Research Article 647
TNF-induced noncanonical NF-B activation is attenuated by RIP1 through stabilization of TRAF2
Joo-Young Kim1, Michael Morgan2, Dong-Gun Kim1, Ju-Yeon Lee1, Lang Bai3, Yong Lin3, Zheng-gang Liu2 and You-Sun Kim1* 1Institute for Medical Sciences, Ajou University School of Medicine, Suwon, 443-749, Korea 2Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892, USA 3Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive, Southeast, Albuquerque, NM 87108, USA *Author for correspondence ([email protected])
Accepted 14 October 2010 Journal of Cell Science 124, 647-656 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.075770
Summary The current paradigm of noncanonical NF-B signaling suggests that the loss of TRAF2, TRAF3 or cIAP1 and cIAP2 leads to stabilization of NF-B-inducing kinase (NIK) to activate the noncanonical pathway. Although a crucial role of RIP1 in the TNF- induced canonical NF-B pathway has been well established, its involvement in noncanonical activation of NF-B through the TNFR1 receptor, is unknown. Here we show that TNF is capable of activating the noncanonical NF-B pathway, but that activation of this pathway is negatively regulated by RIP1. In the absence of RIP1, TNFR1 stimulation leads to activation of the noncanonical NF-B pathway through TRAF2 degradation, leading to NIK stabilization, IKK phosphorylation and the processing of p100 to generate p52. Thus although RIP1–/– mouse embryonic fibroblasts are sensitive at early time points to cell death induced by TNF, probably as a result of lack of canonical NF-B activation, the late activation of the noncanonical NF-B pathway protects the remaining cells from further cell death. The TNFR1-dependent noncanonical NF-B activation in RIP1–/– cells suggests that there is functional interplay between the two NF-B pathways during TNFR1 signaling, which might regulate the number and kinds of NF-B transcription factors and thus finely control NF-B-dependent gene transcription.
Key words: TNF, TNFR1, RIP1, TRAF2, Noncanonical NF-B
Journal of Cell Science Introduction is associated with RelB and enters the nucleus and binds to B Two different NF-B pathways have been identified that lead to sites in certain NF-B-responsive genes to activate transcription transcription from B sites, and are referred to as the canonical and (Dejardin et al., 2002; Senftleben et al., 2001; Xiao et al., 2001). noncanonical (or alternative) pathways (Beinke and Ley, 2004; Processing of p100 requires its phosphorylation by IKK, which Bonizzi and Karin, 2004; Hayden and Ghosh, 2008; Vallabhapurapu in turn requires phosphorylation by NF-B-inducing kinase (NIK) and Karin, 2009). The canonical NF-B pathway is activated (Senftleben et al., 2001; Xiao et al., 2004; Xiao et al., 2001). Rapid following stimulation with a broad range of stimuli such as TNF, turnover of NIK prevents constitutive activation of the noncanonical IL-1, lipopolysaccharide (LPS) and genotoxic agents, and has a NF-B pathway in resting cells (Liao et al., 2004). Most current major role in the control of innate immunity and inflammation data in the literature with regard to stimuli-dependent stabilization (Beinke and Ley, 2004; Bonizzi and Karin, 2004; Hayden and of NIK are consistent with a simple model (Zarnegar et al., 2008; Ghosh, 2008; Vallabhapurapu and Karin, 2009). The canonical Vallabhapurapu et al., 2008), in which TRAF3 recruits NIK to a pathway involves phosphorylation of IB by IB kinase (IKK) complex containing TRAF2 and inhibitor of apoptosis proteins components (primarily IKK) and subsequent degradation of IB, cIAP1 and/or cIAP2 (cIAP1/2), where NIK ubiquitylation by which typically holds the RelA–p50 heterodimer in the cytoplasm. cIAP1 or cIAP2 promotes its proteasomal degradation. The role of IB degradation allows NF-B to translocate to the nucleus TRAF2 in this complex is to recruit TRAF3 and the cIAP proteins where it binds to B sites, leading to gene transcription (Beinke through direct interactions. This model predicts that the loss of and Ley, 2004; Bonizzi and Karin, 2004; Hayden and Ghosh, TRAF2, TRAF3 or both cIAP1 and cIAP2 would lead to 2008; Vallabhapurapu and Karin, 2009). noncanonical NF-B activation by the resulting stabilization of the The noncanonical NF-B pathway is activated by particular NIK protein. Thus, cells deficient in TRAF2 or TRAF3 have high TNF receptor family members that bind to the TNF receptor- basal levels of NIK and p52 (Gardam et al., 2008; Grech et al., associated factors TRAF2 and/or TRAF3, such as LTR, CD40, 2004; He et al., 2007; He et al., 2006; Vallabhapurapu et al., 2008; CD27, CD30, BAFF-R and RANK, which are mainly involved in Xie et al., 2007), whereas inhibition of cIAP proteins by small- secondary lymphoid organ development, B cell survival and molecule antagonists results in activation of the noncanonical NF- maturation and osteoclastogenesis (Beinke and Ley, 2004; Bonizzi B pathway in a manner that is dependent on NIK stabilization, and Karin, 2004; Hayden and Ghosh, 2008; Vallabhapurapu and thereby enhancing B lymphocyte survival and proliferation Karin, 2009). The noncanonical pathway requires IKK-dependent (Varfolomeev et al., 2007; Vince et al., 2007). High levels of NIK NF-B2 (p100) processing in the proteasome to generate p52. p52 and p52 in multiple myelomas might also be explained by several 648 Journal of Cell Science 124 (4)
