grindelwald: Biological Overview | References
Gene name - grindelwald
Synonyms - CG10176
Cytological map position - 36F5-36F5
Function - transmembrane receptor
Keywords - tumour necrosis factor receptor, JNK signalling, integrates apoptotic functions from Eiger with signaling from apical polarity determinants to induce JNK-dependent cell death or tumour growth
Symbol - grnd
FlyBase ID: FBgn0032682
Genetic map position - chr2L:18485417-18487710
Classification - tumour necrosis factor receptor (TNFR) superfamily
Cellular location - cytoplasmic
|Recent literature||Agrawal, N., Delanoue, R., Mauri, A., Basco, D., Pasco, M., Thorens, B. and Leopold, P. (2016). The Drosophila TNF Eiger is an adipokine that acts on insulin-producing cells to mediate nutrient response. Cell Metab 23: 675-684. PubMed ID: 27076079
Adaptation of organisms to ever-changing nutritional environments relies on sensor tissues and systemic signals. Identification of these signals would help understand the physiological crosstalk between organs contributing to growth and metabolic homeostasis. This study shows that Eiger, the Drosophila TNF-alpha, is a metabolic hormone that mediates nutrient response by remotely acting on insulin-producing cells (IPCs). In the condition of nutrient shortage, a metalloprotease of the TNF-alpha converting enzyme (TACE) family is active in fat body (adipose-like) cells, allowing the cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger activates its receptor Grindelwald, leading to JNK-dependent inhibition of insulin production. Therefore, this study has identified a humoral connexion between the fat body and the brain insulin-producing cells relying on TNF-alpha that mediates adaptive response to nutrient deprivation.
Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth, this study identified grindelwald (grnd; CG10176), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. This study shows that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of RasV12/scrib-/- tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth (Andersen, 2015).
A genome-wide screen was carried to identify molecules that are required for neoplastic growth. The condition used for this screen was the disc-specific knockdown of avalanche, also known as syntaxin 7), a gene encoding a syntaxin that functions in the early step of endocytosis2. avl-RNAi results in ectopic Wingless (Wg) expression, neoplastic disc overgrowth, and a 2-day delay in larva-to-pupa transition. A collection of 10,100 transgenic RNA interference (RNAi) lines were screened for their ability to rescue the pupariation delay, and 121 candidate genes were identified. Interestingly, only eight candidate genes also rescued ectopic Wg expression and neoplastic overgrowth. These included five lines targeting core components of the JNK pathway (Bendless, Tab2, Tak1, Hemipterous and Basket. Using a puckered enhancer trap (puc-lacZ) as a readout for JNK activity, it was confirmed that JNK signalling is highly upregulated in avl-RNAi discs. One of the remaining lines targets CG10176, a gene encoding a transmembrane protein. Reducing expression of CG10176 by using two different RNAi lines was as efficient as tak1 silencing to restore normal Wg pattern and suppresses JNK signalling and neoplastic growth in the avl-RNAi background. Sequence analysis of GC10176 identified a cysteine-rich domain (CRD) in the extracellular part with homology to vertebrate TNFRs harbouring a glycosphingolipid-binding motif (GBM) characteristic of many TNFRs including Fas. CG10176 was named grindelwald (grnd) , after a village at the foot of Eiger, a Swiss mountain that lent its name to the unique Drosophila TNF, Egr. Immunostaining and subcellular fractionation of disc extracts confirmed that Grnd localizes to the membrane. Moreover, co-immunoprecipitation experiments showed that both Grnd full-length and Grnd-intra, a form lacking its extracellular domain, directly associate with Traf2, the most upstream component of the JNK pathway. This interaction is disrupted by a single amino acid substitution within a conserved Traf6-binding motif (human TRAF6 is the closest homologue to Traf2. Overexpression of Grnd-intra, but not full-length Grnd, is sufficient to induce JNK signalling, ectopic Wg expression and apoptosis, and Grnd-intra-induced apoptosis is efficiently suppressed in a hep (JNKK) mutant background, confirming that Grnd acts upstream of the JNK signalling cascade (Andersen, 2015).
