A JNK-Dependent Pathway Is Required for TNF -Induced Apoptosis

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1 Cell, Vol. 115, 61 70, October 3, 2003, Copyright 2003 by Cell Press A JNK-Dependent Pathway Is Required for TNF -Induced Apoptosis Yibin Deng, 1 Xiaoyang Ren, 1 Lin Yang, Yahong Lin, and Xiangwei Wu* Huffington Center on Aging and Department of Molecular and Cellular Biology Baylor College of Medicine Houston, Texas Summary Tumor necrosis factor (TNF ) receptor signaling can simultaneously activate caspase 8, the transcription factor, NF- B and the kinase, JNK. While activation of caspase 8 is required for TNF -induced apoptosis, and induction of NF- B inhibits cell death, the precise function of JNK activation in TNF signaling is not clearly understood. Here, we report that TNF -medi- ated caspase 8 cleavage and apoptosis require a se- quential pathway involving JNK, Bid, and Smac/DIA- BLO. Activation of JNK induces caspase 8-independent cleavage of Bid at a distinct site to generate the Bid cleavage product jbid. Translocation of jbid to mito- chondria leads to preferential release of Smac/DIABLO, but not cytochrome c. The released Smac/DIABLO then disrupts the TRAF2-cIAP1 complex. We propose that the JNK pathway described here is required to relieve the inhibition imposed by TRAF2-cIAP1 on caspase 8 activation and induction of apoptosis. Further, our findings define a mechanism for crosstalk between intrinsic and extrinsic cell death pathways. Introduction Activation of death receptor TNFR1 (tumor necrosis fac- tor receptor 1) by TNF leads to the recruitment of TNFR1-associated death domain protein (TRADD), which serves as a platform to form various signaling complexes involved in different biological processes (Baud and Karin, 2001). For instance, TRADD can recruit FADD and lead to caspase 8 activation and apoptosis through the extrinsic pathway (Hsu et al., 1996b). How- ever, TNF is not cytotoxic to most cells, because TRADD can recruit TRAF2 (TNF -receptor-associated factor 2) and RIP to form distinct complexes leading to the activation of NF- B and JNK (Hsu et al., 1996a, 1996b; Lee et al., 1997). It has been well established that activation of NF- B serves as a primary mechanism to protect cells against apoptotic stimuli such as TNF (Beg and Baltimore, 1996; Van Antwerp et al., 1996; Wang et al., 1996). Therefore, TNF -induced apoptosis requires NF- B inhibition. The involvement of JNK in TNF -mediated apoptosis is highly controversial (De Smaele et al., 2001; Liu et al., 1996b; Natoli et al., 1997; Tang et al., 2001). Recently, induction of NF- B has been shown to inhibit TNF mediated JNK activation and blocking NF- B results in sustained activation of JNK, which may directly promote TNF -mediated apoptosis (De Smaele et al., 2001; Tang et al., 2001). Despite the fact that sustained activation of JNK promotes cell death, the molecular basis of how JNK contributes to TNF -mediated apoptosis remains to be addressed. The intrinsic apoptotic pathway is the result of activation of mitochondria-mediated cell death events, including changes in mitochondrial membrane permeability and subsequent release of proapoptotic factors involved in various aspects of apoptosis (Wang, 2001). The released factors include cytochrome c (cyto c; Liu et al., 1996a), apoptosis inducing factor (AIF) (Susin et al., 1999), second mitochondria-derived activator of cas- pase (Smac or DIABLO) (Du et al., 2000; Verhagen et al., 2000), Omi/HtrA2 (Suzuki et al., 2001), and endonu- clease G (Li et al., 2001). Cytosolic cyto c and Apaf-1 form an essential part of apoptosis complex and lead to the activation of caspase 9, which then processes and activates other caspases to orchestrate the biochemical execution of cells (Li et al., 1997). Although the apoptotic pathways through death re- ceptors and mitochondria are capable of operating independently, accumulating evidence suggests that a crosstalk exists between the two pathways. One link between death receptor signaling and mitochondrial pathway comes from the finding that the BH3-domain only protein Bid is cleaved and activated by active cas- pase 8. The truncated Bid (tbid) translocates to mitochondria and triggers cyto c release (Li et al., 1998; Luo et al., 1998). We have shown that caspase 8-Bid-Bax- depedent intrinsic pathway is required for TRAIL- and FasL-mediated apoptosis in human cancer cells (Deng et al., 2002). Studies on the function of mitochondria in TNF induced apoptosis have been complicated by the fact that TNF -induced apoptosis requires the inhibition of NF- B. Most of previous studies achieved this by blocking RNA or protein synthesis using actinomycin D (Act D) or cyclohexamide (CHX), which inevitably affect many other biological processes. To better address the function of mitochondria in TNF -mediated apoptosis and to elucidate the role of JNK in TNF signaling, we used small interfering RNA (sirna) to block TNF mediated JNK activation in p65 / cells and in cells expressing a mutant of i B (Reuther and Baldwin, 1999). We report that unlike TRAIL- and FasL-induced apoptosis where mitochondria act downstream of cas- pase 8 (Deng et al., 2002), TNF -mediated caspase 8 activation requires activation of mitochondria pathway. We further show that a pathway involving TNF -mediated activation of JNK, JNK-induced caspase 8-inde- pendent cleavage of Bid, and Bid-induced preferential release of Smac from mitochondria, is required for TNF -mediated caspase 8 activation and apoptosis. Results Mitochondria Pathway Is Critical for TNF -Induced Caspase 8 Activation and Apoptosis To test the function of mitochondria in TNF -mediated *Correspondence: xiangwei@bcm.tmc.edu 1 These authors contributed equally to this work. apoptosis, a mutant of i B (m-i B) was used to inhibit

