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Death Receptors 4 and 5 Activate Nox1 NADPH Oxidase through Riboflavin Kinase to Induce Reactive Oxygen Species-mediated Apoptotic Cell Death*

  • Kyung-Jin Park
    Affiliations
    Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea
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  • Chang-Han Lee
    Affiliations
    Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea
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  • Aeyung Kim
    Affiliations
    Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea
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  • Ki Jun Jeong
    Affiliations
    Department of Chemical and Biomolecular Engineering, KAIST, Daejeon 305-701, Korea
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  • Chul-Ho Kim
    Affiliations
    Department of Otolaryngology, Ajou University School of Medicine, Suwon 443-749, Korea
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  • Yong-Sung Kim
    Correspondence
    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
    Affiliations
    Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea
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  • Author Footnotes
    * 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.
Open AccessPublished:December 09, 2011DOI:https://doi.org/10.1074/jbc.M111.309021
      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.

      Introduction

      The proapoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL,
      The abbreviations used are: TRAIL
      TNF-related apoptosis-inducing ligand
      Nox
      NADPH oxidase
      ROS
      reactive oxygen species
      DR
      death receptor
      DD
      death domain
      DISC
      death-inducing signaling complex
      TNFR
      TNF receptor
      TRADD
      TNFR-associated death domain
      RFK
      riboflavin kinase
      KD
      Kringle domain
      Z
      benzyloxycarbonyl
      fmk
      fluoromethyl ketone
      NAC
      N-acetyl-l-cysteine
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      MPR
      membrane-proximal region.
      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 (
      • Pennarun B.
      • Meijer A.
      • de Vries E.G.
      • Kleibeuker J.H.
      • Kruyt F.
      • de Jong S.
      Playing the DISC. Turning on TRAIL death receptor-mediated apoptosis in cancer.
      ,
      • Wilson N.S.
      • Dixit V.
      • Ashkenazi A.
      Death receptor signal transducers. Nodes of coordination in immune signaling networks.
      ). 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) (
      • Pennarun B.
      • Meijer A.
      • de Vries E.G.
      • Kleibeuker J.H.
      • Kruyt F.
      • de Jong S.
      Playing the DISC. Turning on TRAIL death receptor-mediated apoptosis in cancer.
      ). Currently, many agonists against DR4 and/or DR5, including recombinant TRAIL and receptor-specific monoclonal antibodies (mAbs), are in various stages of clinical trials for cancer therapy (
      • Pennarun B.
      • Meijer A.
      • de Vries E.G.
      • Kleibeuker J.H.
      • Kruyt F.
      • de Jong S.
      Playing the DISC. Turning on TRAIL death receptor-mediated apoptosis in cancer.
      ).
      Like other DRs, such as TNF receptor 1 (TNFR1) and CD95 (Fas), the cytoplasmic DD of DR4 and DR5 (DR4/DR5) acts as a docking site for intracellular adaptor proteins for downstream signaling (
      • Wilson N.S.
      • Dixit V.
      • Ashkenazi A.
      Death receptor signal transducers. Nodes of coordination in immune signaling networks.
      ,
      • Park H.H.
      Structural analyses of death domains and their interactions.
      ). 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) (
      • Kischkel F.C.
      • Lawrence D.A.
      • Chuntharapai A.
      • Schow P.
      • Kim K.J.
      • Ashkenazi A.
      Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5.
      ,
      • Sprick M.R.
      • Weigand M.A.
      • Rieser E.
      • Rauch C.T.
      • Juo P.
      • Blenis J.
      • Krammer P.H.
      • Walczak H.
      FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2.
      ). Activated caspase-8/10 in DISC directly or indirectly activate effector caspases, such as caspase-3/6/7, triggering caspase-dependent apoptotic cell death (
      • Pennarun B.
      • Meijer A.
      • de Vries E.G.
      • Kleibeuker J.H.
      • Kruyt F.
      • de Jong S.
      Playing the DISC. Turning on TRAIL death receptor-mediated apoptosis in cancer.
      ). Activated CD95 also forms the DISC at the intracellular DD to trigger mainly caspase-dependent apoptotic cell death (
      • Wilson N.S.
      • Dixit V.
