receptors that detect conserved microbial patterns. Among the cellular processes stimulated downstream of pattern recognition machinery is the initiation of autophagy, which plays protective roles against intracellular microbes. We have shown recently that Dictyostelium discoideum, which takes up bacteria for nutritive purposes, may employ pattern recognition machinery to respond to bacterial prey, as D. discoideum cells upregulate bactericidal activity upon stimulation by lipopolysaccharide (LPS). Here we extend these findings, showing that LPS treatment leads to induction of autophagosomal maturation in cells responding to the bacteria Staphylococcus aureus. Cells treated with the autophagy-inducing drug rapamycin clear internalized bacteria at an accelerated rate, while LPS-enhanced clearance of bacteria is reduced in cells deficient for the autophagy-related genes atg1 and atg9. These findings link microbial pattern recognition with autophagy in the social amoeba D. discoideum. PMID:22575510

  16. Current questions and possible controversies in autophagy

    PubMed Central

    Lindqvist, L M; Simon, A K; Baehrecke, E H

    2015-01-01

    Interest in autophagy has exploded over the last decade, with publications highlighting crosstalk with several other cellular processes including secretion, endocytosis, and cell suicide pathways including apoptosis. Autophagy proteins have also been implicated in other cellular processes independently of their roles in autophagy, creating complexities in the interpretation of autophagy (Atg) mutant gene data. Interestingly, this self-eating process is a survival mechanism that can also promote cell death, but when and how autophagy may ‘switch’ its function is still under debate. Indeed, there are currently many models of how autophagy actually influences cell death. In this review, we highlight some outstanding questions and possible controversies in the autophagy field. PMID:26682061

  17. Far-infrared protects vascular endothelial cells from advanced glycation end products-induced injury via PLZF-mediated autophagy in diabetic mice

    PubMed Central

    Chen, Cheng-Hsien; Chen, Tso-Hsiao; Wu, Mei-Yi; Chou, Tz-Chong; Chen, Jia-Rung; Wei, Meng-Jun; Lee, San-Liang; Hong, Li-Yu; Zheng, Cai-Mei; Chiu, I-Jen; Lin, Yuh-Feng; Hsu, Ching-Min; Hsu, Yung-Ho

    2017-01-01

    The accumulation of advanced glycation end products (AGEs) in diabetic patients induces vascular endothelial injury. Promyelocytic leukemia zinc finger protein (PLZF) is a transcription factor that can be activated by low-temperature far-infrared (FIR) irradiation to exert beneficial effects on the vascular endothelium. In the present study, we investigated the influence of FIR-induced PLZF activation on AGE-induced endothelial injury both in vitro and in vivo. FIR irradiation inhibited AGE-induced apoptosis in human umbilical vein endothelial cells (HUVECs). PLZF activation increased the expression of phosphatidylinositol-3 kinases (PI3K), which are important kinases in the autophagic signaling pathway. FIR-induced PLZF activation led to autophagy in HUVEC, which was mediated through the upregulation of PI3K. Immunofluorescence staining showed that AGEs were engulfed by HUVECs and localized to lysosomes. FIR-induced autophagy promoted AGEs degradation in HUVECs. In nicotinamide/streptozotocin-induced diabetic mice, FIR therapy reduced serum AGEs and AGEs deposition at the vascular endothelium. FIR therapy also reduced diabetes-induced inflammatory markers in the vascular endothelium and improved vascular endothelial function. These protective effects of FIR therapy were not found in PLZF-knockout mice. Our data suggest that FIR-induced PLZF activation in vascular endothelial cells protects the vascular endothelium in diabetic mice from AGE-induced injury. PMID:28071754

  18. Role and regulation of autophagy in cancer

    PubMed Central

    Chen, Ning; Karantza-Wadsworth, Vassiliki

    2011-01-01

    Autophagy is an evolutionarily conserved process whereby cytoplasm and cellular organelles are degraded in lysosomes for amino acid and energy recycling. Autophagy is a survival pathway activated in response to nutrient deprivation and other stressful stimuli, such as metabolic stress and exposure to anticancer drugs. However, autophagy may also result in cell death, if it proceeds to completion. Defective autophagy is implicated in tumorigenesis, as the essential autophagy regulator beclin 1 is monoallelically deleted in human breast, ovarian and prostate cancers, and beclin 1+/− mice are tumor-prone. How autophagy suppresses tumorigenesis is under intense investigation. Cell-autonomous mechanisms, involving protection of genome integrity and stability, and a non-cell-autonomous mechanism, involving suppression of necrosis and inflammation, have been discovered so far. The role of autophagy in treatment responsiveness is also complex. Autophagy inhibition concurrently with chemotherapy or radiotherapy has emerged as a novel approach in cancer treatment, as autophagy-competent tumor cells depend on autophagy for survival under drug- and radiation-induced stress. Alternatively, autophagy stimulation and preservation of cellular fitness bymaintenance of protein and organelle quality control, suppression of DNA damage and genomic instability, and limitation of necrosis-associated inflammation may play a critical role in cancer prevention. PMID:19167434

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

    PubMed

    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.

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

    PubMed Central

    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

  1. Autophagy inhibitors.

    PubMed

    Pasquier, Benoit

    2016-03-01

    Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives.

  2. The role of STAT3 in autophagy.

    PubMed

    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.

  3. Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication.

    PubMed

    Guo, Longjun; Yu, Haidong; Gu, Weihong; Luo, Xiaolei; Li, Ren; Zhang, Jian; Xu, Yunfei; Yang, Lijun; Shen, Nan; Feng, Li; Wang, Yue

    2016-03-31

    Autophagy is an evolutionarily ancient pathway that has been shown to be important in the innate immune defense against several viruses. However, little is known about the regulatory role of autophagy in transmissible gastroenteritis virus (TGEV) replication. In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy. In addition, virus replication was required for the increased amount of the autophagosome marker protein LC3-II. Autophagic flux occurred in TGEV-infected cells, suggesting that TGEV infection triggered a complete autophagic response. When autophagy was pharmacologically inhibited by wortmannin or LY294002, TGEV replication increased. The increase in virus yield via autophagy inhibition was further confirmed by the use of siRNA duplexes, through which three proteins required for autophagy were depleted. Furthermore, TGEV replication was inhibited when autophagy was activated by rapamycin. The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy. Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication.

  4. Activation of AMPK protects against hydrogen peroxide-induced osteoblast apoptosis through autophagy induction and NADPH maintenance: new implications for osteonecrosis treatment?

    PubMed

    She, Chang; Zhu, Lun-qing; Zhen, Yun-fang; Wang, Xiao-dong; Dong, Qi-rong

    2014-01-01

    Elevated hydrogen peroxide (H2O2) causes osteoblast dysfunction and apoptosis, serving as an important contributor to the development of osteonecrosis. Here we aimed to understand the role of AMP-activated protein kinase (AMPK) in the process. We observed a high level of AMPK activation in surgery isolated patients' osteonecrosis tissues. In cultured osteoblastoma MG63 cells, H2O2 stimulation induced significant AMPK activation, oxidative stress, cell death and apoptosis. Inhibition of AMPK by its inhibitor (compound C) or by shRNA-mediated knockdown dramatically enhanced H2O2-induced MG63 cell apoptosis, while over-expression of AMPK in HEK-293 cells alleviated H2O2-induced cell damage. These results confirmed that H2O2-activated AMPK is pro-cell survival. We observed that H2O2 induced protective autophagy in MG63 cells, and AMPK-dependent Ulk1 activation and mTORC1 (mTOR complex 1) inactivation might involve autophagy activation. Further, AMPK activation inhibited H2O2-induced oxidative stress, probably through inhibiting NADPH (nicotinamide adenine dinucleotide phosphate) depletion, since more NADPH depletion and oxidative stress were induced by H2O2 in AMPK deficient MG63 cells. Finally, we observed a significant AMPK activation in H2O2-treated primary cultured and transformed (MC3T3-E1) osteoblasts, and AMPK inhibitor compound C enhanced death by H2O2 in these cells. Based on these results, we concluded that H2O2-induced AMPK activation is pro-survival and anti-apoptosis in osteoblasts. Autophagy induction and NADPH maintenance are involved in AMPK-mediated pro-survival effects. AMPK might represent a novel molecular target for osteonecrosis treatment.

  5. Allicin induces anti-human liver cancer cells through the p53 gene modulating apoptosis and autophagy.

    PubMed

    Chu, Yung-Lin; Ho, Chi-Tang; Chung, Jing-Gung; Raghu, Rajasekaran; Lo, Yi-Chen; Sheen, Lee-Yan

    2013-10-16

    Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer globally and ranks first among the cancer-related mortalities in Taiwan. This study aims to understand the modes of cell death mechanism induced by allicin, a major phytochemical of crushed garlic, in human hepatoma cells. Our earlier study indicated that allicin induced autophagic cell death in human HCC Hep G2 (p53(wild type)) cells, whereas in the present study, allicin induced apoptotic cell death through caspase-dependent and caspase-independent pathways by reactive oxygen species (ROS) overproduction in human HCC Hep 3B (p53(mutation)) cells. To gain insight into the cell death mechanism in p53 knocked down Hep G2, we silenced the p53 gene using siRNA-mediated silencing. Allicin treatment induced apoptotic cell death in p53 knocked down Hep G2 cells similar to that of Hep 3B cells. These results suggest that allicin induced cell death in human hepatoma cells through either autophagy or apoptosis and might be a potential novel complementary gene therapeutic agent for the treatment of apoptosis-resistant cancer cells.

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

    SciTech Connect

    Malaviya, Rama; Laskin, Jeffrey D.; Laskin, Debra L.

    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.

  7. Autophagy Promotes the Repair of Radiation-Induced DNA Damage in Bone Marrow Hematopoietic Cells via Enhanced STAT3 Signaling.

    PubMed

    Xu, Fei; Li, Xin; Yan, Lili; Yuan, Na; Fang, Yixuan; Cao, Yan; Xu, Li; Zhang, Xiaoying; Xu, Lan; Ge, Chaorong; An, Ni; Jiang, Gaoyue; Xie, Jialing; Zhang, Han; Jiang, Jiayi; Li, Xiaotian; Yao, Lei; Zhang, Suping; Zhou, Daohong; Wang, Jianrong

    2017-03-01

    Autophagy protects hematopoietic cells from radiation damage in part by promoting DNA damage repair. However, the molecular mechanisms by which autophagy regulates DNA damage repair remain largely elusive. Here, we report that this radioprotective effect of autophagy depends on STAT3 signaling in murine bone marrow mononuclear cells (BM-MNCs). Specifically, we found that STAT3 activation and nuclear translocation in BM-MNCs were increased by activation of autophagy with an mTOR inhibitor and decreased by knockout of the autophagy gene Atg7. The autophagic regulation of STAT3 activation is likely mediated by induction of KAP1 degradation, because we showed that KAP1 directly interacted with STAT3 in the cytoplasm and knockdown of KAP1 increased the phosphorylation and nuclear translocation of STAT3. Subsequently, activated STAT3 transcriptionally upregulated the expression of BRCA1, which increased the ability of BM-MNCs to repair radiation-induced DNA damage. This novel finding that activation of autophagy can promote DNA damage repair in BM-MNCs via the ATG-KAP1-STAT3-BRCA1 pathway suggests that autophagy plays an important role in maintaining genomic integrity of BM-MNCs and its activation may confer protection of BM-MNCs against radiation-induced genotoxic stress.

  8. Autophagy: cellular and molecular mechanisms.

    PubMed

    Glick, Danielle; Barth, Sandra; Macleod, Kay F

    2010-05-01

    Autophagy is a self-degradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress. Autophagy also plays a housekeeping role in removing misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria, endoplasmic reticulum and peroxisomes, as well as eliminating intracellular pathogens. Thus, autophagy is generally thought of as a survival mechanism, although its deregulation has been linked to non-apoptotic cell death. Autophagy can be either non-selective or selective in the removal of specific organelles, ribosomes and protein aggregates, although the mechanisms regulating aspects of selective autophagy are not fully worked out. In addition to elimination of intracellular aggregates and damaged organelles, autophagy promotes cellular senescence and cell surface antigen presentation, protects against genome instability and prevents necrosis, giving it a key role in preventing diseases such as cancer, neurodegeneration, cardiomyopathy, diabetes, liver disease, autoimmune diseases and infections. This review summarizes the most up-to-date findings on how autophagy is executed and regulated at the molecular level and how its disruption can lead to disease.

  9. Feedback regulation between autophagy and PKA

    PubMed Central

    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. PMID:26046386

  10. Feedback regulation between autophagy and PKA.

    PubMed

    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.

  11. Basal Autophagy Is Required for Herpes simplex Virus-2 Infection

    PubMed Central

    Yakoub, Abraam M.; Shukla, Deepak

    2015-01-01

    Autophagy is a conserved catabolic process of the cell, which plays an important role in regulating plethora of infections. The role of autophagy in Herpes simplex virus-2 (HSV-2) infection is unknown. Here, we found that HSV-2 does not allow induction of an autophagic response to infection, but maintains basal autophagy levels mostly unchanged during productive infection. Thus, we investigated the importance of basal autophagy for HSV-2 infection, using pharmacological autophagy suppression or cells genetically deficient in an autophagy-essential gene (ATG5). Interference with basal autophagy flux in cells significantly reduced viral replication and diminished the infection. These results indicate that basal autophagy plays an indispensable role required for a productive infection. Importantly, this study draws a sharp distinction between induced and basal autophagy, where the former acts as a viral clearance mechanism abrogating infection, while the latter supports infection. PMID:26248741

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

    PubMed Central

    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

  13. Molecular characterization and expression analysis of three novel autophagy-related genes from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

    PubMed

    Flores Fernández, José Miguel; Barragán Álvarez, Carla Patricia; Sánchez Hernández, Carla Vanessa; Padilla Camberos, Eduardo; González Castillo, Celia; Ortuño Sahagún, Daniel; Martínez Velázquez, Moisés

    2016-11-01

    The cattle tick Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite of major importance for the livestock industry. It shows a remarkable ability to survive over long periods without feeding. However, the mechanisms used to endure long-term starvation are poorly understood. It is believed that autophagy, a process of intracellular protein degradation, may play a significant role to confront adverse environmental conditions. To advance our understanding of autophagy in R. microplus, in the present study we report the molecular characterization of three autophagy-related (ATG) genes, namely, RmATG3, RmATG4 and RmATG6, as well as their expression profiles in different developmental stages and organs of the parasite. The deduced amino acid sequences derived from the characterized gene sequences were subjected to Basic Local Alignment Search Tool analysis. The testing produced significant alignments with respective ATG proteins from Haemaphysalis longicornis and Ixodes scapularis ticks. Real-time polymerase chain reaction assays revealed that RmATG4 and RmATG6 transcripts were elevated in egg and ovary tissue, when compared with larva and midgut samples, while RmATG3 expression in midgut was 2-fold higher than in egg, larva and ovary samples.

  14. Molecular cloning and characterization of two novel autophagy-related genes belonging to the ATG8 family from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

    PubMed

    Flores Fernández, José Miguel; Gutiérrez Ortega, Abel; Rosario Cruz, Rodrigo; Padilla Camberos, Eduardo; Alvarez, Angel H; Martínez Velázquez, Moisés

    2014-12-01

    Rhipicephalus (Boophilus) microplus is an obligate haematophagous arthropod and the major problem for cattle industry due to economic losses it causes. The parasite shows a remarkable adaptability to changing environmental conditions as well as an exceptional ability to survive long-term starvation. This ability has been related to a process of intracellular protein degradation called autophagy. This process in ticks is still poorly understood and only few autophagy-related (ATG) genes have been characterized. The aim of the present study was to examine the ESTs database, BmiGI, of R. microplus searching for ATG homologues. We predicted five putative ATG genes, ATG3, ATG4, ATG6 and two ATG8s. Further characterization led to the identification of RmATG8a and RmATG8b, homologues of GABARAP and MAP1LC3, respectively, and both of them belonging to the ATG8 family. PCR analyses showed that the expression level of RmATG8a and RmATG8b was higher in egg and larval stages when compared to ovary and midgut from adult ticks. This up-regulation coincides with the period in which ticks are in a starvation state, suggesting that autophagy is active in R. microplus.

  15. Large-scale inference of gene function through phylogenetic annotation of Gene Ontology terms: case study of the apoptosis and autophagy cellular processes

    PubMed Central

    Feuermann, Marc; Gaudet, Pascale; Mi, Huaiyu; Lewis, Suzanna E.; Thomas, Paul D.

    2016-01-01

    We previously reported a paradigm for large-scale phylogenomic analysis of gene families that takes advantage of the large corpus of experimentally supported Gene Ontology (GO) annotations. This ‘GO Phylogenetic Annotation’ approach integrates GO annotations from evolutionarily related genes across ∼100 different organisms in the context of a gene family tree, in which curators build an explicit model of the evolution of gene functions. GO Phylogenetic Annotation models the gain and loss of functions in a gene family tree, which is used to infer the functions of uncharacterized (or incompletely characterized) gene products, even for human proteins that are relatively well studied. Here, we report our results from applying this paradigm to two well-characterized cellular processes, apoptosis and autophagy. This revealed several important observations with respect to GO annotations and how they can be used for function inference. Notably, we applied only a small fraction of the experimentally supported GO annotations to infer function in other family members. The majority of other annotations describe indirect effects, phenotypes or results from high throughput experiments. In addition, we show here how feedback from phylogenetic annotation leads to significant improvements in the PANTHER trees, the GO annotations and GO itself. Thus GO phylogenetic annotation both increases the quantity and improves the accuracy of the GO annotations provided to the research community. We expect these phylogenetically based annotations to be of broad use in gene enrichment analysis as well as other applications of GO annotations. Database URL: http://amigo.geneontology.org/amigo PMID:28025345

  16. Autophagy in inflammation, infection, neurodegeneration and cancer.

    PubMed

    Arroyo, Daniela S; Gaviglio, Emilia A; Peralta Ramos, Javier M; Bussi, Claudio; Rodriguez-Galan, Maria C; Iribarren, Pablo

    2014-01-01

    In its classical form, autophagy is an essential, homeostatic process by which cytoplasmic components are degraded in a double-membrane-bound autophagosome in response to starvation. Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. The roles of autophagy bridge both the innate and adaptive immune systems and autophagic dysfunction is associated with inflammation, infection, neurodegeneration and cancer. In this review, we discuss the contribution of autophagy to inflammatory, infectious and neurodegenerative diseases, as well as cancer.

  17. The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mitochondrial fission and mitochondrial-derived ROS signaling

    PubMed Central

    Saez-Atienzar, S; Bonet-Ponce, L; Blesa, J R; Romero, F J; Murphy, M P; Jordan, J; Galindo, M F

    2014-01-01

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been associated with Parkinson's disease, and its inhibition opens potential new therapeutic options. Among the drug inhibitors of both wild-type and mutant LRRK2 forms is the 2-arylmethyloxy-5-subtitutent-N-arylbenzamide GSK257815A. Using the well-established dopaminergic cell culture model SH-SY5Y, we have investigated the effects of GSK2578215A on crucial neurodegenerative features such as mitochondrial dynamics and autophagy. GSK2578215A induces mitochondrial fragmentation of an early step preceding autophagy. This increase in autophagosome results from inhibition of fusion rather than increases in synthesis. The observed effects were shared with LRRK2-IN-1, a well-described, structurally distinct kinase inhibitor compound or when knocking down LRRK2 expression using siRNA. Studies using the drug mitochondrial division inhibitor 1 indicated that translocation of the dynamin-related protein-1 has a relevant role in this process. In addition, autophagic inhibitors revealed the participation of autophagy as a cytoprotective response by removing damaged mitochondria. GSK2578215A induced oxidative stress as evidenced by the accumulation of 4-hydroxy-2-nonenal in SH-SY5Y cells. The mitochondrial-targeted reactive oxygen species scavenger MitoQ positioned these species as second messengers between mitochondrial morphologic alterations and autophagy. Altogether, our results demonstrated the relevance of LRRK2 in mitochondrial-activated pathways mediating in autophagy and cell fate, crucial features in neurodegenerative diseases. PMID:25118928

  18. Involvement of autophagy in T cell biology.

    PubMed

    Oral, Ozlem; Yedier, Ozlem; Kilic, Seval; Gozuacik, Devrim

    2017-01-01

    Autophagy is an essential cellular pathway that sequesters various cytoplasmic components, including accumulated proteins, damaged organelles or invading microorganisms and delivers them to lysosomes for degradation. The function of autophagy has been reported in various tissues and systems, including its role in the regulation of cellular immunity. Autophagy plays a fundamental role at various stages of T cell maturation. It regulates the thymocyte selection and the generation of T cell repertoire by presenting intracellular antigens to MHC class molecules. Autophagy is crucial for metabolic regulation of T cells, and therefore supports cell survival and homeostasis, particularly in activated mature T cells. Furthermore, deletion of specific autophagy-related genes induces several immunological alterations including differentiation of activated T cells into regulatory, memory or natural killer T cells. In this review, we emphasize the impact of autophagy on T cell development, activation and differentiation, which is pivotal for the adaptive immune system.

  19. Autophagy genes Smatg8 and Smatg4 are required for fruiting-body development, vegetative growth and ascospore germination in the filamentous ascomycete Sordaria macrospora

    PubMed Central

    Voigt, Oliver; Pöggeler, Stefanie

    2013-01-01

    Autophagy is a tightly controlled degradation process involved in various developmental aspects of eukaryotes. However, its involvement in developmental processes of multicellular filamentous ascomycetes is largely unknown. Here, we analyzed the impact of the autophagic proteins SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. A Saccharomyces cerevisiae complementation assay demonstrated that the S. macrospora Smatg8 and Smatg4 genes can functionally replace the yeast homologs. By generating homokaryotic deletion mutants, we showed that the S. macrospora SmATG8 and SmATG4 orthologs were associated with autophagy-dependent processes. Smatg8 and Smatg4 deletions abolished fruiting-body formation and impaired vegetative growth and ascospore germination, but not hyphal fusion. We demonstrated that SmATG4 was capable of processing the SmATG8 precursor. SmATG8 was localized to autophagosomes, whereas SmATG4 was distributed throughout the cytoplasm of S. macrospora. Furthermore, we could show that Smatg8 and Smatg4 are not only required for nonselective macroautophagy, but for selective macropexophagy as well. Taken together, our results suggest that in S. macrospora, autophagy seems to be an essential and constitutively active process to sustain high energy levels for filamentous growth and multicellular development even under nonstarvation conditions. PMID:23064313

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

    PubMed

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

    2016-01-01

    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.

  1. Suppression of T Cell Autophagy Results in Decreased Viability and Function of T Cells Through Accelerated Apoptosis in a Murine Sepsis Model*

    PubMed Central

    Oami, Takehiko; Hatano, Masahiko; Sunahara, Satoshi; Fujimura, Lisa; Sakamoto, Akemi; Ito, Chizuru; Toshimori, Kiyotaka; Oda, Shigeto

    2017-01-01

    Objective: While type 1 programmed cell death (apoptosis) of T cells leads to immunosuppression in sepsis, a crosstalk between apoptosis and autophagy (type 2 programmed cell death) has not been shown. The aim of this study is to elucidate the details of the interaction between autophagy and immunosuppression. Design: Laboratory investigation in the murine sepsis model. Setting: University laboratory. Subjects: Six- to 8-week-old male mice. Interventions: We investigated the kinetics of autophagy in T cells from spleen in a cecal ligation and puncture model with green fluorescent protein-microtubule-associated protein light chain 3 transgenic mice. We analyzed apoptosis, mitochondrial homeostasis and cytokine production in T cells, and survival rate after cecal ligation and puncture using T cell–specific autophagy-deficient mice. Measurements and Main Results: We observed an increase of autophagosomes, which was assessed by flow cytometry. However, an autophagy process in CD4+ T cells during sepsis was insufficient including the accumulation of p62. On the other hand, a blockade of autophagy accelerated T cell apoptosis compared with the control mice, augmenting the gene expression of Bcl-2-like 11 and programmed cell death 1. Furthermore, mitochondrial accumulation in T cells occurred via a blockade of autophagy during sepsis. In addition, interleukin-10 production in CD4+ T cells from the cecal ligation and puncture–operated knockout mice was markedly increased. Consequently, deficiency of autophagy in T cells significantly decreased the survival rate in the murine sepsis model. Conclusions: We demonstrated that blocking autophagy accelerated apoptosis and increased mortality in concordance with the insufficient autophagy process in CD4+ T cells in the murine sepsis model, suggesting that T cell autophagy plays a protective role against apoptosis and immunosuppression in sepsis. PMID:27618275

  2. Targeted cytoplasmic irradiation and autophagy.

    PubMed

    Wu, Jinhua; Zhang, Bo; Wuu, Yen-Ruh; Davidson, Mercy M; Hei, Tom K

    2017-03-01

    The effect of ionizing irradiation on cytoplasmic organelles is often underestimated because the general dogma considers direct DNA damage in the nuclei to be the primary cause of radiation induced toxicity. Using a precision microbeam irradiator, we examined the changes in mitochondrial dynamics and functions triggered by targeted cytoplasmic irradiation with α-particles. Mitochondrial dysfunction induced by targeted cytoplasmic irradiation led to activation of autophagy, which degraded dysfunctional mitochondria in order to maintain cellular energy homeostasis. The activation of autophagy was cytoplasmic irradiation-specific and was not detected in nuclear irradiated cells. This autophagic process was oxyradical-dependent and required the activity of the mitochondrial fission protein dynamin related protein 1 (DRP1). The resultant mitochondrial fission induced phosphorylation of AMP activated protein kinase (AMPK) which leads to further activation of the extracellular signal-related kinase (ERK) 1/2 with concomitant inhibition of the mammalian target of rapamycin (mTOR) to initiate autophagy. Inhibition of autophagy resulted in delayed DNA damage repair and decreased cell viability, which supports the cytoprotective function of autophagy. Our results reveal a novel mechanism in which dysfunctional mitochondria are degraded by autophagy in an attempt to protect cells from toxic effects of targeted cytoplasmic radiation.

  3. Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis

    PubMed Central

    Iannaccone, Alessandro; Giorgianni, Francesco; New, David D.; Hollingsworth, T. J.; Umfress, Allison; Alhatem, Albert H.; Neeli, Indira; Lenchik, Nataliya I.; Jennings, Barbara J.; Calzada, Jorge I.; Satterfield, Suzanne; Mathews, Dennis; Diaz, Rocio I.; Harris, Tamara; Johnson, Karen C.; Charles, Steve; Kritchevsky, Stephen B.; Gerling, Ivan C.; Beranova-Giorgianni, Sarka; Radic, Marko Z.