2 Cell 62 Figure 1. Mitochondria Pathway Is Critical for TNF -Mediated Caspase 8 Activation and Apoptosis (A) HeLa cells were transfected with either m-i B or m-i B and Bcl-xl (HA-tagged) for 48 hr. A green fluorescent protein (GFP) expressing plasmid (pegfp-c1, Clontech) was cotransfected to monitor the transfection efficiency and to mark the transfected cells. The cells were treated with TNF (50 ng/ml) for 8 hr and pictures were taken. (B) HeLa cell extracts were prepared from cells described in (A) and analyzed with antibodies specifically recognizing cleaved human caspase 8 (Casp 8) and cleaved PARP (C-PARP), respectively. Transfected Bcl-xl was detected by anti-ha antibody. Endogenous i B and transfected m-i B were detected by an anti-i B antibody. Expression of tubulin was used as loading controls. (C) p65 / MEFs were transfected with either control vector or Bcl-xl and stable cell lines were established. Expression of Bcl-xl was analyzed by Western blots using an anti Bcl-xl antibody and tubulin was used as loading control. (D) p65 / and p65 / /Bcl-xl cells were treated by TNF (10 ng/ml) for indicted time points. The apoptotic cells were counted by trypan blue exclusion and percentages of cell death were plotted. The data are derived from two independent experiments. (E) Western blot analysis of procaspase 8 expression in TNF -treated cells using an anticaspase 8 antibody. Three independent cell lines were tested and similar results were obtained. NF- B activation in HeLa cells. Transfection of m-i B structed a plasmid expressing sirna for human MKK7 alone had no effect on cell viability but resulted in cell (MKK7siRNA) in the psuper sirna vector (Brummel- death after TNF treatment as cells became rounded, kamp et al., 2002). MKK7, also known as JNKK2, has shrank, and detached (Figure 1A). Coexpression of Bclxl been shown to be the JNK upstream kinase required significantly blocked the TNF toxicity (Figure 1A). for TNF -induced JNK activation (Tournier et al., 2001). Western blotting showed that TNF -induced caspase 8 The MKK7siRNA was transfected into HeLa cells excleavage in cells transfected with m-i B, but the cleav- pressing m-i B (HeLa-m-i B) and TNF -mediated JNK age was inhibited by the expression of Bcl-xl (Figure activation was analyzed. While expression of MKK7siRNA 1B). Cleavage of Poly (ADP-ribose) polymerase (PARP) has no effect on total JNK (JNK1 and JNK2) expression, correlated with caspase 8 cleavage and apoptosis (Figure it greatly reduced MKK7 protein expression and pre- 1B). Analysis of caspase 8 and caspase 3 enzyme vented JNK activation in response to TNF as TNF - activities in these cells correlated with the Western blotting induced phosphorylation of JNK was inhibited (Figure results (data not shown). The ability of Bcl-xl to 2A). To better mark the MKK7siRNA expressing cells, inhibit TNF -mediated caspase 8 activation and apoptosis we cloned a green fluorescent protein (GFP) expression was also tested in mouse embryonic fibroblast (MEF) cassette into the MKK7siRNA vector. This MKK7siRNA- cells lacking the endogenous p65 or RelA subunit of GFP plasmid was transfected into HeLa-m-i B cells. NF- B (p65 / ). Consisting with the results in HeLa Expression of GFP alone had no effect on TNF -induced cells, expression of Bcl-xl significantly inhibited TNF induced cell death as cells turned apoptotic, while coexpressing apoptosis in p65 / cells (Figures 1C and 1D). MKK7siRNA inhibited cell death induced by TNF (Fig- Furthermore, Bcl-xl also blocked TNF -induced caspase ure 2B). Expression of MKK7siRNA also blocked 8 cleavage even after prolonged treatment (Figure TNF -induced cleavage of caspase 8 (Figure 2C). To 1E). Similar experiments were also performed in human further strengthen this observation, p65 / MEF stable colon cancer HCT116 cells and identical results were cell lines expressing mouse MKK7siRNA were established. obtained (data not shown). These data suggest that the Stably expressing MKK7siRNA effectively blocked intrinsic pathway is required not only for TNF -mediated the expression of endogenous MKK7 and TNF -induced apoptosis, but also for TNF -induced caspase 8 activation. JNK activation (Figure 2D). Furthermore, MKK7siRNA The later is distinct to TRAIL and FasL-induced significantly inhibited TNF -mediated apoptosis in caspase 8 activation, which is independent of mitochondria p65 / cells (Figures 2E and 2F). More importantly, (Deng et al., 2002). MKK7siRNA expression blocked TNF -induced cas- pase 8 cleavage and activation (Figure 2G). These data TNF -Mediated Caspase 8 Activation and strongly support that JNK activation is required for Apoptosis Require JNK Activation TNF -induced caspase-8 activation and apoptosis. Since mitochondria events are required for TNF -mediated caspase 8 activation and apoptosis, it raised two Induction of Preferential Smac Release important questions: what is the molecular basis of by TNF -Mediated JNK Activation TNF -induced activation of mitochondria pathway and To explore the role of TNF -mediated JNK activation in how do mitochondria signals regulate TNF -mediated regulating mitochondria events, we proceeded to inves- caspase 8 activation? To address the first question, we tigate whether JNK activation by TNF affected the subcellular focused on TNF -induced JNK activation. We con- localization of cyto c and Smac. To exclude the

3 JNK, Bid, and Smac/DIABLO in TNF Signaling 63 Figure 2. Inhibition of JNK Blocks TNF -Mediated Caspase 8 Activation and Apoptosis (A) MKK7siRNA blocks TNF -mediated JNK activation. HeLa cells stably expressing m-i B were transfected with either vector psuper or MKK7siRNA. Cells were treated with TNF (50 ng/ml) for time indicated after 48 hr transfection and expression of MKK7; total JNK (T-JNK1/2) and phosphorylated JNK (P-JNK1/2) were analyzed by Western blots. (B) HeLa-m-i B cells were transfected with either GFP or MKK7siRNA-GFP for 48 hr and treated by TNF (50 ng/ml). Pictures were taken 12 hr after TNF treatment. (C) Western analysis of MKK7 and cleaved caspase 8 in HeLa-m-i B cells transfected with GFP and MKK7siRNA-GFP. (D) p65 / and p65 / stably expressing MKK7siRNA were treated with TNF (10 ng/ml) for time indicated. Whole-cell extracts were analyzed by Western blots for MKK7, P-JNK1/2, and T-JNK1/2. (E) The control p65 / and p65 / stably expressing MKK7siRNA were treated with TNF. The pictures were taken 8 hr after TNF treatment. (F) Apoptotic cells were counted