CYLD encodes a tumor suppressor that is mutated in familial cylindromatosis. Despite biochemical and cell culture studies, the physiological functions of CYLD in animal development and tumorigenesis remain poorly understood. To address these questions, Drosophila CYLD (dCYLD) mutant and transgenic flies were generated expressing wild-type and mutant dCYLD proteins. dCYLD is essential for JNK-dependent oxidative stress resistance and normal lifespan. Furthermore, dCYLD regulates TNF-induced JNK activation and cell death through dTRAF2, which acts downstream of the TNF receptor Wengen and upstream of the JNKK kinase dTAK1. dCYLD encodes a deubiquitinating enzyme that deubiquitinates dTRAF2 and prevents dTRAF2 from ubiquitin-mediated proteolytic degradation. These data provide a molecular mechanism for the tumor suppressor function of this evolutionary conserved molecule by indicating that dCYLD plays a critical role in modulating TNF-JNK-mediated cell death (Xue, 2007).
Shortened animal lifespan may result from compromised oxidative stress tolerance. To examine the oxidative stress resistance, 3-day-old flies were challenged with paraquat for a prolonged period of time and their survival rates were measured. It was found that dCYLD mutants exhibited a significant reduction in survival rate as compared with wild-type or heterozygous dCYLD flies after 24 hr or 36 hr of exposure to paraquat, suggesting dCYLD plays a pivotal role in regulating oxidative stress resistance (Xue, 2007).
JNK signaling has been reported to play an important role in regulating oxidative stress resistance and lifespan in Drosophila (Wang, 2003). Ubiquitous expression of Bsk, the Drosophila JNK ortholog, under the control of tubulin promoter, rescues both lifespan and oxidative stress resistance defects in dCYLD mutants, suggesting that dCYLD regulates these two physiological effects through the JNK signaling pathway (Xue, 2007).
This study was extended to other stress conditions and it was found that dCYLD mutants are less resistant to dry starvation (no food and water), a phenotype that has been associated with reduced JNK activity (Wang, 2003). In contrast, dCYLD mutants do not affect animal survival at high and low temperature conditions (Xue, 2007).
To further examine the role of dCYLD in regulating JNK signaling in animal development, the genetic interactions between dCYLD and Eiger (Egr), the Drosophila ortholog of TNF that triggers the JNK pathway, was tested. Ectopic expression of Egr, under the control of the GMR promoter (GMR > Egr) and using the Gal4/UAS binary system, induces JNK activation and cell death in the developing eye that results in vastly reduced adult eye size. The Egr-induced JNK activation and small-eye phenotype was suppressed modestly by deleting one copy of dCYLD and suppressed soundly by removing both copies. The strong suppression of the Egr eye phenotype in homozygous dCYLD mutants was partially reverted by adding one copy of dCYLDRes. These results indicate that dCYLD is required for Egr-triggered JNK activation and cell death (Xue, 2007).
dCYLD encodes a protein of 640 amino acids, containing in its N terminal portion a cytoskeleton-associated protein (CAP) domain that is present in proteins associated with microtubules and the cytoskeletal network, two ubiquitin carboxyl-terminal hydrolases (UCH) domains that are commonly associated with deubiquitinating enzyme activity, and three CXXC zinc-finger (ZF) motifs with potential protein-protein interaction ability. To functionally characterize these motifs, UAS transgenes were generated expressing the wild-type or three mutant versions of dCYLD that delete the CAP domain, the two UCH domains, or the three ZF motifs. When expressed under the control of the GMR promoter, neither the full-length nor the dCYLD mutants displayed any detectable phenotype. When introduced into the GMR > Egr; dCYLD−/− background, wild-type dCYLD released the suppression of the Egr eye phenotype, confirming that the suppressive effect was due to the loss of dCYLD functions. In contrast, dCYLDΔUCH had no effect on the suppression of the Egr eye phenotype, and dCYLDΔCAP could only partially relieve this suppression, implying that the UCH domains are necessary for dCYLD functions and that the CAP domain is essential for dCYLD to execute its full activity in vivo. Interestingly, expression of dCYLDΔZF completely abolished the suppression effect, suggesting that the ZF motifs are dispensable in dCYLD regulation of Egr-induced cell death (Xue, 2007).
Ubiquitous expression of the full-length dCYLD, but not dCYLDΔUCH, rescues both shortened lifespan and hypersensitivity to paraquat in dCYLD mutants, suggesting that the deubiquitinating activity is indispensable for dCYLD to regulate JNK-dependent oxidative stress resistance and lifespan (Xue, 2007).
TNF receptor-associated factors (TRAFs) are important adaptor proteins that bind to TNF receptors and relay TNF signals to the JNK and NF-κB pathways in mammals. In Drosophila, Egr signal is mediated exclusively by the JNK pathway. However, the role of Drosophila TRAF proteins in Egr-JNK signaling remains unclear. The Drosophila genome encodes two TRAFs: dTRAF1, the TRAF2 ortholog; and dTRAF2, the TRAF6 ortholog. To determine the role of dTRAF1 and dTRAF2 in Egr-JNK signaling, the effects were examined of loss-of-function mutations and RNAi-mediated downregulation of dTRAF1 or dTRAF2 on the Egr eye phenotype. The Egr-induced small-eye phenotype was not suppressed by either deletion of one copy of the dTRAF1 gene or coexpression of a dTRAF1 RNAi. In contrast, the Egr eye phenotype was suppressed strongly by removing half of the dosage of dTRAF2 and suppressed completely by deleting the dTRAF2 gene. Consistently, coexpression of a dTRAF2 RNAi significantly suppressed the Egr eye phenotype. In agreement with genetic data, dTRAF2 exhibited a much stronger physical interaction with Wgn and dTAK1 than did dTRAF1 . Together, the above results point to dTRAF2, but not dTRAF1, as the adaptor protein that mediates Egr signaling in Drosophila (Xue, 2007).
To investigate the physiological functions of dCYLD and dTRAF2 in JNK activation, the expression pattern of puckered (puc) was checked in dCYLD or dTRAF2 mutants. puc encodes a JNK phosphatase whose expression is positively regulated by the JNK pathway, and thus, the puc-LacZ expression of the pucE69 enhancer-trap allele can be used as a readout of JNK activity in vivo. puc is weakly expressed in wild-type third-instar eye discs, and can be detected by prolonged staining. It has been previously shown that puc expression posterior to the morphogenetic furrow (MF) depends on endogenous Egr signaling. This study found that such expression patterns are reduced dramatically in dCYLD mutants and dTRAF2 RNAi animals. In contrast, puc expression in the disc margin, which is independent of Egr signaling, was not affected. GMR > Egr strongly activated puc transcription posterior to the MF. This ectopic Egr-induced puc expression was largely blocked by loss of dCYLD or by expression of dTRAF2 RNAi. Taken together, these observations indicate that both dCYLD and dTRAF2 are physiologically required by the endogenous JNK pathway (Xue, 2007).
The role of CYLD in modulating JNK signaling in mammalian cells has remained controversial. Consistent with the current observation, it was reported that JNK activity diminished in Cyld−/− thymocytes, which implies that CYLD is physiologically required for JNK activation. However, CYLD was also reported to negatively regulate JNK signaling in culture cells and macrophages. Thus, CYLD could positively or negatively regulate JNK signaling in a cell-type-specific manner (Xue, 2007).
To genetically map the epistasis of dCYLD and dTRAF2 in the Egr-JNK pathway, the genetic interaction between dTAK1 (JNKKK) and dCYLD or dTRAF2 was examined in the developing eyes. Expression of dTAK1 under the control of the GMR promoter resulted in pupa lethality, while dTAK1 expression under the control of the sevenless (sev) promoter (sev > dTAK1) induced extensive cell death in larval eye discs and gave rise to rough eyes with a reduced size. Loss of dCYLD or dTRAF2, or coexpression of a dTRAF2 RNAi, had no effect on the sev > dTAK1 phenotype, while removal of one copy of hep (JNKK) or bsk (JNK) partially suppressed the sev > dTAK1 phenotype, suggesting that dCYLD and dTRAF2 operate upstream of dTAK1 in the Egr-JNK pathway (Xue, 2007).