    2015-01-01

    role of inflammation and the immune system in AMD pathogenesis, AAbs were identified in AMD sera, including early-stage disease. Identified targets may be mechanistically linked to AMD pathogenesis because the identified proteins are implicated in autophagy, immunomodulation, and protection from oxidative stress and apoptosis. In particular, a role in autophagy activation is shared by all five autoantigens, raising the possibility that the detected AAbs may play a role in AMD via autophagy compromise and downstream activation of the inflammasome. Thus, we propose that the detected AAbs provide further insight into AMD pathogenesis and have the potential to contribute to disease biogenesis and progression. PMID:26717306

  4. Recent insights into the function of autophagy in cancer

    PubMed Central

    Amaravadi, Ravi; Kimmelman, Alec C.; White, Eileen

    2016-01-01

    Macroautophagy (referred to here as autophagy) is induced by starvation to capture and degrade intracellular proteins and organelles in lysosomes, which recycles intracellular components to sustain metabolism and survival. Autophagy also plays a major homeostatic role in controlling protein and organelle quality and quantity. Dysfunctional autophagy contributes to many diseases. In cancer, autophagy can be neutral, tumor-suppressive, or tumor-promoting in different contexts. Large-scale genomic analysis of human cancers indicates that the loss or mutation of core autophagy genes is uncommon, whereas oncogenic events that activate autophagy and lysosomal biogenesis have been identified. Autophagic flux, however, is difficult to measure in human tumor samples, making functional assessment of autophagy problematic in a clinical setting. Autophagy impacts cellular metabolism, the proteome, and organelle numbers and quality, which alter cell functions in diverse ways. Moreover, autophagy influences the interaction between the tumor and the host by promoting stress adaptation and suppressing activation of innate and adaptive immune responses. Additionally, autophagy can promote a cross-talk between the tumor and the stroma, which can support tumor growth, particularly in a nutrient-limited microenvironment. Thus, the role of autophagy in cancer is determined by nutrient availability, microenvironment stress, and the presence of an immune system. Here we discuss recent developments in the role of autophagy in cancer, in particular how autophagy can promote cancer through suppressing p53 and preventing energy crisis, cell death, senescence, and an anti-tumor immune response. PMID:27664235

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

    PubMed Central

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

    2014-01-01

    A physiological sequence called autophagy qualitatively determines cellular viability by removing protein aggregates and damaged cytoplasmic constituents, and contributes significantly to the degree of myocardial ischemia-reperfusion (I/R) injury. This tightly orchestrated catabolic 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 protective mechanism or contributes to the injurious effects of I/R injury in the heart. This controversy may stem from several factors including the variability 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 manipulation of autophagy in the challenged myocardium may benefit post-infarction cardiac healing and remodeling. PMID:25593583

  6. FGF signalling regulates bone growth through autophagy.

    PubMed

    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.

  7. Histone deacetylase inhibitors induce autophagy through FOXO1-dependent pathways.

    PubMed

    Zhang, Jianbin; Ng, Shukie; Wang, Jigang; Zhou, Jing; Tan, Shi-Hao; Yang, Naidi; Lin, Qingsong; Xia, Dajing; Shen, Han-Ming

    2015-04-03

    Autophagy is a catabolic process in response to starvation or other stress conditions to sustain cellular homeostasis. At present, histone deacetylase inhibitors (HDACIs) are known to induce autophagy in cells through inhibition of mechanistic target of rapamycin (MTOR) pathway. FOXO1, an important transcription factor regulated by AKT, is also known to play a role in autophagy induction. At present, the role of FOXO1 in the HDACIs-induced autophagy has not been reported. In this study, we first observed that HDACIs increased the expression of FOXO1 at the mRNA and protein level. Second, we found that FOXO1 transcriptional activity was enhanced by HDACIs, as evidenced by increased FOXO1 nuclear accumulation and transcriptional activity. Third, suppression of FOXO1 function by siRNA knockdown or by a chemical inhibitor markedly blocked HDACIs-induced autophagy. Moreover, we found that FOXO1-mediated autophagy is achieved via its transcriptional activation, leading to a dual effect on autophagy induction: (i) enhanced expression of autophagy-related (ATG) genes, and (ii) suppression of MTOR via transcription of the SESN3 (sestrin 3) gene. Finally, we found that inhibition of autophagy markedly enhanced HDACIs-mediated cell death, indicating that autophagy serves as an important cell survival mechanism. Taken together, our studies reveal a novel function of FOXO1 in HDACIs-mediated autophagy in human cancer cells and thus support the development of a novel therapeutic strategy by combining HDACIs and autophagy inhibitors in cancer therapy.

  8. SIRT6 reduces macrophage foam cell formation by inducing autophagy and cholesterol efflux under ox-LDL condition.

    PubMed

    He, Jiangping; Zhang, Guangya; Pang, Qi; Yu, Cong; Xiong, Jie; Zhu, Jing; Chen, Fengling

    2017-03-09

    SIRT6 is a pivotal regulator of lipid metabolism. It is also closely connected to cardiovascular diseases, which are the main cause of death in diabetic patients. We observed a decrease in the expression of SIRT6 and key autophagy effectors (ATG5, LC3B, and LAMP1) in ox-LDL-induced foam cells, a special form of lipid-laden macrophages. In these cells, SIRT6 WT but not SIRT6 H133Y overexpression markedly reduced foam cell formation, as shown by Oil Red O staining, while inducing autophagy flux, as determined by both mRFP-GFP-LC3 labeling and transmission electron microscopy. Silencing the key autophagy initiation gene ATG5, reversed the autophagy-promoting effect of SIRT6 in ox-LDL-treated THP1 cells, as evidenced by an increase in foam cells. Cholesterol efflux assays indicated that SIRT6 overexpression in foam cells promoted cholesterol efflux, increased the levels of ABCA1 and ABCG1, and reduced miR-33 levels. By transfecting miR-33 into cells overexpressing SIRT6, we observed that reduced foam cell formation and autophagy flux induction were largely reversed. These data imply that SIRT6 plays an essential role in protecting against atherosclerosis by reducing foam cell formation through an autophagy-dependent pathway. This article is protected by copyright. All rights reserved.

  9. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  10. Mosaic tetracycline resistance genes encoding ribosomal protection proteins

    PubMed Central

    Warburton, Philip J.; Amodeo, Nina; Roberts, Adam P.

    2016-01-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria. PMID:27494928

  11. Autophagy in the regulation of pathogen replication and adaptive immunity

    PubMed Central

    Randow, Felix; Münz, Christian

    2012-01-01

    Autophagy is an evolutionary conserved homeostatic process by which cells deliver cytoplasmic material for degradation into lysosomes. Autophagy may have evolved as a nutrient-providing homeostatic pathway induced upon starvation, but with the acquisition of cargo-receptors autophagy has become an important cellular defence mechanism as well as a generator of antigenic peptides for MHC presentation. We propose that autophagy efficiently protects against microbes encountering the cytosolic environment accidentally, for example upon phagosomal damage, while pathogens routinely accessing the host cytosol have evolved to avoid or even benefit from autophagy. PMID:22796170

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

    SciTech Connect

    Sobhakumari, Arya; Schickling, Brandon M.; Love-Homan, Laurie; Raeburn, Ayanna; Fletcher, Elise V.M.; Case, Adam J.; Domann, Frederick E.; Miller, Francis J.; 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.

  13. Activation of autophagy by globular adiponectin attenuates ethanol-induced apoptosis in HepG2 cells: involvement of AMPK/FoxO3A axis.

    PubMed

    Nepal, Saroj; Park, Pil-Hoon

    2013-10-01

    Hepatocellular apoptosis is an important pathological entity of alcoholic liver disease. Previously, we have shown that globular adiponectin (gAcrp) protects liver cells from ethanol-induced apoptosis by modulating an array of signaling pathways. In the present study, we investigated the role of autophagy induction by gAcrp in the suppression of ethanol-induced apoptosis and its potential mechanism(s) in liver cells. Here, we demonstrated that gAcrp significantly restores ethanol-induced suppression of autophagy-related genes, including Beclin-1 and microtubule-associated protein light chain (LC3B) both in primary rat hepatocytes and human hepatoma cell line (HepG2). Globular adiponectin also restored autophagosome formation suppressed by ethanol treatment in HepG2. Furthermore, inhibition of gAcrp-induced autophagic process by knock-down of LC3B prevented protection from ethanol-induced apoptosis. In particular, the autophagic process induced by gAcrp was involved in the suppression of ethanol-induced activation of caspase-8 and expression of Bax. Moreover, knock-down of AMPK by small interfering RNA (siRNA) blocked gAcrp-induced expression of genes related to autophagy, which in turn prevented protection from ethanol-induced apoptosis, suggesting that AMPK plays an important role in the induction of autophagy and protection of liver cells by gAcrp. Finally, we also showed that gAcrp treatment induces translocation of the forkhead box O member protein, FoxO3A, into the nucleus, which may play a role in the induction of autophagy-related genes. Taken together, our data demonstrated that gAcrp protects liver cells from ethanol-induced apoptosis via induction of autophagy. Further, the AMPK-FoxO3A axis plays a cardinal role in gAcrp-induced autophagy and subsequent inhibition of ethanol-induced apoptosis.

  14. Beyond autophagy

    PubMed Central

    Liang, Chengyu; Sir, Donna; Lee, Steven; Ou, Jing-hsiung James; Jung, Jae U.

    2009-01-01

    Autophagy is a lysosome-directed membrane trafficking event for the degradation of cytoplasmic components, including organelles. The past few years have seen a great advance in our understanding of the cellular machinery of autophagosome biogenesis, the hallmark of autophagy. However, our global understanding of autophagosome maturity remains relatively poor and fragmented. The topological similarity of autophagosome and endosome delivery to lysosomes suggests that autophagic and endosomal maturation may have evolved to share associated machinery to promote the lysosomal delivery of their cargoes. We have recently discovered that UVRAG, originally identified as a Beclin 1-binding autophagy protein, appears to be an important factor in autophagic and endosomal trafficking through its interaction with the class C Vps tethering complex. Given the ability of UVRAG to bind Beclin 1 and the class C Vps complex in a genetically and functionally separable manner, it may serve as an important regulator for the spatial and/or temporal control of diverse cellular trafficking events. As more non-autophagic functions of UVRAG are unveiled, our understanding of seemingly different cellular processes may move a step further. PMID:18612260

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Functions of Autophagy in Pathological Cardiac Hypertrophy

    PubMed Central

    Li, Zhenhua; Wang, Jian; Yang, Xiao

    2015-01-01

    Pathological cardiac hypertrophy is the response of heart to various biomechanical and physiopathological stimuli, such as aging, myocardial ischemia and hypertension. However, a long-term exposure to the stress makes heart progress to heart failure. Autophagy is a dynamic self-degradative process necessary for the maintenance of cellular homeostasis. Accumulating evidence has revealed a tight link between cardiomyocyte autophagy and cardiac hypertrophy. Sophisticatedly regulated autophagy protects heart from various physiological and pathological stimuli by degradating and recycling of protein aggregates, lipid drops, or organelles. Here we review the recent progresses concerning the functions of autophagy in cardiac hypertrophy induced by various hypertrophic stimuli. Moreover, the therapeutic strategies targeting autophagy for cardiac hypertrophy will also be discussed. PMID:25999790

  17. Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway.

    PubMed

    Yang, Yue-Hua; Chen, Ke; Li, Bo; Chen, Jiang-Wei; Zheng, Xin-Feng; Wang, Yu-Ren; Jiang, Sheng-Dan; Jiang, Lei-Sheng

    2013-11-01

    Estradiol could protect osteoblast against apoptosis, and apoptosis and autophagy were extensively and intimately connected. The aim of the present study was to test the hypothesis that autophagy was present in osteoblasts under serum deprivation and estrogen protected against osteoblast apoptosis via promotion of autophagy. MC3T3-E1 osteoblastic cells were cultured in a serum-free and phenol red-free minimal essential medium (α-MEM). Ultrastructural analysis, lysosomal activity assessment and monodansycadaverine (MDC) staining were employed to determine the presence of autophagy, and real time PCR was used to evaluate the expression of autophagic markers. Meanwhile, the osteoblasts were transferred in a serum-free and phenol red-free α-MEM containing either vehicle or estradiol. Apoptosis and autophagy was assessed by using the techniques of real-time PCR, Western blot, immunofluorescence assay, and flow cytometry. The possible pathway through which estrogen promoted autophagy in the serum-deprived osteoblasts was also investigated. Real-time PCR demonstrated the expression of LC3, beclin1 and ULK1 genes in osteoblasts under serum deprivation, and immunofluorescence assay verified high expression of proteins of these three autophagic bio-markers. Lysosomes and autolysosomes accumulated in the cytoplasm of osteoblasts were also detected under transmission electron microscopy, MDC staining and lysosomal activity assessment. Meanwhile, estradiol significantly decreased the expression of proteins of the bio-markers of apoptosis, and at the same time increased the expression of proteins of the bio-markers of autophagy in the serum-deprived osteoblasts. Furthermore, the estradiol-promoted autophagy in serum-deprived osteoblasts could be blocked by estrogen receptor (ER) antagonist (ICI 182780), and estradiol failed to rescue the cells pretreated with an inhibitor of vacuolar ATPase (bafilomycin A) from apoptosis. Serum deprivation resulted in apoptosis through

  18. Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy.

    PubMed

    Ossareh-Nazari, Batool; Niño, Carlos A; Bengtson, Mario H; Lee, Joong-Won; Joazeiro, Claudio A P; Dargemont, Catherine

    2014-03-17

    Autophagy, the process by which proteins or organelles are engulfed by autophagosomes and delivered for vacuolar/lysosomal degradation, is induced to ensure survival under starvation and other stresses. A selective autophagic pathway for 60S ribosomal subunits elicited by nitrogen starvation in yeast-ribophagy-was recently described and requires the Ubp3-Bre5 deubiquitylating enzyme. This discovery implied that an E3 ligases act upstream, whether inhibiting the process or providing an initial required signal. In this paper, we show that Ltn1/Rkr1, a 60S ribosome-associated E3 implicated in translational surveillance, acts as an inhibitor of 60S ribosomal subunit ribophagy and is antagonized by Ubp3. The ribosomal protein Rpl25 is a relevant target. Its ubiquitylation is Ltn1 dependent and Ubp3 reversed, and mutation of its ubiquitylation site rendered ribophagy less dependent on Ubp3. Consistently, the expression of Ltn1-but not Ubp3-rapidly decreased after starvation, presumably to allow ribophagy to proceed. Thus, Ltn1 and Ubp3-Bre5 likely contribute to adapt ribophagy activity to both nutrient supply and protein translation.

  19. Volume-sensitive outwardly rectifying chloride channel blockers protect against high glucose-induced apoptosis of cardiomyocytes via autophagy activation.

    PubMed

    Wang, Lin; Shen, Mingzhi; Guo, Xiaowang; Wang, Bo; Xia, Yuesheng; Wang, Ning; Zhang, Qian; Jia, Lintao; Wang, Xiaoming

    2017-03-16

    Hyperglycemia is a well-characterized contributing factor for cardiac dysfunction and heart failure among diabetic patients. Apoptosis of cardiomyocytes plays a major role during the onset and pathogenesis of diabetic cardiomyopathy (DCM). Nonetheless, the molecular machinery underlying hyperglycemia-induced cardiac damage and cell death remains elusive. In the present study, we found that chloride channel blockers, 4,4'-diisothiocya-natostilbene-2,2'- disulfonic acid (DIDS) and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), inhibited high glucose-activated volume-sensitive outwardly rectifying (VSOR) Cl(-) channel and improved the viability of cardiomyocytes. High glucose induced cardiomyocyte apoptosis by suppressing the autophagic stress, which can be reversed via blockade of VSOR Cl(-) channel. VSOR activation in high glucose-treated cardiomyocytes was attributed to increased intracellular levels of reactive oxygen species (ROS). Taken together, our study unraveled a role of VSOR chloride currents in impaired autophagy and increased apoptosis of high glucose-exposed cardiomyocyte, and has implications for a therapeutic potential of VSOR chloride channel blockers in DCM.

  20. Volume-sensitive outwardly rectifying chloride channel blockers protect against high glucose-induced apoptosis of cardiomyocytes via autophagy activation

    PubMed Central

    Wang, Lin; Shen, Mingzhi; Guo, Xiaowang; Wang, Bo; Xia, Yuesheng; Wang, Ning; Zhang, Qian; Jia, Lintao; Wang, Xiaoming

    2017-01-01

    Hyperglycemia is a well-characterized contributing factor for cardiac dysfunction and heart failure among diabetic patients. Apoptosis of cardiomyocytes plays a major role during the onset and pathogenesis of diabetic cardiomyopathy (DCM). Nonetheless, the molecular machinery underlying hyperglycemia-induced cardiac damage and cell death remains elusive. In the present study, we found that chloride channel blockers, 4,4′-diisothiocya-natostilbene-2,2′- disulfonic acid (DIDS) and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), inhibited high glucose-activated volume-sensitive outwardly rectifying (VSOR) Cl− channel and improved the viability of cardiomyocytes. High glucose induced cardiomyocyte apoptosis by suppressing the autophagic stress, which can be reversed via blockade of VSOR Cl− channel. VSOR activation in high glucose-treated cardiomyocytes was attributed to increased intracellular levels of reactive oxygen species (ROS). Taken together, our study unraveled a role of VSOR chloride currents in impaired autophagy and increased apoptosis of high glucose-exposed cardiomyocyte, and has implications for a therapeutic potential of VSOR chloride channel blockers in DCM. PMID:28300155

  1. Inhibition of Atg6 and Pi3K59F autophagy genes in neurons decreases lifespan and locomotor ability in Drosophila melanogaster.

    PubMed

    M'Angale, P G; Staveley, B E

    2016-10-24

    Autophagy is a cellular mechanism implicated in the pathology of Parkinson's disease. The proteins Atg6 (Beclin 1) and Pi3K59F are involved in autophagosome formation, a key step in the initiation of autophagy. We first used the GMR-Gal4 driver to determine the effect of reducing the expression of the genes encoding these proteins on the developing Drosophila melanogaster eye. Subsequently, we inhibited their expression in D. melanogaster neurons under the direction of a Dopa decarboxylase (Ddc) transgene, and examined the effects on longevity and motor function. Decreased longevity coupled with an age-dependent loss of climbing ability was observed. In addition, we investigated the roles of these genes in the well-studied α-synuclein-induced Drosophila model of Parkinson's disease. In this context, lowered expression of Atg6 or Pi3K59F in Ddc-Gal4-expressing neurons results in decreased longevity and associated age-dependent loss of locomotor ability. Inhibition of Atg6 or Pi3K59F together with overexpression of the sole pro-survival Bcl-2 Drosophila homolog Buffy in Ddc-Gal4-expressing neurons resulted in further decrease in the survival and climbing ability of Atg6-RNAi flies, whereas these measures were ameliorated in Pi3K59F-RNAi flies.

  2. Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

    PubMed Central

    Chen, Song; Wang, Chenran; Yeo, Syn; Liang, Chun-Chi; Okamoto, Takako; Sun, Shaogang; Wen, Jian; Guan, Jun-Lin

    2016-01-01

    Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582–585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo. PMID:27013233

  3. Endocytosis and Autophagy: Exploitation or Cooperation?

    PubMed Central

    Tooze, Sharon A.; Abada, Adi; Elazar, Zvulun

    2014-01-01

    Autophagy is a lysosome-mediated degradative system that is a highly conserved pathway present in all eukaryotes. In all cells, double-membrane autophagosomes form and engulf cytoplasmic components, delivering them to the lysosome for degradation. Autophagy is essential for cell health and can be activated to function as a recycling pathway in the absence of nutrients or as a quality-control pathway to eliminate damaged organelles or even to eliminate invading pathogens. Autophagy was first identified as a pathway in mammalian cells using morphological techniques, but the Atg (autophagy-related) genes required for autophagy were identified in yeast genetic screens. Despite tremendous advances in elucidating the function of individual Atg proteins, our knowledge of how autophagosomes form and subsequently interact with the endosomal pathway has lagged behind. Recent progress toward understanding where and how both the endocytotic and autophagic pathways overlap is reviewed here. PMID:24789822

  4. Endocytosis and autophagy: exploitation or cooperation?

    PubMed

    Tooze, Sharon A; Abada, Adi; Elazar, Zvulun

    2014-05-01

    Autophagy is a lysosome-mediated degradative system that is a highly conserved pathway present in all eukaryotes. In all cells, double-membrane autophagosomes form and engulf cytoplasmic components, delivering them to the lysosome for degradation. Autophagy is essential for cell health and can be activated to function as a recycling pathway in the absence of nutrients or as a quality-control pathway to eliminate damaged organelles or even to eliminate invading pathogens. Autophagy was first identified as a pathway in mammalian cells using morphological techniques, but the Atg (autophagy-related) genes required for autophagy were identified in yeast genetic screens. Despite tremendous advances in elucidating the function of individual Atg proteins, our knowledge of how autophagosomes form and subsequently interact with the endosomal pathway has lagged behind. Recent progress toward understanding where and how both the endocytotic and autophagic pathways overlap is reviewed here.

  5. Autophagy: molecular machinery for self-eating

    PubMed Central

    Yorimitsu, T; Klionsky, DJ

    2006-01-01

    Autophagy is a highly conserved process in eukaryotes in which the cytoplasm, including excess or aberrant organelles, is sequestered into double-membrane vesicles and delivered to the degradative organelle, the lysosome/vacuole, for breakdown and eventual recycling of the resulting macromolecules. This process has an important role in various biological events such as adaptation to changing environmental conditions, cellular remodeling during development and differentiation, and determination of lifespan. Auto-phagy is also involved in preventing certain types of disease, although it may contribute to some pathologies. Recent studies have identified many components that are required to drive this complicated cellular process. Autophagy-related genes were first identified in yeast, but homologs are found in all eukaryotes. Analyses in a range of model systems have provided huge advances toward understanding the molecular basis of autophagy. Here we review our current knowledge on the machinery and molecular mechanism of autophagy. PMID:16247502

  6. Autophagy regulates sphingolipid levels in the liver.

    PubMed

    Alexaki, Aikaterini; Gupta, Sita D; Majumder, Saurav; Kono, Mari; Tuymetova, Galina; Harmon, Jeffrey M; Dunn, Teresa M; Proia, Richard L

    2014-12-01

    Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well-understood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.

  7. Metformin protects against apoptosis and senescence in nucleus pulposus cells and ameliorates disc degeneration in vivo

    PubMed Central

    Chen, Deheng; Xia, Dongdong; Pan, Zongyou; Xu, Daoliang; Zhou, Yifei; Wu, Yaosen; Cai, Ningyu; Tang, Qian; Wang, Chenggui; Yan, Meijun; Zhang, Jing Jie; Zhou, Kailiang; Wang, Quan; Feng, Yongzeng; Wang, Xiangyang; Xu, Huazi; Zhang, Xiaolei; Tian, Naifeng

    2016-01-01

    Intervertebral disc degeneration (IDD) is a complicated process that involves both cellular apoptosis and senescence. Metformin has been reported to stimulate autophagy, whereas autophagy is shown to protect against apoptosis and senescence. Therefore, we hypothesize that metformin may have therapeutic effect on IDD through autophagy stimulation. The effect of metformin on IDD was investigated both in vitro and in vivo. Our study showed that metformin attenuated cellular apoptosis and senescence induced by tert-butyl hydroperoxide in nucleus pulposus cells. Autophagy, as well as its upstream regulator AMPK, was activated by metformin in nucleus pulposus cells in a dose- and time-dependent manner. Inhibition of autophagy by 3-MA partially abolished the protective effect of metformin against nucleus pulposus cells' apoptosis and senescence, indicating that autophagy was involved in the protective effect of metformin on IDD. In addition, metformin was shown to promote the expression of anabolic genes such as Col2a1 and Acan expression while inhibiting the expression of catabolic genes such as Mmp3 and Adamts5 in nucleus pulposus cells. In vivo study illustrated that metformin treatment could ameliorate IDD in a puncture-induced rat model. Thus, our study showed that metformin could protect nucleus pulposus cells against apoptosis and senescence via autophagy stimulation and ameliorate disc degeneration in vivo, revealing its potential to be a therapeutic agent for IDD. PMID:27787519

  8. Ketogenic diet change cPLA2/clusterin and autophagy related gene expression and correlate with cognitive deficits and hippocampal MFs sprouting following neonatal seizures.

    PubMed

    Ni, Hong; Zhao, Dong-Jing; Tian, Tian

    2016-02-01

    Because the ketogenic diet (KD) was affecting expression of energy metabolism- related genes in hippocampus and because lipid membrane peroxidation and its associated autophagy stress were also found to be involved in energy depletion, we hypothesized that KD might exert its neuroprotective action via lipid membrane peroxidation and autophagic signaling. Here, we tested this hypothesis by examining the long-term expression of lipid membrane peroxidation-related cPLA2 and clusterin, its downstream autophagy marker Beclin-1, LC3 and p62, as well as its execution molecule Cathepsin-E following neonatal seizures and chronic KD treatment. On postnatal day 9 (P9), 48 Sprague-Dawley rats were randomly assigned to two groups: flurothyl-induced recurrent seizures group and control group. On P28, they were further randomly divided into the seizure group without ketogenic diet (RS+ND), seizure plus ketogenic diet (RS+KD), the control group without ketogenic diet (NS+ND), and the control plus ketogenic diet (NS+KD). Morris water maze test was performed during P37-P43. Then mossy fiber sprouting and the protein levels were detected by Timm staining and Western blot analysis, respectively. Flurothyl-induced RS+ND rats show a long-term lower amount of cPLA2 and LC3II/I, and higher amount of clusterin, Beclin-1, p62 and Cathepsin-E which are in parallel with hippocampal mossy fiber sprouting and cognitive deficits. Furthermore, chronic KD treatment (RS+KD) is effective in restoring these molecular, neuropathological and cognitive changes. The results imply that a lipid membrane peroxidation and autophagy-associated pathway is involved in the aberrant hippocampal mossy fiber sprouting and cognitive deficits following neonatal seizures, which might be a potential target of KD for the treatment of neonatal seizure-induced brain damage.

  9. Therapeutic targeting of autophagy in neurodegenerative and infectious diseases

    PubMed Central

    Bento, Carla F.

    2015-01-01

    Autophagy is a conserved process that uses double-membrane vesicles to deliver cytoplasmic contents to lysosomes for degradation. Although autophagy may impact many facets of human biology and disease, in this review we focus on the ability of autophagy to protect against certain neurodegenerative and infectious diseases. Autophagy enhances the clearance of toxic, cytoplasmic, aggregate-prone proteins and infectious agents. The beneficial roles of autophagy can now be extended to supporting cell survival and regulating inflammation. Autophagic control of inflammation is one area where autophagy may have similar benefits for both infectious and neurodegenerative diseases beyond direct removal of the pathogenic agents. Preclinical data supporting the potential therapeutic utility of autophagy modulation in such conditions is accumulating. PMID:26101267

  10. Tumor Suppression and Promotion by Autophagy

    PubMed Central

    Ávalos, Yenniffer; Canales, Jimena; Criollo, Alfredo; Quest, Andrew F. G.

    2014-01-01

    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. PMID:25328887

  11. Tumor suppression and promotion by autophagy.

    PubMed

    Ávalos, Yenniffer; Canales, Jimena; Bravo-Sagua, Roberto; Criollo, Alfredo; Lavandero, Sergio; Quest, Andrew F G

    2014-01-01

    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.