different genetic alterations, including deletion of TRAF2 or TRAF3 or bi-allelic deletions of both cIAP1 and cIAP2 (Annunziata et al., 2007; Keats et al., 2007). However, very few receptor- mediated stimuli have been described that cause noncanonical activation dependent solely upon degradation of TRAF2 in the absence of TRAF3 degradation. Receptor-interacting protein 1 (RIP1) has emerged as an essential molecule that is involved in death receptor signaling and cellular stress (Festjens et al., 2007). The C-terminal death domain of RIP1 is capable of binding to death receptors with a similar death domain motif in death receptors, such as TNFR1 and Fas, and the TRAIL (TNF-related apoptosis-inducing ligand) receptors (Chaudhary et al., 1997), and also interacts with other death domain-containing adaptor proteins, such as TRADD (TNFR1-associated DEATH domain protein) and FADD (FAS-associated death domain protein) (Varfolomeev et al., 1996). As a TRADD- and TNFR1-binding molecule, RIP1 is a key effector molecule in the TNF-induced activation of the transcription factor NF-B (Kelliher et al., 1998). This is largely because the Lys63-linked polyubiquitylation of RIP1 by cIAP1/2 (Lin et al., 2010) leads to its interaction with regulatory subunit of the IKK complex, IKK–NEMO, and thus RIP1 is essential for robust TNF activation of IKK and of the canonical NF-B pathway (Ea et al., 2006; Poyet et al., 2000; Wu et al., 2006). Although RIP1 has a role in death receptor apoptosis (Morgan et al., 2009; O’Donnell et al., 2007; Wang et al., 2008), it is essential for death receptor-mediated necrotic cell death by Fas and TNF (Holler et al., 2000; Lin et al., 2004), where it is required for the generation of reactive oxygen species (ROS) (Lin et al., 2004). We have recently reported that RIP1 and TRADD interact with NOXO1 to recruit and activate the NADPH oxidase Nox1, resulting in superoxide production during TNF-induced necrotic cell death (Kim et al., 2007). Therefore, RIP1 is a crucial component of TNF signaling that controls the divergence of distinct pathways and cellular outcomes in TNF-exposed cells.
Journal of Cell Science However, the involvement of RIP1 in the noncanonical NF-B Fig. 1. TNF induces activation of the noncanonical but not canonical NF- pathway has not been examined. –/– Our previous studies demonstrated that TRAF2, which is an B pathway in RIP1 MEFs. (A)Western blots showing p100 processing in TNF-treated wild-type and RIP1–/– MEFs. Cells were treated with TNF (30 important effector molecule of TNF signaling, is also a crucial, ng/ml) for indicated times and cell lysates were applied for western blot with non-redundant component in LTR signaling (Kim et al., 2005). indicated antibodies. (B)(Top) Electrophoretic mobility shift assay (EMSA) in However, we found that RIP1 is not involved in LTR-mediated nuclear cell lysates from wild-type, TRAF2–/– and RIP1–/– MEFs treated with activation of NF-B and JNK (Kim et al., 2005). In the current TNF showing binding to NF-B probe. (Bottom) As a control, an anti-Sp-1 study, we found that RIP1 is a negative regulator of the antibody was used in western blot of the same nuclear lysates. (C)Western noncanonical NF-B pathway in TNFR1 signaling. The absence of blots of lysates from TNF-treated wild-type and RIP1–/– MEFs showing RIP1 in cells treated with TNFleads to TRAF2 degradation, NIK reduced IB degradation in RIP1–/– MEFs. (D)Western blots from RIP1–/– –/– stability, phosphorylation of IKK and p100 processing, resulting MEFs and stably transfected RIP1 (FLAG–RIP1) MEFs showing recovery of in noncanonical NF-B activation. IB degradation during TNF (30 ng/ml) treatment. (E)Western blots of lysates from wild-type MEFs transfected with non-target control siRNA (NC) or RIP1 siRNA and treated with TNF for indicated times, showing increased Results IB stability in RIP1-deficient cells. TNF activates the noncanonical NF-B pathway in RIP1–/– MEFs Although a crucial role of RIP1 in the canonical TNF-induced NF-B pathway has been well established (Devin et al., 2000; Ea MEFs with human recombinant TNF, which only binds to TNFR1 et al., 2006; Kelliher et al., 1998; Poyet et al., 2000; Wu et al., in murine cells (Lewis et al., 1991). A similar pattern of p100 2006), its involvement in noncanonical NF-B activation is processing was observed when treated with human TNF unknown. We carefully compared TNF-induced p100 processing (supplementary material Fig. S1A), suggesting that p52 generation in MEF cells and found that treatment with murine TNF led to a is mediated by TNFR1, but not TNFR2. Similar results were significant increase in processing of p100 and the formation of p52 obtained in RIP1–/– MEFs treated with an agonistic TNFR1-specific in RIP1–/– mouse embryonic fibroblasts (MEFs), but not in wild- antibody (supplementary material Fig. S1B), and the p52 to p100 type (WT) MEFs (Fig. 1A). Unlike canonical NF-B activation, ratio decreased substantially at the 4-hour time point when a the TNF-induced processing of p100 in these cells appears slowly TNFR1-specific blocking antibody was added (Michael Morgan, and only becomes evident after 4 hours. We treated the RIP1–/– unpublished results). Next, we compared the role of RIP1 in early RIP1 attenuates noncanonical NF-B 649
TNF-induced NF-B activation, which is proposed to be solely mediated by the canonical pathway (Rauert et al., 2009) and LTR- induced NF-B activation, which substantially activates the noncanonical pathway (Dejardin et al., 2002). TNF treatment led to potent NF-B activation in WT MEFs, with a partial reduction in TRAF2–/– MEFs and little activation in RIP1–/– MEFs, as determined by electrophoretic mobility shift assay (EMSA) at early time points (Fig. 1B). Some activation by EMSA was observed in RIP1–/– MEFs at high exposure. Nevertheless, this activation was substantially less than in WT cells. Since a recent report has questioned the role of RIP1 in early canonical NF-B activation using different methodology (Wong et al., 2010), we also looked at IB degradation in the same cells. IB degradation was substantially decreased in RIP1–/– cells when compared with WT cells (Fig. 1C). Because our MEFs were immortalized after going