A novel role for the apoptosis inhibitor ARC in suppressing death receptor-mediated necrosis

Abstract

Apoptosis and necrosis are two morphologically distinct forms of cell death that have been described. Apoptosis has long been recognized as a form of regulated cell death. In contrast, necrosis has traditionally been viewed as cell death that is unregulated and passive that occurs due to excessive trauma or stress. Yet, increasing evidence has emerged to indicate that necrotic cell death can be actively controlled. Regulated necrosis can be stimulated by the same death ligands that activate apoptosis, such as tumor necrosis factor alpha (TNFalpha) and Fas ligand. Programmed necrosis initiated by the ligation of TNF receptor 1 (TNFR1) has been most extensively characterized. Activation of TNFR1 can promote cell survival mediated by Nuclear Factor-kappaB (NF-kappaB) activation, apoptosis, or necrosis depending on cell type and the environment. Although the mechanism that mediates the selection amongst these outcomes is not well understood, a basic tenet of this decision-making is that inhibition of apoptosis obligates the cell to undergo necrosis. ARC (Apoptosis Repressor with a CARD (Caspase Recruitment Domain)) is an endogenous apoptosis inhibitor that antagonizes both the mitochondrial and death receptor apoptosis pathways through protein-protein interactions involving its CARD, a death-fold motif. In keeping with the existing paradigm, we hypothesized that ARC would inhibit apoptosis and thereby promote necrosis induced by the death receptor pathway.;To test this hypothesis, we employed a well-defined system utilizing L929 murine fibrosarcoma cells, in which TNFalpha can induce either apoptosis or necrosis. In these cells, necrosis is elicited through the administration of TNFalpha alone or in conjunction with pan-caspase inhibitor z-VAD-fmk. Conversely, the application of TNFalpha along with the protein synthesis inhibitor cycloheximide results in apoptosis. Assays that detect cellular release of high mobility group box protein 1 and lactate dehydrogenase and entry of propidium iodide were used to monitor cellular loss of plasma membrane integrity, a defining feature of necrosis. Poly ADP-ribose polymerase cleavage and caspase-3 cleavage are markers of apoptosis that were used to assess apoptotic cell death. Surprisingly, we found that overexpression of ARC suppresses, rather than promotes, TNFalpha-induced programmed necrosis, while retaining its ability to block apoptosis. Overexpression of the CARD-defective L31F; G69R double-point mutant form of ARC that is deficient in inhibition of apoptosis, abrogates the ability of ARC to suppress TNFalpha-induced necrosis. In line with this finding, the knockdown of ARC exacerbates TNFalpha-induced necrosis, an effect that can be rescued by reconstitution of knockdown cells with wild type, but not the CARD-defective mutant, ARC. Moreover, using fractionation and NE-kappaB p65 ELISA binding assays, we found that ARC inhibits NF-kappaB activation, another event downstream of TNFalpha stimulation, in a CARD-dependent manner. Together, these data indicate that the CARD of ARC is required in suppressing downstream TNFalpha-TNFR1 signaling. Furthermore, through immunoprecipitations, we discovered an interaction between ARC and TNFR that inhibited the recruitment of RIP1, a critical mediator of TNFalpha-induced necrosis.;To test the relevance of the effect of ARC in TNFalpha-induced necrosis pathway in vivo, we infected wild type mice and mice lacking ARC with vaccinia virus, which has been demonstrated previously to induce necrosis through a pathway mediated by TNFalpha. We found that mice lacking ARC exhibited markedly increased necrosis/inflammation in the adipose tissue compared with wild type controls, paralleling our results that indicate the role of ARC in limiting necrosis.;These studies demonstrate that ARC suppresses TNFalpha-induced necrosis and apoptosis by interacting with TNFR1 and disrupting TNFalpha signaling. Given that both apoptosis and necrosis play important roles in multiple diseases, such as myocardial infarction and stroke, ARC may provide pharmacological strategies to maintain tissue viability in these ischemic syndromes.