  12. Dihydroptychantol A, a macrocyclic bisbibenzyl derivative, induces autophagy and following apoptosis associated with p53 pathway in human osteosarcoma U2OS cells

    SciTech Connect

    Li Xia; Wu, William K.K.; Sun Bin; Cui Min; Liu Shanshan; Gao Jian; Lou Hongxiang

    2011-03-01

    Dihydroptychantol A (DHA), a novel macrocyclic bisbibenzyl compound extracted from liverwort Asterella angusta, has antifungal and multi-drug resistance reversal properties. Here, the chemically synthesized DHA was employed to test its anti-cancer activities in human osteosarcoma U2OS cells. Our results demonstrated that DHA induced autophagy followed by apoptotic cell death accompanied with G{sub 2}/M-phase cell cycle arrest in U2OS cells. DHA-induced autophagy was morphologically characterized by the formation of double membrane-bound autophagic vacuoles recognizable at the ultrastructural level. DHA also increased the levels of LC3-II, a marker of autophagy. Surprisingly, DHA-mediated apoptotic cell death was potentiated by the autophagy inhibitor 3-methyladenine, suggesting that autophagy may play a protective role that impedes the eventual cell death. Furthermore, p53 was shown to be involved in DHA-meditated autophagy and apoptosis. In this connection, DHA increased nuclear expression of p53, induced p53 phosphorylation, and upregulated p53 target gene p21{sup Waf1/Cip1}. In contrast, cytoplasmic p53 was reduced by DHA, which contributed to the stimulation of autophagy. In relation to the cell cycle, DHA decreased the expression of cyclin B{sub 1}, a cyclin required for progression through the G{sub 2}/M phase. Taken together, DHA induces G{sub 2}/M-phase cell cycle arrest and apoptosis in U2OS cells. DHA-induced apoptosis was preceded by the induction of protective autophagy. DHA-mediated autophagy and apoptosis are associated with the cytoplasmic and nuclear functions of p53.

  13. Inhibition of autophagy via activation of PI3K/Akt pathway contributes to the protection of ginsenoside Rb1 against neuronal death caused by ischemic insults.

    PubMed

    Luo, Tianfei; Liu, Guiying; Ma, Hongxi; Lu, Bin; Xu, Haiyang; Wang, Yujing; Wu, Jiang; Ge, Pengfei; Liang, Jianmin

    2014-09-01

    Lethal autophagy is a pathway leading to neuronal death caused by transient global ischemia. In this study, we examined the effect of Ginsenoside Rb1 (GRb1) on ischemia/reperfusion-induced autophagic neuronal death and investigated the role of PI3K/Akt. Ischemic neuronal death in vitro was induced by using oxygen glucose deprivation (OGD) in SH-SY5Y cells, and transient global ischemia was produced by using two vessels occlusion in rats. Cellular viability of SH-SY5Y cells was assessed by MTT assay, and CA1 neuronal death was evaluated by Hematoxylin-eosin staining. Autophagic vacuoles were detected by using both fluorescent microscopy in combination with acridine orange (AO) and Monodansylcadaverine (MDC) staining and transmission electronic microscopy. Protein levels of LC3II, Beclin1, total Akt and phosphor-Akt at Ser473 were examined by western blotting analysis. GRb1 inhibited both OGD and transient ischemia-induced neuronal death and mitigated OGD-induced autophagic vacuoles in SH-SY5Y cells. By contrast, PI3K inhibitor LY294002 counteracted the protection of GRb1 against neuronal death caused by either OGD or transient ischemia. LY294002 not only mitigated the up-regulated protein level of phosphor Akt at Ser473 caused by GRb1, but also reversed the inhibitory effect of GRb1 on OGD and transient ischemia-induced elevation in protein levels of LC3II and Beclin1.

  14. Autophagy in lung disease pathogenesis and therapeutics.

    PubMed

    Ryter, Stefan W; Choi, Augustine M K

    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, and immune system functions. Selective pathways for autophagic degradation of cargoes may have variable significance in disease pathogenesis. Among these, the autophagic clearance of bacteria (xenophagy) may represent a crucial host defense mechanism in the pathogenesis of sepsis and inflammatory diseases. Our recent studies indicate that the autophagic clearance of mitochondria, a potentially protective program, may aggravate the pathogenesis of chronic obstructive pulmonary disease by activating cell death programs. We report similar findings with respect to the autophagic clearance of cilia components, which can contribute to airways dysfunction in chronic lung disease. In certain diseases such as pulmonary hypertension, autophagy may confer protection by modulating proliferation and cell death. In other disorders, such as idiopathic pulmonary fibrosis and cystic fibrosis, impaired autophagy may contribute to pathogenesis. In lung cancer, autophagy has multiple consequences by limiting carcinogenesis, modulating therapeutic effectiveness, and promoting tumor cell survival. In this review we highlight the multiple functions of autophagy and its selective autophagy subtypes that may be of significance to the pathogenesis of human disease, with an emphasis on lung disease and therapeutics.

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

    PubMed

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

    2014-04-04

    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.

  16. Effect of 1,25-dihydroxyvitamin D3 on the expression of mannose receptor, DC-SIGN and autophagy genes in pulmonary tuberculosis.

    PubMed

    Afsal, K; Selvaraj, P

    2016-07-01

    1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a powerful immuno-modulator, which enhances expression of antimicrobial peptides and induces autophagy in monocytes/macrophages. Since 1,25(OH)2D3 increases the phagocytic potential of monocytes/macrophages, we have explored the effect of 1,25(OH)2D3 on the expression of receptors such as mannose receptor (CD206) and DC-SIGN (CD209) as well as autophagy genes such as ATG5 and Beclin-1 (BECN1) in monocytes/macrophages of healthy controls (HCs) and pulmonary tuberculosis (PTB) patients with and without cavitary disease. Peripheral blood mononuclear cells (PBMCs) were isolated from 40 HCs and 40 PTB patients and were cultured for 72 h with Mtb in the presence or absence of 1,25(OH)2D3 at 10(-7) M concentration. 1,25(OH)2D3 significantly upregulated the expression of mannose receptor, ATG5 and BECN1; whereas DC-SIGN expression was suppressed in Mtb infected cells of both study groups (p < 0.05). The 1,25(OH)2D3-induced expression of CD206, ATG5 and BECN1 genes was lower in PTB patients compared to HCs, whereas expression of these genes was impaired in PTB patients with cavitary disease. Moreover, the relative expression of ATG5 and BECN1 was positively correlated with monocyte/macrophage phagocytosis and cathelicidin antimicrobial peptide gene expression in HCs and PTB patients (p < 0.05). Our study results suggest that vitamin D supplementation in PTB patients without cavitary disease could enhance innate immune functions and may help to control intracellular growth of mycobacteria in macrophages.

  17. Transcriptional regulation of autophagy by an FXR/CREB axis

    PubMed Central

    Seok, Sunmi; Fu, Ting; Choi, Sung-E; Li, Yang; Zhu, Rong; Kumar, Subodh; Sun, Xiaoxiao; Yoon, Gyesoon; Kang, Yup; Zhong, Wenxuan; Ma, Jian; Kemper, Byron; Kemper, Jongsook Kim

    2014-01-01

    Lysosomal degradation of cytoplasmic components by autophagy is essential for cellular survival and homeostasis under nutrient-deprived conditions1–4. Acute regulation of autophagy by nutrient-sensing kinases is well defined3, 5–7, but longer-term transcriptional regulation is relatively unknown. Here we show that the fed-state sensing nuclear receptor FXR8, 9 and the fasting transcriptional activator CREB10, 11 coordinately regulate the hepatic autophagy gene network. Pharmacological activation of FXR repressed many autophagy genes and inhibited autophagy even in fasted mice and feeding-mediated inhibition of macroautophagy was attenuated in FXR-knockout mice. From mouse liver ChIP-seq data12–15, FXR and CREB binding peaks were detected at 178 and 112, respectively, of 230 autophagy-related genes, and 78 genes showed shared binding, mostly in their promoter regions. CREB promoted lipophagy, autophagic degradation of lipids16, under nutrient-deprived conditions, and FXR inhibited this response. Mechanistically, CREB upregulated autophagy genes, including Atg7, Ulk1, and Tfeb, by recruiting the coactivator CRTC2. After feeding or pharmacological activation, FXR trans-repressed these genes by disrupting the functional CREB/CRTC2 complex. This study identifies the novel FXR/CREB axis as a key physiological switch regulating autophagy, resulting in sustained nutrient regulation of autophagy during feeding/fasting cycles. PMID:25383523

  18. Reduced autophagy in livers of fasted, fat-depleted, ghrelin-deficient mice: Reversal by growth hormone

    PubMed Central

    Zhang, Yuanyuan; Fang, Fei; Goldstein, Joseph L.; Brown, Michael S.; Zhao, Tong-Jin

    2015-01-01

    Plasma growth hormone (GH) and hepatic autophagy each have been reported to protect against hypoglycemia in the fasted state, but previous data have not linked the two. Here we demonstrate a connection using a mouse model of fasting in a fat-depleted state. Mice were subjected to 1 wk of 60% calorie restriction, causing them to lose nearly all body fat. They were then fasted for 23 h. During fasting, WT mice developed massive increases in plasma GH and a concomitant increase in hepatic autophagy, allowing them to maintain viable levels of blood glucose. In contrast, lethal hypoglycemia occurred in mice deficient in the GH secretagogue ghrelin as a result of knockout of the gene encoding ghrelin O-acyltransferase (GOAT), which catalyzes a required acylation of the peptide. Fasting fat-depleted Goat−/− mice showed a blunted increase in GH and a marked decrease in hepatic autophagy. Restoration of GH by infusion during the week of calorie restriction maintained autophagy in the Goat−/− mice and prevented lethal hypoglycemia. Acute injections of GH after 7 d of calorie restriction also restored hepatic autophagy, but failed to increase blood glucose, perhaps owing to ATP deficiency in the liver. These data indicate that GH stimulation of autophagy is necessary over the long term, but not sufficient over the short term to maintain blood glucose levels in fasted, fat-depleted mice. PMID:25583513

  19. Autophagy in ethanol-exposed liver disease.

    PubMed

    Wang, Li-Ren; Zhu, Gui-Qi; Shi, Ke-Qing; Braddock, Martin; Zheng, Ming-Hua

    2015-01-01

    Ethanol metabolism in hepatocytes causes the generation of reactive oxygen species, endoplasmic reticulum stress and alterations in mitochondrial energy and REDOX metabolism. In ethanol-exposed liver disease, autophagy not only acts as a cleanser to remove damaged organelles and cytosolic components, but also selectively clears specific targets such as lipid droplets and damaged mitochondria. Moreover, ethanol appears to play a role in protecting hepatocytes from apoptosis at certain concentrations. This article describes the evidence, function and potential mechanism of autophagy in ethanol-exposed liver disease and the controversy surrounding the effects of ethanol on autophagy.

  20. Long Non-coding RNA H19 Induces Cerebral Ischemia Reperfusion Injury via Activation of Autophagy

    PubMed Central

    Wang, Jue; Cao, Bin; Han, Dong; Sun, Miao; Feng, Juan

    2017-01-01

    Long non-coding RNA H19 (lncRNA H19) was found to be upregulated by hypoxia, its expression and function have never been tested in cerebral ischemia and reperfusion (I/R) injury. This study intended to investigate the role of lncRNA H19 and H19 gene variation in cerebral I/R injury with focusing on its relationship with autophagy activation. Cerebral I/R was induced in rats by middle cerebral artery occlusion followed by reperfusion. SH-SY5Y cells were subjected to oxygen and glucose deprivation and reperfusion (OGD/R) to simulate I/R injury. Real-time PCR, flow cytometry, immunofluorescence and Western blot were used to evaluate the level of lncRNA H19, apoptosis, autophagy and some related proteins. The modified multiple ligase reaction was used to analyze the gene polymorphism of six SNPs in H19, rs217727, rs2067051, rs2251375, rs492994, rs2839698 and rs10732516 in ischemic stroke patients. We found that the expression of lncRNA H19 was upregulated by cerebral I/R in rats, as well as by OGD/R in vitro in the cells. Inhibition of lncRNA H19 and autophagy protected cells from OGD/R-induced death, respectively. Autophagy activation induced by OGD/R was prevented by H19 siRNA. Autophagy inducer, rapamycin, abolished lncRNA H19 effect. Furthermore, we found that lncRNA H19 inhibited autophagy through DUSP5-ERK1/2 axis. The result from blood samples of ischemic patients revealed that the variation of H19 gene increased the risk of ischemic stroke. Taken together, the results of present study suggest that LncRNA H19 could be a new therapeutic target of ischemic stroke. PMID:28203482

  1. Long Non-coding RNA H19 Induces Cerebral Ischemia Reperfusion Injury via Activation of Autophagy.

    PubMed

    Wang, Jue; Cao, Bin; Han, Dong; Sun, Miao; Feng, Juan

    2017-02-01

    Long non-coding RNA H19 (lncRNA H19) was found to be upregulated by hypoxia, its expression and function have never been tested in cerebral ischemia and reperfusion (I/R) injury. This study intended to investigate the role of lncRNA H19 and H19 gene variation in cerebral I/R injury with focusing on its relationship with autophagy activation. Cerebral I/R was induced in rats by middle cerebral artery occlusion followed by reperfusion. SH-SY5Y cells were subjected to oxygen and glucose deprivation and reperfusion (OGD/R) to simulate I/R injury. Real-time PCR, flow cytometry, immunofluorescence and Western blot were used to evaluate the level of lncRNA H19, apoptosis, autophagy and some related proteins. The modified multiple ligase reaction was used to analyze the gene polymorphism of six SNPs in H19, rs217727, rs2067051, rs2251375, rs492994, rs2839698 and rs10732516 in ischemic stroke patients. We found that the expression of lncRNA H19 was upregulated by cerebral I/R in rats, as well as by OGD/R in vitro in the cells. Inhibition of lncRNA H19 and autophagy protected cells from OGD/R-induced death, respectively. Autophagy activation induced by OGD/R was prevented by H19 siRNA. Autophagy inducer, rapamycin, abolished lncRNA H19 effect. Furthermore, we found that lncRNA H19 inhibited autophagy through DUSP5-ERK1/2 axis. The result from blood samples of ischemic patients revealed that the variation of H19 gene increased the risk of ischemic stroke. Taken together, the results of present study suggest that LncRNA H19 could be a new therapeutic target of ischemic stroke.

  2. Autophagy-related prognostic signature for breast cancer.

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25906080

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

    PubMed

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

    2015-04-03

    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.

  5. Ribozyme genes protecting transgenic melon plants against potyviruses.

    PubMed

    Huttner, E; Tucker, W; Vermeulen, A; Ignart, F; Sawyer, B; Birch, R

    2001-04-01

    Potyviruses are the most important viral pathogens of crops worldwide. Under a contract with Gene Shears Pty Limited, we are using ribozyme genes to protect melon plants against two potyviruses: WMV2 and ZYMV. Different polyribozyme genes were designed, built and introduced into melons plants. Transgenic melon plants containing a resistance gene were obtained and their progeny was challenged by the appropriate virus. Most of the genes tested conferred some degree of resistance to the viruses in glasshouse trials. Melon plants from one family containing one gene directed against WMV2 were also field-trialed on small plots under natural infection pressure and were found immune to WMV2. Field trial is in progress for plants containing genes against ZYMV. Some of the ribozyme genes used in the plants were also assayed in a transient expression system in tobacco cells. This enabled us to study the sequence discrimination capacity of the ribozyme in the case of one ribozyme target site. We found that a mutated target GUG (non cleavable) was less susceptible to inhibition by the ribozyme gene than the corresponding wild type target GUA (cleavable). Work is now in progress to incorporate multiple resistance genes in melon plants, in constructs designed in compliance with the evolving European regulations concerning transgenic plants. The use of ribozyme genes to protect plants against viruses provides an alternative to the technologies currently used for protecting crops against viruses, based on the concept of Pathogen Derived Resistance (see for example 14). In the light of concerns expressed by some plant virologists (13) about the use of viral genes in transgenic plants, it may be that ribozyme genes will find many uses in this area of agricultural biotechnology.

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

    SciTech Connect

    Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo; Koya, Daisuke

    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

  7. Autophagy Is a Protective Response to the Oxidative Damage to Endplate Chondrocytes in Intervertebral Disc: Implications for the Treatment of Degenerative Lumbar Disc

    PubMed Central

    Yu, Fei; Ma, Junxuan

    2017-01-01

    Low back pain (LBP) is the leading cause of disability in the elderly. Intervertebral disc degeneration (IDD) was considered as the main cause for LBP. Degeneration of cartilaginous endplate was a crucial harmful factor during the initiation and development of IDD. Oxidative stress was implicated in IDD. However, the underlying molecular mechanism for the degeneration of cartilaginous endplate remains elusive. Herein, we found that oxidative stress could induce apoptosis and autophagy in endplate chondrocytes evidenced by western blot analysis, flow cytometry, immunofluorescence staining, GFP-LC3B transfection, and MDC staining. In addition, we also found that the apoptosis of endplate chondrocytes was significantly increased after the inhibition of autophagy by bafilomycin A1 shown by flow cytometry. Furthermore, mTOR pathway upstream autophagy was greatly suppressed suggested by western blot assay. In conclusion, our study strongly revealed that oxidative stress could increase autophagy and apoptosis of endplate chondrocytes in intervertebral disc. The increase of autophagy activity could prevent endplate chondrocytes from apoptosis. The autophagy in endplate chondrocytes induced by oxidative stress was mTOR dependent. These findings might shed some new lights on the mechanism for IDD and provide new strategies for the treatments of IDD. PMID:28321270

  8. Regulation of autophagy by the inositol trisphosphate receptor.

    PubMed

    Criollo, A; Maiuri, M C; Tasdemir, E; Vitale, I; Fiebig, A A; Andrews, D; Molgó, J; Díaz, J; Lavandero, S; Harper, F; Pierron, G; di Stefano, D; Rizzuto, R; Szabadkai, G; Kroemer, G

    2007-05-01

    The reduction of intracellular 1,4,5-inositol trisphosphate (IP(3)) levels stimulates autophagy, whereas the enhancement of IP(3) levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP(3) receptor (IP(3)R) with small interfering RNAs and pharmacological IP(3)R blockade is a strong stimulus for the induction of autophagy. The IP(3)R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER-mitochondrial contact sites, and IP(3)R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP(3)R blockade was inhibited by Bcl-2 and Bcl-X(L) specifically targeted to ER but not Bcl-2 or Bcl-X(L) proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-X(L). Autophagy promoted by IP(3)R inhibition could not be attributed to a modulation of steady-state Ca(2+) levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP(3)R exerts a major role in the physiological control of autophagy.

  9. 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

    PubMed Central

    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

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

    PubMed

    Morselli, Eugenia; Mariño, Guillermo; Bennetzen, Martin V; Eisenberg, Tobias; Megalou, Evgenia; Schroeder, Sabrina; Cabrera, Sandra; Bénit, Paule; Rustin, Pierre; Criollo, Alfredo; Kepp, Oliver; Galluzzi, Lorenzo; Shen, Shensi; Malik, Shoaib Ahmad; Maiuri, Maria Chiara; Horio, Yoshiyuki; López-Otín, Carlos; Andersen, Jens S; Tavernarakis, Nektarios; Madeo, Frank; Kroemer, Guido

    2011-02-21

    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 the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.

  11. Autophagy mediates degradation of nuclear lamina

    PubMed Central

    Dou, Zhixun; Xu, Caiyue; Donahue, Greg; Shimi, Takeshi; Pan, Ji-An; Zhu, Jiajun; Ivanov, Andrejs; Capell, Brian C.; Drake, Adam M.; Shah, Parisha P.; Catanzaro, Joseph M.; Ricketts, M. Daniel; Lamark, Trond; Adam, Stephen A.; Marmorstein, Ronen; Zong, Wei-Xing; Johansen, Terje; Goldman, Robert D.; Adams, Peter D.; Berger, Shelley L.

    2015-01-01

    Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents, and is associated with human diseases1–3. While extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known regarding the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery4–6, is present in the nucleus and directly interacts with the nuclear lamina protein Lamin B1, and binds to lamin-associated domains (LADs) on chromatin. This LC3-Lamin B1 interaction does not downregulate Lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated Ras. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers Lamin B1 to the lysosome. Inhibiting autophagy or the LC3-Lamin B1 interaction prevents activated Ras-induced Lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests this new function of autophagy as a guarding mechanism protecting cells from tumorigenesis. PMID:26524528

  12. Autophagy mediates degradation of nuclear lamina.

    PubMed

    Dou, Zhixun; Xu, Caiyue; Donahue, Greg; Shimi, Takeshi; Pan, Ji-An; Zhu, Jiajun; Ivanov, Andrejs; Capell, Brian C; Drake, Adam M; Shah, Parisha P; Catanzaro, Joseph M; Ricketts, M Daniel; Lamark, Trond; Adam, Stephen A; Marmorstein, Ronen; Zong, Wei-Xing; Johansen, Terje; Goldman, Robert D; Adams, Peter D; Berger, Shelley L

    2015-11-05

    Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.

  13. Autophagy: from basic research to its application in food biotechnology.

    PubMed

    Cebollero, Eduardo; Gonzalez, Ramon

    2007-01-01

    Autophagy is a catabolic process by which the cytoplasm is sequestered into double-membrane vesicles and delivered to the lysosome/vacuole for breaking down and recycling of the low molecular weight degradation products. The isolation in the yeast Saccharomyces cerevisiae of many of the genes involved in autophagy constituted a milestone in understanding the molecular bases of this pathway. The identification of ortholog genes in other eukaryotic models revealed that the mechanism of autophagy is conserved among all eukaryotes. This pathway has been shown to be involved in a growing number of physiological processes and conversely, its deregulation may contribute to the development of several diseases. Recent reports have also shown that autophagy may play an important role in biotechnological processes related with the food industry. In this review we discuss current knowledge of the molecular mechanism of autophagy, including some applied aspects of autophagy in the field of food biotechnology.

  14. Nucleotide Sequence of the Protective Antigen Gene of Bacillus Anthracis

    DTIC Science & Technology

    1988-02-02

    transcription and translation of the Bacillus megaterium protein C gene. J. Bacteriol. 158:e09-813. 9. Friedlander, A, M. 1986. Macrophages are sensitive to...of the Protective Antigen Gene of Bacillus anthracis 6. pEaltranalO opl. AMPOA’T B*u~iA S. L. Welkos, J. R. Lowe, F. Eden-McCutchan, M. Vodkin, S. M... Bacillus anthracls and the 5’ and 3’ flanking sequences were determined. Protective antigen ie one of three proteins comprising anthrax toxin. The open

  15. On the role of autophagy in human diseases: a gender perspective

    PubMed Central

    Lista, Pasquale; Straface, Elisabetta; Brunelleschi, Sandra; Franconi, Flavia; Malorni, Walter

    2011-01-01

    Abstract Cytopathological features of cells from males and females, i.e. XX and XY isolated cells, have been demonstrated to represent a key variable in the mechanism underlying gender disparity in human diseases. Major insights came from the studies of gender differences in cell fate, e.g. in apoptotic susceptibility. We report here some novel insights recently emerged from literature that are referred as to a cytoprotection mechanism by which cells recycle cytoplasm and dispose of excess or defective organelles, i.e. autophagy. Autophagy and related genes have first been identified in yeast. Orthologue genes have subsequently been found in other organisms, including human beings. This stimulated the research in the field and, thanks to the use of molecular genetics and cell biology in different model systems, autophagy gained the attention of several research groups operating to analyse the pathogenetic mechanisms of human diseases. It remains unclear, however, whether autophagy can exert a protective effect or instead contribute to the pathogenesis of important human diseases. On the basis of the growing importance of sex/gender as key determinant of human pathology and of the known differences between males and females in the onset, progression, drug susceptibility and outcome of a plethora of diseases, the idea that autophagy could represent key and critical factor should be taken into account. In the review, we summarize our current knowledge about the role of autophagy in some paradigmatic human diseases (cancer, neurodegenerative, autoimmune, cardiovascular) and the role of ‘cell sex’ differences in this context. PMID:21362130

  16. Neferine attenuates the protein level and toxicity of mutant huntingtin in PC-12 cells via induction of autophagy.

    PubMed

    Wong, Vincent Kam Wai; Wu, An Guo; Wang, Jing Rong; Liu, Liang; Law, Betty Yuen-Kwan

    2015-02-18

    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.

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

    PubMed

    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.

  18. A systems biology approach to learning autophagy.

    PubMed

    Klionsky, Daniel J; Kumar, Anuj

    2006-01-01

    With its relevance to our understanding of eukaryotic cell function in the normal and disease state, autophagy is an important topic in modern cell biology; yet, few textbooks discuss autophagy beyond a two- or three-sentence summary. Here, we report an undergraduate/graduate class lesson for the in-depth presentation of autophagy using an active learning approach. By our method, students will work in small groups to solve problems and interpret an actual data set describing genes involved in autophagy. The problem-solving exercises and data set analysis will instill within the students a much greater understanding of the autophagy pathway than can be achieved by simple rote memorization of lecture materials; furthermore, the students will gain a general appreciation of the process by which data are interpreted and eventually formed into an understanding of a given pathway. As the data sets used in these class lessons are largely genomic and complementary in content, students will also understand first-hand the advantage of an integrative or systems biology study: No single data set can be used to define the pathway in full-the information from multiple complementary studies must be integrated in order to recapitulate our present understanding of the pathways mediating autophagy. In total, our teaching methodology offers an effective presentation of autophagy as well as a general template for the discussion of nearly any signaling pathway within the eukaryotic kingdom.

  19. Guidelines for monitoring autophagy in Caenorhabditis elegans.

    PubMed

    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.

  20. Guidelines for monitoring autophagy in Caenorhabditis elegans

    PubMed Central

    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. PMID:25569839

  1. Role of Autophagy in the Maintenance of Intestinal Homeostasis

    PubMed Central

    Baxt, Leigh A.; Xavier, Ramnik J.

    2015-01-01

    Genome-wide association studies of inflammatory bowel disease have identified several risk loci in genes that regulate autophagy, and studies have provided insight into the functional effects of these polymorphisms. We review the mechanisms by which autophagy contributes to intestinal homeostasis, focusing on its cell type-specific roles in regulating gut ecology, restricting pathogenic bacteria, and controlling inflammation. Based on this information, we are beginning to understand how alterations in autophagy can contribute to intestinal inflammation. PMID:26170139

  2. Autophagy in liver diseases.

    PubMed

    Rautou, Pierre-Emmanuel; Mansouri, Abdellah; Lebrec, Didier; Durand, François; Valla, Dominique; Moreau, Richard

    2010-12-01

    Autophagy, or cellular self-digestion, is a cellular pathway crucial for development, differentiation, survival, and homeostasis. Its implication in human diseases has been highlighted during the last decade. Recent data show that autophagy is involved in major fields of hepatology. In liver ischemia reperfusion injury, autophagy mainly has a prosurvival activity allowing the cell for coping with nutrient starvation and anoxia. During hepatitis B or C infection, autophagy is also increased but subverted by viruses for their own benefit. In hepatocellular carcinoma, the autophagy level is decreased. In this context, autophagy has an anti-tumor role and therapeutic strategies increasing autophagy, as rapamycin, have a beneficial effect in patients. Moreover, in hepatocellular carcinoma, Beclin-1 level, an autophagy protein, has a prognostic significance. In α-1-antitrypsin deficiency, the aggregation-prone ATZ protein accumulates in the endoplasmic reticulum. This activates the autophagic response which aims at degrading mutant ATZ. Some FDA-approved drugs which enhance autophagy and the disposal of aggregation-prone proteins may be useful in α-1-antitrypsin deficiency. Following alcohol consumption, autophagy is decreased in liver cells, likely due to a decrease in intracellular 5'-AMP-activated protein kinase (AMPk) and due to an alteration in vesicle transport in hepatocytes. This decrease in autophagy contributes to the formation of Mallory-Denk bodies and to liver cell death. Hepatic autophagy is defective in the liver in obesity and its upregulation improves insulin sensitivity.

