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Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched Inflammatory High /Autophagy Low TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8 + T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

The (macro)autophagy field is facing a paradigm shift after the recent discovery that cytosolic cargoes can still be selectively targeted to phagophores (the precursors to autophagosomes) even in the absence of LC3 or other Atg8-protein family members. Several in vitro studies have indeed reported on the existence of an unconventional selective autophagic pathway that involves the in-situ formation of an autophagosome around the cargo through the direct selective autophagy receptor-mediated recruitment of RB1CC1/FIP200, thereby bypassing the requirement of LC3. In an article recently published in Science , we demonstrate the physiological importance of this unconventional autophagic pathway in the context of TNF (tumor necrosis factor) signaling. We show that it promotes the degradation of the cytotoxic TNFRSF1A/TNFR1 (TNF receptor superfamily member 1A) complex II that assembles upon TNF sensing and thereby protects mice from TNFRSF1A-driven embryonic lethality and skin inflammation. Abbreviations: ATG: autophagy related; CASP: caspase; FIR: RB1CC1/FIP200-interacting region; LIR: LC3-interacting region; M1: linear; PAS: phagophore assembly site; PtdIns3K: phosphatidylinositol 3-kinase; TNF: tumor necrosis factor; TNFRSF1A: TNF receptor superfamily member 1A.

Objectives: Type 1 diabetes (T1D) is caused by progressive immune-mediated loss of insulin-producing β-cells. Inflammation is detrimental to β-cell function and survival, moreover, both apoptosis and necrosis have been implicated as mechanisms of β-cell loss in T1D. The receptor interacting serine/threonine protein kinase 1 (RIPK1) promotes inflammation by serving as a scaffold for NF-κB and MAPK activation, or by acting as a kinase that triggers apoptosis or necroptosis. It is unclear whether RIPK1 kinase activity is involved in T1D pathology. In the present study, we investigated if absence of RIPK1 activation would affect the susceptibility to immune-mediated diabetes or diet induced obesity (DIO)., Methods: The RIPK1 knockin mouse line carrying a mutation mimicking serine 25 phosphorylation (Ripk1 S25D/S25D ), which abrogates RIPK1 kinase activity, was utilized to assess the in vivo role of RIPK1 in immune-mediated diabetes or diet induced obesity (DIO). In vitro, β-cell death and RIPK1 kinase activity was analysed in conditions known to induce RIPK1-dependent apoptosis/necroptosis., Results: We demonstrate that Ripk1 S25D/S25D mice presented normal glucose metabolism and β-cell function. Furthermore, immune-mediated diabetes and DIO were not different between Ripk1 S25D/S25D and Ripk1 +/+ mice. Despite strong activation of RIPK1 kinase and other necroptosis effectors (RIPK3 and MLKL) by TNF+BV6+zVAD, no cell death was observed in mouse islets nor human β-cells., Conclusion: Our results contrast recent literature showing that most cell types undergo necroptosis following RIPK1 kinase activation. This peculiarity may reflect an adaptation to the inability of β-cells to proliferate and self-renewal., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Pre-hospital delays have been associated with poor outcomes in patients with acute ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI). It is currently unknown how environmental variables affect treatment delays in these patients., Methods and Results: The association between environmental variables, time to treatment including transportation times and adverse in-hospital events was assessed in 1828 consecutive patients with STEMI undergoing primary PCI between 2010 and 2014 in the Montreal metropolitan area. Median[Q1;Q3] total ischemia time was significantly longer during summer season (April-September) as compared to winter season (October-March, 201[140;305] min vs 187[126;266] min, p = 0.022). This difference between seasons was due to a significant increase in median decision time to seek treatment for symptoms during summer (90[46;185] min vs 78[40;156], p = 0.004). The former peaked during July and August and was most pronounced in men. Hence, outside temperature and summer season were identified as strong predictors of prolonged decision time in patients with STEMI (p < 0.001 and p = 0.002, respectively). Transportation times slightly increased during winter season and snow fall, this difference, however, was not significant (p = 0.46). A significant increase in in-hospital adverse outcomes following primary PCI was observed during summer season as compared to winter season (7.2% vs 4.8%, p = 0.032). Accordingly, multivariate logistic regression models adjusted for baseline variables identified summer season as a strong predictor of periprocedural adverse events (OR 1.83, 95% CI 1.2-3.11, p = 0.037)., Conclusion: Contrary to our initial hypothesis, pre-hospital delays in patients with STEMI are considerably longer and associated with adverse in-hospital outcomes during summer season. Considering the consequences of global warming, it is imperative that educational efforts targeting patients' perception are implemented to counter treatment delays., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study aimed to evaluate the feasibility and accuracy of a technique for atherosclerosis imaging using local delivery of relatively small quantities (0.04-0.4 mg/kg) of labeled-specific imaging tracers targeting ICAM-1 and unpolymerized type I collagen or negative controls in 13 rabbits with atheroma induced by balloon injury in the abdominal aorta and a 12-week high-cholesterol diet. Immediately after local infusion, in vivo intravascular ultrasonography (IVUS)-NIRF imaging was performed at different time-points over a 40-minute period. The in vivo peak NIRF signal was significantly higher in the molecular tracer-injected rabbits than in the control-injected animals (P < 0.05). Ex vivo peak NIRF signal was significantly higher in the ICAM-1 probe-injected rabbits than in controls (P = 0.04), but not in the collagen probe-injected group (P = 0.29). NIRF signal discrimination following dual-probe delivery was also shown to be feasible in a single animal and thus offers the possibility of combining several distinct biological imaging agents in future studies. This innovative imaging strategy using in vivo local delivery of low concentrations of labeled molecular tracers followed by IVUS-NIRF catheter-based imaging holds potential for detection of vulnerable human coronary artery plaques.

