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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 material The online version of this article (doi:10.1007/s00018-013-1381-6) contains supplementary material, which is available to authorized users.

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 TNFα, Toso promotes the activation of MAPK and NF-κB signaling pathways. Because of this relative augmentation of survival versus apoptotic signals, Toso raises the threshold for death receptor–mediated apoptosis. Our analysis of Toso-deficient mice revealed that Toso is essential for TNFα-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.

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.

FAN (factor associated with neutral sphingomyelinase [N-SMase] activation) exhibits striking structural homologies to Lyst (lysosomal trafficking regulator), a BEACH protein whose inactivation causes formation of giant lysosomes/Chediak-Higashi syndrome. Here, we show that cells lacking FAN show a statistically significant increase in lysosome size (although less pronounced as Lyst), pointing to previously unrecognized functions of FAN in regulation of the lysosomal compartment. Since FAN regulates activation of N-SMase in complex with receptor for activated C-kinase (RACK)1, a scaffolding protein that recruits and stabilizes activated protein kinase C (PKC) isotypes at cellular membranes, and since an abnormal (calpain-mediated) downregulation/membrane recruitment of PKC has been linked to the defects observed in Lyst-deficient cells, we assessed whether PKC is also of relevance in FAN signaling. Our results demonstrate that activation of PKC is not required for regulation of N-SMase by FAN/RACK1. Conversely, activation of PKC and recruitment/stabilization by RACK1 occurs uniformly in the presence or absence of FAN (and equally, Lyst). Furthermore, regulation of lysosome size by FAN is not coupled to an abnormal downregulation/membrane recruitment of PKC by calpain. Identical results were obtained for Lyst, questioning the previously reported relevance of PKC for formation of giant lysosomes and in Chediak-Higashi syndrome. In summary, FAN mediates activation of N-SMase as well as regulation of lysosome size by signaling pathways that operate independent from activation/membrane recruitment of PKC.

Death receptors such as the 55 kDa tumor necrosis factor (TNF) receptor (TNF-R55) or Fas can initiate both apoptotic (caspase-dependent) and caspase-independent routes to programmed cell death (PCD). Here, we demonstrate for the first time that the single murine receptor for (TNF)-related apoptosis-inducing ligand (mTRAIL-R2) can induce a caspase-independent form of PCD with necrosis-like features in addition to apoptosis. Analysis of morphological and cellular features of caspase-independent PCD in response to TRAIL and TNF suggests that mTRAIL-R2 and TNF-R55 elicit caspase-independent PCD through similar pathways, although without participation of cathepsins. Cells overexpressing acid ceramidase (AC), an enzyme that metabolizes the sphingolipid ceramide, show enhanced survival from TRAIL-induced caspase-independent PCD but not from apoptosis, implicating a function of ceramide as a key mediator in caspase-independent PCD (but not apoptosis) induced by mTRAIL-R2. In concert with the enhanced resistance of AC-overexpressing cells against caspase-independent PCD induced by TNF, our results suggest that ceramide acts as a common mediator of caspase-independent PCD caused by death receptors such as mTRAIL-R2 and TNF-R55.

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.

Tumor necrosis factor (TNF) can induce caspase-dependent (apoptotic) and caspase-independent pathways to programmed cell death (PCD). Here, we demonstrate that stable transfection of a cDNA encompassing the C-terminal apoptosis inhibitory domain (AID) of FE65-like protein 1 into mouse L929 fibrosarcoma cells protects from caspase-independent as well as from apoptotic PCD induced by TNF. We show that the AID does not protect from caspase-independent PCD elicited by 1-methyl-3-nitro-1-nitrosoguanidine, suggesting that the AID might prevent cell death by affecting assembly of the death inducing signaling complex of the 55 kDa TNF receptor or clustering of the receptor itself. Interference with caspase-independent PCD mediated by the sphingolipid ceramide further increases protection conferred by the AID, as does the antioxidant butylated hydroxyanisole, implicating ceramide and reactive oxygen species as potential factors interacting with caspase-independent PCD regulated by the AID.

Tumor necrosis factor (TNF) contributes to insulin resistance by binding to the 55 kDa TNF receptor (TNF-R55), resulting in serine phosphorylation of proteins such as insulin receptor (IR) substrate (IRS)-1, followed by reduced tyrosine phosphorylation of IRS-1 through the IR and, thereby, diminished IR signal transduction. Through independent receptor domains, TNF-R55 activates a neutral (N-SMase) and an acid sphingomyelinase (A-SMase), that both generate the sphingolipid ceramide. Multiple candidate kinases have been identified that serine-phosphorylate IRS-1 in response to TNF or ceramide. However, due to the fact that the receptor domain of TNF-R55 mediating inhibition of the IR has not been mapped, it is currently unknown whether TNF exerts these effects with participation of N-SMase or A-SMase. Here, we identify the death domain of TNF-R55 as responsible for the inhibitory effects of TNF on tyrosine phosphorylation of IRS-1, implicating ceramide generated by A-SMase as a downstream mediator of inhibition of IR signaling.