  3. Measurement of autophagy flux in the nervous system in vivo

    PubMed Central

    Castillo, K; Valenzuela, V; Matus, S; Nassif, M; Oñate, M; Fuentealba, Y; Encina, G; Irrazabal, T; Parsons, G; Court, F A; Schneider, B L; Armentano, D; Hetz, C

    2013-01-01

    Accurate methods to measure autophagic activity in vivo in neurons are not available, and most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations. Autophagy is an essential homeostatic process involved in the degradation of diverse cellular components including organelles and protein aggregates. Autophagy impairment is emerging as a relevant factor driving neurodegeneration in many diseases. Moreover, strategies to modulate autophagy have been shown to provide protection against neurodegeneration. Here we describe a novel and simple strategy to express an autophagy flux reporter in the nervous system of adult animals by the intraventricular delivery of adeno-associated viruses (AAV) into newborn mice. Using this approach we efficiently expressed a monomeric tandem mCherry-GFP-LC3 construct in neurons of the peripheral and central nervous system, allowing the measurement of autophagy activity in pharmacological and disease settings. PMID:24232093

  4. New roles for autophagy and spermidine in T cells

    PubMed Central

    Puleston, D. J.; Simon, A. K.

    2015-01-01

    The conserved lysosomal degradation pathway autophagy is now recognised as an essential cog in immune function. While functionally widespread in the innate immune system, knowledge of its roles in adaptive immunity is more limited. Although autophagy has been implicated in naïve T cell homeostasis, its requirement in antigen-specific T cells during infection was unknown. Using a murine model where the essential autophagy gene Atg7 is deleted in the T cell lineage, we have shown that autophagy is dispensable for effector CD8+ T cell responses, but crucial for the formation of memory CD8+ T cells. Here, we suggest reasons why autophagy might be important for the formation of long-lasting immunity. Like in the absence of autophagy, T cell memory formation during ageing is also defective. We observed diminished autophagy levels in T cells from aged mice, linking autophagy to immunosenescence. Importantly, T cell responses to influenza vaccination could be significantly improved using the autophagy-inducing compound spermidine. These results suggest the autophagy pathway as a desirable target to improve aged immunity and modulate T cell function. PMID:28357282

  5. Autophagy in the liver: functions in health and disease.

    PubMed

    Ueno, Takashi; Komatsu, Masaaki

    2017-03-01

    The concept of macroautophagy was established in 1963, soon after the discovery of lysosomes in rat liver. Over the 50 years since, studies of liver autophagy have produced many important findings. The liver is rich in lysosomes and possesses high levels of metabolic-stress-induced autophagy, which is precisely regulated by concentrations of hormones and amino acids. Liver autophagy provides starved cells with amino acids, glucose and free fatty acids for use in energy production and synthesis of new macromolecules, and also controls the quality and quantity of organelles such as mitochondria. Although the efforts of early investigators contributed markedly to our current knowledge of autophagy, the identification of autophagy-related genes represented a revolutionary breakthrough in our understanding of the physiological roles of autophagy in the liver. A growing body of evidence has shown that liver autophagy contributes to basic hepatic functions, including glycogenolysis, gluconeogenesis and β-oxidation, through selective turnover of specific cargos controlled by a series of transcription factors. In this Review, we outline the history of liver autophagy study, and then describe the roles of autophagy in hepatic metabolism under healthy and disease conditions, including the involvement of autophagy in α1-antitrypsin deficiency, NAFLD, hepatocellular carcinoma and viral hepatitis.

  6. The V471A Polymorphism in Autophagy-Related Gene ATG7 Modifies Age at Onset Specifically in Italian Huntington Disease Patients

    PubMed Central

    Metzger, Silke; Walter, Carolin; Riess, Olaf; Roos, Raymund A. C.; Nielsen, Jørgen E.; Craufurd, David; Nguyen, Huu Phuc

    2013-01-01

    The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently, 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 independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the “REGISTRY” cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients). In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis. PMID:23894380

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

    PubMed

    Metzger, Silke; Walter, Carolin; Riess, Olaf; Roos, Raymund A C; Nielsen, Jørgen E; Craufurd, David; Nguyen, Huu Phuc

    2013-01-01

    The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently, 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 independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the "REGISTRY" cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients). In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis.

  8. Autophagy in freshwater planarians.

    PubMed

    González-Estévez, Cristina

    2008-01-01

    Planarians provide a new and emergent in vivo model organism to study autophagy. On the whole, maintaining the normal homeostatic balance in planarians requires continuous dynamic adjustment of many processes, including proliferation, apoptosis, differentiation, and autophagy. This makes them very different from other models where autophagy only occurs at very specific times and/or in very specific organs. This chapter aims to offer a general vision of planarians as a model organism, placing more emphasis on those characteristics related to autophagy and describing how autophagy fits into the processes of body remodeling during regeneration and starvation. We also define exactly what is known about autophagy in these organisms and we discuss the techniques available to study the relevant processes, as well as the techniques that are currently being developed. As such, this chapter will serve as a compilation of the techniques available to investigate autophagy in planarians.

  9. [Autophagy in liver diseases].

    PubMed

    Harada, Masaru

    2011-12-01

    Two major degradation systems exist in cells: the lysosome and proteasome. In the lysosome system, extracellular materials are degraded via endocytosis. Intracellular materials are degraded by autophagy, a cellular pathway crucial for various intracellular events. It has recently been demonstrated that autophagy is involved in the pathogenesis of various liver diseases. In hepatitis B or hepatitis C virus infection, autophagy is enhanced in hepatocytes. In hepatic steatosis, hepatocyte autophagy is inhibited. The expression of the autophagy protein is disrupted in hepatocellular carcinoma. I summarize recent advances in the study of the involvement of autophagy in various liver diseases. The regulation of autophagy in the liver may be a useful therapeutic strategy for various liver diseases.

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

    PubMed

    Bernard, Amélie; Klionsky, Daniel J

    2015-04-03

    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.

  11. Rubicon deficiency enhances cardiac autophagy and protects mice from lipopolysaccharide-induced lethality and reduction in stroke volume.

    PubMed

    Zi, Zhenguo; Song, Zongpei; Zhang, Shasha; Ye, Yong; Li, Can; Xu, Mingqing; Zou, Yunzeng; He, Lin; Zhu, Hongxin

    2015-03-01

    : Rubicon has been suggested to suppress autophagosome maturation by negatively regulating PI3KC3/Vps34 activity. However, the physiological function of Rubicon remains elusive. We hypothesized that Rubicon deficiency enhances autophagic flux in the heart and affects cardiac function. Rubicon knockout (KO) mice were generated by piggyBac transposition. Loss of Rubicon was demonstrated at both mRNA and protein levels. Rubicon KO mice were born in Mendelian ratios. Autophagic flux, assessed by bafilomycin A1-induced changes in LC3 II protein abundance, was enhanced in the heart of Rubicon KO mice compared with wild-type (WT) controls. Hematoxylin-eosin staining and picrosirius red staining showed that Rubicon KO mice exhibited normal baseline cardiac morphology. Echocardiography revealed that ejection fraction and fractional shortening, 2 indices of cardiac function, were comparable between Rubicon KO mice at 2, 8, and 12 months of age (n = 6-8 for each age group) and the corresponding WT controls (n = 6-8 for each age group). In a mouse model of lipopolysaccharide (LPS)-induced sepsis, the survival time of LPS-treated Rubicon KO mice (n = 10) was prolonged compared with LPS-treated WT controls (n = 11). Echocardiography revealed that Rubicon deficiency partially normalized LPS-induced reduction in stroke volume and cardiac output 12 hours after LPS administration compared with LPS-treated WT controls (n = 6 for each group). Autophagic flux was enhanced in Rubicon-deficient hearts 12 hours after LPS treatment compared with LPS-treated WT controls. Real-time quantitative polymerase chain reaction suggested that proinflammatory cytokine expression was not significantly different between LPS-treated Rubicon KO mice and WT controls (n = 3 for each group). Our data demonstrate for the first time that Rubicon deficiency enhances autophagic flux in the heart and protects mice from lethality and reduction in stroke volume induced by LPS.

  12. Strange bedfellows expose ancient secrets of autophagy in immunity.

    PubMed

    Deretic, Vojo

    2009-04-17

    Autophagy has many roles in immunity, including the control of intracellular microbes by a cell-autonomous mechanism. In this issue of Immunity, Shelly et al. (2009) use VSV infection in Drosophila to show the role of autophagy genes in controlling viruses.

  13. Systems biology of the autophagy-lysosomal pathway.

    PubMed

    Jegga, Anil G; Schneider, Lonnie; Ouyang, Xiaosen; Zhang, Jianhua

    2011-05-01

    The mechanisms of the control and activity of the autophagy-lysosomal protein degradation machinery are emerging as an important theme for neurodevelopment and neurodegeneration. However, the underlying regulatory and functional networks of known genes controlling autophagy and lysosomal function and their role in disease are relatively unexplored. We performed a systems biology-based integrative computational analysis to study the interactions between molecular components and to develop models for regulation and function of genes involved in autophagy and lysosomal function. Specifically, we analyzed transcriptional and microRNA-based post-transcriptional regulation of these genes and performed functional enrichment analyses to understand their involvement in nervous system-related diseases and phenotypes. Transcriptional regulatory network analysis showed that binding sites for transcription factors, SREBP1, USF, AP-1 and NFE2, are common among autophagy and lysosomal genes. MicroRNA enrichment analysis revealed miR-130, 98, 124, 204 and 142 as the putative post-transcriptional regulators of the autophagy-lysosomal pathway genes. Pathway enrichment analyses revealed that the mTOR and insulin signaling pathways are important in the regulation of genes involved in autophagy. In addition, we found that glycosaminoglycan and glycosphingolipid pathways also make a major contribution to lysosomal gene regulation. The analysis confirmed the known contribution of the autophagy-lysosomal genes to Alzheimer and Parkinson diseases and also revealed potential involvement in tuberous sclerosis, neuronal ceroidlipofuscinoses, sepsis and lung, liver and prostatic neoplasms. To further probe the impact of autophagy-lysosomal gene deficits on neurologically-linked phenotypes, we also mined the mouse knockout phenotype data for the autophagylysosomal genes and found them to be highly predictive of nervous system dysfunction. Overall this study demonstrates the utility of systems

  14. AEG-1/MTDH-activated autophagy enhances human malignant glioma susceptibility to TGF-β1-triggered epithelial-mesenchymal transition

    PubMed Central

    Zou, Meijuan; Zhu, Wei; Wang, Li; Shi, Lei; Gao, Rui; Ou, Yingwei; Chen, Xuguan; Wang, Zhongchang; Jiang, Aiqin; Liu, Kunmei; Xiao, Ming; Ni, Ping; Wu, Dandan; He, Wenping; Sun, Geng; Li, Ping; Zhai, Sulan; Wang, Xuerong; Hu, Gang

    2016-01-01

    Autophagy is a tightly regulated process activated in response to metabolic stress and other microenvironmental changes. Astrocyte elevated gene 1 (AEG-1) reportedly induces protective autophagy. Our results indicate that AEG-1 also enhances the susceptibility of malignant glioma cells to TGF-β1-triggered epithelial-mesenchymal transition (EMT) through induction of autophagy. TGF-β1 induced autophagy and activated AEG-1 via Smad2/3 phosphorylation in malignant glioma cells. Also increased was oncogene cyclin D1 and EMT markers, which promoted tumor progression. Inhibition of autophagy using siRNA-BECN1 and siRNA-AEG-1 suppressed EMT. In tumor samples from patients with malignant glioma, immunohistochemical assays showed that expression levels of TGF-β1, AEG-1, and markers of autophagy and EMT, all gradually increase with glioblastoma progression. In vivo siRNA-AEG-1 administration to rats implanted with C6 glioma cells inhibited tumor growth and increased the incidence of apoptosis among tumor cells. These findings shed light on the mechanisms underlying the invasiveness and progression of malignant gliomas. PMID:26909607

  15. Cloning of the Protective Antigen Gene of Bacillus anthracis

    DTIC Science & Technology

    1983-09-01

    of the complicated precedents of duplicate toxin genes in chro- muumm mosomall and plasmid DNA of B. thuringiensis (Schnepf and Whitely, 1981; Klier...OiL V4. 34. S-W7. SW 1v 99 CwI 0193 by MT 0 009-7483/06O-002.00/0 mU"- - 1*;)-0Cloning of the Protective Antigen Gene OCT 19 MI L Sof Bacillus ...Sumnler uncertain, it is probably caused by other Bacillus antigens, 4 t which may include LF and EF. PA produced from recom- A The - "w t of a

  16. Neuropeptide Y stimulates autophagy in hypothalamic neurons

    PubMed Central

    Aveleira, Célia A.; Botelho, Mariana; Carmo-Silva, Sara; Ferreira-Marques, Marisa; Nóbrega, Clévio; Cortes, Luísa; Valero, Jorge; Sousa-Ferreira, Lígia; Álvaro, Ana R.; Santana, Magda; Kügler, Sebastian; Pereira de Almeida, Luís

    2015-01-01

    Aging is characterized by autophagy impairment that contributes to age-related disease aggravation. Moreover, it was described that the hypothalamus is a critical brain area for whole-body aging development and has impact on lifespan. Neuropeptide Y (NPY) is one of the major neuropeptides present in the hypothalamus, and it has been shown that, in aged animals, the hypothalamic NPY levels decrease. Because caloric restriction (CR) delays aging, at least in part, by stimulating autophagy, and also increases hypothalamic NPY levels, we hypothesized that NPY could have a relevant role on autophagy modulation in the hypothalamus. Therefore, the aim of this study was to investigate the role of NPY on autophagy in the hypothalamus. Using both hypothalamic neuronal in vitro models and mice overexpressing NPY in the hypothalamus, we observed that NPY stimulates autophagy in the hypothalamus. Mechanistically, in rodent hypothalamic neurons, NPY increases autophagy through the activation of NPY Y1 and Y5 receptors, and this effect is tightly associated with the concerted activation of PI3K, MEK/ERK, and PKA signaling pathways. Modulation of hypothalamic NPY levels may be considered a potential strategy to produce protective effects against hypothalamic impairments associated with age and to delay aging. PMID:25775546

  17. Inhibition of the Hedgehog pathway induces autophagy in pancreatic ductal adenocarcinoma cells.

    PubMed

    Xu, Yonghua; An, Yong; Wang, Xuehao; Zha, Wenzhang; Li, Xiangcheng

    2014-02-01

    The HH signaling pathway is a 'core' signal transduction pathway in pancreatic cancer that promotes the tumorigenesis of pancreatic cancers via enhancing cell proliferation, increasing invasion and metastasis and protecting against apoptosis. In the present study, we found that HH signaling regulates autophagy in pancreatic cancer cells. Activation of HH signaling inhibits autophagy, while inhibition of the HH pathway induces autophagy. Although the role of autophagy in cell survival and apoptosis may depend on tumor type and the microenvironment, our data clearly demonstrated that GANT61-induced autophagy contributed to reduced viability and increased apoptosis in pancreatic cancer cells both in vivo and in vitro, and these effects were reversed by the autophagy inhibitor, 3-MA. We propose that HH signaling by regulating autophagy plays an important role in determining the cellular response to HH-targeted therapy in pancreatic cancer and further investigation of the interaction between autophagy and HH signaling is particularly important.

  18. A Targeted Genetic Modifier Screen Links the SWI2/SNF2 Protein Domino to Growth and Autophagy Genes in Drosophila melanogaster

    PubMed Central

    Kwon, Matt Hyoung; Callaway, Heather; Zhong, Jim; Yedvobnick, Barry

    2013-01-01

    Targeted genetic studies can facilitate phenotypic analyses and provide important insights into development and other complex processes. The SWI2/SNF2 DNA-dependent ATPase Domino (Dom) of Drosophila melanogaster, a component of the Tip60 acetyltransferase complex, has been associated with a wide spectrum of cellular processes at multiple developmental stages. These include hematopoiesis, cell proliferation, homeotic gene regulation, histone exchange during DNA repair, and Notch signaling. To explore the wider gene network associated with Dom action, we used RNAi directed against domino (dom) to mediate loss-of-function at the wing margin, a tissue that is readily scored for phenotypic changes. Dom RNAi driven through GAL4-UAS elicited dominant wing nicking that responded phenotypically to the dose of dom and other loci known to function with dom. We screened for phenotypic modifiers of this wing phenotype among 2500 transpositions of the EP P element and found both enhancers and suppressors. Several classes of modifier were obtained, including those encoding transcription factors, RNA regulatory proteins, and factors that regulate cell growth, proliferation and autophagy, a lysosomal degradation pathway that affects cell growth under conditions of starvation and stress. Our analysis is consistent with prior studies, suggesting that Dom acts pleiotropically as a positive effector of Notch signaling and a repressor of proliferation. This genetic system should facilitate screens for additional loci associated with Dom function, and complement biochemical approaches to their regulatory activity. PMID:23550128

  19. RUFY4: Immunity piggybacking on autophagy?

    PubMed

    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.

  20. AGEs Induced Autophagy Impairs Cutaneous Wound Healing via Stimulating Macrophage Polarization to M1 in Diabetes

    PubMed Central

    Guo, Yuanyuan; Lin, Cai; Xu, Peng; Wu, Shan; Fu, Xiujun; Xia, Weidong; Yao, Min

    2016-01-01

    Autophagy is essential in physiological and pathological processes, however, the role of autophagy in cutaneous wound healing and the underlying molecular mechanism remain elusive. We hypothesized that autophagy plays an important role in regulating wound healing. Here, we show that enhanced autophagy negatively impacts on normal cutaneous healing process and is related to chronic wounds as demonstrated by the increased LC3 in diabetic mice skin or patients’ chronic wounds. In addition, inhibition of autophagy by 3-MA restores delayed healing in C57BL/6 or db/db mice, demonstrating that autophagy is involved in regulating wound healing. Furthermore, we identify that macrophage is a major cell type underwent autophagy in wounds and increased autophagy induces macrophages polarization into M1 with elevated CD11c population and gene expressions of proinflammatory cytokines. To explore the mechanism underlying autophagy-impaired wound healing, we tested the role of IRF8, a regulator of autophagy, in autophagy-modulated macrophages polarization. IRF8 activation is up-regulating autophagy and M1 polarization of macrophages after AGEs (advanced glycation endproducts) treatment, blocking the IRF8 with shIRF8 inhibits autophagic activity and M1 polarization. In summary, this study elucidates that AGEs induces autophagy and modulates macrophage polarization to M1 via IRF8 activation in impairment of cutaneous wound healing. PMID:27805071

  1. Targeting the MIR34C-5p-ATG4B-autophagy axis enhances the sensitivity of cervical cancer cells to pirarubicin.

    PubMed

    Wu, Yaran; Ni, Zhenhong; Yan, Xiaojing; Dai, Xufang; Hu, Changjiang; Zheng, Yingru; He, Fengtian; Lian, Jiqin

    2016-07-02

    Pirarubicin (THP) is a newer generation anthracycline anticancer drug. In the clinic, THP and THP-based combination therapies have been demonstrated to be effective against various tumors without severe side effects. However, previous clinical studies have shown that most patients with cervical cancer are not sensitive to THP treatment, and the associated mechanisms are not clear. Consistent with the clinical study, we confirmed that cervical cancer cells were resistant to THP in vitro and in vivo. Our data demonstrated that THP induced a protective macroautophagy/autophagy response in cervical cancer cells, and suppression of this autophagy dramatically enhanced the cytotoxicity of THP. By scanning the mRNA level change of autophagy-related genes, we found that the upregulation of ATG4B (autophagy-related 4B cysteine peptidase) plays an important role in THP-induced autophagy. Moreover, THP increased the mRNA level of ATG4B in cervical cancer cells by promoting mRNA stability without influencing its transcription. Furthermore, THP triggered a downregulation of MIR34C-5p, which was associated with the upregulation of ATG4B and autophagy induction. Overexpression of MIR34C-5p significantly decreased the level of ATG4B and attenuated autophagy, accompanied by enhanced cell death and apoptosis in THP-treated cervical cancer cells. These results for the first time reveal the presence of a MIR34C-5p-ATG4B-autophagy signaling axis in THP-treated cervical cancer cells in vitro and in vivo, and the axis, at least partially, accounts for the THP nonsensitivity in cervical cancer patients. This study may provide a new insight for improving the chemotherapeutic effect of THP, which may be beneficial to the further clinical application of THP in cervical cancer treatment.

  2. Inhibition of Hedgehog signaling pathway impedes cancer cell proliferation by promotion of autophagy.

    PubMed

    Tang, Xiaoli; Deng, Libin; Chen, Qi; Wang, Yao; Xu, Rong; Shi, Chao; Shao, Jia; Hu, Guohui; Gao, Meng; Rao, Hai; Luo, Shiwen; Lu, Quqin

    2015-05-01

    Multiple lines of evidence implicate that aberrant activation of Hedgehog (Hh) signaling is involved in a variety of human cancers. However, the molecular mechanisms underlying how cancer cells respond to Hh inhibition remain to be elucidated. In this study, we found that blockade of Hh signaling suppresses cell proliferation in human cancer cells. Microarray analysis revealed that differentially expressed genes (DEGs) in human cancer cells are enriched in autophagy pathway in response to the inhibition of Hh signaling. Interestingly, inhibition of Hh signaling induced autophagy, whereas activation of Hh signaling by ligand treatments prevented the induction of autophagy. In addition, inhibition of autophagy by 3-methyladenine (3-MA) partially suppressed cytotoxicity induced by inhibition of Hh signaling. Finally, in autophagy deficient cells, cytotoxic effect triggered by inhibition of Hh signaling was partially reversed, indicating the modulation of autophagy by Hh signaling is autophagy-specific. These results suggest that inhibition of Hh signaling impedes cancer cell proliferation in part through induction of autophagy.

  3. Autophagy in the Eye: Implications for Ocular Cell Health

    PubMed Central

    Frost, Laura S.; Mitchell, Claire H.; Boesze-Battaglia, Kathleen

    2014-01-01

    Autophagy, a catabolic process by which a cell “eats” itself, turning over its own cellular constituents, plays a key role in cellular homeostasis. In an effort to maintain normal cellular function, autophagy is often up-regulated in response to environmental stresses and excessive organelle damage to facilitate aggregated protein removal. In the eye, virtually all cell types from those comprising the cornea in the front of the eye to the retinal pigment epithelium (RPE) providing a protective barrier for the retina at the back of the eye, rely on one or more aspects of autophagy to maintain structure and/or normal physiological function. In the lens autophagy plays a critical role in lens fiber cell maturation and the formation of the organelle free zone. Numerous studies delineating the role of Atg5, Vsp34 as well as FYCO1 in maintenance of lens transparency are discussed. Corneal endothelial dystrophies are also characterized as having elevated levels of autophagic proteins. Therefore, novel modulators of autophagy such as lithium and melatonin are proposed as new therapeutic strategies for this group of dystrophies. In addition, we summarize how corneal Herpes Simplex Virus (HSV-1) infection subverts the cornea’s response to infection by inhibiting the normal autophagic response. Using glaucoma models we analyze the relative contribution of autophagy to cell death and cell survival. The cytoprotective role of autophagy is further discussed in an analysis of photoreceptor cell heath and function. We focus our analysis on the current understanding of autophagy in photoreceptor and RPE health, specifically on the diverse role of autophagy in rods and cones as well as its protective role in light induced degeneration. Lastly, in the RPE we highlight hybrid phagocytosis-autophagy pathways. This comprehensive review allows us to speculate on how alterations in various stages of autophagy contribute to glaucoma and retinal degenerations. PMID:24810222

  4. Measuring autophagy in macrophages.

    PubMed

    Harris, James; Hanrahan, Orla; De Haro, Sergio A

    2009-11-01

    Macroautophagy is a conserved intracellular homeostatic mechanism for the degradation of cytosolic constituents. Autophagy can promote cell survival by providing essential amino acids from the breakdown of macromolecules during periods of nutrient deprivation, and can remove damaged or excess organelles, such as mitochondria and peroxisomes. More recently, autophagy has been shown to play an important role in innate and adaptive immune responses to pathogenic bacteria in macrophages and dendritic cells. This unit presents protocols for the measurement of autophagy in macrophages.

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

    PubMed

    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.

  6. Autophagy inhibition sensitizes hepatocellular carcinoma to the multikinase inhibitor linifanib.

    PubMed

    Pan, Hongming; Wang, Zhanggui; Jiang, Liming; Sui, Xinbing; You, Liangkun; Shou, Jiawei; Jing, Zhao; Xie, Jiansheng; Ge, Weiting; Cai, Xiujun; Huang, Wendong; Han, Weidong

    2014-10-20

    Autophagy is a critical survival pathway for cancer cells under conditions of stress. Thus, induction of autophagy has emerged as a drug resistance mechanism. This study is to determine whether autophagy is activated by a novel multikinase inhibitor linifanib, thereby impairing the sensitivity of hepatocellular carcinoma (HCC) cells to this targeted therapy. Here, we found that linifanib induced a high level of autophagy in HCC cells, which was accompanied by suppression of phosphorylation of PDGFR-β and its downstream Akt/mTOR and Mek/Erk signaling pathways. Cell death induced by linifanib was greatly enhanced after autophagy inhibition by the pharmacological inhibitors or siRNAs against autophagy related genes, ATG5 and ATG7, in vitro. Moreover, HCQ, an FDA-approved drug used to inhibit autophagy, could significantly augment the anti-HCC effect of linifanib in a mouse xenograft model. In conclusion, linifanib can induce cytoprotective autophagy by suppression of PDGFR-β activities in HCC cells. Thus, autophagy inhibition represents a promising approach to improve the efficacy of linifanib in the treatment of HCC patients.

  7. Regulation of cardiac autophagy by insulin-like growth factor 1.

    PubMed

    Troncoso, Rodrigo; Díaz-Elizondo, Jessica; Espinoza, Sandra P; Navarro-Marquez, Mario F; Oyarzún, Alejandra P; Riquelme, Jaime A; Garcia-Carvajal, Ivonne; Díaz-Araya, Guillermo; García, Lorena; Hill, Joseph A; Lavandero, Sergio

    2013-07-01

    Insulin-like growth factor-1 (IGF-1) signaling is a key pathway in the control of cell growth and survival. Three critical nodes in the IGF-1 signaling pathway have been described in cardiomyocytes: protein kinase Akt/mammalian target of rapamycin (mTOR), Ras/Raf/extracellular signal-regulated kinase (ERK), and phospholipase C (PLC)/inositol 1,4,5-triphosphate (InsP3 )/Ca(2+) . The Akt/mTOR and Ras/Raf/ERK signaling arms govern survival in the settings of cardiac stress and hypertrophic growth. By contrast, PLC/InsP3 /Ca(2+) functions to regulate metabolic adaptability and gene transcription. Autophagy is a catabolic process involved in protein degradation, organelle turnover, and nonselective breakdown of cytoplasmic components during nutrient starvation or stress. In the heart, autophagy is observed in a variety of human pathologies, where it can be either adaptive or maladaptive, depending on the context. We proposed the hypothesis that IGF-1 protects the heart by rescuing the mitochondrial metabolism and the energetics state, reducing cell death and controls the potentially exacerbate autophagic response to nutritional stress. In light of the importance of IGF-1 and autophagy in the heart, we review here IGF-1 signaling and autophagy regulation in the context of cardiomyocyte nutritional stress.

