Agonist Antibody Activates Death Receptor 6 Downstream Signaling Involving TRADD Recruitment

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FEBS Letters 588 (2014) 401–407

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Agonist antibody activates death receptor 6 downstream signaling involving TRADD recruitment ⇑ Rui Hu a,b, Qiumei Du a,b, Xiangyun Yin a,b, Jingyun Li a,b, Tingting Wang c, Liguo Zhang a,

a CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China b University of Chinese Academy of Sciences, Beijing 100049, China c Department of Neurology, General Hospital of PLA Shenyang Military Area Command, Shenyang 110015, China

article info abstract

Article history: Death receptor 6 (DR6) is a member of the death domain-containing receptors that belong to the Received 3 October 2013 TNFR superfamily. To date, the ligand for DR6 is still not clearly defined. Here, we developed a func- Revised 5 December 2013 tional agonist monoclonal antibody (DQM3) against DR6, which bound to the first cysteine-rich Accepted 7 December 2013 domain. Importantly, DR6 signaling could be clearly activated by DQM3, which was dependent on Available online 24 December 2013 its intracellular death domain. In addition, we demonstrated that the association between DR6 Edited by Angel Nebreda and TRADD was enhanced upon DQM3 stimulation and TRADD was involved in DR6-induced signaling activation. Taken together, our findings provide new insight into a novel mechanism by which DR6 induces downstream signaling in response to an agonist antibody. Keywords: DR6 Agonist antibody Structured summary of protein interactions: Death domain DR6 physically interacts with TRADD by anti bait coimmunoprecipitation (View interaction) TRADD Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Death receptor 6 (DR6, TNFRSF21) was identified as a death domain-containing receptor and the least characterized member of Death receptors are a subset of the tumor necrosis factor (TNF) the TNFR superfamily [5]. DR6 is widely expressed in human tis- receptor superfamily characterized by the presence of a conserved sues, including lymphoid organs, brain, pancreas and several death domain (DD) in their cytoplasmic regions [1,2]. There are non-lymphoid cancer cell lines [5,6]. Upon overexpression in some eight death receptors which have been identified in humans, cell lines, DR6 was reported to induce apoptosis as well as activa- including TNFR1, Fas (DR2, CD95 or APO-1), DR3, TRAIL-R1 tion of NF-jB and c-Jun N-terminal kinase (JNK) pathways [5]. (DR4), TRAIL-R2 (DR5), DR6, p75NTR (NGFR) and EDAR. In addition Knockout studies in mice suggest that DR6 serves as an important to primarily inducing apoptosis, engagement of death receptor un- regulator for T cell and B cell functions [7–9]. In addition, DR6/ der certain circumstances may initiate multiple non-apoptotic sig- mice were found to be resistant to EAE and allergic airway hyper- naling pathways, including activation of nuclear factor-jB (NF-jB) sensitivity [10,11]. and mitogen-activated protein kinase (MAPK) cascades, which pro- It has been recently shown that a cleaved N-terminal fragment mote the regulation of cell differentiation, proliferation, cytokine of the b-amyloid precursor protein (N-APP) may act as a ligand for and chemokine production, and the modulation of immune DR6, triggering axon pruning and neuron death [12]. However, the responses [3,4]. N-APP did not bind to DR6 and trigger the activation of NF-jB and JNK [13]. Moreover, DR6 regulates oligodendrocyte survival, maturation and myelination through a mechanism independent of N- APP [14]. To date, DR6 still remains as an orphan TNFR superfamily Abbreviations: CRD, cysteine-rich domain; DD, death domain; DR, death receptor; FADD, Fas-associated death domain; JNK, c-Jun N-terminal kinase; N- member and the mechanism by which DR6 induces downstream APP, N-terminal b-amyloid precursor protein; siRNA, small interfering RNA; TNFR, signaling is still largely unknown. tumor necrosis factor receptor; TRADD, TNFR-associated death domain protein; In the present study, we prove that the first cysteine-rich do- TRAF, tumor necrosis factor receptor-associated factor main of DR6 is responsible for the binding of the agonist antibody ⇑ Corresponding author. Address: CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing (DQM3) against DR6. Notably, DR6 signaling can be activated by 100101, China. DQM3. Furthermore, we characterize the death domain of DR6 E-mail address: [email protected] (L. Zhang). and the adaptor molecule TRADD involved in DR6 activation.