at multiple fields and percentages of survival cells were plotted. The data are the average of two independent experiments. (G) TNF treated cells were analyzed for procaspase 8 expression by Western blots. Tubulin was used as loading control. Three independent cell lines were tested and similar results were obtained. induced redistribution of Smac-GFP to diffused cytosolic pattern (Figure 3C). While less than 20% of Smac- GFP expressing cells showed cytosolic GFP before TNF treatment, more than 75% of the Smac-GFP be- came cytosolic after TNF stimulation (Figure 3D). More importantly, expression of MKK7siRNA inhibited Smac translocation by more than 4-fold (Figures 3C and 3D). These results indicate that TNF -activated JNK, functioning upstream of mitochondria, is required for TNF induced preferential release of Smac. It has been shown that inhibition of NF- B resulted in sustained activation of JNK induced by TNF (De Smaele et al., 2001; Tang et al., 2001). To test whether sustained JNK activation is sufficient to induce preferen- tial release of Smac from mitochondria, a constitutive active JNK expression vector JNK-MKK7-wt and its ki- nase inactive mutant JNK-MKK7-kd were transfected into HeLa-m-i B-FADD-DN cells along with either Smac-GFP or a Cyto C-GFP fusion protein (Figure 3E). Expression of JNK-MKK7-wt, but not the JNK-MKK7- kd induces sustained JNK activation in HeLa cells as reported (Rennefahrt et al., 2002 and data not shown). More importantly, JNK-MKK7-wt, but not JNK-MKK7- kd specifically resulted in mitochondrial release of Smac-GFP without affecting Cyto C-GFP distribution (Figure 3F), suggesting that sustained JNK activation is capable of inducing preferential release of Smac from mitochondria. possibility that activation of mitochondria pathway is the result of TNF -induced caspase 8 activation, we established stable cell lines expressing a dominant-negative mutant of FADD (FADD-DN) using p65 / MEFs (Chinnaiyan et al., 1995). Consistent with previous reports, expression of FADD-DN blocked TNF -mediated caspase 8 activation and apoptosis without affecting TNF -induced JNK activation (Liu et al., 1996b and data not shown). Immunostaining of p65 / FADD-DN cells showed that both cyto c and Smac displayed punctuated perinuclear pattern before TNF treatment, indicative of mitochondria localization. The mitochondria staining pattern was confirmed using MitoTracker (data not shown). The staining pattern of the Smac protein after TNF treatment became diffused, indicative of cytosol localization, while cyto c was not affected (Figure 3A). These results suggest that TNF signaling leads to preferential release of Smac from mitochondria in a caspase 8-independent manner. To test whether Smac is also preferentially released in TNF signaling pathway in other cell types, we established HCT116 cell lines stably expressing both m-i B and FADD-DN (data not shown). The subcellular fraction results showed that after TNF stimulation, Smac was released from mitochondria to cytosol, but majority of cyto c remained in mitochondria (Figure 3B). To investigate if JNK activation acts upstream of Smac redistribution, a Smac-GFP fusion protein was cotransfected with either vector control or MKK7siRNA into HeLa cell lines stably expressing both m-i B and FADD-DN (Figure 3C). Similar to endogenous Smac, the Smac-GFP was expressed in mitochondria in untreated HeLa-m-i B- FADD-DN cells as Smac-GFP showed punctuated perinuclear mitochondrial pattern (Figure 3C). TNF treatment Bid Acts Downstream of JNK in TNF Signaling to Mitochondria Since Bid has been shown to act as a link between mitochondria and death receptor pathways induced by FasL and TRAIL (Deng et al., 2002), it is possible that

4 Cell 64 Figure 3. TNF -Induced Mitochondria Smac Release Requires JNK Activation (A) p65 / FADD-DN cells were treated for 4 hr with or without TNF (10 ng/ml). The cells were stained for either cytochrome c or Smac proteins. DNA was visualized by staining with Hoechst (B) Mitochondria (Mito) and cytosolic (Cyto) extracts of TNF -treated (50 ng/ml) and untreated HCT116-m-i B-FADD-DN cells were blotted for Smac and cytochrome c expression. Mitochondria CoxIV and cytosolic actin were used as subcellular fraction controls. (C) HeLa-m-i B-FADD-DN cells were transiently transfected with plasmids psuper and Smac-GFP, or Smac-GFP and MKK7siRNA. Distribution of Smac-GFP was viewed under a fluorescent microscope before and after TNF treatment for 12 hr (50 ng/ml). (D) GFP positive cells were counted at multiple fields and percentages of cytosolic GFP cells were plotted. The data are derived from three independent experiments. (E) JNK-MKK7-wt and JNK-MKK7-kd (Myc-tagged) were transiently transfected into HeLa-m-i B-FADD-DN cells along with either Smac-GFP or a Cyto C-GFP. Expression of JNK-MKK7 proteins was analyzed using anti-myc antibody. (F) GFP positive cells were counted at multiple fields and percentages of cytosolic GFP cells were plotted. The data are derived from three independent experiments. Bid may function downstream of JNK to transduce TNF expression had no effect on TNF -induced decrease signal to mitochondria. To test this hypothesis, we in Bid protein levels, while expression of MKK7siRNA first proceeded to examine Bid expression in TNF - prevented Bid reduction (Figure 4A), suggesting that mediated apoptosis. In p65 / cells, TNF treatment led TNF is capable of inducing caspase 8-independent to a reduction in full-length Bid protein levels as Bid can but JNK-dependent Bid cleavage. Similar results were be cleaved by activated caspase 8 as reported (Li et al., obtained in HeLa cells (Figure 4B). To explore whether 1998; Luo et al., 1998) (Figure 4A). However, inhibition JNK-induced Bid cleavage leads to translocation of of TNF -mediated caspase 8 cleavage by FADD-DN cleaved Bid to mitochondria, we generated a Bid and GFP fusion protein at the C-terminal of Bid. Transfection of Bid-GFP into HeLa-m-i B-FADD-DN cells resulted in most cells in diffused cytosolic GFP expression pattern, consistent with the fact that full-length Bid resides in cytosol (Figure 4C). After TNF