Programmed necrosis is important in many (patho)physiological settings. For specific therapeutic intervention, however, a better knowledge is required whether necrosis occurs through one single “core program” or through several independent pathways. Previously, the poly(ADP-ribose) polymerase (PARP) pathway has been suggested as a crucial element of tumor necrosis factor (TNF)-mediated necroptosis. Here, we show that TNF-induced necroptosis and the PARP pathway represent distinct and independent routes to programmed necrosis. First, DNA-alkylating agents such as 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) or methyl methanesulfonate rapidly activate the PARP pathway, whereas this is a late and secondary event in TNF-induced necroptosis. Second, inhibition of the PARP pathway does not protect against TNF-induced necroptosis, e.g., the PARP-1 inhibitor 3-AB prevented MNNG- but not TNF-induced adenosine-5′-triposphate depletion, translocation of apoptosis-inducing factor, and necrosis. Likewise, olaparib, a more potent and selective PARP-1 inhibitor failed to block TNF-induced necroptosis, identical to knockdown/knockout of PARP-1, pharmacologic and genetic interference with c-Jun N-terminal kinases and calpain/cathepsin proteases as further components of the PARP pathway. Third, interruption of TNF-induced necroptosis by interference with ceramide generation, RIP1 or RIP3 function or by the radical scavenger butylated hydroxyanisole did not prevent programmed necrosis through the PARP pathway. In summary, our results suggest that the currently established role of the PARP pathway in TNF-induced necroptosis needs to be revised, with consequences for the design of future therapeutic strategies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-013-1381-6) contains supplementary material, which is available to authorized users.

Although numerous studies have implicated the sphingolipid ceramide in the induction of cell death, a causative function of ceramide in caspase-dependent apoptosis remains a highly debated issue. Here, we show that ceramide is a key mediator of a distinct route to programmed cell death (PCD), i.e., caspase-independent PCD. Under conditions where apoptosis is either not initiated or actively inhibited, TNF induces caspase-independent PCD in L929 fibrosarcoma cells, NIH3T3 fibroblasts, human leukemic Jurkat T cells, and lung fibroblasts by increasing intracellular ceramide levels prior to the onset of cell death. Survival is significantly enhanced when ceramide accumulation is prevented, as demonstrated in fibroblasts genetically deficient for acid sphingomyelinase, in L929 cells overexpressing acid ceramidase, by pharmacological intervention, or by RNA interference. Jurkat cells deficient for receptor-interacting protein 1 (RIP1) do not accumulate ceramide and therefore are fully resistant to caspase-independent PCD whereas Jurkat cells overexpressing the mitochondrial protein Bcl-2 are partially protected, implicating RIP1 and mitochondria as components of the ceramide death pathway. Our data point to a role of caspases (but not cathepsins) in suppressing the ceramide death pathway under physiological conditions. Moreover, clonogenic survival of tumor cells is clearly reduced by induction of the ceramide death pathway, promising additional options for the development of novel tumor therapies.

The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer's, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2. In yeast, the N terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize and physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1•FAN•RACK1-complex in a timeframe concurrent with activation of nSMase2. After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2, and which therefore represents the "missing link" that completes one of the last unresolved signaling pathways of TNF-R1.embryonic ectodermal development | immune response | inflammation N eutral sphingomyelinases (N-SMases) mediate stress-induced ceramide generation and participate in inflammation, development, cellular growth, differentiation and death, heart failure, ischemia/reperfusion damage, atherosclerosis, and Alzheimer's disease (1, 2). They are acutely activated by TNF, a major mediator of inflammatory and immunoregulatory responses (3, 4). Out of the three N-SMase genes cloned in mammals, nSMase2 corresponds to the biochemically characterized N-SMase. It is a membrane-bound protein with two putative N-terminal hydrophobic membrane-anchoring domains, a collagen-like linker region, and a C-terminal catalytic domain (Fig. 1A) (5). nSMase2 has been linked to cell cycle regulation and contact inhibition (1), late embryonal and postnatal development (5, 6), exosome secretion (7), and Alzheimer's disease and combined pituitary hormone deficiency (1, 6). In response to TNF, nSMase2 is important for inflammatory signaling, cell adhesion and migration, endothelial regulation, cell death, and cutaneous barrier repair (5,(8)(9)(10)(11). Even though N-SMase activation by TNF has been reported since almost 20 years ago (3), the corresponding signaling pathway is not fully resolved. In response to TNF, nSMase2 is activated exclusively by the 55-kDa receptor TNF receptor 1 (TNF-R1) (10). We have previously defined a neutral sphingomyelinase activation domain (NSD) within TNF-R1 (12) that serves as a binding site for the protein FAN (factor associated with N-SMase activation) (13). FAN recruits the WD repeat protein...