      • Ashkenazi A.
      Death receptor signal transducers. Nodes of coordination in immune signaling networks.
      ). 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 (
      • Wilson N.S.
      • Dixit V.
      • Ashkenazi A.
      Death receptor signal transducers. Nodes of coordination in immune signaling networks.
      ). 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 (
      • Shen H.M.
      • Pervaiz S.
      TNF receptor superfamily-induced cell death. Redox-dependent execution.
      ). Stimulation of TNFR1, CD95, and DR4/DR5 generate intracellular ROS, the major source of which is mitochondria (
      • Shen H.M.
      • Pervaiz S.
      TNF receptor superfamily-induced cell death. Redox-dependent execution.
      ,
      • Morgan M.J.
      • Liu Z.G.
      Reactive oxygen species in TNFα-induced signaling and cell death.
      ,
      • Lee M.W.
      • Park S.C.
      • Kim J.H.
      • Kim I.K.
      • Han K.S.
      • Kim K.Y.
      • Lee W.B.
      • Jung Y.K.
      • Kim S.S.
      The involvement of oxidative stress in tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in HeLa cells.
      ). 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 (
      • Shen H.M.
      • Pervaiz S.
      TNF receptor superfamily-induced cell death. Redox-dependent execution.
      ). 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 (
      • Kim Y.S.
      • Morgan M.J.
      • Choksi S.
      • Liu Z.G.
      TNF-induced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death.
      ,
      • Reinehr R.
      • Becker S.
      • Eberle A.
      • Grether-Beck S.
      • Häussinger D.
      Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis.
      ,
      • Suzuki Y.
      • Ono Y.
      • Hirabayashi Y.
      Rapid and specific reactive oxygen species generation via NADPH oxidase activation during Fas-mediated apoptosis.
      ).
      NADPH oxidases are integral membrane proteins that transfer electrons from NADPH to oxygen across biological membranes, generating superoxide anion (O2̇̄) and its downstream ROS, such as H2O2 (
      • Petry A.
      • Weitnauer M.
      • Görlach A.
      Receptor activation of NADPH oxidases.
      ). The Nox family encompasses seven different enzymes, Nox1 to -5, DUOX1, and DUOX2, with distinct cellular distribution and functions in human cells (
      • Petry A.
      • Weitnauer M.
      • Görlach A.
      Receptor activation of NADPH oxidases.
      ). NADPH oxidase activity is controlled by regulatory subunits, including the cytosolic subunits of Rac1, Noxo1, and Noxa1 and membrane-bound p22phox for Nox1 to -3 (
      • Petry A.
      • Weitnauer M.
      • Görlach A.
      Receptor activation of NADPH oxidases.
      ,
      • Jiang F.
      • Zhang Y.
      • Dusting G.J.
      NADPH oxidase-mediated redox signaling. Roles in cellular stress response, stress tolerance, and tissue repair.
      ). TNFα-induced activation of Nox1 is mediated by recruiting riboflavin kinase (RFK) to TNFR1 (
      • Yazdanpanah B.
      • Wiegmann K.
      • Tchikov V.
      • Krut O.
      • Pongratz C.
      • Schramm M.
      • Kleinridders A.
      • Wunderlich T.
      • Kashkar H.
      • Utermöhlen O.
      • Brüning J.C.
      • Schütze S.
      • Krönke M.
      Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.
      ). RFK is a cytosolic enzyme that catalyzes the phosphorylation of riboflavin to form flavin mononucleotide (FMN), a precursor to FAD (
      • Karthikeyan S.
      • Zhou Q.
      • Mseeh F.
      • Grishin N.V.
      • Osterman A.L.
      • Zhang H.
      Crystal structure of human riboflavin kinase reveals a β barrel fold and a novel active site arch.
      ). FAD is an essential prosthetic group of Nox1 to -4 for electron transport (
      • Karthikeyan S.
      • Zhou Q.
      • Mseeh F.
      • Grishin N.V.
      • Osterman A.L.
      • Zhang H.
      Crystal structure of human riboflavin kinase reveals a β barrel fold and a novel active site arch.
      ). Thus, RFK activation enhances FAD incorporation into Nox1, facilitating the assembly and activation of Nox1 NADPH oxidase (
      • Yazdanpanah B.