Ectopic Egr expression in the dorsal thorax driven by the potent pannier-GAL4 driver resulted in pupa lethality. However, when reared at 18°C, these animals survived to adulthood, presumably due to lessened Egr expression caused by reduced Gal4 activity, and produced a small-scutellum phenotype. This phenotype could be suppressed by RNAi inactivation of JNK signaling components, e.g., wgn, dTRAF2, dTAK1, hep, or bsk, suggesting that the phenotype is caused by activation of JNK signaling. Ectopic expression of dCYLD driven by pannier-GAL4 produced a similar but weaker phenotype, which could be fully suppressed by the coexpression of an RNAi of dTRAF2 and dTAK1, but not that of wgn. These results indicate that dCYLD functions downstream of Wgn, but upstream of dTRAF2 and dTAK1, in modulating JNK signaling (Xue, 2007).
RNAi-mediated downregulation of dTRAF2, but not dTRAF1, resulted in compromised oxidative stress resistance and shortened lifespan, suggesting that the role of dTRAF2 in dCYLD-JNK signaling has been conserved in different physiological contexts (Xue, 2007).
Previous studies have reported that the Shark tyrosine kinase and Src42A regulate JNK signaling in epidermal closure during embryogenesis and metamorphosis. However, null mutants for egr and dCYLD are fully viable and do not display the epidermal closure defect, implying that Shark and Src42A act in parallel to Egr and dCYLD in modulating JNK signaling. Consistent with this interpretation, loss of shark or src42A failed to suppress the GMR > Egr or pnr > dCYLD phenotype. In addition, it was found that loss of the transcription factor dFOXO could suppress both GMR > Egr and pnr > dCYLD phenotypes, suggesting that dFOXO acts downstream of dCYLD in JNK signaling. Consistent with this observation, dFOXO is required downstream of JNK in modulating cell death, oxidative stress resistance, and lifespan (Xue, 2007).
Overexpression of CYLD in mammalian tissue culture cells negatively regulates NF-κB signaling by deubiquitinating TRAF2/6. dCYLD, like its mammalian counterpart, contains two UCH deubiquitinating domains. Indeed, genetic analysis revealed that the UCH deubiquitinating domains are crucial for the in vivo functions of dCYLD. Furthermore, genetic epistasis data show that dCYLD acts upstream of dTRAF2 in the JNK pathway. Thus, it was hypothesized that dCYLD might act in the JNK pathway to deubiquitinate and subsequently stabilize dTRAF2 by preventing its ubiquitination-mediated proteolytic degradation. To examine this hypothesis in vivo, a FLAG-tagged dTRAF2 transgene (GMR > FLAG-dTRAF2) was introduced into dCYLD mutants and transgenic flies. Proteins were extracted from the heads of these flies for biochemical analyses. It was found that loss of dCYLD resulted in a significant reduction in dTRAF2 protein level, while the ubiquitination of dTRAF2 was markedly enhanced. Both changes were suppressed by dCYLDRes. Consistently, overexpression of dCYLD, but not dCYLDΔUCH, increased dTRAF2 protein level and decreased its ubiquitination. These results show that dCYLD functions as a deubiquitinating enzyme that deubiquitinates dTRAF2 and promotes dTRAF2 accumulation in vivo (Xue, 2007).
Polyubiquitination chains are usually formed on two lysine residues, K48 and K63. It is generally believed that the K48-linked polyubiquitination mediates proteasome-dependent protein degradation, while the K63-linked polyubiquitination mediates endocytosis and signal transduction. Previous work in mammalian culture cells has implicated that CYLD encodes a deubiquitinating enzyme that preferentially cleaves K63-linked polyubiquitin chain from its target proteins for NF-κB signaling. However, a recent in vivo study in Cyld−/− mice has reported that CYLD could regulate the stability of its target protein by effectively removing K48-linked polyubiquitin chain in thymocytes. Interestingly, JNK activity also diminished in Cyld−/− thymocytes. Thus, the role of CYLD in regulating protein stability and positively modulating JNK signaling could be conserved in mammals (Xue, 2007).
CYLD mutations in human patients cause dramatic skin tumors. However, the physiological function of CYLD and the mechanism underlying CYLD deficiency-induced tumorigenesis remain largely unknown. By generating the null dCYLD mutation and dCYLD transgenic animals and performing genetic analysis, this study has shown that dCYLD is a critical regulatory component of the JNK signaling pathway. Genetic epistasis and biochemical analysis further reveal that dCYLD modulates JNK signaling by deubiquitinating dTRAF2 and thus preventing dTRAF2 from ubiquitination-mediated proteolytic degradation. Loss of dCYLD results in augmented ubiquitination and degradation of dTRAF2, which renders cells resistant to apoptosis triggered by JNK signaling. Deregulation of apoptosis has been implicated as a major cause of tumorigenesis. Consistently, mice deficient for both jnk1 and jnk2 were resistant to apoptosis induced by UV irradiation, anisomycin, and MMS, and jnk1−/− mice exhibited enhanced skin tumor development, a phenotype that is pathogenically similar to cylindromatosis in CYLD human patients. Together these data argue that modulation of JNK signaling could be a conserved mechanism underlying familial cylindromatosis in CYLD patients (Xue, 2007).
Wengen has been identified as the first member of the Drosophila tumor necrosis factor receptor (TNFR) superfamily. Wengen is a type III membrane protein with conserved cysteine-rich residues (TNFR homology domain) in the extracellular domain, a hallmark of the TNFR superfamily. Wengen mRNA is expressed at all stages of Drosophila development. The small-eye phenotype caused by an eye-specific overexpression of a Drosophila TNF superfamily ligand, Eiger, is dramatically suppressed by downregulation of Wengen using RNA interference. In addition, Wengen and Eiger physically interact with each other through their TNFR homology domain and TNF homology domain, respectively. These results suggest that Wengen can act as a component of a functional receptor for Eiger. This identification of Wengen and further genetic analysis should provide increased understanding of the evolutionarily conserved roles of TNF/TNFR superfamily proteins in normal development, as well as in some pathophysiological conditions (Kanda, 2002).
In a Drosophila dominant-modifier screen using the chromosomal deficiency lines that covered more than 70% of the genome, several lines were obtained that suppress the small-eye phenotype caused by Eiger overexpression (GMR>eigerregg1). Through the analysis of these deficiency lines, a line, Df(1)E128/FM7c, was identified in which the deficiency spans the coding region of a predicted gene, CG6531. CG6531 encodes a protein with a cysteine-rich domain (TNFR homology domain), the hallmark of the TNFR superfamily. This gene was named wengen for a village at the foot of Switzerland's Mt. Eiger. Analysis of the wengen nucleotide sequence revealed an open reading frame of 343 amino acids with a predicted relative molecular mass of 40 kDa. Alignment analysis revealed that Wengen harbors a TNFR homology domain in the extracellular region and a membrane-spanning region without signal sequence. This is a characteristic of the type III membrane protein of TNFR superfamily (extracellular N terminus, intracellular C terminus, lacking a signal peptide). The TNFR homology domain of Wengen has significant structural and amino acid homology with the TNFR domains of human EDAR (hXEDAR) and human TNFR1 (hTNFR1). The TNFR homology domains of Wengen, hXEDAR, and hTNFR1 share the topologically distinctive modules (termed A1 and B2 module, except for the TNFR homology domain of hTNFR1 [amino acids 168-195], which is composed of the A1 and C2 modules). To investigate whether Wengen is indeed a type III membrane protein like the other TNFR superfamily members, its subcellular localization was examined. S2 cells were transiently transfected with expression vectors for C-terminally HA-tagged Wengen (Wengen-HA) and GFP. In GFP-positive cells Wengen-HA is detected on the surface membrane with anti-HA antibody when the S2 cells were permeabilized. These data suggest that the Wengen C-terminal domain is indeed cytoplasmic. These results suggest that Wengen is a member of the type III TNFR superfamily. The expression of wengen was examined in flies. RT-PCR analysis revealed that wengen mRNA is expressed at all stages of development. Its putative ligand, Eiger, is also expressed at all stages of Drosophila development (Kanda, 2002).
