Evolution of TNF signaling mechanisms: JNK-dependent apoptosis triggered by Eiger,the Drosophila homolog of the TNF superfamily

Much of what we know 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. Here, we describe the cloning and characterization of Eiger, a unique TNF homolog in Drosophila. 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. Our 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.     

Wengen,a member of the Drosophila tumor necrosis factor receptor superfamily,is required for Eiger signaling

We identified Wengen, 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, was dramatically suppressed by down-regulation of Wengen using RNA interference. In addition, Wengen and Eiger physically interacted 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. Our 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.     

TWEAK,a member of the TNF superfamily,is a multifunctional cytokine that binds the TweakR/Fn14 receptor

The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) was initially described as a member of the tumor necrosis factor (TNF) superfamily in 1997. TWEAK is a cell surface-associated type II transmembrane protein, but a smaller, biologically active form can also be shed into the extracellular milieu. There is one receptor currently known to bind TWEAK with physiological affinity, and it is a type I transmembrane protein that is referred to in the literature as either TWEAK receptor (TweakR) or fibroblast growth factor-inducible 14 (Fn14). TweakR/Fn14 is the smallest member of the TNF receptor (TNFR) superfamily described to date, and it appears to signal via recruitment of several different TNFR-associated factors. TWEAK has multiple biological activities, including stimulation of cell growth and angiogenesis, induction of inflammatory cytokines, and under some experimental conditions, stimulation of apoptosis. In this report, we summarize the results from recent studies focused on the TWEAK cytokine. Although these studies have contributed a significant amount of new information, numerous questions still remain regarding the role of TWEAK in both normal physiology and the pathogenesis of human disease.     

Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand

The characterisation of ligands that activate the JAK/STAT pathway has the potential to throw light onto a comparatively poorly understood aspect of this important signal transduction cascade. Here, we describe our analysis of the only invertebrate JAK/STAT pathway ligands identified to date, the Drosophila unpaired-like family. We show that upd2 is expressed in a pattern essentially identical to that of upd and demonstrate that the proteins encoded by this region activate JAK/STAT pathway signalling. Mutational analysis demonstrates a mutual semi-redundancy that can be visualised in multiple tissues known to require JAK/STAT signalling. In order to better characterise the in vivo function of these ligands, we developed a reporter based on a natural JAK/STAT pathway responsive enhancer and show that ectopic upd2 expression can effectively activate the JAK/STAT pathway. While both Upd and Upd2 are secreted JAK/STAT pathway agonists, tissue culture assays show that the signal-sequences of Upd and Upd2 confer distinct properties, with Upd associated primarily with the extracellular matrix and Upd2 secreted into the media. The differing biophysical characteristics identified for Upd-like molecules have implications for their function in vivo and adds another aspect to our understanding of cytokine signalling in Drosophila.     

Roles of the JNK signaling pathway in Drosophila morphogenesis.

Epithelial cell differentiation and morphogenesis are crucial in many aspects of metazoan development. Recent genetic studies in Drosophila have revealed that the conserved Jun amino-terminal kinase (JNK) signaling pathway regulates epithelial morphogenesis during the process of embryonic dorsal closure and participates in the control of planar polarity in several tissues. Importantly, these studies have linked the JNK pathway to the decapentaplegic and Frizzled pathways in these processes, suggesting a high degree of integrative signaling during epithelial morphogenesis.     

DCX, a new mediator of the JNK pathway

Mutations in the X-linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c-Jun N-terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus-end directed molecular motor) versus dynein (a minus-end directed molecular motor).     

Odd-skipped genes specify the signaling center that triggers retinogenesis in Drosophila

Although many of the factors responsible for conferring identity to the eye field in Drosophila have been identified, much less is known about how the expression of the retinal ;trigger', the signaling molecule Hedgehog, is controlled. Here, we show that the co-expression of the conserved odd-skipped family genes at the posterior margin of the eye field is required to activate hedgehog expression and thereby the onset of retinogenesis. The fly Wnt1 homologue wingless represses the odd-skipped genes drm and odd along the anterior margin and, in this manner, spatially restricts the extent of retinal differentiation within the eye field.     