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.12.010 402 R. Hu et al. / FEBS Letters 588 (2014) 401–407

2. Materials and methods sequences are: sense, 50-GGAGGAUGCGCUGCGAAAUUU-30; anti- sense, 50-AAAUUUCGCAGCGCAUCCUCC-30 [15]. The siRNA with a 2.1. Reagents and plasmid construction scrambled sequence was used as a negative control. HEK293T cells were transfected with siRNA using Lipofectamine 2000 according The full-length DR6 (DR6-FL) and DR6 mutants including extra- to the manufacturer’s instructions. cellular DR6 (DR6-EX), DR6 (1-414), DR6 (1-498), DTRAF (D371- 414) and DDD (D415-498) were cloned into mammalian expres- 2.6. Co-immunoprecipitation and Western blot analysis sion vector. DR6 mutants DR6DCRD3-4, DR6DCRD2-1, DR6DCRD2, DR6DCRD1, DR6D67-88 and DR6D50-66 were generated from For co-immunoprecipitation, 2 106 of HEK293T cells were pIRES2-EGFP-DR6-FL. A 5 kbp fragment of the human IL-6 gene pro- transfected with the indicated expression plasmids for DR6-FL or moter was amplified from human genomic DNA and ligated into the DR6-EX and flag-tagged TRADD. 24 h later, anti-DR6 mAb was vector pGL4.20 (Promega). pNF-jB-Luc was purchased from Clon- added for stimulation for the indicated times. After treatment, cells tech. The full-length TRADD and dominant negative form of TRADD were washed twice with cold PBS and then lysed for 30 min on ice (TRADD-DN) lacking the death domain were epitope tagged with an in 500 ll of lysis buffer. The lysates were precipitated with anti- N-terminal Flag tag and cloned into the vector pTT3. DR6 mAb overnight at 4 °C. Then, Protein G beads (GE Health) were Mouse monoclonal antibodies against human DR6 were gener- added and incubated for 4 h at 4 °C under gentle rotation. The ated using the standard techniques from splenocytes of mice beads were washed five times with lysis buffer and eluted by boil- immunized with the protein DR6-EX fused with human IgG1-Fc ing the beads for 5 min in 2 SDS loading buffer. The eluted pro- expressed in HEK293T cells. The rabbit polyclonal anti-TRADD teins were separated by SDS–polyacrylamide gel electrophoresis antibody was purchased from Abcam. The mouse monoclonal (SDS–PAGE) and transferred to PVDF membranes followed by anti-Flag M2 antibody was from Sigma. Antibodies against phos- immunoblot analysis with the indicated antibodies. pho-SAPK/JNK (Thr183/Tyr185) and SAPK/JNK were purchased from Cell Signaling Technology. Antibody against phospho-IjBa 2.7. Statistical analysis was obtained from Santa Cruz Biotechnology. GraphPad Prism software was used for statistical analysis. All 2.2. Cell culture and transfection samples were performed in at least duplicate, with each experi- ment repeated at least three times. Data are shown as The human embryonic kidney cell line HEK293T was cultured in means ± standard errors of mean (S.E.M.) of three independent a5%CO2 incubator at 37 °C with complete DMEM (Gibco) medium experiments. Differences between groups were determined using supplemented with 10% fetal bovine serum, 2 mM L-Glutamine and an unpaired, two-tailed, Student’s t test. P-values < 0.05 (⁄), 100 U/ml penicillin/streptomycin. For transfection, HEK293T cells P < 0.01 (⁄⁄) and P < 0.001 (⁄⁄⁄) were considered statistically were transfected with indicated plasmids using the Lipofectamine significant. 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Empty vector was used to equalize the total amount of DNA in all transfections. Subsequently, reporter gene assays, flow 3. Results cytometry analysis and co-IP experiments were preformed. 3.1. DR6 induces the activation of NF-jB 2.3. Flow cytometry analysis In addition to inducing apoptosis in some cell lines, DR6 was The cells for flow cytometry analysis are harvested and incubated also reported to induce NF-jB activation [5]. Overexpression of on ice with blocking buffer for 20 min, and then incubate the cells death receptors can mimic ligand activation and trigger receptor with anti-DR6 mAbs (5 lg/ml) or isotype control antibodies (BioLeg- activation and downstream signaling events. Consistent with pre- end) on ice for 30 min. Add the PE-conjugated goat anti-mouse IgG vious observation, we found that in transfection studies, DR6-FL in- (BioLegend) to the cells and incubate in the dark on ice for 30 min. duced NF-jB activation in a dose dependent manner, but DR6-EX Add the viability dye 7-AAD (Invitrogen) to each sample to exclude lacking the intracellular domain did not (Fig. 1A). IL-6 represents dead cells from analysis. Wash the stained cells once and resuspend a pleiotropic inflammatory cytokine, which is predominantly regu- in staining buffer prior to flow cytometry analysis on FACSCalibur lated by NF-jB. We wondered whether DR6 could induce the acti- (BD Biosciences). Data were analyzed using Summit software. vation of target gene regulated by NF-jB. As shown in Fig. 1B, overexpression of DR6-FL resulted in significantly upregulation of 2.4. Luciferase assay IL-6 luciferase activity (up to 6-fold) in a dose dependent manner. However, in contrast to DR6-FL, DR6-EX had no obvious effect on HEK293T cells in 24-well plates were transiently transfected IL-6 activation, indicating that DR6-induced downstream signaling with reporter plasmid encoding the firefly luciferase gene under activation is dependent on cytoplasmic domain. control of the NF-jB or IL-6 promoter together with various expression plasmids for DR6 or DR6 mutants. The pRL-TK encoding 3.2. DR6 mAbs bind to different antigenic epitopes in CRD domain Renilla luciferase served as an internal transfection control. Anti- DR6 mAb was added 24 h after transfection, and isotype control Although the N-APP has been reported to function as a DR6 li- antibody also served as a negative control. 16 h later, luciferase gand, triggering axon pruning, neuron death and axonal degenera- activity was measured with the Dual-Luciferase Reporter Assay tion, results from two other groups indicate that the N-APP may System (Promega). not activate DR6 in other cell types [13,14]. Accordingly, we also demonstrated that N-APP had no effect on DR6-induced NF-jB 2.5. RNA interference and IL-6 activation in HEK293T cells (data not shown). Therefore, we generated three monoclonal antibodies (DQM2, DQM3 and Double strand siRNA oligonucleotides targeting human TRADD DQM5) against DR6, which could bind HEK293T cells transfected were purchased from GeneChem (Shanghai, China), and the with DR6-FL (Fig. 2A). R. Hu et al. / FEBS Letters 588 (2014) 401–407 403