To assess whether Wengen is required for Eiger to induce the small-eye phenotype, RNA interference (RNAi) was used to down-regulate the endogenous expression of Wengen. A head-to-head inverted repeat construct for wengen, pUAS-wengen-IR, was generated, and its ability to knock down the wengen expression was examined. Co-transfection of pUAS-HA-wengen together with pUAS-wengen-IR into S2 cells dramatically reduces Wengen expression but has no effect on the expression of HA-CARD, suggesting that wengen-IR works as a specific inhibitor of Wengen expression. To assess the biological functions of Wengen in Drosophila, transgenic flies were generated that misexpress wengen-IR in the developing retina. The small-eye phenotype induced by the eye-specific ectopic expression of Eiger (GMR>eigerregg1) was suppressed by the coexpression of wengen-IR. These results strongly suggest that Wengen is required as a functional transducer of Eiger signaling (Kanda, 2002).
The physical interactions between Wengen and Eiger were assessed using various deletion mutants. Immunoprecipitation assays revealed that full-length Wengen and Eiger physically interact with each other. Eiger interacts with WengenDeltacyt (lacking the cytoplasmic domain) but not with WengenDeltaTNFR (lacking the homology TNFR domain). In addition, full-length Wengen could not interact with Eiger DeltaTNF. These results suggest that Wengen can interact with Eiger, and this interaction is mediated through the TNFR homology domain of Wengen and the TNF homology domain of Eiger (Kanda, 2002).
This study has identified the first Drosophila member of the TNFR superfamily, Wengen. Most of the genes for the TNFR superfamily encode type I or III membrane proteins with one or more extracellular ligand-binding domains and a cytoplasmic region that activates cell functions. In general, the extracellular domain of this family of proteins shows a relatively low level of sequence conservation, despite sharing a common fundamental structure. The cytoplasmic regions of the receptors show considerably more diversity in sequence and size than the extracellular regions. There are no common intracellular motifs found in all members of the TNFR superfamily except for some domains such as the TRAF2-binding domain [(P/S/A/T)X(Q/E)E or PXQXXD], which is required for both NF-kappaB activation and JNK activation, or a domain of ~80 amino acids called the 'death domain', for caspase activation. However, the amino acid sequence of Wengen reveals that it has neither a TRAF2-binding domain nor a death domain in the cytoplasmic region, suggesting that there should be another mechanism to transduce signals (Kanda, 2002).
Whereas Eiger can stimulate the JNK pathway, the stimulation of the JNK pathway in response to the overexpression of Wengen in S2 cells or the Drosophila compound eye could not be detected. It is possible that because the amount of ligand is limited, overexpression of Wengen is not sufficient to activate the downstream signals. It is also possible that intracellular adapter proteins, which are required for transducing signals, are not expressed or limited in Wengen expressing cells. Otherwise, Wengen may require one or more co-receptors that transduce signals to the cytoplasm. For instance, heteromeric receptor complex is used to transduce Hedgehog signaling. Hedgehog binds to its receptor Patched, and then the inhibitory function of Patched against its binding partner, Smoothened, is cancelled. In this way, Hedgehog signaling is transduced into the cells. Because the heteromeric complex of receptors has never been reported to transduce TNF family signaling, it is possible that Eiger/Wengen may use the novel type of TNF signaling mechanisms. In any case, further genetic and biochemical studies of Eiger/Wengen should help to elucidate the unique signaling mechanisms that include the caspase-independent pathway triggered by Eiger (Kanda, 2002).
Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).
Analysis of the Drosophila genome sequence reveals a single predicted transcript that encodes a type II membrane protein with structural similarities to members of the TNF superfamily. This protein is referred to as Eiger, in memory of the numerous mountaineers that have been killed by the Eiger Nordwand, the 'wall of death.' The Eiger protein contains a cytoplasmic domain, a transmembrane region between amino acid residues 36 and 62, and an extracellular domain of 353 amino acids. The C-terminal TNF homology domain (THD) of Eiger shows comparable homology to several human TNF family members (20%–25% identity). In situ hybridization has revealed a weak expression in imaginal discs with a pronounced pattern in the eye (Moreno, 2002).
Like a subset of human TNF ligands, Eiger can induce caspase-dependent apoptosis. Targeted expression of Eiger in the eyes and wings of Drosophila causes a severe ablation of these organs, and Eiger-expressing cell clones are rapidly eliminated. Both of these effects can be suppressed by coexpression of the pan-caspase inhibitor p35 (Moreno, 2002).
Caspase-8 is the key initiator caspase of death ligand-induced apoptosis in mammals. Upon stimulation by TNF, the adaptor protein FADD recruits and aggregates several molecules of procaspase-8 that mutually cleave and activate each other. Due to the involvement of an extracellular ligand, this pathway has been referred to as the 'extrinsic death pathway'. DREDD is the Drosophila caspase most similar to caspase-8 and has been shown to physically interact with Drosophila FADD. Surprisingly, complete removal of DREDD function fails to block Eiger-induced apoptosis, indicating that Eiger triggers cell death by a DREDD/caspase-8-independent pathway (Moreno, 2002).
Another pathway activated by mammalian TNF death factors is the JNK pathway, although its role in inducing apoptosis upon TNF signaling is less well defined. JNK signaling in Drosophila is reflected by the expression levels of puckered (puc), a gene encoding a dual-specificity phosphatase that forms a negative feedback loop by downregulating the activity of JNK. High levels of puc expression are induced by Eiger. Due to its function as a negative regulator of JNK, Puc can also be used as a powerful means to repress JNK activity if overexpressed by a constitutive promoter that is no longer dependent on this activity. Coexpression of Eiger and Puc completely blocks Eiger activity, strikingly reverting the eye and wing phenotypes to wild-type and blocking Eiger-induced elimination of cell clones. Forced expression of Puc does not prevent all forms of cell death. For example, when tested in the situation of polyglutamine repeat-induced neurodegeneration, which is also caused by apoptosis, coexpression of puc has no discernible protective effect. Taken together, these results are interpreted as firm evidence that Eiger induces the JNK signaling pathway and that Eiger-induced apoptosis is critically dependent on JNK activity (Moreno, 2002).
Consistent with this interpretation, several components of the Drosophila JNK pathway are rate limiting in mediating or preventing Eiger-induced apoptosis. The removal of one wild-type copy of either DTRAF1 (encoding the homolog of human TRAF2), misshapen (encoding a Ste20 kinase that binds to DTRAF1), or basket (encodes Drosophila JNK) suppresses Eiger-induced apoptosis. Conversely, animals heterozygous for a mutation in puc display an enhanced phenotype (Moreno, 2002).
The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).
Unlike the situation in mammals, Drosophila TNF appears to activate a linear pathway to induce apoptosis, involving JNK and the apoptosome as landmark components. Thus, in Drosophila, the signaling module formed by FADD and DREDD appears to be dedicated to signaling by the Toll/IMD system, whereas, in mammals, this 'extrinsic' apoptotic pathway can be activated both by some members of the TNF superfamily and by ligands of Toll-like receptors. It is proposed that, at some point during vertebrate evolution, the Toll signaling modules, comprising at least the FADD/caspase-8 branch, but presumably also the NF-kappaB pathway, were coopted by some members of the TNF/TNFR superfamilies, expanding their ancestral function of JNK-mediated induction of apoptosis. Other members of the mammalian TNF/TNFR superfamilies may have retained the primordial Eiger mode of signaling with a linear 'JNK/apoptosome-only' output (Moreno, 2002).
In mammals, members of the tumor necrosis factor (TNF) family play an important role in the regulation of cellular proliferation, differentiation and programmed cell death. This study describes isolation and characterization of an orthologous ligand/receptor axis in Drosophila. The ligand, designated Eiger, is a type II membrane glycosylated protein, which can be cleaved at residue 145 and released from the cell surface as a soluble factor, thereby representing the first potential cytokine to be described in Drosophila. Eiger exists in two alternatively spliced isoforms, Eiger long (Eiger-L) and Eiger short (Eiger-s), both of which are expressed throughout development and in the adult. A novel Drosophila member of the TNF receptor family, designated Wengen, is a type I membrane protein that can physically interact with the recently described TRAF2 homolog dTRAF2. Both Eiger and Wengen are expressed in distinctive patterns during embryogenesis and Eiger is responsive to genotoxic stress. Forced expression of Eiger-L, Eiger-s or Wengen, caused apoptotic cell death which could be rescued by caspase inhibitors or the JNK phosphatase Puckered. In addition, Eiger-induced cell killing is attenuated by RNAi-mediated suppression of Wengen. These results illustrate that Eiger and Wengen represent proximal components of an evolutionarily conserved TNF-like signaling pathway in Drosophila (Kauppila, 2003).