through crisis, we wanted to verify that the effect was due to loss of RIP1 and not other molecules. We therefore stably transfected FLAG–RIP1 into the RIP1–/– MEFs and observed that IB degradation was restored when RIP1 expression was reconstituted (Fig. 1D). In addition, siRNA knockdown of RIP1 in WT MEFs also led to a decrease in IB degradation (Fig. 1E). This data suggests that although RIP1 might not be absolutely required for any TNF-induced canonical NF-B activation per se, it has a significant and positive role in this process. By contrast, and consistent with our previous finding (Kim et al., 2005), LTR- mediated noncanonical NF-B activation was similar in WT and RIP1–/– MEFs (supplementary material Fig. S1C), suggesting that RIP1 is not important in the noncanonical NF-B activation initiated through the LT receptor. Taken together, these results suggest that RIP1 is required for activation of the canonical pathway by TNFR1 and it might negatively affect activation of the noncanonical pathway specifically through TNFR1 when the cells are treated with TNF. Fig. 2. Deficiency of RIP1 leads to p100 processing and TRAF2 –/– Journal of Cell Science TNF induces TRAF2 degradation in RIP1 MEFs degradation in response to TNF. (A)Western blots of lysates from TNF- The noncanonical NF-B pathway is constitutively active in treated wild-type, RIP1–/– and TRAF2–/– MEFs showing TRAF2 degradation TRAF2–/– B cells and MEFs (Grech et al., 2004; Vince et al., and p52 generation in RIP1–/– MEFs. (B)Western blots of lysates from short 2007), and is degraded upon CD40 engagement in B cells (Brown time course of TNF-treated RIP1–/– MEFs showing a correlation between TRAF2 degradation and p52 generation. (C)Western blots of lysates from et al., 2001; Liao et al., 2004), suggesting that TRAF2 functions as –/– a negative regulator of p100 processing. We therefore carefully wild-type and RIP1 MEFs showing TRAF2 degradation in TNF-treated RIP1–/– MEFs cells, but lack of TRAF3 degradation. (D) Western blots of examined TRAF2 protein levels in TNF -treated RIP1–/– MEFs. lysates from wild-type MEFs transfected with non-target control siRNA (NC) Upon TNF treatment, there was marked reduction of TRAF2 in or RIP1 siRNA and treated with TNF for indicated times, showing –/– RIP1 MEFs, but not in WT MEFs (Fig. 2A and supplementary degradation of TRAF2 in RIP1-knockdown wild-type MEFs. (E)Western blot material Fig. S2). Notably, reduction of TRAF2 protein levels in showing protein expression levels of RIP1 in wild-type, RIP1–/– and RIP1–/– RIP1–/– MEFs correlated with p100 processing, whereas p52 protein (Flag-RIP1) MEFs. (F)Western blots of lysates from RIP1–/– MEFs levels were constitutively higher in TRAF2–/– MEFs, irrespective transfected with FLAG or FLAG–RIP1 plasmid, respectively, showing no of TNF treatment. A closer kinetic study revealed that TRAF2 TRAF2 degradation and p52 generation in RIP1-reconstituted RIP1–/– MEFs. degradation began 30 minutes after TNF treatment and TRAF2 levels continued to decrease until 4 hours after treatment, which is when we observed processing of p100 to p52 (Fig. 2B). Treatment TRAF3 is degraded upon engagement of BAFF-R or CD40, and with both murine and human TNF led to a reduction in TRAF2 its degradation is therefore thought to be important in activation of protein levels to the same extent, suggesting that TNF-induced the noncanonical NF-B pathway (Brown et al., 2001; Liao et al., reduction of TRAF2 is mediated by TNFR1 (supplementary 2004). TRAF3-deficient cells show constitutive processing of p100 material Fig. S3A). TRAF2 was not degraded in WT or RIP1–/– to p52 (Gardam et al., 2008). However, we failed to observe much MEFs in response to treatment with TRAIL and no increase in change in TRAF3 protein levels in TNF-treated RIP1–/– MEFs p52 was observed in response to this ligand (supplementary (Fig. 2C). We previously saw little change in TRAF3 protein levels material Fig. S3B,C). However, treatment of either WT or RIP1–/– during noncanonical NF-B activation induced by anti-LTR MEFs with LIGHT led to generation of p52 in the absence of antibody (Kim et al., 2005). Therefore, although TRAF3 TRAF2 degradation, suggesting that another mechanism for p52 degradation might be involved in noncanonical NF-B activation generation is present in this pathway (supplementary material Fig. downstream of some TNF receptor family members in certain cell S3C). types, it appears to not be required for activation of the noncanonical 650 Journal of Cell Science 124 (4)
pathway downstream of TNFR1 or LTR in MEFs, and it is attributable to activation of the noncanonical pathway. No reduction TRAF2 degradation that is associated with the TNF-induced in NF-B-driven activity was seen in control cells transfected with activation of the noncanonical NF-B pathway in the RIP1–/– IKK siRNA, suggesting that the noncanonical pathway is activated MEFs. only in the absence of RIP1. To verify that TRAF2 degradation and noncanonical NF-B activation in the RIP1–/– MEFs is the authentic effect of RIP1 Degradation of TRAF2 leads to accumulation of NIK deficiency, we first used siRNA to knock down RIP1 in wild-type We next examined the signaling events downstream of TRAF2 MEFs, and verified that TRAF2 degradation accompanied RIP1 degradation. Activation of the noncanonical NF-B pathway deficiency in TNF-treated cells (Fig. 2D). We next examined the requires NF-B-inducing kinase (NIK) activity to induce p100 effect of RIP1 reconstitution in RIP1–/– MEFs in response to TNF processing (Xiao et al., 2004). TRAF3 recruits NIK to a complex treatment. Transfection of FLAG-tagged RIP1 into RIP1–/– MEFs that promotes its proteasomal-mediated degradation, whereas restored the RIP1 protein level to endogenous levels (Fig. 2E). TRAF2 recruits cIAP1 or cIAP2, which