Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila

BACKGROUND: Cell growth arrest and autophagy are required for autophagic cell death in Drosophila. Maintenance of growth by expression of either activated Ras, Dp110, or Akt is sufficient to inhibit autophagy and cell death in Drosophila salivary glands, but the mechanism that controls growth arrest is unknown. Although the Warts (Wts) tumor suppressor is a critical regulator of tissue growth in animals, it is not clear how this signaling pathway controls cell growth. RESULTS: Here, we show that genes in the Wts pathway are required for salivary gland degradation and that wts mutants have defects in cell growth arrest, caspase activity, and autophagy. Expression of Atg1, a regulator of autophagy, in salivary glands is sufficient to rescue wts mutant salivary gland destruction. Surprisingly, expression of Yorkie (Yki) and Scalloped (Sd) in salivary glands fails to phenocopy wts mutants. By contrast, misexpression of the Yki target bantam was able to inhibit salivary gland cell death, even though mutations in bantam fail to suppress the wts mutant salivary gland-persistence phenotype. Significantly, wts mutant salivary glands possess altered phosphoinositide signaling, and decreased function of the class I PI3K-pathway genes chico and TOR suppressed wts defects in cell death. CONCLUSIONS: Although we have previously shown that salivary gland degradation requires genes in the Wts pathway, this study provides the first evidence that Wts influences autophagy. Our data indicate that the Wts-pathway components Yki, Sd, and bantam fail to function in salivary glands and that Wts regulates salivary gland cell death in a PI3K-dependent manner.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.

Addendum to: Berry DL, Baehrecke EH. Growth arrest and autophagy are required for programmed salivary gland cell degradation in Drosophila. Cell 2007; 131:1137-48.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.     