The Drosophila genomic database was searched for sequences with homology to the extracellular domain of human TNFR1, and a candidate sequence was identified. This sequence encoded a cDNA of 993 nt and was identical to the sequence of a recently isolated Drosophila receptor, designated Wengen (Kanda, 2002). Sequence analysis revealed that the extracellular domain of Wengen contained a single cysteine-rich pseudorepeat with significant homology to other members of the TNFR family. However, Wengen possessed a unique cytoplasmic domain with no sequence homology to any TNFR family member (Kauppila, 2003).
It was reported that Wengen is a type III membrane protein with a single hydrophobic transmembrane domain (Kanda, 2002). However, sequence analysis revealed that, in addition to the transmembrane domain between residues 202 and 222, Wengen cDNA also encoded for a hydrophobic stretch of amino acids between residues 54 and 59, which could potentially represent a signal peptide. Such a topology is characteristic of type I membrane proteins, including most mammalian receptors of the TNFR family. In order to determine whether Wengen is a type I or type III membrane protein, several constructs were generated with a Flag epitope tag placed at different locations in the deduced open reading frame. Construct Wengen-A positions the Flag epitope tag at the N-terminus of the full-length Wengen protein-coding region. The construct designated Wengen-B encoded a murine Igkappa chain signal peptide followed by the Flag epitope tag and amino acids 14-343 of Wengen. Construct C resembled construct B in topology except that it included the Wengen protein-coding region downstream of the hydrophobic stretch of amino acids representing the putative signal peptide (i.e. amino-acid residues 60-343). A construct was generated encoding amino acids 1-318 of human XEDAR fused to the murine Igkappa chain signal peptide and the Flag epitope. XEDAR is a recently isolated receptor of the TNFR family and a well-characterized type III membrane protein. The above constructs were transfected into 293 T cells along with a GFP reporter construct, and the expression of the Flag epitope tag was analysed on the surface of GFP-expressing cells using FACS analysis. If Wengen corresponds to a type I membrane protein, the epitope tag will be absent from the mature proteins predicted from constructs A and B since it is positioned upstream of the signal peptide cleavage site in these plasmids and, as such, the Flag tag would not be detected on the surface of the cells expressing these constructs (Wengen-A and Wengen-B). In contrast, the Flag epitope tag will be expected to be expressed on the surface of cells expressing the construct C since, in this construct, the Flag epitope is located downstream of the heterologous signal peptide. The cell surface expression of the Flag epitope was not detected in the case of cells transfected with constructs A and B, but readily detected Flag-positive cells in the construct C transfectants. Hence, the epitope was found on the surface of cells only if it was positioned on the C-terminal side of the predicted Wengen signal peptide. In parallel studies with a known type 1 receptor, cell surface expression of the Flag tag was also detected in the case of cells transfected with the comparable Flag-XEDAR construct. Collectively, the above results lend strong support for the hypothesis that Wengen is a type I membrane protein (Kauppila, 2003).
To begin a functional characterization of Eiger and Wengen, the embryonic expression patterns of these genes were examined by in situ hybridization. Eiger mRNA was detected in early embryonic stages in a pattern that was highly localized to the dorsal surface of pregastrulating embryos. Localization of mRNAs to the dorsal side probably reflects maternally derived transcripts that become positioned during oogenesis and could reflect an important role in early pattern formation. By contrast, expression of Wengen appears to be very low or undetectable in pregastrulating embryos. During gastrulation, Wengen-positive cells are detected in the inner layer of embryonic tissue corresponding to the presumptive mesoderm while, at the same stage, Eiger expression occurs in the epidermal layer of the embryo and is most prominent at the surface of dorsal folds that will later form the amnioserosa. At germ band extended stages, Wengen transcripts continue to accumulate in the mesodermal segments of the embryo while its ligand, Eiger, is prominent in the adjacent neurogenic region. In later-staged embryos (stages 15/16) both Eiger and Wengen are detected in subsets of cells within the condensing nerve cord (Kauppila, 2003).
Since TNF and its receptors are implicated in some vertebrate models of damage-induced cell death, tests were performed to see whether Eiger or Wengen might be responsive during genotoxic stress. Eiger RNAs were consistently induced by gamma radiation between two- and fourfold while, in these same samples, levels of Wengen transcripts were unchanged. These findings were confirmed using RT-PCR methods and also it was also determined that both Eiger-s and Eiger-L isoforms are radiation responsive (Kauppila, 2003).
Using transient transfection assays, Wengen and Eiger were tested for apoptosis induction in Drosophila S2 cells. Both the long and short isoforms of Eiger ligand are equally effective at promoting cell death (~40% survival). Similar levels of cell death are provoked by the receptor, Wengen. No overt synergistic effects were detected in cotransfections of Eiger together with Wengen, but cell death induced by both isoforms of the ligand -- as well as the receptor -- were partially reversed by caspase inhibitor peptides, zDEVD and zVAD (Kauppila, 2003).
To test the hypothesis that Wengen is a receptor for Eiger, the effects of dsRNA-mediated silencing of Wengen upon Eiger-induced cell killing were tested. Silencing of Wengen effectively attenuates cell killing triggered by either of the Eiger isoforms. Moreover, these effects are clearly target specific since dsRNA-mediated silencing of either hid or rpr had no influence upon Eiger-induced cell killing. As expected, in converse experiments, it was found that silencing of Eiger had no influence upon Wengen-induced cell killing. Together, these results support a ligand/receptor relationship for Eiger and Wengen (Kauppila, 2003).
Since the JNK phosophatase Puckered is an effective suppressor of Eiger-dependent phenotypes in the animal (Igaki, 2002), the effects of forced puckered expression were examined in cultured cells. This phosphatase clearly protected against cell killing via the Eiger/Wengen axis. In these assays, Puckered had pronounced suppressive effects upon killing by Wengen and the short isoform of Eiger and significant -- but less potent effects -- against the long Eiger isoform. These effects were specific for Eiger/Wengen signaling since, in parallel assays, it was found that puckered had no effect upon levels of cell killing elicited by grim or rpr (Kauppila, 2003).
Each of the two recently published studies on Eiger found only a single isoform, each of which differ by five amino-acid residues. This report has clarified that both isoforms are authentic and expressed at the mRNA level. However, so far no differential expression of these two isoforms during embryogenesis or following genotoxic stress has been discovered. In addition, using the assays undertaken, no overt difference was found in killing activity conferred by the two isoforms. However, the above results are not surprising since the difference between the two isoforms of Eiger is located outside the TNF homology domain, which is usually the main determinant of receptor binding (Kauppila, 2003).
Unlike mammalian TNF family receptors, only a single cysteine-rich domain is present in Wengen. It is conceivable that multiple copies of cysteine-rich domains contribute to increase in affinity and/or specificity of receptor-ligand interaction. Consistent with the above hypothesis, no significant physical interactions were found between soluble Eiger and Wengen (Kauppila, 2003).
The in vivo roles of Eiger and its receptor during development await further characterization but the expression patterns, particularly for the ligand Eiger, raise intriguing possibilities. Eiger is highly localized to a narrow stripe along the dorsal surface of pregastrulating embryos at a time coincident with specification of ventral cell fates via the Toll/Dorsal pathway. Few precedents exist for this unusual transcript distribution. One such precedent, zen, is localized to the dorsal surface as a consequence of repression by dorsal proteins and, in a recent genome-wide analysis, the Eiger locus was also identified as dorsal target. Together with the fact that Eiger can be cleaved to form a soluble ligand, these observations raise the possibility that Eiger may function during early embryonic patterning along the dorsal ventral axis and/or in the specification of dorsal structures such as the amnioserosa. In subsequent embryonic stages, Eiger and Wengen are expressed in nonoverlapping but neighboring tissues. For example, in germ-band-extended embryos, Wengen is expressed in mesodermal tissues, while Eiger is expressed in the adjacent neurogenic ectoderm. Hence, it is possible that TNF-like signaling occurs among these tissues as they develop in the embryo (Kauppila, 2003).