BackgroundIn apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis.ResultsThe serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis.ConclusionsThe proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic properties of HtrA2/Omi and UCH-L1 may prove beneficial for the treatment of patients, e.g. in kidney failure.

The regulation of cellular survival and apoptosis is of critical importance for the immune system to maintain immune homeostasis and to establish tolerance. Here, we demonstrate that the immune specific cell surface molecule Toso exhibits antiapoptotic effects on death receptor signaling by a novel regulatory mechanism involving the adaptor kinase RIP1.The antiapoptotic function of Toso depends on RIP1 ubiquitination and involves the recruitment of the death adaptor FADD to a Toso/RIP1 protein complex. In response to CD95L and TNF␣, Toso promotes the activation of MAPK and NF-B signaling pathways. Because of this relative augmentation of survival versus apoptotic signals, Toso raises the threshold for death receptormediated apoptosis. Our analysis of Tosodeficient mice revealed that Toso is essential for TNF␣-mediated liver damage. Furthermore, the antiapoptotic function of Toso could be blocked by a Toso-specific monoclonal antibody, opening up new therapeutic prospects for the treatment of immune disorders and hematologic malignancies. (Blood. 2011;118(3):598-608) IntroductionOne of the most well-characterized death receptors in the immune system is CD95 (Fas, APO-1). CD95 belongs to the TNF receptor superfamily. Expressed as preassociated homotrimers, CD95 on ligand binding directly conveys apoptotic signals. On ligand binding, CD95 assembles the death inducing signaling complex (DISC). The DISC consists of the death adaptor FADD (Fas associated death domain protein), procaspase-8, and procaspase-10. 1,2 Efficient DISC formation provides a molecular scaffold concentrating cysteine proteases to induce autoproteolytic cleavage of caspase-8 and release of active caspase-8 that initiates the apoptotic program. Mutations of CD95 or its ligand have been found in autoimmune strains of mice and in patients with autoimmune lymphoproliferative syndrome (ALPS). 3 These genetic abnormalities in CD95 result in the development of massive lymphoadenopathy and disruption of lymphocyte homeostasis because of increased survival of activated lymphocytes. 4,5 Although CD95 has been mainly perceived as a death inducing receptor, recent findings paint a far more complex picture of CD95 and the DISC components by providing evidence that they can transmit both, deathinducing and cellular activation signals in response to the same ligand. 6,7 Such nonapoptotic signaling activities of CD95 are thought to mediate proinflammatory responses in immune cells, neuronal tissue remodeling and promote tumor progression. [7][8][9] The execution and regulation of CD95-mediated nonapoptotic signaling, in particular with respect to its contribution to tumorigenesis, is however still largely unclear.Toso, also known as FAIM3, is a type I transmembrane protein belonging to the immunoglobulin gene superfamily. Toso was originally identified as a surface molecule with negative regulatory function on lymphocyte apoptosis. 10 Recent studies have also implicated Toso in IgM binding. 11 The expression of Toso is restricted to lymphoid organs, where it is ...