  8. Impairing autophagy in retinal pigment epithelium leads to inflammasome activation and enhanced macrophage-mediated angiogenesis

    PubMed Central

    Liu, Jian; Copland, David A.; Theodoropoulou, Sofia; Chiu, Hsi An Amy; Barba, Miriam Durazo; Mak, Ka Wang; Mack, Matthias; Nicholson, Lindsay B.; Dick, Andrew D.

    2016-01-01

    Age-related decreases in autophagy contribute to the progression of age-related macular degeneration (AMD). We have now studied the interaction between autophagy impaired in retinal pigment epithelium (RPE) and the responses of macrophages. We find that dying RPE cells can activate the macrophage inflammasome and promote angiogenesis. In vitro, inhibiting rotenone-induced autophagy in RPE cells elicits caspase-3 mediated cell death. Co-culture of damaged RPE with macrophages leads to the secretion of IL-1β, IL-6 and nitrite oxide. Exogenous IL-6 protects the dysfunctional RPE but IL-1β causes enhanced cell death. Furthermore, IL-1β toxicity is more pronounced in dysfunctional RPE cells showing reduced IRAK3 gene expression. Co-culture of macrophages with damaged RPE also elicits elevated levels of pro-angiogenic proteins that promote ex vivo choroidal vessel sprouting. In vivo, impaired autophagy in the eye promotes photoreceptor and RPE degeneration and recruitment of inflammasome-activated macrophages. The degenerative tissue environment drives an enhanced pro-angiogenic response, demonstrated by increased size of laser-induced choroidal neovascularization (CNV) lesions. The contribution of macrophages was confirmed by depletion of CCR2+ monocytes, which attenuates CNV in the presence of RPE degeneration. Our results suggest that the interplay between perturbed RPE homeostasis and activated macrophages influences key features of AMD development. PMID:26847702

  9. Oxidized low-density lipoproteins upregulate proline oxidase to initiate ROS-dependent autophagy.

    PubMed

    Zabirnyk, Olga; Liu, Wei; Khalil, Shadi; Sharma, Anit; Phang, James M

    2010-03-01

    Epidemiological studies showed that high levels of oxidized low-density lipoproteins (oxLDLs) are associated with increased cancer risk. We examined the direct effect of physiologic concentrations oxLDL on cancer cells. OxLDLs were cytotoxic and activate both apoptosis and autophagy. OxLDLs have ligands for peroxisome proliferator-activated receptor gamma and upregulated proline oxidase (POX) through this nuclear receptor. We identified 7-ketocholesterol (7KC) as a main component responsible for the latter. To elucidate the role of POX in oxLDL-mediated cytotoxicity, we knocked down POX via small interfering RNA and found that this (i) further reduced viability of cancer cells treated with oxLDL; (ii) decreased oxLDL-associated reactive oxygen species generation; (iii) decreased autophagy measured via beclin-1 protein level and light-chain 3 protein (LC3)-I into LC3-II conversion. Using POX-expressing cell model, we established that single POX overexpression was sufficient to activate autophagy. Thus, it led to autophagosomes accumulation and increased conversion of LC3-I into LC3-II. Moreover, beclin-1 gene expression was directly dependent on POX catalytic activity, namely the generation of POX-dependent superoxide. We conclude that POX is critical in the cellular response to the noxious effects of oxLDL by activating protective autophagy.

  10. Ursolic Acid Suppresses Hepatitis B Virus X Protein-mediated Autophagy and Chemotherapeutic Drug Resistance.

    PubMed

    Chang, Ching-Dong; Lin, Ping-Yuan; Hsu, Jue-Liang; Shih, Wen-Ling

    2016-10-01

    Hepatitis B virus X (HBx) protein is a multifunctional oncoprotein that affects diverse cell activities via regulation of various host cell signaling pathways. The current investigation demonstrated that ursolic acid (UA), a pentacyclic triterpenoid, protected hepatoma cells and reduced HBx-mediated autophagy through modulation of Ras homolog gene family member A (RhoA). Low-level ectopic HBx expression in Huh7 cells induced more significant autophagosome formation than high-level HBx expression. HBx activated beclin-1 promoter and enhanced the beclin-1 protein expression under low HBx expression. Transcription factor AP-1 played an essential function in HBx-mediated beclin-1 promoter activation. Inhibition of RhoA and its downstream effector Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) alleviated HBx-mediated autophagy significantly. Transiently-expressed HBx elicited an increased RhoA-GTP level, as well as phospho-ROCK1 transient accumulation. Utilization of transactivation-deficient HBx demonstrated that the transactivation activity of HBx is required for autophagy induction. Furthermore, UA suppressed HBx-mediated RhoA activation, beclin-1 promoter activation and subsequent autophagy induction, while, most importantly, reversed HBx-induced anti-cancer drug resistance.

  11. Pharmacologic agents targeting autophagy

    PubMed Central

    Vakifahmetoglu-Norberg, Helin; Xia, Hong-guang; Yuan, Junying

    2015-01-01

    Autophagy is an important intracellular catabolic mechanism critically involved in regulating tissue homeostasis. The implication of autophagy in human diseases and the need to understand its regulatory mechanisms in mammalian cells have stimulated research efforts that led to the development of high-throughput screening protocols and small-molecule modulators that can activate or inhibit autophagy. Herein we review the current landscape in the development of screening technology as well as the molecules and pharmacologic agents targeting the regulatory mechanisms of autophagy. We also evaluate the potential therapeutic application of these compounds in different human pathologies. PMID:25654545

  12. Autophagy: machinery and regulation

    PubMed Central

    Yin, Zhangyuan; Pascual, Clarence; Klionsky, Daniel J.

    2016-01-01

    Macroautophagy/autophagy is an evolutionarily conserved cellular degradation process that targets cytoplasmic materials including cytosol, macromolecules and unwanted organelles. The discovery and analysis of autophagy-related (Atg) proteins have unveiled much of the machinery of autophagosome formation. Although initially autophagy was regarded as a survival response to stress, recent studies have revealed its significance in cellular and organismal homeostasis, development and immunity. Autophagic dysfunction and dysregulation are implicated in various diseases. In this review, we briefly summarize the physiological roles, molecular mechanism, regulatory network, and pathophysiological roles of autophagy. PMID:28357331

  13. Morphological analysis of autophagy.

    PubMed

    Tabata, Keisuke; Hayashi-Nishino, Mitsuko; Noda, Takeshi; Yamamoto, Akitsugu; Yoshimori, Tamotsu

    2013-01-01

    Autophagy is a bulk intracellular degradation process that is ubiquitous in eukaryotic cells and helps to recycle nutrients from catabolites by degrading proteins, lipids, and glycans, including organelles. Since autophagy has divergent physiological roles in cancer, infection, immunity, and other processes, it is important to accurately analyze autophagic activity. In this chapter, we describe methods that can be used to monitor autophagy in cultured mammalian cells by immunostaining and using fluorescently tagged autophagy-related proteins such as GFP- or mRFP-GFP-tandem-tagged proteins as well as electron microscopic methods, including electron tomography and immuno-electron microscopy.

  14. Autophagy, nutrition and immunology

    PubMed Central

    Cuervo, Ana Maria; Macian, Fernando

    2014-01-01

    Turnover of cellular components in lysosomes or autophagy is an essential mechanism for cellular quality control. Added to this cleaning role, autophagy has recently been shown to participate in the dynamic interaction of cells with the surrounding environment by acting as a point of integration of extracellular cues. In this review, we focus on the relationship between autophagy and two types of environmental factors: nutrients and pathogens. We describe their direct effect on autophagy and discuss how the autophagic reaction to these stimuli allows cells to accommodate the requirements of the cellular response to stress, including those specific to the immune responses. PMID:21982744

  15. Nucleofection of Rat Pheochromocytoma PC-12 Cells with Human Mutated Beta-Amyloid Precursor Protein Gene (APP-sw) Leads to Reduced Viability, Autophagy-Like Process, and Increased Expression and Secretion of Beta Amyloid

    PubMed Central

    Pająk, Beata; Kania, Elżbieta

    2015-01-01

    Pheochromocytoma PC-12 cells are immune to physiological stimuli directed to evoke programmed cell death. Besides, metabolic inhibitors are incapable of sensitizing PC-12 cells to extrinsic or intrinsic apoptosis unless they are used in toxic concentrations. Surprisingly, these cells become receptive to cell deletion after human APP-sw gene expression. We observed reduced cell viability in GFP vector + APP-sw-nucleofected cells (drop by 36%) but not in GFP vector − or GFP vector + APP-wt-nucleofected cells. Lower viability was accompanied by higher expression of Aβ 1-16 and elevated secretion of Aβ 1-40 (in average 53.58 pg/mL). At the ultrastructural level autophagy-like process was demonstrated to occur in APP-sw-nucleofected cells with numerous autophagosomes and multivesicular bodies but without autolysosomes. Human APP-sw gene is harmful to PC-12 cells and cells are additionally driven to incomplete autophagy-like process. When stimulated by TRAIL or nystatin, CLU protein expression accompanies early phase of autophagy. PMID:25821818

  16. miR-124 regulates cell apoptosis and autophagy in dopaminergic neurons and protects them by regulating AMPK/mTOR pathway in Parkinson’s disease

    PubMed Central

    Gong, Xin; Wang, Huiqing; Ye, Yongyi; Shu, Yugao; Deng, Yongwen; He, Xiaozheng; Lu, Guohui; Zhang, Shizhong

    2016-01-01

    The important roles of miR-124 in the development and progression of various diseases are being increasing recognized. This study was aimed to investigate the potential roles of miR-124 in dopaminergic (DA) neuronal apoptosis and autophagy in Parkinson’s disease (PD) and to explore their mechanisms. Human SH-SY5Y cells that are treated with MPTP were transfected with mature miR-124 vector and control empty vector. The effect of MPTP on miR-124 mRNA level was analyzed using RT-PCR analysis. Furthermore, the effects of miR-124 expression on neuronal apoptosis and autophagy, as well as the expression of proteins in the AMPK/mTOR pathway, were analyzed using RT-PCR and western blotting. This study found that miR-124 was down-regulated in the MPTP-treated (100 μM) neurons, and miR-124 suppression significantly increased cell apoptosis and induced autophagy-associated protein expression, including that of Beclin 1 and increased the ratio of LC3 II/LC3 I compared with that in controls. In addition, in vitro rescue of miR-124 significantly decreased the percentage of apoptotic cells and the ratio of LC3 II/LC3 I, findings that were approximately equal to the controls. Moreover, miR-124 suppression increased p-AMPK but decreased p-mTOR levels in neurons. Our study suggested that miR-124 functions as a protector of DA neurons during PD through the involvement of cell apoptosis and autophagy by regulating the AMPK/mTOR pathway. PMID:27347320

  17. ASPP2 attenuates triglycerides to protect against hepatocyte injury by reducing autophagy in a cell and mouse model of non-alcoholic fatty liver disease.

    PubMed

    Xie, Fang; Jia, Lin; Lin, Minghua; Shi, Ying; Yin, Jiming; Liu, Yin; Chen, Dexi; Meng, Qinghua

    2015-01-01

    ASPP2 is a pro-apoptotic member of the p53 binding protein family. ASPP2 has been shown to inhibit autophagy, which maintains energy balance in nutritional deprivation. We attempted to identify the role of ASPP2 in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In a NAFLD cell model, control treated and untreated HepG2 cells were pre-incubated with GFP-adenovirus (GFP-ad) for 12 hrs and then treated with oleic acid (OA) for 24 hrs. In the experimental groups, the HepG2 cells were pre-treated with ASPP2-adenovirus (ASPP2-ad) or ASPP2-siRNA for 12 hrs and then treated with OA for 24 hrs. BALB/c mice fed a methionine- and choline-deficient (MCD) diet were used to generate a mouse model of NAFLD. The mice with fatty livers in the control group were pre-treated with injections of GFP-ad for 10 days. In the experimental group, the mice that had been pre-treated with ASPP2-ad were fed an MCD diet for 10 days. ASPP2-ad or GFP-ad was administered once every 5 days. Liver tissue from fatty liver patients and healthy controls were used to analyse the role of ASPP2. Autophagy, apoptosis markers and lipid metabolism mediators, were assessed with confocal fluorescence microscopy, immunohistochemistry, western blot and biochemical assays. ASPP2 overexpression decreased the triglyceride content and inhibited autophagy and apoptosis in the HepG2 cells. ASPP2-ad administration suppressed the MCD diet-induced autophagy, steatosis and apoptosis and decreased the previously elevated alanine aminotransferase levels. In conclusion, ASPP2 may participate in the lipid metabolism of non-alcoholic steatohepatitis and attenuate liver failure.

  18. Protective effect of fucoidan from Fucus vesiculosus on liver fibrosis via the TGF-β1/Smad pathway-mediated inhibition of extracellular matrix and autophagy

    PubMed Central

    Li, Jingjing; Chen, Kan; Li, Sainan; Feng, Jiao; Liu, Tong; Wang, Fan; Zhang, Rong; Xu, Shizan; Zhou, Yuqing; Zhou, Shunfeng; Xia, Yujing; Lu, Jie; Zhou, Yingqun; Guo, Chuanyong

    2016-01-01

    Liver fibrosis is a dynamic reversible pathological process in the development of chronic liver disease to cirrhosis. However, the current treatments are not administered for a long term due to their various side effects. Autophagy is initiated to decompose damaged or excess organelles, which had been found to alter the progression of liver fibrosis. In this article, we hypothesized that fucoidan from Fucus vesiculosus may attenuate liver fibrosis in mice by inhibition of the extracellular matrix and autophagy in carbon tetrachloride- and bile duct ligation-induced animal models of liver fibrosis. The results were determined using enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemical staining. Fucoidan from F. vesiculosus could inhibit the activation of hepatic stellate cells and the formation of extracellular matrix and autophagosomes, and its effect may be associated with the downregulation of transforming growth factor beta 1/Smads pathways. Fucoidan, as an autophagy and transforming growth factor beta 1 inhibitor, could be a promising potential therapeutic agent for liver fibrosis. PMID:26929597

  19. Protective effect of fucoidan from Fucus vesiculosus on liver fibrosis via the TGF-β1/Smad pathway-mediated inhibition of extracellular matrix and autophagy.

    PubMed

    Li, Jingjing; Chen, Kan; Li, Sainan; Feng, Jiao; Liu, Tong; Wang, Fan; Zhang, Rong; Xu, Shizan; Zhou, Yuqing; Zhou, Shunfeng; Xia, Yujing; Lu, Jie; Zhou, Yingqun; Guo, Chuanyong

    2016-01-01

    Liver fibrosis is a dynamic reversible pathological process in the development of chronic liver disease to cirrhosis. However, the current treatments are not administered for a long term due to their various side effects. Autophagy is initiated to decompose damaged or excess organelles, which had been found to alter the progression of liver fibrosis. In this article, we hypothesized that fucoidan from Fucus vesiculosus may attenuate liver fibrosis in mice by inhibition of the extracellular matrix and autophagy in carbon tetrachloride- and bile duct ligation-induced animal models of liver fibrosis. The results were determined using enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemical staining. Fucoidan from F. vesiculosus could inhibit the activation of hepatic stellate cells and the formation of extracellular matrix and autophagosomes, and its effect may be associated with the downregulation of transforming growth factor beta 1/Smads pathways. Fucoidan, as an autophagy and transforming growth factor beta 1 inhibitor, could be a promising potential therapeutic agent for liver fibrosis.

  20. Survival by self-destruction: a role for autophagy in the placenta?

    PubMed

    Bildirici, I; Longtine, M S; Chen, B; Nelson, D M

    2012-08-01

    Autophagy is a burgeoning area of research from yeast to humans. Although previously described as a death pathway, autophagy is now considered an important survival phenomenon in response to environmental stressors to which most organs are exposed. Despite an ever expanding literature in non-placental cells, studies of autophagy in the placenta are lagging. We review the regulation of autophagy, summarize available placental studies of autophagy, and highlight potential areas for future research. We believe that such studies will yield novel insights into how placentas protect the survival of the species by "self-eating".

  1. Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control[W

    PubMed Central

    Ramundo, Silvia; Casero, David; Mühlhaus, Timo; Hemme, Dorothea; Sommer, Frederik; Crèvecoeur, Michèle; Rahire, Michèle; Schroda, Michael; Rusch, Jannette; Goodenough, Ursula; Pellegrini, Matteo; Perez-Perez, Maria Esther; Crespo, José Luis; Schaad, Olivier; Civic, Natacha; Rochaix, Jean David

    2014-01-01

    Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria. PMID:24879428

  2. Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control.

    PubMed

    Ramundo, Silvia; Casero, David; Mühlhaus, Timo; Hemme, Dorothea; Sommer, Frederik; Crèvecoeur, Michèle; Rahire, Michèle; Schroda, Michael; Rusch, Jannette; Goodenough, Ursula; Pellegrini, Matteo; Perez-Perez, Maria Esther; Crespo, José Luis; Schaad, Olivier; Civic, Natacha; Rochaix, Jean David

    2014-05-01

    Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Parkinson's Disease and Autophagy

    PubMed Central

    Sánchez-Pérez, Ana María; Claramonte-Clausell, Berta; Sánchez-Andrés, Juan Vicente; Herrero, María Trinidad

    2012-01-01

    It is generally accepted that a correlation between neurodegenerative disease and protein aggregation in the brain exists; however, a causal relationship has not been elucidated. In neurons, failure of autophagy may result in the accumulation of aggregate-prone proteins and subsequent neurodegeneration. Thus, pharmacological induction of autophagy to enhance the clearance of intracytoplasmic aggregate-prone proteins has been considered as a therapeutic strategy to ameliorate pathology in cell and animal models of neurodegenerative disorders. However, autophagy has also been found to be a factor in the onset of these diseases, which raises the question of whether autophagy induction is an effective therapeutic strategy, or, on the contrary, can result in cell death. In this paper, we will first describe the autophagic machinery, and we will consider the literature to discuss the neuroprotective effects of autophagy. PMID:23125941

  5. Parkinson's disease and autophagy.

    PubMed

    Sánchez-Pérez, Ana María; Claramonte-Clausell, Berta; Sánchez-Andrés, Juan Vicente; Herrero, María Trinidad

    2012-01-01

    It is generally accepted that a correlation between neurodegenerative disease and protein aggregation in the brain exists; however, a causal relationship has not been elucidated. In neurons, failure of autophagy may result in the accumulation of aggregate-prone proteins and subsequent neurodegeneration. Thus, pharmacological induction of autophagy to enhance the clearance of intracytoplasmic aggregate-prone proteins has been considered as a therapeutic strategy to ameliorate pathology in cell and animal models of neurodegenerative disorders. However, autophagy has also been found to be a factor in the onset of these diseases, which raises the question of whether autophagy induction is an effective therapeutic strategy, or, on the contrary, can result in cell death. In this paper, we will first describe the autophagic machinery, and we will consider the literature to discuss the neuroprotective effects of autophagy.

  6. Egr-1 regulates irradiation-induced autophagy through Atg4B to promote radioresistance in hepatocellular carcinoma cells

    PubMed Central

    Peng, W-x; Wan, Y-y; Gong, A-h; Ge, L; Jin, J; Xu, M; Wu, C-y

    2017-01-01

    Although hepatocellular carcinoma (HCC) is usually response to radiation therapy, radioresistance is still the major obstacle that limits the efficacy of radiotherapy for HCC patients. Therefore, further investigation of underlying mechanisms in radioresistant HCC cells is warranted. In this study, we determined the effect of early growth response factor (Egr-1) on irradiation-induced autophagy and radioresistance in HCC cell lines SMMC-7721 and HepG2. We showed that autophagy-related gene 4B (Atg4B) is induced by Egr-1 upon ionizing radiation (IR) in HCC cells. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) revealed that Egr-1 binds to the Atg4B promoter to upregulate its expression in HCC cells. Suppression of Egr-1 function by dominant-negative Egr-1 dampens IR-induced autophagy, cell migration, and increases cell sensitivity to radiotherapy. Together, these results suggest that Egr-1 contributes to HCC radioresistance through directly upregulating target gene Atg4B, which may serve as a protective mechanism by preferential activation of the autophagy. PMID:28134935

  7. Autophagy: a new target for nonalcoholic fatty liver disease therapy

    PubMed Central

    Mao, Yuqing; Yu, Fujun; Wang, Jianbo; Guo, Chuanyong; Fan, Xiaoming

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) has gained importance in recent decades due to drastic changes in diet, especially in Western countries. NAFLD occurs as a spectrum from simple hepatic steatosis, steatohepatitis to cirrhosis, and even hepatocellular carcinoma. Although the molecular mechanisms underlying the development of NAFLD have been intensively investigated, many issues remain to be resolved. Autophagy is a cell survival mechanism for disposing of excess or defective organelles, and has become a hot spot for research. Recent studies have revealed that autophagy is linked to the development of NAFLD and regulation of autophagy has therapeutic potential. Autophagy reduces intracellular lipid droplets by enclosing them and fusing with lysosomes for degradation. Furthermore, autophagy is involved in attenuating inflammation and liver injury. However, autophagy is regarded as a double-edged sword, as it may also affect adipogenesis and adipocyte differentiation. Moreover, it is unclear as to whether autophagy protects the body from injury or causes diseases and even death, and the association between autophagy and NAFLD remains controversial. This review is intended to discuss, comment, and outline the progress made in this field and establish the possible molecular mechanism involved. PMID:27099536

  8. Autophagy: a new target for nonalcoholic fatty liver disease therapy.

    PubMed

    Mao, Yuqing; Yu, Fujun; Wang, Jianbo; Guo, Chuanyong; Fan, Xiaoming

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) has gained importance in recent decades due to drastic changes in diet, especially in Western countries. NAFLD occurs as a spectrum from simple hepatic steatosis, steatohepatitis to cirrhosis, and even hepatocellular carcinoma. Although the molecular mechanisms underlying the development of NAFLD have been intensively investigated, many issues remain to be resolved. Autophagy is a cell survival mechanism for disposing of excess or defective organelles, and has become a hot spot for research. Recent studies have revealed that autophagy is linked to the development of NAFLD and regulation of autophagy has therapeutic potential. Autophagy reduces intracellular lipid droplets by enclosing them and fusing with lysosomes for degradation. Furthermore, autophagy is involved in attenuating inflammation and liver injury. However, autophagy is regarded as a double-edged sword, as it may also affect adipogenesis and adipocyte differentiation. Moreover, it is unclear as to whether autophagy protects the body from injury or causes diseases and even death, and the association between autophagy and NAFLD remains controversial. This review is intended to discuss, comment, and outline the progress made in this field and establish the possible molecular mechanism involved.

  9. Microgravity control of autophagy modulates osteoclastogenesis.

    PubMed

    Sambandam, Yuvaraj; Townsend, Molly T; Pierce, Jason J; Lipman, Cecilia M; Haque, Azizul; Bateman, Ted A; Reddy, Sakamuri V

    2014-04-01

    Evidence indicates that astronauts experience significant bone loss during space mission. Recently, we used the NASA developed rotary cell culture system (RCCS) to simulate microgravity (μXg) conditions and demonstrated increased osteoclastogenesis in mouse bone marrow cultures. Autophagy is a cellular recycling process of nutrients. Therefore, we hypothesize that μXg control of autophagy modulates osteoclastogenesis. Real-time PCR analysis of total RNA isolated from mouse bone marrow derived non-adherent cells subjected to modeled μXg showed a significant increase in autophagic marker Atg5, LC3 and Atg16L mRNA expression compared to ground based control (Xg) cultures. Western blot analysis of total cell lysates identified an 8.0-fold and 7.0-fold increase in the Atg5 and LC3-II expression, respectively. Confocal microscopy demonstrated an increased autophagosome formation in μXg subjected RAW 264.7 preosteoclast cells. RT(2) profiler PCR array screening for autophagy related genes identified that μXg upregulates intracellular signaling molecules associated with autophagy, autophagosome components and inflammatory cytokines/growth factors which coregulate autophagy in RAW 264.7 preosteoclast cells. Autophagy inhibitor, 3-methyladenine (3-MA) treatment of mouse bone marrow derived non-adherent mononuclear cells showed a significant decrease in μXg induced Atg5 and LC3 mRNA expression in the presence or absence of RANK ligand (RANKL) stimulation. Furthermore, RANKL treatment significantly increased (8-fold) p-CREB transcription factor levels under μXg as compared to Xg cultures and 3-MA inhibited RANKL increased p-CREB expression in these cells. Also, 3-MA suppresses μXg elevated osteoclast differentiation in mouse bone marrow cultures. Thus, our results suggest that μXg induced autophagy plays an important role in enhanced osteoclast differentiation and could be a potential therapeutic target to prevent bone loss in astronauts during space flight missions.

  10. Induction of Protective Genes Leads to Islet Survival and Function

    PubMed Central

    Wang, Hongjun; Ferran, Christiane; Attanasio, Chiara; Calise, Fulvio; Otterbein, Leo E.

    2011-01-01

    Islet transplantation is the most valid approach to the treatment of type 1 diabetes. However, the function of transplanted islets is often compromised since a large number of β cells undergo apoptosis induced by stress and the immune rejection response elicited by the recipient after transplantation. Conventional treatment for islet transplantation is to administer immunosuppressive drugs to the recipient to suppress the immune rejection response mounted against transplanted islets. Induction of protective genes in the recipient (e.g., heme oxygenase-1 (HO-1), A20/tumor necrosis factor alpha inducible protein3 (tnfaip3), biliverdin reductase (BVR), Bcl2, and others) or administration of one or more of the products of HO-1 to the donor, the islets themselves, and/or the recipient offers an alternative or synergistic approach to improve islet graft survival and function. In this perspective, we summarize studies describing the protective effects of these genes on islet survival and function in rodent allogeneic and xenogeneic transplantation models and the prevention of onset of diabetes, with emphasis on HO-1, A20, and BVR. Such approaches are also appealing to islet autotransplantation in patients with chronic pancreatitis after total pancreatectomy, a procedure that currently only leads to 1/3 of transplanted patients being diabetes-free. PMID:22220267

  11. Translational Control of Autophagy by Orb in the Drosophila Germline.

    PubMed

    Rojas-Ríos, Patricia; Chartier, Aymeric; Pierson, Stéphanie; Séverac, Dany; Dantec, Christelle; Busseau, Isabelle; Simonelig, Martine

    2015-12-07

    Drosophila Orb, the homolog of vertebrate CPEB, is a key translational regulator involved in oocyte polarity and maturation through poly(A) tail elongation of specific mRNAs. orb also has an essential function during early oogenesis that has not been addressed at the molecular level. Here, we show that orb prevents cell death during early oogenesis, thus allowing oogenesis to progress. It does so through the repression of autophagy by directly repressing, together with the CCR4 deadenylase, the translation of Autophagy-specific gene 12 (Atg12) mRNA. Autophagy and cell death observed in orb mutant ovaries are reduced by decreasing Atg12 or other Atg mRNA levels. These results reveal a role of Orb in translational repression and identify autophagy as an essential pathway regulated by Orb during early oogenesis. Importantly, they also establish translational regulation as a major mode of control of autophagy, a key process in cell homeostasis in response to environmental cues.