Signalling pathways of the TNF superfamily: a double-edged sword

Two different tumour-necrosis factors (TNFs), first isolated in 1984, were found to be cytotoxic to tumour cells and to induce tumour regression in mice. Research during the past two decades has shown the existence of a superfamily of TNF proteins consisting of 19 members that signal through 29 receptors. These ligands, while regulating normal functions such as immune responses, haematopoiesis and morphogenesis, have also been implicated in tumorigenesis, transplant rejection, septic shock, viral replication, bone resorption, rheumatoid arthritis and diabetes; so indicating their role as 'double-edged swords'. These cytokines either induce cellular proliferation, survival, differentiation or apoptosis. Blockers of TNF have been approved for human use in treating TNF-linked autoimmune diseases in the United States and other countries.     

Drosophila DREF acting via the JNK pathway is required for thorax development

The Drosophila Jun N-terminal kinase (JNK) gene basket (bsk) promoter contains a DNA replication-related element (DRE)-like sequence, raising the possibility of regulation by the DNA replication-related element-binding factor (DREF). Chromatin immunoprecipitation assays with anti-DREF IgG showed the bsk gene promoter region to be effectively amplified. Luciferase transient expression assays revealed the DRE-like sequence to be important for bsk gene promoter activity, and knockdown of DREF decreased the bsk mRNA level and the bsk gene promoter activity. Furthermore, knockdown of DREF in the notum compartment of wing discs by pannier-GAL4 and UAS-DREFIR resulted in a split thorax phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc)-LacZ enhancer trap line revealed the reduction in DREF knockdown clones. These findings indicate that DREF is involved in regulation of Drosophila thorax development via actions on the JNK pathway.     

Powerful Drosophila screens that paved the wingless pathway

The Wnt/Wingless (Wg) signaling cascade controls a number of biological processes in animal development and adult life; aberrant Wnt/Wg signaling can cause diseases. In the 1980s genes were discovered that encode core Wnt/Wg pathway components: their mutant phenotypes were similar and an outline of a signaling cascade emerged. Over the years our knowledge of this important signaling system increased and more components were uncovered that are instrumental for Wnt/Wg secretion and transduction. Here we provide an overview of these discoveries, the technologies involved, with a particular focus on the important role Drosophila screens played in this process.     

Powerful Drosophila screens that paved the wingless pathway

The Wnt/Wingless (Wg) signaling cascade controls a number of biological processes in animal development and adult life; aberrant Wnt/Wg signaling can cause diseases. In the 1980s genes were discovered that encode core Wnt/Wg pathway components: their mutant phenotypes were similar and an outline of a signaling cascade emerged. Over the years our knowledge of this important signaling system increased and more components were uncovered that are instrumental for Wnt/Wg secretion and transduction. Here we provide an overview of these discoveries, the technologies involved, with a particular focus on the important role Drosophila screens played in this process.     

肿瘤坏死因子受体超家族成员TROY的研究进展

TROY(TNFRSF expressed on the mouse embryo)是近年新发现的表达于小鼠胚胎的肿瘤坏死因子受体超家族成员.TROY在体内分布广泛,尤其高表达于胚胎和成熟的中枢神经系统.研究表明,TROY能与髓鞘抑制因子Nogo NgR1及脑内神经再生抑制因子(LINGO-1)形成功能受体复合物,参与中枢神经系统轴突生长抑制因子的信号转导;TROY还能诱导细胞副凋亡,促进某些细胞的增殖分化.本文就TROY在上述相关领域的研究进展作一综述.     

Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection

Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.     