Both Eiger and Wengen triggered cell death which could be blocked by Puckered, an inhibitor of the Drosophila JNK pathway. These results confirm earlier reports from Igaki (2002) and Moreno (2002) who also demonstrated a requirement for JNK signaling. Interestingly, a mammalian TNFR family member has been described that also lacks a death domain, TAJ, that similarly activates the JNK pathway and promotes cell death (Eby, 2000). Thus, it seems plausible that Wengen and TAJ could share evolutionarily conserved mechanisms to trigger cell death. However, unlike TAJ, Eiger- and Wengen-induced cell death was partially blocked by caspase inhibitors. Further characterization of Eiger/Wengen-induced cell death may shed light on this novel evolutionary conserved pathway of apoptosis and open up new therapeutic opportunities for the treatment of cancer (Kauppila, 2003).
Death by infection is often as much due to the host's reaction as it is to the direct result of microbial action. This study identifies genes in both the host and microbe that are involved in the pathogenesis of infection and disease in Drosophila challenged with Salmonella enterica serovartyphimurium. Wild-type Salmonella causes a lethal systemic infection when injected into the hemocoel of Drosophila. Deletion of the gene encoding the secreted bacterial effector Salmonella leucine-rich (PslrP) changes an acute and lethal infection to one that is persistent and less deadly. A model is proposed in which Salmonella secreted effectors stimulate the fly and thus cause an immune response that is damaging both to the bacteria and, subsequently, to the host. In support of this model, mutations in the fly gene eiger, a TNF homolog, are shown to delay the lethality of Salmonella infection. These results suggest that Salmonella-infected flies die from a condition that resembles TNF-induced metabolic collapse in vertebrates. This idea provides a new model to study shock-like biology in a genetically manipulable host. In addition, it allows the study of difference in pathways followed by a microbe when producing an acute or persistent infection (Brandt, 2005).
Attempts were made to infect Drosophila with Salmonella by feeding bacteria to flies and by injecting bacteria into the hemocoel. Flies were resistant to feeding, but direct injection of approximately 10,000 bacterial cells resulted in an infection that caused death in 7–9 d, while injection of sterile medium led to a mean time of death of approximately 20 d (Brandt, 2005).
The most thoroughly characterized immune response in the fly is the humoral immune response, which involves the secretion of antimicrobial peptides into the hemocoel by an organ called the fat body. Two highly conserved signaling systems, the Toll and IMD pathways, have been shown to control the transcription of the antimicrobial peptide genes. The IMD pathway is implicated in raising a response to gram-negative bacteria such as Salmonella. Flies homozygous for an imd mutation succumbed to Salmonella infections rapidly, dying within 12 h of infection. Bacterial numbers exceeded 100,000,000 per fly as the flies died, and Salmonella in dying flies were found circulating in the hemolymph. Circulating bacteria were not seen in wild-type flies. In contrast to imd homozygotes, flies homozygous for a mutation in Dif, a transcription factor in the Toll pathway, did not die rapidly. This experiment shows that mutations in the IMD but not the Toll pathway sensitize the fly to Salmonella. Further, these results suggest that the fly's humoral immune response limits the growth of free Salmonella in the hemocoel (Brandt, 2005).
In mammals and birds, Salmonella is largely an intracellular pathogen. To determine whether Salmonella was indeed growing in a protected intracellular niche in the fly, the in vivo sensitivity of Salmonella to gentamicin, an antibiotic that does not cross host-cell plasma membranes was measured. This experiment showed that bacteria that had been in the fly for 7 d were protected from the antibiotic and suggests that the bacteria were located in an intracellular location (Brandt, 2005).
In other bacterial infection models in the fly, death of the host occurs only when bacterial numbers increase beyond 106 bacteria per fly. This is the case for infections both of wild-type flies with pathogens such as Pseudomonas aeruginosa and L. monocytogenes and of mutant flies with nonpathogenic bacteria such as Escherichia coli. This is not the case for Salmonella in a wild-type fly. To determine the growth characteristics of Salmonella in Drosophila, bacteria were injected over a 1,000-fold dilution range; 7 d after infection, flies were homogenized and plated. At this time, bacteria were found to cover only a 5-fold range. Bacteria injected at low densities grew to levels between 10,000 and 100,000 bacteria per fly, but those injected at higher levels did not pass this threshold. This final number is at least 1,000 fold lower than the number of bacteria found during fatal L. monocytogenes and E. coli infections in the fly. Salmonella thus reaches a population ceiling in flies, and the flies die containing relatively small numbers of bacteria (Brandt, 2005).
To determine the location of bacteria within the fly, flies infected with Salmonella expressing green fluorescent protein (GFP) were examined. Bacteria carrying the plasmid containing the macrophage-inducible gene (pmig-1), which induces GFP expression upon entry into the phagosome, or the strain smo22, which constitutively expresses GFP, were injected into larvae and flies. GFP-expressing Salmonella could be seen within hemocytes bled from infected larvae. Hemocytes in adults are mostly sessile and cannot be easily removed from the fly. However, these cells can be observed through the cuticle, and clusters of these cells are found on the dorsal surface of the abdomen, along the dorsal vessel. Salmonella were found associated with these cells. The distribution of bacteria seen using the two GFP constructs was similar. This experiment suggests that, similar to the situation in mice, Salmonella are found within phagocytes. Furthermore, the Salmonella in fly hemocytes induced the expression of GFP from a Salmonella promoter normally induced upon phagocytosis by a mouse macrophage. This demonstrates that a Salmonella infection-related gene is induced even when the bacteria are infecting an animal at 29°C instead of 37°C (Brandt, 2005).
Salmonella is naturally infectious to mice through an oral route and causes a systemic disease resembling human typhoid fever. Salmonella crosses the gut epithelium by entering and then killing M cells of the Peyer's patch. Once across the epithelial barrier, Salmonella infect macrophages and spread to the mesenteric lymph nodes and subsequently to other organs. S. typhimurium has two type III secretory apparatuses (TTSAs) that translocate effector proteins across both bacterial and host membranes into the host cell cytoplasm. One TTSA encoded by Salmonella pathogenicity island 1 (SPI1) plays an important role in cell entry, whereas a second TTSA, SPI2, alters the intracellular environment of the host cell to permit Salmonella growth. Salmonella likely does not find itself in the hemolymph of Drosophila in nature, but by placing it there some questions can be answered that are difficult to approach using other techniques (Brandt, 2005).
Salmonella carrying mutations in a gene encoding a regulator of virulence, phosphatase P (phoP), or blocking the function of either SPI1 (orgA::Tn10) or SPI2 (ssrA::miniTn5), were injected into flies. All of these mutants killed flies more slowly than wild-type bacteria. However, analysis of bacterial growth within infected flies revealed a more complicated story. PhoP mutants produced infections that were less lethal than wild-type Salmonella, but the infecting bacteria grew to the same levels seen in wild-type infections. In contrast, a SPI1 or SPI2 mutant caused little death, and the bacteria grew to an average of 149,000 cfu/fly (SPI2 mutants) instead of the average of 40,000 cfu per fly found in wild-type infections. Thus, the level of bacterial growth of this mutant is almost 4-fold higher than for wild-type Salmonella. This suggests that if Salmonella cannot manipulate Drosophila cells by secreting effectors, the survival of both pathogen and host is dramatically improved (Brandt, 2005).
To determine which of the known TTSA effector proteins contributed to this phenotype, flies were injected with Salmonella carrying in-frame deletions of these genes as well as several others suspected to be involved in virulence in the fly. Deletions of spiC, Salmonella secreted effector A (sseA), sseB, sseC, and sseD all resembled a SPI2 knockout as expected because the proteins encoded by these genes are implicated in building the SPI2 translocation machinery. Deletion of the gene encoding the Salmonella leucine-rich repeat-containing protein (PSlrP) produced a phenotype similar to that of the knockout of the entire SPI2 TTSA in terms of fly death. Wild-type virulence could be restored to the slrP mutant by expressing slrP, controlled by its native promoter, in trans on a single-copy plasmid, p.slrP. Because SPI1 and SPI2 bacteria accumulate to higher levels than wild-type strains, in the fruit fly, growth was measured of slrP-knockout bacteria. Mutation of slrP did not significantly alter the growth of Salmonella. These experiments suggest that slrP is a determinant of Salmonella virulence in the fruit fly, but that mutation of slrP alone is not sufficient to alter Salmonella growth. The protein encoded by slrP is suspected to be a specific effector translocated directly into the host cytoplasm. Its function there remains unknown (Brandt, 2005).