The Canadian Organization of Medical Physicists (COMP), in close partnership with the Canadian Partnership for Quality Radiotherapy (CPQR) has developed a series of Technical Quality Control (TQC) guidelines for radiation treatment equipment. These guidelines outline the performance objectives that equipment should meet in order to ensure an acceptable level of radiation treatment quality. The TQC guidelines have been rigorously reviewed and field tested in a variety of Canadian radiation treatment facilities. The development process enables rapid review and update to keep the guidelines current with changes in technology (the most updated version of this guideline can be found on the CPQR website). This particular TQC details recommended quality control testing of conventional radiotherapy simulators., (© 2018 The Authors. Journal of Applied Clinical Medical Physics published by Wiley Periodicals, Inc. on behalf of American Association of Physicists in Medicine.)

Background: The association between diagnosed acute ST-elevation myocardial infarction (STEMI) and hockey games in the Canadian population is unknown., Methods: We retrospectively analyzed the association between hockey games of the National Hockey League Montreal Canadiens and daily hospital admissions for acute STEMI at the Montreal Heart Institute, Canada., Results: Between June 2010 and December 2014, a total of 2199 patients (25.9% women; mean age, 62.6 ± 12.4 years) were admitted for acute STEMI. An increase in STEMI admissions was observed the day after a hockey game of the Montreal Canadiens in the overall population (from 1.3 ± 1.2 to 1.5 ± 1.3), however, this difference was not significant (P = 0.1). The number of STEMI admissions increased significantly from 0.9 ± 1.0 to 1.2 ± 1.0 per day in men (P = 0.04), but not in women (P = 0.7). The association between ice hockey matches and STEMI admission rates was strongest after a victory of the Montreal Canadiens. Accordingly, an increased risk for the occurrence of STEMI was observed in the overall population (hazard ratio [HR], 1.15; 95% confidence interval [CI], 1.0-1.3; P = 0.037) when the Montreal Canadiens won a match. This association was present in men (HR, 1.2; 95% CI, 1.03-1.4; P = 0.02) but not in women (P = 0.87), with a most pronounced effect seen in younger men (younger than 55 years; HR, 1.4; 95% CI, 1.1-1.8; P = 0.009)., Conclusions: Although a weak association between hockey games and hospital admissions for STEMI was found in our overall population, the event of a hockey game significantly increased the risk for STEMI in younger men. Preventive measures targeting behavioural changes could positively affect this risk., (Copyright © 2018 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Background: During the last decade, the incidence and mortality rates of ST-elevation myocardial infarction (STEMI) has been steadily increasing in young women but not in men. Environmental variables that contribute to cardiovascular events in women remain ill-defined., Methods and Results: A total of 2199 consecutive patients presenting with acute ST-elevation myocardial infarction (STEMI, 25.8% women, mean age 62.6±12.4 years) were admitted at the Montreal Heart Institute between June 2010 and December 2014. Snow fall exceeding 2cm/day was identified as a positive predictor for STEMI admission rates in the overall population (RR 1.28, 95% CI 1.07-1.48, p = 0.005), with a significant effect being seen in men (RR 1.30, 95% CI 1.06-1.53, p = 0.01) but not in women (p = NS). An age-specific analysis revealed a significant increase in hospital admission rates for STEMI in younger women ≤55 years, (n = 104) during days with higher outside temperature (p = 0.004 vs men ≤55 years) and longer daylight hours (p = 0.0009 vs men ≤55 years). Accordingly, summer season, increased outside temperature and sunshine hours were identified as strong positive predictors for STEMI occurrence in women ≤55 years (RR 1.66, 95% CI 1.1-2.5, p = 0.012, RR 1.70, 95% CI 1.2-2.5, p = 0.007, and RR 1.67, 95% CI 1.2-2.5, p = 0.011, respectively), while an opposite trend was observed in men ≤55 years (RR for outside temperature 0.8, 95% CI 0.73-0.95, p = 0.01)., Conclusion: The impact of environmental variables on STEMI is age- and sex-dependent. Higher temperature may play an important role in triggering such acute events in young women.

