Search

Necroptosis is a pro-inflammatory cell death program executed by the terminal effector, mixed lineage kinase domain-like (MLKL). Previous studies suggested a role for the necroptotic machinery in platelets, where loss of MLKL or its upstream regulator, RIPK3 kinase, impacted thrombosis and haemostasis. However, it remains unknown whether necroptosis operates within megakaryocytes, the progenitors of platelets, and whether necroptotic cell death might contribute to or diminish platelet production. Here, we demonstrate that megakaryocytes possess a functional necroptosis signalling cascade. Necroptosis activation leads to phosphorylation of MLKL, loss of viability and cell swelling. Analyses at steady state and post antibody-mediated thrombocytopenia revealed that platelet production was normal in the absence of MLKL, however, platelet activation and haemostasis were impaired with prolonged tail re-bleeding times. We conclude that MLKL plays a role in regulating platelet function and haemostasis and that necroptosis signalling in megakaryocytes is dispensable for platelet production.

Cell death is a vital process that occurs in billions of cells in the human body every day. This process helps maintain tissue homeostasis, supports recovery from acute injury, deals with infection and regulates immunity. Cell death can also provoke inflammatory responses, and lytic forms of cell death can incite inflammation. Loss of cell membrane integrity leads to the uncontrolled release of damage-associated molecular patterns (DAMPs), which are normally sequestered inside cells. Such DAMPs increase local inflammation and promote the production of cytokines and chemokines that modulate the innate immune response. Cell death can be both a consequence and a cause of inflammation, which can be difficult to distinguish in chronic diseases. Despite this caveat, excessive or poorly regulated cell death is increasingly recognized as a contributor to chronic inflammation in rheumatic disease and other inflammatory conditions. Drugs that inhibit cell death could, therefore, be used therapeutically for the treatment of these diseases, and programmes to develop such inhibitors are already underway. In this Review, we outline pathways for the major cell death programmes (apoptosis, necroptosis, pyroptosis and NETosis) and their potential roles in chronic inflammation. We also discuss current and developing therapies that target the cell death machinery.

Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. Precisely how phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. Here, we reveal the existence of a basal monomeric MLKL conformer present in human cells prior to exposure to a necroptotic stimulus. Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer. In contrast to mouse MLKL, substitution of RIPK3 substrate sites in the human MLKL pseudokinase domain completely abrogated necroptotic signaling. Therefore, while the pseudokinase domains of mouse and human MLKL function as molecular switches to control MLKL activation, the underlying mechanism differs between species.

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death 2-8 . In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype 9-11 . Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1 -/- (also known as Rbck1 -/- ) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3 -/- Casp8 -/- Hoil-1 -/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Inhibitor of apoptosis proteins (IAPs) are critical regulators of cell death and survival pathways. Mice lacking cIAP1 and either cIAP2 or XIAP die in utero, and myeloid lineage-specific deletion of all IAPs causes sterile inflammation, but their role in the skin is unknown. We generated epidermal-specific IAP-deficient mice and found that combined genetic deletion of cIAP1 (epidermal knockout [EKO]) in keratinocytes and ubiquitous cIAP2 deletion (cIap1 EKO/EKO .cIap2 -/- ) caused profound skin inflammation and keratinocyte death, lethal by postpartum day 10. To investigate their role in skin homeostasis, we injected an IAP antagonist compound subcutaneously into wild-type and knockout mice. This induced a toxic epidermal necrolysis-like local inflammation, which mirrored the phenotype seen in cIap1 EKO/EKO .cIap2 -/- mice. Loss of one Ripk1 allele limited lesion formation and significantly extended the lifespan of cIap1 EKO/EKO .cIap2 -/- mice. cIAP activities are important for recruitment of LUBAC to signaling complexes, and loss of LUBAC component SHARPIN, induces dermatitis in mice. Consistent with this relationship between cIAPs and LUBAC, Ripk1 heterozygosity also protected against development of dermatitis in Sharpin-deficient mice. This work therefore refines our molecular understanding of inflammatory signaling in the skin and defines potential targets for treating skin inflammation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Malignant cells must circumvent endogenous cell death pathways to survive and develop into cancers. Acquired cell death resistance also sets up malignant cells to survive anticancer therapies. Acute Myeloid Leukemia (AML) is an aggressive blood cancer characterized by high relapse rate and resistance to cytotoxic therapies. Recent collaborative profiling projects have led to a greater understanding of the 'fearful symmetry' of the genomic landscape of AML, and point to the development of novel potential therapies that can overcome factors linked to chemoresistance. We review here the most recent research in the genetics of AML and how these discoveries have led, or might lead, to therapies that specifically activate cell death pathways to substantially challenge this 'fearful' disease., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

