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To whom correspondence should be addressed: Dept. of Molecular Science and Technology, Ajou University, San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-749, Korea. Tel.: 82-31-219-2662; Fax: 82-31-219-1610
* This work was supported by grants from the Korean Health Technology R&D Project (A101800) from the Ministry for Health, Welfare & Family Affairs, the Converging Research Center Program (2009-0093653) and the Priority Research Center Program (2011-0022978) from the National Research Foundation of MEST, and the “GRRC” Project of the Gyeonggi Provincial Government, Republic of Korea. This article contains supplemental Experimental Procedures and Figs. S1–S6.
Stimulation of the proapoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, death receptors 4 (DR4) and 5 (DR5), conventionally induces caspase-dependent apoptosis in tumor cells. Here we report that stimulation of DR4 and/or DR5 by the agonistic protein KD548-Fc, an Fc-fused DR4/DR5 dual-specific Kringle domain variant, activates plasma membrane-associated Nox1 NADPH oxidase to generate superoxide anion and subsequently accumulates intracellular reactive oxygen species (ROS), leading to sustained c-Jun N-terminal kinase activation and eventual apoptotic cell death in human HeLa and Jurkat tumor cells. KD548-Fc treatment induces the formation of a DR4/DR5 signaling complex containing riboflavin kinase (RFK), Nox1, the Nox1 subunits (Rac1, Noxo1, and Noxa1), TNF receptor-associated death domain (TRADD), and TNF receptor-associated factor 2 (TRAF2). Depletion of RFK, but not the Nox1 subunits, TRADD and TRAF2, failed to recruit Nox1 and Rac1 to DR4 and DR5, demonstrating that RFK plays an essential role in linking DR4/DR5 with Nox1. Knockdown studies also reveal that RFK, TRADD, and TRAF2 play critical, intermediate, and negligible roles, respectively, in the KD548-Fc-mediated ROS accumulation and downstream signaling. Binding assays using recombinantly expressed proteins suggest that DR4/DR5 directly interact with cytosolic RFK through RFK-binding regions within the intracellular death domains, and TRADD stabilizes the DR4/DR5-RFK complex. Our results suggest that DR4 and DR5 have a capability to activate Nox1 by recruiting RFK, resulting in ROS-mediated apoptotic cell death in tumor cells.
Apo2L) receptors, death receptors 4 (DR4, TRAIL-R1) and 5 (DR5, TRAIL-R2), are members of the DR family and are attractive anti-cancer targets because their stimulation with the cognate ligand TRAIL induces apoptosis in various tumor cells without significant cytotoxicity on normal cells (
). The additional TRAIL membrane receptors, death decoy receptor 1 (DcR1, TRAIL-R3) and DcR2 (TRAIL-R4), also bind to TRAIL but cannot induce apoptosis due to a lack of fully functional intracellular death domain (DD) (
). Activation of DR4/DR5 by homotrimeric TRAIL results in the oligomerization of the intracellular DD to recruit the primary adaptor protein Fas-associated DD (FADD) via homotypic interactions between the DDs and then caspase-8/-10, forming the so-called death-inducing signaling complex (DISC) (
). However, TNFα-bound TNFR1 primarily recruits TNFR-associated death domain (TRADD), rather than FADD, via homotypic DD interactions and then other second adaptor molecules, mediating mainly proinflammatory and necrotic cell death signaling (
). Thus, depending on which adaptor protein binds to the DDs of activated DRs, the outcome of the downstream signaling varies. However, the structural and molecular mechanism(s) by which the DDs of DRs distinguish the respective primary adaptor protein is not clear yet.
Reactive oxygen species (ROS), such as superoxide anion (O2̇̄) and hydrogen peroxide (H2O2), are known to induce a wide range of responses, depending on the cell types and ROS levels within the cell, including apoptotic and necrotic cell death (
). The external cell death stimuli mediated by the DRs cause uncoupling of the mitochondrial electron transport chain and/or collapse of the mitochondrial outer membrane potential, producing intracellular ROS (
). For an additional source of ROS, however, recent studies have shown that TNFR1 and CD95 can activate plasma membrane-associated NADPH oxidases (Nox enzymes) to generate superoxide anion and eventually accumulate intracellular ROS (
). The Fc-fused form in its C terminus, KD548-Fc (Fig. 1A), induced cell death in several cancer cell lines in vitro and suppressed tumor growth in xenograft mouse models even for TRAIL-resistant cells (
). However, the detailed cell death mechanism of KD548-Fc has not been reported yet.