The Canadian Organization of Medical Physicists (COMP), in close partnership with the Canadian Partnership for Quality Radiotherapy (CPQR) has developed a series of Technical Quality Control (TQC) guidelines for radiation treatment equipment. These guidelines outline the performance objectives that equipment should meet in order to ensure an acceptable level of radiation treatment quality. The TQC guidelines have been rigorously reviewed and field tested in a variety of Canadian radiation treatment facilities. The development process enables rapid review and update to keep the guidelines current with changes in technology. This announcement provides an introduction to the guidelines, describing their scope and how they should be interpreted. Details of recommended tests can be found in separate, equipment specific TQC guidelines published in the JACMP (COMP Reports), or the website of the Canadian Partnership for Quality Radiotherapy (www.cpqr.ca)., (© 2018 Royal Victoria Regional Health Centre. Journal of Applied Clinical Medical Physics published by Wiley Periodicals, Inc. on behalf of American Association of Physicists in Medicine.)

Introduction: Cardiovascular (CV) atherosclerotic disease remains the leading cause of morbidity and mortality worldwide, despite the advances in contemporary therapies. Inflammation is an important process in atherosclerosis, leading to plaque rupture and acute coronary syndrome. Although statin therapy has substantially reduced CV events in primary and secondary prevention, many treated patients will have recurrent adverse CV events despite the standard of care. Thus, drug development aiming to target inflammatory pathways seems a promising avenue for novel therapies in atherosclerosis. Areas covered: Statins have been extensively studied and are the most effective lipid-lowering drugs available for CV prevention. Novel anti-inflammatory drugs are being tested in Phase II and III trials, targeting pathways like interleukin-1, leukotrienes, TNF-α, P-selectin, CCL2-CCR2 and MAP Kinase. Expert opinion: Novel anti-inflammatory therapies seem promising additions to address the residual CV risk present despite the current standard of care, but large clinical trials have not yet shown beneficial effects on clinical events. PCSK9 inhibitors have been shown to substantially reduce LDL-C, however their long-term safety and effects on CV risk are currently being investigated. Pharmacogenomics holds great potential in future lipid trials, enabling the identification of patients who will respond with greater benefits and smaller risk to therapies and to decrease failure rates in drug development, as genotype-dependent effects of the CETP inhibitor dalcetrapib were shown in the dal-OUTCOMES and dal-PLAQUE-2 trials.

Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology., Competing Interests: The authors declare no conflict of interest.

Purpose of Review: Randomized clinical outcome trials are costly, long, and often yield neutral or modestly positive results, and these issues have impeded cardiovascular drug development in the past decade. Despite the significant reduction of cardiovascular morbidity and mortality with statins, substantial residual risk of major cardiovascular events remains. This could be because of the difficulty of demonstrating benefits of new drugs in addition to the current standard of care in unselected populations as well as the interindividual variability in drug response. Pharmacogenomics is a promising avenue for the development of novel or failed drugs and for the repurposing of other medications., Recent Findings: Several variants were identified in genes that were associated with the effects of statins on plasma lipids. Genomic studies of mutations in genes that encode drug targets have the potential to inform on the link between drug therapy acting on those targets and clinical outcomes. Recently, ADCY9 gene variants were shown to be significantly associated with responses to dalcetrapib in terms of clinical outcomes, atherosclerosis imaging, cholesterol efflux, and inflammation, which provided support for the conduct of a new prospective clinical trial in a genetically determined population., Summary: Pharmacogenomics hold great potential in future lipid trials to decrease failure rates in drug development and to identify patients who will respond with greater benefits and smaller risk.