A B pNF-κB-Luc pIL6-Luc Fold induction Fold induction

DR6-FL −−−−DR6-FL −−−−− DR6-EX −−−− DR6-EX −−−−−

Fig. 1. Overexpression of DR6 induces NF-jB and IL-6 activation. (A) HEK293T cells were transiently transfected with NF-jB promoter-driven luciferase reporter plasmid alone or together with increasing amounts of expression vectors for DR6-FL or DR6-EX. Renilla was co-transfected as an internal transfecion control. Promoter activity was measured 24 h post transfection by luciferase assay. (B) Similar assay was done with IL-6 reporter plasmid. Data are shown as the mean ± S.E.M. Representative results from at least three independent experiments are shown.

DR6, a type I transmembrane protein, has four highly conserved located on CDR1. To narrow down the binding sites of these mAbs, cysteine-rich domains (CRDs) and a stalk region in the extracellular further studies showed that DQM3 and DQM5 could still bind portion [16]. To investigate the binding sites of anti-DR6 mAbs, we DR6D67-88, while DQM2 could not (ﬁfth panel). It was worth to constructed a set of DR6 mutants with deletion of certain extracell- point out that the mutant DR6D67-88 severely decreased the cell uar domains (Fig. 2B). As shown in Fig. 2C, in ﬂow cytometry anal- surface localization of DR6 and led to its intracellular retention ysis with HEK293T cells expressing DR6 mutants, all three mAbs (data not shown). Moreover, all three anti-DR6 mAbs could not could still bind DCRD3-4, but not DCDR1-2 (top two panels). Addi- bind DR6D50-66 (bottom panel). Additionally, the deletion of stalk tionally, we found these mAbs could bind DCDR2, but not DCDR1 region between CRDs and the transmembrane domain did not af- (third and fourth panels), suggesting that the binding sites were fect their binding (data not shown). Taken together, these results

A C DQM2 DQM3 DQM5 DQM2 DQM3 DQM5 CRD3-4 CRD3-4 EV Δ ΔCRD1-2 DR6-FL CRD2

DR6 Δ B

DR6-FL (1-655) ΔCRD1

DR6ΔCRD3-4 88 DR6ΔCRD1-2 Δ67- DR6ΔCRD2

DR6ΔCRD1 -66

DR6Δ67-88 50 Δ

DR6Δ50-66 DR6

Fig. 2. Mapping binding epitopes of anti-DR6 mAbs. (A) HEK293T cells were transiently transfected with empty vector or expression vector encoding DR6-FL. Transfected cells were indicated by the expression of GFP. DR6 expression was analyzed by flow cytometry with anti-DR6 mAbs (DQM2, DQM3 and DQM5) or isotype control antibodies. EV, empty vector; black line, anti-DR6 mAbs; grey fill, isotype control antibodies. (B) Schematic representation of the DR6 deletion mutants used in this study (intracellular domain is not shown). The tested DR6 deletion mutants targeting extracellular domain include DCRD3-4, DCRD1-2, DCRD2, DCRD1, D67-88 and D50-66. (C) DR6 and its mutants were transfected into HEK293T cells. Cells were collected after 36 h, and FACS analysis was performed to detect the binding of DQM2, DQM3 and DQM5. Data are representative of three independent experiments. 