are necessary for the Reconstitution of RIP1 blocked TRAF2 degradation and degradation to take place (Vallabhapurapu et al., 2008; Zarnegar et significantly reduced processing of p100 that was induced by al., 2008). Therefore, we hypothesized that TRAF2 degradation TNF (Fig. 2F), supporting the notion that RIP1 negatively should lead to the stabilization of NIK and the subsequent activation regulates noncanonical NF-B activation in response to TNF of the noncanonical NF-B pathway. TNF did induce NIK treatment by preventing TRAF2 degradation. accumulation in RIP1–/– MEFs, whereas no increase occurred in To further substantiate the role of RIP1 in prevention of WT MEFs (Fig. 4A). As expected, NIK protein levels were high noncanonical NF-B activation, we infected A549 lung cancer in TRAF2–/– MEFs, even in the absence of TNF (Fig. 4A). To cells with a lentivirus expressing RIP1 shRNA. When RIP1 was verify that this was a TNFR1-specific effect, we treated RIP1–/– knocked down, TNF-induced p100 processing to p52 was clearly MEFs with an agonistic TNFR1-specific antibody (supplementary detected (Fig. 3A), confirming that TNF-stimulated activation of material Fig. S4A) and NIK accumulated similarly as with TNF the noncanonical pathway is not restricted to MEFs, and that RIP1 treatment. To further eliminate the effect of TNFR2, we transfected negatively regulates this pathway. TNF-stimulated NF-B activity TNFR2–/– MEFs with siRNA to knock down RIP1 and treated as measured by a NF-B luciferase reporter was significantly these cells with TNF. We observed NIK accumulation in these reduced in the RIP1-knockdown cells (Fig. 3B); however, some cells at 1 and 4 hours after TNF treatment (supplementary material the remaining NF-B activity was additionally reduced by Fig. S4B). Because NIK activates IKK by phosphorylation to transfection of IKK siRNA to block noncanonical NF-B trigger p100 processing (Senftleben et al., 2001; Xiao et al., 2004; activation (Fig. 3B). Since IKK is required upstream of the Xiao et al., 2001), we looked at whether IKK is phosphorylated noncanonical pathway, this probably indicates that at least some of when NIK levels increase in the absence of RIP1. There was a the remaining NF-B activity in the RIP1-deficient cells is transient increase in IKK phosphorylation in TNF-treated RIP1–/– MEFs that followed the increase of NIK (Fig. 4B). Although NIK stability usually peaked between 1and 4 hours, after that the NIK stability and the IKK phosphorylation decreased, and the
Journal of Cell Science levels of TRAF2 began to rise again. By 8 hours, NIK levels had
Fig. 3. NF-B activation in stable RIP-knockdown cells is dependent on IKK. (A)Western blots of lysates from stable A549-NC and A549-RIP1 KD cells treated with TNF (10 ng/ml) for the indicated times showing p52 generation in response to TNF in the RIP-deficient A549 cells. (B)(Top) NF- B luciferase assay from A549-NC and A549-RIP1 KD cells mock transfected Fig. 4. Degradation of TRAF2 induces NIK accumulation and or transfected with 5 nM IKK siRNA or negative control siRNA (NC) phosphorylation of IKK. (A)Western blots of NIK and TRAF2 in TNF showing decrease in NF-B activity in response to IKK knockdown 48 hours (30 ng/ml)-treated wild-type, RIP1–/– and TRAF2–/– MEFs for the indicated after transfection (6 hours after TNF treatment). Data are means ± s.e.m.; times. (B)Western blots of longer time course showing NIK accumulation and *P<0.05. (Bottom) Knockdown of IKK was confirmed by western blot. IKK phosphorylation in response to TNF in RIP1–/– MEFs. RIP1 attenuates noncanonical NF-B 651
returned to that of the untreated cells despite the fact that the RIP1 binding to the receptor complex limits the TRAF2 levels did not completely recover. This might suggest a recruitment of TRAF2 to TNFR1 secondary feedback inhibition on the stability of NIK in addition When stimulated, TNFR1 recruits adaptor proteins, such as TRADD, to TRAF2. Nevertheless, these results indicate that the degradation RIP1 and TRAF2 to the receptor complex at the cell membrane, of TRAF2 in the absence of RIP1 precedes the appearance of NIK which is then internalized as a whole to form secondary signaling stabilization and phosphorylation of IKK, which precedes the complexes (Schneider-Brachert et al., 2004). Although studies by generation of p52. Thus TRAF2 degradation is probably the crucial our group and others have found that TRAF2 recruitment to TNFR1 event that leads to the activation of the noncanonical NF-B is TRADD dependent, the binding of RIP1 within the TNFR1 pathway by TNF. complex does not absolutely require TRADD, but TRADD substantially enhances the RIP1 recruitment and is required for TRAF2 degradation in TNF-stimulated RIP1–/– MEFs is ubiquitylation of RIP1 in the complex (Ermolaeva et al., 2008; blocked by proteasomal inhibition Pobezinskaya et al., 2008). We performed co-immunoprecipitation Because our data indicate that TNF-induced degradation of experiments in WT and RIP1–/– MEFs with an anti-TNFR1 antibody TRAF2 is a crucial step in p100 processing in these cells, we and examined the recruitment of TRAF2. Both TRAF2 and RIP1 examined the mechanism of TRAF2 degradation. Neither NIK were quickly recruited to TNFR1 following TNF treatment in WT stabilization and p52 generation, nor TRAF2 degradation was MEFs (Fig. 5A). In RIP1–/– MEFs, TNF-induced TRAF2 inhibited in the presence of cycloheximide (supplementary recruitment was dramatically increased by nearly 100-fold, whereas material Fig. S5A), indicating that de novo protein synthesis is the levels of TNFR1 protein precipitated were similar in both cell not required for induction of the upstream noncanonical pathway. types (Fig. 5A). The TRAF2 interaction was rapid and transient, TRAF2 degradation and p100 processing was