  12. Autophagy in Drosophila: From Historical Studies to Current Knowledge

    PubMed Central

    Mulakkal, Nitha C.; Nagy, Peter; Takats, Szabolcs; Tusco, Radu; Juhász, Gábor; Nezis, Ioannis P.

    2014-01-01

    The discovery of evolutionarily conserved Atg genes required for autophagy in yeast truly revolutionized this research field and made it possible to carry out functional studies on model organisms. Insects including Drosophila are classical and still popular models to study autophagy, starting from the 1960s. This review aims to summarize past achievements and our current knowledge about the role and regulation of autophagy in Drosophila, with an outlook to yeast and mammals. The basic mechanisms of autophagy in fruit fly cells appear to be quite similar to other eukaryotes, and the role that this lysosomal self-degradation process plays in Drosophila models of various diseases already made it possible to recognize certain aspects of human pathologies. Future studies in this complete animal hold great promise for the better understanding of such processes and may also help finding new research avenues for the treatment of disorders with misregulated autophagy. PMID:24949430

  13. HMGB1-dependent and -independent autophagy.

    PubMed

    Sun, Xiaofang; Tang, Daolin

    2014-10-01

    HMGB1 (high mobility group box 1) is a multifunctional, ubiquitous protein located inside and outside cells that plays a critical role in various physiological and pathological processes including cell development, differentiation, inflammation, immunity, metastasis, metabolism, and death. Increasing evidence demonstrates that HMGB1-dependent autophagy promotes chemotherapy resistance, sustains tumor metabolism requirements and T cell survival, prevents polyglutamine aggregates and excitotoxicity, and protects against endotoxemia, bacterial infection, and ischemia-reperfusion injury in vitro or in vivo. In contrast, HMGB1 may not be required for autophagy in some organs such as the liver and heart. Understanding HMGB1-dependent and -independent autophagy in more detail will provide insight into the integrated stress response and guide HMGB1-based therapeutic intervention.

  14. Autophagy, prion infection and their mutual interactions.

    PubMed

    Heiseke, Andreas; Aguib, Yasmine; Schatzl, Hermann M

    2010-01-01

    Prion diseases are infectious and fatal neurodegenerative disorders of man and animals which are characterized by spongiform degeneration in the central nervous system. Prion propagation involves the endocytic pathway and endosomal and lysosomal compartments are implicated in trafficking and re-cycling as well as final degradation of prions. Shifting the equilibrium between propagation and lysosomal clearance to the latter impairs cellular prion load. This and earlier findings of autophagic vacuoles in correlation to prion infections both in in vitro and in vivo studies prompted us and others to analyze the role of autophagy in prion infection. Autophagy is a fundamental cellular bulk degradation process for e.g. organelles or cytoplasmic proteins which has many implications for physiology and patho-physiology of cells and whole organisms. In various neurodegenerative disease models mainly protective functions of autophagy were recently described. In this review, we focus on recent findings which correlate autophagy and its manipulations with prion infection scenarios, and discuss perspectives and future directions. The findings summarized here add to the knowledge of the role of autophagy in neurodegeneration and provide interesting new insight into how non-cytosolic aggregated proteins might be subjected to autophagic clearance.

  15. Autophagy and Liver Ischemia-Reperfusion Injury

    PubMed Central

    2015-01-01

    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. PMID:25861623

  16. Links of autophagy dysfunction to inflammatory bowel disease onset

    PubMed Central

    El-Khider, Faris; McDonald, Christine

    2017-01-01

    Introduction Autophagy is a cellular stress response that plays key roles in physiological processes, such as adaptation to starvation, degradation of aberrant proteins or organelles, anti-microbial defense, protein secretion, and innate and adaptive immunity. Dysfunctional autophagy is recognized as a contributing factor in many chronic inflammatory diseases, including inflammatory bowel disease (IBD). Genetic studies have identified multiple IBD-associated risk loci that include genes required for autophagy, and several lines of evidence demonstrate that autophagy is impaired in IBD patients. How dysfunctional autophagy contributes to IBD onset is currently under investigation by researchers. Key messages Dysfunctional autophagy has been identified to play a role IBD pathogenesis by altering processes that include: (1) intracellular bacterial killing, (2) anti-microbial peptide secretion by Paneth cells, (3) pro-inflammatory cytokine production by macrophages, (4) antigen presentation by dendritic cells, (5) goblet cell function, and (6) the endoplasmic reticulum stress response in enterocytes. The overall effect of dysregulation of these processes varies by cell type, stimulus, as well as cellular context. Manipulation of the autophagic pathway may provide a new avenue in the search for effective therapies for IBD. Conclusion Autophagy plays multiple roles in IBD pathogenesis. A better understanding of the role of autophagy in IBD patients may provide better subclassification of IBD phenotypes and novel approaches to disease management. PMID:26982478

  17. Autophagy Is Associated with Pathogenesis of Haemophilus parasuis

    PubMed Central

    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

  18. Sestrin 1 ameliorates cardiac hypertrophy via autophagy activation.

    PubMed

    Xue, Ruicong; Zeng, Junyi; Chen, Yili; Chen, Cong; Tan, Weiping; Zhao, Jingjing; Dong, Bin; Sun, Yu; Dong, Yugang; Liu, Chen

    2017-02-09

    Cardiac hypertrophy is one of the major risk factors of cardiovascular morbidity and mortality. Autophagy is acknowledged to be an important mechanism regulating cardiac hypertrophy. Sestrin 1, a downstream target gene of p53, has been proven to regulate autophagy. However, the role of Sestrin 1 in cardiac hypertrophy remains unknown. Our study showed that Sestrin 1 mRNA and protein expression declined in pressure overload cardiac hypertrophy and phenylephrine (PE)-induced cardiac hypertrophy. Knockdown of Sestrin 1 by RNAi deteriorated PE-induced cardiac hypertrophy, whereas the overexpression of Sestrin 1 by adenovirus transfection blunted hypertrophy. We discovered that knockdown of Sestrin 1 resulted in impaired autophagy while overexpression of Sestrin 1 resulted in increased autophagy without affecting lysosomal function. In addition, the antihypertrophic effect of Sestrin 1 overexpression was eliminated by autophagy blockade. Importantly, Sestrin 1 targets at the AMPK/mTORC1/autophagy pathway to inhibit cardiac hypertrophy by interaction with AMPK which is responsible for autophagy regulation. Taken together, our data indicate that Sestrin 1 regulates AMPK/mTORC1/autophagy axis to attenuate cardiac hypertrophy.

  19. Role of autophagy in the pathogenesis of multiple sclerosis.

    PubMed

    Liang, Peizhou; Le, Weidong

    2015-08-01

    Autophagy plays an important role in maintaining the cellular homeostasis. One of its functions is to degrade unnecessary organelles and proteins for energy recycling or amino-acids for cell survival. Ablation of autophagy leads to neurodegeneration. Multiple sclerosis (MS), a permanent neurological impairment typical of chronic inflammatory demyelinating disorder, is an auto-immune disease of the central nervous system (CNS). Autophagy is tightly linked to the innate and adaptive immune systems during the autoimmune process, and several studies have shown that autophagy directly participates in the progress of MS or experimental autoimmune encephalomyelitis (EAE, a mouse model of MS). Dysfunction of mitochondria that intensively influences the autophagy pathway is one of the important factors in the pathogenesis of MS. Autophagy-related gene (ATG) 5 and immune-related GTPase M (IRGM) 1 are increased, while ATG16L2 is decreased, in T-cells in EAE and active relapsing-remitting MS brains. Administration of rapamycin, an inhibitor of mammalian target of rapamycin ( mTOR), ameliorates relapsing-remitting EAE. Inflammation and oxidative stress are increased in MS lesions and EAE, but Lamp2 and the LC3-II/LC3-I ratio are decreased. Furthermore, autophagy in various glial cells plays important roles in regulating neuro-inflammation in the CNS, implying potential roles in MS. In this review, we discuss the role of autophagy in the peripheral immune system and the CNS in neuroinflammation associated with the pathogenesis of MS.

  20. MTOR inhibition attenuates DNA damage and apoptosis through autophagy-mediated suppression of CREB1.

    PubMed

    Wang, Ying; Hu, Zhongdong; Liu, Zhibo; Chen, Rongrong; Peng, Haiyong; Guo, Jing; Chen, Xinxin; Zhang, Hongbing

    2013-12-01

    Hyperactivation of mechanistic target of rapamycin (MTOR) is a common feature of human cancers, and MTOR inhibitors, such as rapamycin, are thus becoming therapeutics in targeting certain cancers. However, rapamycin has also been found to compromise the efficacy of chemotherapeutics to cells with hyperactive MTOR. Here, we show that loss of TSC2 or PTEN enhanced etoposide-induced DNA damage and apoptosis, which was blunted by suppression of MTOR with either rapamycin or RNA interference. cAMP response element-binding protein 1 (CREB1), a nuclear transcription factor that regulates genes involved in survival and death, was positively regulated by MTOR in mouse embryonic fibroblasts (MEFs) and cancer cell lines. Silencing Creb1 expression with siRNA protected MTOR-hyperactive cells from DNA damage-induced apoptosis. Furthermore, loss of TSC2 or PTEN impaired either etoposide or nutrient starvation-induced autophagy, which in turn, leads to CREB1 hyperactivation. We further elucidated an inverse correlation between autophagy activity and CREB1 activity in the kidney tumor tissue obtained from a TSC patient and the mouse livers with hepatocyte-specific knockout of PTEN. CREB1 induced DNA damage and subsequent apoptosis in response to etoposide in autophagy-defective cells. Reactivation of CREB1 or inhibition of autophagy not only improved the efficacy of rapamycin but also alleviated MTOR inhibition-mediated chemoresistance. Therefore, autophagy suppression of CREB1 may underlie the MTOR inhibition-mediated chemoresistance. We suggest that inhibition of MTOR in combination with CREB1 activation may be used in the treatment of cancer caused by an abnormal PI3K-PTEN-AKT-TSC1/2-MTOR signaling pathway. CREB1 activators should potentiate the efficacy of chemotherapeutics in treatment of these cancers.

  1. Autophagy and Lipid Droplets in the Liver.

    PubMed

    Martinez-Lopez, Nuria; Singh, Rajat

    2015-01-01

    Autophagy is a conserved quality-control pathway that degrades cytoplasmic contents in lysosomes. Autophagy degrades lipid droplets through a process termed lipophagy. Starvation and an acute lipid stimulus increase autophagic sequestration of lipid droplets and their degradation in lysosomes. Accordingly, liver-specific deletion of the autophagy gene Atg7 increases hepatic fat content, mimicking the human condition termed nonalcoholic fatty liver disease. In this review, we provide insights into the molecular regulation of lipophagy, discuss fundamental questions related to the mechanisms by which autophagosomes recognize lipid droplets and how ATG proteins regulate membrane curvature for lipid droplet sequestration, and comment on the possibility of cross talk between lipophagy and cytosolic lipases in lipid mobilization. Finally, we discuss the contribution of lipophagy to the pathophysiology of human fatty liver disease. Understanding how lipophagy clears hepatocellular lipid droplets could provide new ways to prevent fatty liver disease, a major epidemic in developed nations.

  2. Autophagy in unicellular eukaryotes.

    PubMed

    Kiel, Jan A K W

    2010-03-12

    Cells need a constant supply of precursors to enable the production of macromolecules to sustain growth and survival. Unlike metazoans, unicellular eukaryotes depend exclusively on the extracellular medium for this supply. When environmental nutrients become depleted, existing cytoplasmic components will be catabolized by (macro)autophagy in order to re-use building blocks and to support ATP production. In many cases, autophagy takes care of cellular housekeeping to sustain cellular viability. Autophagy encompasses a multitude of related and often highly specific processes that are implicated in both biogenetic and catabolic processes. Recent data indicate that in some unicellular eukaryotes that undergo profound differentiation during their life cycle (e.g. kinetoplastid parasites and amoebes), autophagy is essential for the developmental change that allows the cell to adapt to a new host or form spores. This review summarizes the knowledge on the molecular mechanisms of autophagy as well as the cytoplasm-to-vacuole-targeting pathway, pexophagy, mitophagy, ER-phagy, ribophagy and piecemeal microautophagy of the nucleus, all highly selective forms of autophagy that have first been uncovered in yeast species. Additionally, a detailed analysis will be presented on the state of knowledge on autophagy in non-yeast unicellular eukaryotes with emphasis on the role of this process in differentiation.

  3. Autophagy in protists

    PubMed Central

    Duszenko, Michael; Ginger, Michael L; Brennand, Ana; Gualdrón-López, Melisa; Colombo, Maria-Isabel; Coombs, Graham H; Coppens, Isabelle; Jayabalasingham, Bamini; Langsley, Gordon; de Castro, Solange Lisboa; Menna-Barreto, Rubem; Mottram, Jeremy C; Navarro, Miguel; Rigden, Daniel J; Romano, Patricia S; Stoka, Veronika; Turk, Boris

    2011-01-01

    Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles and defense against parasitic invaders. During the past 10–20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target. PMID:20962583

  4. Induction of autophagy by second-fermentation yeasts during elaboration of sparkling wines.

    PubMed

    Cebollero, Eduardo; Gonzalez, Ramon

    2006-06-01

    Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.

  5. Induction of Autophagy by Second-Fermentation Yeasts during Elaboration of Sparkling Wines

    PubMed Central

    Cebollero, Eduardo; Gonzalez, Ramon

    2006-01-01

    Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed. PMID:16751523

  6. Environmental Correlation Analysis for Genes Associated with Protection against Malaria.

    PubMed

    Mackinnon, Margaret J; Ndila, Carolyne; Uyoga, Sophie; Macharia, Alex; Snow, Robert W; Band, Gavin; Rautanen, Anna; Rockett, Kirk A; Kwiatkowski, Dominic P; Williams, Thomas N

    2016-05-01

    Genome-wide searches for loci involved in human resistance to malaria are currently being conducted on a large scale in Africa using case-control studies. Here, we explore the utility of an alternative approach-"environmental correlation analysis, ECA," which tests for clines in allele frequencies across a gradient of an environmental selection pressure-to identify genes that have historically protected against death from malaria. We collected genotype data from 12,425 newborns on 57 candidate malaria resistance loci and 9,756 single nucleotide polymorphisms (SNPs) selected at random from across the genome, and examined their allele frequencies for geographic correlations with long-term malaria prevalence data based on 84,042 individuals living under different historical selection pressures from malaria in coastal Kenya. None of the 57 candidate SNPs showed significant (P < 0.05) correlations in allele frequency with local malaria transmission intensity after adjusting for population structure and multiple testing. In contrast, two of the random SNPs that had highly significant correlations (P < 0.01) were in genes previously linked to malaria resistance, namely, CDH13, encoding cadherin 13, and HS3ST3B1, encoding heparan sulfate 3-O-sulfotransferase 3B1. Both proteins play a role in glycoprotein-mediated cell-cell adhesion which has been widely implicated in cerebral malaria, the most life-threatening form of this disease. Other top genes, including CTNND2 which encodes δ-catenin, a molecular partner to cadherin, were significantly enriched in cadherin-mediated pathways affecting inflammation of the brain vascular endothelium. These results demonstrate the utility of ECA in the discovery of novel genes and pathways affecting infectious disease.

  7. Environmental Correlation Analysis for Genes Associated with Protection against Malaria

    PubMed Central

    Mackinnon, Margaret J.; Ndila, Carolyne; Uyoga, Sophie; Macharia, Alex; Snow, Robert W.; Band, Gavin; Rautanen, Anna; Rockett, Kirk A.; Kwiatkowski, Dominic P.; Williams, Thomas N.

    2016-01-01

    Genome-wide searches for loci involved in human resistance to malaria are currently being conducted on a large scale in Africa using case-control studies. Here, we explore the utility of an alternative approach—“environmental correlation analysis, ECA,” which tests for clines in allele frequencies across a gradient of an environmental selection pressure—to identify genes that have historically protected against death from malaria. We collected genotype data from 12,425 newborns on 57 candidate malaria resistance loci and 9,756 single nucleotide polymorphisms (SNPs) selected at random from across the genome, and examined their allele frequencies for geographic correlations with long-term malaria prevalence data based on 84,042 individuals living under different historical selection pressures from malaria in coastal Kenya. None of the 57 candidate SNPs showed significant (P < 0.05) correlations in allele frequency with local malaria transmission intensity after adjusting for population structure and multiple testing. In contrast, two of the random SNPs that had highly significant correlations (P < 0.01) were in genes previously linked to malaria resistance, namely, CDH13, encoding cadherin 13, and HS3ST3B1, encoding heparan sulfate 3-O-sulfotransferase 3B1. Both proteins play a role in glycoprotein-mediated cell-cell adhesion which has been widely implicated in cerebral malaria, the most life-threatening form of this disease. Other top genes, including CTNND2 which encodes δ-catenin, a molecular partner to cadherin, were significantly enriched in cadherin-mediated pathways affecting inflammation of the brain vascular endothelium. These results demonstrate the utility of ECA in the discovery of novel genes and pathways affecting infectious disease. PMID:26744416

  8. Boning up on autophagy

    PubMed Central

    Shapiro, Irving M; Layfield, Robert; Lotz, Martin; Settembre, Carmine; Whitehouse, Caroline

    2014-01-01

    From an evolutionary perspective, the major function of bone is to provide stable sites for muscle attachment and affording protection of vital organs, especially the heart and lungs (ribs) and spinal cord (vertebrae and intervertebral discs). However, bone has a considerable number of other functions: serving as a store for mineral ions, providing a site for blood cell synthesis and participating in a complex system-wide endocrine system. Not surprisingly, bone and cartilage cell homeostasis is tightly controlled, as is the maintenance of tissue structure and mass. While a great deal of new information is accruing concerning skeletal cell homeostasis, one relatively new observation is that the cells of bone (osteoclasts osteoblasts and osteocytes) and cartilage (chondrocytes) exhibit autophagy. The focus of this review is to examine the significance of this process in terms of the functional demands of the skeleton in health and during growth and to provide evidence that dysregulation of the autophagic response is involved in the pathogenesis of diseases of bone (Paget disease of bone) and cartilage (osteoarthritis and the mucopolysaccharidoses). Delineation of molecular changes in the autophagic process is uncovering new approaches for the treatment of diseases that affect the axial and appendicular skeleton. PMID:24225636

  9. Purinergic receptor X7 is a key modulator of metabolic oxidative stress-mediated autophagy and inflammation in experimental nonalcoholic steatohepatitis.

    PubMed

    Das, Suvarthi; Seth, Ratanesh Kumar; Kumar, Ashutosh; Kadiiska, Maria B; Michelotti, Gregory; Diehl, Anna Mae; Chatterjee, Saurabh

    2013-12-01

    Recent studies indicate that metabolic oxidative stress, autophagy, and inflammation are hallmarks of nonalcoholic steatohepatitis (NASH) progression. However, the molecular mechanisms that link these important events in NASH remain unclear. In this study, we investigated the mechanistic role of purinergic receptor X7 (P2X7) in modulating autophagy and resultant inflammation in NASH in response to metabolic oxidative stress. The study uses two rodent models of NASH. In one of them, a CYP2E1 substrate bromodichloromethane is used to induce metabolic oxidative stress and NASH. Methyl choline-deficient diet feeding is used for the other NASH model. CYP2E1 and P2X7 receptor gene-deleted mice are used to establish their roles in regulating metabolic oxidative stress and autophagy. Autophagy gene expression, protein levels, confocal microscopy based-immunolocalization of lysosome-associated membrane protein (LAMP)2A and histopathological analysis were performed. CYP2E1-dependent metabolic oxidative stress induced increases in P2X7 receptor expression and chaperone-mediated autophagy markers LAMP2A and heat shock cognate 70 but caused depletion of light chain 3 isoform B (LC3B) protein levels. P2X7 receptor gene deletion significantly decreased LAMP2A and inflammatory indicators while significantly increasing LC3B protein levels compared with wild-type mice treated with bromodichloromethane. P2X7 receptor-deleted mice were also protected from NASH pathology as evidenced by decreased inflammation and fibrosis. Our studies establish that P2X7 receptor is a key regulator of autophagy induced by metabolic oxidative stress in NASH, thereby modulating hepatic inflammation. Furthermore, our findings presented here form a basis for P2X7 receptor as a potential therapeutic target in the treatment for NASH.

  10. Autophagy regulates pancreatic beta cell death in response to Pdx1 deficiency and nutrient deprivation.

    PubMed

    Fujimoto, Kei; Hanson, Piia T; Tran, Hung; Ford, Eric L; Han, Zhiqiang; Johnson, James D; Schmidt, Robert E; Green, Karen G; Wice, Burton M; Polonsky, Kenneth S

    2009-10-02

    There are three types of cell death; apoptosis, necrosis, and autophagy. The possibility that activation of the macroautophagy (autophagy) pathway may increase beta cell death is addressed in this study. Increased autophagy was present in pancreatic islets from Pdx1(+/-) mice with reduced insulin secretion and beta cell mass. Pdx1 expression was reduced in mouse insulinoma 6 (MIN6) cells by delivering small hairpin RNAs using a lentiviral vector. The MIN6 cells died after 7 days of Pdx1 deficiency, and autophagy was evident prior to the onset of cell death. Inhibition of autophagy prolonged cell survival and delayed cell death. Nutrient deprivation increased autophagy in MIN6 cells and mouse and human islets after starvation. Autophagy inhibition partly prevented amino acid starvation-induced MIN6 cell death. The in vivo effects of reduced autophagy were studied by crossing Pdx1(+/-) mice to Becn1(+/-) mice. After 1 week on a high fat diet, 4-week-old Pdx1(+/-) Becn1(+/-) mice showed normal glucose tolerance, preserved beta cell function, and increased beta cell mass compared with Pdx1(+/-) mice. This protective effect of reduced autophagy had worn off after 7 weeks on a high fat diet. Increased autophagy contributes to pancreatic beta cell death in Pdx1 deficiency and following nutrient deprivation. The role of autophagy should be considered in studies of pancreatic beta cell death and diabetes and as a target for novel therapeutic intervention.

  11. Autophagy Is an Innate Mechanism Associated with Leprosy Polarization

    PubMed Central

    Andrade, Priscila Ribeiro; Ferreira, Helen; Nery, José Augusto da Costa; Côrte-Real, Suzana; da Silva, Gilberto Marcelo Sperandio; Rosa, Patricia Sammarco; Fabri, Mario; Sarno, Euzenir Nunes

    2017-01-01

    Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages. PMID:28056107

  12. Autophagy drives epidermal deterioration in a Drosophila model of tissue aging.

    PubMed

    Scherfer, Christoph; Han, Violet C; Wang, Yan; Anderson, Aimee E; Galko, Michael J

    2013-04-01

    Organismal lifespan has been the primary readout in aging research. However, how longevity genes control tissue-specific aging remains an open question. To examine the crosstalk between longevity programs and specific tissues during aging, biomarkers of organ-specific aging are urgently needed. Since the earliest signs of aging occur in the skin, we sought to examine skin aging in a genetically tractable model. Here we introduce a Drosophila model of skin aging. The epidermis undergoes a dramatic morphological deterioration with age that includes membrane and nuclear loss. These changes were decelerated in a long-lived mutant and accelerated in a short-lived mutant. An increase in autophagy markers correlated with epidermal aging. Finally, the epidermis of Atg7 mutants retained younger characteristics, suggesting that autophagy is a critical driver of epidermal aging. This is surprising given that autophagy is generally viewed as protective during aging. Since Atg7 mutants are short-lived, the deceleration of epidermal aging in this mutant suggests that in the epidermis healthspan can be uncoupled from longevity. Because the aging readout we introduce here has an early onset and is easily visualized, genetic dissection using our model should identify other novel mechanisms by which lifespan genes feed into tissue-specific aging.

  13. Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase-Dependent Cell Death and Leads to Mild Growth Retardation.

    PubMed

    Vuppalapati, Karuna K; Bouderlique, Thibault; Newton, Phillip T; Kaminskyy, Vitaliy O; Wehtje, Henrik; Ohlsson, Claes; Zhivotovsky, Boris; Chagin, Andrei S

    2015-12-01

    Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter-driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes.

  14. PTPRO-mediated autophagy prevents hepatosteatosis and tumorigenesis.

    PubMed

    Zhang, Wenjie; Hou, Jiajie; Wang, Xiaochen; Jiang, Runqiu; Yin, Yin; Ji, Jie; Deng, Lei; Huang, Xingxu; Wang, Ke; Sun, Beicheng

    2015-04-20

    Autophagy plays a critical role in the progression of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Protein tyrosine phosphatase receptor type O (PTPRO) was recently identified as a tumor suppressor, but little is known about its role in NASH. Here, we investigated the role of PTPRO-dependent autophagy in insulin resistance, lipid metabolism, and hepatocarcinogenesis. Wild-type (WT) and ptpro-/- mice were fed a high-fat diet (HFD) for another 16 weeks after diethylnitrosamine (DEN) injection to induce NASH. Ptpro-/- mice exhibited severe liver injury, insulin resistance, hepatosteatosis and autophagy deficiency compared with WT littermates. PTPRO deletion also promoted the induction of lipogenic target genes and decreases in β-oxidation-related genes. Increased activation of AKT and accumulation of cytoplasmic p53 was detected in ptpro-/- mice, which in combination repressed autophagy. Intriguingly, hyperinsulinemia involving AKT activation was also exacerbated in HFD-fed mice due to PTPRO deletion. Activation of AKT induced stabilization of the MDMX/MDM2 heterocomplex, thus promoting p53 accumulation in the cytoplasm. Inhibition of AKT restored autophagy and p53 accumulation in hepatocytes, indicating that AKT acts upstream of p53. Due to hyperinsulinemia and autophagy deficiency, a HFD could aggravate steatohepatitis in ptpro-/- mice. Importantly, the expression of PTPRO was much decreased in human steatohepatitis, which was associated with increased p62 accumulation. Together, these data indicate that PTPRO regulates insulin and lipid metabolism via the PI3K/Akt/MDM4/MDM2/P53 axis by affecting autophagy.

  15. Ceramide metabolism regulates autophagy and apoptotic cell death induced by melatonin in liver cancer cells.

    PubMed

    Ordoñez, Raquel; Fernández, Anna; Prieto-Domínguez, Néstor; Martínez, Laura; García-Ruiz, Carmen; Fernández-Checa, José C; Mauriz, José L; González-Gallego, Javier

    2015-09-01

    Autophagy is a process that maintains homeostasis during stress, although it also contributes to cell death under specific contexts. Ceramides have emerged as important effectors in the regulation of autophagy, mediating the crosstalk with apoptosis. Melatonin induces apoptosis of cancer cells; however, its role in autophagy and ceramide metabolism has yet to be clearly elucidated. This study was aimed to evaluate the effect of melatonin administration on autophagy and ceramide metabolism and its possible link with melatonin-induced apoptotic cell death in hepatocarcinoma (HCC) cells. Melatonin (2 mm) transiently induced autophagy in HepG2 cells through JNK phosphorylation, characterized by increased Beclin-1 expression, p62 degradation, and LC3II and LAMP-2 colocalization, which translated in decreased cell viability. Moreover, ATG5 silencing sensitized HepG2 cells to melatonin-induced apoptosis, suggesting a dual role of autophagy in cell death. Melatonin enhanced ceramide levels through both de novo synthesis and acid sphingomyelinase (ASMase) stimulation. Serine palmitoyltransferase (SPT) inhibition with myriocin prevented melatonin-induced autophagy and ASMase inhibition with imipramine-impaired autophagy flux. However, ASMase inhibition partially protected HepG2 cells against melatonin, while SPT inhibition significantly enhanced cell death. Findings suggest a crosstalk between SPT-mediated ceramide generation and autophagy in protecting against melatonin, while specific ASMase-induced ceramide production participates in melatonin-mediated cell death. Thus, dual blocking of SPT and autophagy emerges as a potential strategy to potentiate the apoptotic effects of melatonin in liver cancer cells.

