Abstract SY27-02: In vitro and in vivo regulation of regulated necrosis induced by TNF
Authors: Peter Vandenabeele
Journal: Cancer Research (2012)

Abstract Cells can die by multiple ways. Two prototypes of regulated cell death have been put forward viz. apoptosis and programmed necrosis. Nowadays, programmed necrosis, also called regulated necrosis or necroptosis (1), is recognized as a highly controlled form of necrotic cell death (2). Using the L929 fibrosarcoma cellular system of TNF-induced necrotic cell death, we identified crucial subcellular events during necroptosis such as mitochondrial ROS production and lysosomal membrane permeabilization (LMP) (3). The initial events that lead to TNF-induced necrosis involve the activation of the serine threonine kinases RIPK1 and RIPK3, which initiate the formation of the socalled necrosome complex due to homotypic interaction between the RHIM domains. Also other stimuli such as TLR3 and TLR4 ligands have been shown to require RIPK3 for regulated necrosis in macrophages, while in this cellular context RIPK1 fullfils a survival function (4). Although a direct link between RIPK1/RIPK3 kinase activity and the downstream subcellular events at the mitochondrial and lysosomal level remain to be established, at the moment more is known on the regulation of the formation of necrosome complex containing FADD, caspase-8, RIPK1 and RIPK3. This TNF-induced complex formation is highly regulated by ubiquitylation/deubiquitylation balances involving ubiquitylating (a.o. IAP, A20) and deubiquitylating enzymes (CYLD), kinases (a.o. RIP1, RIP3, TAK1) and caspase activating platforms (FADD, FLIPL, caspase-8) (5,6). Recently, other stimuli like etoposide and Smac mimetics where shown to induce spontaneously a cytosolic complex called complex called ripoptosome independent of endogenous TNF production and containing FADD, caspase-8, RIPK1 and RIPK3 (7,8). These regulatory mechanisms involving ubiquitylation, phosphorylation and caspase-8-mediated proteolysis desensitize/sensitize and retard/accellerate necroptosis, like brakes and gears on the necrotic program. They determine the cellulular outcome being survival, apoptosis or necroptosis, and apparently also operate in vivo (9,10,11) and are involved in hyperinflammation in gut (12,13) and skin (14). We studied whether the regulatory mechanisms of TNF-induced necroptosis identified in vitro were also applicable for in vivo. Therefore we injected high dose of TNF causing systemic inflammatory response syndrome (SIRS), resulting in septic shock. We demonstrated that deletion of apoptotic executioner or inflammatory caspases had no impact on lethal SIRS despite lowered levels of apoptosis or circulating IL-1β. In contrast, deletion of the RIPK3 gene conferred complete survival and protection against necrotic cell death, reflected by reduced levels in the serum of mitochondrial DNA, lysosomal enzymes and other markers of organ damage. Interesting, also circulating inflammatory cytokines such as IL-6 and IL-1 were dramatically lowered in RIPK3 knockout mice, demonstrating that induction of necroptosis is preceding excessive inflammatory cytokine production. Pretreatment with the RIPK1 kinase inhibitor, necrostatin-1, had a similar protective effect on mortality and resulted in reduced levels of markers of organ damage and circulating inflammatory cytokines. These results demonstrate that RIPK1/RIPK3-mediated necroptosis plays an indispensable role in TNF-induced SIRS as the determinant between life and death. The crucial role of necroptosis in SIRS in infectious sepsis was further underscored by the protective effect of RIPK3 deficiency in caecal ligation puncture (CLP) model. Altogether, our findings demonstrate that regulated necrosis or necroptosis in vivo is a crucial process and that components of the necroptotic cell death pathway are potential therapeutic targets for treatment of SIRS and sepsis (15). References 1. Galluzi et al. Cell Death Differ. 19 (2012), 107-120. 2. Vandenabeele et al. Nat. Rev. Mol. Cell. Biol. 11 (2010), 700-714. 3. Vanden Berghe et al. Cell Death Differ. 17 (2010), 922-930. 4. He et al. Proc. Natl. Acad. Sci. 108, (2011), 20054-20059. 5. Vanlangenakker et al. Cell Death Differ. 18 (2011), 656-665. 6. Vanlangenakker et al. Cell Death Dis. 2 (2011), e230. 7. Tenev et al. Mol. Cell 43 (2011), 432-448. 8. Feoktistova et al. Mol. Cell 43 (2011), 443-469. 9. Kaiser et al. Nature 471 (2011), 368-371. 10. Oberst et al. Nature 471 (2011), 363-367. 11. Zhang et al. Nature 471 (2011), 373-376. 12. Welz et al. Nature 477 (2011), 330-334. 13. Günther et al. Nature 477 (2011), 335-339. 14. Bonnet et al. Immunity 35 (2011), 572-582. 15. Duprez et al., Immunity 35 (2011), 908-918. Acknowledgement Peter Vandenabeele has got a Methusalem Grant (BOF09/01M00709) from the Flemish Government. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY27-02. doi:1538-7445.AM2012-SY27-02