The results obtained during fly infection differ from what is found in mammalian and avian hosts. SPI1 is required by Salmonella for breaching the gut barrier but is dispensable if the bacteria are introduced systemically by intraperitoneal or intravenous injection. Both phoP- and SPI2-mutant Salmonella are highly attenuated even when injected into mammalian hosts; host survival increases, and the mutant bacteria do not readily replicate and are cleared during infections. In the fly, not only does host survival increase during infection by SPI1 or SPI2 mutants but, paradoxically, the bacteria are not cleared and replicate better than their wild-type parents. This is an interesting difference, because it separates mere bacterial presence from pathogenesis and disease. It remains to be determined why the fly does not clear the mutant bacteria in the manner that is seen in the mammalian host (Brandt, 2005).
The analysis of adult hemocytes in the fly is difficult, because good molecular markers for them are lacking, and the cells are sessile and rare enough to make them difficult to find regularly in tissue sections. Therefore a functional assay was used to monitor the behavior of these infected cells by determining their ability to carry out one of their definitive behaviors, phagocytosis. Flies were infected with wild-type and mutant Salmonella and then assayed for the phagocytic capacity of the hemocytes on their dorsal surface. Fluorescein isothiocyanate (FITC)-labeled dead E. coli were injected into flies and incubated for 60 min to allow time for phagocytosis to occur. Trypan blue was then injected into the flies to quench the fluorescence of extracellular bacteria. Uptake of the E. coli was then monitored by observing the flies under FITC illumination with a dissecting microscope. At day 1 post-Salmonella infection, hemocytes infected with wild-type, SPI1-mutant, SPI2-mutant, and SlrP bacteria showed similar phagocytic activities. Following 7 d of infection, flies infected with wild-type or slrP-mutant bacteria had greatly reduced numbers of phagocytic cells. In contrast, phagocytes remained active during the course of infection by SPI1 or SPI2 mutants. This experiment is interpreted to show that the wild-type Salmonella infection results in death of phagocytes or a reduced capacity for phagocytosis. This reduction is not dependent on SlrP function (Brandt, 2005).
In mammals, cytokines such as TNF and interferon relay information about infection through the body. Cytokine expression, in particular TNF expression, is responsible directly and indirectly for a significant degree of the pathology observed during microbial infection. Flies have one known TNF-like protein, encoded by the gene eiger. Overexpression of eiger can induce cell death, but no phenotype had been identified for loss-of-function mutants thus far. Wild-type and eiger– flies were infected with Salmonella to determine whether this fly TNF homolog plays a role in the pathogenesis of Salmonella infections in Drosophila. The mean time to death was lengthened by 3 d in the two eiger mutants tested. This experiment suggests that eiger/TNF signaling during a Salmonella infection contributes to the rate of death of the fly. Accumulation of Salmonella does not differ between eiger mutants and the background fly strain. This suggests that eiger mutants do not directly affect Salmonella growth, but do markedly affect host survival. RNA transcripts of eiger were not significantly altered during Salmonella infection. This suggests that if eiger is regulated during Salmonella infection, the transcriptional changes are too small to be seen relative to expression in whole flies or the changes are posttranscriptional (Brandt, 2005).
In human disease, morbidity and mortality are often the result of immune responses to invading pathogens rather than to direct action of the pathogens themselves. Fever, inflammation, and shock are examples of such processes. In plants, a model is emerging in which host cells monitor important cell functions and respond to their perturbations by pathogens. It is suggested that infection caused by Salmonella in the fly has attributes of both models. Salmonella growth appears to be restricted to hemocytes by action of the humoral immune response. Perhaps the manipulation of these hemocytes by secreted effectors induces additional immune responses that limit bacterial growth. This resembles what is seen in a resistant plant infected by a bacterium expressing the appropriate avirulence protein. Such proteins are secreted into the plant cell's cytoplasm via a TTSA, and they alter the host cell's physiology, presumably to make growth conditions for the bacteria more favorable. Resistant plants can sense this physiological change and raise a type of immune reaction called a hypersensitive response. In the case of Drosophila, it is suggested that the fly's immune response not only limits the growth of the pathogen but also damages the fly and ultimately leads to its death. When Salmonella is prevented from using its secreted effectors, the bacteria survive in the phagocyte and grow to higher numbers, possibly because the bacteria are not being attacked as intensely by the host. The result is that there are more bacteria in the fly and host death is greatly delayed. These two properties appear to be separable. The mutations in eiger suggest that there are signaling events that increase the death rate in the fly but do not alter the numbers of Salmonella. These experiments provide a new genetic model to explore microbial choice of pathogenic lifestyles in addition to a potential new genetic model for the study of TNF-induced metabolic collapse (Brandt, 2005).
The spatial patterns of eiger expression during embryogenesis and in larval tissues were analyzed by in situ hybridization. Weak signals were detected in pre-blastoderm embryos (stage 1-3), indicating a low level of maternal contribution. After stage 10, eiger transcripts are predominantly detected in the nervous system of the embryos. In the third-instar larva, eiger is strongly expressed in the brain hemispheres and at the morphogenetic furrow in the eye disc; it is also expressed at significant levels in many cells posterior to the furrow. Double staining of the eye disc with the eiger RNA probe and an anti-ELAV antibody, which recognizes terminally differentiated neuronal nuclei, has revealed that eiger is strongly expressed in the proliferating cells at the furrow. RT-PCR analysis demonstrated that eiger mRNA is expressed at all stages of Drosophila development (Igaki, 2002).
Many mammalian TNF superfamily proteins activate both the NF-kappaB and the JNK pathway, and activation of the latter pathway facilitates cell death (Davis, 2000). To examine whether Eiger activates the JNK pathway, the genetic interactions of Eiger with the components of the Drosophila JNK cascade were examined. The reduced-eye phenotype induced by Eiger is strongly suppressed in basket (bsk), a heterozygous mutant of Drosophila JNK. In addition, overexpression of a dominant-negative form of Bsk almost completely suppresses the eye phenotype. Moreover, heterozygosity at the hemipterous (hep) locus, which encodes Drosophila JNKK, suppresses the reduced-eye phenotype much as does bsk, and its hemizygosity (null background) rescues the phenotype almost completely. Furthermore, the co-expression of a dominant-negative form of dTAK1 (TGF-ß activated kinase 1; Drosophila JNKKK) also rescues the Eiger-induced phenotype completely. Misshapen (Msn) is a MAPKKKK that is genetically upstream of the JNK pathway in Drosophila. A half dosage of the msn gene strongly suppresses the Eiger-induced phenotype. Heterozygosity of Drosophila jun, a target of the JNK pathway, did not show any genetic interaction with Eiger, raising the possibility that the Eiger-stimulated death-inducing JNK pathway may not require new transcripts that are controlled by Jun (Igaki, 2002).
Puckered (Puc) is a dual-specificity phosphatase, the expression of which is induced by the Drosophila JNK pathway to inactivate Bsk, so that puc expression can be used to monitor the extent of activation of the JNK pathway. To confirm that the JNK pathway is actually activated by Eiger, puc expression level was assessed in the eye disc of GMR-GAL4 activated regg1GS9830 flies using a puc-LacZ enhancer-trap allele. The strong induction of puc-LacZ was observed in the region posterior to the morphogenetic furrow of the eye disc compared with the control eye disc. Furthermore, Western blot analysis with an anti-phospho-JNK antibody has revealed that Bsk is phosphorylated by Eiger overexpression. These genetic and biochemical data led to a model in which Eiger activates Msn, thereby triggering the JNK signaling pathway, sequentially stimulating dTAK1, Hep and Bsk. Using RT-PCR analysis, whether Eiger could stimulate the NF-kappaB pathway was tested; however, no obvious upregulation of the antimicrobial peptide genes, the target genes of the Drosophila NF-kappaB pathway, was detected (Igaki, 2002).
To analyze the physiological role of Eiger, a loss-of-function mutant for eiger was generated by imprecise excision of the P element in the Regg1GS9830 strain. Nine mutants were isolated harboring deletions that removed parts of the eiger genomic sequences, but leaving wild-type sequences downstream of the P element intact. egr1 and egr3 mutants were homozygous viable with greatly reduced eiger expressions. In the eye disc of the puc-LacZ enhancer-trap line, endogenous JNK activity could be detected in the region posterior to the morphogenetic furrow by long-time (20 h) X-gal staining. The puc-LacZ expression is dramatically reduced in the eye disc of the eiger mutant. Notably, the JNK activities at the marginal region of the eye disc, where endogenous eiger expression was not detected, were not affected in the eiger mutant. These findings indicate that Eiger functions as a physiological ligand for the Drosophila JNK pathway (Igaki, 2002).