Factor associated with neutral sphingomyelinase activation (FAN) represents a p55 TNFR (TNF-R55)-associated protein essential for the activation of neutral sphingomyelinase. By means of the yeast interaction trap system, we have identified the scaffolding protein receptor for activated C-kinase (RACK)1 as an interaction partner of FAN. Mapping studies in yeast revealed that RACK1 is recruited to the C-terminal WD-repeat region of FAN and binds to FAN through a domain located within WD repeats V to VII of RACK1. Our data indicate that binding of both proteins is not mediated by linear motifs but requires folding into a secondary structure, such as the multibladed propeller characteristic of WD-repeat proteins. The interaction of FAN and RACK1 was verified in vitro by glutathione S-transferase-based coprecipitation assays as well as in eukaryotic cells by coimmunoprecipitation experiments. Colocalization studies in transfected cells suggest that TNF-R55 forms a complex with FAN and that this complex recruits RACK1 to the plasma membrane. Furthermore, activation of N-SMase by TNF was strongly enhanced when RACK1, FAN, and a noncytotoxic TNF-R55 mutant were expressed concurrently, suggesting RACK1 as a modulator of N-SMase activation. Together, these findings implicate RACK1 as a novel component of the signaling pathways of TNF-R55.

The gap between American and European growth rates vanishes. But there is little evidence that the old countries can go ahead in the near future. During the past three years American investors have done a lot. Housebuilding and the service sector fully benefited by it, but many manufacturing activities, under pressure of competition, also gained in strength. Out from the big european countries, Germany alone strove to modernize accordingly and displays some optimism. Would the dollar fall be confirmed, the external constraint still imposed to several countries will be relieved, and consumption stimulated through reduced inflation. In return competition will increase, demand from developing countries will drop and there will be little ground left for exports. French growth, albeit more sustained next year, will still show signs of lagging. Consumption is drawn by tax cuts and a weakening saving propensity. Net borrowing will be narrowed with curtailed expenses of the central government and still low purchases of new dwellings by households. As for firms, debt funding and cautious investment will be carried on. A lower inflation and some linkage of financial and monetary markets will help the easing of interest rates. Trade gap could be reduced and long term foreign debt would stop growing., Le différentiel de croissance entre les États-Unis et l'Europe s'estompe, mais il n'est pas pour autant assuré que les pays du vieux continent prennent prochainement les devants. Au cours des trois dernières années les Américains ont massivement investi. Bien que le logement et les activités tertiaires y aient pris une large part, l'industrie, pressée par la concurrence, s'est aussi renforcée en de nombreux points. Parmi les grands pays européens seule la RFA a pu conduire un effort de modernisation comparable et affiche un certain optimisme. Le baisse du dollar, si elle se confirme, allégera la contrainte extérieure qui s'imposait encore à plusieurs pays et, en ralentissant l'inflation, stimulera la consommation. Par contre la concurrence va s'intensifier sur le marché européen, la demande des pays en développement fléchira, et les places à l'exportation seront chères. En France la croissance restera en retrait de celle de nos partenaires, quoique plus soutenue l'an prochain. La consommation bénéficie des allégements fiscaux et d'une propension à épargner qui s'affaiblit. Les besoins de financement de l'économie se réduisent car l'Etat freine ses dépenses et les ménages achètent peu de logements neufs. Quant aux entreprises elles consolident leurs bilans et investiront encore prudemment en 1986. La baisse des taux d'intérêt sera encouragée par celle de l'inflation et le décloisonnement des marchés financiers et monétaires. Le déficit commercial pourrait être réduit et la dette extérieure à long terme stabilisée., Département des diagnostics de l'OFCE, Sigogne Philippe, Fouet Monique, Mathieu Sabine, Milewski Françoise, Cocker Michael, Dujust Catherine, Gubian Alain. Revenus et concurrence accrus. In: Observations et diagnostics économiques : revue de l'OFCE, n°13, 1985. pp. 5-88.