IAPs were named as inhibitors of apoptosis, programmed cell death, but it has become apparent that they are regulators of other types of cell death too. Because they inhibit cell death in cancer cells there has been an intense interest in developing inhibitors of these proteins to induce or sensitise cancer cells to death. In this article, we will discuss the involvement of IAPs in the apoptosis, necroptosis and pyroptosis programmed cell death paradigms. All these types of cell death are intimately involved with causing or repressing inflammation and it should perhaps therefore come as no surprise that IAPs are also involved in regulating inflammation directly. To come full circle, the IAP antagonist drugs that were developed to sensitise cancer cells to apoptosis have led to some of these insights.

Cell death, one of the most fundamental biological processes, has not made it into the public consciousness in the same way that genetic inheritance, cell division, or DNA replication has. Everyone knows they get their genes from their parents, but few would be aware that even before they were born a lot of essential cell death has shaped their development. The greater population, for the most part, is blissfully unaware that every day millions of their own cells die in a programmed way and that this is essential for normal human physiology-their well-being, in fact. Nowhere is the burial liturgy, "In the midst of life we are in death," more apt. Despite this public underappreciation, cell death research is a major industry. A search in PubMed for "apoptosis," a special form of cell death that is caused by caspases, returns approximately 280,000 hits. The intense research interest arises from the realization that abnormal cell death responses play an important role in two of the biggest killers in the western world: cancer and cardio/cerebrovascular disease. Furthermore, the manner in which cells die can also influence the development of autoimmune and autoinflammatory diseases. It is therefore of paramount importance to ensure that experiments accurately quantitate and correctly identify cell death in all its guises. That is the goal of this protocol collection., (© 2016 Cold Spring Harbor Laboratory Press.)

The kinases RIPK1 and RIPK3 and the pseudo-kinase MLKL have been identified as key regulators of the necroptotic cell death pathway, although a role for MLKL within the whole animal has not yet been established. Here, we have shown that MLKL deficiency rescued the embryonic lethality caused by loss of Caspase-8 or FADD. Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice were viable and fertile but rapidly developed severe lymphadenopathy, systemic autoimmune disease, and thrombocytopenia. These morbidities occurred more rapidly and with increased severity in Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice compared to Casp8(-/-)Ripk3(-/-) or Fadd(-/-)Ripk3(-/-) mice, respectively. These results demonstrate that MLKL is an essential effector of aberrant necroptosis in embryos caused by loss of Caspase-8 or FADD. Furthermore, they suggest that RIPK3 and/or MLKL may exert functions independently of necroptosis. It appears that non-necroptotic functions of RIPK3 contribute to the lymphadenopathy, autoimmunity, and excess cytokine production that occur when FADD or Caspase-8-mediated apoptosis is abrogated., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Growth and survival of hematopoietic cells is regulated by growth factors and cytokines, such as interleukin 3 (IL-3). When cytokine is removed, cells dependent on IL-3 kill themselves by a mechanism that is inhibited by overexpression of Bcl-2 and is likely to be mediated by proapoptotic Bcl-2 family members. Bad and Bim are 2 such BH3-only Bcl-2 family members that have been implicated as key initiators in apoptosis following growth factor withdrawal, particularly in IL-3-dependent cells. To test the role of Bad, Bim, and other proapoptotic Bcl-2 family members in IL-3 withdrawal-induced apoptosis, we generated IL-3-dependent cell lines from mice lacking the genes for Bad, Bim, Puma, both Bad and Bim, and both Bax and Bak. Surprisingly, Bad was not required for cell death following IL-3 withdrawal, suggesting changes to phosphorylation of Bad play only a minor role in apoptosis in this system. Deletion of Bim also had no effect, but cells lacking Puma survived and formed colonies when IL-3 was restored. Inhibition of the PI3 kinase pathway promoted apoptosis in the presence or absence of IL-3 and did not require Bad, Bim, or Puma, suggesting IL-3 receptor survival signals and PI3 kinase survival signals are independent.