The Drosophila TNF Egr activates JNK signalling and triggers cell death or proliferation, depending on the cellular context. Therefore tests were performed to see whether Grnd is required for the small-eye phenotype generated by Egr-induced apoptosis in the retinal epithelium (via Egr overexpression). Inhibition of JNK signalling by reducing tak1 or traf2 expression, or by overexpressing puckered, blocks Egr-induced apoptosis and rescues the small-eye phenotype. In contrast to a previous report, RNAi silencing of wengen (wgn) , a gene encoding a presumptive receptor for Egr, does not rescue the small-eye phenotype. Furthermore, the small-eye phenotype is not modified in a wgn-null mutant background, confirming that Wgn is not required for Egr-induced apoptosis in the eye. By contrast, reducing grnd levels partially rescues the Egr-induced small-eye phenotype, producing a 'hanging-eye' phenotype that is not further rescued in a wgn-knockout mutant background. A similar phenotype was previously reported as a result of non-autonomous cell death induced by a diffusible form of Egr. This suggests that Grnd prevents Egr from diffusing outside of its expression domain. Co-immunoprecipitation experiments show that both full-length Grnd and Grnd-extra, a truncated form of Grnd lacking the cytoplasmic domain, associate with Egr through its TNF-homology domain. Although Grnd-extra can bind Egr, it cannot activate JNK signalling. Therefore, it was reasoned that Grnd-extra expression might prevent both cell-autonomous and non-autonomous apoptosis by trapping Egr and preventing its diffusion and binding to endogenous Grnd. Indeed, GMR-Gal4-mediated expression of grnd-extra fully rescues the Egr small-eye phenotype. To confirm that the removal of Grnd induces Egr-mediated non-autonomous cell death, wing disc clones were generated expressing egr alone, egr + tak1 RNAi, or egr + grnd RNAi. As expected, reducing tak1 levels in egr-expressing clones prevents their elimination by apoptosis. Similarly, reducing grnd levels prevents autonomous cell death, but also induces non-autonomous apoptosis. This suggests that Egr, like its mammalian counterpart TNF-α, can be processed into a diffusible form in vivo whose interaction with Grnd limits the potential to act at a distance. Flies carrying homozygous (grndMinos/Minos) or transheterozygous (grndMinos/Df) combinations of a transposon inserted in the grnd locus express no detectable levels of Grnd protein and are equally resistant to Egr-induced cell death. In addition, grndMinos/Minos mutant flies are viable and display no obvious phenotype, suggesting that Grnd, like Egr, participates in a stress response to limit organismal damage. Collectively, these data demonstrate that Grnd is a new Drosophila TNF receptor that mediates most, if not all, Egr-induced apoptosis (Andersen, 2015).
TNFs probably represent a danger signal produced in response to tissue damage to rid the organism of premalignant tissue or to facilitate wound healing. Disc clones mutant for the polarity gene scribbled (scrib) induce an Egr-dependent response resulting in the elimination of scrib mutant cells by JNK-mediated apoptosis. To test the requirement for Grnd in this process, scrib-RNAi and scrib-RNAi + grnd-RNAi clones obtained 72 h after heat shock induction were compared. As expected, scrib-RNAi cells undergo apoptosis and detach from the epithelium. By contrast, scrib-RNAi clones with reduced grnd expression survive, indicating that Grnd is required for Egr-dependent elimination of scrib-RNAi cells. Similar results were obtained by generating scrib mutant clones in the eye disc (Andersen, 2015).
In both mammals and flies, TNFs are double-edged swords that also have the capacity to promote tumorigenesis in specific cellular contexts. Indeed, scrib minus eye disc cells expressing an activated form of Ras (RasV12) exhibit a dramatic tumour-like overgrowth and metastatic behaviour, a process that critically relies on Egr. RasV12/scrib-/- metastatic cells show a strong accumulation of Grnd and Mmp1, and invade the ventral nerve cord. Primary tumour cells reach peripheral tissues such as the fat body and the gut, where they form micro-metastases expressing high levels of Grnd. Reducing grnd levels in RasV12/scrib-/- clones is sufficient to restore normal levels of Mmp1 and abolish invasiveness in a way similar to that observed in an egr mutant background. Therefore, Grnd is required for the Egr-induced metastatic behaviour of RasV12/scrib-/- tumorous cells. Similarly, reducing grnd, but not wgn levels, strongly suppresses Mmp1 expression in RasV12/dlg-RNAi cells and limits tumour invasion, indicating that Wgn does not have a major role in the progression of these tumours (Andersen, 2015).