The engulfment receptor Draper is required for autophagy during cell death

Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.Key words: autophagy, Draper, programmed cell death, engulfment, developmentProgrammed cell death is required for animal development and tissue homeostasis. Improper cell death leads to pathologies including autoimmunity and cancer. Several morphological forms of cell death occur during animal development, including apoptosis and autophagic cell death. Autophagic cell death is characterized by the presence of autophagosomes in dying cells that are not known to be engulfed by phagocytes. Autophagic cell death is observed during several types of mammalian developmental cell death, including regression of the corpus luteum and involution of mammary and prostate glands.During macroautophagy (autophagy), cytoplasmic components are sequestered by autophagosomes and delivered to the lysosome for degradation. Autophagy is a cellular response to stress required for survival in response to starvation. Whereas autophagy has been associated with cell death, it is unknown how autophagy is distinguished during cell death and cell survival. Autophagy is induced in Drosophila in response to starvation in the fat body where it promotes cell survival, while autophagy is induced by the steroid hormone ecdysone in salivary glands where it promotes cell death. This allows studies of autophagy in different cell types and in response to different stimuli.Drosophila larval salivary glands die with autophagic cell death morphology and autophagy is required for their degradation. Expression of the caspase inhibitor p35 enhances salivary gland persistence in Atg mutants, suggesting that caspases and autophagy function in parallel during salivary gland degradation. Either activation of caspases or Atg1 misexpression is sufficient to induce ectopic salivary gland clearance. We queried genome-wide microarray data from purified dying salivary glands and noted the induction of engulfment genes, those required for a phagocyte to consume and degrade a dying cell. We also noted few detectable changes in engulfment genes in Drosophila larvae during starvation.We found that Drpr, the Drosophila orthologue of C. elegans engulfment receptor CED-1, is enriched in dying salivary glands, and drpr null mutants have persistent salivary glands. Interestingly, whereas knockdown of drpr in phagocytic blood cells fails to influence salivary gland clearance, expression of drpr-RNAi in salivary glands prevents gland clearance. Drosophila drpr is alternatively spliced to produce three isoforms. We found that drpr-I-specific knockdown prevents salivary gland degradation and Drpr-I expression in salivary glands of drpr null mutants rescues salivary gland persistence. Therefore, drpr is autonomously required for salivary gland clearance. However, how Drpr is induced or activated during hormone-regulated cell death remains to be determined.drpr knockdown fails to influence caspase activation, and caspase inhibitor p35 expression in drpr null mutants enhances salivary gland persistence, suggesting that Drpr functions downstream or parallel to caspases in dying salivary glands. Interestingly, we found that drpr knockdown in salivary glands prevents the formation of GFP-LC3 puncta. Further, Atg1 misexpression in salivary glands of drpr null mutants suppresses salivary gland persistence. drpr is therefore required for autophagy induction in salivary glands, and Atg1 functions downstream of Drpr in this tissue. We found that several other engulfment genes are required for salivary gland degradation. However, the Drpr signaling mechanism leading to autophagy induction in salivary glands remains to be elucidated.We tested whether drpr is a general regulator of autophagy. The Drosophila fat body is a nutrient storage and mobilization organ akin to the mammalian liver, and is a well-established model to study starvation-induced autophagy. We found that drpr-RNAi expression in fat body clone cells fails to prevent GFP-Atg8 puncta formation in response to starvation. Similarly, drpr null fat body clone cells form Cherry-Atg8 puncta after starvation. Strikingly, drpr-RNAi expression in salivary gland clone cells inhibits the formation of GFP-Atg8 puncta. Therefore, drpr is cell-autonomously required for autophagy induction in dying salivary gland cells, but not for autophagy induction in fat body cells after starvation. These findings suggest that distinct signaling mechanisms regulate autophagy in response to nutrient deprivation compared to steroid hormone induction. Little is known about what distinguishes autophagy function in cell survival versus death. It is possible that varying levels of autophagy are induced during specific cell contexts and that high levels of autophagy could overwhelm a cell—leading to cell death. Autophagic degradation of specific cargo, such as cell death inhibitors, could also contribute to cell death.Given recent interest in manipulation of autophagy for therapies, it is possible that factors such as Drpr could be used as biomarkers to distinguish autophagy leading to cell death versus cell survival. While it is generally accepted that augmentation of protein clearance by autophagy during neurodegeneration would be beneficial, the role of autophagy in tumor progression is less clear. For example, monoallelic loss of the human Atg6 homolog beclin 1 is prevalent in human cancers, suggesting that autophagy is a tumorsuppressive mechanism. Thus, autophagy enhancers have been proposed for cancer prevention. However, autophagy occurs in tumor cells as a survival mechanism, and autophagy inhibitors have been proposed for anti-cancer therapies. Understanding how autophagy is regulated in different contexts is critical for appropriate therapeutic strategies.     

Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death.     

Caspases function in autophagic programmed cell death in Drosophila

Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Here we show that autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes beta FTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death.     

Autophagy as a trigger for cell death: autophagic degradation of inhibitor of apoptosis dBruce controls DNA fragmentation during late oogenesis in Drosophila

Autophagy has been reported to contribute to cell death, but the underlying mechanisms remain largely unknown and controversial. We have: been studying oogenesis in Drosophila melanogaster as a model system to understand the interplay between autophagy and cell death. Using a novel autophagy reporter we found that autophagy occurs during developmental cell death of nurse cells in late oogenesis. Genetic inhibition: of autophagy-related genes atg1, atg13 and vps34 results in late-stage egg chambers containing persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. We found that Drosophila inhibitor of apoptosis dBruce is degraded by autophagy and this degradation promotes DNA fragmentation and subsequent nurse cell death. These studies demonstrate that autophagic degradation of an inhibitor: of apoptosis is a novel mechanism of triggering cell death.     