In the present work, we demonstrate that DR4/DR5-specific KD548-Fc induces apoptotic cell death via the ROS-dependent sustained c-Jun N-terminal kinase (JNK) activation pathway without caspase activation in HeLa and Jurkat cells. We found that KD548-Fc-induced ROS accumulation resulted from superoxide anion generation from Nox1 NADPH oxidase, which was mediated by the formation of a KD548-Fc-induced DR4/DR5-signaling complex containing RFK, Nox1, Rac1, TRADD, and TNF receptor-associated factor 2 (TRAF2). RFK is an essential component in linking DR4/DR5 with Nox1. We have identified RFK-binding regions within the DDs of DR4/DR5 by mapping the intracellular domains. Our results provide a new signaling pathway of DR4/DR5, directly activating Nox1 via RFK, which is distinct from conventional DISC-mediated apoptosis.
Conventionally activated DR4 and DR5, in response to the cognate ligand TRAIL or agonistic mAbs, form the canonical DISC, which activates initiator caspase-8/10 and then effector caspase-3/6/7, triggering apoptosis in various tumor cells (
). In this study, we have shown that KD548-Fc-stimulated DR4/DR5 activate plasma membrane-associated Nox1 NADPH oxidase to generate superoxide anion by forming a unique signaling complex, including RFK, Nox1, the Nox1 cytosolic subunits (Rac1, Noxo1, and Noxa1), TRADD, and TRAF2. The signaling complex was formed within 30 min after cells were treated with KD548-Fc, coincident with the O2̇̄ generation. Although other Nox enzymes, such as Nox2, Nox3, and Nox4, are expressed in HeLa and HCT116 cells, our data suggest that only Nox1 is activated by KD548-Fc-mediated DR4/DR5 stimulation in the cells. Nox1 knockdown substantially abolished intracellular ROS accumulation, suggesting that O2̇̄ generated outside the cells is the main source of ROS by conversion into plasma membrane-permeable ROS species, such as H2O2 (
). Our data also suggest that the Nox1-mediated ROS accumulation is responsible for sustained JNK activation-dependent apoptotic cell death in response to KD548-Fc.
Nox1 recruitment to the intracellular DDs of DR4/DR5 seems to be directly mediated by the adaptor protein RFK rather than the Nox1 cytosolic subunits, like in the case of RFK-mediated linking of TNFR1 with Nox1 (
). In the RFK-deficient HeLashRFK cells, KD548-Fc-induced ROS accumulation, sustained JNK activation, and apoptotic cell death were dramatically inhibited, supporting the essential role of RFK in the DR4/DR5-mediated Nox1 activation. TRADD knockdown did not prevent the recruitment of RFK, Nox1, and Rac1 to KD548-Fc-stimulated DR4/DR5, but it substantially attenuated ROS accumulation, sustained JNK activation, and apoptotic cell death in HeLa cells. These results are similar to the role of TRADD in TNFα-induced Nox1 activation in mouse and human cells, where TRADD was dispensable in the formation of the TNFα-induced signaling complex of TNFR1-RFK-Nox1 (
). In the study with recombinantly purified proteins, TRADD improved ∼5-fold the interactions of RFK with the DDs of DR4/DR5, suggesting that the ternary complex of DR4/DR5-DD·TRADD·RFK is more stable than binary complexes consisting of two of the three molecules. These data suggest that RFK can physically and functionally couple KD548-Fc-stimulated DR4/DR5 to Nox1, the complex of which is stabilized by TRADD, enhancing the activity of RFK and/or Nox1. TRAF2 played a negligible role in the formation of the KD548-Fc-induced signaling complex and subsequent downstream signaling. TRADD contains an N-terminal TRAF2-binding domain and a C-terminal DD interacting with other DDs (
) (supplemental Fig. S6C). Thus, it seems that TRAF2 is recruited to the activated complex indirectly via the adaptor protein TRADD, but its effect on the downstream signaling is not clear at this point.