      • Wiegmann K.
      • Tchikov V.
      • Krut O.
      • Pongratz C.
      • Schramm M.
      • Kleinridders A.
      • Wunderlich T.
      • Kashkar H.
      • Utermöhlen O.
      • Brüning J.C.
      • Schütze S.
      • Krönke M.
      Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.
      ). CD95L-bound CD95 also produces superoxide anion and ROS by activating Nox enzymes, such as Nox1, Nox3, and Nox4 (
      • Reinehr R.
      • Becker S.
      • Eberle A.
      • Grether-Beck S.
      • Häussinger D.
      Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis.
      ,
      • Suzuki Y.
      • Ono Y.
      • Hirabayashi Y.
      Rapid and specific reactive oxygen species generation via NADPH oxidase activation during Fas-mediated apoptosis.
      ), although the molecular mechanism(s) remain obscure. There have been no reports yet for DR4/DR5-mediated RFK recruiting or Nox activation.
      Recently, we have developed the Kringle domain (KD) scaffold based on the KD2 of human plasminogen as an antibody surrogate (
      • Lee C.H.
      • Park K.J.
      • Sung E.S.
      • Kim A.
      • Choi J.D.
      • Kim J.S.
      • Kim S.H.
      • Kwon M.H.
      • Kim Y.S.
      Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo.
      ). From a KD synthetic library on the yeast cell surface, we isolated a variant, KD548, with DR4/DR5 dual-specific binding activity (
      • Lee C.H.
      • Park K.J.
      • Sung E.S.
      • Kim A.
      • Choi J.D.
      • Kim J.S.
      • Kim S.H.
      • Kwon M.H.
      • Kim Y.S.
      Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo.
      ,
      • Lee C.H.
      • Park K.J.
      • Kim S.J.
      • Kwon O.
      • Jeong K.J.
      • Kim A.
      • Kim Y.S.
      Generation of bivalent and bispecific kringle single domains by loop grafting as potent agonists against death receptors 4 and 5.
      ). 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 (
      • Lee C.H.
      • Park K.J.
      • Sung E.S.
      • Kim A.
      • Choi J.D.
      • Kim J.S.
      • Kim S.H.
      • Kwon M.H.
      • Kim Y.S.
      Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo.
      ). However, the detailed cell death mechanism of KD548-Fc has not been reported yet.
      Figure thumbnail gr1
      FIGURE 1KD548-Fc induces the apoptotic cell death of tumor cells by specifically binding to DR4 and/or DR5. A, schematic diagram of KD548-Fc, the homodimeric Fc-fused form of KD548, generated by its C-terminal fusion to Fc of human IgG1. B, ELISA to analyze the binding specificity of KD548-Fc for the extracellular domains of the indicated TNF family receptors, in comparison with TRAIL and TNFα. C, competition ELISA. Shown is binding activity of KD548-Fc for the plate-coated extracellular domain of DR4 or DR5 in the absence or presence of 1 μm TRAIL. D, colocalization of KD548-Fc with cell surface-expressed DR4 and DR5. TRITC-labeled KD548-Fc was incubated for 1 h at 4 °C with HeLa cells transiently transfected with DR4ΔCD-YFP or DR5ΔCD-YFP fusion protein, respectively, and then visualized by confocal fluorescence microscopy. Nuclei were costained with DAPI. Bar, 20 μm. E and F, knockdown of DR4, DR5, or TNFR1 by siRNA transfection (E) and the effects on TRAIL- or KD548-Fc-mediated cell death (F) in HeLa cells. Cells untransfected (control) or transfected with the indicated siRNA for 36 h (E) were incubated with the indicated concentrations of TRAIL or KD548-Fc for 40 h prior to the MTT assay (F). DR4/DR5 siRNA indicates the cotransfection of DR4 and DR5 siRNAs. G, representative transmission electron microscopy images of HeLa cells, which were left untreated (control) or treated with either TRAIL (30 nm) or KD548-Fc (0.8 μm) for 20 h. Bar, 2 μm. H, oligonucleosomal DNA fragmentation assay for HeLa cells, pretreated for 1 h with Z-VAD-fmk, SP600125, or NAC and further left untreated (control) or treated with TRAIL (30 nm) for 15 h or KD548-Fc (0.8 μm) for 40 h. B, C, and F, data represent mean ± S.E. (error bars) of three independent experiments carried out in triplicate.
