FN14 is expressed by most tumor cell lines
To understand how endogenous cIAPs regulate TNFRSF signaling, we used TNFSF ligands to screen for cell lines containing detectable levels of endogenous TNFSF receptors. To facilitate the screen, we generated TNFSF ligands as recombinant proteins tagged with the Fc portion of human IgG (Fig. S1A, available at http://www.jcb.org/cgi/content/full/jcb.200801010/DC1
; Bossen et al., 2006
). These molecules are cross-linked via the Fc portion that promotes higher order aggregation of the corresponding receptors, closely mimicking engagement by membrane-bound ligands (Holler et al., 2003
). The Fc portion also facilitates reliable detection of these proteins by Western blot (Fig. S1 B) and allows for their simple purification with protein A (Fig. S1 B, right) and for immunoprecipitation of interacting protein complexes (see ).
Figure 1. TWEAK specifically binds to endogenous FN14 in many tumor cell lines and cIAP1 binds to FN14 via its TRAF2 binding domain. (A) Cells were harvested and incubated with Fc-CD70 or Fc-TWEAK, Tricolor-labeled anti-Fc, and analyzed by flow cytometry. (B) FN14 (more ...)
We tested the purified Fc ligands for specific binding to their cognate receptor using FlpIn stable cell lines inducibly expressing different TNFSF receptors and only observed binding when the ligand was added to cells in which expression of the corresponding cognate receptor was induced, i.e., CD27/CD70 and FN14/TWEAK (Fig. S1 C). Satisfied with the specificity of the ligands, we used them to screen a panel of tumor cell lines (including those from kidney, brain, colon, melanoma, breast, and ovarian cancers). Only one of the ligands, TWEAK, bound to a high proportion of the tumor cell lines examined (, Fig. S1 D, and not depicted), suggesting that in culture, many tumor cells constitutively express the TWEAK receptor FN14.
Some studies have suggested that TWEAK binds other receptors in addition to FN14 (Polek et al., 2003
; Bover et al., 2007
). To confirm that the signal caused by binding of TWEAK correlated with expression of FN14, we used a commercial antibody against FN14. The specificity of this FN14 antibody was demonstrated by flow cytometry using cells inducible for FN14 expression (). Importantly, cell lines that bound TWEAK also stained strongly with the antibody to FN14 (). These results demonstrate that a large number of transformed cell lines of both human and mouse origin constitutively express the TWEAK receptor FN14.
TWEAK binding to FN14 recruits TRAF2 and cIAP1
Because yeast two-hybrid screens suggested a potential interaction between TRAF2 and FN14 (Brown et al., 2003
), we tested whether TRAF2 could interact with FN14 in vivo. Recombinant Fc-TWEAK successfully immunoprecipitated endogenous TRAF2 and cIAP1 in D645 glioma cells, whereas in the absence of TWEAK, no TRAF2 or cIAP1 was detected ().
To test whether cIAP1 binding to FN14 was indirectly mediated through TRAF2, we transiently transfected cIAP1 ΔC6 (a stable mutant of cIAP1 lacking the last six residues) and cIAP1 ΔBIR1 constructs into D645 glioma cells and immunoprecipitated endogenous FN14 with Fc-TWEAK. Consistent with previous observations (Samuel et al., 2006
; Varfolomeev et al., 2006
), cIAP1 constructs that lacked a BIR1 domain were unable to bind endogenous TRAF2, whereas mutations to other regions of cIAP1 did not affect the TRAF2 interaction (Fig. S1 E and not depicted). As for endogenous cIAP1 (), transfected cIAP1 ΔC6 could be immunoprecipitated by Fc-TWEAK (), whereas ΔBIR1 cIAP1 could not be detected under the same conditions, even though endogenous TRAF2 was immunoprecipitated (). Similarly, when vesicular stomatitis virus (VSV)–tagged FN14 was induced and immunoprecipitated with anti-VSV in the presence of TWEAK, cIAP1 ΔC6 could be detected, whereas neither ΔBIR1 cIAP1 nor ΔBIR1 cIAP1 ΔC6 associated with FN14 (Fig. S1 F). These results strongly suggest that endogenous cIAP1 binds indirectly to FN14 through its association with TRAF2.
Signaling from FN14 induces the degradation of both TRAF2 and cIAP1
The role of TRAF2 and cIAP1 in TNFSF signaling is still unclear. Several studies suggest that TRAF2 is the ubiquitin E3 ligase that ubiquitylates RIP (Lee et al., 2004
), thereby promoting activation of canonical NF-κB (Chen et al., 2006
). Other studies suggest cIAP1 is the ubiquitin E3 ligase for RIP (Park et al., 2004
) and for TRAF2 itself (Li et al., 2002
). Indeed, the most widely accepted model for cIAP1 function, based largely on overexpression data, states that cIAP1 ubiquitylates TRAF2 causing its proteasomal degradation (Li et al., 2002
). A followup study using primary B cells also showed that TRAF2 degradation after stimulation with agonistic TNF-R2 antibodies did not occur in cIAP1 knockout cells (Zhao et al., 2007
), lending credence to this model. We therefore tested whether TWEAK induced TRAF2 degradation. OVCAR4, SKOV3, Kym1 (), and transformed MEF cells ( and not depicted) were treated with TWEAK for 0–6 h and cellular levels of endogenous cIAP1 and TRAF2 analyzed by Western blot (). After 1–6 h of TWEAK addition, cellular levels of both TRAF2 and cIAP1 were reduced in all cell lines examined.