Cell death of hepatocytes is a prominent characteristic in the pathogenesis of liver disease, while hepatolysis is a starting point of inflammation in hepatitis and loss of hepatic function. However, the precise molecular mechanisms of hepatocyte cell death, the role of the cytokines of hepatic microenvironment and the involvement of intracellular kinases, remain unclear. Tumor necrosis factor alpha (TNF-α) is a key cytokine involved in cell death or survival pathways and the role of RIPK1 has been associated to the TNF-α-dependent signaling pathway. We took advantage of two different deficient mouse lines, the RIPK1 kinase dead knock-in mice (Ripk1 K45A ) and the conditional knockout mice lacking RIPK1 only in liver parenchymal cells (Ripk1 LPC-KO ), to characterize the role of RIPK1 and TNF-α in hepatitis induced by concanavalin A (ConA). Our results show that RIPK1 is dispensable for liver homeostasis under steady-state conditions but in contrast, RIPK1 kinase activity contributes to caspase-independent cell death induction following ConA injection and RIPK1 also serves as a scaffold, protecting hepatocytes from massive apoptotic cell death in this model. In the Ripk1 LPC-KO mice challenged with ConA, TNF-α triggers apoptosis, responsible for the observed severe hepatitis. Mechanism potentially involves both TNF-independent canonical NF-κB activation, as well as TNF-dependent, but canonical NF-κB-independent mechanisms. In conclusion, our results suggest that RIPK1 kinase activity is a pertinent therapeutic target to protect liver against excessive cell death in liver diseases.

The endoplasmic reticulum (ER) has a crucial role in the proper folding of proteins that are synthesized in the secretory pathway. Physiological and pathological conditions can induce accumulation of mis- or unfolded proteins in the ER lumen and thereby generate a state of cellular stress known as ER stress. The unfolded protein response aims at restoring protein-folding homeostasis, but turns into a toxic signal when ER stress is too severe or prolonged. ER stress-induced cellular dysfunction and death is associated with several human diseases, but the molecular mechanisms regulating death under unresolved ER stress are still unclear. We performed a siRNA-based screen to identify new regulators of ER stress-induced death and found that repression of the Carney complex-associated protein PRKAR1A specifically protected the cells from ER stress-induced apoptosis, and not from apoptosis induced by etoposide or TNF. We demonstrate that the protection results from PKA activation and associate it, at least in part, with the phosphorylation-mediated inhibition of the PKA substrate Drp1 (dynamin-related protein 1). Our results therefore provide new information on the complex regulation of cellular death under ER stress conditions and bring new insights on the conditions that regulate the pro- versus anti-death functions of PKA.

Several cell death assays have been developed based on a single biochemical parameter such as caspase activation or plasma membrane permeabilization. Our fluorescent apoptosis/necrosis (FAN) assay directly measures cell death and distinguishes between caspase-dependent apoptosis and caspase-independent necrosis of cells grown in any multiwell plate. Cell death is monitored in standard growth medium as an increase in fluorescence intensity of a cell-impermeable dye (SYTOX Green) after plasma membrane disintegration, whereas apoptosis is detected through caspase-mediated release of a fluorophore from its quencher (DEVD-amc). The assay determines the normalized percentage of dead cells and caspase activation per condition as an end-point measurement or in real time (automated). The protocol can be applied to screen drugs, proteins or siRNAs for interference with cell death while simultaneously detecting cell death modality switching between apoptosis and necrosis. Initial preparation may take up to 5 d, but the typical hands-on time is ∼2 h.

Tumor necrosis factor (TNF) is a master pro-inflammatory cytokine, and inappropriate TNF signaling is implicated in the pathology of many inflammatory diseases. Ligation of TNF to its receptor TNFR1 induces the transient formation of a primary membrane-bound signaling complex, known as complex I, that drives expression of pro-survival genes. Defective complex I activation results in induction of cell death, in the form of apoptosis or necroptosis. This switch occurs via internalization of complex I components and assembly and activation of secondary cytoplasmic death complexes, respectively known as complex II and necrosome. In this review, we discuss the crucial regulatory functions of ubiquitination-a post-translational protein modification consisting of the covalent attachment of ubiquitin, and multiples thereof, to target proteins-to the various steps of TNFR1 signaling leading to necroptosis.

