Eiger,a TNF superfamily ligand that triggers the Drosophila JNK pathway

Drosophila provides a powerful genetic model for studying the in vivo regulation of cell death. In our large-scale gain-of-function screen, we identified Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death. Eiger is a type II transmembrane protein with a C-terminal TNF homology domain. It is predominantly expressed in the nervous system. Genetic evidence shows that Eiger induces cell death by activating the Drosophila JNK pathway. Although this cell death process is blocked by Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), it does not require caspase activity. We also show genetically that Eiger is a physiological ligand for the Drosophila JNK pathway. Our findings demonstrate that Eiger can initiate cell death through an IAP-sensitive cell death pathway via JNK signaling.     

A genetic screen targeting the tumor necrosis factor/Eiger signaling pathway: identification of Drosophila TAB2 as a functionally conserved component

Signaling by tumor necrosis factors (TNFs) plays a prominent role in mammalian development and disease. To fully understand this complex signaling pathway it is important to identify all regulators and transduction components. A single TNF family member, Eiger, is encoded in the Drosophila genome, offering the possibility of applying genetic approaches for pursuing this goal. Here we present a screen for the isolation of novel genes involved in the TNF/Eiger pathway. On the basis of Eiger's ability to potently activate Jun-N-terminal kinase (JNK) and trigger apoptosis, we used the Drosophila eye to establish an assay for dominant suppressors of this activity. In a large-scale screen the Drosophila homolog of TAB2/3 (dTAB2) was identified as an essential component of the Eiger-JNK pathway. Genetic epistasis and biochemical protein-protein interaction assays assign an adaptor role to dTAB2, linking dTRAF1 to the JNKKK dTAK1, demonstrating a conserved mechanism of TNF signal transduction in mammals and Drosophila. Thus, in contrast to morphogenetic processes, such as dorsal closure of the embryo, in which the JNK pathway is activated by the JNKKK Slipper, Eiger uses the dTAB2-dTAK1 module to induce JNK signaling activity.     

Loss of Rab5 drives non-autonomous cell proliferation through TNF and Ras signaling in Drosophila

Deregulation of the endocytic machinery has been implicated in human cancers. However, the mechanism by which endocytic defects drive cancer development remains to be clarified. Here, we find through a genetic screen in Drosophila that loss of Rab5, a protein required for early endocytic trafficking, drives non-autonomous cell proliferation in imaginal epithelium. Our genetic data indicate that dysfunction of Rab5 leads to cell-autonomous accumulation of Eiger (a TNF homolog) and EGF receptor (EGFR), which causes activation of downstream JNK and Ras signaling, respectively. JNK signaling and its downstream component Cdc42 cooperate with Ras signaling to induce upregulation of a secreted growth factor Upd (an IL-6 homolog) through inactivation of the Hippo pathway. Such non-autonomous tissue growth triggered by Rab5 defect could contribute to epithelial homeostasis as well as cancer development within heterogeneous tumor microenvironment.     

Delineation of the cell-extrinsic apoptosis pathway in the zebrafish

The mammalian extrinsic apoptosis pathway is triggered by Fas ligand (FasL) and Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL). Ligand binding to cognate receptors activates initiator caspases directly in a death-inducing signaling complex. In Drosophila, TNF ligand binding activates initiator caspases indirectly, through JNK. We characterized the extrinsic pathway in zebrafish to determine how it operates in a nonmammalian vertebrate. We identified homologs of FasL and Apo2L/TRAIL, their receptors, and other components of the cell death machinery. Studies with three Apo2L/TRAIL homologs demonstrated that they bind the receptors zHDR (previously linked to hematopoiesis) and ovarian TNFR (zOTR). Ectopic expression of these ligands during embryogenesis induced apoptosis in erythroblasts and notochord cells. Inhibition of zHDR, zOTR, the adaptor zFADD, or caspase-8-like proteases blocked ligand-induced apoptosis, as did antiapoptotic Bcl-2 family members. Thus, the extrinsic apoptosis pathway in zebrafish closely resembles its mammalian counterpart and cooperates with the intrinsic pathway to trigger tissue-specific apoptosis during embryogenesis in response to ectopic Apo2L/TRAIL expression.     

Transgenic Expression of the Helicobacter pylori Virulence Factor CagA Promotes Apoptosis or Tumorigenesis through JNK Activation in Drosophila

Gastric cancer development is strongly correlated with infection by Helicobacter pylori possessing the effector protein CagA. Using a transgenic Drosophila melanogaster model, we show that CagA expression in the simple model epithelium of the larval wing imaginal disc causes dramatic tissue perturbations and apoptosis when CagA-expressing and non-expressing cells are juxtaposed. This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells. We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis. In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner. These data suggest a potential role for CagA-mediated JNK pathway activation in promoting gastric cancer progression.     

