Starvation-induced Hyperacetylation of Tubulin Is Required for the Stimulation of Autophagy by Nutrient Deprivation

The molecular mechanisms underlying microtubule participation in autophagy are not known. In this study, we show that starvation-induced autophagosome formation requires the most dynamic microtubule subset. Upon nutrient deprivation, labile microtubules specifically recruit markers of autophagosome formation like class III-phosphatidylinositol kinase, WIPI-1, the Atg12-Atg5 conjugate, and LC3-I, whereas mature autophagosomes may bind to stable microtubules. We further found that upon nutrient deprivation, tubulin acetylation increases both in labile and stable microtubules and is required to allow autophagy stimulation. Tubulin hyperacetylation on lysine 40 enhances kinesin-1 and JIP-1 recruitment on microtubules and allows JNK phosphorylation and activation. JNK, in turn, triggers the release of Beclin 1 from Bcl-2-Beclin 1 complexes and its recruitment on microtubules where it may initiate autophagosome formation. Finally, although kinesin-1 functions to carry autophagosomes in basal conditions, it is not involved in motoring autophagosomes after nutrient deprivation. Our results show that the dynamics of microtubules and tubulin post-translational modifications play a major role in the regulation of starvation-induced autophagy.     

Autophagy Controls an Intrinsic Host Defense to Bacteria by Promoting Epithelial Cell Survival: A Murine Model

Cell death is a critical host response to regulate the fate of bacterial infections, innate immune responses, and ultimately, disease outcome. Shigellaspp. invade and colonize gut epithelium in human and nonhuman primates but adult mice are naturally resistant to intra-gastric Shigella infection. In this study, however, we found Shigella could invade the terminal ileum of the mouse small intestine by 1 hour after infection and be rapidly cleared within 24 h. These early phase events occurred shortly after oral infection resulting in epithelial shedding, degranulation of Paneth cells, and cell death in the intestine. During this process, autophagy proceeded without any signs of inflammation. In contrast, blocking autophagy in epithelial cells enhanced host cell death, leading to tissue destruction and to inflammation, suggesting that autophagic flow relieves cellular stress associated with host cell death and inflammation. Herein we propose a new concept of “epithelial barrier turnover” as a general intrinsic host defense mechanism that increases survival of host cells and inhibits inflammation against enteric bacterial infections, which is regulated by autophagy.     

Nuclear Receptor Nur77 Facilitates Melanoma Cell Survival under Metabolic Stress by Protecting Fatty Acid Oxidation

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Interleukin-21 Is a Critical Regulator of CD4 and CD8 T Cell Survival during Priming under Interleukin-2 Deprivation Conditions

Optimal T cell activation and expansion require binding of the common gamma-chain (γc) cytokine Interleukin-2 (IL-2) to its cognate receptor that in turn engages a γc/Janus tyrosine kinase (Jak)3 signaling pathway. Because of its restricted expression by antigen-activated T cells and its obligatory role in promoting their survival and proliferation, IL-2 has been considered as a selective therapeutic target for preventing T cell mediated diseases. However, in order to further explore IL-2 targeted therapy, it is critical to precisely understand its role during early events of T cell activation. In this study, we delineate the role of IL-2 and other γc cytokines in promoting the survival of CD4 and CD8 T cells during early phases of priming. Under IL-2 inhibitory conditions (by neutralizing anti-IL-2 mAbs), the survival of activated CD8⁺ T cells was reduced, whereas CD4⁺ T cells remained much more resistant. These results correlated with reduced Bcl-2 expression, and mitochondrial membrane potential in CD8⁺ T cells in comparison to CD4⁺ T cells. However, using transwell co-culture assays we have found that CD4⁺ T cells could rescue the survival of CD8⁺ T cells even under IL-2 deprived conditions via secretion of soluble factors. A cytokine screen performed on CD8⁺ T cells cultured alone revealed that IL-21, another γc cytokine, was capable of rescuing their survival under IL-2 deprivation. Indeed, blocking the IL-21 signaling pathway along with IL-2 neutralization resulted in significantly reduced survival of both CD4⁺ and CD8⁺ T cells. Taken together, we have shown that under IL-2 deprivation conditions, IL-21 may act as the major survival factor promoting T cell immune responses. Thus, investigation of IL-2 targeted therapies may need to be revisited to consider blockade of the IL-21 signaling pathways as an adjunct to provide more effective control of T cell immune responses.     

