The Drosophila retinoblastoma protein induces apoptosis in proliferating but not in post-mitotic cells

International audienceThe retinoblastoma protein, pRb, plays important roles in many processes implicated in cell fate decisions, including cell cycle, differentiation and apoptosis. In cell cycle regulation, pRb interacts principally with the E2F transcription factor family members to inhibit the transcription of many genes controlling cell cycle progression. In this study, we focused on the role of pRb in apoptosis, which is much less clear than its role in cell cycle regulation. Indeed, pRb has been found to be either pro- or anti-apoptotic. To clarify how the proliferative status of the cells impacts the role of pRb in apoptosis, we used Drosophila to induce RBF (the pRb fly homologue) expression in different cellular and developmental contexts. We found that RBF expression induces apoptosis in different proliferative tissues in a caspase-dependent manner, whereas this effect was not observed in differentiated post-mitotic cells. Furthermore, RBF-induced apoptosis in proliferating cells was inhibited by co-expression of dE2F1, an antagonistic partner of RBF in cell cycle regulation. These results are in agreement with the view that the apoptotic properties of pRb are tightly linked to, and are probably a consequence of, an effect on cell cycle progression. Moreover, we show for the first time that RBF has a direct anti-apoptotic effect on Dmp53-induced cell death in post-mitotic cells only. Taken together, these data clearly show that RBF can exert a dual role in the control of apoptotic processes, and that its properties depend on the proliferative status of the cells

Le come-back de la mitochondrie dans l'apoptose chez la drosophile

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Two different specific JNK activators are required to trigger apoptosis or compensatory proliferation in response to Rbf1 in Drosophila

International audienceThe Jun Kinase (JNK) signaling pathway responds to diverse stimuli by appropriate and specific cellular responses such as apoptosis, differentiation or proliferation. The mechanisms that mediate this specificity remain largely unknown. The core of this signaling pathway, composed of a JNK protein and a JNK kinase (JNKK), can be activated by various putative JNKK kinases (JNKKK) which are themselves downstream of different adaptor proteins. A proposed hypothesis is that the JNK pathway specific response lies in the combination of a JNKKK and an adaptor protein upstream of the JNKK. We previously showed that the Drosophila homolog of pRb (Rbf1) and a mutant form of Rbf1 (Rbf1 D253A) have JNK-dependent pro-apoptotic properties. Rbf1 D253A is also able to induce a JNK-dependent abnormal proliferation. Here, we show that Rbf1-induced apoptosis triggers proliferation which depends on the JNK pathway activation. Taking advantage of these phenotypes, we investigated the JNK signaling involved in either Rbf1-induced apoptosis or in proliferation in response to Rbf1-induced apoptosis. We demonstrated that 2 different JNK pathways involving different adaptor proteins and kinases are involved in Rbf1-apoptosis (i.e. Rac1-dTak1-dMekk1-JNK pathway) and in proliferation in response to Rbf1-induced apoptosis (i.e., dTRAF1-Slipper-JNK pathway). Using a transient induction of rbf1, we show that Rbf1-induced apoptosis activates a compensatory proliferation mechanism which also depends on Slipper and dTRAF1. Thus, these 2 proteins seem to be key players of compensatory proliferation in Drosophila

Apoptosis in Drosophila: which role for mitochondria?

International audienceIt is now well established that the mitochon-drion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mam-malian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mito-chondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human

The Drosophila retinoblastoma protein, Rbf1, induces a Debcl-and Drp1-dependent mitochondrial apoptosis

International audienceIn accordance with its tumor suppressor role, the retinoblastoma protein pRb can ensure pro-apoptotic functions. Rbf1, the Drosophila homolog of Rb, also displays a pro-apoptotic activity in proliferative cells. We have previously shown that the Rbf1 pro-apoptotic activity depends on its ability to decrease the level of anti-apoptotic proteins such as the Bcl-2 family protein Buffy. Buffy often acts in an opposite manner to Debcl, the other Drosophila Bcl-2-family protein. Both proteins can localize at the mitochondrion, but the way they control apoptosis still remains unclear. Here, we demonstrate that Debcl and the pro-fission gene Drp1 are necessary downstream of Buffy to trigger a mitochondrial fragmentation during Rbf1-induced apoptosis. Interestingly, Rbf1-induced apoptosis leads to a Debcl-and Drp1-dependent reactive oxygen species production, which in turn activates the Jun Kinase pathway to trigger cell death. Moreover, we show that Debcl and Drp1 can interact and that Buffy inhibits this interaction. Notably, Debcl modulates Drp1 mitochondrial localization during apoptosis. These results provide a mechanism by which Drosophila Bcl-2 family proteins can control apoptosis, and shed light on a link between Rbf1 and mitochondrial dynamics in vivo