Cellular inhibitor of apoptosis proteins (cIAPs) are RING‐containing E3 ubiquitin ligases that ubiquitylate receptor‐interacting protein kinase 1 (RIPK1) to regulate TNF signalling. Here, we established mice simultaneously expressing enzymatically inactive cIAP1/2 variants, bearing mutations in the RING domains of cIAP1/2 (cIAP1/2 mutant RING, cIAP1/2MutR). cIap1/2MutR/MutR mice died during embryonic development due to RIPK1‐mediated apoptosis. While expression of kinase‐inactive RIPK1D138N rescued embryonic development, Ripk1D138N/D138N/cIap1/2MutR/MutR mice developed systemic inflammation and died postweaning. Cells expressing cIAP1/2MutR and RIPK1D138N were still susceptible to TNF‐induced apoptosis and necroptosis, implying additional kinase‐independent RIPK1 activities in regulating TNF signalling. Although further ablation of Ripk3 did not lead to any phenotypic improvement, Tnfr1 gene knock‐out prevented early onset of systemic inflammation and premature mortality, indicating that cIAPs control TNFR1‐mediated toxicity independent of RIPK1 and RIPK3. Beyond providing novel molecular insights into TNF‐signalling, the mouse model established in this study can serve as a useful tool to further evaluate ongoing therapeutic protocols using inhibitors of TNF, cIAPs and RIPK1. Synopsis: cIAPs control RIPK1‐dependent and ‐independent TNF toxicity in mouse. This study finds ubiquitylation and inhibition of RIPK1 by cIAPs to be required for mouse embryonic development, while RIPK1‐independent cIAP inhibition of TNF‐induced cell death regulates tissue inflammation in adult mice. cIAP1/2 E3 ubiquitin ligase activity is required for mouse embryonic development.Catalytic activity of cIAPs regulates RIPK1 kinase activity to promote embryonic survival.TNF induces apoptosis and necroptosis in cells lacking the cIAPs‐RIPK1 regulatory axis.cIAPs control non‐overlapping signalling processes downstream of RIPK1 and TNFR1. [ABSTRACT FROM AUTHOR]

Genetic lesions in X‐linked inhibitor of apoptosis (XIAP) pre‐dispose humans to cell death–associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency–associated inflammatory bowel disease display increased inflammatory IL‐1β maturation as well as cell death–associated caspase‐8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase‐8‐driven cell death and bioactive IL‐1β release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase‐8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase‐1, ‐3, ‐7, ‐11 and BID), while caspase‐8 can still cause cell death in the absence of both GSDMD and GSDME when caspase‐3 and caspase‐7 are present. Neither caspase‐3 and caspase‐7‐mediated activation of the pannexin‐1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase‐1 and IL‐1β maturation downstream of XIAP inhibition and caspase‐8 activation, even though the pannexin‐1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co‐opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency. Synopsis: XIAP deficiency causes hemophagocytic lymphohistiocytosis and Crohn's disease. Analysis of XIAP‐deficient patients and cellular studies show that apoptotic caspases and GSDMD act redundantly downstream of caspase‐8 to cause excess cell death and IL‐1β release. XIAP deficiency increases cleaved caspase‐8 and GSDMD in mucosal tissue and cellsCrosstalk in apoptosis and pyroptosis sensitises cells to caspase‐8‐driven death and IL‐1β activation upon XIAP inhibitionPannexin‐1 and GSDMD are dispensable for NLRP3 inflammasome formation downstream of caspase‐8Pannexin‐1 cleavage by caspase‐3 and caspase‐7 is required for efficient NLRP3 activation following intrinsic apoptosis [ABSTRACT FROM AUTHOR]

This suggests that while impairment of necroptosis or apoptosis attenuates airway inflammation, only impairment of necroptosis attenuates airway remodeling and emphysema in COPD. Now, a new study by Lu I et al i .1 looking at the role of necroptosis in chronic obstructive pulmonary disease (COPD) adds further support to the idea that necroptotic cell death might play an important role in lung pathologies. Recent work by Lu et al. identified elevated levels of the necroptotic effectors and markers of active necroptosis signaling in COPD patient lung samples and in experimental COPD mouse models induced by exposure to cigarette smoke. Recent studies, reviewed here, using a cigarette smoke exposure model for chronic obstructive pulmonary disease (COPD) in I Ripk3 i and I Mlkl i knock-out mice, and correlation with patient samples, suggest necroptosis plays a pathophysiological role in COPD by promoting inflammation, airway remodeling and emphysema. [Extracted from the article]

