Nele Vanlangenakker

Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features

Necroptosis, necrosis and secondary necrosis following apoptosis represent different modes of cell death that eventually result in similar cellular morphology including rounding of the cell, cytoplasmic swelling, rupture of the plasma membrane and spilling of the intracellular content. Subcellular events during tumor necrosis factor (TNF)-induced necroptosis, H2O2-induced necrosis and anti-Fas-induced secondary necrosis were studied using high-resolution time-lapse microscopy. The cellular disintegration phase of the three types of necrosis is characterized by an identical sequence of subcellular events, including oxidative burst, mitochondrial membrane hyperpolarization, lysosomal membrane permeabilization and plasma membrane permeabilization, although with different kinetics. H2O2-induced necrosis starts immediately by lysosomal permeabilization. In contrast, during TNF-mediated necroptosis and anti-Fas-induced secondary necrosis, this is a late event preceded by a defined signaling phase. TNF-induced necroptosis depends on receptor-interacting protein-1 kinase, mitochondrial complex I and cytosolic phospholipase A2 activities, whereas H2O2-induced necrosis requires iron-dependent Fenton reactions.

Many stimuli pull the necrotic trigger, an overview

The lab of Jürg Tschopp was the first to report on the crucial role of receptor-interacting protein kinase 1 (RIPK1) in caspase-independent cell death. Because of this pioneer finding, regulated necrosis and in particular RIPK1/RIPK3 kinase-mediated necrosis, referred to as necroptosis, has become an intensively studied form of regulated cell death. Although necrosis was identified initially as a backup cell death program when apoptosis is blocked, it is now recognized as a cellular defense mechanism against viral infections and as being critically involved in ischemia-reperfusion damage. The observation that RIPK3 ablation rescues embryonic lethality in mice deficient in caspase-8 or Fas-associated-protein-via-a-death-domain demonstrates the crucial role of this apoptotic platform in the negative control of necroptosis during development. Here, we review and discuss commonalities and differences of the increasing list of inducers of regulated necrosis ranging from cytokines, pathogen-associated molecular patterns, to several forms of physicochemical cellular stress. Since the discovery of the crucial role of RIPK1 and RIPK3 in necroptosis, these kinases have become potential therapeutic targets. The availability of new pharmacological inhibitors and transgenic models will allow us to further document the important role of this form of cell death in degenerative, inflammatory and infectious diseases.

MOLECULAR MECHANISMS AND PATHOPHYSIOLOGY OF NECROTIC CELL DEATH

Necrotic cell death has long been considered an accidental and uncontrolled mode of cell death. But recently it has become clear that necrosis is a molecularly regulated event that is associated with pathologies such as ischemia-reperfusion (IR) injury, neurodegeneration and pathogen infection. The serine/threonine kinase receptor-interacting protein 1 (RIP1) plays a crucial role during the initiation of necrosis induced by ligand-receptor interactions. On the other hand, ATP depletion is an initiating factor in ischemia-induced necrotic cell death. Common players in necrotic cell death irrespective of the stimulus are calcium and reactive oxygen species (ROS). During necrosis, elevated cytosolic calcium levels typically lead to mitochondrial calcium overload, bioenergetics effects, and activation of proteases and phospholipases. ROS initiates damage to lipids, proteins and DNA and consequently results in mitochondrial dysfunction, ion balance deregulation and loss of membrane integrity. Membrane destabilization during necrosis is also mediated by other factors, such as acid-sphingomyelinase (ASM), phospholipase A(2) (PLA(2)) and calpains. Furthermore, necrotic cells release immunomodulatory factors that lead to recognition and engulfment by phagocytes and the subsequent immunological response. The knowledge of the molecular mechanisms involved in necrosis has contributed to our under-standing of necrosis-associated pathologies. In this review we will focus on the intracellular and intercellular signaling events in necrosis induced by different stimuli, such as oxidative stress, cytokines and pathogen-associated molecular patterns (PAMPs), which can be linked to several pathologies such as stroke, cardiac failure, neurodegenerative diseases, and infections.

cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production

Three members of the IAP family (X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins-1/-2 (cIAP1 and cIAP2)) are potent suppressors of apoptosis. Recent studies have shown that cIAP1 and cIAP2, unlike XIAP, are not direct caspase inhibitors, but block apoptosis by functioning as E3 ligases for effector caspases and receptor-interacting protein 1 (RIP1). cIAP-mediated polyubiquitination of RIP1 allows it to bind to the pro-survival kinase transforming growth factor-β-activated kinase 1 (TAK1) which prevents it from activating caspase-8-dependent death, a process reverted by the de-ubiquitinase CYLD. RIP1 is also a regulator of necrosis, a caspase-independent type of cell death. Here, we show that cells depleted of the IAPs by treatment with the IAP antagonist BV6 are greatly sensitized to tumor necrosis factor (TNF)-induced necrosis, but not to necrotic death induced by anti-Fas, poly(I:C) oxidative stress. Specific targeting of the IAPs by RNAi revealed that repression of cIAP1 is responsible for the sensitization. Similarly, lowering TAK1 levels or inhibiting its kinase activity sensitized cells to TNF-induced necrosis, whereas repressing CYLD had the opposite effect. We show that this sensitization to death is accompanied by enhanced RIP1 kinase activity, increased recruitment of RIP1 to Fas-associated via death domain and RIP3 (which allows necrosome formation), and elevated RIP1 kinase-dependent accumulation of reactive oxygen species (ROS). In conclusion, our data indicate that cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent ROS production.

TNF-induced necroptosis in L929 cells is tightly regulated by multiple TNFR1 complex I and II members

TNF receptor 1 signaling induces NF-κB activation and necroptosis in L929 cells. We previously reported that cellular inhibitor of apoptosis protein-mediated receptor-interacting protein 1 (RIP1) ubiquitination acts as a cytoprotective mechanism, whereas knockdown of cylindromatosis, a RIP1-deubiquitinating enzyme, protects against tumor necrosis factor (TNF)-induced necroptosis. We report here that RIP1 is a crucial mediator of canonical NF-κB activation in L929 cells, therefore questioning the relative cytoprotective contribution of RIP1 ubiquitination versus canonical NF-κB activation. We found that attenuated NF-κB activation has no impact on TNF-induced necroptosis. However, we identified A20 and linear ubiquitin chain assembly complex as negative regulators of necroptosis. Unexpectedly, and in contrast to RIP3, we also found that knockdown of RIP1 did not block TNF cytotoxicity. Cell death typing revealed that RIP1-depleted cells switch from necroptotic to apoptotic death, indicating that RIP1 can also suppress apoptosis in L929 cells. Inversely, we observed that Fas-associated protein via a death domain, cellular FLICE inhibitory protein and caspase-8, which are all involved in the initiation of apoptosis, counteract necroptosis induction. Finally, we also report RIP1-independent but RIP3-mediated necroptosis in the context of TNF signaling in particular conditions.

Programmed necrosis from molecules to health and disease.

During the past decade, cell death researchers have witnessed a gradual but deep conceptual revolution: it has been unequivocally shown that necrosis, which for long had been considered as a purely accidental cell death mode, can also be induced by finely regulated signal transduction pathways. In particular, when caspases are inhibited by pharmacological or genetic means, the ligation of death receptors such as the tumor necrosis factor receptor 1 (TNFR1) can lead to the assembly of a supramolecular complex containing the receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) that delivers a pronecrotic signal. Such complex has recently been dubbed necrosome and mediates the execution of a specific instance of regulated necrosis, necroptosis. Soon, it turned out that programmed necrosis occurs in nonmammalian model organisms and that it is implicated in human diseases including ischemia and viral infection. In this review, we first describe the historical evolution of the concept of programmed necrosis and the molecular mechanisms that underlie necroptosis initiation and execution. We then provide evidence suggesting that necroptosis represents an ancient and evolutionarily conserved cell death modality that may be targeted for drug development.

Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis

In human cells, the RIPK1–RIPK3–MLKL–PGAM5–Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types.

