Distinct mechanisms of apoptosis-induced compensatory proliferation in proliferating and differentiating tissues in the Drosophila eye

In multicellular organisms, apoptotic cells induce compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. Here we show that a second mechanism of apoptosis-induced compensatory proliferation exists. This mechanism is dependent on effector caspases which trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Furthermore, whereas Dpp and Wg signaling is preferentially employed in apoptotic proliferating tissues, Hh signaling is activated in differentiating eye tissues. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell-cycle reentry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent nonapoptotic function.     

The role of apoptosis-induced proliferation for regeneration and cancer

Genes dedicated to killing cells must have evolved because of their positive effects on organismal survival. Positive functions of apoptotic genes have been well established in a large number of biological contexts, including their role in eliminating damaged and potentially cancerous cells. More recently, evidence has suggested that proapoptotic proteins-mostly caspases-can induce proliferation of neighboring surviving cells to replace dying cells. This process, that we will refer to as "apoptosis-induced proliferation," may be critical for stem cell activity and tissue regeneration. Depending on the caspases involved, at least two distinct types of apoptosis-induced proliferation can be distinguished. One of these types have been studied using a model in which cells have initiated cell death, but are prevented from executing it because of effector caspase inhibition, thereby generating "undead" cells that emit persistent mitogen signaling and overgrowth. Such conditions are likely to contribute to certain forms of cancer. In this review, we summarize the current knowledge of apoptosis-induced proliferation and discuss its relevance for tissue regeneration and cancer.     

Recognition-dependent signaling events in response to apoptotic targets inhibit epithelial cell viability by multiple mechanisms: implications for non-immune tissue homeostasis

Apoptosis allows for the removal of damaged, aged, and/or excess cells without harm to surrounding tissue. To accomplish this, cells undergoing apoptosis acquire new activities that enable them to modulate the fate and function of nearby cells. We have shown that receptor-mediated recognition of apoptotic versus necrotic target cells by viable kidney proximal tubular epithelial cells (PTEC) modulates the activity of several signaling pathways critically involved in regulation of proliferation and survival. Generally, apoptotic and necrotic targets have opposite effects with apoptotic targets inhibiting and necrotic targets stimulating the activity of these pathways. Here we explore the consequences of these signaling differences. We show that recognition of apoptotic targets induces a profound decrease in PTEC viability through increased responder cell death and decreased proliferation. In contrast, necrotic targets promote viability through decreased death and increased proliferation. Both target types mediate their effects through a network of Akt-dependent and -independent events. Apoptotic targets modulate Akt-dependent viability in part through a reduction in cellular β-catenin and decreased inactivation of Bad. In contrast, Akt-independent modulation of viability occurs through activation of caspase-8, suggesting that death receptor-dependent pathways are involved. Apoptotic targets also activate p38, which partially protects responders from target-induced death. The response of epithelial cells varies depending on their tissue origin. Some cell lines, like PTEC, demonstrate decreased viability, whereas others (e.g. breast-derived) show increased viability. By acting as sentinels of environmental change, apoptotic targets allow neighboring cells, especially non-migratory epithelial cells, to monitor and potentially adapt to local stresses.     

A dp53/JNK-dependant feedback amplification loop is essential for the apoptotic response to stress in Drosophila

Programmed cell death (apoptosis) is a conserved process aimed to eliminate unwanted cells. The key molecules are a group of proteases called caspases that cleave vital proteins, which leads to the death of cells. In Drosophila, the apoptotic pathway is usually represented as a cascade of events in which an initial stimulus activates one or more of the proapoptotic genes (hid, rpr, grim), which in turn activate caspases. In stress-induced apoptosis, the dp53 (Drosophila p53) gene and the Jun N-terminal kinase (JNK) pathway function upstream in the activation of the proapoptotic genes. Here we demonstrate that dp53 and JNK also function downstream of proapoptotic genes and the initiator caspase Dronc (Drosophila NEDD2-like caspase) and that they establish a feedback loop that amplifies the initial apoptotic stimulus. This loop plays a critical role in the apoptotic response because in its absence there is a dramatic decrease in the amount of cell death after a pulse of the proapoptotic proteins Hid and Rpr. Thus, our results indicate that stress-induced apoptosis in Drosophila is dependant on an amplification loop mediated by dp53 and JNK. Furthermore, they also demonstrate a mechanism of mutual activation of proapoptotic genes.     