404 R. Hu et al. / FEBS Letters 588 (2014) 401–407 demonstrate that all three antibodies bind to the CRD1 of DR6, and 3.4. Death domain is essential for DR6-induced signaling upon the the binding sites of DQM3 and DQM5 are located on amino acids agonist antibody stimulation 50-66 of CRD1. The death domain is an essential protein-binding domain that 3.3. DQM3, but not DQM2 and DQM5, activates DR6 downstream accounts for the ability of TNFR1 to induce the activation of the signaling NF-jB pathway and activation of the apoptotic caspase cascade [17]. In order to investigate the mechanism by which DR6 trans- We next asked whether DR6 could induce increased activation mits signals upon the agonist antibody stimulation, a panel of of downstream signaling upon anti-DR6 mAbs stimulation, and DR6 mutants were constructed (Fig. 4A and Supplementary we sought to compare the effects of three anti-DR6 mAbs on Fig. 2). They were designed to delete large portions of DR6 intracel- DR6-induced signaling activation by reporter gene assays. As lular domains responsible for cytosolic signal propagation. Signal- shown in Fig. 3A, only DQM3 could upregulate IL-6 promoter-dri- ing activation induced by DR6 truncations was assessed by ven luciferase activity, while DQM2 and DQM5 could not. In addi- luciferase reporter assay. In response to DQM3 stimulation, DR6- tion, DR6-induced IL-6 promoter activation was shown in a dose- FL resulted in a 3.6-fold increase of IL-6 promoter activity over re- dependent manner in response to DQM3 stimulation (Fig. 3B). moval of cytoplasmic domain (DR6-EX) (Fig. 4B). In contrast to These results indicate that the DQM3 may serve as an agonist anti- DR6-EX, DR6 (1-414) had no effect on induction of IL-6 activity body specific to DR6, which will be used in further dissecting the in cells treated with DQM3. However, removal of the N-terminal downstream signaling of DR6. fragment following DD termed DR6 (1-498), and deletion of the To further understand the potential mechanism through which TNFR-associated factor (TRAF) binding motif (DTRAF) had almost DR6 mediates downstream signaling, we next examined the im- the same ability as DR6-FL to induce IL-6 promoter activation upon pact of DR6 on NF-jB and JNK activation. DR6-FL induced activa- DQM3 stimulation (Fig. 4B). Interestingly, deletion of the DD tion of both JNK and NF-jB, as indicated by the phosphorylation (DDD) showed considerably impaired induction of IL-6 promoter of JNK and NF-jB inhibitor IjBa (Fig. 3C). However, DR6-EX activity comparing to DR6-FL. These results suggest that death do- showed considerably impaired induction of NF-jB, but not JNK main is essential for DR6-induced downstream signaling upon the activation (Fig. 3C and Supplementary Fig. 1A), suggesting that agonist antibody stimulation. DR6-induced NF-jB activation is intracellular domain dependent, whereas JNK activation is not. Furthermore, DQM3 stimulation 3.5. TRADD is involved in DR6-induced signaling activation could enhance DR6-induced NF-jB activation (Fig. 3D, third panel). In contrast, the JNK pathway was not affected upon ligation of Ligation of TNFR1 by TNF recruits the TNFR associated death do- DQM3, as demonstrated by the similar level of phosphorylated main (TRADD), which serves as the adaptor protein [17]. Pan et al. JNK (Fig. 3D and Supplementary Fig. 1B). have demonstrated that DR6 doesn’t interact with the adaptor