Background: Three pregnancies with male offspring in one family were complicated by severe polyhydramnios and prematurity. One fetus died; the other two had transient massive salt-wasting and polyuria reminiscent of antenatal Bartter's syndrome., Methods: To uncover the molecular cause of this possibly X-linked disease, we performed whole-exome sequencing of DNA from two members of the index family and targeted gene analysis of other members of this family and of six additional families with affected male fetuses. We also evaluated a series of women with idiopathic polyhydramnios who were pregnant with male fetuses. We performed immunohistochemical analysis, knockdown and overexpression experiments, and protein-protein interaction studies., Results: We identified a mutation in MAGED2 in each of the 13 infants in our analysis who had transient antenatal Bartter's syndrome. MAGED2 encodes melanoma-associated antigen D2 (MAGE-D2) and maps to the X chromosome. We also identified two different MAGED2 mutations in two families with idiopathic polyhydramnios. Four patients died perinatally, and 11 survived. The initial presentation was more severe than in known types of antenatal Bartter's syndrome, as reflected by an earlier onset of polyhydramnios and labor. All symptoms disappeared spontaneously during follow-up in the infants who survived. We showed that MAGE-D2 affects the expression and function of the sodium chloride cotransporters NKCC2 and NCC (key components of salt reabsorption in the distal renal tubule), possibly through adenylate cyclase and cyclic AMP signaling and a cytoplasmic heat-shock protein., Conclusions: We found that MAGED2 mutations caused X-linked polyhydramnios with prematurity and a severe but transient form of antenatal Bartter's syndrome. MAGE-D2 is essential for fetal renal salt reabsorption, amniotic fluid homeostasis, and the maintenance of pregnancy. (Funded by the University of Groningen and others.).

Successful immunogenic apoptosis in experimental cancer therapy depends on the induction of strong host anti-tumor responses. Given that tumors are often resistant to apoptosis, it is important to identify alternative molecular mechanisms that elicit immunogenic cell death. We have developed a genetic model in which direct dimerization of FADD combined with inducible expression of RIPK3 promotes necroptosis. We report that necroptotic cancer cells release damage-associated molecular patterns and promote maturation of dendritic cells, the cross-priming of cytotoxic T cells, and the production of IFN-γ in response to tumor antigen stimulation. Using both FADD-dependent and FADD-independent RIPK3 induction systems, we demonstrate the efficient vaccination potential of immunogenic necroptotic cells. Our study broadens the current concept of immunogenic cell death and opens doors for the development of new strategies in cancer therapy., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

NF-κB-inducing kinase (NIK) is well-known for its role in promoting p100/NF-κB2 processing into p52, a process defined as the alternative, or non-canonical, NF-κB pathway. Here we reveal an unexpected new role of NIK in TNFR1-mediated RIP1-dependent apoptosis, a consequence of TNFR1 activation observed in c-IAP1/2-depleted conditions. We show that NIK stabilization, obtained by activation of the non-death TNFRs Fn14 or LTβR, is required for TNFα-mediated apoptosis. These apoptotic stimuli trigger the depletion of c-IAP1/2, the phosphorylation of RIP1 and the RIP1 kinase-dependent assembly of the RIP1/FADD/caspase-8 complex. In the absence of NIK, the phosphorylation of RIP1 and the formation of RIP1/FADD/caspase-8 complex are compromised while c-IAP1/2 depletion is unaffected. In vitro kinase assays revealed that recombinant RIP1 is a bona fide substrate of NIK. In vivo, we demonstrated the requirement of NIK pro-death function, but not the processing of its substrate p100 into p52, in a mouse model of TNFR1/LTβR-induced thymus involution. In addition, we also highlight a role for NIK in hepatocyte apoptosis in a mouse model of virus-induced TNFR1/RIP1-dependent liver damage. We conclude that NIK not only contributes to lymphoid organogenesis, inflammation and cell survival but also to TNFR1/RIP1-dependent cell death independently of the alternative NF-κB pathway.

TNF is a master pro-inflammatory cytokine. Activation of TNFR1 by TNF can result in both RIPK1-independent apoptosis and RIPK1 kinase-dependent apoptosis or necroptosis. These cell death outcomes are regulated by two distinct checkpoints during TNFR1 signaling. TNF-mediated NF-κB-dependent induction of pro-survival or anti-apoptotic molecules is a well-known late checkpoint in the pathway, protecting cells from RIPK1-independent death. On the other hand, the molecular mechanism regulating the contribution of RIPK1 to cell death is far less understood. We demonstrate here that the IKK complex phosphorylates RIPK1 at TNFR1 complex I and protects cells from RIPK1 kinase-dependent death, independent of its function in NF-κB activation. We provide in vitro and in vivo evidence that inhibition of IKKα/IKKβ or its upstream activators sensitizes cells to death by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We therefore report on an unexpected, NF-κB-independent role for the IKK complex in protecting cells from RIPK1-dependent death downstream of TNFR1., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Our current knowledge of the molecular mechanisms regulating the signaling pathways leading to cell survival, cell death, and inflammation has shed light on the tight mutual interplays between these processes. Moreover, the fact that both apoptosis and necrosis can be molecularly controlled has greatly increased our interest in the roles that these types of cell death play in the control of general processes such as development, homeostasis, and inflammation. In this review, we provide a brief update on the different cell death modalities and describe in more detail the intracellular crosstalk between survival, apoptotic, necroptotic, and inflammatory pathways that are activated downstream of death receptors. An important concept is that the different cell death processes modulate each other by mutual inhibitory mechanisms, serve as alternative back-up death routes in the case of a defect in the first-line cell death response, and are controlled by multiple feedback loops. We conclude by discussing future perspectives and challenges in the field of cell death and inflammation research.

