Mechanistic Events in Necrosis

Evidence is accumulating to support the notion that necrosis, like apoptosis, is a mechanistic cell-death process. For instance, necrosis can also be induced by death ligands such as TNF and Fas, redox signaling pathways involving ROS generation, stress-activated protein kinases such as JNK and p38, and release of mitochondrial factors [77, 100, 104, 105]. Anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-xL delay or protect against necrosis induced by a variety of insults in different cell lines [77]. Recent studies conducted in our laboratory indicate that the transcription co-activator and stress-regulated protein lens epithelium-derived growth factor p75 (LEDGF/p75) protects cultured mammalian cells from necrosis induced by tert-butyl hydroperoxide (unpublished observations). This protection appears to be mediated by transcriptional upregula-tion of antioxidant proteins.

As in apoptosis, proteases also play a major role in the execution of necrosis. While the role of caspases in apoptosis is well established, it was not until recently that the morphological changes associated with necrosis have been linked to the activation of non-caspase proteases such as the calcium-dependent cal-pains and the lysosomal cathepsins [65, 74]. The elevation of intracellular free calcium during certain pathological processes leads to activation of calpains, phospholipases, and endonucleases; alteration of membrane protein and lipid; generation of toxic ROS; and mitochondrial disruption [65]. Excessive activation of calpains has been associated with lysosomal membrane disruption, leading to the release of cathepsins into the cytoplasm with the resultant cell autolysis [65].

Important insights into biochemical mechanisms associated with necrotic cell death have been obtained using the murine L929 fibrosarcoma cell line. Upon exposure to TNF, L929 cells die preferentially via a slow necrotic process [100]. This necrosis is dependent on the death domain of TNFR-55 [114]. Treatment with TNF in the presence of broad caspase inhibitors such as Z-VAD-fmk or inhibitors of caspase-8 and caspase-3 dramatically sensitizes these cells to necrotic cell death [100, 115]. These inhibitors also sensitize L929 cells overexpressing the human Fas receptor to necrosis, although less dramatically, if these cells are treated with agonistic anti-Fas antibody [115]. Anti-Fas antibody and forced mul-timerization of the adaptor protein FADD (Fas-associated protein with death domain), which is required for initiating the caspase-8-mediated apoptotic pathway, have also been shown to induce necrosis in a caspase-8-deficient subline (JB6) of Jurkat T cells [114-117]. While there is no evidence that caspase-8 activation is required for death receptor-induced caspase-independent cell death with a necrotic phenotype, it appears that inactivation of caspase-8 or deficiency of this caspase does favor this death process [114-118]. Whether inactivation of other initiator caspases favors death receptor-induced necrosis remains to be established, although inhibition of the effector caspase-3 has been implicated in this process [100]. Other apoptosis-inducing ligands such as TRAIL have also been shown to cause necrosis [90]. Death receptor-induced necrosis is mediated by the RIP kinase, which is thought to phosphorylate and regulate a yet unidentified key factor involved in the necrotic process [11, 90].

Death receptor-induced necrotic cell death under caspase-inhibition conditions appears to be mediated by the generation of ROS in the mitochondria because it can be partially inhibited by antioxidants [77, 110]. Although the mechanisms by which caspase inhibition potentiates the death receptor signaling in L929 cells are not entirely clear, various studies have demonstrated that poly (ADP-ribose) polymerase (PARP) acts as a molecular switch between apoptosis and necrosis in these cells [119, 120]. According to these studies, excessive ROS generation leads to profound DNA damage, which in turn leads to excessive PARP activation and increased poly-ADP-ribosylation. The use of ATP for the synthesis of the PARP substrate NAD+ then leads to a dramatic depletion of intracellular ATP, which results in necrotic cell death [120]. It should be noted that PARP is unable to respond in a similar fashion to DNA damage caused by apoptotic stimuli because it is cleaved and inactivated by effector caspases very early on in the apoptotic process, which prevents ATP depletion and ensures a well-controlled, non-inflammatory cell demise [121, 122].

How do the events mentioned above lead to lysosomal rupture, which appears to be the critical event for triggering the typical necrotic morphology? Recent studies show that under persistent oxidative stress conditions, intralysosomal labile iron catalyzes Fenton reactions, which result in rupture of lysosomal membranes and subsequent efflux of iron and cathepsins into the cytoplasm [123, 124]. From there, cathepsins can relocate to the cytoplasm and nucleus where they cause proteolysis of limited substrates, leading to cytoplasmic destruction and plasma membrane permeabilization. Consistent with this, Ono et al. [74] demonstrated that TNF induces rupture of lysosomes in L929 cells, leading to plasma membrane disruption. The rupture of lysosomes that precedes the appearance of the necrotic morphology is a molecular event that can be regulated. A recent study demonstrated that heat shock protein 70 (HSP70), a stress protein that is known to inhibit apoptosis, also inhibits necrotic cell death by concentrating in lysosomes and preventing their rupture through interactions with components of lysosomal membranes [125]. Taken together, these observations indicate that apoptosis and necrosis share some biochemical mechanisms and strengthen the notion that the capability of the cell to die via these pathways and their variants is essential to ensure its elimination when absolutely necessary.

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