A B Control DQM2 DQM3 DQM5 Fold induction Fold induction

Vector + −−−− − − Vector + −−−− DR6-FL − + + + + + + DR6-FL − + + + + DQM3 − − 0.3 1 3 10 30 (μg/ml) C D DR6-FL − − − DR6-EX −− − DQM30 5 1530 60 Time (min) Vector + −−− − p-JNK p-JNK

JNK JNK

p-IκBα p-IκBα

actin actin

Fig. 3. DQM3 stimulates DR6-induced IL-6 luciferase activity. (A) HEK293T cells were co-transfected with DR6-FL, IL-6 luciferase reporter and Renilla. Anti-DR6 mAbs (DQM2, DQM3 and DQM5, 10 lg/ml) were added 24 h after transfection, and isotype control antibody alone also served as a negative control. Luciferase assays were performed to measure promoter activity after 16 h of antibody incubation. (B) Transfected cells in (A) were stimulated with increasing amounts of DQM3. Luciferase activity was determined as described in (A). (C) HEK293T cells were transfected with increasing amounts of vectors for DR6-FL or DR6-EX. 36 h later, cell lysates were prepared and subjected to Western blot analysis using antibodies to phospho-JNK, total JNK and phospho-IjBa. b-Actin was used as loading control. (D) Cells transfected with DR6-FL in (C) were left untreated or treated with DQM3 (10 lg/ml) for the indicated times. Western blot analysis was performed as described in (C). Data are representative of at least three independent experiments. R. Hu et al. / FEBS Letters 588 (2014) 401–407 405

A B

CRDs DD N.S. DR6-FL (1-655) *** ** DR6-EX (1-370)

DR6 (1-414)

DR6 (1-498) Fold induction Fold induction

ΔTRAF (Δ371-414)

ΔDD (Δ415-498) .

