Structural Biochemistry/The Molecular Regulation of Programmed Necrotic Cell Injury
Proper regulation of cell death is essential for human development and cell function. Unlike apoptosis, necrosis is a more inflammatory form of cell death that might contribute to anti-viral immunity. Indeed, necrotic cell injury is distinguished from apoptosis by extensive organelle and cell swelling and plasma membrane rupture. Recent evidence indicates that an elaborate biochemical network emanating from receptors in the TNF superfamily can induce apoptosis as well as necrotic cell death. The induction of necrosis by TNF-like cytokines requires biochemical components that are distinct from those involved in apoptosis. Specifically, serine/threonine protein kinases in the receptor interacting protein (RIP) family are required for “programmed” necrotic cell injury. The paper discusses the newly discovered pathway towards necrosis and the molecular crosstalk between apoptosis and programmed necrosis, with a special emphasis on how caspases, protein ubiquitylation, and phosphorylation regulate the induction of necrotic cell injury.
Programmed Necrotic Cell Death 
The balance between cellular proliferation and cell death is critical for homeostasis of higher organisms. Pathologists have long relied on morphology alone to distinguish different forms of cell death. The advent of molecular biology greatly enhanced our knowledge of the biochemical regulation of apoptosis. By comparison, our understanding of the biochemical pathways that regulate non-apoptotic cell death programs such as necrosis remained scarce. Until recently, the prevalent view was that cellular necrosis is the consequence of nonspecific cell injury from trauma.
However, with the identification of dedicated molecular machinery regulating the process, the study of necrosis has greatly advanced. “Programmed necrosis” is the new term deemed, in order to distinguish necrosis induced by tumor necrosis factor (TNF) family death cytokines from those induced by non-specific trauma or injury (e.g. heat shock). Other terms, including “necroptosis”, have also been used to describe receptor interacting protein 1 (RIP1)-dependent, death cytokine-induced programmed necrosis [1,2]. This paper focuses on the recent advances in understanding the molecular regulation of programmed necrosis induced by TNF-like cytokines.
Molecular Crosstalk Between Apoptosis and Necrosis 
TNF-like death cytokines can signal and cause three different outcomes: nuclear factor kappa-B (NF-κB) activation, apoptosis, or programmed necrosis. Evidence indicates that the activation of one response often opposes the others.
Under most circumstances, TNF stimulation results in NF-κB activation rather than cell death. However, when NF-κB activation is inhibited, either by macro-molecular synthesis inhibitors or by expression of a dominant negative mutant of the negative regulator IκBα, TNF induces apoptosis. These systems were widely used to study the mechanism of TNF-induced apoptosis and contributed to the perception that TNF predominantly induces apoptotic cell death.
However, a very early study of TNF showed that it causes solid tumor regression in the form of necrotic cell death. In the late 1990s, it was realized that caspase inhibition does not always inhibit TNF or Fas ligand (FasL)-induced cell death in certain cell types. Rather, caspase inhibition often led to necrosis marked by cell organelle swelling and rupture of the plasma membrane. In addition, expression of a dimerized FADD death domain (FADD-DD), which inhibits FasL- and TNF-induced apoptosis, triggered caspase-independent cell death with characteristic “necrotic” morphology. This reveals that FADD and caspase 8 deficiencies potently sensitize cells to programmed necrosis. These results clearly illustrate that the molecular pathways regulating cell death by apoptosis and programmed necrosis are intimately intertwined.
RIP1: a pleiotropic kinase controlling cell survival and cell death signals 
A breakthrough in the study of programmed necrosis came when several groups described that the serine/threonine kinase RIP1 plays an obligate role in mediating programmed necrotic cell death induced by FasL, TNF, TRAIL (TNF-related apoptosis-inducing ligand). Early studies indicated that RIP1 plays an important role in the activation of NF-κB.
Nonetheless, RIP1 polyubiquitylation at lysine 377, which lies in the intermediate domain appears to be essential for NF-κB activation. By contrast, an intact kinase domain is crucial for RIP1-mediated programmed necrosis, but dispensable for NF-κB activation. Thus, RIP1 utilizes distinct domains to activate NF-κB and programmed necrosis.
Although RIP1 is normally dispensable for death cytokine-induced apoptosis, recent evidence indicates that RIP1 can facilitate apoptosis under certain circumstances. In addition to programmed necrosis, RIP1 kinase activity is also required for apoptosis induced by IAP antagonists.
RIP1-RIP3 Complex 
The fact that RIP1 activates signaling pathways other than programmed necrosis suggests that additional mechanisms must exist to specifically regulate or mediate its pro-necrotic pathway. Recently, two separate RNA interference (RNAi) screens identified another RIP family kinase, RIP3, as an essential mediator for TNF-, FasL- and TRAIL-mediated programmed necrosis. New studies demonstrate the importance of RIP3 in programmed necrosis and are consistent with an early report which showed that RIP3 over-expression induces apoptotic as well as necrotic cell death. Thus, RIP3 is an essential inducer of programmed necrosis.