Mixed lineage kinase domain‐like (MLKL) is the executioner in the caspase‐independent form of programmed cell death called necroptosis. Receptor‐interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis‐specific multi‐mono‐ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin‐insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane‐located deubiquitylating enzyme. Oligomerization‐induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome‐ and lysosome‐dependent turnover. Using a MLKL‐DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto‐activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL‐induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death. SYNOPSIS: RIPK3 phosphorylates the necroptotic effector molecule MLKL leading to its oligomerization and translocation to the plasma membrane to kill cells. MLKL becomes multi mono‐ubiquitylated in an oligomerization dependent manner. Forced de‐ubiquitylation of MLKL increases MLKL's cytotoxic potential and confers RIPK3 and necroptotic stimulus independent activation and cell death. UbiCRest analysis shows that MLKL becomes multi mono‐ubiquitylated contemporaneously with activating phosphorylation and translocation to membranes.Analysis of gain and loss of function MLKL mutants indicates that MLKL oligomerization is required for necroptosis induced ubiquitylation.MLKL‐deubiquitylating enzyme (DUB) fusions kill cells more rapidly than MLKL‐catalytically dead DUB fusions and can induce necroptosis without a necroptotic stimulus, suggesting that ubiquitylation serves as a brake on MLKL's cytotoxic potential. [ABSTRACT FROM AUTHOR]

Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption, although the precise choreography of events is incompletely understood. Here, we use single-cell imaging approaches to map the chronology of endogenous human MLKL activation during necroptosis. During the effector phase of necroptosis, we observe that phosphorylated MLKL assembles into higher order species on presumed cytoplasmic necrosomes. Subsequently, MLKL co-traffics with tight junction proteins to the cell periphery via Golgi-microtubule-actin-dependent mechanisms. MLKL and tight junction proteins then steadily co-accumulate at the plasma membrane as heterogeneous micron-sized hotspots. Our studies identify MLKL trafficking and plasma membrane accumulation as crucial necroptosis checkpoints. Furthermore, the accumulation of phosphorylated MLKL at intercellular junctions accelerates necroptosis between neighbouring cells, which may be relevant to inflammatory bowel disease and other necroptosis-mediated enteropathies. Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. Here the authors show that MLKL trafficking and plasma membrane accumulation are crucial necroptosis checkpoints, and that accumulation of phosphorylated MLKL at intercellular junctions promotes necroptosis. [ABSTRACT FROM AUTHOR]

One of the hallmarks of cancer cells is their ability to evade cell death via apoptosis. The inhibitor of apoptosis proteins (IAPs) are a family of proteins that act to promote cell survival. For this reason, upregulation of IAPs is associated with a number of cancer types as a mechanism of resistance to cell death and chemotherapy. As such, IAPs are considered a promising therapeutic target for cancer treatment, based on the role of IAPs in resistance to apoptosis, tumour progression and poor patient prognosis. The mitochondrial protein smac (second mitochondrial activator of caspases), is an endogenous inhibitor of IAPs, and several small molecule mimetics of smac (smac-mimetics) have been developed in order to antagonise IAPs in cancer cells and restore sensitivity to apoptotic stimuli. However, recent studies have revealed that smac-mimetics have broader effects than was first attributed. It is now understood that they are key regulators of innate immune signalling and have wide reaching immuno-modulatory properties. As such, they are ideal candidates for immunotherapy combinations. Pre-clinically, successful combination therapies incorporating smac-mimetics and oncolytic viruses, as with chimeric antigen receptor (CAR) T cell therapy, have been reported, and clinical trials incorporating smac-mimetics and immune checkpoint blockade are ongoing. Here, the potential of IAP antagonism to enhance immunotherapy strategies for the treatment of cancer will be discussed. [ABSTRACT FROM AUTHOR]

Inflammasomes sense cellular danger to activate the cysteine-aspartic protease caspase-1, which processes precursor interleukin-1β (IL-1β) and IL-18 into their mature bioactive fragments. In addition, activated caspase-1 or the related inflammatory caspase, caspase-11, can cleave gasdermin D to induce a lytic cell death, termed pyroptosis. The intertwining of IL-1β activation and cell death is further highlighted by research showing that the extrinsic apoptotic caspase, caspase-8, may, like caspase-1, directly process IL-1β, activate the NLRP3 inflammasome itself, or bind to inflammasome complexes to induce apoptotic cell death. Similarly, RIPK3- and MLKL-dependent necroptotic signaling can activate the NLRP3 inflammasome to drive IL-1β inflammatory responses in vivo. Here, we review the mechanisms by which cell death signaling activates inflammasomes to initiate IL-1β-driven inflammation, and highlight the clinical relevance of these findings to heritable autoinflammatory diseases. We also discuss whether the act of cell death can be separated from IL-1β secretion and evaluate studies suggesting that several cell death regulatory proteins can directly interact with, and modulate the function of, inflammasome and IL-1β containing protein complexes. [ABSTRACT FROM AUTHOR]