Regulation and Function of Autophagy during Cell Survival and Cell Death

Autophagy is an important catabolic process that delivers cytoplasmic material to the lysosome for degradation. Autophagy promotes cell survival by elimination of damaged organelles and proteins aggregates, as well as by facilitating bioenergetic homeostasis. Although autophagy has been considered a cell survival mechanism, recent studies have shown that autophagy can promote cell death. The core mechanisms that control autophagy are conserved between yeast and humans, but animals also possess genes that regulate autophagy that are not present in yeast. These regulatory differences may be explained by the need to control autophagy in a cell context-specific manner in multicellular animals, such as during cell survival and cell death. Autophagy was thought to be a bulk cytoplasmic degradation mechanism, but recent studies have shown that specific cargo is recruited for degradation. This suggests the possibility that either cell survival or death may be regulated by selective autophagic clearance of cytoplasmic material. Here we summarize the mechanisms that regulate autophagy and how they may contribute to cell survival and death.Autophagy (self-eating) is an evolutionarily conserved catabolic process that is used to deliver cytoplasmic materials, including organelles and proteins, to the lysosome for degradation. Three types of autophagy have been described, including macroautophagy, microautophagy, and chaperone-mediated autophagy (Mizushima and Komatsu 2011). Although macroautophagy involves the fusion of the double membrane autophagosome and lysosomes, microautophagy is poorly understood and thought to involve direct uptake of material by the lysosome via a process that appears similar to pinocytosis. By contrast, chaperone-mediated autophagy is a biochemical mechanism to import proteins into the lysosome; it depends on a signature sequence and interaction with protein chaperones. Here we will focus on macroautophagy (hereafter called autophagy) because of our knowledge of this process in cell survival and cell death.Autophagy was likely first observed when electron microscopy was used to observe “dense bodies” containing mitochondria in mouse kidneys (Clark 1957). Five years later, it was reported that rat hepatocytes exposed to glucagon possessed membrane-bound vesicles that were rich in mitochondria and endoplasmic reticulum (Ashford and Porter 1962). Almost simultaneously, it was shown that these membrane-bound vesicles contained lysosomal hydrolases (Novikoff and Essner 1962). In 1965 de Duve coined the term “autophagy” (Klionsky 2008).The delivery of cytoplasmic material to the lysosome by autophagy involves membrane formation and fusion events (Fig. 1). First an isolation membrane, also known as a phagophore, must be initiated from a membrane source known as the phagophore assembly site (PAS). de Duve suggested that the smooth endoplasmic reticulum could be the source of autophagosome membrane (de Duve and Wattiaux 1966), and subsequent studies have supported this possibility (Dunn 1990; Axe et al. 2008). Although controversial, mitochondria and plasma membrane could also supply membranes for the formation of the autophagosomes under different conditions (Hailey et al. 2010; Ravikumar et al. 2010). The elongating isolation membrane surrounds cargo that is ultimately enclosed in the double membrane autophagosome. Once the autophagosome is formed, it fuses with lysosomes (known as the vacuole in yeasts and plants) to form autolysosomes in which the cargo is degraded by lysosomal hydrolases. At this stage lysosomes must reform so that subsequent autophagy may occur (Yu et al. 2010).Open in a separate windowFigure 1.Macroautophagy (autophagy) delivers cytoplasmic cargo to lysosomes for degradation, and involves membrane formation and fusion. The isolation membrane is initiated from a membrane source known as the from the phagophore assembly site (PAS). The isolation membrane surrounds cargo, including organelles and proteins, to form a double membrane autophagosome. Autophagosomes fuse with lysosomes to form autolysosomes in which the cargo is degraded by lysosomal hydrolases.     

Regulation of Mitochondrial Integrity,Autophagy and Cell Survival by BNIP3

Understanding the role of BNIP3 in the systemic response to hypoxia has been complicated by conflicting results that indicate on the one hand that BNIP3 promotes cell death, and other data, including our own that BNIP3 is not sufficient for cell death, but rather plays a critical role in hypoxia-induced autophagy. This work suggests that rather than promoting death, BNIP3 may actually allow survival either by preventing ATP depletion or by eliminating damaged mitochondria. However, the function of BNIP3 may be subverted under unusual conditions associated with acidosis that arise following extended periods of hypoxia and anaerobic glycolysis. Despite this novel insight into BNIP3 function, much remains to be done in terms of pinning down a molecular activity for BNIP3 that explains both its role in autophagy and how this may be subverted to induce cell death. As a target of the RB tumor suppressor, our work also places BNIP3 at the center of efforts to exploit autophagy to better treat human cancers in which tumor hypoxia is implicated as a progression factor.