Our results suggest that KD548-Fc-stimulated DR4/DR5 simultaneously interacts with RFK and TRADD via the DDs. The DD is a homotypic protein interaction module composed of a bundle of six α-helices, the tertiary structures of which are closely superimposed despite the low sequence homology below 30% (
). The DDs of DR5 and DR4 share ∼63% identity with each other (supplemental Fig. S6B), indicative of their redundant functions. Our data have shown that the DR4RFK and DR5RFK regions, structurally equivalent to the RFK-binding regions of the DD of TNFR1 (TNFR1RFK) (
The question is how TRAIL and KD548-Fc recruit different signaling adaptors to the same DDs of DR4/DR5. We hypothesize two possible reasons: different binding regions and valency between TRAIL and KD548-Fc. KD548-Fc recognizes partially overlapping but different extracellular regions of DR5 and DR4 from those of TRAIL. Recently, we reported that the KD548 single domain without Fc fusion has bivalent binding ability, with each binding site possessing independent DR4/DR5 dual-specific binding ability (
). Thus, homodimeric KD548-Fc has tetravalent binding sites, each site of which has DR4/DR5 dual binding specificity. Tetravalent KD548-Fc might induce higher oligomeric states of DR4/DR5 than those induced by homotrimeric TRAIL. The higher valency and distinct binding sites of KD548-Fc from those of TRAIL raise the possibility that KD548-Fc induces a distinct conformation and/or oligomeric states in the intracellular DDs of DR4/DR5, which may provide preferential binding surfaces to RFK and TRADD over the primary adaptor FADD of TRAIL. Thus, it seems that, depending on the external stimuli, the DD of activated DR4 and/or DR5 may adopt distinct conformations, which provide preferential interaction sites for the adaptor proteins, such as FADD, TRADD, and/or RFK.
ROS play a critical role in apoptotic and necrotic cell death by oxidatively damaging many molecular targets and thus affecting various signaling cascades (
). The apoptotic morphological features of dying cells caused by exposure to KD548-Fc and little effects of the necrosis inhibitor necrostatin-1 and autophagy inhibitors (3-methyladenine and chloroquine) on KD54-Fc-induced cell death exclude necrosis and autophagy as the primary cell death mechanisms of KD548-Fc. In good agreement with our results, ROS-mediated sustained JNK activation was responsible for the caspase-independent apoptotic cell death of Jurkat cells mediated by an agonistic mAb against DR5 (
). However, RIP1 was absent in the DR4/DR5-associated signaling complex induced by KD548-Fc in HeLa and Jurkat cells. Thus, the different signaling complexes induced between TNFα and KD548-Fc might explain the distinct cell death mode. However, further study is required to elucidate the exact mechanism by which Nox1-mediated ROS accumulation and JNK activation triggered by KD548-Fc-stimulated DR4/DR5 induce apoptosis.
DR4/DR5 and Nox1 are expressed in a wide range of cells (
), suggesting that the alternative cell death pathway of KD548-Fc to that of TRAIL might overcome some of the TRAIL resistance mechanism. Because there have been no reports of physiological agonists mimicking KD548-Fc, however, the physiological relevance of the KD548-Fc-induced DR4/DR5 signaling pathway remains to be determined. In conclusion, our results propose that DR4 and/or DR5 have the capability to activate Nox1 via RFK recruitment, resulting in intracellular ROS accumulation, sustained JNK activation, and eventually apoptotic cell death in tumor cells.
We thank Prof. Martin Krönke (University of Cologne) for providing HeLashRFK cells and the bacterial plasmid encoding RFK-GST. We also thank Dr. Avi Ashkenazi (Genentech Inc.) and You-Sun Kim (Ajou University, Korea) for helpful discussions.