The dark side of fly TNF

The fruit fly Drosophila is an important model for biological research; however, due to its relatively short lifespan its relevance in cancer research is often questioned. Nevertheless, among many other intriguing Drosophila models, scribble group mutants provided early evidence for the existence of tumour suppressor genes and their importance in mammalian systems is beginning to emerge. In this review, I discuss recent advances in our understanding of the phenotypes of scrib group gene mutants, in which the activation of JNK signaling plays a crucial role. Several mechanisms can account for the activation of JNK within scrib group mutant cells, including a mechanical stress triggered by the loss of polarity, cell competition, intrinsic tumour suppression by autonomous production of Eiger, and an inflammatory response mediated by Eiger-producing haemocytes. Eiger, the sole Drosophila homolog of tumour necrosis factor, is emerging as a 'danger signal' initiated upon the presence of external pathogens, damaged tissues, and the appearance of pre-malignant cells. Remarkably, in the presence of the Ras oncoprotein Eiger can act as a tumor promoter by stimulating invasive migration and delaying the onset of metamorphosis.     

Deltex interacts with Eiger and consequently influences the cell death in Drosophila melanogaster

TNF-JNK signaling is one of the highly conserved signaling pathways that regulate a broad spectrum of cellular processes including proliferation and apoptosis. Eiger, the sole homologue of TNF in Drosophila, initiates the TNF-JNK pathway to induce cell death. Previously, Deltex (Dx) has been identified as a Notch signaling component that regulates vesicular trafficking of Notch. In the present study, we have investigated the interaction between these two proteins in order to identify how Dx influences the activity of Eiger. Dx is found to act as a novel modulator of JNK-mediated cell death inducing activity of Eiger. Additionally, we observe that dx genetically interacts with eiger during wing development, and these two proteins, Dx and Eiger, colocalize in the cytoplasm. Our analysis reveals that Dx is involved in the cytoplasmic relocalization of Eiger from the cell membrane, thereby influencing Eiger-mediated JNK-activation process. Moreover, we demonstrate that Dx potentiates the activity of Eiger to downregulate Notch signaling pathway by retaining the Notch protein in the cytoplasm. Together, our findings reveal a novel role of Dx to modulate the signaling activity of Eiger and subsequent JNK-mediated cell death.     

POSH is involved in Eiger-Basket （TNF-JNK） signaling and embryogenesis in Drosophila

TNFα can trigger different signaling pathways, including the JNK pathway, to regulate various biological functions such as cell death, differentiation and proliferation. The scaffold protein POSH(Plenty of SH3 Domains)has been shown to be an important regulator of the JNK pathway, but whether it is involved in TNF-signaling has not been reported.Although POSH has been implicated to play a role in development in zebrafish,it has not been studied in null mutants and the underlying mechanism of its effects is still not clear.In this study,we provide evidence that the JNK pathway scaffold protein,POSH,is involved in TNF(Eiger)signaling in Drosophila.POSH is likely to act downstream of dTAB2 and upstream of dTAK1 in the TNF-JNK signaling pathway.In addition,we found that POSH is essential during Drosophila embryogenesis,including epidermal dorsal closure,similar to other JNK pathway components such as Slipper,Hemipterous,and Basket. We observed defects in F-actin accumulation and adherens junction formation during dorsal closure in different posh null mutants, suggesting that POSH is required for epidermal cell migration and cell-shape change during epidermal dorsal closure.     

Deltex cooperates with TRAF6 to promote apoptosis and cell migration through Eiger-independent JNK activation in Drosophila

JNK signaling is a highly conserved signaling pathway that regulates a broad spectrum of cellular processes including cell proliferation, migration, and apoptosis. In Drosophila, JNK signaling is activated by binding of the tumor necrosis factor (TNF) Eiger to its receptor Wengen, and a conserved signaling cascade operates that culminates into activation of dual phosphatase Puckered thereby triggering apoptosis. The tumor necrosis factor receptor (TNFR) associated factor 6 (TRAF6) is an adaptor protein, which transduces the signal from TNFRs and Toll-like receptor/interleukin-1 receptor superfamily to induce a wide spectrum of cellular responses. TRAF6 also acts as the adaptor protein that mediates Eiger/JNK signaling in Drosophila. In a genetic interaction study, deltex (Dx) was identified as a novel interactor of TRAF6. Dx is well known to regulate Notch signaling in a context-dependent manner. Our data suggest that combinatorial action of Dx and TRAF6 enhances the Dx-induced wing nicking phenotype by inducing caspase-mediated cell death. Co-expression of Dx and TRAF6 also results in enhanced invasive behavior and perturbs the normal morphology of cells. The cooperative action of Dx and TRAF6 is attributed to JNK activation, which also leads to ectopic wingless (Wg) and decapentaplegic (Dpp) expression. Our results also reveal that the endocytic pathway component Rab7 may play a pivotal role in the regulation of Dx–TRAF6-mediated activation of JNK signaling. Here, we present the fact that Dx and TRAF6 together activate JNK signaling in an Eiger-independent mechanism.     