TNFR1 (TNF receptor 1) signaling is mediated through two spatially and temporally separate signaling complexes: a transient and unstable receptor-associated signaling complex termed “Complex I”, and a slow-forming receptor-independent cytoplasmic complex termed “Complex II." Whereas RIP1 is recruited to both complexes, RIP3 only binds RIP1 within a “pro-necrotic” Complex II. The assembly of the pro-necrotic RIP1–RIP3 complex is specifically induced during TNF-induced programmed necrosis, but not during apoptosis or NF-κB activation. The RIP1–RIP3 interaction results in the induction of their kinase activities. The paper shows different studies that emphasize the central role of RIP3 in the necrotic signaling pathway and indicate that programmed necrosis can proceed in a RIP1-independent manner. By contrast, RIP1-driven necrosis that is independent of RIP3 has not been observed.
The assembly of the pro-necrotic RIP1–RIP3 complex is mediated through the “RIP homotypic interaction motif” (RHIM). The RHIM represents an emerging protein–protein interaction motif whose structure is undefined at present. Questions remain regarding whether RIP1 or RIP3 is the upstream activator in the necrotic signaling cascade, but several lines of evidence favor RIP1 as the upstream kinase. These results argue that RIP3 also regulates RIP1 function. Additional experiments will be needed to unequivocally determine the order in which RIP1 and RIP3 are activated.
Role of FADD and Caspases 
Caspase inhibition has been observed in malignant diseases and during certain viral infections. Under these conditions, TNF-like cytokines might preferentially induce programmed necrosis. However, it is important to remember that programmed necrosis can proceed in the absence of caspase inhibition.
Although further work is needed to validate whether RIP3 is cleaved and inactivated by caspase 8 within Complex II, these results are in agreement with the model that preservation of the integrity of both RIP1 and RIP3 is important for optimal induction of programmed necrosis. Because the pro-necrotic RIP1–RIP3 complex is formed as a consequence of the receptor-associated complex, FADD deficiency might preferentially inhibit necrotic signaling by Fas and TRAIL receptors.
Similar to the cell variants that lack FADD expression, expression of FADD death domain alone (FADD-DD), which dominantly inhibits caspase-dependent apoptosis, also sensitizes cells to programmed necrosis.
Protein Ubiquitylation Role in Programmed Necrosis 
Protein ubiquitylation is an important process that regulates numerous signal transduction pathways. Many proteins in the TNF signaling pathway are targets of ubiquitylation. RIP1 is heavily modified through polyubiquitylation. Polyubiquitylated RIP1 mediates activation of the pro-survival transcription factor NF-κB. Several interesting recent reports indicate that polyubiquitylated RIP1 provides an early anti-death signal that is independent of NF-κB activation. The protection conferred by polyubiquitylated RIP1 appears to act through preventing the RIP1–caspase 8 interaction.
Could polyubiquitylated RIP1 similarly protect cells against programmed necrosis? It is tempting to speculate that RIP1 polyubiquitylation sterically hinders the recruitment of downstream pro-necrotic proteins such as RIP3. However, it remains unknown whether RIP3 polyubiquitylation is an important regulatory mechanism in programmed necrosis.
Effector Mechanisms in Programmed Necrosis 
Although it is clear that caspase-mediated cleavage of cellular proteins causes apoptotic death, much less is known about the mechanisms by which programmed necrosis kills cells. The most remarkable morphological feature of programmed necrosis is the organelle and cell swelling that culminates in rupture of the plasma membrane. The increase in cell volume and extensive intracellular vacuole formation implies an imbalance in osmotic pressure. Reactive oxygen species (ROS) production is an important effector killing mechanism for programmed necrosis. ROS can induce lipid peroxidation or alter the function of certain channel proteins, both of which can lead to necrotic cell injury.
The mitochondria are major producers of ROS. Consistent with a role for mitochondrial ROS in programmed necrosis, inhibition of the mitochondrial complex I and complex II protects against TNF-induced programmed necrosis. RIP1 and RIP3 act upstream to regulate ROS production during programmed necrosis [27,29]. Although the results are controversial, both RIP1 and RIP3 were reported to localize to the mitochondria. RIP3 overexpression activates the activities of these enzymes and enhances mitochondrial energy metabolism, which correlates with enhanced ROS production and programmed necrosis. Thus, the RIP1–RIP3 complex might directly regulate ROS production from the mitochondria by engaging the mitochondrial metabolism machinery.
In contrast to the evidence supporting mitochondrial production of ROS, several recent reports indicate that the plasma membrane-associated NADPH oxidase 1 (NOX1) forms a complex with TRADD, RIP1 and the small GTPase RAC1 to regulate ROS production at the plasma membrane. The disparate results might be reconciled by the TNF-induced shuttling of NADPH oxidases between the cytoplasm and the plasma membrane.
Rather, a RIP1-dependent loss of cellular ATP was reported to precede programmed necrosis in U937 cells. Thus, the cellular metabolic “fitness level” might greatly influence the outcome of pro-necrotic cell signaling, a notion that seems to be supported by the correlation between reduced cellular ATP level and increased susceptibility to necrotic cell injury in older individuals. These recent findings suggest that different effector mechanisms operate in different cell types to mediate programmed necrosis.