Fig. 4. Death domain is essential for DR6-induced signaling upon DQM3 stimulation. (A) Schematic representation of DR6 mutants used in this study. The deletion mutants targeting intracellular domain include DR6-EX (1-370), DR6 (1-414), DR6 (1-498), DR6-DTRAF (D371-414) and DR6-DDD (D415-498). (B) HEK293T cells were transfected with IL-6 reporter plasmid alone or together with a series of expression vectors encoding DR6 or its mutants as indicated above. 24 h later, cells were stimulated with DQM3 or isotype control antibody. Cell lysates were prepared 16 h after antibody incubation and relative luciferase activity of stimulus versus control was calculated as the mean ± S.E.M. Similar results were obtained in three independent experiments. ⁄⁄⁄P < 0.001; ⁄⁄P < 0.01; N.S., not significant. protein FADD, but has a weak interaction with TRADD [5]. How- activity could not be further upregulated upon DQM3 stimulation, ever, whether TRADD is involved in DR6 signaling is still unknown. suggesting TRADD was sufficient for the signaling pathway in- We showed that transfection of TRADD alone resulted in induction duced by DR6 (Fig. 5C). of IL-6 promoter activity, but TRADD-DN did not (Fig. 5A). Further- To bolster the above findings, we examined whether TRADD more, in cells with co-transfection of DR6 and TRADD, the IL-6 pro- deficiency had effects on DR6-induced signaling activation. We moter activity increased significantly up to 6-fold over transfection used siRNA targeting TRADD to suppress its expression in HEK293T of DR6 alone, but the cells co-transfected with DR6 and TRADD-DN cells (Fig. 5D) and then examined the ability of DR6 to activate IL-6 did not have clear effect (Fig. 5B). We found that DR6-induced IL-6 luciferase reporter in response to DQM3 stimulation. As shown in

A B C Control DQM3

** Fold induction Fold induction Fold induction

− Vector −− − − DR6-FL + + + + + + + DR6-FL −−+ + + + −− −−−− TRADD −− −− Vector TRADD − + −−+ + −−−− −− TRADD-DN −− −− TRADD TRADD-DN −−−−−− D E si-NC si-TRADD ** TRADD

actin * Fold induction

Control DQM3

Fig. 5. TRADD is involved in DR6-induced IL-6 activation. (A and B) HEK293T cells were transiently transfected with IL-6 reporter plasmid, Renilla and increasing amounts of expression vectors for TRADD or TRADD-DN (A) or together with DR6-FL (B). 24 h later, promoter activity was measured by luciferase assay. (C) HEK293T cells were co- transfeced with IL-6-Luc, Renilla, DR6-FL and TRADD as described in (B), and then stimulated with DQM3. 16 h later, luciferase activity was measured. (D) HEK293T cells were co-transfected with TRADD siRNA (si-TRADD) or negative control siRNA (si-NC) and expression plasmid for TRADD. Western blot analysis was performed to verify the efﬁciency of siRNA. (E) TRADD siRNA-treated cells were transfected with IL-6 reporter plasmid and DR6-FL. 24 h after transfection, cells were subjected to DQM3 stimulation for 16 h, and then luciferase activity was monitored as described above. Results are shown as the mean ± S.E.M. ⁄P < 0.05; ⁄⁄P < 0.01. 406 R. Hu et al. / FEBS Letters 588 (2014) 401–407

Fig. 5E, silencing the expression of TRADD could signiﬁcantly study indicated that DR6–TRADD interaction could be enhanced inhibit the DR6-induced IL-6 promoter activity in cells treated with as soon as 15 min of DQM3 stimulation (second panel). Taken to- control antibody or DQM3. The effect of DQM3 ligation on siRNA- gether, these ﬁndings demonstrate that there is a possibility that control group (1.6-fold) was comparable to that of siRNA-TRADD TRADD serves as an adaptor molecule that mediates DR6 down- group (1.4-fold). These data further support our conclusion that stream signaling. TRADD is critically involved in DR6 signaling both in the presence and absence of DQM3. 4. Discussion