Bendless modulates JNK-mediated cell death and migration in Drosophila

The TNF–JNK pathway is a highly conserved signaling pathway that regulates a wide spectrum of biological processes including cell death and migration. To further delineate this pathway, we carried out a genetic screen for dominant modifiers of the cell death phenotype triggered by ectopic expression of Eiger (Egr), the Drosophila TNF ortholog. Here we show that Bendless (Ben), an E2 ubiquitin-conjugating enzyme, modulates Egr-induced JNK activation and cell death through dTRAF2. Furthermore, Ben physically interacts with dTRAF2 and regulates Egr-induced dTRAF2 polyubiquitination. Finally, Ben is required for JNK-dependent tumor progression, cell migration, oxidative stress resistance and longevity. Our results indicate that Ben constitutes an essential component of the evolutionarily conserved TNF–JNK pathway that modulates cell death and invasion, tumor progression, stress response and lifespan in metazoans.     

Activation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway by overexpression of caspase-8 and its homologs.

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases. A point mutant in the first DED of caspase-8 can block JNK activation induced by several death domain receptors. Inhibition of JNK activation blocks apoptosis mediated by caspase-10, Mach-related inducer of toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of this pathway in the mediation of caspase-induced apoptosis.     

Caenorhabditis elegans innate immune response triggered by Salmonella enterica requires intact LPS and is mediated by a MAPK signaling pathway

Compared to mammals, insects, and plants, relatively little is known about innate immune responses in the nematode Caenorhabditis elegans. Previous work showed that Salmonella enterica serovars cause a persistent infection in the C. elegans intestine that triggers gonadal programmed cell death (PCD) and that C. elegans cell death (ced) mutants are more susceptible to Salmonella-mediated killing. To further dissect the role of PCD in C. elegans innate immunity, we identified both C. elegans and S. enterica factors that affect the elicitation of Salmonella-induced PCD. Salmonella-elicited PCD was shown to require the C. elegans homolog of the mammalian p38 mitogen-activated protein kinase (MAPK) encoded by the pmk-1 gene. Inactivation of pmk-1 by RNAi blocked Salmonella-elicited PCD, and epistasis analysis showed that CED-9 lies downstream of PMK-1. Wild-type Salmonella lipopolysaccharide (LPS) was also shown to be required for the elicitation of PCD, as well as for persistence of Salmonella in the C. elegans intestine. However, a presumptive C. elegans TOLL signaling pathway did not appear to be required for the PCD response to Salmonella. These results establish a PMK-1-dependant PCD pathway as a C. elegans innate immune response to Salmonella.     

Distinct stress and cell destruction pathways are engaged by TNF and ceramide during apoptosis of MCF-7 cells

Ceramide has been proposed to be an important signaling intermediate in tumor necrosis factor (TNF)-induced apoptosis in human MCF-7 breast adenocarcinoma cells. We compared cell death and signal transduction pathways induced by TNF and ceramide in TNF-sensitive, parental MCF-7 cells to those in TNF-resistant, MCF-7 cells (3E9). TNF caused proteolysis of the caspase substrate, polyADP-ribose polymerase (PARP) in parental cells, but not in 3E9 cells. Both apoptosis and PARP cleavage were strongly prevented by co-incubation with caspase inhibitors. In contrast, ceramide-induced cell death was neither affected by TNF resistance nor was it associated with PARP cleavage, and death could not be prevented by co-incubation with caspase inhibitors in either cell line. TNF was able to activate JNK/SAPK approximately 30-fold and approximately 5-fold in parental MCF-7 and 3E9 cells, respectively; in contrast, cell-permeable ceramide only weakly stimulated JNK/SAPK activity in either cell type. Although JNK was activated by TNF, pharmacological blockade of the JNK pathway did not inhibit TNF- or ceramide-mediated cell death. Using mass spectroscopic analysis for ceramide, no increase, rather, a decrease in total ceramide content in TNF-treated parental cells was observed. These results suggest that the cell death signaling and execution pathways utilized by ceramide are distinct from those activated by TNF in MCF-7 cells.     