As indicated by their name, members of the Inhibitor of APoptosis (IAP) family were first believed to be functionally restricted to apoptosis inhibition. It is now clear that IAPs have a much wider spectrum of action, and recent studies even suggest that some of its members primarily regulate inflammatory responses. Inflammation, the first response of the immune system to infection or tissue injury, is highly regulated by ubiquitylation - a posttranslational modification of proteins with various consequences. In this review, we focus on the recently reported functions of XIAP, cIAP1 and cIAP2 as ubiquitin ligases regulating innate immunity and inflammation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Endoplasmic reticulum (ER) stress-induced cellular dysfunction and death is associated with several human diseases. It has been widely reported that ER stress kills through activation of the intrinsic mitochondrial apoptotic pathway. Here we demonstrate that ER stress can also induce necroptosis, an receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain-like protein (MLKL)-dependent form of necrosis. Remarkably, we observed that necroptosis induced by various ER stressors in L929 cells is dependent on tumor necrosis factor receptor 1 (TNFR1), but occurs independently of autocrine TNF or lymphotoxin α production. Moreover, we found that repression of either TNFR1, RIPK1 or MLKL did not protect the cells from death but instead allowed a switch to ER stress-induced apoptosis. Interestingly, while caspase inhibition was sufficient to protect TNFR1- or MLKL-deficient cells from death, rescue of the RIPK1-deficient cells additionally required RIPK3 depletion, indicating a switch back to RIPK3-dependent necroptosis in caspase-inhibited conditions. The finding that ER stress also induces necroptosis may open new therapeutic opportunities for the treatment of pathologies resulting from unresolved ER stress.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and results in the activation of the unfolded protein response (UPR), which aims at restoring ER homeostasis. However, when the stress is too severe the UPR switches from being a pro-survival response to a pro-death one, and the molecular mechanisms underlying ER stress-mediated death have remained incompletely understood. In this study, we identified receptor interacting protein kinase 1 (RIPK1)-a kinase at the crossroad between life and death downstream of various receptors-as a new regulator of ER stress-induced death. We found that Ripk1-deficient MEFs are protected from apoptosis induced by ER stressors, which is reflected by reduced caspase activation and PARP processing. Interestingly, the pro-apoptotic role of Ripk1 is independent of its kinase activity, is not regulated by its cIAP1/2-mediated ubiquitylation, and does not rely on the direct regulation of JNK or CHOP, two reportedly main players in ER stress-induced death. Instead, we found that ER stress-induced apoptosis in these cells relies on death receptor-independent activation of caspase-8, and identified Ripk1 upstream of caspase-8. However, in contrast to RIPK1-dependent apoptosis downstream of TNFR1, we did not find Ripk1 associated with caspase-8 in a death-inducing complex upon unresolved ER stress. Our data rather suggest that RIPK1 indirectly regulates caspase-8 activation, in part via interaction with the ER stress sensor inositol-requiring protein 1 (IRE1).

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

Although mixed lineage kinase domain-like (MLKL) protein has emerged as a specific and crucial protein for necroptosis induction, how MLKL transduces the death signal remains poorly understood. Here, we demonstrate that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death. Moreover, we found that a patch of positively charged amino acids on the surface of the 4HBD binds to phosphatidylinositol phosphates (PIPs) and allows recruitment of MLKL to the plasma membrane. Importantly, we found that recombinant MLKL, but not a mutant lacking these positive charges, induces leakage of PIP-containing liposomes as potently as BAX, supporting a model in which MLKL induces necroptosis by directly permeabilizing the plasma membrane. Accordingly, we found that inhibiting the formation of PI(5)P and PI(4,5)P2 specifically inhibits tumor necrosis factor (TNF)-mediated necroptosis but not apoptosis., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The final decision on cell fate, survival versus cell death, relies on complex and tightly regulated checkpoint mechanisms. The caspase-3 protease is a predominant player in the execution of apoptosis. However, recent progress has shown that this protease paradoxically can also protect cells from death. Here, we discuss the underappreciated, protective, and prosurvival role of caspase-3 and detail the evidence showing that caspase-3, through differential processing of p120 Ras GTPase-activating protein (RasGAP), can modulate a given set of proteins to generate, depending on the intensity of the input signals, opposite outcomes (survival vs death)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.