likewise not reaching its peak at 5 minutes, whereas only a small fraction of affected by the pan-caspase inhibitor zVAD (supplementary TRAF2 was found interacting with TNFR1 at 30 minutes (Fig. 5A). material Fig. S5B), indicating that caspases are not the source Also consistent with previous data (Devin et al., 2000), there was an of cleavage or degradation. Although TRAF2 degradation in enhanced level of TRADD pulled down by TNFR1 in RIP1–/– MEFs RIP1–/– MEFs was not inhibited by zVAD, it was completely compared with levels in WT MEFs (Fig. 5A), indicating that RIP1 inhibited by MG132 (supplementary material Fig. S6A), might compete somewhat with TRADD in its interaction with suggesting that the reduction of TRAF2 protein is due to TNFR1 in response to TNF. When co-immunoprecipitation proteasome-dependent degradation, rather than caspase-mediated experiments were performed with an anti-TRADD antibody, cleavage. Stabilization of TRAF2 by MG132 treatment resulted significantly more TRAF2 interacted with TRADD in RIP1–/– MEFs in the suppression of p100 processing in response to TNF than in WT MEFs (Fig. 5B), suggesting that that RIP1 also limits (supplementary material Fig. S6B), which might further suggest that TRAF2 negatively regulates the TNF-induced noncanonical NF-B pathway in RIP1–/– MEFs. However, p100 processing is thought to also be dependent on the proteasome, so we examined the effect of short-term MG132 treatment on the basal levels of –/– –/– –/– –/–
Journal of Cell Science p52 in TRAF2 , TRAF2 TRAF5 and TRAF3 MEFs, which have constitutive p52 processing (supplementary material Fig. S6C,D,E). Short-term treatment of MG132 did not substantially reduce p52 levels in these cell types. Interestingly, although these cells have stable NIK levels because of a lack of TRAFs, the NIK protein was more highly upregulated in all of these cell types upon MG132 treatment (supplementary material Fig. S6C,D,E), suggesting that NIK expression is tightly negatively controlled by other mechanisms in addition to the TRAF-dependent ubiquitylation that targets NIK to the proteasome. The increased NIK in the MG132-treated cells would be predicted lead to higher p52 generation. However, we saw no such generation, suggesting either that MG132 blocked additional p52 generation, or that maximal p52 generation was already occurring. These data would be consistent with the hypothesis of Demchenko and colleagues, who, based on results from multiple myeloma cell lines with mutant TRAF proteins, postulated that an initial NIK stabilization event substantially activates noncanonical NF-B, whereas further stabilization of NIK has little cumulative effect on NF-B activation (Demchenko et al., 2010). These data indicate that although it is clear that TRAF2 is being degraded in a proteasomal-dependent manner in TNF-stimulated RIP1–/– MEFs, because proteasomal inhibition can block both TRAF2 degradation and p100 processing, we could not conclude, based Fig. 5. RIP1 reduces the recruitment of TRAF2 to TNFR1 and TRADD. on these experiments, that p52 generation was dependent on (A,B) Western blots of immunoprecipitates in TNF (30 ng/ml)-treated wild- TRAF2 degradation in this case. We therefore sought other data type or RIP1–/– MEFs immunoprecipitated with (A) anti-TNFR1 antibody or that would be consistent with this hypothesis. (B) anti-TRADD antibody. Input: 1% of total lysates. 652 Journal of Cell Science 124 (4)
TRAF2 recruitment to TRADD in response to TNF. Importantly, using nonmutant FLAG–RIP1 (Fig. 2F). It is not clear whether this the level of ubiquitylation in the receptor complex increased was due to less RIP1 transfection efficiency or expression, use of a considerably (supplementary material Fig. S7), and this corresponded different epitope tag or lack of kinase activity. Nevertheless, the with substantially higher levels of high molecular weight TRAF2 in partial protection seen in this experiment might indicate that the the complex (Fig. 5 and supplementary material Fig. S7), indicating kinase activity of RIP1 is not required for its protection from TRAF2 that TRAF2 is more highly ubiquitylated in the receptor complex in degradation. the RIP1–/– MEFs, which is consistent with data in supplementary material Fig. S6 showing that the proteasome inhibition blocks Noncanonical NF-B activation protects against TNF- TRAF2 degradation. These data thus suggest that RIP1 has a induced death in RIP1–/– MEFs previously unidentified function: to limit the recruitment of TRAF2 RIP1 has important roles in death-receptor-induced cell death in to TNFR1, thus stabilizing TRAF2 levels and subsequently both apoptosis and necrosis (Holler et al., 2000; Kim et al., 2007; preventing the activation of the noncanonical NF-B pathway. To Lin et al., 2004; Morgan et al., 2009; O’Donnell et al., 2007; Wang determine whether the kinase activity of RIP1 was important in et al., 2008). Owing to activation of the NF-B pathway, which preventing TRAF2, we transfected RIP1–/– MEFs with kinase- results in production of pro-survival proteins, most cells are resistant deficient Xpress-RIP1 (K45A). Transfection of this plasmid to TNF in the absence of protein synthesis inhibitors. We decreased TRAF2 degradation, NIK stability and generation of p52 examined TNF-induced cell death in the RIP1–/– MEFs, which in response to TNF (supplementary material Fig. S8), although lack the strong canonical NF-B activation induced in WT MEFs these decreases were not as substantial as in our previous experiment (Kelliher et al., 1998). As shown in Fig. 6A, TNF alone was Journal of Cell Science