treatment, most of GFP displayed punctuated mitochondria pattern (Figure 4C) and colocalized with mitochondria marker MitoTracker (data not shown). Cotransfecting with MKK7siRNA significantly inhibited GFP fluorescence distribution to mitochondria (Figure 4C). These data suggest that TNF induces a caspase 8-independent and JNK-dependent Bid cleavage and subsequent translocation of cleaved Bid to mitochondria. Figure 4. Caspase 8-Independent and JNK-Dependent Bid Cleavage in TNF Signaling Induction of Preferential Smac Release (A) p65 / and p65 / stable cell lines expressing MKK7siRNA or by JNK-Activated Bid FADD-DN were treated with TNF (10 ng/ml) for time indicated. Caspase 8 cleaved product tbid has been shown to Expression of full-length Bid was analyzed using a mouse specific induce apoptosis via mitochondria (Li et al., 1998; Luo anti-bid antibody. (B) HeLa-m-i B-FADD-DN cells were transiently transfected with et al., 1998). However, our results illustrate that JNKeither psuper vector or MKK7siRNA. After 48 hr, the transfected induced Bid cleavage is incapable of killing cells, sug- cells were treated with TNF (50 ng/ml) for 12 hr. HeLa cells were gesting that the Bid cleavage product generated by JNK treated with TRAIL (100 ng/ml) for 6 hr. Expression of Bid and MKK7 activation is different. As a first step to test this possibilwas examined by Western blots. ity, an in vitro Bid cleavage assay was established. Cyto- (C) HeLa-m-i B-FADD-DN cells were transiently transfected with sol extracts were prepared from TNF -treated HeLa-meither Bid-GFP or Bid-GFP and MKK7siRNA. The transfected cells were treated with TNF (50 ng/ml) for 12 hr. Mitochondrial GFP i B-FADD-DN cells and cells expressing MKK7siRNA. fluorescence was counted and plotted and pictures were taken. The The extracts were incubated with an in vitro translated data represents three independent experiments. Bid protein. While full-length Bid migrates around 24

5 JNK, Bid, and Smac/DIABLO in TNF Signaling 65 Figure 5. Identification and Characterization of JNK-Mediated Bid Cleavage (A) In vitro JNK-mediated Bid cleavage. In vitro translated Bid protein labeled with 35 S-methionine was incubated with cytosol extracts prepared from either TNF -treated or nontreated HeLa-m-i B-FADD-DN and HeLa-m-i B-FADD-DN cell expressing MKK7siRNA. The reaction mix was separated on a 12% SDS-PAGE and subject to autoradiography. In vitro translated tbid protein labeled with 35 S-methionine was used as size comparison. (B) In vivo JNK-mediated Bid cleavage. HeLa-m-i B and HeLa-m-i B-FADD-DN cells were transfected with Bid-GFP and treated with TNF (50 ng/ml) for 8 hr. Transfected Bid proteins were detected by an anti-gfp monoclonal antibody (Santa Cruz). HeLa cells transfected with tbid-gfp were used as size comparison. (C) Detection of endogenous jbid. HeLa-m-i B-FADD cells were transfected with either MKK7siRNA or psuper vector. After 48 hr, cells were treated with or without TNF (50 ng/ml) for 6 hr. Expression of Bid and MKK7 were detected by Western blots. Tubulin was used as loading control. The experiments were repeated multiple times and representative results were presented. (D) Bid-GFP or GFP fused deletion mutants of Bid was transfected into HeLa cells. Subcellular localization of transfected proteins was observed using GFP fluorescence and mitochondria localization was verified by colocalizing with MitoTracker-Red (Molecular Probes). Apoptosis were plotted as described in Figure 1D. Data represents the average of two independent experiments. (E) Size comparison of jbid with Bid deletion mutants. Extracts from HeLa cells transfected with either Bid 12-GFP, or Bid 25-GFP, or Bid 30- GFP, or Bid 40-GFP, and extract from TNF treated HeLa-m-i B-FADD-DN cells transfected with Bid-GFP were separated on a 7% SDS- PAGE. The transfected proteins were detected by an anti-gfp monoclonal antibody. (F) In vitro cleavage of Bid mutants. Wild-type full-length Bid and alanine scanning mutants of Bid for amino acid were transcripted and translated and labeled with 35 S-methionine (top image) and subject to in vitro Bid cleavage assay using extracts of TNFa-treated HeLam-i B-FADD-DN cells as described in Figure 5A (bottom image). The nonspecific band generated by in vitro translation was indicated by an *. (G) Bid 25-GFP was transfected into HeLa cells and cells were stained for either cytochrome c or Smac proteins. DNA was visualized by staining with Hoechst (H) Bid-GFP, Bid 25-GFP, and tbid-gfp were separately transfected into HeLa cells. Smac and Cyto C localization were examined by immunostaining. Data from three independent experiments were plotted. kda on a SDS-PAGE as predicated, reaction with HeLa-m-i B-FADD-DN and HeLa-m-i B cells. The TNF -treated HeLa-m-i B-FADD-DN extract generated cleavage of Bid-GFP was analyzed by Western blotting a specific Bid cleavage product migrating approximately using antibody against GFP (Figure 5B). Expression of at 21 kda (jbid for JNK-mediated cleaved Bid) (Figure Bid-GFP in HeLa-m-i B cells resulted in the generation 5A). The in vitro translated tbid (caspase 8 cleaved) of tbid-gfp after TNF treatment, comparable to transfected displayed molecular weight around 15 kda (Figure 5A). tbid-gfp in HeLa cells (Figure 5B). A larger jbiddisplayed These results suggest that TNF -JNK activation results GFP cleavage product was detected when Bid-GFP expressing in the generation of a unique Bid cleavage product jbid, in HeLa-m-i B-FADD-DN cells were treated which is distinct to caspase 8 cleaved product tbid. by TNF (Figure 5B). To demonstrate the existence of More importantly, addition of extracts prepared from endogenous jbid in TNF signaling, HeLa-m-i B-FADD- MKK7siRNA expressing cells failed to induce Bid cleav- DN cells were transiently transfected with either age (Figure 5A), suggesting that generation of jbid requires MKK7siRNA or vector control and cell extracts were ana- the activation of JNK. To further confirm the in lyzed by Western blotting. Transfection of MKK7siRNA vitro cleavage results, Bid-GFP was transfected into significantly inhibited MKK7 expression (Figure 5C).