Cross-regulation between herpesviruses and the TNF superfamily members

Herpesviruses have evolved numerous strategies to subvert host immune responses so they can coexist with their host species. These viruses 'co-opt' host genes for entry into host cells and then express immunomodulatory genes, including mimics of members of the tumour-necrosis factor (TNF) superfamily, that initiate and alter host-cell signalling pathways. TNF superfamily members have crucial roles in controlling herpesvirus infection by mediating the direct killing of infected cells and by enhancing immune responses. Despite these strong immune responses, herpesviruses persist in a latent form, which suggests a dynamic relationship between the host immune system and the virus that results in a balance between host survival and viral control.     

The Drosophila JNK pathway controls the morphogenesis of imaginal discs during metamorphosis.

In Drosophila, the Jun-N-terminal Kinase-(JNK) signaling pathway is required for epithelial cell shape changes during dorsal closure of the embryo. In the absence of JNK pathway activity, as in the DJNKK/hemipterous (hep) mutant, the dorsolateral ectodermal cells fail both to elongate and move toward the dorsal midline, leading to dorsally open embryos. We show here that hep and the JNK pathway are required later in development, for correct morphogenesis of other epithelia, the imaginal discs. During metamorphosis, the imaginal discs undergo profound morphological changes, giving rise to the adult head and thoracic structures, including the cuticle and appendages. hep mutant pupae and pharate adults show severe defects in discs morphogenesis, especially in the fusion of the two lateral wing discs. We show that these defects are accompanied by a loss of expression of puckered (puc), a JNK phosphatase-encoding gene, in a subset of peripodial cells that ultimately delineates the margins of fusing discs. In further support of a role of puc in discs morphogenesis, pupal and adult hep phenotypes are suppressed by reducing puc function, indicative of a negative role of puc in disc morphogenesis. Furthermore, we show that the small GTPase Dcdc42, but not Drac1, is an activator of puc expression in a hep-dependent manner in imaginal discs. Altogether, these results demonstrate a new role for the JNK pathway in epithelial morphogenesis, and provide genetic evidence for a role of the peripodial membrane in disc morphogenesis. We discuss a general model whereby the JNK pathway regulates morphogenesis of epithelia with differentiated edges.     

Thorax closure in Drosophila: involvement of Fos and the JNK pathway.

Dorsal closure, a morphogenetic movement during Drosophila embryogenesis, is controlled by the Drosophila JNK pathway, D-Fos and the phosphatase Puckered (Puc). To identify principles of epithelial closure processes, we studied another cell sheet movement that we term thorax closure, the joining of the parts of the wing imaginal discs which give rise to the adult thorax during metamorphosis. In thorax closure a special row of margin cells express puc and accumulate prominent actin fibres during midline attachment. Genetic data indicate a requirement of D-Fos and the JNK pathway for thorax closure, and a negative regulatory role of Puc. Furthermore, puc expression co-localises with elevated levels of D-Fos, is reduced in a JNK or D-Fos loss-of-function background and is ectopically induced after JNK activation. This suggests that Puc acts downstream of the JNK pathway and D-Fos to mediate a negative feed-back loop. Therefore, the molecular circuitry required for thorax closure is very similar to the one directing dorsal closure in the embryo, even though the tissues are not related. This finding supports the hypothesis that the mechanism controlling dorsal closure has been co-opted for thorax closure in the evolution of insect metamorphosis and may represent a more widely used functional module for tissue closure in other species as well.     

The molecular architecture of the TNF superfamily.

Ligands of the TNF (tumour necrosis factor) superfamily have pivotal roles in the organization and function of the immune system, and are implicated in the aetiology of several acquired and genetic diseases. TNF ligands share a common structural motif, the TNF homology domain (THD), which binds to cysteine-rich domains (CRDs) of TNF receptors. CRDs are composed of structural modules, whose variation in number and type confers heterogeneity upon the family. Protein folds reminiscent of the THD and CRD are also found in other protein families, raising the possibility that the mode of interaction between TNF and TNF receptors might be conserved in other contexts.