  16. Lithium and Autophagy

    PubMed Central

    2014-01-01

    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

  17. The Autophagy-Senescence Connection in Chemotherapy: Must Tumor Cells (Self) Eat Before They Sleep?

    PubMed Central

    Goehe, Rachel W.; Di, Xu; Sharma, Khushboo; Bristol, Molly L.; Henderson, Scott C.; Valerie, Kristoffer; Rodier, Francis; Davalos, Albert R.

    2012-01-01

    Exposure of MCF-7 breast tumor cells or HCT-116 colon carcinoma cells to clinically relevant concentrations of doxorubicin (Adriamycin; Farmitalia Research Laboratories, Milan, Italy) or camptothecin results in both autophagy and senescence. To determine whether autophagy is required for chemotherapy-induced senescence, reactive oxygen generation induced by Adriamycin was suppressed by N-acetyl cysteine and glutathione, and the induction of ataxia telangiectasia mutated, p53, and p21 was modulated pharmacologically and/or genetically. In all cases, autophagy and senescence were collaterally suppressed. The close association between autophagy and senescence indicated by these experiments reflects their collateral regulation via common signaling pathways. The potential relationship between autophagy and senescence was further examined through pharmacologic inhibition of autophagy with chloroquine and 3-methyl-adenine and genetic ablation of the autophagy-related genes ATG5 and ATG7. However, inhibition of autophagy by pharmacological and genetic approaches could not entirely abrogate the senescence response, which was only reduced and/or delayed. Taken together, our findings suggest that autophagy and senescence tend to occur in parallel, and furthermore that autophagy accelerates the development of the senescent phenotype. However, these responses are not inexorably linked or interdependent, as senescence can occur when autophagy is abrogated. PMID:22927544

  18. The autophagy-senescence connection in chemotherapy: must tumor cells (self) eat before they sleep?

    PubMed

    Goehe, Rachel W; Di, Xu; Sharma, Khushboo; Bristol, Molly L; Henderson, Scott C; Valerie, Kristoffer; Rodier, Francis; Davalos, Albert R; Gewirtz, David A

    2012-12-01

    Exposure of MCF-7 breast tumor cells or HCT-116 colon carcinoma cells to clinically relevant concentrations of doxorubicin (Adriamycin; Farmitalia Research Laboratories, Milan, Italy) or camptothecin results in both autophagy and senescence. To determine whether autophagy is required for chemotherapy-induced senescence, reactive oxygen generation induced by Adriamycin was suppressed by N-acetyl cysteine and glutathione, and the induction of ataxia telangiectasia mutated, p53, and p21 was modulated pharmacologically and/or genetically. In all cases, autophagy and senescence were collaterally suppressed. The close association between autophagy and senescence indicated by these experiments reflects their collateral regulation via common signaling pathways. The potential relationship between autophagy and senescence was further examined through pharmacologic inhibition of autophagy with chloroquine and 3-methyl-adenine and genetic ablation of the autophagy-related genes ATG5 and ATG7. However, inhibition of autophagy by pharmacological and genetic approaches could not entirely abrogate the senescence response, which was only reduced and/or delayed. Taken together, our findings suggest that autophagy and senescence tend to occur in parallel, and furthermore that autophagy accelerates the development of the senescent phenotype. However, these responses are not inexorably linked or interdependent, as senescence can occur when autophagy is abrogated.

  19. Autophagy in Trypanosomatids

    PubMed Central

    Brennand, Ana; Rico, Eva; Michels, Paul A. M.

    2012-01-01

    Autophagy is a ubiquitous eukaryotic process that also occurs in trypanosomatid parasites, protist organisms belonging to the supergroup Excavata, distinct from the supergroup Opistokontha that includes mammals and fungi. Half of the known yeast and mammalian AuTophaGy (ATG) proteins were detected in trypanosomatids, although with low sequence conservation. Trypanosomatids such as Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for serious tropical diseases in humans. The parasites are transmitted by insects and, consequently, have a complicated life cycle during which they undergo dramatic morphological and metabolic transformations to adapt to the different environments. Autophagy plays a major role during these transformations. Since inhibition of autophagy affects the transformation, survival and/or virulence of the parasites, the ATGs offer promise for development of drugs against tropical diseases. Furthermore, various trypanocidal drugs have been shown to trigger autophagy-like processes in the parasites. It is inferred that autophagy is used by the parasites in an—not always successful—attempt to cope with the stress caused by the toxic compounds. PMID:24710480

  20. Autophagy in Cancer Metastasis

    PubMed Central

    Mowers, Erin E.; Sharifi, Marina N.; Macleod, Kay F.

    2016-01-01

    Autophagy is a highly conserved self-degradative process that plays a key role in cellular stress responses and survival. Recent work has begun to explore the function of autophagy in cancer metastasis, which is of particular interest given the dearth of effective therapeutic options for metastatic disease. Autophagy is induced upon progression of various human cancers to metastasis and together with data from genetically engineered mice and experimental metastasis models, a role for autophagy at nearly every phase of the metastatic cascade has been identified. Specifically, autophagy has been shown to be involved in modulating tumor cell motility and invasion, cancer stem cell viability and differentiation, resistance to anoikis, epithelial-to-mesenchymal transition, tumor cell dormancy and escape from immune surveillance, with emerging functions in establishing the pre-metastatic niche and other aspects of metastasis. In this review, we provide a general overview of how autophagy modulates cancer metastasis and discuss the significance of new findings for disease management. PMID:27593926

  1. PINK1 deficiency enhances autophagy and mitophagy induction

    PubMed Central

    Gómez-Sánchez, Rubén; Yakhine-Diop, Sokhna M S; Bravo-San Pedro, José M; Pizarro-Estrella, Elisa; Rodríguez-Arribas, Mario; Climent, Vicente; Martin-Cano, Francisco E; González-Soltero, María E; Tandon, Anurag; Fuentes, José M; González-Polo, Rosa A

    2016-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder with poorly understood etiology. Increasing evidence suggests that age-dependent compromise of the maintenance of mitochondrial function is a key risk factor. Several proteins encoded by PD-related genes are associated with mitochondria including PTEN-induced putative kinase 1 (PINK1), which was first identified as a gene that is upregulated by PTEN. Loss-of-function PINK1 mutations induce mitochondrial dysfunction and, ultimately, neuronal cell death. To mitigate the negative effects of altered cellular functions cells possess a degradation mechanism called autophagy for recycling damaged components; selective elimination of dysfunctional mitochondria by autophagy is termed mitophagy. Our study indicates that autophagy and mitophagy are upregulated in PINK1-deficient cells, and is the first report to demonstrate efficient fluxes by one-step analysis. We propose that autophagy is induced to maintain cellular homeostasis under conditions of non-regulated mitochondrial quality control. PMID:27308585

  2. PINK1 deficiency enhances autophagy and mitophagy induction.

    PubMed

    Gómez-Sánchez, Rubén; Yakhine-Diop, Sokhna M S; Bravo-San Pedro, José M; Pizarro-Estrella, Elisa; Rodríguez-Arribas, Mario; Climent, Vicente; Martin-Cano, Francisco E; González-Soltero, María E; Tandon, Anurag; Fuentes, José M; González-Polo, Rosa A

    2016-03-01

    Parkinson's disease (PD) is a neurodegenerative disorder with poorly understood etiology. Increasing evidence suggests that age-dependent compromise of the maintenance of mitochondrial function is a key risk factor. Several proteins encoded by PD-related genes are associated with mitochondria including PTEN-induced putative kinase 1 (PINK1), which was first identified as a gene that is upregulated by PTEN. Loss-of-function PINK1 mutations induce mitochondrial dysfunction and, ultimately, neuronal cell death. To mitigate the negative effects of altered cellular functions cells possess a degradation mechanism called autophagy for recycling damaged components; selective elimination of dysfunctional mitochondria by autophagy is termed mitophagy. Our study indicates that autophagy and mitophagy are upregulated in PINK1-deficient cells, and is the first report to demonstrate efficient fluxes by one-step analysis. We propose that autophagy is induced to maintain cellular homeostasis under conditions of non-regulated mitochondrial quality control.

  3. Here, there be dragons: charting autophagy-related alterations in human tumors.

    PubMed

    Lebovitz, Chandra B; Bortnik, Svetlana B; Gorski, Sharon M

    2012-03-01

    Macroautophagy (or autophagy) is a catabolic cellular process that is both homeostatic and stress adaptive. Normal cells rely on basal levels of autophagy to maintain cellular integrity (via turnover of long-lived proteins and damaged organelles) and increased levels of autophagy to buoy cell survival during various metabolic stresses (via nutrient and energy provision through lysosomal degradation of cytoplasmic components). Autophagy can function in both tumor suppression and tumor progression, and is under investigation in clinical trials as a novel target for anticancer therapy. However, its role in cancer pathogenesis has yet to be fully explored. In particular, it remains unknown whether in vitro observations will be applicable to human cancer patients. Another outstanding question is whether there exists tumor-specific selection for alterations in autophagy function. In this review, we survey reported mutations in autophagy genes and key autophagy regulators identified in human tumor samples and summarize the literature regarding expression levels of autophagy genes and proteins in various cancer tissues. Although it is too early to draw inferences from this collection of in vivo studies of autophagy-related alterations in human cancers, their results highlight the challenges that must be overcome before we can accurately assess the scope of autophagy's predicted role in tumorigenesis.

  4. Autophagy supports survival and phototransduction protein levels in rod photoreceptors

    PubMed Central

    Zhou, Z; Doggett, T A; Sene, A; Apte, R S; Ferguson, T A

    2015-01-01

    Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod

  5. Intestinal autophagy activity is essential for host defense against Salmonella typhimurium infection in Caenorhabditis elegans.

    PubMed

    Curt, Alexander; Zhang, Jiuli; Minnerly, Justin; Jia, Kailiang

    2014-08-01

    Salmonella typhimurium infects both intestinal epithelial cells and macrophages. Autophagy is a lysosomal degradation pathway that is present in all eukaryotes. Autophagy has been reported to limit the Salmonella replication in Caenorhabditis elegans and in mammals. However, it is unknown whether intestinal autophagy activity plays a role in host defense against Salmonella infection in C. elegans. In this study, we inhibited the autophagy gene bec-1 in different C. elegans tissues and examined the survival of these animals following Salmonella infection. Here we show that inhibition of the bec-1 gene in the intestine but not in other tissues confers susceptibility to Salmonella infection, which is consistent with recent studies in mice showing that autophagy is involved in clearance of Salmonella in the intestinal epithelial cells. Therefore, the intestinal autophagy activity is essential for host defense against Salmonella infection from C. elegans to mice, perhaps also in humans.

  6. An expanded role for mTORC1 in autophagy.

    PubMed

    Kim, Young-Mi; Park, Ji-Man; Grunwald, Douglas; Kim, Do-Hyung

    2016-01-01

    Mechanistic target of rapamycin complex 1 (mTORC1) negatively regulates autophagy at early stages by phosphorylating Unc51-like kinase 1 (ULK1). Our recent study expanded the roles of mTORC1 in autophagy by identifying ultraviolet radiation resistance-associated gene product (UVRAG) as a substrate of mTORC1. This finding has provided new insight into the roles of mTORC1 in cellular membrane processes and cancer.

  7. The autophagy-related genes BbATG1 and BbATG8 have different functions in differentiation, stress resistance and virulence of mycopathogen Beauveria bassiana

    PubMed Central

    Ying, Sheng-Hua; Liu, Jing; Chu, Xin-Ling; Xie, Xue-Qin; Feng, Ming-Guang

    2016-01-01

    Autophagy-related proteins play significantly different roles in eukaryotes. In the entomopathogenic fungus Beauveria bassiana, autophagy is associated with fungal growth and development. BbATG1 (a serine/threonine protein kinase) and BbATG8 (a ubiquitin-like protein) have similar roles in autophagy, but different roles in other processes. Disruption mutants of BbATG1 and BbATG8 had impaired conidial germination under starvation stress. The mutant ΔBbATG8 exhibited enhanced sensitivity to oxidative stress, while a ΔBbATG1 mutant did not. BbATG1 and BbATG8 showed different roles in spore differentiation. The blastospore yield was reduced by 70% and 92% in ΔBbATG1 and ΔBbATG8 mutants, respectively, and the double mutant had a reduction of 95%. Conidial yield was reduced by approximately 90% and 50% in ΔBbATG1 and ΔBbATG8 mutants, respectively. A double mutant had a reduction similar to ΔBbATG1. Additionally, both BbATG1 and BbATG8 affected the levels of conidial protein BbCP15p required for conidiation. The virulence of each autophagy-deficient mutant was considerably weakened as indicated in topical and intrahemocoel injection assays, and showed a greater reduction in topical infection. However, BbATG1 and BbATG8 had different effects on fungal virulence. Our data indicate that these autophagy-related proteins have different functions in fungal stress response, asexual development and virulence. PMID:27197558

  8. The autophagy-related genes BbATG1 and BbATG8 have different functions in differentiation, stress resistance and virulence of mycopathogen Beauveria bassiana.

    PubMed

    Ying, Sheng-Hua; Liu, Jing; Chu, Xin-Ling; Xie, Xue-Qin; Feng, Ming-Guang

    2016-05-20

    Autophagy-related proteins play significantly different roles in eukaryotes. In the entomopathogenic fungus Beauveria bassiana, autophagy is associated with fungal growth and development. BbATG1 (a serine/threonine protein kinase) and BbATG8 (a ubiquitin-like protein) have similar roles in autophagy, but different roles in other processes. Disruption mutants of BbATG1 and BbATG8 had impaired conidial germination under starvation stress. The mutant ΔBbATG8 exhibited enhanced sensitivity to oxidative stress, while a ΔBbATG1 mutant did not. BbATG1 and BbATG8 showed different roles in spore differentiation. The blastospore yield was reduced by 70% and 92% in ΔBbATG1 and ΔBbATG8 mutants, respectively, and the double mutant had a reduction of 95%. Conidial yield was reduced by approximately 90% and 50% in ΔBbATG1 and ΔBbATG8 mutants, respectively. A double mutant had a reduction similar to ΔBbATG1. Additionally, both BbATG1 and BbATG8 affected the levels of conidial protein BbCP15p required for conidiation. The virulence of each autophagy-deficient mutant was considerably weakened as indicated in topical and intrahemocoel injection assays, and showed a greater reduction in topical infection. However, BbATG1 and BbATG8 had different effects on fungal virulence. Our data indicate that these autophagy-related proteins have different functions in fungal stress response, asexual development and virulence.

  9. A dual role of p53 in the control of autophagy.

    PubMed

    Tasdemir, Ezgi; Chiara Maiuri, M; Morselli, Eugenia; Criollo, Alfredo; D'Amelio, Marcello; Djavaheri-Mergny, Mojgan; Cecconi, Francesco; Tavernarakis, Nektarios; Kroemer, Guido

    2008-08-01

    Genotoxic stress can induce autophagy in a p53-dependent fashion and p53 can transactivate autophagy-inducing genes. We have observed recently that inactivation of p53 by deletion, depletion or inhibition can trigger autophagy. Thus, human and mouse cells subjected to knockout, knockdown or pharmacological inhibition of p53 manifest signs of autophagy such as depletion of p62/SQSTM1, LC3 lipidation, redistribution of GFP-LC3 in cytoplasmic puncta, and accumulation of autophagosomes and autolysosomes, both in vitro and in vivo. Inhibition of p53 causes autophagy in enucleated cells, indicating that the cytoplasmic, non-nuclear pool of p53 can regulate autophagy. Accordingly, retransfection of p53(-/-) cells with wild-type p53 as well as a p53 mutant that is excluded from the nucleus (due to the deletion of the nuclear localization sequence) can inhibit autophagy, whereas retransfection with a nucleus-restricted p53 mutant (in which the nuclear localization sequence has been deleted) does not inhibit autophagy. Several distinct autophagy inducers (e.g., starvation, rapamycin, lithium, tunicamycin and thapsigargin) stimulate the rapid degradation of p53. In these conditions, inhibition of the p53-specific E3 ubiquitin ligase HDM2 can avoid p53 depletion and simultaneously prevent the activation of autophagy. Moreover, a p53 mutant that lacks the HDM2 ubiquitinylation site and hence is more stable than wild-type p53 is particularly efficient in suppressing autophagy. In conclusion, p53 plays a dual role in the control of autophagy. On the one hand, nuclear p53 can induce autophagy through transcriptional effects. On the other hand, cytoplasmic p53 may act as a master repressor of autophagy.

  10. Autophagy and Transporter-Based Multi-Drug Resistance

    PubMed Central

    Kumar, Priyank; Zhang, Dong-Mei; Degenhardt, Kurt; Chen, Zhe-Sheng

    2012-01-01

    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. PMID:24710490

  11. Autophagy and microRNA dysregulation in liver diseases.

    PubMed

    Kim, Kyu Min; Kim, Sang Geon

    2014-01-01

    Autophagy is a catabolic process through which organelles and cellular components are sequestered into autophagosomes and degraded via fusion with lysosomes. Autophagy plays a role in many physiological processes, including stress responses, energy homeostasis, elimination of cellular organelles, and tissue remodeling. In addition, autophagy capacity changes in various disease states. A series of studies have shown that autophagy is strictly controlled to maintain homeostatic balance of energy metabolism and cellular organelle and protein turnover. These studies have also shown that this process is post-transcriptionally controlled by small noncoding microRNAs that regulate gene expression through complementary base pairing with mRNAs. Conversely, autophagy regulates the expression of microRNAs. Therefore, dysregulation of the link between autophagy and microRNA expression exacerbates the pathogenesis of various diseases. In this review, we summarize the roles of autophagy and microRNA dysregulation in the course of liver diseases, with the aim of understanding how microRNAs modify key autophagic effector molecules, and we discuss how this dysregulation affects both physiological and pathological conditions. This article may advance our understanding of the cellular and molecular bases of liver disease progression and promote the development of strategies for pharmacological intervention.

  12. Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

    PubMed

    Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I

    2016-07-25

    Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes.

  13. Autophagy in osteoblasts is involved in mineralization and bone homeostasis

    PubMed Central

    Nollet, Marie; Santucci-Darmanin, Sabine; Breuil, Véronique; Al-Sahlanee, Rasha; Cros, Chantal; Topi, Majlinda; Momier, David; Samson, Michel; Pagnotta, Sophie; Cailleteau, Laurence; Battaglia, Séverine; Farlay, Delphine; Dacquin, Romain; Barois, Nicolas; Jurdic, Pierre; Boivin, Georges; Heymann, Dominique; Lafont, Frank; Lu, Shi Shou; Dempster, David W; Carle, Georges F; Pierrefite-Carle, Valérie

    2014-01-01

    Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies. PMID:25484092

  14. Toxic metals and autophagy.

    PubMed

    Chatterjee, Sarmishtha; Sarkar, Shuvasree; Bhattacharya, Shelley

    2014-11-17

    The earth's resources are finite, and it can no longer be considered a source of inexhaustible bounty for the human population. However, this realization has not been able to contain the human desire for rapid industrialization. The collateral to overusing environmental resources is the high-level contamination of undesirable toxic metals, leading to bioaccumulation and cellular damage. Cytopathological features of biological systems represent a key variable in several diseases. A review of the literature revealed that autophagy (PCDII), a high-capacity process, may consist of selective elimination of vital organelles and/or proteins that intiate mechanisms of cytoprotection and homeostasis in different biological systems under normal physiological and stress conditions. However, the biological system does survive under various environmental stressors. Currently, there is no consensus that specifies a particular response as being a dependable biomarker of toxicology. Autophagy has been recorded as the initial response of a cell to a toxic metal in a concentration- and time-dependent manner. Various signaling pathways are triggered through cellular proteins and/or protein kinases that can lead to autophagy, apoptosis (or necroptosis), and necrosis. Although the role of autophagy in tumorigenesis is associated with promoting tumor cell survival and/or acting as a tumor suppressive mechanism, PCDII in metal-induced toxicity has not been extensively studied. The aim of this review is to analyze the comparative cytotoxicity of metals/metalloids and nanoparticles (As, Cd, Cr, Hg, Fe, and metal-NP) in cells enduring autophagy. It is noted that metals/metalloids and nanoparticles prefer ATG8/LC3 as a potent inducer of autophagy in several cell lines or animal cells. MAP kinases, death protein kinases, PI3K, AKT, mTOR, and AMP kinase have been found to be the major components of autophagy induction or inhibition in the context of cellular responses to metals/metalloids and

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

    PubMed Central

    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

  16. The Autophagy Enhancer Spermidine Reverses Arterial Aging

    PubMed Central

    LaRocca, Thomas J.; Gioscia-Ryan, Rachel A.; Hearon, Christopher M.; Seals, Douglas R.

    2013-01-01

    Arterial aging, characterized by stiffening of large elastic arteries and the development of arterial endothelial dysfunction, increases cardiovascular disease (CVD) risk. We tested the hypothesis that spermidine, a nutrient associated with the anti-aging process autophagy, would improve arterial aging. Aortic pulse wave velocity (aPWV), a measure of arterial stiffness, was ~20% greater in old (O, 28 months) compared with young C57BL6 mice (Y, 4 months, P < 0.05). Arterial endothelium-dependent dilation (EDD), a measure of endothelial function, was ~25% lower in O (P < 0.05 vs. Y) due to reduced nitric oxide (NO) bioavailability. These impairments were associated with greater arterial oxidative stress (nitrotyrosine), superoxide production, and protein cross-linking (advanced glycation end-products, AGEs) in O (all P < 0.05). Spermidine supplementation normalized aPWV, restored NO-mediated EDD and reduced nitrotyrosine, superoxide, AGEs and collagen in O. These effects of spermidine were associated with enhanced arterial expression of autophagy markers, and in vitro experiments demonstrated that vascular protection by spermidine was autophagy-dependent. Our results indicate that spermidine exerts a potent anti-aging influence on arteries by increasing NO bioavailability, reducing oxidative stress, modifying structural factors and enhancing autophagy. Spermidine may be a promising nutraceutical treatment for arterial aging and prevention of age-associated CVD. PMID:23612189

  17. The renoprotective role of autophagy activation in proximal tubular epithelial cells in diabetic nephropathy.

    PubMed

    Xu, Ying; Liu, Lei; Xin, Wei; Zhao, Xu; Chen, Liyong; Zhen, Junhui; Wan, Qiang

    2015-01-01

    With intensive investigations recently, autophagy is hoped to be a potential therapeutic target to prevent or alleviate diabetic nephropathy (DN). Our previous study revealed that lipotoxicity participated in epithelial-to-mesenchymal transition (EMT) of proximal tubular epithelial cells (PTECs) under diabetic conditions. Based on evidences that autophagy and lipid metabolism are closely related, we investigated autophagy under diabetic conditions and how it contributed in the lipotoxicity and EMT. In high-glucose-cultured PTECs, we found that Beclin1 and LC3-II were elevated, while p62 was decreased. These results indicate that autophagy activity was elevated under diabetic conditions. Autophagy deficiency induced by autophagy inhibitors, chloroquine diphosphate (CQ) and 3-Methyladenine (3-MA), and by Atg5 siRNA transfection exacerbated lipid accumulation and EMT. This supports that the elevated autophagy activity acts as a renoprotective response under diabetic conditions. Treatment of rapamycin, which is a mammalian target of rapamycin (mTOR) receptor-specific inhibitor and a known autophagy activator, attenuated high-glucose-induced lipid accumulation and EMT. The Atg5 silence counteracted the protective effect of rapamycin. The present study deepens our understanding of the role of autophagy in DN, suggesting a complex interplay of autophagy, metabolic pathways, lipotoxicity and EMT.

  18. Intestinal Autophagy and Its Pharmacological Control in Inflammatory Bowel Disease

    PubMed Central

    Ke, Ping; Shao, Bo-Zong; Xu, Zhe-Qi; Chen, Xiong-Wen; Liu, Chong

    2017-01-01

    Intestinal mucosal barrier, mainly composed of the intestinal mucus layer and the epithelium, plays a critical role in nutrient absorption as well as protection from pathogenic microorganisms. It is widely acknowledged that the damage of intestinal mucosal barrier or the disturbance of microorganism balance in the intestinal tract contributes greatly to the pathogenesis and progression of inflammatory bowel disease (IBD), which mainly includes Crohn’s disease and ulcerative colitis. Autophagy is an evolutionarily conserved catabolic process that involves degradation of protein aggregates and damaged organelles for recycling. The roles of autophagy in the pathogenesis and progression of IBD have been increasingly studied. This present review mainly describes the roles of autophagy of Paneth cells, macrophages, and goblet cells in IBD, and finally, several potential therapeutic strategies for IBD taking advantage of autophagy. PMID:28119697

  19. Resveratrol inhibits TIGAR to promote ROS induced apoptosis and autophagy.

    PubMed

    Kumar, Bhupender; Iqbal, Mohammad Askandar; Singh, Rajnish Kumar; Bamezai, Rameshwar N K

    2015-11-01

    Resveratrol has been shown to exhibit its anti-cancer effect through a variety of mechanisms. Here, TIGAR (TP53-Induced Glycolysis and Apoptosis Regulator) was identified as an important target of resveratrol for exhibiting ROS-dependent-consequences on apoptosis and autophagy. Resveratrol treatment decreased TIGAR protein irrespective of cell line used. Down-regulated TIGAR protein triggered a drop in reduced-glutathione levels which resulted in sustained ROS, responsible for apoptosis and autophagy. Over-expression and silencing experiments demonstrated the importance of TIGAR in affecting the ROS-dependent anti-cancer effects of resveratrol. Resveratrol treated cells exhibited autophagy to escape apoptosis, however, chloroquine treatment along with resveratrol, blocked protective autophagy and facilitated apoptosis. Collectively, results unravel the effects of resveratrol on TIGAR in mediating its ROS dependent influence and suggest a better combination therapy of resveratrol and chloroquine for probable cancer treatment.

  20. Oxidative stress contributes to autophagy induction in response to endoplasmic reticulum stress in Chlamydomonas reinhardtii.

    PubMed

    Pérez-Martín, Marta; Pérez-Pérez, María Esther; Lemaire, Stéphane D; Crespo, José L

    2014-10-01

    The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) results in the activation of stress responses, such as the unfolded protein response or the catabolic process of autophagy to ultimately recover cellular homeostasis. ER stress also promotes the production of reactive oxygen species, which play an important role in autophagy regulation. However, it remains unknown whether reactive oxygen species are involved in ER stress-induced autophagy. In this study, we provide evidence connecting redox imbalance caused by ER stress and autophagy activation in the model unicellular green alga Chlamydomonas reinhardtii. Treatment of C. reinhardtii cells with the ER stressors tunicamycin or dithiothreitol resulted in up-regulation of the expression of genes encoding ER resident endoplasmic reticulum oxidoreductin1 oxidoreductase and protein disulfide isomerases. ER stress also triggered autophagy in C. reinhardtii based on the protein abundance, lipidation, cellular distribution, and mRNA levels of the autophagy marker ATG8. Moreover, increases in the oxidation of the glutathione pool and the expression of oxidative stress-related genes were detected in tunicamycin-treated cells. Our results revealed that the antioxidant glutathione partially suppressed ER stress-induced autophagy and decreased the toxicity of tunicamycin, suggesting that oxidative stress participates in the control of autophagy in response to ER stress in C. reinhardtii In close agreement, we also found that autophagy activation by tunicamycin was more pronounced in the C. reinhardtii sor1 mutant, which shows increased expression of oxidative stress-related genes.