The Role of Intercellular Communication in Cancer Progression

Although understanding of the molecular biology of cancer has advanced and medicine has an impressive arsenal of chemotherapeutic drugs, the problem of tumor resistance to individual drugs and drug combinations has not yet been resolved. Known mechanisms of cancer chemoresistance do not explain the reason for such a phenomenon as “apoptosis-induced proliferation,” where cells dying under the effect of the therapy secrete some signaling molecules into the extracellular medium to promote proliferation, survival, and acquisition of a more aggressive phenotype of neighboring cancer cells. The nature of this unexpected phenomenon is only now beginning to be partially clarified, but the nature of such signaling between apoptotic cancer cells and their neighboring cells remains largely unknown. For this reason, in this review we discuss currently known types of intercellular communication of tumor cells, give specific examples of important secreted molecules involved in signaling between cancer cells, and describe possible cell interactions contributing to the progress of cancer outgrowth.     

ROS-Induced JNK and p38 Signaling Is Required for Unpaired Cytokine Activation during Drosophila Regeneration

Upon apoptotic stimuli, epithelial cells compensate the gaps left by dead cells by activating proliferation. This has led to the proposal that dying cells signal to surrounding living cells to maintain homeostasis. Although the nature of these signals is not clear, reactive oxygen species (ROS) could act as a signaling mechanism as they can trigger pro-inflammatory responses to protect epithelia from environmental insults. Whether ROS emerge from dead cells and what is the genetic response triggered by ROS is pivotal to understand regeneration of Drosophila imaginal discs. We genetically induced cell death in wing imaginal discs, monitored the production of ROS and analyzed the signals required for repair. We found that cell death generates a burst of ROS that propagate to the nearby surviving cells. Propagated ROS activate p38 and induce tolerable levels of JNK. The activation of JNK and p38 results in the expression of the cytokines Unpaired (Upd), which triggers the JAK/STAT signaling pathway required for regeneration. Our findings demonstrate that this ROS/JNK/p38/Upd stress responsive module restores tissue homeostasis. This module is not only activated after cell death induction but also after physical damage and reveals one of the earliest responses for imaginal disc regeneration.     

Apoptosis-induced compensatory proliferation. The Cell is dead. Long live the Cell!

In multi-cellular organisms, activation of apoptosis can trigger compensatory proliferation in surrounding cells to maintain tissue homeostasis. Genetic studies in Drosophila have indicated that distinct mechanisms of compensatory proliferation are employed in apoptotic tissues of different developmental states. In proliferating eye and wing tissues, the initiator caspase Dronc coordinates cell death and compensatory proliferation through the Jun N-terminal kinase and p53. The mitogens Decapentaplegic and Wingless are induced in this process. By contrast, in differentiating eye tissues, the effector caspases DrICE and Dcp-1 activate the Hedgehog signaling pathway to induce compensatory proliferation. In this review, we summarize these findings and discuss how activation of apoptosis is linked to the process of compensatory proliferation. The developmental and pathological relevance of compensatory proliferation is also discussed.     

Puckered, a Drosophila MAPK phosphatase, ensures cell viability by antagonizing JNK-induced apoptosis

MAPK phosphatases (MKPs) are important negative regulators of MAPKs in vivo, but ascertaining the role of specific MKPs is hindered by functional redundancy in vertebrates. Thus, we characterized MKP function by examining the function of Puckered (Puc), the sole Drosophila Jun N-terminal kinase (JNK)-specific MKP, during embryonic and imaginal disc development. We demonstrate that Puc is a key anti-apoptotic factor that prevents apoptosis in epithelial cells by restraining basal JNK signaling. Furthermore, we demonstrate that JNK signaling plays an important role in gamma-irradiation-induced apoptosis, and examine how JNK signaling fits into the circuitry regulating this process. Radiation upregulates both JNK activity and puc expression in a p53-dependent manner, and apoptosis induced by loss of Puc can be suppressed by p53 inactivation. JNK signaling acts upstream of both Reaper and effector caspases. Finally, we demonstrate that JNK signaling directs normal developmentally regulated apoptotic events. However, if cell death is prevented, JNK activation can trigger tissue overgrowth. Thus, MKPs are key regulators of the delicate balance between proliferation, differentiation and apoptosis during development.     

