BIOLOGICAL OVERVIEW

Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death, was identified in a large-scale gain-of-function screen. 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, Thread), it does not require caspase activity. Genetically, Eiger has been shown to be a physiological ligand for the Drosophila JNK pathway. These findings demonstrate that Eiger can initiate cell death through an IAP-sensitive cell death pathway via JNK signaling (Igaki, 2002).

To discover the cell death triggers encoded in the Drosophila genome, a misexpression screen was conducted using the GAL4/UAS system. The GS vector is a P element-based gene search vector with UAS enhancers. A collection of 5000 lines harboring the GS vector (GS lines) was crossed with an eye-specific GAL4 (GMR-GAL4) strain to screen for genes that generate the reduced-eye phenotype. Six GS lines resulted in greatly reduced eyes in a GAL4-dependent manner; these are called Regg strains (Regg1-6, for reduced-eye generator with a GMR-GAL4 driver) (Igaki, 2002).

The F₁ progeny generated by mating the GMR-GAL4 strain and Regg1 (GS9830) strain (GMR-GAL4 driven regg1^GS9830) display a strong reduced-eye phenotype when compared with the wild-type eye. To assess whether the small eye phenotype of GMR-GAL4 driven regg1^GS9830 flies are generated by the acceleration of cell death, eye discs were stained with acridine orange to detect dying cells. Acridine orange is commonly used as a marker for apoptotic cell death in Drosophila. In GMR-GAL4 driven regg1^GS9830 discs, numerous acridine orange-staining cells were observed, indicating that regg1^GS9830 induces massive apoptotic cell death, leading to the reduced-eye phenotype. Using the inverse PCR method, the insertion site of the P element in the Regg1^GS9830 strain was determined; a predicted gene, CG12919, was discovered adjacent to the insertion site. The GS9830 strain was generated by inserting the GS6 vector, which has a green fluorescent protein (GFP) trailer (UAS-GFP-SV40 terminator) near the 3'P end and a UAS enhancer near the 5'P end, so that a misexpression of vector-flanking sequence occurs at the 5'P side only. As expected, CG12919 was the only gene with an elevated expression level in a GAL4-dependent manner. A Drosophila EST clone, LP03784, which included the nucleotide sequence of CG12919 was sequenced, and the open reading frame (ORF) of a novel gene that has been named eiger (EDA-like cell death trigger) was identified (Igaki, 2002).

To examine whether Eiger is indeed a membrane protein, S2 cells were transfected with expression vectors for an N-terminal hemagglutinin (HA)-tagged Eiger and GFP. In permeabilized cells, cell surface staining was observed in GFP-positive cells by anti-HA immunostaining. When immunostained prior to fixation and permeabilization, however, the staining was not detected, consistent with Eiger being a type II transmembrane protein with an extracellular C-terminus and a cytoplasmic N-terminus (Igaki, 2002).

Ectopic expression of Eiger in the eye results in a reduced-eye phenotype similar to that of Reaper, a Drosophila 'intrinsic' cell death trigger. The effects of Eiger overexpression in other tissues using different GAL4 drivers was further analyzed. When overexpressed in the dorsoventral compartments of the wing discs by a vg-GAL4 driver, Eiger entirely blocks wing formation, whereas Reaper induces only a regional defect. Ectopic expression of Eiger in precursor cells for the external sensory organs by a sca-GAL4 driver results in disorganized macrochaetae in the notum and scutellum, whereas Reaper induces a complete loss of bristles in these regions. In the abdomen, in contrast, sca-GAL4 driven regg1^GS9830 results in a severe developmental defect, whereas reaper causes a loss of bristles in the tergum. Thus, Eiger has the potential to induce developmental defects distinct from the defects caused by the cell-autonomous killer protein Reaper (Igaki, 2002).

