TRADD is critical for resistance to TRAIL-induced cell death through NF-κB activation

Abstract

TRAIL physically interacts with FADD and RIP1 by pull down (View interaction) TNF Receptor-Associated Death Domain (TRADD) is an essential mediator of TNF receptor-1 (TNFR1)-mediated TNF signaling and is responsible for recruitment of other effector proteins, such as RIP1 (Receptor Interacting Protein 1) and TRAF2 (TNF Receptor-Associated Factor 2) to the TNFR1 signaling complex [1-4], and FADD (Fas-Associated protein with Death Domain) to TNFR1-induced secondary complexes [5]. Recruitment of these adaptor proteins leads to the activation of MAP kinases and NF-κB, as well as other signaling pathways, including cell death [2, 6]. Like TNFR1, TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 and 2 contain a cytoplasmic death domain (DD) that associates with DD-containing adaptor proteins such as FADD, TRADD, or RIP1 to trigger cell signaling [7], although FADD may be involved in the recruitment of secondary adaptors. TRAIL can activate the NF-κB and MAPK signaling pathways [7, 8], and NF-κB-mediated induction of anti-apoptotic genes, such as inhibitors of apoptosis (IAPs) and Bcl-2 homologues may determine cellular susceptibility toward apoptosis induction [9, 10]. Here we show that TRADD deficiency renders cells more susceptible to TRAIL-induced cell death. This is likely due to impairment of the pro-survival NF-κB activation since TRAIL does not induce NF-κB activation in TRADD−/− MEFs, while ectopic expression of p65 or IKKβ prevents TRAIL-induced cell death in these cells. Despite the fact that TRADD−/− MEFs lack TRAIL-induced ERK and JNK activation, the loss of these pathways does not contribute to TRAIL-induced cell death in the knockout MEFs. Depletion of TRADD in cancer cells potentiates TRAIL-induced cell death, suggesting that TRADD is a potential target for sensitization to TRAIL-mediated cancer chemotherapy. Anti-phospho-ERK, anti-phospho-p38, anti-phospho-IκBα, anti-ERK, anti-p38, anti-caspase 3, anti-caspase 8, and anti-PARP1 antibodies were from Cell-Signaling. Anti-phospho-JNK antibody was purchased from Biosource. Anti-TRADD, anti-FADD, and anti-IκBα antibodies were from Santa Cruz. Anti-TRAIL-R and RIP1 antibodies were from BD. Cycloheximide, SP 600125, PD 98059, SB 202190, and IKK-2 inhibitor V were from Calbiochem. Glutathione S-transferase (GST)-TRAIL was described previously [11]. Recombinant murine TRAIL and recombinant murine TNFα were from R&D. Wild type and TRADD−/− MEFs [1-4] and HepG2 cells were cultured in DMEM with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. TRADD−/− (Flag-mTRADD) stable cell lines were selected in media containing 2 μg/ml puromycin. Upon treatment, cells were lysed in M2 buffer (20 mM Tris at pH 7, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, 1 μg/ml leupeptin). Equal amounts of protein from each cell lysates were resolved by 12% SDS–PAGE and visualized by enhanced chemiluminescence following the manufacturer's instruction (ECL, Amersham). Cell death was determined using tetrazolium dye colorimetric tests (MTT), and also measured by using a FITC-Annexin V Apoptosis Detection kit (BD Phamingen) according to the manufacturer's protocol. Cells were transfected with HA-IκBα super suppression plasmid, HA-IKKβ, and GFP-p65 or Mock vector with Lipofectamine PLUS reagent according to manufacturer's protocol (Invitrogen, Inc.). HepG2 cells plated in 12-well plates were transfected with 50 pmol of TRADD or non-targeting control (NC) RNAi oligo (Dharmacon) using Lipofectamine 2000 reagent (Invitrogen, Inc.). After 48 h, cells were treated with GST-TRAIL for 12 h and LDH leakage was quantified using a cytotoxicity detection kit (Promega). Cell death was also