Metabolic regulation of Drosophila apoptosis through inhibitory phosphorylation of Dronc

Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome‐wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc‐mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII‐mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death‐inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non‐phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.     

Injury-induced activation of the MAPK/CREB pathway triggers apoptosis-induced compensatory proliferation in hydra head regeneration

After bisection, Hydra polyps regenerate their head from the lower half thanks to a head-organizer activity that is rapidly established at the tip. Head regeneration is also highly plastic as both the wild-type and the epithelial Hydra (that lack the interstitial cell lineage) can regenerate their head. In the wild-type context, we previously showed that after mid-gastric bisection, a large subset of the interstitial cells undergo apoptosis, inducing compensatory proliferation of the surrounding progenitors. This asymmetric process is necessary and sufficient to launch head regeneration. The apoptotic cells transiently release Wnt3, which promotes the formation of a proliferative zone by activating the beta-catenin pathway in the adjacent cycling cells. However the injury-induced signaling that triggers apoptosis is unknown. We previously reported an asymmetric immediate activation of the mitogen-activated protein kinase/ribosomal S6 kinase/cAMP response element binding protein (MAPK/RSK/CREB) pathway in head-regenerating tips after mid-gastric bisection. We show here that pharmacological inhibition of the MAPK/ERK pathway or RNAi knockdown of the RSK, CREB, CREB binding protein (CBP) genes prevents apoptosis, compensatory proliferation and blocks head regeneration. As the activation of the MAPK pathway upon injury plays an essential role in regenerating bilaterian species, these results suggest that the MAPK-dependent activation of apoptosis-induced compensatory proliferation represents an evolutionary-conserved mechanism to launch a regenerative process.     

Mitogenic signaling from apoptotic cells in Drosophila

Apoptotic cells of Drosophila not only activate caspases, but also are able to secrete developmental signals like Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) before dying. Since Dpp and Wg are secreted in growing tissues and behave as growth factors, it was proposed that they play a role in compensatory proliferation, the process by which a growing blastema can restore normal size after massive apoptosis. We discuss recent results showing that there is normal compensatory proliferation in the absence of Dpp/Wg signaling, thus indicating it has no significant role in the process. Furthermore, we argue that Dpp/Wg signaling is not a resident feature of apoptotic cells, but a side effect of the necessary activation of the JNK pathway. Nevertheless, the ectopic JNK/Dpp/Wg signaling may have an important role in tissue regeneration. Recent work in other organisms suggests that paracrine signaling from apoptotic cells may be of general significance in wound healing and tissue regeneration in metazoans.     

Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis.

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.     

Dronc caspase exerts a non-apoptotic function to restrain phospho-Numb-induced ectopic neuroblast formation in Drosophila

Drosophila neuroblasts have served as a model to understand how the balance of stem cell self-renewal versus differentiation is achieved. Drosophila Numb protein regulates this process through its preferential segregation into the differentiating daughter cell. How Numb restricts the proliferation and self-renewal potentials of the recipient cell remains enigmatic. Here, we show that phosphorylation at conserved sites regulates the tumor suppressor activity of Numb. Enforced expression of a phospho-mimetic form of Numb (Numb-TS4D) or genetic manipulation that boosts phospho-Numb levels, attenuates endogenous Numb activity and causes ectopic neuroblast formation (ENF). This effect on neuroblast homeostasis occurs only in the type II neuroblast lineage. We identify Dronc caspase as a novel binding partner of Numb, and demonstrate that overexpression of Dronc suppresses the effects of Numb-TS4D in a non-apoptotic and possibly non-catalytic manner. Reduction of Dronc activity facilitates ENF induced by phospho-Numb. Our findings uncover a molecular mechanism that regulates Numb activity and suggest a novel role for Dronc caspase in regulating neural stem cell homeostasis.     