Addendum to:

BNIP3 is an RB/E2F Target Gene Required for Hypoxia-Induced Autophagy

K. Tracy, B.C. Dibling, BT. Spike, J. Knabb, P. Schumacker and K.F. Macleod

Mol Cell Biol 2007; In press     

Coibamide A Induces mTOR-Independent Autophagy and Cell Death in Human Glioblastoma Cells

Coibamide A is an N-methyl-stabilized depsipeptide that was isolated from a marine cyanobacterium as part of an International Cooperative Biodiversity Groups (ICBG) program based in Panama. Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and “COMPARE-negative” profile indicative of a unique mechanism of action. We report that coibamide A is a more potent and efficacious cytotoxin than was previously appreciated, inducing concentration- and time-dependent cytotoxicity (EC₅₀<100 nM) in human U87-MG and SF-295 glioblastoma cells and mouse embryonic fibroblasts (MEFs). This activity was lost upon linearization of the molecule, highlighting the importance of the cyclized structure for both anti-proliferative and cytotoxic responses. We show that coibamide A induces autophagosome accumulation in human glioblastoma cell types and MEFs via an mTOR-independent mechanism; no change was observed in the phosphorylation state of ULK1 (Ser-757), p70 S6K1 (Thr-389), S6 ribosomal protein (Ser-235/236) and 4EBP-1 (Thr-37/46). Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type. SF-295 glioblastoma cells showed caspase-3 activation and evidence of apoptotic cell death in a pattern that was also seen in wild-type and autophagy-deficient (ATG5-null) MEFs. In contrast, cell death in U87-MG glioblastoma cells was characterized by extensive cytoplasmic vacuolization and lacked clear apoptotic features. Cell death was attenuated, but still triggered, in Apaf-1-null MEFs lacking a functional mitochondria-mediated apoptotic pathway. From the study of ATG5-null MEFs we conclude that a conventional autophagy response is not required for coibamide A-induced cell death, but likely occurs in dying cells in response to treatment. Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death mechanisms in apoptotic-resistant cancer cells.     

COX10-AS1 Facilitates Cell Proliferation and Inhibits Cell Apoptosis in Glioblastoma Cells at Post-Transcription Level

Neurochemical Research - Glioblastoma (GBM) is an invasive cancer with poor prognosis in patients. Researching on molecular functions in GBM has attracted more and more attention. Actin gamma 1...     

Loss of PINK1 Impairs Stress-Induced Autophagy and Cell Survival

The mitochondrial kinase PINK1 and the ubiquitin ligase Parkin are participating in quality control after CCCP- or ROS-induced mitochondrial damage, and their dysfunction is associated with the development and progression of Parkinson''s disease. Furthermore, PINK1 expression is also induced by starvation indicating an additional role for PINK1 in stress response. Therefore, the effects of PINK1 deficiency on the autophago-lysosomal pathway during stress were investigated. Under trophic deprivation SH-SY5Y cells with stable PINK1 knockdown showed downregulation of key autophagic genes, including Beclin, LC3 and LAMP-2. In good agreement, protein levels of LC3-II and LAMP-2 but not of LAMP-1 were reduced in different cell model systems with PINK1 knockdown or knockout after addition of different stressors. This downregulation of autophagic factors caused increased apoptosis, which could be rescued by overexpression of LC3 or PINK1. Taken together, the PINK1-mediated reduction of autophagic key factors during stress resulted in increased cell death, thus defining an additional pathway that could contribute to the progression of Parkinson''s disease in patients with PINK1 mutations.     