The dark side of fly TNF: An ancient developmental proofreading mechanism turned into tumor promoter

The fruit fly Drosophila is an important model for biological research; however, due to its relatively short lifespan its relevance in cancer research is often questioned. Nevertheless, among many other intriguing Drosophila models, scribble group mutants provided early evidence for the existence of tumor suppressor genes, and their importance in mammalian systems is beginning to emerge. In this extra view, I discuss recent advances in our understanding of the phenotypes of scrib-group mutants, in which the activation of JNK signaling plays a crucial role. Several mechanisms can account for the activation of JNK within scrib-group mutant cells, including a mechanical stress triggered by the loss of polarity, cell competition, intrinsic tumor suppression by autonomous production of Eiger and an inflammatory response mediated by Eiger-producing hemocytes. Eiger, the sole Drosophila homolog of tumor necrosis factor, is emerging as a “danger signal” initiated upon the presence of external pathogens, damaged tissues and the appearance of pre-malignant cells. Remarkably, in the presence of the Ras oncoprotein, Eiger can act as a tumor promoter by stimulating invasive migration and delaying the onset of metamorphosis.Key words: Drosophila, tumor necrosis factor, scribble, lgl, dlg     

Tumor necrosis factor-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway

In vitro studies of hepatocytes have implicated over-activation of c-Jun N-terminal kinase (JNK) signaling as a mechanism of tumor necrosis factor-alpha (TNF)-induced apoptosis. However, the functional significance of JNK activation and the role of specific JNK isoforms in TNF-induced hepatic apoptosis in vivo remain unclear. JNK1 and JNK2 function was, therefore, investigated in the TNF-dependent, galactosamine/lipopolysaccharide (GalN/LPS) model of liver injury. The toxin GalN converted LPS-induced JNK signaling from a transient to prolonged activation. Liver injury and mortality from GalN/LPS was equivalent in wild-type and jnk1-/- mice but markedly decreased in jnk2-/- mice. This effect was not secondary to down-regulation of TNF receptor 1 expression or TNF production. In the absence of jnk2, the caspase-dependent, TNF death pathway was blocked, as reflected by the failure of caspase-3 and -7 and poly(ADP-ribose) polymerase cleavage to occur. JNK2 was critical for activation of the mitochondrial death pathway, as in jnk2-/- mice Bid cleavage and mitochondrial translocation and cytochrome c release were markedly decreased. This effect was secondary to the failure of jnk2-/- mice to activate caspase-8. Liver injury and caspase activation were similarly decreased in jnk2 null mice after GalN/TNF treatment. Ablation of jnk2 did not inhibit GalN/LPS-induced c-Jun kinase activity, although activity was completely blocked in jnk1-/- mice. Toxic liver injury is, therefore, associated with JNK over-activation and mediated by JNK2 promotion of caspase-8 activation and the TNF mitochondrial death pathway through a mechanism independent of c-Jun kinase activity.     

POSH promotes cell survival in Drosophila and in human RASF cells

In Drosophila, Eiger, a tumor necrosis factor α (TNFα) superfamily ligand, induces cell death by activating the c-Jun N-terminal kinase (JNK) pathway. Here, we report that overexpression of Plenty of SH3s (POSH) suppresses Eiger-induced cell death and produces highly deformed tissues. These results imply that high levels of POSH protect tissues from cell death. In humans, rheumatoid arthritis synovial fibroblasts (RASF) are generally resistant to apoptosis. We show that POSH is expressed at relatively high levels in RASF, and its reduction by RNAi sensitizes these cells to Fas-mediated apoptosis. Thus, we demonstrate that POSH promotes cell survival in Drosophila and in human RASF.     

Apoptosome-independent activation of the lysosomal cell death pathway by caspase-9

The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using a large panel of genetically modified murine embryonic fibroblasts, we show here that, in response to tumor necrosis factor (TNF), caspase-8 cleaves and activates caspase-9 in an apoptosome-independent manner. Interestingly, caspase-8-cleaved caspase-9 induced lysosomal membrane permeabilization but failed to activate the effector caspases whereas apoptosome-dependent activation of caspase-9 could trigger both events. Consistent with the ability of TNF to activate the intrinsic apoptosis pathway and the caspase-9-dependent lysosomal cell death pathway in parallel, their individual inhibition conferred only a modest delay in TNF-induced cell death whereas simultaneous inhibition of both pathways was required to achieve protection comparable to that observed in caspase-9-deficient cells. Taken together, the findings indicate that caspase-9 plays a dual role in cell death signaling, as an activator of effector caspases and lysosomal membrane permeabilization.