Hirotaka Kanuka

Eiger, a TNF superfamily ligand that triggers the Drosophila JNK pathway

Drosophila provides a powerful genetic model for studying the in vivo regulation of cell death. In our large‐scale gain‐of‐function screen, we identified Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death. 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), it does not require caspase activity. We also show genetically that Eiger is a physiological ligand for the Drosophila JNK pathway. Our findings demonstrate that Eiger can initiate cell death through an IAP‐sensitive cell death pathway via JNK signaling.

Androgen-dependent neurodegeneration by polyglutamine-expanded human androgen receptor in Drosophila.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset, neurodegenerative disorder affecting only males and is caused by expanded polyglutamine (polyQ) stretches in the N-terminal A/B domain of human androgen receptor (hAR). Although no overt phenotype was detected in adult fly eye photoreceptor neurons expressing mutant hAR (polyQ 52), ingestion of androgen or its known antagonists caused marked neurodegeneration with nuclear localization and structural alteration of the hAR mutant. Ligand-independent toxicity was detected with a truncated polyQ-expanded A/B domain alone, which was attenuated with cytosolic trapping by coexpression of the unliganded hAR E/F ligand binding domain. Thus, our findings suggest that the full binding of androgen to the polyQ-expanded hAR mutants leads to structural alteration with nuclear translocation that eventually results in the onset of SBMA in male patients.

Drosophila IKK-Related Kinase Regulates Nonapoptotic Function of Caspases via Degradation of IAPs

Caspase activation has been extensively studied in the context of apoptosis. However, caspases also control other cellular functions, although the mechanisms regulating caspases in nonapoptotic contexts remain obscure. Drosophila IAP1 (DIAP1) is an endogenous caspase inhibitor that is crucial for regulating cell death during development. Here we describe Drosophila IKK-related kinase (DmIKKvarepsilon) as a regulator of caspase activation in a nonapoptotic context. We show that DmIKKvarepsilon promotes degradation of DIAP1 through direct phosphorylation. Knockdown of DmIKKvarepsilon in the proneural clusters of the wing imaginal disc, in which nonapoptotic caspase activity is required for proper sensory organ precursor (SOP) development, stabilizes endogenous DIAP1 and affects Drosophila SOP development. Our results demonstrate that DmIKKvarepsilon is a determinant of DIAP1 protein levels and that it establishes the threshold of activity required for the execution of nonapoptotic caspase functions.

Reaper-mediated inhibition of DIAP1-induced DTRAF1 degradation results in activation of JNK in Drosophila

Although Jun amino-terminal kinase (JNK) is known to mediate a physiological stress signal that leads to cell death, the exact role of the JNK pathway in the mechanisms underlying intrinsic cell death is largely unknown. Here we show through a genetic screen that a mutant of Drosophila melanogaster tumour-necrosis factor receptor-associated factor 1 (DTRAF1) is a dominant suppressor of Reaper-induced cell death. We show that Reaper modulates the JNK pathway through Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), which negatively regulates DTRAF1 by proteasome-mediated degradation. Reduction of JNK signals rescues the Reaper-induced small eye phenotype, and overexpression of DTRAF1 activates the Drosophila ASK1 (apoptosis signal-regulating kinase 1; a mitogen-activated protein kinase kinase kinase) and JNK pathway, thereby inducing cell death. Overexpresson of DIAP1 facilitates degradation of DTRAF1 in a ubiquitin-dependent manner and simultaneously inhibits activation of JNK. Expression of Reaper leads to a loss of DIAP1 inhibition of DTRAF1-mediated JNK activation in Drosophila cells. Taken together, our results indicate that DIAP1 may modulate cell death by regulating JNK activation through a ubiquitin–proteasome pathway.

Legionella Metaeffector Exploits Host Proteasome to Temporally Regulate Cognate Effector

Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of “metaeffector,” a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein.

The skin is an important bulwark of acquired immunity against intestinal helminths

IL-4–producing basophils promote the trapping of N. brasiliensis in the skin during secondary infection, a process critical for limiting the spread of infection to the lungs.