6 Cell 66 When cleavage of Bid was analyzed, a faster migrating or Bid / MEFs (Figure 6A). As expected, no Bid protein band than the full-length Bid was detected in cells transfected expression was detected in Bid / cells (Figure 6A). Ex- with vector control after TNF treatment with cor- pression of GFP-m-i B in wild-type MEFs, but not in responding decrease in the levels of full-length Bid, but Bid / MEFs, resulted in cell death after TNF treatment not in MKK7siRNA-transfected cells (Figure 5C). The as GFP-m-i B expressing cells became apoptotic (Figure endogenous jbid band migrated around 21 kda and is 6B), suggesting that Bid is required for TNF endogenous similar in size as the in vitro cleaved jbid (Figure 5A and mediated apoptosis. To further explore function of Bid data not shown). on TNF -mediated caspase 8 activation, we con- It has been shown that caspase 8 cleaves human Bid structed a plasmid expressing both Bid small interfering at amino acid position 60 (D60) to generated tbid. The RNA (BidsiRNA) and GFP to inhibit Bid expression in finding that jbid is larger than tbid suggests that jbid is HeLa-m-i B cells. Expression of BidsiRNA-GFP greatly generated by a cleavage closer to Bid s N terminus. reduced the endogenous full-length Bid expression Furthermore, since the generation of jbid is not sufficient while cells transfected with only GFP contained readily to induce cell death, it suggests that the difference in detectable full-length Bid (Figure 6C). Furthermore, cas- Bid cleavage site at Bid N-terminal region may lead to pase 8 was activated as cleaved caspase 8 appeared the generation of Bid cleavage products possessing and Bid was cleaved as the level of the full-length Bid distinct proapoptotic potentials. To test this hypothesis, protein was greatly reduced in GFP transfected HeLam-i B we generated a series N-terminal deletion mutants of cells after TNF treatment (Figure 6C). Expression Bid. Deletion of the first 12 amino acids of Bid had little of BidsiRNA in HeLa-m-i B cells inhibited TNF -mediated effect on its protein localization and cell death inducing caspase 8 activation, as there was a significant capability when transfected into HeLa cells (Figure 5D). reduction in the level of cleaved caspase 8 (Figure 6C). Removal of 25 amino acid or more of Bid s N-terminal The mitochondrial release of Smac was also blocked sequences resulted in Bid translocation from cytosol to (data not shown). Counting apoptosis of GFP expressing mitochondria. Of these mutants, only the mutant with cells showed that GFP alone had no effect on TNF - deletion of the first 25 amino acids (Bid 25) showed induced apoptosis, while coexpression of BidsiRNA significantly potential to translocate to mitochondria, but did not inhibited TNF -induced cell death (Figure 6D). induce cell death (Figure 5D). More importantly, this These results indicate that Bid acts upstream of caspase mutant was similar in size as the jbid protein (Figure 8 and is required for TNF -mediated caspase 8 activa- 5E), suggesting that activation of JNK resulted in Bid tion and apoptosis. cleavage around amino acid position 25. To further narrow To test the function of JNK-dependent cleavage defiscanning down the JNK-mediated Bid cleavage site, alanine cient mutant Bid (BidL25A) in TNF pathway, BidL25A mutants around amino acid position 25 were (GFP fusion protein) was transfected into HeLa-m-i B- generated and subject to in vitro cleavage reactions. FADD-DN cells and no Bid cleavage was detected after Serendipitously, mutation in amino acid position 25 resulted TNF treatment (Figure 6E), consistent with in vitro Bid in a completely loss of Bid cleavage, while muta- cleavage assay (Figure 5F). However, BidL25A still can tions of other amino acids had no effect on JNK-mediated be cleaved by active caspase 8 (data not shown). While Bid cleavage (Figure 5F). These results suggest transfection of wild-type Bid restored TNF -mediated that jbid is generated by a proteolytic cleavage at amino apoptosis in Bid / MEFs, expression of BidL25A had acid position 25 and Bid 25 is or very similar to jbid. To no effect on cell death (Figure 6F), suggesting that JNKmediated investigate if Bid 25 is capable of inducing preferential Bid cleavage at position 25 is critical for TNF Smac release, HeLa cells transfected with GFP-Bid 25 signaling to apoptosis. were immunostained with either an anti-smac or an anticyto c antibody (Figure 5G). The results showed that Activation of Smac Bypasses JNK and expression of Bid 25 lead to redistribution of Smac pro- Mitochondria Requirement in Promoting tein staining patterns from mitochondria to cytosol (Fig- TNF -Mediated Caspase 8 Activation ures 5G and 5H). Consistent with the fact Bid 25 is To test whether Smac may function as the mitochondria not cytotoxic, no cyto c release was observed in cells signal to regulate TNF -mediated caspase 8 activation, expressing Bid 25 (Figures 5G and 5H). In contrast, full- a recently developed expression system using ubiquitin length Bid had little effect on the redistribution of either fusion to produce mature and