The tightening of monetary policy in industrialised countries from 1980 onwards is one of the causes of the subsequent slowdown in economic growth along with inflation and the reduction of international liquidity. This trend will continue in the near future. Despite a smooth reduction in the growth rate, the needs of the american economy in foreign capital will remain substantial. This drain on foreign savings is likely to hamper recovery elsewhere and maintain the strong dollar. Since the second oil shock, the underlying trend of growth has been 1 % p. a. higher in the US than in Europe as a whole and France in particular. The French economy, which was in phase with the rest of the EEC in 1983, shows signs of lagging. This should be more evident in 1985 than 1984 but insufficient to close the trade gap. There will be a gradual easing of economic policy in 1985. Consumer expenditure, which has been maintained over recent months only by a fall in the saving ratio, should pick up with the reduction of tax levels. Investment should continue to recover thanks to public funding and an improvement in profits but will be restricted to the modernisation of industry. With fewer jobs on offer and an increasing labour force, the rise in unemployment is unlikely to be stemmed., Les politiques monétaires des pays industrialisés sont devenues restrictives au début des années quatre-vingt, ce qui a été l'une des causes du ralentissement tant de l'activité que de l'inflation mondiale et de la raréfaction des liquidités disponibles pour des financements internationaux. Elles demeureront rigoureuses au cours des prochains trimestres. Les États-Unis continueront à faire largement appel aux capitaux étrangers, malgré un ralentissement ordonné de leur croissance. Cette ponction sur l'épargne mondiale risque de maintenir le taux de change du dollar à haut niveau et de freiner la reprise économique des autres pays. Depuis le second choc pétrolier la croissance tendancielle européenne est inférieure de 1 % par an à celle des États-Unis. Il en est de même pour la France, qui en 1983 était en phase conjoncturelle avec ses partenaires de la CEE et accuse un léger retard en 1984. Celui-ci serait plus sensible en 1985, insuffisant toutefois pour supprimer le déficit extérieur. En 1985 la politique de rigueur ira en s' atténuant : les allégements fiscaux permettraient aux ménages d'accroître à nouveau leurs dépenses de consommation, qui s'étaient maintenues ces derniers mois au prix d'un recul de l'épargne. Le redressement de l'investissement pourrait se poursuivre grâce à l'amélioration de la situation financière des entreprises et aux financements publics, mais il serait encore limité à la seule modernisation de l'industrie. La montée du chômage sera difficilement contenue à cause du retour à des taux d'activité plus élevés et de la réduction des offres d'emploi., Département des diagnostics de l'OFCE, Sigogne Philippe, Mathieu Sabine, Radisson Vincent, Aroyo Philippe, Fouet Monique, Cocker Michael, Gubian Alain, Roy Daniel, Milewski Françoise. Différentiel de croissance entre les États-Unis et l'Europe. In: Observations et diagnostics économiques : revue de l'OFCE, n°9, 1984. pp. 5-80.

After the first oil shock, the Italian economy experienced strong growth. There was extensive investment for the modernization of plant and equipment. Since 1981, Italy has been in a recession. In the first half of 1983, activity did not benefit from the support of consummer spending since the growth of disposable income was limited by economic policy ; at the same time foreign demand remained weak. Higher consumer spending the end of the fall in investment and inventories and stronger world demand probably account for the apparent improvement in the second half of 1983. Over the 1980-1983 period, certain conditions of a healthy economy have been obtained. The pace of inflation has slowed. The adjustment of production structures is under way. The foreign trade deficit has been reduced. However, the extent of the budget deficit still weighs on activity. Also, despite the support of foreign demand, the expected improvement for 1984 will remain modest. The external trade deficit nonetheless will be unchanged in 1984 because of the growth of imports. In the long term, the balance is likely to deteriorate if penetration of the domestic market is not slowed or counterbalanced by a geographic and product-based reorientation of exports. Appendices : forecast results and basic charts., L'économie italienne avait connu après le premier choc pétrolier, une croissance forte. Des investissements importants étaient orientés vers la rationalisation de l'appareil productif. Depuis 1981 l'Italie est en récession. Au premier semestre 1983 l'activité n'a pas bénéficié du soutien de la consommation des ménages, la croissance du revenu disponible étant limitée par la politique économique ; la demande extérieure est restée faible. Au deuxième semestre 1983 un rattrapage a sans doute eu lieu : la situation des ménages s'est améliorée, la chute des investissements et le déstockage se sont arrêtés, la demande extérieure s'est redressée. L'évolution des années 1981 à 1983 aura permis de créer certaines des conditions de l'assainissement. Le rythme de la hausse des prix s'est ralenti. L'ajustement des structures productives est en cours. Le rééquilibrage des échanges avec l'extérieur est engagé. Cependant, l'ampleur du déficit budgétaire pèse encore sur l'activité. Aussi l'amélioration attendue pour 1984 restera modeste, malgré le soutien de la demande externe. Le déficit des échanges extérieurs sera toutefois inchangé en 1984, en raison de la croissance des importations. A plus long terme, le solde risque de se détériorer si la pénétration du marché intérieur n'est pas freinée ou contrebalancée par une réorientation géographique et sectorielle des exportations. En annexes : prévisions quantitatives et diagrammes de base., Mathieu Sabine. Italie : difficile sortie de récession. In: Observations et diagnostics économiques : revue de l'OFCE, n°6, 1984. pp. 39-67.

Background In 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. Results The 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. Conclusions The 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.