TNF-α is a pleiotropic cytokine that induces diverse biological responses ranging from inflammation to cell death. In most cells, TNF-α induces apoptosis, but under certain conditions it stimulates programmed necrosis (13
). The mechanism by which the RIP1-RIP3 necroptotic complex triggers ATP loss has remained elusive since the discovery of TNF-α-mediated programmed necrosis. Here, we demonstrated that the TNF-α-dependent RIP1-RIP3 necroptotic complex alone is insufficient to elicit mitochondrial dysfunction, ATP loss, and programmed necrosis. TCZ-mediated necroptosis is dependent on the TNF-α signaling molecules FADD, RIP1, RIP3, and NEMO. This necroptotic complex leads to ROS production and mitochondrial dysfunction. This finding is important because mitochondria not only participate in apoptosis but also are involved in necroptosis. The recently described proinflammatory cytokine-induced necroptosis requires a unique RIP1-RIP3 pathway that impinges on cell function (10
). The system described in our study might serve as a useful system for monitoring the translocation of a cytosolic factor(s) to the mitochondria that induces mitochondrial dysfunction during necroptosis.
Cells of the human Jurkat cell line lacking FADD undergo necroptosis via RIP1 and RIP3 by TNF-α (10
). Primary T cells lacking either FADD or caspase 8 undergo autophagy, and RIP1 inhibition prevents cell death (4
). Interestingly, necroptosis of FADD-deficient T cells failed to induce RIP1 cleavage or a RIP1-RIP3 complex formation (57
). In contrast, MEFs lacking FADD are resistant to TNF-α-induced necrosis, and reconstitution of FADD restored both the apoptotic and necrotic phenotype induced by TNF-α (39
). Here, we extend these studies to show that FADD is crucial for formation of the RIP1-RIP3 complex in fibroblasts. The bioenergetic parameters, including the mitochondrial respiration rate, ΔΨm
, and ATP levels, are preserved in TCZ-challenged FADD−/−
MEFs (, , , and ). RIP-family proteins play important roles in both cell survival and cell death (50
). However, genetic ablation of RIP3 in mice had no physiological phenotype (54
), but deletion of RIP1 caused severe death in lymphoid and adipose tissue and mice exhibited postnatal lethality (32
). RIP1 but not RIP3 is indispensable for NF-κB activation in response to TNF-α (10
). Genetic ablation of TNF-α signaling downstream candidates RIP1−/−
(RIP KO) and RIP3 (siRNA-mediated knockdown) prevents the TCZ-induced mitochondrial dysfunction (, , , , and ), indicating the contribution of the RIP1-RIP3 complex in mitochondrion-dependent necroptosis. In addition, cytosol exchange experiments also demonstrate that RIP1 is essential for ΔΨm
loss (). TNF-α-induced RIP1-FADD-caspase 8 complex formation and the following recruitment of RIP3 exquisitely potentiate necroptotic cell death, while disruption of this complex by necrostatin-1 effectively inhibits necroptosis (28
). Consistent with previous findings, prevention of the necroptotic complex by the RIP1 inhibitor Nec-1 inhibits ΔΨm
loss and cell death (A, I, J, and K). TNF-α-induced caspase 8 activation results in RIP1 cleavage and halts the necrotic cell death but facilitates apoptosis (40
). Our study suggests that cells lacking caspase 8 undergo a delayed ΔΨm
loss in a permeabilized system and considerable loss of ΔΨm
and ATP levels in an intact system after TCZ treatment (, , , and ). Though TCZ-induced dissipation of ΔΨm
and ATP levels occurs in caspase 8−/−
MEFs, the plasma membrane integrity failed to deteriorate, suggesting that the NEMO-FADD-RIP1-RIP3 complex could still cause bioenergetic collapse ( and ). TNF-α–CHX-induced apoptosis i s defective in caspase 8−/−
MEFs (data not shown). It is possible that the absence of caspase 8 could delay the plasma membrane permeability in necroptosis (F, J, and K). On the basis of less ROS burst in caspase 8−/−
MEFs, we speculate that ROS-mediated lipid peroxidation may contribute to plasma membrane integrity loss in necroptosis.
TNF-α-induced NF-κB activation requires NEMO, IKKβ-dependent IκB phosphorylation and degradation and dissociation of cytosolic NF-κB to form a heterodimer. Cells lacking NEMO still maintain modest NF-κB2 activity via a noncanonical pathway that requires IKKα and NF-κB-inducing kinase (NIK) for cell survival (11
). The results presented here indicate that cells lacking NEMO are insensitive to TCZ challenge and that the mitochondrial bioenergetics remain intact (, , , , and ). Further, pharmacologic blockage of NF-κB with an IKKβ inhibitor prevented the reduction of ATP levels by necroptosis (F). Although the involvement of IκBα kinases in TNF-α-induced necroptosis is an interesting area to be investigated, our findings suggest that upstream NF-κB molecules IKKβ and NEMO but not RelA likely participate in TNF-α-induced mitochondrial dysfunction.