The pseudokinase MLKL (mixed lineage kinase domain-like), has recently emerged as a critical component of the necroptosis cell death pathway. Although it is clear that phosphorylation of the activation loop in the MLKL pseudokinase domain by the upstream protein kinase RIPK3 (receptor-interacting protein kinase-3), is crucial to trigger MLKL activation, it has remained unclear whether other phosphorylation events modulate MLKL function. By reconstituting Mlkl-/-, Ripk3-/- and Mlkl-/-Ripk3-/- cells withMLKLphospho-site mutants, we compared the function of known MLKL phosphorylation sites in regulating necroptosis with three phospho-sites that we identified by MS, Ser158, Ser228 and Ser248. Expression of a phosphomimetic S345D MLKL activation loop mutant-induced stimulus-independent cell death in all knockout cells, demonstrating that RIPK3 phosphorylation of the activation loop of MLKL is sufficient to induce cell death. Cell death was also induced by S228A, S228E and S158A MLKL mutants in the absence of death stimuli, but was most profound in Mlkl-/- Ripk3-/- double knockout fibroblasts. These data reveal a potential role for RIPK3 as a suppressor of MLKL activation and indicate that phosphorylation can fine-tune the ability of MLKL to induce necroptosis. [ABSTRACT FROM AUTHOR]

SHARPIN regulates signaling from the tumor necrosis factor (TNF) superfamily and pattern-recognition receptors. An inactivating Sharpin mutation in mice causes TNF-mediated dermatitis. Blocking cell death prevents the phenotype, implicating TNFR1-induced cell death in causing the skin disease. However, the source of TNF that drives dermatitis is unknown. Immune cells are a potent source of TNF in vivo and feature prominently in the skin pathology; however, T cells, B cells, and eosinophils are dispensable for the skin phenotype. We use targeted in vivo cell ablation, immune profiling, and extensive imaging to identify immune populations driving dermatitis. We find that systemic depletion of Langerin+ cells significantly reduces disease severity. This is enhanced in mice that lack Langerhans cells (LCs) from soon after birth. Reconstitution of LC-depleted Sharpin mutant mice with TNF-deficient LCs prevents dermatitis, implicating LCs as a potential cellular source of pathogenic TNF and highlighting a T cell-independent role in driving skin inflammation. [Display omitted] • In vivo depletion of LCs in Sharpin cpdm mice prevents TNF-mediated dermatitis • The Sharpin cpdm dermatitis phenotype is microbiota independent • Sharpin mutation on LCs is necessary for the dermatitis-causing pathological effect • Sharpin mutant LCs need to express TNF for the effect to occur TNF can be a potent cause of skin inflammation, but the origin of disease-causing TNF often remains elusive. Anderton et al. identify Langerhans cells (LCs) as the source of TNF responsible for dermatitis in Sharpin cpdm mice, highlighting a T cell-independent, microbiota-independent role for LCs in driving TNF-mediated skin inflammation. [ABSTRACT FROM AUTHOR]

Inflammation is a healthy response to infection or danger and should be rapid, specific and terminated once the threat has passed. Inflammatory diseases, where this regulation fails, cause considerable human suffering. Treatments have successfully targeted pro-inflammatory cytokines, such as tumor-necrosis factor (TNF), that directly induce genes encoding inflammatory products. Inflammatory signals, including TNF, may also directly induce caspase-independent cell death (necroptosis), which can also elicit inflammation. Necroptosis was originally defined as being dependent on the kinase RIPK1 but is now known to be dependent on RIPK3 and the pseudo-kinase MLKL. Therefore, RIPK1, RIPK3 and MLKL are potential therapeutic targets. RIPK1 and RIPK3 also directly regulate inflammatory signaling, which complicates interpretation of their function but might alter their therapeutic utility. This Review examines the role of cell death, particularly necroptosis, in inflammation, in the context of recent insights into the roles of the key necroptosis effector molecules RIPK1, RIPK3 and MLKL. [ABSTRACT FROM AUTHOR]

