RIP3 was originally identified as a NF-κB and apoptosis regulator (Kasof et al., 2000
; Pazdernik et al., 1999
; Sun et al., 1999
; Yu et al., 1999
). However, RIP3−/−
mice exhibit no remarkable phenotypes and responded normally to apoptosis and NF-κB activation signals (Newton et al., 2004
). Thus, the biological role of RIP3 was unknown until now. Although RIP3 was not identified as a necrosis mediator in a recent genome-wide RNAi screen (Hitomi et al., 2008
), we show that formation of a unique pro-necrotic Complex II composed of RIP1 and RIP3 is a crucial first step in the induction of programmed necrosis. RIP3 acts upstream to regulate necrosis-specific RIP1 phosphorylation. However, since the level of RIP1 phosphorylation by ectopically expressed RIP3 was low, it remains possible that RIP3 may activate another kinase that directly phosphorylates RIP1.
Interestingly, the RIP1 kinase activity is also required for RIP3 phosphorylation during programmed necrosis. Because ectopically expressed RIP1 did not phosphorylate RIP3, RIP1 may facilitate RIP3 phosphorylation by activating another downstream kinase. In this regard, it is tempting to speculate that the other kinases we identified in the siRNA screen might fulfill this function. Alternatively, RIP1 within the pro-necrotic Complex II might directly phosphorylate RIP3, since in vitro
phosphorylation of RIP3 by Complex II was inhibited by the RIP1-specific inhibitor Nec-1 (Fig. S4
). In addition, ectopically expressed RIP3, but not RIP3 present within the endogenous FADD complex, efficiently phosphorylated the artificial substrate MBP. Thus, the kinase activities of RIP1 and RIP3 are tightly controlled within the context of the pro-necrotic Complex II.
Our results implicate a crucial role for RIP1 and RIP3 phosphorylation in the stable assembly of the pro-necrotic RIP1-RIP3 complex. Intriguingly, transfection of KD-RIP1 or KD-RIP3 did not dominantly inhibit programmed necrosis in cells expressing endogenous RIP1 and RIP3, but rather enhanced TNF-induced programmed necrosis (unpublished observation). The lack of dominant inhibition by KD-RIP1 or KD-RIP3 might suggest oligomerization as a crucial first step in activating the pro-necrotic kinase complex. In such scenario, the RHIM might facilitate oligomerization of RIP1 and RIP3. Phosphorylation of RIP1 and RIP3 may stabilize the structural scaffold of the pro-necrotic complex. However, once the oligomer is formed, a single copy of kinase active RIP1 and RIP3 within the oligomer might be sufficient to activate downstream function. Consistent with this model, RIP3 RHIM mutant was kinase inactive and failed to sensitize programmed necrosis in wild type Jurkat cells (unpublished observation).
The mitochondria generate ROS as a result of oxidative respiration. Disruption of mitochondrial function can further exacerbate ROS release. Interestingly, several components of the mitochondria permeability transition pore (mPTP) are kinases substrates (Le Mellay et al., 2002
; Pastorino et al., 2005
). It is therefore tempting to speculate that RIP1, RIP3 or other downstream kinases may phosphorylate components of the mPTP to disrupt their functions and to trigger an increase in ROS during programmed necrosis. Alternatively, the NADPH oxidase Nox-1 has been shown to mediate ROS production (Kim et al., 2007
). Although Nox-1 was implicated to signal via the TNFR-1 associated complex, it is possible that RIP3 might also interact with Nox-1 within the pro-necrotic Complex II to mediate ROS generation.
TNF has long been known to be an important innate immune effector cytokine against bacterial and viral infections. TNF exerts its anti-microbial effects through induction of apoptosis and NF-κB. Using vaccinia virus infection as a model, we have now established RIP3-dependent programmed necrosis as a third mechanism by which TNF contributes to innate immune control of pathogens. TNF expression was rapidly induced upon VV infection in multiple cell types. The expression of TNF coincided with the formation of the pro-necrotic RIP1-RIP3 complex in the liver of infected mice. In RIP3−/−
mice, virus-induced tissue necrosis and inflammation was severely compromised. Strikingly, the resultant increase in viral replication was comparable to that observed in the TNFR1−/−
mice (Chan et al., 2003
; Ruby et al., 1997
) and much higher than that observed in MyD88−/−
mice (Zhu et al., 2007
). These results are consistent with the normal TLR responses of the RIP3−/−
mice (Newton et al., 2004
) and indicate that the viral disease observed in RIP3−/−
mice was caused by defective TNF signaling rather than abnormal TLR signaling. Our results also support a role for RIP3-dependent programmed necrosis in promoting the subsequent virus-induced inflammation. An important role for RIP1/RIP3-mediated programmed necrosis in anti-viral responses is further bolstered by our previous discovery of certain viral FLIPs that potently inhibit programmed necrosis (Chan et al., 2003
). More recently, the M45 viral cell death inhibitor from murine cytomegalovirus was shown to interact with RIP1 and RIP3 via the RHIM (Upton et al., 2008
). These results suggest that like apoptosis, inhibition of programmed necrosis might be an emerging viral immune evasion strategy.
In addition to virus-induced inflammation, necrotic cell death may have broader roles in regulating other inflammatory processes through the release of “endogenous adjuvants” into the tissue milieu (Kono and Rock, 2008
). For example, RIP3 expression was upregulated during wound healing (Adams et al., 2007
), a biological process that shares some hallmarks of inflammation. In addition, programmed necrosis can directly induce cancer cell death or promote cancer growth and metastasis through its pro-inflammatory effects (Coussens and Werb, 2002
). In this light, it is noteworthy that Non-Hodgkin's lymphomas-associated RIP3 SNPs have been identified (Cerhan et al., 2007
). Thus, RIP3-dependent programmed necrosis may be a significant component in determining the outcome of viral diseases, trauma/injury-induced inflammation and cancers.