InteractiveFly: GeneBrief

wengen: Biological Overview | References

Wengen has been identified as the first member of the Drosophila tumor necrosis factor receptor (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>eiger^regg1). 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>eiger^regg1) 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).

Eiger and its receptor, Wengen, comprise a TNF-like system in Drosophila

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 (Igaki, 2002; Moreno, 2002). 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).

Search PubMed for articles about Drosophila Wengen

Eby, M. T., Jasmin, A., Kumar, A., Sharma, K. and Chaudhary, P. M. (2000). TAJ, a novel member of the tumor necrosis factor receptor family, activates the c-Jun N-terminal kinase pathway and mediates caspase-independent cell death. J. Biol. Chem. 275(20): 15336-42. PubMed citation: 10809768

Igaki, T., et al. (2002). Eiger, a TNF superfamily ligand that triggers the Drosophila JNK pathway. EMBO J. 21: 3009-3018. PubMed citation: 12065414

Kanda, H., Igaki, T., Kanuka, H., Yagi, T. and Miura, M. (2002). Wengen, a member of the Drosophila tumor necrosis factor receptor superfamily, is required for Eiger signaling. J. Biol. Chem. 277: 28372-28375. PubMed citation; Online text

Kauppila, S., et al. (2003). Eiger and its receptor, Wengen, comprise a TNF-like system in Drosophila. Oncogene 22(31): 4860-7. PubMed citation: 12894227

Moreno, E., Yan, M. and Basler, K. (2002). Evolution of TNF signaling mechanisms: JNK-dependent apoptosis triggered by Eiger, the Drosophila homolog of the TNF superfamily. Curr. Biol. 12: 1263-1268. PubMed citation: 12176339

date revised: 6 May 2008

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