3.6. TRADD is a putative adaptor molecule in DR6 downstream Death receptor 6, characterized by the intracellular death do- signaling main, belongs to the TNF receptor superfamily. DR6 has been shown to play an apparent role in regulating transcription factor Previous studies demonstrate that TRADD is a crucial signal activation, apoptosis, maturation and immune responses. Upon li- adaptor molecule that mediates intracellular responses from gand binding and activation, members of the death receptor family TNFR1 [18,19]. To further investigate a potential role for TRADD trigger different signaling pathways. Previous studies demonstrate in DR6 signaling, we sought to confirm the possible interaction of that DR6 is highly expressed in human brain and it has been shown DR6 with TRADD by co-immunoprecipitation. As shown in to induce axon pruning, neuron death and axonal degeneration Fig. 6A, TRADD was only co-immunoprecipitated with DR6 in cells through binding to its recently described ligand N-APP [12]. How- induced for DR6-FL expression (lane 3, second panel), but not in ever, Klíma and colleagues showed that N-APP could not bind to cells with induction of DR6-EX expression (lane 4). These data sug- DR6 and mediate the activation of NF-jB and JNK [13]. In particu- gest that DR6 interacts with TRADD in HEK293T cells. lar, a recently published report proposed a potential role of DR6 in It has been shown that the death domain-containing C-termi- the regulation of oligodendrocyte survival, maturation and myeli- nus of TRADD (195-312) was essential and sufficient for interac- nation through a mechanism independent of N-APP [14]. We also tion with the DD of TNFR1 and induction of apoptosis and NF-jB found that N-APP had no effect on DR6-induced NF-jB and IL-6 activation [17]. To further validate the interaction of TRADD with promoter activation, and it was difficult to definitively address DR6, HEK293T cells were transfected with DR6 and TRADD or the molecular signaling mechanisms of this receptor. Thus, we TRADD-DN, and the association was determined as described generated a functional agonist antibody against DR6, which could above. As shown in Fig. 6B, the results demonstrated that TRADD, serve as a useful tool in the studies on DR6-induced signaling path- but not TRADD-DN co-immunoprecipitated with DR6 (compare ways. Importantly, we showed that DR6 signaling could be clearly lane 1 with lane 2, second panel), indicating that the DD of TRADD activated by this agonist antibody, which was dependent on the was necessary for the interaction between DR6 and TRADD. death domain. Next, we asked whether DQM3 could enhance the association Upon binding of their specific ligands, DD-containing adaptor between DR6 and TRADD. As shown in Fig. 6C, the time course proteins are recruited by death receptors leading to the assembly A B DR6-FL − + + − Vector −−−+ DR6-EX −−−+ DR6-FL + + −− Vector + −−− DR6-EX −−+ − Flag-TRADD + − + + Flag-TRADD + −−− Flag-TRADD-DN − + + + DR6-FL, 110kD DR6-FL, 90kD DR6-FL, 110kD IB: DR6 IB: DR6 DR6-FL, 90kD IP: DR6 * DR6-EX * IP: DR6 DR6-EX IB: TRADD TRADD IB: Flag TRADD DR6-FL, 110kD DR6-FL, 90kD DR6-FL, 110kD IB: DR6 DR6-FL, 90kD IB: DR6 Input DR6-EX Input DR6-EX IB: TRADD TRADD TRADD IB: Flag IB: β-actin actin TRADD-DN C IB: β-actin actin DQM30 5 15 30 Time (min) DR6-FL, 110kD IB: DR6 DR6-FL, 90kD IP: DR6 IB: TRADD TRADD