Drosophila caspase transduces Shaggy/GSK‐3β kinase activity in neural precursor development

Caspases are well known for their role in the execution of apoptotic programs, in which they cleave specific target proteins, leading to the elimination of cells, and for their role in cytokine maturation. In this study, we identified a novel substrate, which, through cleavage by caspases, can regulate Drosophila neural precursor development. Shaggy (Sgg)46 protein, an isoform encoded by the sgg gene and essential for the negative regulation of Wingless signaling, is cleaved by the Dark‐dependent caspase. This cleavage converts it to an active kinase, which contributes to the formation of neural precursor (sensory organ precursor (SOP)) cells. Our evidence suggests that caspase regulation of the wingless pathway is not associated with apoptotic cell death. These results imply a novel role for caspases in modulating cell signaling pathways through substrate cleavage in neural precursor development.

Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia

In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. Here, we show the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerated as development proceeded, and completed a full 360° rotation. This acceleration was impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced. Acceleration was induced by two distinct subcompartments of the A8 segment that formed a ring shape and surrounded the male genitalia: the inner ring rotated with the genitalia and the outer ring rotated later, functioning as a ‘moving walkway’ to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking showed that the timing and degree of apoptosis correlated with the movement of the outer ring, and upregulation of the apoptotic signal increased the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame.

Progesterone is a cell death suppressor that downregulates Fas expression in rat corpus luteum

In female rats, apoptotic cell death in the corpus luteum is induced by the prolactin (PRL) surge occurring in the proestrous afternoon during the estrous cycle. We have previously shown that this luteolytic action of PRL is mediated by the Fas/Fas ligand (FasL) system. During pregnancy or pseudopregnancy, apoptosis does not occur in the corpus luteum. Progesterone (P4), a steroid hormone secreted from luteal steroidogenic cells, attenuated PRL‐induced apoptosis in cultured luteal cells in a dose‐dependent manner. P4 significantly decreased the expression of mRNA of Fas, but not FasL, in cultured luteal cells prepared from both proestrous and mid‐pseudopregnant rats. These data indicate that P4 suppresses PRL‐induced luteal cell apoptosis via reduction of the expression level of Fas mRNA in the corpus luteum, suggesting that P4 acts as an important factor that can change the sensitivity of corpus luteum to PRL.

GATA-1 regulates the generation and function of basophils

Significance The GATA-1 transcription factor has been extensively characterized and shown to play crucial roles in the development of erythroid cells, magakaryocytes, eosinophils, and mast cells. However, the role of GATA-1 in basophils remained unidentified. We demonstrate that knockdown of Gata1 gene expression in basophils results in impaired cytokine production upon allergen-mediated activation. Moreover, ΔdblGATA mice carrying the mutated Gata1 promoter have reduced numbers of basophils and their progenitors and show impaired responses in basophil-mediated protective immunity against parasitic infections. Thus, GATA-1 plays an important role in both generation and activation of basophils, and ΔdblGATA mice display numerical and functional aberrancy in basophils, in addition to the well-known eosinophil deficiency. Developmental processes of hematopoietic cells are orchestrated by transcriptional networks. GATA-1, the founding member of the GATA family of transcription factors, has been demonstrated to play crucial roles in the differentiation of erythroid cells, magakaryocytes, eosinophils, and mast cells. However, the role of GATA-1 in basophils remains elusive. Here we show that basophils abundantly express Gata1 mRNAs, and that siRNA-mediated knockdown of Gata1 resulted in impaired production of IL-4 by basophils in response to the stimulation with IgE plus antigens. ΔdblGATA mice that carry the mutated Gata1 promoter and are widely used for functional analysis of eosinophils owing to their selective loss of eosinophils showed a decreased number of basophils with reduced expression of Gata1 mRNAs. The number of basophil progenitors in bone marrow was reduced in these mice, and the generation of basophils from their bone marrow cells in culture with IL-3 or thymic stromal lymphopoietin was impaired. ΔdblGATA basophils responded poorly ex vivo to stimulation with IgE plus antigens compared with wild-type basophils as assessed by degranulation and production of IL-4 and IL-6. Moreover, ΔdblGATA mice showed impaired responses in basophil-mediated protective immunity against intestinal helminth infection. Thus, ΔdblGATA mice showed numerical and functional aberrancy in basophils in addition to the known deficiency of eosinophils. Our findings demonstrate that GATA-1 plays a key role in the generation and function of basophils and underscore the need for careful distinction of the cell lineage responsible for each phenotype observed in ΔdblGATA mice.