      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.

      DISCUSSION

      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 (
      • Pennarun B.
      • Meijer A.
      • de Vries E.G.
      • Kleibeuker J.H.
      • Kruyt F.
      • de Jong S.
      Playing the DISC. Turning on TRAIL death receptor-mediated apoptosis in cancer.
      ). 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 (
      • Kim Y.S.
      • Morgan M.J.
      • Choksi S.
      • Liu Z.G.
      TNF-induced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death.
      ). 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 (
      • Yazdanpanah B.
      • Wiegmann K.
      • Tchikov V.
      • Krut O.
      • Pongratz C.
      • Schramm M.
      • Kleinridders A.
      • Wunderlich T.
      • Kashkar H.
      • Utermöhlen O.
      • Brüning J.C.
      • Schütze S.
      • Krönke M.
      Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.
      ). 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 (
      • Yazdanpanah B.
      • Wiegmann K.
      • Tchikov V.
      • Krut O.
      • Pongratz C.
      • Schramm M.
      • Kleinridders A.
      • Wunderlich T.
      • Kashkar H.
      • Utermöhlen O.
      • Brüning J.C.
      • Schütze S.
      • Krönke M.
      Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.
      ) but essential for TNFα-induced ROS accumulation mediated by Nox1 activation (
      • Kim Y.S.
      • Morgan M.J.
      • Choksi S.
      • Liu Z.G.
      TNF-induced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death.
      ). 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 (
      • Kieser A.
      Pursuing different “TRADDes”. TRADD signaling induced by TNF receptor 1 and the Epstein-Barr virus oncoprotein LMP1.
      ) (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% (
      • Park H.H.
      Structural analyses of death domains and their interactions.
      ). 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) (
      • Yazdanpanah B.
      • Wiegmann K.
      • Tchikov V.
      • Krut O.
      • Pongratz C.
      • Schramm M.
      • Kleinridders A.
      • Wunderlich T.
      • Kashkar H.
      • Utermöhlen O.
      • Brüning J.C.
      • Schütze S.
      • Krönke M.
      Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.
      ), are indeed responsible for the interactions of DR4 and DR5 with RFK. The RFK has 6-stranded antiparallel β-barrel structures (
      • Karthikeyan S.
      • Zhou Q.
      • Mseeh F.
      • Grishin N.V.
      • Osterman A.L.
      • Zhang H.
      Crystal structure of human riboflavin kinase reveals a β barrel fold and a novel active site arch.
      ), which is much different from the DD structure. Accordingly, the DDs of DR4/DR5 have a capability to interact heterotypically with RFK, like the interactions of TNFR1-DD with RFK (
      • Yazdanpanah B.
      • Wiegmann K.
      • Tchikov V.
      • Krut O.
      • Pongratz C.
      • Schramm M.
      • Kleinridders A.
      • Wunderlich T.
      • Kashkar H.
      • Utermöhlen O.
      • Brüning J.C.
      • Schütze S.
      • Krönke M.
      Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.
      ) and CD95-DD with the caspase recruitment domain (
      • Nam Y.J.
      • Mani K.
      • Ashton A.W.
      • Peng C.F.
      • Krishnamurthy B.
      • Hayakawa Y.
      • Lee P.
      • Korsmeyer S.J.
      • Kitsis R.N.
      Inhibition of both the extrinsic and intrinsic death pathways through nonhomotypic death fold interactions.
      ). Although DR4/DR5 activated by TRAIL primarily recruit FADD via the DD interactions (
      • Wilson N.S.
      • Dixit V.
      • Ashkenazi A.
      Death receptor signal transducers. Nodes of coordination in immune signaling networks.
      ), KD548-Fc-stimulated DR4/DR5 interact with TRADD through the homotypic DD interactions. Previous reports have also shown that DR4 and/or DR5 interact directly with TRADD rather than FADD (
      • Park K.J.
      • Lee S.H.
      • Kim T.I.
      • Lee H.W.
      • Lee C.H.
      • Kim E.H.