measured by using a FITC-Annexin V Apoptosis Detection kit (BD Phamingen). Wild type and TRADD−/− MEFs (1 × 108) cells were treated with 10 μg/ml GST-TRAIL for 15 min or left untreated. Cells were then washed with PBS and lysed with M2 buffer. The soluble fraction was incubated with 30 μl of 50% glutathione-Sepharose beads (GE healthcare) for 3 h on a rotator at 4 °C. Bound proteins were eluted, resolved in 12% SDS–PAGE, and analyzed by immunoblotting. Treatment of TRADD−/− MEFs with recombinant murine TRAIL showed the TRADD−/− MEFs were sensitive to TRAIL-induced cell death, while wild type (WT) MEFs were resistant, suggesting TRADD prevented TRAIL-mediated cell death under normal circumstances (Fig. 1 A). Cycloheximide sensitized WT MEFs to TRAIL, however, TRADD−/− MEFs continued to be more sensitive than the WT MEFs to TRAIL as shown by contrast microscopy or MTT assay (Fig. 1B) or by PI/Annexin V staining (Fig. 1C). Though TRADD−/− MEFs were more sensitive to CHX plus TRAIL than WT cells, they were completely refractory to CHX plus TNFα (Fig. 2 A), consistent with the previous reports [1]. This suggests that TRADD−/− MEFs are not more generally susceptible to cell death, and that TRAIL-induced sensitivity is stimulus specific. Cell death induced by TRAIL was completely blocked by zVAD, indicating that only apoptotic cell death was occurring in the TRADD−/− MEFs (Fig. 2B). Consistent with this, we detected fully processed caspase 8, caspase 3, and PARP-1 cleavage in the TRAIL-treated TRADD−/− MEFs, but not in WT MEFs (Fig. 2C), and zVAD efficiently blocked these events (Fig. 2D). We verified the involvement of TRADD in these events via stable TRADD reconstitution in TRADD−/− MEFs (Fig. 3 A). Reconstitution of TRADD expression restored TNFα-induced cell death in TRADD−/− MEFs (Fig. 3B), but substantially blocked TRAIL-induced cell death (Fig. 3C), confirming that increased sensitivity of TRADD−/− MEFs to TRAIL was due to loss of TRADD. TRAIL can activate the transcription factor NF-κB, which can protect from TRAIL-induced apoptosis [12-14]. To investigate whether TRAIL-induced NF-κB activation requires TRADD, we treated WT and TRADD−/− MEFs with GST-TRAIL because we were able to increase the activity in MEFs compared to rmTRAIL (see Fig. 4 A for cell death activity [11]). While GST-TRAIL activated IκBα phosphorylation and degradation in WT MEFs, GST-TRAIL-induced NF-κB activation was non-existent in TRADD−/− MEFs (Fig. 4B). Reconstitution of TRADD expression restored NF-κB activation in response to TRAIL as well as to TNFα in TRADD−/− MEFs (Fig. 4C). These results strongly suggest that TRADD is crucial for TRAIL-induced NF-κB activation and impaired NF-κB activation could explain the observed sensitization to TRAIL-induced apoptosis in TRADD-deficient cells. Inhibition of NF-κB activation by IκBα super suppressor in WT MEFs bestowed sensitivity to TRAIL (Fig. 4D), while ectopic expression of IKKβ or p65 rescued TRADD−/− MEFs (Fig. 4E) or HepG2 cells (Fig. 4F) in response to TRAIL. Taken together, our data suggest that TRADD protects cells from TRAIL-induced cell death through NF-κB activation. MAPKs are readily activated in response to various stresses, and play key roles in deciding cell fate [15, 16]. TRADD−/− MEFs lacked TRAIL-induced activation of ERK and JNK, while the activation of p38 was not affected (Fig. 5 A). However, the MAPK inhibitors, SP 600125 for JNK, PD 98059 for ERK, and SB 202190 for p38, respectively, did not influence TRAIL-induced cell death in either WT and TRADD−/− MEFs (Fig. 5B), indicating that TRAIL-induced cell death in MEFs is independent of MAPK activity. In contrast, when NF-κB activation was blocked in WT MEFs or HepG2 cells through IKK inhibition, TRAIL-induced cell death