达格列净通过增强营养剥夺信号促进糖尿病肾病大鼠足细胞自噬的研究

摘要 目的：探究达格列净是否通过增强营养剥夺信号促进糖尿病大鼠肾脏足细胞自噬,延缓糖尿病肾病进展。方法：采用高脂饮食和低剂量链脲佐菌素（STZ）腹腔注射诱导糖尿病模型。将成模大鼠随机分为糖尿病组（n=6）,达格列净组（n=7）,另设正常对照组（n=7）。达格列净组和糖尿病组分别给予达格列净和生理盐水灌胃4周。灌胃结束后收集大鼠24 h尿液以及血液、肾脏。记录体重、肾重、肾周脂肪重并测定空腹血糖、空腹血清胰岛素、血肌酐、尿素氮、甘油三酯以及尿白蛋白、尿总蛋白、尿白蛋白/肌酐比值;ELISA法检测血清酮体水平;HE染色、PAS染色以及电镜观察肾脏组织病理学改变;免疫组化和免疫荧光检测各组自噬相关蛋白7（ATG7）和足细胞标记蛋白NPHS1的表达情况; Western blot检测各组肾皮质中ATG7、沉默信息调节因子同源蛋白1（SIRT1）、过氧化物酶体增殖物激活受体γ共激活物-1α（PGC-1α）、成纤维细胞生长因子21（FGF21）、磷酸烯醇丙酮酸羧激酶（PEPCK）、过氧化物酶体增殖物激活受体α（PPARα）的蛋白水平。结果：与糖尿病组相比,达格列净组空腹血糖、24 h尿白蛋白、24 h尿总蛋白、尿白蛋白/肌酐比值降低,肾重/体重比、肾周脂肪重/体重比减小（P<0.05）;HE和PAS染色和电镜观察到糖尿病组较达格列净组肾小球基底膜增厚,足细胞足突增宽、融合（P<0.05）;达格列净组甘油三酯、空腹血清胰岛素以及胰岛素抵抗指数低于糖尿病组,血清酮体高于糖尿病组（P<0.05）;电镜下观察到达格列净组足细胞内自噬溶酶体数密度高于糖尿病组（P<0.05）;免疫组化和免疫荧光显示达格列净组足细胞NPHS1和ATG7的表达高于糖尿病组（P<0.05）; Western blot显示达格列净组肾皮质ATG7、SIRT1/PGC-1α/FGF21信号通路以及PEPCK、PPARα的表达较糖尿病组明显升高（P<0.05）。结论：达格列净增强营养剥夺信号的同时,观察到糖尿病大鼠肾脏足细胞自噬增强,这种自噬的增强可能是通过营养剥夺信号诱导的,其中的机制可能与达格列净上调SIRT1/PGC-1α /FGF21信号通路有关。     

Attenuated AMPA Receptor Expression Allows Glioblastoma Cell Survival in Glutamate-Rich Environment

Background

Glioblastoma multiforme (GBM) cells secrete large amounts of glutamate that can trigger AMPA-type glutamate receptors (AMPARs). This commonly results in Na⁺ and Ca²⁺-permeability and thereby in excitotoxic cell death of the surrounding neurons. Here we investigated how the GBM cells themselves survive in a glutamate-rich environment.

Methods and Findings

In silico analysis of published reports shows down-regulation of all ionotropic glutamate receptors in GBM as compared to normal brain. In vitro, in all GBM samples tested, mRNA expression of AMPAR subunit GluR1, 2 and 4 was relatively low compared to adult and fetal total brain mRNA and adult cerebellum mRNA. These findings were in line with primary GBM samples, in which protein expression patterns were down-regulated as compared to the normal tissue. Furthermore, mislocalized expression of these receptors was found. Sequence analysis of GluR2 RNA in primary and established GBM cell lines showed that the GluR2 subunit was found to be partly unedited.

Conclusions

Together with the lack of functional effect of AMPAR inhibition by NBQX our results suggest that down-regulation and afunctionality of AMPARs, enable GBM cells to survive in a high glutamate environment without going into excitotoxic cell death themselves. It can be speculated that specific AMPA receptor inhibitors may protect normal neurons against the high glutamate microenvironment of GBM tumors.     

Hsc70 Contributes to Cancer Cell Survival by Preventing Rab1A Degradation under Stress Conditions

Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology.     

Enhanced Arginine Methylation of Programmed Cell Death 4 Protein during Nutrient Deprivation Promotes Tumor Cell Viability

The role of programmed cell death 4 (PDCD4) in tumor biology is context-dependent. PDCD4 is described as a tumor suppressor, but its coexpression with protein arginine methyltransferase 5 (PRMT5) promotes accelerated tumor growth. Here, we report that PDCD4 is methylated during nutrient deprivation. Methylation occurs because of increased stability of PDCD4 protein as well as increased activity of PRMT5 toward PDCD4. During nutrient deprivation, levels of methylated PDCD4 promote cell viability, which is dependent on an enhanced interaction with eIF4A. Upon recovery from nutrient deprivation, levels of methylated PDCD4 are regulated by phosphorylation, which controls both the localization and stability of methylated PDCD4. This study reveals that, in response to particular environmental cues, the role of PDCD4 is up-regulated and is advantageous for cell viability. These findings suggest that the methylated form of PDCD4 promotes tumor viability during nutrient deprivation, ultimately allowing the tumor to grow more aggressively.     