      • Jang J.Y.
      • Choi K.S.
      • Kwon M.H.
      • Kim Y.S.
      A human scFv antibody against TRAIL receptor 2 induces autophagic cell death in both TRAIL-sensitive and TRAIL-resistant cancer cells.
      ,
      • Chaudhary P.M.
      • Eby M.
      • Jasmin A.
      • Bookwalter A.
      • Murray J.
      • Hood L.
      Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-κB pathway.
      ,
      • Cao X.
      • Pobezinskaya Y.L.
      • Morgan M.J.
      • Liu Z.G.
      The role of TRADD in TRAIL-induced apoptosis and signaling.
      ).
      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 (
      • Lee C.H.
      • Park K.J.
      • Kim S.J.
      • Kwon O.
      • Jeong K.J.
      • Kim A.
      • Kim Y.S.
      Generation of bivalent and bispecific kringle single domains by loop grafting as potent agonists against death receptors 4 and 5.
      ). 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 (
      • Shen H.M.
      • Pervaiz S.
      TNF receptor superfamily-induced cell death. Redox-dependent execution.
      ,
      • Morgan M.J.
      • Liu Z.G.
      Reactive oxygen species in TNFα-induced signaling and cell death.
      ). One of the prominent effects of ROS is the direct activation of JNK and then sustaining the activation by inhibiting mitogen-activated protein kinase phosphatases (
      • Kamata H.
      • Honda S.
      • Maeda S.
      • Chang L.
      • Hirata H.
      • Karin M.
      Reactive oxygen species promote TNFα-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases.
      ). ROS generated by KD548-Fc-mediated Nox1 activation leads to prolonged JNK activation, as does ROS produced by TNFR1- or CD95-mediated Nox activation (
      • Kim Y.S.
      • Morgan M.J.
      • Choksi S.
      • Liu Z.G.
      TNF-induced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death.
      ,
      • Reinehr R.
      • Becker S.
      • Eberle A.
      • Grether-Beck S.
      • Häussinger D.
      Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis.
      ). Interestingly, in the sustained JNK activation-dependent cell death induced by Nox1-mediated ROS, TNFα induces necrotic cell death (
      • Kim Y.S.
      • Morgan M.J.
      • Choksi S.
      • Liu Z.G.
      TNF-induced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death.
      ), whereas DR4/DR5-specific KD548-Fc induces apoptotic cell death, like CD95-mediated apoptosis (
      • Reinehr R.
      • Becker S.
      • Eberle A.
      • Grether-Beck S.
      • Häussinger D.
      Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis.
      ). 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 (
      • Chen C.
      • Liu Y.
      • Zheng D.
      An agonistic monoclonal antibody against DR5 induces ROS production, sustained JNK activation and Endo G release in Jurkat leukemia cells.
      ). The protein RIP1 is required for CD95L-, TNFα-, and TRAIL-induced necrotic cell death in human cells (
      • Holler N.
      • Zaru R.
      • Micheau O.
      • Thome M.
      • Attinger A.
      • Valitutti S.
      • Bodmer J.L.
      • Schneider P.
      • Seed B.
      • Tschopp J.
      Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.
      ) and the ROS-mediated necrotic cell death by TNFα in mouse fibroblasts (
      • Kim Y.S.
      • Morgan M.J.
      • Choksi S.
      • Liu Z.G.
      TNF-induced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death.
      ). 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 (
      • Pennarun B.
      • Meijer A.
      • de Vries E.G.
      • Kleibeuker J.H.
      • Kruyt F.
      • de Jong S.
      Playing the DISC. Turning on TRAIL death receptor-mediated apoptosis in cancer.
      ,
      • Petry A.
      • Weitnauer M.
      • Görlach A.
      Receptor activation of NADPH oxidases.
      ). KD548-Fc killed a variety of tumor cells in vitro and efficiently inhibited tumor growth in mouse models, including TRAIL-resistant cells (
      • Lee C.H.
      • Park K.J.
      • Sung E.S.
      • Kim A.
      • Choi J.D.
      • Kim J.S.
      • Kim S.H.
      • Kwon M.H.
      • Kim Y.S.
      Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo.
      ), 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.

      Acknowledgments

      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.

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