RIP1 is required for IAP inhibitor-mediated sensitization of childhood acute leukemia cells to chemotherapy-induced apoptosis

Evasion of apoptosis may contribute to poor treatment response in pediatric acute lymphoblastic leukemia (ALL), calling for novel treatment strategies. Here, we report that inhibitors of apoptosis (IAPs) at subtoxic concentrations cooperate with various anticancer drugs (that is, AraC, Gemcitabine, Cyclophosphamide, Doxorubicin, Etoposide, Vincristine and Taxol) to induce apoptosis in ALL cells in a synergistic manner as calculated by combination index and to reduce long-term clonogenic survival. Importantly, we identify RIP1 as a critical regulator of this synergism of IAP inhibitors and AraC that mediates the formation of a RIP1/FADD/caspase-8 complex via an autocrine/paracrine loop of tumor necrosis factor-α (TNFα). Knockdown of RIP1 abolishes formation of this complex and subsequent activation of caspase-8 and -3, mitochondrial perturbations and apoptosis. Similarly, inhibition of RIP1 kinase activity by Necrostatin-1 or blockage of TNFα by Enbrel inhibits IAP inhibitor- and AraC-triggered interaction of RIP1, FADD and caspase-8 and apoptosis. In contrast to malignant cells, IAP inhibitors and AraC at equimolar concentrations are non-toxic to normal peripheral blood lymphocytes or mesenchymal stromal cells. Thus, our findings provide first evidence that IAP inhibitors present a promising strategy to prime childhood ALL cells for chemotherapy-induced apoptosis in a RIP1-dependent manner. These data have important implications for developing apoptosis-targeted therapies in childhood leukemia.

Regulated IRE1-dependent mRNA decay sets the threshold for dendritic cell survival

The IRE1–XBP1 signalling pathway is part of a cellular programme that protects against endoplasmic reticulum (ER) stress, but also controls development and survival of immune cells. Loss of XBP1 in splenic type 1 conventional dendritic cells (cDC1s) results in functional alterations without affecting cell survival. However, in mucosal cDC1s, loss of XBP1 impaired survival in a tissue-specific manner—while lung cDC1s die, intestinal cDC1s survive. This was not caused by differential activation of ER stress cell-death regulators CHOP or JNK. Rather, survival of intestinal cDC1s was associated with their ability to shut down protein synthesis through a protective integrated stress response and their marked increase in regulated IRE1-dependent messenger RNA decay. Furthermore, loss of IRE1 endonuclease on top of XBP1 led to cDC1 loss in the intestine. Thus, mucosal DCs differentially mount ATF4- and IRE1-dependent adaptive mechanisms to survive in the face of ER stress.

Opposing regulation and roles for PHD3 in lung dendritic cells and alveolar macrophages

The prolyl hydroxylase domain‐containing enzymes (PHDs) are important metabolic sensors of the cell and its environment, which might be employed to alert cells of the immune system. These enzymes regulate the expression of the hypoxia inducible factor (HIF) isoforms and NF‐κB, crucial transcription factors controlling cellular metabolism and inflammation. PHD/HIF signaling is activated in the allergic lung and is proposed as a potential druggable pathway. Here, we investigated the regulation and role of the PHD isoforms in CD11c‐expressing dendritic cells (DCs) and macrophages (Mϕ), sensors of the environment and crucial antigen‐presenting cells in the pathogenesis of asthma. Although PHD2 and PHD3 were expressed in baseline, stimulation with house dust mite (HDM) allergen, hypoxia, and TLR4 ligands induced the expression of PHD3 in DCs. Conditional deletion or overexpression of PHD3 in CD11chi cells had minor effects on DCs and alveolar Mϕ biology in steady state. However, when put into competition with wild‐type counterparts in mixed chimeric mice, alveolar Mϕ uniquely required PHD3 for optimal reconstitution of the alveolar space. Using genetic and chemical approaches, we were unable to find a clear role for PHD3 or the other PHD isoforms in DCs in asthma development. These data show cell‐specific competitive advantage of PHD3 expression in antigen‐presenting cells, but question whether therapeutic manipulation of PHDs in DCs would offer therapeutic benefit in asthma.