Contribution of Atg1-Dependent Autophagy to TOR-Mediated Cell Growth and Survival

The Ser/Thr kinase Atg1 (Ulk1/Unc51) appears to act as a convergence point for multiple signals that regulate autophagy, and in turn interacts with a large number of autophagy-related (Atg) proteins. Working in the Drosophila system, we recently found that overexpression of Atg1 is sufficient to induce autophagy, independent of upstream nutrient signals. We exploited this finding to examine the roles of autophagy in cell growth and death, and to test the interaction of Atg1 with the TOR signaling pathway. These studies provided genetic evidence that autophagy is a potent inhibitor of cell growth, and that high levels of autophagy lead to caspase-dependent apoptotic cell death in vivo. Atg1 also has an inhibitory effect on TOR signaling, indicating the existence of a positive feedback mechanism that may amplify the nutrient-dependent signals that control autophagy.

Addendum to:

Direct Induction of Autophagy by Atg1 Inhibits Cell Growth and Induces Apoptotic Cell Death

R.C. Scott, G. Juhász and T.P. Neufeld

Curr Biol 2007; 17:1-11     

Nucleolar Follistatin Promotes Cancer Cell Survival under Glucose-deprived Conditions through Inhibiting Cellular rRNA Synthesis

Solid tumor development is frequently accompanied by energy-deficient conditions such as glucose deprivation and hypoxia. Follistatin (FST), a secretory protein originally identified from ovarian follicular fluid, has been suggested to be involved in tumor development. However, whether it plays a role in cancer cell survival under energy-deprived conditions remains elusive. In this study, we demonstrated that glucose deprivation markedly enhanced the expression and nucleolar localization of FST in HeLa cells. The nucleolar localization of FST relied on its nuclear localization signal (NLS) comprising the residues 64–87. Localization of FST to the nucleolus attenuated rRNA synthesis, a key process for cellular energy homeostasis and cell survival. Overexpression of FST delayed glucose deprivation-induced apoptosis, whereas down-regulation of FST exerted the opposite effect. These functions depended on the presence of an intact NLS because the NLS-deleted mutant of FST lost the rRNA inhibition effect and the cell protective effect. Altogether, we identified a novel nucleolar function of FST, which is of importance in the modulation of cancer cell survival in response to glucose deprivation.     

Ulk1/FUNDC1 Prevents Nerve Cells from Hypoxia-Induced Apoptosis by Promoting Cell Autophagy

Cell autophagy and cell apoptosis are both observed in the process of hypoxia-induced ischemic cerebral infarction (ICI). Unc-51 like autophagy activating kinase 1 (Ulk1) and FUN14 Domain-containing Protein 1 (FUNDC1) are both involved in the regulation of cell autophagy. This study aimed to investigate the regulatory effects of Ulk1 and FUNDC1 on hypoxia-induced nerve cell autophagy and apoptosis. Cell viability was measured using cell counting kit-8 (CCK-8) assay. Cell apoptosis was detected using Annexin V-PE/7-ADD staining assay. qRT-PCR was used to quantify the mRNA levels of Ulk1 and FUNDC1 in PC-12 cells. Cell transfection was performed to up-regulate the expression of Ulk1. 3-Methyladenine (3-MA) was used as autophagy inhibitor and rapamycin was used as autophagy activator in our experiments. SP600125 was used as c-Jun N-terminal kinase (JNK) inhibitor. Western blotting was performed to analyze the expression levels of key factors that are related to cell autophagy, apoptosis and JNK pathway. We found that hypoxia simultaneously induced apoptosis and autophagy of PC-12 cells. The activation of Ulk1 and FUNDC1 were also found in PC-12 cells after hypoxia induction. Overexpression of Ulk1 promoted the activation of FUNDC1 and prevented PC-12 cells from hypoxia-induced apoptosis. Suppression of Ulk1 had opposite effects. Furthermore, we also found that JNK pathway participated in the effects of Ulk1 overexpression on PC-12 cell apoptosis reduction. To conclude, Ulk1/FUNDC1 played critical regulatory roles in hypoxia-induced nerve cell autophagy and apoptosis. Overexpression of Ulk1 prevented nerve cells from hypoxia-induced apoptosis by promoting cell autophagy.