Tissue transglutaminase triggers oligomerization and activation of dual leucine zipper-bearing kinase in calphostin C-treated cells to facilitate apoptosis

Although tissue transglutaminase (tTG) has been recognized as a mediator of apoptosis in various experimental models, little is currently known about the molecular mechanisms by which this protein modulates cell death. Recent work from our laboratory has shown that activation of tTG in cells exposed to the apoptotic inducer calphostin C triggers the crosslinking of dual leucine zipper-bearing kinase (DLK), a proapoptotic kinase acting as an essential component of the c-Jun amino-terminal kinase (JNK) signaling pathway. As a consequence of this observation, we have undertaken experiments to investigate the functional relevance of DLK oligomerization in tTG-mediated apoptosis. Our results indicate that, in cells undergoing calphostin C-induced apoptosis, tTG-dependent DLK oligomerization occurs early in the apoptotic response. Both immunocomplex kinase assays and immunoblotting with phosphospecific antibodies revealed that oligomer formation by tTG-mediated crosslinking reactions significantly enhanced the kinase activity of DLK and its ability to activate the JNK pathway. Moreover, functional studies demonstrate that tTG-mediated oligomerization of wild-type DLK sensitizes cells to calphostin C-induced apoptosis, while crosslinking of a kinase-inactive variant of DLK does not. Collectively, these data strongly suggest that tTG facilitates apoptosis, at least partly, by oligomerization and activation of the proapoptotic kinase DLK.     

The impact of trisomy 21 on early human hematopoiesis

Mitogen-activated protein kinases (MAPKs) are components of signaling cascades regulated by environmental stimuli. In addition to participating in the stress response, the MAPKs c-Jun N-terminal Kinases JNK1 and JNK2 regulate the proliferation of normal and neoplastic cells. JNKs contribute to these processes largely by phosphorylating c-Jun and thus contributing to the activation of the AP-1 complex. We here report that JNKs control entry into mitosis. We have observed that JNK activity and phosphorylation of c-Jun become elevated during the G2/M transition of the cell cycle in immortalized fibroblasts and ovarian granulosa cells. Pharmacological inhibition of JNK causes a profound cell cycle arrest at the G2/M transition in both cell types. This effect is specific as it occurs with two distinct small molecule compounds. Inactivation of JNK prior to mitosis prevents expression of Aurora B and phosphorylation of Histone-H3 at Ser 10. Silencing of JNK1 and 2 causes a similar effect, whereas overexpression of JNK1 and 2 causes the opposite effect. Inhibition of JNK delays activation of cdc-2 and prevents downregulation of Cyclin B1. We conclude that JNK signaling promotes entry into mitosis by promoting expression of Aurora B and thereby phosphorylation of Histone-H3.     

RNF13, a RING Finger Protein,Mediates Endoplasmic Reticulum Stress-induced Apoptosis through the Inositol-requiring Enzyme (IRE1α)/c-Jun NH2-terminal Kinase Pathway

Disturbance of homeostasis at endoplasmic reticulum (ER) causes stress to cells that in turn triggers an adaptive signaling pathway termed unfolded protein response for the purpose of restoring normal cellular physiology or initiating signaling events leading to apoptosis. Identification of those genes that are involved in the unfolded protein response-mediated apoptotic signaling pathway would be valuable toward elucidating the molecular mechanism underlying the relationship between ER stress and apoptosis. We initiated a genetic screen by using the retroviral insertion mutation system to search for genes whose inactivation confers resistance to apoptosis induction by staurosporine. Using this approach, RING finger protein 13 (RNF13) was identified. Interestingly, RNF13 is highly enriched in ER. RNF13 knockdown cells are resistant to apoptosis and JNK activation triggered by ER stress. Conversely, overexpression of RNF13 induces JNK activation and caspase-dependent apoptosis. The RING and transmembrane domains of RNF13 are both required for its effects on JNK activation and apoptosis. Moreover, systematic analysis of the involvement of individual signaling components in the ER stress pathway using knockdown approach reveals that RNF13 acts upstream of the IRE1α-TRAF2 signaling axis for JNK activation and apoptosis. Finally, RNF13 co-immunoprecipitates with IRE1α, and the intact RING domain is also required for mediating its interaction. Together, our data support a model that RNF13 is a critical mediator for facilitating ER stress-induced apoptosis through the activation of the IRE1α-TRAF2-JNK signaling pathway.