Progranulin (proepithelin, granulin precursor) has been recently suggested to exhibit anti-inflammatory properties by directly binding to tumour necrosis factor (TNF) receptors and thereby inhibiting TNF signalling by Tang et al. This finding was challenged by Chen et al. and no interaction between progranulin and TNF receptor (TNFR) 1 or 2 was observed. We tested the ability of recombinant progranulin from different commercial sources to inhibit TNF- or lymphotoxin-α-induced signalling through TNFR1. We observed that progranulin does not affect signalling and cell death induction downstream of TNF or lymphotoxin-α. Our results suggest that the anti-inflammatory role of progranulin is not mediated through direct inhibition of TNFR1. [ABSTRACT FROM AUTHOR]

The Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death, whose amplification has been correlated with tumor progression. Due to the presence of a RING domain, IAP proteins are classed as ubiquitin ligases and regulate cell survival by orchestrating a variety of ubiquitin modifications. Ubiquitin protein modification is fundamental in cell signaling and different ubiquitin modifications may label proteins for destruction, relocalization or provide a recruitment platform for ubiquitin binding proteins. Ubiquitin performs a myriad of different functions because it can be conjugated to a large range of target proteins through numerous different types of ubiquitin linkages. Despite the fact that ubiquitin is extremely versatile, the E3s such as the IAPs provide an important level of control due to their specificity for certain substrates. Several recent reviews have discussed the role of IAPs in regulating immune signaling so we have therefore focused our review on the interplay between IAPs and ubiquitin and discussed the importance of this relationship for the regulation of themselves, specific substrates and various cell death and survival signaling pathways. © 2012 IUBMB Life, 2012 [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a ''death ligand''-a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-κB and JNK signalling pathways. To determine the role of TGF-β-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1-/- MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-κB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-κB, protected TAK1-/- MEFs against TRAIL killing, suggesting that TAK1 activation of NF-κB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-κB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK12/2 MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1-NF-κB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance. [ABSTRACT FROM AUTHOR]

Simple Summary: The incidence of pancreatic cancer is increasing but there has been little progress in the diagnosis and survival rates of this lethal cancer for decades. Understanding the biological mechanisms and defining how pancreatic cancer develops from normal pancreas tissue, to early lesions and then tumor is vital in developing better treatment options. Unrelenting inflammation in the pancreas significantly increases the risk of developing precursor lesions which result in pancreatic cancer. This inflammatory environment can result in the "switching on" of signaling pathways in the pancreas that influences many factors such as cell survival and turnover involved in tumor initiation, development and spread. In this review we discuss in detail how components of one such pathway, the NF-κB signaling network, are involved at various stages of pancreatic cancer development and in the cellular milieu of this cancer. We also discuss how this signaling pathway could be potentially "switched off" or regulated using new inhibitors and how these agents may be coupled with conventional and/or other therapies for better patient treatment outcomes. Pancreatic Ductal Adenocarcinoma (PDAC) is one of the world's most lethal cancers. An increase in occurrence, coupled with, presently limited treatment options, necessitates the pursuit of new therapeutic approaches. Many human cancers, including PDAC are initiated by unresolved inflammation. The transcription factor NF-κB coordinates many signals that drive cellular activation and proliferation during immunity but also those involved in inflammation and autophagy which may instigate tumorigenesis. It is not surprising therefore, that activation of canonical and non-canonical NF-κB pathways is increasingly recognized as an important driver of pancreatic injury, progression to tumorigenesis and drug resistance. Paradoxically, NF-κB dysregulation has also been shown to inhibit pancreatic inflammation and pancreatic cancer, depending on the context. A pro-oncogenic or pro-suppressive role for individual components of the NF-κB pathway appears to be cell type, microenvironment and even stage dependent. This review provides an outline of NF-κB signaling, focusing on the role of the various NF-κB family members in the evolving inflammatory PDAC microenvironment. Finally, we discuss pharmacological control of NF-κB to curb inflammation, focussing on novel anti-cancer agents which reinstate the process of cancer cell death, the Smac mimetics and their pre-clinical and early clinical trials. [ABSTRACT FROM AUTHOR]