DR6-FL, 110kD IB: DR6 DR6-FL, 90kD Input IB: TRADD TRADD

IB: β-actin actin

Fig. 6. DR6 interacts with TRADD in co-immunoprecipitation assays. (A and B) HEK293T cells in 60-mm dishes co-transfected with DR6-FL/EX and Flag-tagged TRADD or TRADD-DN were harvested 48 h after transfection. Total cell lysates were immunoprecipitated with anti-DR6 (DQM3) and immunoblotted with anti-TRADD (A) or anti-Flag (B). Western blot analysis of DR6, TRADD and Flag was performed on whole cell lysates as a control (Input). (C) HEK293T cells were co-transfected with expression vectors for DR6-FL and Flag-tagged TRADD, and then stimulated or not with DQM3 for the indicated times. The interaction of TRADD with DR6 was assessed after DQM3 stimulation by co-immunoprecipitation. The immunoprecipitates and cell extracts were analyzed by immunoblot. Results representative of at least three independent experiments are shown. b-Actin was used as loading control for each fraction. ⁄, Heavy chain (DQM3); IP, immunoprecipitates; IB, immunoblot. R. Hu et al. / FEBS Letters 588 (2014) 401–407 407 of protein complex that serves as a binding platform for [4] Guicciardi, M.E. and Gores, G.J. (2009) Life and death by death receptors. FASEB signal-transducing proteins. It has been demonstrated that TRADD, J. 23, 1625–1637. [5] Pan, G. et al. (1998) Identification and functional characterization of DR6, a the first protein recruited to TNFR1, serves as a central adaptor to novel death domain-containing TNF receptor. FEBS Lett. 431, 351–356. recruit additional mediators such as the serine/threonine kinase [6] Benschop, R., Wei, T. and Na, S. (2009) Tumor necrosis factor receptor RIP and TRAF2, activating NF-jB and JNK survival pathways superfamily member 21: TNFR-related death receptor-6, DR6. Adv. Exp. Med. Biol. 647, 186–194. [17,20,21]. We hypothesized it was likely that DR6 mediated [7] Liu, J., Na, S., Glasebrook, A., Fox, N., Solenberg, P.J., Zhang, Q., Song, H.Y. and downstream signaling using a similar mechanism as TNFR1. In this Yang, D.D. (2001) Enhanced CD4+ T cell proliferation and Th2 cytokine study, we showed that TRADD was also involved in DR6-induced production in DR6-deficient mice. Immunity 15, 23–34. [8] Zhao, H., Yan, M., Wang, H., Erickson, S., Grewal, I.S. and Dixit, V.M. (2001) signaling, which was significantly inhibited when the expression Impaired c-Jun amino terminal kinase activity and T cell differentiation in of TRADD was silenced (Fig. 5E). To further dissect the role of death receptor 6-deficient mice. J. Exp. Med. 194, 1441–1448. TRADD in DR6-induced signaling activation, we confirmed the [9] Schmidt, C.S. et al. (2003) Enhanced B cell expansion, survival, and humoral responses by targeting death receptor 6. J. Exp. Med. 197, 51–62. physical interaction of DR6 with TRADD. Nevertheless, further [10] Schmidt, C.S. et al. (2005) Resistance to myelin oligodendrocyte glycoprotein- studies are still needed to discover other unknown mediators in- induced experimental autoimmune encephalomyelitis by death receptor 6- volved in DR6 signaling. deficient mice. J. Immunol. 175, 2286–2292. Mimicking ligand induced receptor activation by overexpress- [11] Venkataraman, C., Justen, K., Zhao, J., Galbreath, E. and Na, S. (2006) Death receptor-6 regulates the development of pulmonary eosinophilia and airway ing DR6 in some cell lines such as HeLa and HEK293 cells demon- inflammation in a mouse model of asthma. Immunol. Lett. 106, 42–47. strated that DR6 could induce apoptosis. Moreover, DR6-induced [12] Nikolaev, A., McLaughlin, T., O’Leary, D.D.M. and Tessier-Lavigne, M. (2009) apoptosis was independent of FADD and could be inhibited by APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 457, 981–989. the caspase inhibitor Z-VAD [22]. 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(2008) Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent many tumor cell lines and clinical tumor samples [5,22,26]. There- inflammatory responses. Nat. Immunol. 9, 1037–1046. fore, DR6 signaling may play a crucial role in tumorigenesis by [19] Pobezinskaya, Y.L., Kim, Y.S., Choksi, S., Morgan, M.J., Li, T., Liu, C. and Liu, Z. affecting the generation of anti-tumor activity. Understanding (2008) The function of TRADD in signaling through tumor necrosis factor receptor 1 and TRIF-dependent Toll-like receptors. Nat. Immunol. 9, 1047– the mechanism of DR6-induced signaling pathway makes DR6 to 1054. be a potential therapeutic target for treating inflammatory and [20] Hsu, H., Shu, H.B., Pan, M.G. and Goeddel, D.V. (1996) TRADD-TRAF2 and autoimmune disease or cancer [27,28]. TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84, 299–308. [21] Hsu, H., Huang, J., Shu, H.B., Baichwal, V. and Goeddel, D.V. 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