The mitochondrion is an organelle with two well-defined compartments, the matrix and the intermembrane space, which are bounded by the inner mitochondrial membrane (IMM) and the outer mitochondrial membrane (OMM), respectively. In order to maintain the function and metabolism of the mitochondria, the inner membrane is highly selective and permeable to limited ions and metabolites. The highly selective nature of the inner membrane allows the establishment of a proton-motive electrochemical force across the mitochondrial inner membrane, which can be converted by F0
ATP synthase into ATP. Mitochondrial PTP opening consists of a voltage-dependent anion channel, cyclophilin D, and adenine nucleotide translocase (ANT) and is implicated in necrosis (1
). However, disagreement continues as to the role of the voltage-dependent anion channel (VDAC) in PTP opening (2
). Furthermore, studies demonstrated that mice lacking the Ppif gene (cyclophilin D) are resistant to Ca2+
-dependent PTP opening (1
). The most abundant mitochondrial ANT has two states, the cytosolic state (c state) and the matrix state (m state) (62
). It plays a vital role in ATP flux from mitochondria. However, ANT can switch to a lethal function corresponding to its pore-forming activity. Direct application of zVAD in a permeabilized system did not induce PTP opening or ΔΨm
loss in either control ot TNF-α-stimulated cells. In contrast, ANT inhibitor atractyloside induced a rapid mitochondrial membrane depolarization (data not shown). These results indicate that zVAD may not have a direct effect on the mitochondria. We postulate that manipulation of the ANT isoforms with zVAD has little or no impact on the mitochondrial membrane potential. On the other hand, mitochondrial outer membrane permeabilization plays a crucial role in apoptosis that is regulated via Bcl-2-family proteins (23
). The interaction of proapoptotic effector Bcl-2 members BAK and BAX with cytosolic BH3-only proteins facilitates oligomerization, which leads to OMM permeabilization (35
). Cells lacking both BAK and BAX are insensitive to apoptosis (7
). The loss of ATP is remarkable in TNF-α-induced necroptosis, but the induction of mitochondrial malfunction is unknown. Surprisingly, MEFs deficient in both BAX and BAK failed to undergo necroptosis in response to TCZ (). In addition, our cytosol swap approach further confirmed that BAX/BAK is necessary for mitochondrion-dependent downstream necroptotic signaling. Though it is uncertain how BAX and BAK control necroptotic complex-induced mitochondrial malfunction at the subcellular milieu, interestingly, upstream FADD-RIP1-RIP3 necroptotic complex formation was not disrupted in BAX/BAK-DKO MEFs (). Because necroptotic complex formation remains unaffected, in BAX/BAK-DKO MEFs, it is possible that the necroptotic complex could interact with BAK to initiate mitochondrial membrane depolarization that leads to bioenergetic collapse.
Though mitochondria are biological engines for a multitude of functions such as nutrient oxidation, ATP production, Ca2+
buffering, and ROS production, the mitochondrial dysfunction in pathological settings could be caused by Ca2+
overload, permeability transition pore opening, and overproduction of ROS (56
). Robust ROS production has been implicated in TNF-α-induced necrosis through either members of the NOX family or mitochondria (34
). Disruption of the mitochondrial respiration rate induces proton gradient collapse and electron leakage from the mitochondrial matrix and favors ROS production. In the context of TCZ-induced necroptosis, ROS levels are elevated in wild-type cells but not in FADD-, RIP1-, caspase 8-, and BAX/BAK-deficient cells, which suggests that both NOX-family- and mitochondrion-dependent pathways are involved in ROS production ( and ). Mitochondrial and cytosolic antioxidants are the target of NF-κB at the transcription level (30
). Interestingly, NEMO−/−
MEFs did not prevent the TCZ-induced overproduction of ROS. Cells lacking p65 show lower levels of antioxidant transcripts following TNF-α stimulation (unpublished data). It is conceivable that an imbalance of oxidant/antioxidant levels occurs in NF-κB-deficient cells following TNF-α stimulation. siRNA knockdown of NOXO1 or pretreatment with ROS scavengers attenuates the TNF-α-induced RIP1-dependent ROS elevation and subsequently improves cell survival (34
). Using pharmacologic and genetic approaches, we have now demonstrated that TCZ-induced ROS play a crucial role in mitochondrial dysfunction and that overexpression of mitochondrion-targeted and cytosolic antioxidants or inhibition of ROS production preserves the ATP levels (). Similar to apoptosis, prevention of necroptosis-induced mitochondrial damage might improve the management of organ function.
More generally, the results presented here provide further understanding of the role of mitochondria in inflammation-induced necroptosis. Using multiple TNF-α signaling molecule-knockout cells, our mitochondrial functional studies provide evidence that mitochondria play an amplifying role in the pathophysiology of inflammation-induced necroptosis.