Genetic and microarray analyses have been used to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER. Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. These results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways (Brodsky, 2004).
Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Using a Drosophila genetic model for Ras-induced tumor progression, a molecular link has been shown between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/β-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic RasV12-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, since JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. The finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression (Igaki, 2006).
Most human cancers originate from epithelial tissues. These epithelial tumors, except for those derived from squamous epithelial cells, normally exhibit pronounced apicobasal polarity. However, these tumors commonly show defects in cell polarity as they progress toward malignancy. Although the integrity of cell polarity is essential for normal development, how cell polarity disruption contributes to the signaling mechanisms essential for tumor progression and metastasis is unknown. To address this, a recently established Drosophila model of Ras-induced tumor progression triggered by loss of cell polarity has been used. This fly tumor model exhibits many aspects of metastatic behaviors observed in human malignant cancers, such as basement membrane degradation, loss of E-cadherin expression, migration, invasion, and metastatic spread to other organ sites (Pagliarini, 2003). In the developing eye tissues of these animals, loss of apicobasal polarity is induced by disruption of evolutionarily conserved cell polarity genes such as scribble (scrib), lethal giant larvae (lgl), or discs large (dlg), three polarity genes that function together in a common genetic pathway, as well as other cell polarity genes such as bazooka, stardust, or cdc42. Oncogenic Ras (RasV12), a common alteration in human cancers, causes noninvasive benign overgrowths in these eye tissues (Pagliarini, 2003). Loss of any one of the cell polarity genes somehow strongly cooperates with the effect of RasV12 to promote excess tumor growth and metastatic behavior. However, on their own, clones of scrib mutant cells are eliminated during development in a JNK-dependent manner; expression of RasV12 in these mutant cells prevents this cell death (Igaki, 2006).
To better quantify the metastatic behavior of tumors in different mutant animals, the analysis focused on invasion of the ventral nerve cord (VNC), a process in which tumor cells leave the eye-antennal discs and optic lobes (the areas where they were born) and migrate to and invade a different organ, the VNC. It was further confirmed that the genotypes associated with the invasion of the VNC in this study also resulted in the presence of secondary tumor foci at distant locations, although the number and size of these foci were highly variable (Pagliarini, 2003; Igaki, 2006).
In analyzing the global expression profiles of noninvasive and invasive tumors induced in Drosophila developing eye discs, it was observed that expression of the JNK phosphatase puckered (puc) was strongly upregulated in the invasive tumors. Upregulation of puc represents activation of the JNK pathway in Drosophila. Therefore an enhancer-trap allele, puc-LacZ, was used to monitor the activation of JNK signaling in invasive tumor cells. Strong ectopic JNK activation was present in invasive tumors, while only a slight expression of puc was seen in restricted regions of RasV12-induced noninvasive overgrowth. Intriguingly, more intense JNK activation was seen in tumor cells located in the marginal region of the eye-antennal disc and tumor cells invading the VNC. Analysis of clones of cells with a cell polarity mutation alone revealed that JNK signaling was activated by mutation of cell polarity genes. Notably, JNK signaling was not activated in a strictly cell-autonomous fashion. JNK activation in these cells was further confirmed by anti-phospho-JNK antibody staining that detects activated JNK (Igaki, 2006).
To examine the contribution of JNK activation to metastatic behavior, the JNK pathway was blocked by overexpressing a dominant-negative form of Drosophila JNK (BskDN). As previously reported (Pagliarini, 2003), clones of cells mutant for scrib, lgl, or dlg do not proliferate as well as wild-type clones, while combination of these mutations with RasV12 expression resulted in massive and metastatic tumors. Strikingly, inhibition of JNK activation by BskDN completely blocked the invasion of the VNC, as well as secondary tumor foci formation. Drosophila has two homologs of TRAF proteins (DTRAF1 and DTRAF2), which mediate signals from cell surface receptors to the JNK kinase cascade in mammalian systems. It was found that RNAi-mediated inactivation of DTRAF2, but not DTRAF1, in the tumors strongly suppressed their metastatic behavior. Inactivation of dTAK1, a Drosophila JNK kinase kinase (JNKKK), or Hep, a JNKK, also suppressed metastatic behavior. Drosophila has two known cell surface receptors that act as triggers for the JNK pathway, Wengen (TNF receptor) and PVR (PDGF/VEGF receptor). Intriguingly, it was found that RNAi-mediated inactivation of Wengen partially suppressed tumor invasion. Inactivation of PVR, in contrast, did not show any suppressive effect on metastatic behavior. It was also found that the metastatic behavior of RasV12-expressing tumors that were also mutated for one of three other cell polarity genes, bazooka, stardust, or cdc42, was also blocked by BskDN. These data indicate that loss of cell polarity contributes to metastatic behavior by activating the evolutionarily conserved JNK pathway (Igaki, 2006).
Next, whether JNK activation is sufficient to trigger metastatic behavior in RasV12-induced benign tumors was examined. Two genetic alterations can be used to activate JNK in Drosophila. First, JNK signaling can be activated by overexpression of Eiger, a Drosophila TNF ligand. While mammalian TNF superfamily proteins activate both the JNK and NFκB pathways, Eiger has been shown to specifically activate the JNK pathway through dTAK1 and Hep. Indeed, the eye phenotype caused by Eiger overexpression could be reversed by blocking JNK through Bsk-IR (Bsk-RNAi). Second, overexpression of a constitutively activated form of Hep (HepCA) can also activate JNK signaling. However, the eye phenotype caused by HepCA overexpression was only slightly suppressed by Bsk-IR, suggesting that HepCA overexpression may have additional effects other than JNK activation. Therefore Eiger overexpression was used to activate JNK in RasV12-induced benign tumors, and it was found that the RasV12+Eiger-expressing tumor cells did not result in the invasion of the VNC. This indicates that loss of cell polarity must induce an additional downstream effect(s) essential for metastatic behavior. A strong candidate for the missing event is downregulation of the E-cadherin/catenin adhesion complex, since this complex is frequently downregulated in malignant human cancer cells and is also downregulated by loss of cell polarity genes in Drosophila invasive tumors (Pagliarini, 2003). In addition, it has been recently reported that higher motility of mammalian scrib knockdown cells can be partially rescued by overexpression of E-cadherin-catenin fusion protein, suggesting a role of E-cadherin in preventing polarity-dependent invasion. Furthermore, overexpression of E-cadherin blocks metastatic behavior of RasV12/scrib−/− tumors (Pagliarini, 2003), indicating that loss of E-cadherin is essential for inducing tumor invasion in this model. It was found that loss of the Drosophila E-cadherin homolog shotgun (shg), combined with the expression of both RasV12 and Eiger, induced the invasion of the VNC. Intriguingly, loss of shg in RasV12-expressing clones also showed a weak invasive phenotype at lower penetrance. In agreement with the essential role of JNK in tumor invasion, clones of shg−/− cells weakly upregulated puc expression. It was further found that JNK activation in dlg−/− clones is not blocked by overexpression of E-cadherin, suggesting that mechanism(s) other than loss of E-cadherin also exist for inducing JNK activation downstream of cell polarity disruption. The metastatic behavior of RasV12+Eiger/shg−/− tumors was completely blocked by coexpression of BskDN, indicating a cell-autonomous requirement of JNK activation for this process. Furthermore, it was found that loss of the β-catenin homolog armadillo also induced metastatic behavior in RasV12-induced benign tumors. In contrast, overexpression of HepCA in RasV12/shg−/− cells resulted in neither enhanced tumor growth nor metastatic behavior. Together, these results suggest that, although the RasV12+Eiger/shg−/− does not completely phenocopy the effect of RasV12/scrib−/−, activation of JNK signaling and inactivation of the E-cadherin/catenin complex are the downstream components of cell polarity disruption that trigger metastatic behavior in RasV12-induced benign tumors (Igaki, 2006).
During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC 'buds' off. This compensatory mechanism has been referred to as 'telophase rescue'. The Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (TRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. TRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires TRAF1. These data suggest that TRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway (Wang, 2006).