Steroid regulation of autophagic programmed cell death during development

Apoptosis and autophagy are morphologically distinct forms of programmed cell death. While autophagy occurs during the development of diverse organisms and has been implicated in tumorigenesis, little is known about the molecular mechanisms that regulate this type of cell death. Here we show that steroid-activated programmed cell death of Drosophila salivary glands occurs by autophagy. Expression of p35 prevents DNA fragmentation and partially inhibits changes in the cytosol and plasma membranes of dying salivary glands, suggesting that caspases are involved in autophagy. The steroid-regulated BR-C, E74A and E93 genes are required for salivary gland cell death. BR-C and E74A mutant salivary glands exhibit vacuole and plasma membrane breakdown, but E93 mutant salivary glands fail to exhibit these changes, indicating that E93 regulates early autophagic events. Expression of E93 in embryos is sufficient to induce cell death with many characteristics of apoptosis, but requires the H99 genetic interval that contains the rpr, hid and grim proapoptotic genes to induce nuclear changes diagnostic of apoptosis. In contrast, E93 expression is sufficient to induce the removal of cells by phagocytes in the absence of the H99 genes. These studies indicate that apoptosis and autophagy utilize some common regulatory mechanisms.     

Autophagy gene disruption reveals a non-vacuolar cell death pathway in Dictyostelium

Types of cell death include apoptosis, necrosis, and autophagic cell death. The latter can be defined as death of cells containing autophagosomes, autophagic bodies, and/or vacuoles. Are autophagy and vacuolization causes, consequences, or side effects in cell death with autophagy? Would control of autophagy suffice to control this type of cell death? We disrupted the atg1 autophagy gene in Dictyostelium discoideum, a genetically tractable model for developmental autophagic vacuolar cell death. The procedure that induced autophagy, vacuolization, and death in wild-type cells led in atg1 mutant cells to impaired autophagy and to no vacuolization, demonstrating that atg1 is required for vacuolization. Unexpectedly, however, cell death still took place, with a non-vacuolar and centrally condensed morphology. Thus, a cell death mechanism that does not require vacuolization can operate in this cell death model showing conspicuous vacuolization. The revelation of non-vacuolar cell death in this protist by autophagy gene disruption is reminiscent of caspase inhibition revealing necrotic cell death in animal cells. Thus, hidden alternative cell death pathways may be found across kingdoms and for diverse types of cell death.     

Does autophagy contribute to cell death?

Autophagy (specifically macroautophagy) is an evolutionarily conserved catabolic process where the cytoplasmic contents of a cell are sequestered within double membrane vacuoles, called autophagosomes, and subsequently delivered to the lysosome for degradation. Autophagy can function as a survival mechanism in starving cells. At the same time, extensive autophagy is commonly observed in dying cells, leading to its classification as an alternative form of programmed cell death. The functional contribution of autophagy to cell death has been a subject of great controversy. However, several recent loss-of-function studies of autophagy (atg) genes have begun to address the roles of autophagy in both cell death and survival. Here, we review the emerging evidence in favor of and against autophagic cell death, discuss the possible roles that autophagic degradation might play in dying cells, and identify salient issues for future investigation.     

Inhibitors of phosphatidylinositol 3'-kinases promote mitotic cell death in HeLa cells

The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in many biological processes, including cell cycle progression, cell growth, survival, actin rearrangement and migration, and intracellular vesicular transport. However, the involvement of the PI3K pathway in the regulation of mitotic cell death remains unclear. In this study, we treated HeLa cells with the PI3K inhibitors, 3-methyladenine (3-MA, as well as a widely used autophagy inhibitor) and wortmannin to examine their effects on cell fates using live cell imaging. Treatment with 3-MA decreased cell viability in a time- and dose-dependent manner and was associated with caspase-3 activation. Interestingly, 3-MA-induced cell death was not affected by RNA interference-mediated knockdown (KD) of beclin1 (an essential protein for autophagy) in HeLa cells, or by deletion of atg5 (an essential autophagy gene) in mouse embryonic fibroblasts (MEFs). These data indicate that cell death induced by 3-MA occurs independently of its ability to inhibit autophagy. The results from live cell imaging studies showed that the inhibition of PI3Ks increased the occurrence of lagging chromosomes and cell cycle arrest and cell death in prometaphase. Furthermore, PI3K inhibitors promoted nocodazole-induced mitotic cell death and reduced mitotic slippage. Overexpression of Akt (the downstream target of PI3K) antagonized PI3K inhibitor-induced mitotic cell death and promoted nocodazole-induced mitotic slippage. These results suggest a novel role for the PI3K pathway in regulating mitotic progression and preventing mitotic cell death and provide justification for the use of PI3K inhibitors in combination with anti-mitotic drugs to combat cancer.     