active Smac in cytosol of Smac or cyto c, while tbid induced mitochondrial release transfected cells was utilized to deliver active Smac into of both of these factors (Figure 5H). These results HeLa cells (Hunter et al., 2003). Transfection of Ub-Smac strongly support the notion that TNF activates JNK to (active Smac), but not the Ub- AVPI-Smac (inactive lead to a caspase 8-independent cleavage of Bid at Smac) cooperated with TNF in inducing caspase 8 amino acid position 25 to generate a 21 kda cleavage cleavage and apoptosis (Figures 7A and 7B). Since Ubproduct jbid. The jbid protein then translocates to mito- AVPI-Smac lacks IAP binding sequence, it further sugchondria to induce preferential release of Smac. gests that the activity of Smac in TNF pathway requires its interaction with IAP family proteins. To support this notion, a cell-permeable peptide derived from Smac Bid Acts Upstream of Caspase 8 Activation protein sequence containing the domain required for IAP in TNF Signaling binding (N-7) was added into HeLa-m-i B-MKK7siRNA To test the function of Bid in TNF -signal transduction cells. A noncell permeable Smac peptide (N7-NP) was to cell death, we first constructed a GFP and m-i B used as negative control. Treating HeLa-m-i B cells with fusion protein and transfected it into either the wild-type TNF resulted in caspase 8 activation and coexpressing

7 JNK, Bid, and Smac/DIABLO in TNF Signaling 67 Figure 6. Requirement of Bid for TNF -Mediated Caspase 8 Activation and Apoptosis (A) GFP-m-i B was transiently transfected into Bid / and wild-type MEFs. The cells were treated with TNF (10 ng/ml) for 12 hr and expression of Bid; GFP-m-i B was examined by Western blots. (B) Morphology of GFP positive cells as described in (A). (C) HeLa-m-i B cells were transfected with either GFP or BidsiRNA-GFP for 48 hr. The cells were treated with TNF (50 ng/ml) for 16 hr. Expression of Bid and cleaved caspase 8 were examined by Western blots. (D) Cells were counted at multiple fields and percentages of apoptotic cells were plotted. (E) JNK-mediated cleavage of wild-type and L25A mutant Bid in cells. Bid-GFP or BidL25A-GFP was transfected into HeLa-m-i B-FADD-DN cells and Western analysis was performed as in Figure 5B. (F) Rescue of TNF -mediated apoptosis in Bid / cells. Bid / MEFs were transfected with either Bid-GFP or BidL25A-GFP along with m-i B. The transfected cells were treated with TNFa and percentages of apoptosis were plotted. of MKK7siRNA inhibited caspase 8 cleavage as expected (Figure 7C). Addition of N-7, but not N-7NP, cooperated with TNF to induce caspase 8 activation and apoptosis in HeLa-m-i B-MKK7siRNA cells (Figure 7C). These results suggest that Smac is sufficient to rescue TNF -induced caspase 8 activation and apoptosis in cells deficient in JNK activation and Smac acts downstream of JNK and mitochondria in TNF signaling to caspase 8 activation and cell death. TNF -Mediated Caspase 8 Activation and Apoptosis Require Endogenous Smac To Investigate the involvement of endogenous Smac in TNF signaling, a plasmid expressing both sirna of Smac and GFP (SmacsiRNA-GFP) was transfected into HeLa-m-i B cells. Expression of SmacsiRNA-GFP greatly reduced Smac protein levels (Figure 7D). More importantly, SmacsiRNA-GFP significantly inhibited TNF -mediated caspase 8 cleavage and apoptosis by 4-fold (Figures 7D and 7E). Similar results were obtained in HCT116 cells stably expressing SmacsiRNA (data not shown). These results indicate that Smac plays a pivotal role at downstream of JNK-Bid-mitochondria pathway in regulation of TNF -mediated caspase 8 activation and apoptosis in these cells. It has been shown that ciap1 and ciap2 are recruited to the TNF signaling complex by TRAF2 and coexpression of TRAF2 and ciap1 significantly inhibited TNF - Figure 7. Smac Is Involved in TNF -Mediated Caspase 8 Activation and Apoptosis (A) HeLa cells were transfected with GFP and either Ub-Smac or Ub- AVPI-Smac (HAtagged), and cells were treated with TNF (50 ng/ml) for 16 hr. Expression of Smac and cleaved caspase 8 were detected using anti- HA and anti-human cleaved caspase 8 antibodies, respectively. (B) Quantitation of apoptosis of GFP positive cells in (A) was plotted. (C) HeLa-m-i B (m-i B) and HeLa-m-i B- MKK7siRNA (m-i B-MKK7siRNA) cells were treated with Smac peptide N7 (50 M), control peptiden7-np (50 M), and TNF (50 ng/ml) in various combinations as indicated and cell extracts were analyzed for cleaved caspase 8 by Western blots. (D) GFP and SmacsiRNA-GFP were transfected into HeLa-m-i B cells for 48 hr. Cells were treated with TNF (50 ng/ml) for 16 hr. Expression of Smac and cleaved caspase 8 were analyzed by Western blotting. (E) Percentage of apoptosis in GFP positive cells described in (D) was plotted. Three independent experiments were performed. (F) N7 leads to TRAF2-cIAP1 dissociation. HeLa cells were treated for 1 hr with either TNF (50 ng/ml) or TNF and N7 (50 M). Whole-cell extracts were immunoprecipitated with an antibody against TRAF2 and blotted with anti-ciap1 and anti-traf2 antibodies respectively. Ig: immunoglobulin heavy chain.