Fig. 6. TNF alone induces apoptotic cell death in RIP1–/– MEFs at earlier time points, but death is inhibited at later time points. (A)Viability of TNF (30 ng/ml)-treated wild-type or RIP1–/– MEFs at 8 hours visualized by light microscopy (left) or by MTT assay (right) (*P<0.05 compared with untreated group in RIP1–/– cells; mean ± s.d.). (B)Caspase-3 western blot in lysates from TNF-treated wild-type or RIP1–/– MEFs. (C)Time-course LDH leakage assay of TNF-treated wild-type or RIP1–/– MEFs (mean ± s.d.). (D,E)Western blot analysis over long time course in TNF-treated RIP1–/– MEFs showing cleavage of caspase-3 (D) or PARP1 (E). (F)Apoptotic cell death measured by using a FITC Annexin-V Apoptosis Detection kit in TNF-treated wild-type or RIP1–/– MEFs. RIP1 attenuates noncanonical NF-B 653
sufficient to induce apoptotic cell death in RIP1–/– MEFs at earlier activation of the noncanonical NF-B pathway. RIP1 limits the time points (within 8 hours), but not in WT MEFs. Knockdown of amount of TRADD and TRAF2 proteins recruited to the TNFR1 RIP1 in wild-type cells also led to cell death from TNF alone signaling complex, probably limiting extensive ubiquitylation of (supplementary material Fig. S9A), with corresponding cleavage TRAF2 in the receptor complex. When RIP1 is removed, of caspase-3 (supplementary material Fig. S9B). Conversely, ubiquitylation-dependent and proteasomal-mediated TRAF2 ectopic expression of FLAG–RIP1 in these cells decreased their degradation leads to NIK stabilization and subsequently the sensitivity to TNF (supplementary material Fig. S9C), indicating activation of the noncanonical pathway. From the data in our study, that RIP1 deficiency is the likely reason for cell death. In agreement we propose that RIP1 deficiency leads to a lack of canonical NF- with the cell cytotoxicity assay data, we detected caspase-3 cleavage B upon TNF treatment. This sensitizes the cells to cell death at in RIP1–/– MEFs upon TNF treatment, but not in WT MEFs (Fig. earlier time points. However, as a result of the lack of RIP1, 6B). Surprisingly, when we treated these cells for 24 hours, we TRAF2 is degraded and NIK is stabilized, leading to noncanonical found that cell death did not increase after the 8-hour time point, NF-B activation, and thus protecting the cells from further death. as measured by LDH release (Fig. 6C) or morphological detection RIP1 has a weak binding affinity to TRAF2 through its kinase under a microscope (supplementary material Fig. S10). To confirm and intermediate domains (Hsu et al., 1996), suggesting a possible this phenomenon, we examined caspase-3 activation and PARP1 direct interaction between RIP1 and TRAF2 while in the complex. cleavage over a long time course. Caspase-3 activation increased Because it has a strong affinity for RIP1, TRADD enables the until 4 hours after TNF treatment, and then decreased rapidly efficient recruitment RIP1 to the TNFR1 receptor complex (Fig. 6D). Consistently, PARP1 cleavage started at 1 hour and (Ermolaeva et al., 2008; Pobezinskaya et al., 2008). However, in peaked at 4 hours after TNF treatment and then diminished, the absence of TRADD, RIP1 maintains a weak interaction with accompanying the reduction of caspsase-3 activity (Fig. 6E). The TNFR1, suggesting that there is also a direct interaction between kinetics of cell death and its subsequent reduction was verified by Annexin-V and propidium iodide staining (Fig. 6F). This analysis showed that cell death peaked at about 4 hours and then was subsequently reduced. TNF-induced cell death was sensitized in the presence of CHX, whereas the pan-caspase inhibitor, z-VAD- fmk, completely blocked TNF-induced cell death (supplementary material Fig. S11). The necrosis inhibitor necrostatin-1 had no effect on cell death, indicating that only apoptotic cell death occurred, and consistent with the requirement of RIP1 in necrotic cell death and the fact that this drug acts as an inhibitor of RIP1 kinase activity (supplementary material Fig. S11). The correlation of the decrease in caspase activation with the onset of strong p52 generation 4 hours after TNF treatment in RIP1–/– cells suggests that the noncanonical NF-B pathway
Journal of Cell Science protects these cells from further apoptotic cell death, probably through induction of anti-apoptotic genes. To see whether this was the case, we used siRNA to prevent NIK expression in RIP1–/– MEFs. NIK protein expression was effectively eliminated from occurring in cells transfected with NIK siRNA, although TRAF2 degradation continued to take place (Fig. 7A). The prevention of NIK expression led to an increased sensitivity of the RIP1–/– MEFs to TNF, especially at the 8 hour time point, as observed by light microscopy (Fig. 7B) or by LDH assay (Fig. 7C), thus showing that the activation of the noncanonical NF-B pathway protected RIP1–/– MEFs from TNF-induced cell death. As further evidence that this was the case, the pretreatment of these cells with LIGHT, a well-known inducer of noncanonical NF-B activation prevented RIP1–/– MEFs from dying in response to TNF (Fig. 7D). Thus, although RIP1–/– MEFs undergo cell death early, as a result of RIP1 deficiency, the late activation of the noncanonical NF-B Fig. 7. Inhibition of noncanonical NF-kB pathway causes TNF-induced protects surviving cells from long-term TNF toxicity. cell death in RIP1–/– MEFs. (A)Western blots of lysates from RIP1–/– MEFs transfected with non-target control siRNA or NIK siRNA and treated with Discussion TNF (2 hours) showing that TRAF2 continues to be degraded in NIK- –/– –/– We report here that TNFR1 is capable of activating the knockdown RIP1 MEFs. (B,C)Viability assays of RIP1 MEFs transfected with NIK siRNA or non-targeted control (NC) siRNA and treated with TNF noncanonical NF-B signaling pathway in addition to the canonical –/– pathway. Consistent with previous reports (Dejardin et al., 2002; showing the effect of NIK knockdown on TNF-induced apoptosis in RIP1 MEFs. (B)Representative phase-contrast microscopy images and (C) LDH Rauert et al., 2009), our data suggest that under normal leakage assay data (mean ± s.d.) are shown at indicated times. (D)MTT cell circumstances in MEF cells, TNF does not substantially activate viability assay of RIP1–/– MEFs untreated, or pretreated for 5 hours with this pathway through TNFR1. During TNFR1 signaling, the LIGHT then treated with TNF for 8 hours (*P<0.05 compared with untreated presence of RIP1 in the receptor complex not only serves to group using Student’s t-test analysis; N.S., not significant) showing that potentiate the canonical NF-B pathway, but also prevents LIGHT pretreatment protects RIP1–/– MEFs from TNF-induced cell death. 654 Journal of Cell Science 124 (4)
