TNFα-induced noncanonical NF-κB activation is attenuated by RIP1 through stabilization of TRAF2

Joo-Young Kim 2 Michael Morgan 1 Dong-Gun Kim 2 Ju-Yeon Lee 2 Lang Bai 0 Yong Lin 0 Zheng-gang Liu 1 You-Sun Kim 2 0 Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute , 2425 Ridgecrest Drive, Southeast, Albuquerque, NM 87108 , USA 1 Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health , 37 Convent Drive, Bethesda, MD 20892 , USA 2 Institute for Medical Sciences, Ajou University School of Medicine , Suwon, 443-749 , Korea - Results 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 et al., 2006; Kelliher et al., 1998; Poyet et al., 2000; Wu et al., 2006), its involvement in noncanonical NF-B activation is unknown. We carefully compared TNF-induced p100 processing in MEF cells and found that treatment with murine TNF led to a significant increase in processing of p100 and the formation of p52 in RIP1/ mouse embryonic fibroblasts (MEFs), but not in wildtype (WT) MEFs (Fig. 1A). Unlike canonical NF-B activation, the TNF-induced processing of p100 in these cells appears slowly and only becomes evident after 4 hours. We treated the RIP1/ Fig. 1. TNF induces activation of the noncanonical but not canonical NFB pathway in RIP1/ MEFs. (A)Western blots showing p100 processing in TNF-treated wild-type and RIP1/ MEFs. Cells were treated with TNF (30 ng/ml) for indicated times and cell lysates were applied for western blot with indicated antibodies. (B)(Top) Electrophoretic mobility shift assay (EMSA) in nuclear cell lysates from wild-type, TRAF2/ and RIP1/ MEFs treated with TNF showing binding to NF-B probe. (Bottom) As a control, an anti-Sp-1 antibody was used in western blot of the same nuclear lysates. (C)Western blots of lysates from TNF-treated wild-type and RIP1/ MEFs showing reduced IB degradation in RIP1/ MEFs. (D)Western blots from RIP1/ MEFs and stably transfected RIP1/(FLAGRIP1) MEFs showing recovery of 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 IB stability in RIP1-deficient cells. MEFs with human recombinant TNF, which only binds to TNFR1 in murine cells (Lewis et al., 1991). A similar pattern of p100 processing was observed when treated with human TNF (supplementary material Fig. S1A), suggesting that p52 generation is mediated by TNFR1, but not TNFR2. Similar results were obtained in RIP1/ MEFs treated with an agonistic TNFR1-specific antibody (supplementary material Fig. S1B), and the p52 to p100 ratio decreased substantially at the 4-hour time point when a TNFR1-specific blocking antibody was added (Michael Morgan, unpublished results). Next, we compared the role of RIP1 in early TNF-induced NF-B activation, which is proposed to be solely mediated by the canonical pathway (Rauert et al., 2009) and LTRinduced 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 FLAGRIP1 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 ce consistent with our previous finding (Kim et al., 2005), LTRne mediated noncanonical NF-B activation was similar in WT and ic RIP1/ MEFs (supplementary material Fig. S1C), suggesting that S RIP1 is not important in the noncanonical NF-B activation lle initiated through the LT receptor. Taken together, these results C suggest that RIP1 is required for activation of the canonical pathway fo by TNFR1 and it might negatively affect activation of the l noncanonical pathway specifically through TNFR1 when the cells rna are treated with TNF. u o J TNF induces TRAF2 degradation in RIP1/ MEFs The noncanonical NF-B pathway is constitutively active in TRAF2/ B cells and MEFs (Grech et al., 2004; Vince et al., 2007), and is degraded upon CD40 engagement in B cells (Brown et al., 2001; Liao et al., 2004), suggesting that TRAF2 functions as a negative regulator of p100 processing. We therefore carefully examined TRAF2 protein levels in TNF-treated RIP1/ MEFs. Upon TNF treatment, there was marked reduction of TRAF2 in RIP1/ MEFs, but not in WT MEFs (Fig. 2A and supplementary material Fig. S2). Notably, reduction of TRAF2 protein levels in RIP1/ MEFs correlated with p100 processing, whereas p52 protein levels were constitutively higher in TRAF2/ MEFs, irrespective of TNF treatment. A closer kinetic study revealed that TRAF2 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 with both murine and human TNF led to a reduction in TRAF2 protein levels to the same extent, suggesting that TNF-induced reduction of TRAF2 is mediated by TNFR1 (supplementary material Fig. S3A). TRAF2 was not degraded in WT or RIP1/ MEFs in response to treatment with TRAIL and no increase in p52 was observed in response to this ligand (supplementary material Fig. S3B,C). However, treatment of either WT or RIP1/ MEFs with LIGHT led to generation of p52 in the absence of TRAF2 degradation, suggesting that another mechanism for p52 generation is present in this pathway (supplementary material Fig. S3C). Fig. 2. Deficiency of RIP1 leads to p100 processing and TRAF2 degradation in response to TNF. (A)Western blots of lysates from TNFtreated wild-type, RIP1/ and TRAF2/ MEFs showing TRAF2 degradation and p52 generation in RIP1/ MEFs. (B)Western blots of lysates from short time course of TNF-treated RIP1/ MEFs showing a correlation between TRAF2 degradation and p52 generation. (C)Western blots of lysates from wild-type and RIP1/ MEFs showing TRA (...truncated)