Receptor-interacting protein kinase (RIPK) 1 and RIPK3 have emerged as essential kinases mediating a regulated form of necrosis, known as necroptosis, that can be induced by tumor necrosis factor (TNF) signaling. As a consequence, inhibiting RIPK1 kinase activity and repressing RIPK3 expression levels have become commonly used approaches to estimate the contribution of necroptosis to specific phenotypes. Here, we report that RIPK1 kinase activity and RIPK3 also contribute to TNF-induced apoptosis in conditions of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) depletion or TGF-β-activated kinase 1 (TAK1) kinase inhibition, implying that inhibition of RIPK1 kinase activity or depletion of RIPK3 under cell death conditions is not always a prerequisite to conclude on the involvement of necroptosis. Moreover, we found that, contrary to cIAP1/2 depletion, TAK1 kinase inhibition induces assembly of the cytosolic RIPK1/Fas-associated protein with death domain/caspase-8 apoptotic TNF receptor 1 (TNFR1) complex IIb without affecting the RIPK1 ubiquitylation status at the level of TNFR1 complex I. These results indicate that the recruitment of TAK1 to the ubiquitin (Ub) chains, and not the Ub chains per se, regulates the contribution of RIPK1 to the apoptotic death trigger. In line with this, we found that cylindromatosis repression only provided protection to TNF-mediated RIPK1-dependent apoptosis in condition of reduced RIPK1 ubiquitylation obtained by cIAP1/2 depletion but not upon TAK1 kinase inhibition, again arguing for a role of TAK1 in preventing RIPK1-dependent apoptosis downstream of RIPK1 ubiquitylation. Importantly, we found that this function of TAK1 was independent of its known role in canonical nuclear factor-κB (NF-κB) activation. Our study therefore reports a new function of TAK1 in regulating an early NF-κB-independent cell death checkpoint in the TNFR1 apoptotic pathway. In both TNF-induced RIPK1 kinase-dependent apoptotic models, we found that RIPK3 contributes to full caspase-8 activation independently of its kinase activity or intact RHIM domain. In contrast, RIPK3 participates in caspase-8 activation by acting downstream of the cytosolic death complex assembly, possibly via reactive oxygen species generation.

An inflammatory response is initiated when innate immune pattern-recognition receptors (PRRs) expressed by different cell types detect constituents of invading microorganisms and endogenous intracellular molecules released by dying cells. The intracellular cascades activated by PRRs induce the expression and maturation of inflammatory molecules that coordinate the removal of the infectious agents and of the infected or damaged cells. In this Review, we discuss the findings implicating members of the inhibitor of apoptosis protein (IAP) family in the ubiquitylation-dependent regulation of PRR signalling. Understanding the role of IAPs in innate immunity may open new therapeutic perspectives for the treatment of PRR-dependent inflammatory diseases.

MAGED1, NECDIN and MAGEL2 are members of the MAGE gene family. The latter two of these genes have been involved in Prader-Willi syndrome (PWS), which includes hyperphagia, repetitive and compulsive behaviors, and cognitive impairment. Here, we show that Maged1-deficient mice develop progressive obesity associated with hyperphagia and reduced motor activity. Loss of Maged1 also results in a complex behavioral syndrome that includes reduced social interactions and memory, deficient sexual behavior, as well as increased anxiety and self-grooming. Oxytocin (OT), which is produced in the hypothalamus, can act as a neurotransmitter that reduces anxiety, promotes social behaviors and regulates food intake. Growing evidences indicate that OT is involved in autism. We found that Maged1 mutants showed a severe reduction in the levels of mature OT, but not of its precursors, in the hypothalamus. Moreover, the administration of OT rescued the deficit in social memory of these mice. We conclude that Maged1 is required for OT processing or stability. A decrease in mature OT levels in Maged1 mutants affects social interactions and possibly other behavioral processes. Our observations suggest that, in human, MAGED1 could play a role in autism or cause a neurodevelopmental condition that is reminiscent of the PWS.

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.

One of the early cellular responses to endoplasmic reticulum (ER) stress is the activation of the unfolded protein response (UPR). ER stress and the UPR are both implicated in numerous human diseases and pathologies. In spite of this, our knowledge of the molecular mechanisms that regulate cell fate following ER stress is limited. The UPR is initiated by three ER transmembrane receptors: PKR-like ER kinase (PERK), activating transcription factor (ATF) 6 and inositol-requiring enzyme 1 (IRE1). These proteins sense the accumulation of unfolded proteins and their activation triggers specific adaptive responses to resolve the stress. Intriguingly, the very same receptors can initiate signalling pathways that lead to apoptosis when the attempts to resolve the ER stress fail. In this review, we describe the known pro-apoptotic signalling pathways emanating from activated PERK, ATF6 and IRE1 and discuss how their signalling switches from an adaptive to a pro-apoptotic response., (Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.)