In mammals, the binding of death ligands such as TNF-alpha and FasL to their receptors triggers the activation of caspase-8, leading to the subsequent caspase-dependent cell death cascade (Ashkenazi, 1998). To assess whether Eiger stimulates similar death signaling in Drosophila, Eiger and caspase-inhibitory proteins such as baculovirus P35, Drosophila inhibitor-of-apoptosis protein 1 (DIAP1) or a dominant-negative form of DRONC (DRONC-DN), a P35-resistant Drosophila apical caspase, were co-expressed. P35 and DRONC-DN exhibit only slight suppressive effects on the Eiger-induced eye phenotype, although they strongly suppressed the Reaper-induced eye ablation. These results indicate that Eiger-induced cell death does not require caspase activity differing from the mammalian 'extrinsic' cell death system. In contrast, the co-expression of DIAP1 strongly suppresses the Eiger-induced eye phenotype, suggesting that DIAP1 can block Eiger-activated death signaling through a mechanism that is independent of caspase inhibition. Whether endogenous DIAP1 negatively regulates the Eiger-induced phenotype was assessed genetically. Whereas heterozygous diap1 mutant flies exhibit a normal eye, GMR-GAL4 driven regg1^GS9830 flies with a half dosage of the diap1 gene display a completely ablated eye phenotype compared with the reduced-eye phenotype of GMR-GAL4 driven regg1^GS9830 flies, suggesting that DIAP1 is an endogenous inhibitor of Eiger-induced cell death signaling (Igaki, 2002).

To assess whether Eiger does cause caspase activation, the eye disc was stained with (DMe)₂R, a caspase substrate that contains only an aspartate residue linked to rhodamine-110. The eye disc from GMR-GAL4 driven reaper flies shows a strong rhodamine-110 fluorescence at the region posterior to the morphogenetic furrow, compared with the eye disc from GMR-GAL4 flies. In the GMR-GAL4 driven regg1^GS9830 eye disc, the fluorescence is detected at lower, but still significant, levels in many cells posterior to the furrow. These data suggest that although caspase activation is not essential for cell death execution, Eiger activates both caspase-dependent and -independent signaling pathways (Igaki, 2002).

Thus, Eiger has been identified as a novel cell death trigger molecule in Drosophila. The structure and function of Eiger suggest that the extrinsic cell death-inducing mechanism might be evolutionarily conserved in Drosophila. Genetic evidence reveals that caspase activation is not essential to execute Eiger-induced cell death. The Drosophila extrinsic cell death system might predominantly utilize the caspase-independent pathway, in contrast to the intrinsic cell death system, which is regulated by Reaper, Hid and Grim, and depends completely on caspase activation. Although caspases do take part in the apoptotic effects of most of the mammalian TNF ligand/receptor superfamily members studied so far, there is accumulating evidence that they can also kill the cells in the absence of caspases (Igaki, 2002 and references therein).

The genetic data clearly show that the Eiger-induced small eye phenotype depends strongly on the JNK signaling pathway. In mammals, it has been demonstrated that the JNK pathway is essential for the execution of stress-induced cell death. JNK3, a JNK isoform that is selectively expressed in the nervous system, is required for neuronal cell death caused by excitotoxic stress. The results suggest the possibility that Eiger-induced cell death signaling may be independent of downstream jun expression, similar to the observation that the effect of UV to cause cell death does not require new gene expression. The JNK signaling also mediates heat shock-induced cell death, the execution of which is caspase independent. Furthermore, overexpression of the EDA receptor or TAJ/TROY, a member of the TNF receptor superfamily that exhibits extensive homology to the EDA receptor, results in the activation of the JNK pathway and caspase-independent cell death (Eby, 2000; Kumar, 2001). In some cases, JNK-induced cell death is mediated by the release of mitochondrial apoptogenic factors. Recently, it has been shown that cancer cell death induced by TRAIL, a mammalian TNF superfamily ligand, requires mitochondrial release of Smac. One possible mechanism of Eiger-induced cell death may be JNK-mediated release of mitochondrial caspase-independent cell death factors. In fact, the Drosophila genome also encodes homologs of such molecules: AIF, endo G and HtrA2 (Igaki, 2002 and references therein).

One important feature of Eiger-stimulating cell death signaling is that it can be blocked by DIAP1. It is well understood that IAP family proteins suppress cell death through direct inhibition of caspases. The observations in this study suggest a potential mechanism of IAP that can inhibit caspase-independent cell death. It has been reported that Xenopus cell death induced by TAK1 (see Drosophila TGF-þ activated kinase 1) and TAB1, an activator for TAK1, is blocked by X-chromosome-linked IAP (XIAP). More recently, it has been shown that XIAP attenuates TNF-alpha-mediated JNK activation in HeLa cells and RelA^-/- fibroblasts. These findings and the data presented in this study lead to a model in Drosophila in which DIAP1 regulates caspase-dependent and -independent cell death pathways by blocking both the caspases and the JNK signaling (Igaki, 2002).