  1. Species-specific impact of the autophagy machinery on Chikungunya virus infection.

    PubMed

    Judith, Delphine; Mostowy, Serge; Bourai, Mehdi; Gangneux, Nicolas; Lelek, Mickaël; Lucas-Hourani, Marianne; Cayet, Nadège; Jacob, Yves; Prévost, Marie-Christine; Pierre, Philippe; Tangy, Frédéric; Zimmer, Christophe; Vidalain, Pierre-Olivier; Couderc, Thérèse; Lecuit, Marc

    2013-06-01

    Chikungunya virus (CHIKV) is a recently re-emerged arbovirus that triggers autophagy. Here, we show that CHIKV interacts with components of the autophagy machinery during its replication cycle, inducing a cytoprotective effect. The autophagy receptor p62 protects cells from death by binding ubiquitinated capsid and targeting it to autophagolysosomes. By contrast, the human autophagy receptor NDP52--but not its mouse orthologue--interacts with the non-structural protein nsP2, thereby promoting viral replication. These results highlight the distinct roles of p62 and NDP52 in viral infection, and identify NDP52 as a cellular factor that accounts for CHIKV species specificity.

  2. Essential role for autophagy during invariant NKT cell development

    PubMed Central

    Salio, Mariolina; Puleston, Daniel J.; Mathan, Till S. M.; Shepherd, Dawn; Stranks, Amanda J.; Adamopoulou, Eleni; Veerapen, Natacha; Besra, Gurdyal S.; Hollander, Georg A.; Simon, Anna Katharina; Cerundolo, Vincenzo

    2014-01-01

    Autophagy is an evolutionarily conserved cellular homeostatic pathway essential for development, immunity, and cell death. Although autophagy modulates MHC antigen presentation, it remains unclear whether autophagy defects impact on CD1d lipid loading and presentation to invariant natural killer T (iNKT) cells and on iNKT cell differentiation in the thymus. Furthermore, it remains unclear whether iNKT and conventional T cells have similar autophagy requirements for differentiation, survival, and/or activation. We report that, in mice with a conditional deletion of the essential autophagy gene Atg7 in the T-cell compartment (CD4 Cre-Atg7−/−), thymic iNKT cell development—unlike conventional T-cell development—is blocked at an early stage and mature iNKT cells are absent in peripheral lymphoid organs. The defect is not due to altered loading of intracellular iNKT cell agonists; rather, it is T-cell–intrinsic, resulting in enhanced susceptibility of iNKT cells to apoptosis. We show that autophagy increases during iNKT cell thymic differentiation and that it developmentally regulates mitochondrial content through mitophagy in the thymus of mice and humans. Autophagy defects result in the intracellular accumulation of mitochondrial superoxide species and subsequent apoptotic cell death. Although autophagy-deficient conventional T cells develop normally, they show impaired peripheral survival, particularly memory CD8+ T cells. Because iNKT cells, unlike conventional T cells, differentiate into memory cells while in the thymus, our results highlight a unique autophagy-dependent metabolic regulation of adaptive and innate T cells, which is required for transition to a quiescent state after population expansion. PMID:25512546

  3. Autophagy in alcoholic liver disease, self-eating triggered by drinking.

    PubMed

    Wang, Lin; Khambu, Bilon; Zhang, Hao; Yin, Xiao-Ming

    2015-09-01

    Macroautophagy (autophagy) is an evolutionarily conserved mechanism. It is important for normal cellular function and also plays critical roles in the etiology and pathogenesis of a number of human diseases. In alcohol-induced liver disease, autophagy is a protective mechanism against the liver injury caused by alcohol. Autophagy is activated in acute ethanol treatment but could be suppressed in chronic and/or high dose treatment of alcohol. The selective removal of lipid droplets and/or damaged mitochondria is likely the major mode of autophagy in reducing liver injury. Understanding the dynamics of the autophagy process and the approach to modulate autophagy could help finding new ways to battle against alcohol-induced liver injury.

  4. Avermectin induced autophagy in pigeon spleen tissues.

    PubMed

    Liu, Ci; Zhao, Yanbing; Chen, Lijie; Zhang, Ziwei; Li, Ming; Li, Shu

    2015-12-05

    The level of autophagy is considered as an indicator for monitoring the toxic impact of pesticide exposure. Avermectin (AVM), a widely used insecticide, has immunotoxic effects on the pigeon spleen. The aim of this study was to investigate the status of autophagy and the expression levels of microtubule-associated protein1 light chain 3 (LC3), beclin-1, dynein, autophagy associated gene (Atg) 4B, Atg5, target of rapamycin complex 1 (TORC1) and target of rapamycin complex 2 (TORC2) in AVM-treated pigeon spleens. Eighty two-month-old pigeons were randomly divided into four groups: a control group, a low-dose group, a medium-dose group and a high-dose group, which were fed a basal diet spiked with 0, 20, 40 and 60 mg AVM/kg diet, respectively. Microscopic cellular morphology revealed a significant increase in autophagic structures in the AVM-treated groups. The expression of LC3, beclin-1, dynein, Atg4B and Atg5 increased, while mRNA levels of TORC1 and TORC2 were decreased in the AVM-treated groups relative to the control groups at 30, 60 and 90 days in the pigeon spleen. These results indicated that AVM exposure could up-regulate the level of autophagy in a dose-time-dependent manner in the pigeon spleen.

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

    PubMed

    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. A pathway of targeted autophagy is induced by DNA damage in budding yeast

    PubMed Central

    Eapen, Vinay V.; Waterman, David P.; Bernard, Amélie; Schiffmann, Nathan; Sayas, Enrich; Kamber, Roarke; Lemos, Brenda; Memisoglu, Gonen; Ang, Jessie; Mazella, Allison; Chuartzman, Silvia G.; Loewith, Robbie J.; Schuldiner, Maya; Denic, Vladimir; Klionsky, Daniel J.; Haber, James E.

    2017-01-01

    Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response. PMID:28154131

  7. A pathway of targeted autophagy is induced by DNA damage in budding yeast.

    PubMed

    Eapen, Vinay V; Waterman, David P; Bernard, Amélie; Schiffmann, Nathan; Sayas, Enrich; Kamber, Roarke; Lemos, Brenda; Memisoglu, Gonen; Ang, Jessie; Mazella, Allison; Chuartzman, Silvia G; Loewith, Robbie J; Schuldiner, Maya; Denic, Vladimir; Klionsky, Daniel J; Haber, James E

    2017-02-14

    Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

  8. Hypoxia-induced autophagy mediates cisplatin resistance in lung cancer cells

    PubMed Central

    Wu, Hui-Mei; Jiang, Zi-Feng; Ding, Pei-Shan; Shao, Li-Jie; Liu, Rong-Yu

    2015-01-01

    Hypoxia which commonly exists in solid tumors, leads to cancer cells chemoresistance via provoking adaptive responses including autophagy. Therefore, we sought to evaluate the role of autophagy and hypoxia as well as the underlying mechanism in the cisplatin resistance of lung cancer cells. Our study demonstrated that hypoxia significantly protected A549 and SPC-A1 cells from cisplatin-induced cell death in a Hif-1α- and Hif-2α- dependent manner. Moreover, compared with normoxia, cisplatin-induced apoptosis under hypoxia was markedly reduced. However, when autophagy was inhibited by 3-MA or siRNA targeted ATG5, this reduction was effectively attenuated, which means autophagy mediates cisplatin resisitance under hypoxia. In parallel, we showed that hypoxia robustly augmented cisplatin-induced autophagy activation, accompanying by suppressing cisplatin-induced BNIP3 death pathways, which was due to the more efficient autophagic process under hypoxia. Consequently, we proposed that autophagy was a protective mechanism after cisplatin incubation under both normoxia and hypoxia. However, under normoxia, autophagy activation ‘was unable to counteract the stress induced by cisplatin, therefore resulting in cell death, whereas under hypoxia, autophagy induction was augmented that solved the cisplatin-induced stress, allowing the cells to survival. In conclusion, augmented induction of autophagy by hypoxia decreased lung cancer cells susceptibility to cisplatin-induced apoptosis. PMID:26201611

  9. Autophagy signal transduction by ATG proteins: from hierarchies to networks.

    PubMed

    Wesselborg, Sebastian; Stork, Björn

    2015-12-01

    Autophagy represents an intracellular degradation process which is involved in both cellular homeostasis and disease settings. In the last two decades, the molecular machinery governing this process has been characterized in detail. To date, several key factors regulating this intracellular degradation process have been identified. The so-called autophagy-related (ATG) genes and proteins are central to this process. However, several additional molecules contribute to the outcome of an autophagic response. Several review articles describing the molecular process of autophagy have been published in the recent past. In this review article we would like to add the most recent findings to this knowledge, and to give an overview of the network character of the autophagy signaling machinery.

  10. Role of autophagy in the pathogenesis of inflammatory bowel disease

    PubMed Central

    Iida, Tomoya; Onodera, Kei; Nakase, Hiroshi

    2017-01-01

    Inflammatory bowel disease (IBD) results from a complex series of interactions between susceptibility genes, the environment, and the immune system. Recently, some studies provided strong evidence that the process of autophagy affects several aspects of mucosal immune responses. Autophagy is a cellular stress response that plays key roles in physiological processes, such as innate and adaptive immunity, adaptation to starvation, degradation of aberrant proteins or organelles, antimicrobial defense, and protein secretion. Dysfunctional autophagy is recognized as a contributing factor in many chronic inflammatory diseases, including IBD. Autophagy plays multiple roles in IBD pathogenesis by altering processes that include intracellular bacterial killing, antimicrobial peptide secretion by Paneth cells, goblet cell function, proinflammatory cytokine production by macrophages, antigen presentation by dendritic cells, and the endoplasmic reticulum stress response in enterocytes. Recent studies have identified susceptibility genes involved in autophagy, such as NOD2, ATG16L1, and IRGM, and active research is ongoing all over the world. The aim of this review is a systematic appraisal of the current literature to provide a better understanding of the role of autophagy in the pathogenesis of IBD. Understanding these mechanisms will bring about new strategies for the treatment and prevention of IBD. PMID:28373760

  11. Autophagy functions as an antiviral mechanism against geminiviruses in plants

    PubMed Central

    Haxim, Yakupjan; Ismayil, Asigul; Jia, Qi; Wang, Yan; Zheng, Xiyin; Chen, Tianyuan; Qian, Lichao; Liu, Na; Wang, Yunjing; Han, Shaojie; Cheng, Jiaxuan; Qi, Yijun; Hong, Yiguo; Liu, Yule

    2017-01-01

    Autophagy is an evolutionarily conserved process that recycles damaged or unwanted cellular components, and has been linked to plant immunity. However, how autophagy contributes to plant immunity is unknown. Here we reported that the plant autophagic machinery targets the virulence factor βC1 of Cotton leaf curl Multan virus (CLCuMuV) for degradation through its interaction with the key autophagy protein ATG8. A V32A mutation in βC1 abolished its interaction with NbATG8f, and virus carrying βC1V32A showed increased symptoms and viral DNA accumulation in plants. Furthermore, silencing of autophagy-related genes ATG5 and ATG7 reduced plant resistance to the DNA viruses CLCuMuV, Tomato yellow leaf curl virus, and Tomato yellow leaf curl China virus, whereas activating autophagy by silencing GAPC genes enhanced plant resistance to viral infection. Thus, autophagy represents a novel anti-pathogenic mechanism that plays an important role in antiviral immunity in plants. DOI: http://dx.doi.org/10.7554/eLife.23897.001 PMID:28244873

  12. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    SciTech Connect

    Orfali, Nina; McKenna, Sharon L.; Cahill, Mary R.; Gudas, Lorraine J.; Mongan, Nigel P.

    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.

  13. Autophagy is an adaptive response in desmin-related cardiomyopathy

    PubMed Central

    Tannous, Paul; Zhu, Hongxin; Johnstone, Janet L.; Shelton, John M.; Rajasekaran, Namakkal S.; Benjamin, Ivor J.; Nguyen, Lan; Gerard, Robert D.; Levine, Beth; Rothermel, Beverly A.; Hill, Joseph A.

    2008-01-01

    A missense mutation in the αB-crystallin (CryAB) gene triggers a severe form of desmin-related cardiomyopathy (DRCM) characterized by accumulation of misfolded proteins. We hypothesized that autophagy increases in response to protein aggregates and that this autophagic activity is adaptive. Mutant CryAB (CryABR120G) triggered a >2-fold increase in cardiomyocyte autophagic activity, and blunting autophagy increased the rate of aggregate accumulation and the abundance of insoluble CryABR120G-associated aggregates. Cardiomyocyte-restricted overexpression of CryABR120G in mice induced intracellular aggregate accumulation and systolic heart failure by 12 months. As early as 2 months (well before the earliest declines in cardiac function), we detected robust autophagic activity. To test the functional significance of autophagic activation, we crossed CryABR120G mice with animals harboring heterozygous inactivation of beclin 1, a gene required for autophagy. Blunting autophagy in vivo dramatically hastened heart failure progression with a 3-fold increase in interstitial fibrosis, greater accumulation of polyubiquitinated proteins, larger and more extensive intracellular aggregates, accelerated ventricular dysfunction, and early mortality. This study reports activation of autophagy in DRCM. Further, our findings point to autophagy as an adaptive response in this proteotoxic form of heart disease. PMID:18621691

  14. TRAF3IP3, a novel autophagy up-regulated gene, is involved in marginal zone B lymphocyte development and survival.

    PubMed

    Peng, S; Wang, K; Gu, Y; Chen, Y; Nan, X; Xing, J; Cui, Q; Chen, Y; Ge, Q; Zhao, H

    2015-10-01

    Tumour necrosis factor receptor-associated factor 3 (TRAF3) interacting protein 3 (TRAF3IP3; also known as T3JAM) is expressed specifically in immune organs and tissues. To investigate the impact of TRAF3IP3 on immunity, we generated Traf3ip3 knock-out (KO) mice. Interestingly, these mice exhibited a significant reduction in the number of common lymphoid progenitors (CLPs) and inhibition of B cell development in the bone marrow. Furthermore, Traf3ip3 KO mice lacked marginal zone (MZ) B cells in the spleen. Traf3ip3 KO mice also exhibited a reduced amount of serum natural antibodies and impaired T cell-independent type II (TI-II) responses to trinitrophenol (TNP)-Ficoll antigen. Additionally, our results showed that Traf3ip3 promotes autophagy via an ATG16L1-binding motif, and MZ B cells isolated from mutant mice showed a diminished level of autophagy and a high rate of apoptosis. These results suggest that TRAF3IP3 contributes to MZ B cell survival by up-regulating autophagy, thereby promoting the TI-II immune response.

  15. Mutation in ATG5 reduces autophagy and leads to ataxia with developmental delay.

    PubMed

    Kim, Myungjin; Sandford, Erin; Gatica, Damian; Qiu, Yu; Liu, Xu; Zheng, Yumei; Schulman, Brenda A; Xu, Jishu; Semple, Ian; Ro, Seung-Hyun; Kim, Boyoung; Mavioglu, R Nehir; Tolun, Aslıhan; Jipa, Andras; Takats, Szabolcs; Karpati, Manuela; Li, Jun Z; Yapici, Zuhal; Juhasz, Gabor; Lee, Jun Hee; Klionsky, Daniel J; Burmeister, Margit

    2016-01-26

    Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects' cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy. Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder, in contrast to flies expressing the wild-type human protein. Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health.

  16. Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma.

    PubMed

    Liu, Yuan-Ling; Yang, Pei-Ming; Shun, Chia-Tung; Wu, Ming-Shiang; Weng, Jing-Ru; Chen, Ching-Chow

    2010-11-01

    Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Drug treatments for HCC have been largely unsuccessful. Histone deacetylase inhibitors can reactivate tumor suppressor genes in cancer cells and serve as potential anti-cancer drugs. Two potent HDAC inhibitors OSU-HDAC42 and SAHA induced autophagy in HCC cells as revealed by transmission electron microscopy, immunofluorescence and LC3-II accumulation. We found that SAHA and OSU-HDAC42 induced autophagy through downregulation of Akt/mTOR signaling and induction of ER stress response. Inhibition of autophagy by 3-MA or Atg5 knockout reduced SAHA-induced cytotoxicity, indicating that SAHA-induced autophagy led to cell death. Our results show that the combination of autophagy inducers with SAHA might be attractive for the treatment of HCC and pharmacological targeting of autophagy provides promise for the management of cancer therapy.

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

    SciTech Connect

    Liu, Qinghao; Qin, Yixian; Zhou, Lei; Kou, Qiuwen; Guo, Xin; Ge, Xinna; Yang, Hanchun; Hu, Hongbo

    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.

  18. Loss of STAT1 protects hair cells from ototoxicity through modulation of STAT3, c-Jun, Akt, and autophagy factors

    PubMed Central

    Levano, S; Bodmer, D

    2015-01-01

    Hair cell damage is a side effect of cisplatin and aminoglycoside use. The inhibition or attenuation of this process is a target of many investigations. There is growing evidence that STAT1 deficiency decreases cisplatin-mediated ototoxicity; however, the role of STAT function and the molecules that act in gentamicin-mediated toxicity have not been fully elucidated. We used mice lacking STAT1 to investigate the effect of STAT1 ablation in cultured organs treated with cisplatin and gentamicin. Here we show that ablation of STAT1 decreased cisplatin toxicity and attenuated gentamicin-mediated hair cell damage. More TUNEL-positive hair cells were observed in explants of wild-type mice than that of STAT1−/− mice. Although cisplatin increased serine phosphorylation of STAT1 in wild-type mice and diminished STAT3 expression in wild-type and STAT1−/− mice, gentamicin increased tyrosine phosphorylation of STAT3 in STAT1−/− mice. The early inflammatory response was manifested in the upregulation of TNF-α and IL-6 in cisplatin-treated explants of wild-type and STAT1−/− mice. Expression of the anti-inflammatory cytokine IL-10 was altered in cisplatin-treated explants, upregulated in wild-type explants, and downregulated in STAT1−/− explants. Cisplatin and gentamicin triggered the activation of c-Jun. Activation of Akt was observed in gentamicin-treated explants from STAT1−/− mice. Increased levels of the autophagy proteins Beclin-1 and LC3-II were observed in STAT1−/− explants. These data suggest that STAT1 is a central player in mediating ototoxicity. Gentamicin and cisplatin activate different downstream factors to trigger ototoxicity. Although cisplatin and gentamicin triggered inflammation and activated apoptotic factors, the absence of STAT1 allowed the cells to overcome the effects of these drugs. PMID:26673664

  19. Defective autophagy is a key feature of cerebral cavernous malformations

    PubMed Central

    Marchi, Saverio; Corricelli, Mariangela; Trapani, Eliana; Bravi, Luca; Pittaro, Alessandra; Delle Monache, Simona; Ferroni, Letizia; Patergnani, Simone; Missiroli, Sonia; Goitre, Luca; Trabalzini, Lorenza; Rimessi, Alessandro; Giorgi, Carlotta; Zavan, Barbara; Cassoni, Paola; Dejana, Elisabetta; Retta, Saverio Francesco; Pinton, Paolo

    2015-01-01

    Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3–0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions. PMID:26417067

  20. Defective autophagy is a key feature of cerebral cavernous malformations.

    PubMed

    Marchi, Saverio; Corricelli, Mariangela; Trapani, Eliana; Bravi, Luca; Pittaro, Alessandra; Delle Monache, Simona; Ferroni, Letizia; Patergnani, Simone; Missiroli, Sonia; Goitre, Luca; Trabalzini, Lorenza; Rimessi, Alessandro; Giorgi, Carlotta; Zavan, Barbara; Cassoni, Paola; Dejana, Elisabetta; Retta, Saverio Francesco; Pinton, Paolo

    2015-11-01

    Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3-0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.

  1. Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus.

    PubMed

    Hagiwara, D; Arima, H; Morishita, Y; Wenjun, L; Azuma, Y; Ito, Y; Suga, H; Goto, M; Banno, R; Sugimura, Y; Shiota, A; Asai, N; Takahashi, M; Oiso, Y

    2014-03-27

    Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30-40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy

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

    PubMed

    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.

  3. Celastrol Induces Autophagy by Targeting AR/miR-101 in Prostate Cancer Cells

    PubMed Central

    Guo, Jianquan; Huang, Xuemei; Wang, Hui; Yang, Huanjie

    2015-01-01

    Autophagy is an evolutionarily conserved process responsible for the degradation and recycling of cytoplasmic components through autolysosomes. Targeting AR axis is a standard strategy for prostate cancer treatment; however, the role of AR in autophagic processes is still not fully understood. In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy. Knockdown of AR in AR-positive prostate cancer cells resulted in enhanced autophagy. Ectopic expression of AR in AR-negative prostate cancer cells, or gain of function of the AR signaling in AR-positive cells, led to suppression of autophagy. Since miR-101 is an inhibitor of autophagy and its expression was decreased along with AR in the process of celastrol-induced autophagy, we hypothesize that AR inhibits autophagy through transactivation of miR-101. AR binding site was defined in the upstream of miR-101 gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. The inhibition of celastrol-induced autophagy by AR was compromised by blocking miR-101; while transfection of miR-101 led to inhibition of celastrol-induced autophagy in spite of AR depletion. Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR. Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells. Our results demonstrate that AR inhibits autophagy via transactivation of miR-101, thus combination of miR-101 mimics with celastrol may represent a promising therapeutic approach for treating prostate cancer. PMID:26473737

  4. Celastrol Induces Autophagy by Targeting AR/miR-101 in Prostate Cancer Cells.

    PubMed

    Guo, Jianquan; Huang, Xuemei; Wang, Hui; Yang, Huanjie

    2015-01-01

    Autophagy is an evolutionarily conserved process responsible for the degradation and recycling of cytoplasmic components through autolysosomes. Targeting AR axis is a standard strategy for prostate cancer treatment; however, the role of AR in autophagic processes is still not fully understood. In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy. Knockdown of AR in AR-positive prostate cancer cells resulted in enhanced autophagy. Ectopic expression of AR in AR-negative prostate cancer cells, or gain of function of the AR signaling in AR-positive cells, led to suppression of autophagy. Since miR-101 is an inhibitor of autophagy and its expression was decreased along with AR in the process of celastrol-induced autophagy, we hypothesize that AR inhibits autophagy through transactivation of miR-101. AR binding site was defined in the upstream of miR-101 gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. The inhibition of celastrol-induced autophagy by AR was compromised by blocking miR-101; while transfection of miR-101 led to inhibition of celastrol-induced autophagy in spite of AR depletion. Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR. Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells. Our results demonstrate that AR inhibits autophagy via transactivation of miR-101, thus combination of miR-101 mimics with celastrol may represent a promising therapeutic approach for treating prostate cancer.

  5. Bioenergetic adaptation in response to autophagy regulators during rotenone exposure

    PubMed Central

    Giordano, Samantha; Dodson, Matthew; Ravi, Saranya; Redmann, Matthew; Ouyang, Xiaosen; Usmar, Victor M Darley; Zhang, Jianhua

    2015-01-01

    Parkinson’s disease (PD) is the second most common neurodegenerative disorder with both mitochondrial dysfunction and insufficient autophagy playing a key role in its pathogenesis. Among the risk factors, exposure to the environmental neurotoxin rotenone increases the probability of developing PD. We previously reported that in differentiated SH-SY5Y cells, rotenone-induced cell death is directly related to inhibition of mitochondrial function. How rotenone at nM concentrations inhibits mitochondrial function, and whether it can engage the autophagy pathway necessary to remove damaged proteins and organelles, is unknown. We tested the hypothesis that autophagy plays a protective role against rotenone toxicity in primary neurons. We found that rotenone (10–100 nM) immediately inhibited cellular bioenergetics. Concentrations that decreased mitochondrial function at 2 hr, caused cell death at 24 hr with an LD50 of 10 nM. Overall autophagic flux was decreased by 10 nM rotenone at both 2 and 24 hr, but surprisingly mitophagy, or autophagy of the mitochondria, was increased at 24 hr, suggesting that a mitochondrial-specific lysosomal degradation pathway may be activated. Upregulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3-methyladenine (3-MA) exacerbated cell death. Interestingly, while 3-MA exacerbated the rotenone-dependent effects on bioenergetics, rapamycin did not prevent rotenone-induced mitochondrial dysfunction, but caused reprogramming of mitochondrial substrate usage associated with both complex I and complex II activities. Taken together, these data demonstrate that autophagy can play a protective role in primary neuron survival in response to rotenone; moreover, surviving neurons exhibit bioenergetic adaptations to this metabolic stressor. PMID:25081478

  6. Cytoplasmic sphingosine-1-phosphate pathway modulates neuronal autophagy

    PubMed Central

    Moruno Manchon, Jose Felix; Uzor, Ndidi-Ese; Dabaghian, Yuri; Furr-Stimming, Erin E.; Finkbeiner, Steven; Tsvetkov, Andrey S.

    2015-01-01

    Autophagy is an important homeostatic mechanism that eliminates long-lived proteins, protein aggregates and damaged organelles. Its dysregulation is involved in many neurodegenerative disorders. Autophagy is therefore a promising target for blunting neurodegeneration. We searched for novel autophagic pathways in primary neurons and identified the cytosolic sphingosine-1-phosphate (S1P) pathway as a regulator of neuronal autophagy. S1P, a bioactive lipid generated by sphingosine kinase 1 (SK1) in the cytoplasm, is implicated in cell survival. We found that SK1 enhances flux through autophagy and that S1P-metabolizing enzymes decrease this flux. When autophagy is stimulated, SK1 relocalizes to endosomes/autophagosomes in neurons. Expression of a dominant-negative form of SK1 inhibits autophagosome synthesis. In a neuron model of Huntington’s disease, pharmacologically inhibiting S1P-lyase protected neurons from mutant huntingtin-induced neurotoxicity. These results identify the S1P pathway as a novel regulator of neuronal autophagy and provide a new target for developing therapies for neurodegenerative disorders. PMID:26477494

  7. A vitamin for autophagy.

    PubMed

    Fabri, Mario; Modlin, Robert L

    2009-09-17

    Recent discoveries have revealed the importance of the vitamin D-dependent generation of antimicrobial peptides in human host defense against Mycobacterium tuberculosis. Now, Yuk et al. (2009) show how vitamin D induces autophagy and mediates colocalization of Mycobacterium tuberculosis and antimicrobial peptides within an autophagolysosome, leading to killing of the bacterium.

  8. Autophagy and cancer cell metabolism.

    PubMed

    Lozy, Fred; Karantza, Vassiliki

    2012-06-01

    Autophagy is a catabolic process involving lysosomal turnover of proteins and organelles for maintenance of cellular homeostasis and mitigation of metabolic stress. Autophagy defects are linked to diseases, such as liver failure, neurodegeneration, inflammatory bowel disease, aging and cancer. The role of autophagy in tumorigenesis is complex and likely context-dependent. Human breast, ovarian and prostate cancers have allelic del