This year's Cold Spring Harbor meeting on programmed cell death (September 17-21, 2003), organised by Craig Thompson and Junying Yuan, was proof that the 'golden age' of research in this field is far from over. There was a flurry of fascinating insights into the regulation of diverse apoptotic pathways and unexpected non-apoptotic roles for some of the key apoptotic regulators and effectors. In addition to their role in cell death, components of the apoptotic molecular machinery are now known to also function in a variety of essential cellular processes, such as regulating glucose homeostasis, lipid metabolism, cell proliferation and differentiation. [ABSTRACT FROM AUTHOR]

XIAP is a mammalian inhibitor of apoptosis protein (lAP). To determine residues within the second baculoviral lAP repeat (BIR2) required for inhibition of caspase 3, we screened a library of BIR2 mutants for toss of the ability to inhibit caspase 3 toxicity in the yeast Schizosaccharomyces pombe. Four of the mutations, not predicted to affect the structure of the BIR fold, clustered together on the N-terminal region that flanks BIR2, suggesting that this is a site of interaction with caspase 3. Introduction of these mutations into full-length XIAP reduced caspase 3 inhibitory activity up to 500-fold, but did not affect its ability to inhibit caspase 9 or interact with the lAP antagonist IMABLO. Furthermore, these mutants retained full ability to inhibit apoptosis in transfected cells, demonstrating that although XIAP is able to inhibit caspase 3, this activity is dispensable for inhibition of apoptosis by XIAP in vivo. [ABSTRACT FROM AUTHOR]

Arising from N. Narayan et al. 492, 199-204 (2012)Sirtuins can promote deacetylation of a wide range of substrates in diverse cellular compartments to regulate many cellular processes; recently, Narayan et al. reported that SIRT2 was required for necroptosis on the basis of their findings that SIRT2 inhibition, knockdown or knockout prevented necroptosis. We sought to confirm and explore the role of SIRT2 in necroptosis and tested four different sources of the SIRT2 inhibitor AGK2, three independent short interfering RNAs (siRNAs) against Sirt2, and cells from two independently generated Sirt2−/− mouse strains; however, we were unable to show that inhibiting or depleting SIRT2 protected cells from necroptosis. Furthermore, Sirt2−/− mice succumbed to tumour-necrosis factor (TNF)-induced systemic inflammatory response syndrome (SIRS) more rapidly than wild-type mice, whereas Ripk3−/− mice were resistant. Our results therefore question the importance of SIRT2 in the necroptosis cell death pathway. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B virus (HBV) infection remains a global health threat and affects hundreds of millions worldwide. Small molecule compounds that mimic natural antagonists of inhibitor of apoptosis (IAP) proteins, known as Smac-mimetics (second mitochondria-derived activator of caspases-mimetics), can promote the death of HBV-replicating liver cells and promote clearance of infection in preclinical models of HBV infection. The Smac-mimetic birinapant is a substrate of the multidrug resistance protein 1 (MDR1) efflux pump, and therefore inhibitors of MDR1 increase intracellular concentration of birinapant in MDR1 expressing cells. Liver cells are known to express MDR1 and other drug pump proteins. In this study, we investigated whether combining the clinical drugs, birinapant and the MDR1 inhibitor zosuquidar, increases the efficacy of birinapant in killing HBV expressing liver cells. We showed that this combination treatment is well tolerated and, compared to birinapant single agent, was more efficient at inducing death of HBV-positive liver cells and improving HBV-DNA and HBV surface antigen (HBsAg) control kinetics in an immunocompetent mouse model of HBV infection. Thus, this study identifies a novel and safe combinatorial treatment strategy to potentiate substantial reduction of HBV replication using an IAP antagonist. [ABSTRACT FROM AUTHOR]

It is well accepted that the ability of cancer cells to circumvent the cell death program that untransformed cells are subject to helps promote tumor growth. Strategies designed to reinstate the cell death program in cancer cells have therefore been investigated for decades. Overexpression of members of the Inhibitor of APoptosis (IAP) protein family is one possible mechanism hindering the death of cancer cells. To promote cell death, drugs that mimic natural IAP antagonists, such as second mitochondria-derived activator of caspases (Smac/DIABLO) were developed. Smac-Mimetics (SMs) have entered clinical trials for hematological and solid cancers, unfortunately with variable and limited results so far. This review explores the use of SMs for the treatment of cancer, their potential to synergize with up-coming treatments and, finally, discusses the challenges and optimism facing this strategy. [ABSTRACT FROM AUTHOR]