was dramatically increased (Fig. 5C and 5D) suggesting the NF-κB defect is responsible for the their susceptibility to TRAIL-induced cell death. TRAIL a highly promising cancer therapeutic agent, however, cancer cells can acquire resistance to TRAIL-induced cytotoxicity. We sought to determine whether TRADD knockdown can also sensitize cancer cells to TRAIL. Knockdown of TRADD expression in HepG2 cells (Fig. 6 A) led to an increased sensitivity of HepG2 cells to TRAIL (Fig. 6B and 6C), suggesting that targeting TRADD is able to sensitize human cancer cells to TRAIL-induced cytotoxicity. Finally, we investigated recruitment of signaling adaptors to the TRAIL receptor. We were able to detect RIP1 in the complex in both MEF cell types (Fig. 7 ), though RIP1 recruitment to the receptor complex lasted longer in TRADD−/− MEFs. Although less clear, FADD recruitment to the DISC also appeared to slightly increase in TRADD−/− MEFs, (Fig. 7). We therefore hypothesize that TRADD may compete with FADD and/or RIP1 for DISC recruitment and the additional FADD recruitment in TRADD−/− MEFs may contribute to enhanced sensitivity to TRAIL-induced cell death. TRAIL is selective cytotoxic in cancer cells and therefore a promising anti-cancer therapeutic agent. However, cancer cells often acquire TRAIL resistance and underlying molecular mechanisms are still elusive. To gain better understanding of TRAIL-induced signaling, we investigated that function of TRADD in this pathway. Deficiency of TRADD sensitizes cells to TRAIL's cytotoxicity, and cytotoxicity is blocked when TRADD expression is reconstituted. Thus, TRADD plays a protective role against TRAIL-induced cell death. Our data indicate that TRAIL-induced NF-κB activation has pro-survival role in MEFs and TRADD is required for activation of this signaling pathway. This is consistent with several earlier reports showing the anti-apoptotic function of NF-κB in TRAIL-induced cell death [11]. For instance, Lluis et al. showed that NF-κB negatively regulated TRAIL induced cell death in TAK1 knockout MEFs by increasing c-FLIPL levels [17] and a specific IKK inhibitor BMS-345541 or expression of IκBαM, which inhibits constitutively active NF-κB, confers sensitivity to TRAIL-induced cell death [18]. In addition, IKKγ−/− MEFs are sensitive to TRAIL-induced cytotoxicity [17]. Although our data suggest that lack of NF-κB activation is the primary reason for TRAIL-induced death of TRADD−/− cells, we do not exclude the possibility that TRADD also limits FADD recruitment to the DISC and that TRADD depletion results in greater FADD recruitment, as has been recently suggested [19]. We saw some evidence that this could be the case. Most likely both mechanisms play a role in the sensitivity of TRADD−/− cells to TRAIL. In contrast to this previous study [19], we observed that RIP1 was recruited to the TRAIL receptor complex in the absence of TRADD, and additional RIP1 recruited, suggesting that NF-κB deficiency occurs from loss of another TRADD-recruited component, such as cIAPs, that are necessary for NF-κB activation, and not from loss of RIP1 recruitment. Additional RIP1 recruitment in the absence of NF-κB activation could also play a role in greater caspase-8 activity by TRAIL as has been suggested for Fas [20]. There is mounting evidence that inhibition of NF-κB signaling leads to TRAIL-resistance by upregulating anti-apoptotic genes in multiple human cancer cells [21-23]. Since TRADD depletion in cancer cells increases TRAIL-induced cell death, TRADD could therefore serve as a target for sensitizing cancer cells to TRAIL cytotoxicity. This work was supported by Grant funded by the Korean Government (2009-0067084). This work was also supported by “2010 Research Fund of Myongji University”.