Perturbation of cell polarity is an early hallmark of tumour progression in epithelial cells. In contrast to small patches of polarity-deficient cells, for example, scrib mutant clones, organ compartments or animals fully composed of polarity-deficient cells become refractory to Egr-induced cell death and develop epithelial tumours. The formation of these tumours requires JNK/MAPK signalling, but not Egr, suggesting Egr-independent coupling between loss of polarity and JNK/MAPK-dependent tumour growth. In line with these observations, it was noticed that, in contrast to Grnd, Egr is not required to drive neoplastic growth in avl-RNAi conditions. This suggests that, in addition to its role in promoting Egr-dependent functions, Grnd couples loss of polarity with JNK-dependent growth independently of Egr. Disc immunostainings revealed that Grnd co-localizes with the apical determinant Crb in the marginal zone, apical to the adherens junction protein E-cadherin (E-cad) and the atypical protein kinase C (aPKC). In avl-RNAi discs, Grnd and Crb accumulate in a wider apical domain. Apical accumulation of Crb is proposed to be partly responsible for the neoplastic growth induced by avl knockdown, since overexpression of Crb or a membrane-bound cytoplasmic tail of Crb (Crb-intra) mimics the avl-RNAi phenotype. Therefore whether Grnd might couple the activity of the Crb complex with JNK-mediated neoplastic growth was examined. Indeed, reducing grnd levels, but not wgn, in ectopic crb-intra discs suppresses neoplastic growth as efficiently as inhibiting the activity of the JNK pathway. Notably, Yki activation is not rescued in these conditions, illustrating the ability of Crb-intra to promote growth independently of Grnd by inhibiting Hippo signalling through its FERM-binding motif (FBM). Indeed, neoplastic growth and polarity defects induced by a form of Crb-intra lacking its FBM (CrbΔFBM-intra) are both rescued by Grnd silencing. As expected, the size of ectopic crbΔFBM-intra;grnd-RNAi discs is reduced compared to the size of ectopic crb-intra; grnd-RNAi discs (Andersen, 2015).
Crb, Stardust (Sdt; PALS1 in humans), and Pals1-associated tight junction protein (Patj) make up the core Crb complex, which recruits the adaptor protein Veli (MALS1-3 in humans). In agreement with previous yeast two-hybrid data, this study found that Grnd binds directly and specifically to the PDZ domain of Veli through a membrane-proximal stretch of 28 amino acids in its intracellular domain. Grnd localization is unaffected in crb and veli RNAi mutant clones. However, reducing veli expression rescues the patterning defects and disc morphology of ectopic crb-intra mutant cells, suggesting that Grnd couples Crb activity with JNK signalling through its interaction with Veli. Interestingly, aPKC-dependent activation of JNK signalling also depends on Grnd. aPKC is capable of directly binding and phosphorylating Crb, which is important for Crb function. This suggests that aPKC, either directly or through Crb phosphorylation, activates Grnd-dependent JNK signalling in response to perturbation of apico-basal polarity (Andersen, 2015).
These data are consistent with a model whereby Grnd integrates signals from Egr, the unique fly TNF, and apical polarity determinants to induce JNK-dependent neoplastic growth or apoptosis in a context-dependent manner. Recent work reveals a correlation between mammalian Crb3 expression and tumorigenic potential in mouse kidney epithelial cells. The conserved nature of the Grnd receptor suggests that specific TNFRs might carry out similar functions in vertebrates, in which the link between apical cell polarity and tumour progression remains elusive (Andersen, 2015).
Search PubMed for articles about Drosophila Grindelwald
Andersen, D. S., Colombani, J., Palmerini, V., Chakrabandhu, K., Boone, E., Rothlisberger, M., Toggweiler, J., Basler, K., Mapelli, M., Hueber, A. O. and Leopold, P. (2015). The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth. Nature. PubMed ID: 25874673
date revised: 31 May, 2015
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