RIP1 and TNFR1 (Chen et al., 2008; Ermolaeva et al., 2008; this, our data also indicate that TRAF2 degradation, as induced by Pobezinskaya et al., 2008). Our data here show that loss of this a receptor stimulus, is sufficient for activation of the noncanonical interaction (through a RIP1 deficiency) permits greater TRADD pathway. Based on our data, and the current model, one would recruitment to TNFR1. The comparative amount of TRAF2 increase predict that TNFR2 stimulation would result in the activation of in the receptor complex is even proportionally greater than the the noncanonical pathway, because TRAF2 is degraded by cIAP1 increase in TRADD when compared with levels in WT cells. during TNFR2 signaling (Li et al., 2002). Consistent with our Therefore, it seems likely that RIP1 is not only competing to some model, a new report has been published during the preparation of extent with TRADD for TNFR1 binding, but also slows or obstructs this manuscript showing that membrane-bound TNF can activate TRAF2 binding to TRADD, perhaps by a kinetic constraint that is the noncanonical pathway in primary T-cells and cancer cell lines imposed by forming its own associations with TRADD and TRAF2. through its binding to TNFR2 (Rauert et al., 2009). In the absence of RIP1, not only was more TRAF2 recruited to Both TRAF2- and TRAF3-knockout mice die perinatally of TNFR1 complex, but also a greater abundance of higher molecular uncontrolled noncanonical NF-B activation (He et al., 2006; weight TRAF2 was found in the receptor complex, suggesting that Vallabhapurapu et al., 2008; Yeh et al., 1997). In light of our data, TRAF2 is further ubiquitylated, thus increasing TRAF2 protein and because RIP1–/– mice have similar prenatal lethality (Kelliher degradation. Since cIAP proteins are recruited by TRAF2 to the et al., 1998) that is partially rescued by TNFR1 deficiency (Cusson TNFR1 complex and TRAF2 is degraded by cIAP1 during TNFR2 et al., 2002), it is possible that some of the defects in RIP1–/– mice signaling (Li et al., 2002; Rothe et al., 1995; Shu et al., 1996), we are also due, in part, to hyperactive noncanonical NF-B signaling hypothesize that the loss of RIP1 results in less competition for downstream of TNF. cIAP activity, and therefore more TRAF2 ubiquitylation. Without Previous reports have suggested that activation of the RIP1, cIAP proteins might also be in a less spatially constrained noncanonical pathway serves to downregulate the canonical environment, and therefore the enzymatic sites might be able to pathway in different ways (Basak et al., 2007; Lawrence et al., better interact structurally and therefore more substantially 2005). Our studies show that TNFR1 is capable of inducing both ubiquitylate TRAF2. canonical and noncanonical pathways, but that both pathways are The absence of RIP1 in the complex probably prevents the deliberatively regulated in opposite ways by RIP1. Thus, there is formation of a full IKK complex consisting of all the subunits (i.e. a functional interplay between the two pathways, which might IKK, IKK, IKK), which is necessary for the full activation of allow regulation of the number and kinds of NF-B transcription the canonical pathway. We have previously shown that IKK and factors and thus fine-tuning the regulation of NF-B-dependent IKK can be recruited to an activated TNFR1 complex in the genes. The canonical pathway is clearly the dominant NF-B absence of IKK through interaction with TRAF2 (Devin et al., pathway that ensures an early response of cells to TNF 2000; Devin et al., 2001). However, others have shown that the stimulation, whereas the noncanonical pathway might serve as a polyubiquitylated RIP1-recruited IKK subunit strongly stabilizes compensatory redundant mechanism. It is possible that when the complex formation (Ea et al., 2006; Poyet et al., 2000; Wu et al., canonical pathway is suppressed, the non-canonical pathway is 2006). Although an absolute primary role for RIP1 in activation of activated to maintain a certain level of NF-B activity. For example, the canonical pathway has been recently questioned (Wong et al., in some embryonic developmental stages, the canonical pathway
Journal of Cell Science 2010) based on observation of IB degradation and analysis of is inactivated in certain organs when RIP1 expression is lost (Wong gene expression in response to TNF, in our hands, the deficiency et al., 2010). Although our results demonstrate that the noncanonical of RIP1 in RIP1–/– MEFs or by RIP1 knockdown leads to a pathway is a pro-survival signal that protects cells from TNF- significant decrease in TNF-induced IB degradation and NF- induced cytotoxicity, it would be interesting to determine whether B binding as measured by EMSA, which is in agreement with this pathway has other biological roles during the TNF response. numerous previous studies. Because less IKK is tied up in the canonical pathway, more IKK might be free to form IKK Materials and Methods homodimers to activate the noncanonical pathway downstream of Reagents. Recombinant murine and human TNF, mouse recombinant