Autophagy and apoptosis coordinate physiological cell death in larval salivary glands of Apis mellifera (Hymenoptera: Apidae)

Larval salivary glands of bees provide a good model for the study of hormone-induced programmed cell death in Hymenoptera because they have a well-defined secretory cycle with a peak of secretory activity phase, prior to cocoon spinning, and a degenerative phase, after the cocoon spinning. Our findings demonstrate that there is a relationship between apoptosis and autophagy during physiological cell death in these larval salivary glands, that adds evidence to the hypothesis of overlap in the regulation pathways of both types of programmed cell death. Features of autophagy include cytoplasm vacuolation, acid phosphatase activity, presence of autophagic vacuoles and multi-lamellar structures, as well as a delay in the collapse of many nuclei. Features of apoptosis include bleb formation in the cytoplasm and nuclei, with release of parts of the cytoplasm into the lumen, chromatin compaction, and DNA and nucleolar fragmentation. We propose a model for programmed cell death in larval salivary glands of Apis mellifera where autophagy and apoptosis function cooperatively for a more efficient degeneration of the gland secretory cells.     

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.     

The engulfment receptor Draper is required for autophagy during cell death

Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction

during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.     

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

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.     

Polyphyllin VII Induces an Autophagic Cell Death by Activation of the JNK Pathway and Inhibition of PI3K/AKT/mTOR Pathway in HepG2 Cells

Polyphyllin VII (PP7), a pennogenyl saponin isolated from Rhizoma Paridis, exhibited strong anticancer activities in various cancer types. Previous studies found that PP7 induced apoptotic cell death in human hepatoblastoma cancer (HepG2) cells. In the present study, we investigated whether PP7 could induce autophagy and its role in PP7-induced cell death, and elucidated its mechanisms. PP7 induced a robust autophagy in HepG2 cells as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, formation of punctate LC3-positive structures, and autophagic vacuoles tested by western blot analysis or InCell 2000 confocal microscope. Inhibition of autophagy by treating cells with autophagy inhibitor (chloroquine) abolished the cell death caused by PP7, indicating that PP7 induced an autophagic cell death in HepG2 cells. C-Jun N-terminal kinase (JNK) was activated after treatment with PP7 and pretreatment with SP600125, a JNK inhibitor, reversed PP7-induced autophagy and cell death, suggesting that JNK plays a critical role in autophagy caused by PP7. Furthermore, our study demonstrated that PP7 increased the phosphorylation of AMPK and Bcl-2, and inhibited the phosphorylation of PI3K, AKT and mTOR, suggesting their roles in the PP7-induced autophagy. This is the first report that PP7 induces an autophagic cell death in HepG2 cells via inhibition of PI3K/AKT/mTOR, and activation of JNK pathway, which induces phosphorylation of Bcl-2 and dissociation of Beclin-1 from Beclin-1/Bcl-2 complex, leading to induction of autophagy.     

Real-time observation of autophagic programmed cell death of<Emphasis Type="Italic"> Drosophila</Emphasis> salivary glands in vitro

Autophagy, a form of programmed cell death (PCD) that is morphologically distinguished from apoptosis, is thought to be as prevalent as apoptosis, at least during development. In insect metamorphosis, the steroid hormone 20-hydroxyecdysone (ecdysone) activates autophagic PCD to eliminate larval structures that are no longer needed. However, in comparison with apoptosis, there are not many studies on the regulation mechanisms of autophagy. To provide a useful model for studying autophagic PCD, I established an in vitro culture system that enables real-time observation of the autophagic cell destruction of Drosophila salivary glands. The new system revealed that de novo gene expression was still required for the destruction of salivary glands dissected from phanerocephalic pupae. This indicates the usefulness of the system for exploring genes that participate in the last processes of autophagic PCD.Edited by N. Satoh