8 Cell 68 mediated caspase 8 activation, suggesting that ciap1 Further, our model is consistent with the results that and TRAF2 function as a complex to regulate TNF strongly coexpression of TRAF2 and ciap1, but not individually, mediated caspase 8 cleavage (Park et al., 2000; Wang inhibits TNF -induced apoptosis in p65 null et al., 1998). We observed that IAP binding sequence is cells (Wang et al., 1998). To further support this hypothe- required and sufficient to cooperate with TNF in cell sis, we have shown that active Smac, but not the mutant death signaling (Figures 7A and 7B), thus, it is possible lacking ciap1 binding sequences, sensitizes TNF that Smac acts on the TRAF2-cIAP1 complex to promote induced caspase 8 activation, and a Smac peptide con- TNF -mediated caspase 8 activation. To support this taining the ciap1 binding domain is sufficient to disrupt hypothesis, we found that addition of N-7 peptide disinduce TRAF2-cIAP1 interaction and cooperate with TNF to rupted the association of TRAF-2 and c-iap1 after TNF cell death. treatment (Figure 7F). Therefore, it is likely that release The TNF -JNK-jBid-Smac pathway defines another of Smac from mitochondria disrupts TRAF2-cIAP1 comtrinsic mechanism for the crosstalk between intrinsic and ex- plex and its inhibition on caspase 8 activation. cell death pathways. This pathway is distinct to Fas- and TRAIL-induced apoptosis where Bid acts Discussion downstream of caspase 8 and mitochondrial release of Smac acts on XIAP to facilitate caspase 3 activation We have shown that a sequential pathway involving (Deng et al., 2002). Interestingly, recent reports have JNK, Bid, mitochondria, and Smac is required for TNF perfamily, activates Drosophila homologs of JNK to in- shown that Eiger, the Drosophila homolog of TNF sumediated apoptosis. In wild-type cells, TNF treatment leads to the recruitment ciap1 to the TRADD-FADDal., 2002; Moreno et al., 2002). The later proteins are duce apoptosis through Hid, Reaper, and Grim (Igaki et caspase 8 complex by TRAF2 and TNF -mediated activation of NF- B keeps JNK in check by inhibiting functional homolog of mammalian Smac, suggesting sustained activation of JNK. These events prevent that the TNF -JNK-Smac pathway is evolutionarily con- TNF -mediated caspase 8 cleavage and apoptosis. Inserved. hibition of NF- B allows JNK to induce caspase 8-indefor TNF -induced cell death and MEFs deficient for A recent study has shown that JNK is not required pendent cleavage of Bid at a distinct site to generate a Bid cleavage product jbid. The jbid protein then translo- JNK1 and JNK2 are somewhat more sensitive to cates to mitochondria to induce the selective release of TNF -induced apoptosis, suggesting that JNK plays a role as survival factor (Lamb et al., 2003). However, pro- Smac, but not cyto c. Smac is then required to relieve tein synthesis inhibitors CHX and emetine were used in the inhibition of caspase 8 cleavage and activation. Furthese experiments. We have observed that treating cells thermore, we propose that this inhibition is caused by with TNF and CHX or RNA synthesis inhibitor Act D TRAF2-cIAP1 within the TRADD-FADD-caspase 8 comsignificantly inhibits TRAF2 and ciap1 expressions and plex. In support of this hypothesis, we show that an bypasses JNK-dependent mitochondria requirement active fragment of Smac can disrupt the interaction be- (Y.D. and X.W., unpublished data). It is possible that low tween TRAF2 and ciap1 upon TNF stimulation. Since or basal levels of JNK provide protection against cell ciap1 does not directly bind to caspase 8 and inhibit death while sustained activation of JNK, as the one its activation (Deveraux et al., 1998), the mechanisms generated by TNF in p65 / cells, overrides the antiof TRAF2-cIAP1-mediated inhibition on caspase 8 actiapoptotic signals to promote cell death. vation is likely due to close proximity (TRAF2 brings Although our results indicate that TNF is able to ciap1 close to caspase 8 in the TNF signaling comtransduce signal to mitochondria via JNK, the JNK-Bid plex). This hypothesis is supported by a recently pubpathway results in only selective release of Smac withlished report showing that a single complex containing out affecting cyto c, which is required for caspase 9 TRADD, FADD, caspase 8, TRAF2, and ciap1 exists in activation. Studies using constitutive active JNK mimwild-type cells after TNF stimulation (no cell death), icking sustained JNK activation in TNF signaling to while ciap1 in the complex is greatly reduced in cells apoptosis further support this notion. Thus, TNF -mediexpressing m-i B (cell death) (Micheau and Tschopp, ated JNK activation is insufficient to induce cell death. 2003). This is consistent with our results that no caspase 9 Based on this model, loss of TRAF2 will prevent the cleavage and apoptosis were observed in cells expressrecruitment ciap1 to the TRADD-FADD-caspase 8 com- ing m-i B and FADD-DN after TNF treatment (data not plex thereby leaving caspase 8 free of inhibition. This shown). It is also consistent with the finding that caspase would render TRAF2 null cells hypersensitive to caspase 8 and FADD are required for TNF -mediated apoptosis 8 activation and apoptosis without the necessity for JNK (Varfolomeev et al., 1998; Yeh et al., 1998). Despite our activation. This is consistent with previously published finding on the role of JNK in TNF -mediated apoptosis, results using TRAF2 deficient cells where it was ob- it appears that different means of JNK activation and/or served that TRAF2 / cells were highly sensitive to cell context may lead to different mitochondria effects, TNF -induced apoptosis although JNK activation was since some previous reports have shown that activation defective and NF- B activation was mildly affected in of JNK is sufficient to induce apoptosis via mitochondria these cells (Yeh et al., 1997). Similarly, our hypothesis (Tournier et al., 2000). predicts that inhibition of ciap1 expression should also In addition to caspase 8, other proteases such as sensitize TNF -mediated caspase 8 activation and apo- lysozyme, calpin, and granzyme B, have also been reptosis as is shown to be the case (see Supplemental ported to cleave and activate Bid (Chen et al., 2001; Data available at 114/6/61/DC1). Heibein et al., 2000; Stoka et al., 2001; Sutton et al., 2000), suggesting that multiple pathways exist in cells