It is becoming clear that “apoptotic” caspases can effect cellular processes other than cell death. A recent paper in Cell points to a novel role of the Drosophila caspase inhibitor DIAP1 as a determinant of cell migration. [Copyright &y& Elsevier]

The article presents a study which explores that Tankyrase-mediated ADP-ribosylation, a regulator of Tumor necrosis factor (TNF)-induced death . It mentions that findings of the study suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

Differentiation of naïve CD4+ T cells into effector (Th1, Th2, and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by the Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). Here we show that silencing of the Ifng, Gata3, and Il10 loci in naïve CD4+ T cells is dependent on Ezh2. Naïve CD4+ T cells lacking Ezh2 were epigenetically primed for overproduction of IFN-γ in Th2 and iTreg and IL-10 in Th2 cells. In addition, deficiency of Ezh2 accelerated effector Th cell death via death receptor-mediated extrinsic and intrinsic apoptotic pathways, confirmed in vivo for Ezh2-null IFN-γ-producing CD4+ and CD8+ T cells responding to Listeria monocytogenes infection. These findings demonstrate the key role of PRC2/Ezh2 in differentiation and survival of peripheral T cells and reveal potential immunotherapeutic targets. [ABSTRACT FROM AUTHOR]

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. LAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer, However, addition of either mono-or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways. [ABSTRACT FROM AUTHOR]

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpressioñ of RIPK1 can cause caspase-8-dependent cell death, when RIPK1-/- cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities (1, 2). To determine how RIPK1 promotes cell survival, we compared wild-type and RIPIKF-/- cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1-/- cells caused TRAF2 and clAP 1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIPL, and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1. [ABSTRACT FROM AUTHOR]

Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death that are highly expressed in many cancers. Cell death caused by antagonists that bind to IAP proteins is associated with their ubiquitylation and degradation. The RING domain at the C terminus of IAP proteins is pivotal. Here we report the crystal structures of the cIAP2 RING domain homodimer alone, and bound to the ubiquitin-conjugating (E2) enzyme UbcH5b. These structures show that small changes in the RING domain accompany E2 binding. By mutating residues at the E2-binding surface, we show that autoubiquitylation is required for regulation of IAP abundance. Dimer formation is also critical, and mutation of a single C-terminal residue abrogated dimer formation and E3 ligase activity was diminished. We further demonstrate that disruption of E2 binding, or dimerization, stabilizes IAP proteins against IAP antagonists in vivo. [ABSTRACT FROM AUTHOR]

Death ligands not only activate a death program but also regulate inflammatory signalling pathways, for example, through NF-κB induction. Although tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF both activate NF-κB in human keratinocytes, only TRAIL potently induces apoptosis. However, when induction of NF-κB was inhibited with a kinase dead IKK2 mutant (IKK2-KD), TNF- but not TRAIL-induced apoptosis was dramatically enhanced. Acquired susceptibility to TNF-induced apoptosis was due to increased caspase-8 activation. To investigate the mechanism of resistance of HaCaT keratinocytes to TNF-induced apoptosis, we analyzed a panel of NF-κB-regulated effector molecules. Interestingly, the inhibitor of apoptosis protein (IAP) family member cIAP2, but not cIAP1, X-linked inhibitor of apoptosis, TNF receptor-associated factor (TRAF)-1, or TRAF2, was downregulated in sensitive but not in resistant HaCaT keratinocytes. Surprisingly, however, stable inducible expression of cIAP2 was not sufficient to render IKK2-KD-sensitized keratinocytes resistant to TNF, and reduction of cIAP2 alone did not increase the sensitivity of HaCaT keratinocytes to TNF. In conclusion, we demonstrate that inhibition of NF-κB dramatically sensitizes human keratinocytes to TNF- but not to TRAIL-induced apoptosis and that this sensitization for TNF was largely independent of cIAP2. Our data thus clearly exclude the candidates proposed to date to confer TNF apoptosis resistance and suggest the function of an unanticipated effector of NF-κB critical for the survival of HaCaT keratinocytes upstream or at the level of caspase-8 activation.Journal of Investigative Dermatology (2008) 128, 1134–1147; doi:10.1038/sj.jid.5701141; published online 8 November 2007 [ABSTRACT FROM AUTHOR]

Summary: XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-κB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-κB-stimulated production of TNFα that killed cells in an autocrine fashion. Inhibition of NF-κB reduced TNFα production, and blocking NF-κB activation or TNFα allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFα, suggesting novel uses of these compounds in treating cancer. [Copyright &y& Elsevier]