In telophase NBs, segregation of cell fate determinants, such as Pros, into future GMCs, is critical for their proper development. Telophase rescue appears to be one of the safeguard mechanisms that acts to ensure that GMCs inherit the cell fate determinants and adopt the correct cell identity when the mechanisms, which normally operate during NB divisions, fail (e.g., in insc mutant). Telophase rescue is a phenomenon for which the underlying mechanism involved remains largely unknown. The current data demonstrate that TRAF1 and Egr are two members of the Insc-independent telophase rescue pathway specific for Mira/Pros (Wang, 2006).
Although it is apically enriched in mitotic NBs and can directly interact with Baz in vitro, TRAF1 does not seem to be involved with the functions normally associated with the apical complex proteins. One distinct feature of TRAF1 differs from the other known apical proteins is its localization pattern; it is cytoplasmic in interphase and the apical crescent is prominent only at metaphase. In contrast, proteins of the apical complex are largely undetectable during interphase and form distinct apical crescents, starting from late interphase or early prophase. The protein localization difference between TRAF1 and other apical proteins suggests that TRAF1 and apical proteins are not always colocalized during mitosis. If TRAF1 is a bona fide member of the apical complex, the localization defects of other apical proteins are expected to be observed in TRAF1 mutant, as well as mislocalization of basal proteins, which was not detect. In addition, no spindle orientation or geometry defects were observed in the absence of TRAF1. Based on these observations, it is concluded that TRAF1 is not involved with the functions normally associated with the apical complex proteins (Wang, 2006).
The in vitro GST fusion protein pull-down assay suggests that TRAF1 may physically bind to Baz. This result is consistent with genetic data, indicating that TRAF1 acts downstream of baz and that its apical localization requires baz. These observations are consistent with the view that TRAF1 is recruited to the apical cortex by apical Baz in mitotic NBs. Baz, even at very low levels, can recruit TRAF1 to the apical cortex of the mitotic NBs. For example, in insc mutant NBs, TRAF1 remains apical probably owing to the low levels of Baz that remain localized to the apical cortex. This speculation is supported by Mira/Pros telophase rescue data, which clearly demonstrate that the telophase rescue seen in insc mutant NBs is severely damaged in baz mutant, suggesting that the Baz function required for Mira/Pros (and Pon/Numb) telophase rescue is intact in insc mutant NBs (Wang, 2006).
It has been shown that Pins/Gαi asymmetric cortical localization can be induced at metaphase by the combination of astral microtubules, kinesin Khc-73 and Dlg in the absence of Insc; this coincides with the observation that TRAF1 also forms tight crescent only at metaphase in both WT and insc mutant NBs. Does TRAF1 apical crescent formation also require the functions of astral microtubules, kinesin Khc-73 and Dlg? The data do not favor this hypothesis based on the following observations. (1) In TE35BC-3, a small deficiency uncovering sna family genes insc is not expressed but Pins and Gαi are asymmetrically localized, indicating that the astral microtubules, kinesin Khc-73 and Dlg pathway remain functional. TRAF1 is delocalized and is uniformly cortical in this deficiency line. (2) Similarly, in egr insc NBs, TRAF1 is cytoplasmic whereas the functions of astral microtubules, kinesin Khc-73 and Dlg are intact. (3) In egr NBs TRAF1 is cytoplasmic, whereas the apical complex is normal and astral microtubules, kinesin Khc-73 and Dlg are present. (4) TRAF1 apical localization remains unchanged in dlg mutant NBs. Based on these observations, it is concluded that TRAF1 apical localization is unlikely to share similar mechanism with Pins and Gαi and is likely to be independent of astral microtubules, kinesin Khc-73 and Dlg. TRAF1 apical localization appears to specifically require Egr and Baz (Wang, 2006).
In TRAF1 insc double-mutant embryos, the complete segregation of Mir/Pros into future GMCs occurs only in about 12% of the total population, and in the remaining NBs, only a fraction of Mira/Pros segregate into future GMCs as indicated by the Mira 'tail' extending into the future NBs at telophase. As it is difficult to address the global effect of this partial segregation of Mira/Pros on GMC specification in TRAF1 insc double mutant, focus was place on a well-defined GMC, GMC4-2a in NB4-2 lineage, to evaluate this issue. It is assumed that as long as the RP2 neuron (progeny of GMC4-2a, Even-skipped (Eve)-positive) was identified in a particular hemisegment, the GMC cell fate of GMC4-2a in that hemisegment should have been correctly specified. In insc mutants, almost all hemisegments contain RP2s, indicating that GMC4-2a has adopted the correct GMC cell fate in 99% of the total hemisegments. When TRAF1 insc double-mutant embryos were stained with anti-Eve, it was found the frequency of loss of Eve-positive RP2 neuron increased (to 8%) in late embryos, suggesting that about 8% of the GMCs in TRAF1 insc double mutant did not inherit sufficient Pros to specify the GMC fate in these embryos. The relatively low frequency (8%) of mis-specification of GMCs suggests that the threshold amount of Pros protein needed is sufficiently low such that just a partial inheritance of Pros, even when telophase rescue is compromised, is sufficient for most GMCs to be correctly specified (Wang, 2006).
Although Mira/Pros and Pon/Numb share similar basal localization patterns in insc NBs, further removal of either TRAF1 or Egr compromised telophase rescue only for Mira/Pros, but not for Pon/Numb. This difference between Mira/Pros and Pon/Numb indicates that the detailed mechanisms of basal localization and segregation of Mira/Pros differ from those of Pon/Numb, which is consistent with the observations that the dynamics of Mira/Pros and Pon/Numb localization early in mitosis are different and the basal localization for Mira/Pros and Pon/Numb requires different regions of the Insc coding sequence (Wang, 2006).
Dlg/Lgl/Scrib are required for correct basal localization of Mira/Pros and Pon/Numb in mitotic NBs. Dlg has been shown to be involved in the Mira telophase rescue. In dlg insc double-mutant NBs, not only was spindle geometry symmetric but Mira telophase rescue was also affected. It would be interesting to know if Dlg belongs to the same pathway as TRAF1 and Egr and if Dlg is also involved in Pon/Numb telophase rescue (Wang, 2006).
Two other members of the TRAF family have also been identified in Drosophila: DTRAF2 (DTRAF6) and DTRAF3. In contrast to the specific and strong expression of TRAF1 in the embryonic NBs, only low levels of ubiquitous signals similar to the control background were seen in the NBs with DTRAF2 and DTRAF3 probes. It is likely that DTRAF2 and DTRAF3 are not expressed in NBs and do not play an important role in Mira/Pros telophase rescue pathway as the Mira/Pros telophase rescue is dramatically compromised in TRAF1 insc and egr insc NBs (Wang, 2006).
In mammals, the TNF pathway works as a typical receptor-mediated signal transduction pathway. TNFR is a key player in transducing external signal to the cytoplasm. In the Drosophila compound eyes, ectopic Egr, Wgn and TRAF1 seem to work in a similar receptor-mediated signal pathway to induce apoptosis through the activation of the JNK pathway. Does the same Egr, Wgn and TRAF1 receptor-mediated signal pathway play a role in Mira/Pros telophase rescue? If it does, the coexpression of Egr, Wgn and TRAF1 might be expected to be seen in dividing NBs, along with the potential interaction between TRAF1 and the cytoplasmic domain of Wgn. Three observations argue against this hypothesis: (1) wgn is not expressed in embryonic NBs but in the mesoderm. (2) The domain analysis suggests that the Drosophila Wgn cytoplasmic domain is unique with no sequence homology to any mammalian TNFR family members and has neither a TRAF-binding domain nor a death domain, which is required for the interaction between TNFR and TRAF in mammals. (3) More informatively, Wgn knockdown by a UAS head-to-head inverted repeat construct of wgn (UAS-wgn-IR) driven by a strong maternal driver, mata-gal4 V32A, in WT embryos did not affect TRAF1 apical localization. These observations are consistent with the view that the receptor Wgn may not be involved in Mira/Pros telophase rescue or is redundant in this pathway. If this is the case, then how do TRAF1 and Egr function in Mira/Pros telophase rescue? It has been reported that TRAFs associate with numerous receptors other than the TNFR superfamily in mammals. It is speculated that Egr and TRAF1 may adopt an alternative receptor in NBs for Mira/Pros telophase rescue. However, until an anti-Wgn antibody and wgn mutant alleles are available, the possibility that Wgn is involved in Mira/Pros telophase rescue cannot be ruled out (Wang, 2006).
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date revised: 10 May 2008
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