TRAIL, human NIK (Senftleben et al., 2001). Inhibition of the regulatory IKK recombinant LIGHT, agonistic (AF-425-PB) and blocking (MAB430) TNFR1 subunit binding using peptide inhibitors has previously been shown antibodies, TRAF2 antibody and zVAD were purchased from R&D Systems. TRAF3, to increase the basal level of IKK activity (May et al., 2000). Thus, Sp-1, TRADD, ubiquitin and IB antibodies were purchased from Santa Cruz. RIP1 deficiency could conceivably contribute to noncanonical RIP1 antibody was purchased from Transduction Laboratories. Caspase3 and PARP1 antibodies were purchased from Pharmingen. NIK, phospho-IKK, p52 and IKK activation in two different ways. antibodies were purchased from Cell Signaling. MG132 and CHX were purchased Consistent with our current data regarding TRAF3 expression in from Calbiochem. Agonistic monoclonal anti-murine lymphotoxin receptor (LTR) TNF-treated RIP1–/– cells, we previously reported little change in antibody was kindly provided by J. Browning (Biogene). Actin and FLAG antibodies and Necrostatin-1 were purchased from Sigma. TRAF3 protein levels in response to LIGHT-induced activation of the noncanonical NF-B pathway (Kim et al., 2005). However, we Cell culture observed extensive TRAF2 degradation during TNFR1 activation, Wild-type (WT), RIP1–/– and TRAF2–/– mouse embryonic fibroblast (MEF) cells were previously described (Kelliher et al., 1998; Yeh et al., 1997). MEFs were but not during LIGHT signaling (Kim et al., 2005). These two cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, pathways are therefore distinctly different from those of BAFF and 100 U/ml penicillin and 100 g/ml streptomycin. RIP1–/– (FLAG–RIP1) stable cell CD40, where TRAF3 is degraded (Brown et al., 2001; Liao et al., lines were established by transfecting cells with the designated vector and then maintaining in medium containing 10 g/ml puromycin. A549/NF-B-luc cells were 2004). BAFF does not cause TRAF2 degradation, whereas CD40 purchased from Panomics (Fremont, CA), maintained in RPMI 1640 medium stimulation results in loss of both TRAF2 and TRAF3 (Brown et supplemented with 100 g/ml hygromycin, and were infected with GIPZ-RIP1 al., 2001; Liao et al., 2004). However, TWEAK signaling through shRNA or negative control shRNA lentivirus (Open Biosystems, Huntville, AL). The its receptor, FN14, activates a lysosome-mediated degradation of puromycin-resistant cell clones were selected with puromycin (2 g/ml) medium and verified by RIP1 antibody by western blot, and were designated as A549/RIP1 KD TRAF2 and cIAP1 in a cIAP1-dependent manner, and thus activates (for EIP knockdown) and A459/NC (for negative control). They were maintained in the noncanonical pathway (Vince et al., 2008). Consistent with RPMI 1640 with hygromycin (100 g/ml) and puromycin (2 g/ml). RIP1 attenuates noncanonical NF-B 655
Western blot analysis and co-immunoprecipitation TRADD signaling in toll-like receptors. Proc. Natl. Acad. Sci. USA 105, 12429- Upon treatment, cells were lysed in 0.5% NP-40 Tris M2 buffer (Kim et al., 2007). 12434. Equal amounts of cell extracts were resolved by 12% SDS-PAGE and analyzed by Cusson, N., Oikemus, S., Kilpatrick, E. D., Cunningham, L. and Kelliher, M. (2002). western blot. For immunoprecipitation assays, lysates were mixed and precipitated The death domain kinase RIP protects thymocytes from tumor necrosis factor receptor with antibody and protein-A–Sepharose or protein-G–Agarose beads by incubation type 2-induced cell death. J. Exp. Med. 196, 15-26. at 4°C. The bound proteins were removed by boiling in SDS buffer and resolved in Dejardin, E., Droin, N. M., Delhase, M., Haas, E., Cao, Y., Makris, C., Li, Z. W., 12% SDS-polyacrylamide gels for western blot analysis and visualized by enhanced Karin, M., Ware, C. F. and Green, D. R. (2002). The lymphotoxin-beta receptor chemiluminescence (ECL, Amersham). induces different patterns of gene expression via two NF-kappaB pathways. Immunity 17, 525-535. Demchenko, Y. N., Glebov, O. K., Zingone, A., Keats, J. J., Bergsagel, P. L. and Electrophoretic mobility shift assay Kuehl, W. M. (2010). Classical and/or alternative NF-kappaB pathway activation in Nuclear extracts were prepared and analyzed as previously described (Kim et al., multiple myeloma. Blood 115, 3541-3552. 2005). For the binding reaction, 5 g of nuclear extract was incubated at room Devin, A., Cook, A., Lin, Y., Rodriguez, Y., Kelliher, M. and Liu, Z. (2000). The distinct temperature for 20 minutes with reaction buffer containing 20 mM HEPES (pH 7.9), roles of TRAF2 and RIP in IKK activation by TNF-R1: TRAF2 recruits IKK to TNF- 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 5% glycerol, 200 g/ml BSA, and 2 g R1 while RIP mediates IKK activation. Immunity 12, 419-429. 32 poly(dI-dC)·poly(dI-dC). Then the P-labeled double-stranded oligonucleotide (1 ng, Devin, A., Lin, Y., Yamaoka, S., Li, Z., Karin, M. and Liu, Z. (2001). The alpha and ≥1ϫ105 c.p.m.) containing the NF-B binding consensus sequence (5Ј-GGCAA- beta subunits of IkappaB kinase (IKK) mediate TRAF2-dependent IKK recruitment to CTGGGGACTCTCCCTTT-3Ј) was added to the reaction mixture for an additional tumor necrosis factor (TNF) receptor 1 in response to TNF. Mol. Cell. Biol. 21, 3986- 10 minutes at room temperature. The reaction products were fractionated on a 3994. nondenaturating 5% polyacrylamide gel, which was then dried and subjected to Ea, C. K., Deng, L., Xia, Z. P., Pineda, G. and Chen, Z. J. (2006). Activation of IKK autoradiography. As controls of nuclear protein content, an anti-Sp1 antibody was by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by used in EMSA and western blotting, respectively. NEMO. Mol. Cell 22, 245-257. Ermolaeva, M. A., Michallet, M. 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