Receptor-interacting protein kinase 1 (RIPK1) is an important component of the tumor necrosis factor receptor 1 (TNFR1) signaling pathway. Depending on the cell type and conditions, RIPK1 mediates MAPK and NF-κB activation as well as cell death. Using a mutant form of RIPK1 (RIPK1ΔID) lacking the intermediate domain (ID), we confirm the requirement of this domain for activation of these signaling events. Moreover, expression of RIPK1ΔID resulted in enhanced recruitment of caspase-8 to the TNFR1 complex II component Fas-associated death domain (FADD), which allowed a shift from TNF-induced necroptosis to apoptosis in L929 cells. Addition of the RIPK1 kinase inhibitor necrostatin-1 strongly reduced recruitment of RIPK1 and caspase-8 to FADD and subsequent apoptosis, indicating a role for RIPK1 kinase activity in apoptotic complex formation. Our study shows that RIPK1 has an anti-apoptotic function residing in its ID and demonstrates a cellular system as an elegant genetic model for RIPK1 kinase-dependent apoptosis that, in contrast to the Smac mimetic model, does not rely on depletion of cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2).

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.

Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation) in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.

Members of the family of NOD-like receptors (NLRs) play essential roles in innate immunity by detecting intracellular 'pathogen-associated molecular patterns' (PAMPs) and 'danger-associated molecular patterns' (DAMPs). These molecules reveal the presence of pathogenic infection, abiotic stress, environmental insults, cellular damage, and cell death. NLR family members can be divided in two functional groups. One group consists of intracellular receptors, such as NLRP1, NLRP3, NLRP6 and NLRC4, which mediate the assembly of inflammasome complexes leading to the activation of procaspase-1. The second group includes members such as NOD1 and NOD2, and mediates the assembly of complexes that activate MAPK and NF-κB signaling pathways. We review the roles of NLR family members in health and disease, with emphasis on the signaling mechanisms in cell death and inflammation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In this issue of Molecular Cell, Tenev et al. and Feoktistova et al. describe the Ripoptosome, a cytosolic death-inducing RIP1-, FADD-, and caspase-8-containing complex that spontaneously assembles upon cIAP depletion, challenging the view that such complexes exclusively originate from receptor activation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (© 2011 Macmillan Publishers Limited)

The RIP kinases have emerged as essential mediators of cellular stress that integrate both extracellular stimuli emanating from various cell-surface receptors and signals coming from intracellular pattern recognition receptors. The molecular mechanisms regulating the ability of the RIP proteins to transduce the stress signals remain poorly understood, but seem to rely only partially on their kinase activities. Recent studies on RIP1 and RIP2 have highlighted the importance of ubiquitination as a key process regulating their capacity to activate downstream signaling pathways. In this study, we found that XIAP, cIAP1 and cIAP2 not only directly bind to RIP1 and RIP2 but also to RIP3 and RIP4. We show that cIAP1 and cIAP2 are direct E3 ubiquitin ligases for all four RIP proteins and that cIAP1 is capable of conjugating the RIPs with diverse types of ubiquitin chains, including linear chains. Consistently, we show that repressing cIAP1/2 levels affects the activation of NF-κB that is dependent on RIP1, -2, -3 and -4. Finally, we identified Lys51 and Lys145 of RIP4 as two critical residues for cIAP1-mediated ubiquitination and NF-κB activation.

Background: In normal adult skeletal muscle, cell turnover is very slow. However, after an acute lesion or in chronic pathological conditions, such as primary myopathies, muscle stem cells, called satellite cells, are induced to proliferate, then withdraw definitively from the cell cycle and fuse to reconstitute functional myofibers., Results: We show that Maged1 is expressed at very low levels in normal adult muscle but is strongly induced after injury, during the early phase of myoblast differentiation. By comparing in vitro differentiation of myoblasts derived from wild-type or Maged1 knockout mice, we observed that Maged1 deficiency results in reduced levels of p21CIP1/WAF1, defective cell cycle exit and impaired myotube maturation. In vivo, this defect results in delayed regeneration of injured muscle., Conclusions: These data demonstrate for the first time that Maged1 is an important factor required for proper skeletal myoblast differentiation and muscle healing.