PINK1 and BNIP3 mitophagy inducers have an antagonistic effect on Rbf1-induced apoptosis in Drosophila

The structure and function of the mitochondrial network are finely regulated. Among the proteins involved in these regulations, mitochondrial dynamics actors have been reported to regulate the apoptotic process. We show here in the Drosophila model that the mitophagy inducers, PINK1 (PTENinduced putative kinase 1) and BNIP3 (Bcl-2 Interacting Protein 3), modulate mitochondrial apoptosis differently. If close links between the fission-inducing protein DRP1 and Bcl-2 family proteins, regulators of apoptosis, are demonstrated, the connection between mitophagy and apoptosis is still poorly understood. In Drosophila, we have shown that Rbf1, a homolog of the oncosuppressive protein pRb, induces cell death in proliferating larval tissues through a mechanism involving the interaction of Drp1 with Debcl, a pro-apoptotic protein of the Bcl-2 family. This interaction is necessary to induce mitochondrial fission, ROS production, and apoptosis. To better understand the interactions between the proteins involved in mitochondrial homeostasis and the apoptotic process, we focused on the role of two known players in mitophagy, the proteins PINK1 and BNIP3, during mitochondrial apoptosis induced by Rbf1 and Debcl in a proliferating Drosophila larval tissue. We show that Rbf1-or Debcl-induced apoptosis is accompanied by mitophagy. Interestingly, PINK1 and BNIP3 have distinct effects in regulating cell death. PINK1 promotes rbf1-or debcl-induced apoptosis, whereas BNIP3 protects against Rbf1-induced apoptosis but reduces Debclinduced tissue loss without inhibiting Debcl-induced cell death. Furthermore, our results indicate that BNIP3 is required to induce basal mitophagy while PINK1 is responsible for mitophagy induced by rbf1 overexpression. These results highlight the critical role of mitophagy regulators in controlling homeostasis and cell fate

Mutating RBF Can Enhance Its Pro-Apoptotic Activity and Uncovers a New Role in Tissue Homeostasis

International audienceThe tumor suppressor retinoblastoma protein (pRb) is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD) is found in the Drosophila homologue of pRb (RBF) at a position similar to the site generating the p76Rb form in mammals. Thus, we generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBF D253A , conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. We show that this overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg) ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo

The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the b-TrCP homologue slimb

International audienceThe ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. We then demonstrated that Uba1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of b-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family

Lessons on SpA pathogenesis from animal models

International audienceUnderstanding the complex mechanisms underlying a disorder such as spondyloarthritis (SpA) may benefit from studying animal models. Several suitable models have been developed, in particular to investigate the role of genetic factors predisposing to SpA, including HLA-B27, ERAP1, and genes related to the interleukin (IL)-23/IL-17 axis. One of the best examples of such research is the HLA-B27 transgenic rat model that fostered the emergence of original theories regarding HLA-B27 pathogenicity, including dysregulation of innate immunity, contribution of the adaptive immune system to chronic inflammation, and influence of the microbiota on disease development. Very recently, a new model of HLA-B27 transgenic Drosophila helped to expand further some of those theories in an unexpected direction involving the TGFβ/BMP family of mediators. On the other hand, several spontaneous, inducible, and/or genetically modified mouse models-including SKG mouse, TNFΔARE mouse and IL-23-inducible mouse model of SpA-have highlighted the importance of TNFα and IL-23/IL-17 axis in the development of SpA manifestations. Altogether, those animal models afford not only to study disease mechanism but also to investigate putative therapeutic targets