Loss-of-function study demonstrates that Eiger is a physiological trigger for the JNK pathway in the eye disc. Genetic interaction assays show that Eiger-stimulating cell death signaling is mediated by Msn, dTAK1, Hep and Bsk. Although dominant-negative dTAK1 completely suppresses the Eiger-induced phenotype, it is also possible that many components of MAP kinase pathways expressed as 'dominant negatives' can have a gain-of-function inhibitory activity. In fact, the immune response phenotype of dTAK1 mutants seems to be inconsistent with the idea that dTAK1 participates in the Eiger pathway. Another possible JNKKK family member to mediate Eiger signaling is Slipper. Previous genetic studies in Drosophila have revealed that the JNK signaling pathway regulates epithelial morphogenesis during the process of embryonic dorsal closure, and that it also participates in the control of planar polarity in several tissues. It has also been reported that the JNK signaling regulates cell death to maintain normal morphogenesis of the wing. Eiger might function as a JNK-dependent cell death regulator to facilitate normal morphogenesis of the eye. Further analysis of eiger mutant flies would dissect the physiological role of Eiger in neural development (Igaki, 2002 and references therein).

In mammals, members of the TNF superfamily play crucial roles in the regulation of infections, inflammation, autoimmune diseases and tissue homeostasis. The TNF superfamily ligands bind to their respective receptors leading to the activation of diverse signaling pathways, including the caspase cascade, NF-kappaB, or MAPKs such as JNK or ERK. Thus, TNF-related ligands can trigger either the extrinsic cell death execution, differentiation or proliferation. Although overexpression of Eiger can strongly induce cell death in the Drosophila compound eye, the possibility that Eiger-stimulated signaling may contribute to cellular events other than cell death execution cannot be excluded. In fact, the amino acid sequence of Eiger showed the highest homology (19%) with EDA, a human TNF superfamily ligand, the mutation of which causes impaired ectodermal development. eiger is predominantly expressed in the nervous system, whereas most mammalian TNF/TNF receptor superfamily proteins are expressed in the immune system, raising the possibility that Eiger might regulate proliferation of neural progenitor cells as does TNF-alpha (Arnett, 2001) to maintain normal development of the nervous system. The Drosophila genome has a gene encoding a candidate Eiger receptor with a TNF receptor homology domain and a transmembrane domain (see Wengen). In addition, the Drosophila genome also encodes genes for mediating factors such as TNF-receptor-associated factors (TRAFs: see Traf1 and Traf2), FADD and RIP (IMD), all of which may play a role in Eiger/Eiger receptor signaling. For information about Traf1 see Misshappen Protein Interactions section. For information about Traf2 see Pelle Protein Interactions section. Further genetic study of Eiger and its receptor should help elucidate the universal role of TNF/TNF receptor superfamily proteins in normal development, as well as in some pathophysiological conditions (Igaki, 2002).

GENE STRUCTURE

cDNA clone length - 2159

Bases in 5' UTR - 640

Exons - 5

Bases in 3' UTR - 271

PROTEIN STRUCTURE AND EVOLUTIONARY HOMOLOGS

Amino Acids - 409

Structural Domains

eiger encodes a protein of 409 amino acids with a C-terminal TNF homology domain and a hydrophobic transmembrane domain, indicating that Eiger is the first Drosophila member of the TNF superfamily. The absence of a signal peptide suggests that Eiger is a type II membrane protein, which is typical of the members of the TNF ligand family. The sequence of the C-terminal TNF domain of Eiger shows highest homology with human EDA-A2 (28% identity), and also shows significant homology with all known TNF superfamily members including RANKL, CD40L, FasL, APRIL, TWEAK, TNF-alpha and TRAIL (Igaki, 2002).

For information on vertebrate TNF and its interaction with the JNK pathway, search PubMed for information on tumor necrosis factor and the JNK pathway

date revised: 20 July 2002

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