9 JNK, Bid, and Smac/DIABLO in TNF Signaling 69 scanning mutants were generated using QuickChange site directed mutagenesis kit (Stratagene) and verified by DNA sequencing. The resulting fusion proteins with six histidines were translated in a TNT T7 transcription/translation kit (Promega) in the presence of 35 S- methionine according to the manufacturer s instructions. The trans- lated proteins were added into either reaction buffer (20 mm HEPES- KOH [ph 7.5], 10 mm KCl, 1.5 mm MgCl 2, 1 mm sodium EDTA, 1 mm sodium EGTA, 1 mm dithiothreitol, and 0.1 mm PMSF) or 50 g cytosol extracts prepared with the reaction buffer and incubated in a total volume of 25 lfor2hrat37 C. The samples were subjected to 12% SDS-PAGE and exposed to X-ray film for 24 hr at 80 C. to activate Bid. JNK is not a protease; therefore, it is likely that JNK induced a protease to cleave Bid in TNF signaling. This protease activity is readily detected in TNF -treated cytosol extracts in our in vitro Bid cleavage assay. Interestingly, the sequence around this cleavage site is highly conserved among human, mouse, and rat. The JNK-mediated Bid cleavage site is much closer to its N terminus than other known Bid cleavage, which could explain why jbid only induces selective release of mitochondria factors. Experimental Procedures Construction of Plasmids The m-i B (deleted N-terminal 31 amino acids, S32A and T36A mutations) and cyto c cdna were generated by RT-PCR from total RNA isolated from HeLa cells and cloning the m-i B RT-PCR prod- ucts into the pcdna3.1( )-hygromycin B (Invitrogen) or into BglII site in frame with GFP (green fluorescence protein) in pegfp-c1 vector (Clontech). Cytochrome C RT-PCR products were cloned into EcoRI site in frame with GFP in pegfp-c1 vector (Clontech). Smac, Bid, and Bid deletion mutants were generated by PCR of Smac and Bid cdna and cloned into Hind III site in frame with GFP in pegfp-n1. All inserts were verified by DNA sequencing. All other plasmids for expressing Bid, tbid, Smac, Bcl-xl (Deng et al., 2002), FADD-DN (Chinnaiyan et al., 1995), JNK-MKK7-WT, JNK-MKK7-KD (Rennefahrt et al., 2002), Ub-Smac, and Ub- AVPI-Smac (Hunter et al., 2003) were as described previously. Antibodies and Other Materials We obtained anti-i B, antiphosphorylated i B, anti-jnk, antiphosphorylated JNK, anti-mkk7, anti-myc, anti-ciap1/2, anti-traf-1/2, and anti-tnfr1 from Santa Cruz Biotechnology. Anti-FADD was purchased from Signal Transduction Lab. Anti-TRADD and anti- Smac/DIABLO antibodies were purchased from Upstate Biotechnology, anti-bid (mouse) from Biosource, and anti-bid (human) from Cell Signaling and BD Pharmingen. Other antibodies were described as previously (Deng et al., 2002). Human recombinant TNF was acquired from R&D systems. Smac N7 and N7np peptides were purchased from Calbiochem. Radioactive materials were from Amersham Biosciences. Cell viability was determined by trypan blue exclusion and aspase-8, -3 enzyme activities using enzyme assay kits following by the manufacturer s instructions (Clontech). Subcellular Fraction, Immunofluorescence, Immunoprecipitation, and Western Blot Analysis All the procedures were performed as described previously (Deng et al., 2002; Deng and Wu, 2000). Bid antibody (BD-Pharmingen) and SuperSignal West Femto-kit (Pierce) were used to detect endog- enous jbid. Acknowledgments We thank Dr. H. Zheng and members of her laboratory at Baylor College of Medicine and Dr. Bing Su at MD Anderson Cancer Center for helpful discussions. We also thank Dr. B. Vogelstein at Johns Hopkins Medical School for providing HCT116 cells; Dr. P. Chiao at M.D. Anderson Cancer Center for p65 / cells; and Dr. Xiaoming Yin at University of Pittsburgh for Bid / MEFs. Dr. R. Agami at Netherlands Cancer Institute for providing psuper; Dr. Rennefahrt and Dr. Rapp at University of Wuerzburg in Germany for pmkk7- JNK-wt and -kd plasmids; Dr. Hunter at Children s Hospital of Eastern in Ontario, Canada, for Ub-Smac and Ub- AVPI-Smac cdna; and Dr. C. Vincenz at University of Michigan for FADD-DN cdna. Also, we thank Dr. J. Yuan at Harvard Medical School for Bid, tbid cdna. This work was supported by grants from NCI (to X.W.). Received: April 16, 2003 Revised: September 10, 2003 Accepted: September 16, 2003 Published: October 3, 2003 References Baud, V., and Karin, M. (2001). Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol. 11, Beg, A.A., and Baltimore, D. (1996). An essential role for NF-kappaB Vector-Based sirna Construction in preventing TNF-alpha-induced cell death. Science 274, SiRNA was generated in psuper as described (Brummelkamp et Brummelkamp, T.R., Bernards, R., and Agami, R. (2002). A system al., 2002). 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