Figure 2. FN14 signaling decreases cellular cIAP1 and TRAF2 levels by lysosomal degradation. (A) OVCAR4, SKOV3, or Kym1 cells were treated with 100 ng/ml Fc-TWEAK at 37°C for the indicated time, lysed, and analyzed by Western blot. (B) Proteasome inhibition (more ...)
TWEAK/FN14 promotes cathepsin dependent lysosomal degradation of the cIAP1–TRAF2 complex
Although we observed substantial degradation of TRAF2 and cIAP1 after TWEAK treatment, we were surprised that the degradation of TRAF2 and cIAP1 could not be blocked by preincubating cells with proteasome inhibitors such as MG132 (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200801010/DC1
) or PS341 () before TWEAK stimulation, despite the fact that these inhibitors efficiently blocked proteasome function, as indicated by enhanced levels of total cellular ubiquitylated proteins ().
TNF-R2–induced TRAF2 degradation has been reported to occur by the E3 ubiquitin ligase activity of cIAP1 targeting it for proteasomal degradation (Li et al., 2002
). To examine the requirement of cIAP1 for TWEAK-induced TRAF2 loss, and vice versa, we used gene knockout transformed MEF cell lines and stimulated endogenous FN14 with TWEAK. Although TWEAK stimulation resulted in decreased levels of cIAP1 in wild-type MEFs, it was not degraded in TRAF2−/− knockout MEFs (, left). TRAF2-mediated binding of cIAP1 to FN14 is therefore required for TWEAK-induced degradation of cIAP1. Consistent with previous studies, cIAP1 was required for the degradation of TRAF2 because TWEAK-stimulated TRAF2 depletion did not occur in cIAP1−/− MEFs (, left).
To examine the requirement for cIAP1 in TRAF2 degradation in greater detail, we performed further experiments where we lysed cells in Triton X-100 and examined the detergent soluble and insoluble membrane fractions. Remarkably, TRAF2 disappeared from the Triton X-100–soluble fraction in cIAP1 knockout cells as it did from wild-type cells (, right; and Fig. S2 B). These two results suggest that TRAF2 translocation to an insoluble compartment occurs in the absence of cIAP1 but its degradation requires the activity of cIAP1. This notion is consistent with previous reports demonstrating that TRAF2 relocalizes to a detergent-insoluble fraction and becomes degraded after signaling from other TNFSF receptors (Habelhah et al., 2004
; Wu et al., 2005
Because TWEAK did not induce proteasomal degradation of the cIAP1–TRAF2 complex, we tested other protease inhibitors. Cells preincubated with a protease inhibitor cocktail showed reduced TWEAK-mediated degradation of TRAF2 and a modest protecton of cIAP1 when serum was removed from the medium before addition of the inhibitor (). We therefore tested whether TWEAK-mediated TRAF2 and cIAP1 depletion was dependent upon lysosomal function. Consistent with this hypothesis, inhibitors of lysosomal function, such as chloroquine and ammonium chloride, prevented TWEAK-mediated TRAF2 degradation, whereas ammonium chloride also substantially blocked TWEAK-mediated cIAP1 degradation, although not to the same extent as it blocked TRAF2 depletion ().
To further test a role for lysosomal proteases, we used specific protease inhibitors. The serine protease inhibitor AEBSF failed to block TWEAK-mediated cIAP1–TRAF2 degradation, whereas TLCK, which can inhibit both serine and cysteine proteases, partially blocked TWEAK-mediated TRAF2 loss (). The cathepsin B inhibitor CA-074Me () also provided protection against loss of both cIAP1 and TRAF2, implying that lysosomal cathepsins may be important for the degradation of this complex. Importantly, neither CA-074Me nor the inhibitors of lysosomal function perturbed the proteasomal degradation pathway because they did not prevent the loss of cIAP1 induced by IAP antagonist (compound A) treatment (), which we have previously shown is proteasomal dependent (Vince et al., 2007
Although endogenous TRAF2 and cIAP1 was difficult to detect by confocal microscopy, analysis of D645 cells transiently transfected with FLAG-TRAF2 revealed that in unstimulated cells, TRAF2 was exclusively cytosolic and did not overlap with the acidotropic lysosome marker lysotracker (Fig. S2, C and D). However upon stimulation with TWEAK ligand for 3–6 h, TRAF2 showed a significant redistribution to punctate vesicles (Fig. S2, C and E). TRAF2-containing vesicles were juxtaposed with lysotracker-stained compartments and often directly overlapped (Fig. S2 E), suggesting that TRAF2 degradation occurs in the lysosome or in compartments that are in close association. It is probable that the Triton X-100–insoluble fraction contains MVB/lysosomal membranes because the inhibitors NH4Cl and CA-074Me significantly blocked degradation of TRAF2 and cIAP1 in the Triton X-100–insoluble fraction (unpublished data).
TWEAK activates noncanonical NF-κB by depleting cIAP1 and TRAF2
TWEAK/FN14 signaling has previously been shown to activate both canonical and noncanonical NF-κB (Saitoh et al., 2003
). Because TRAF2 knockout B cells and either immortalized cIAP1 or TRAF2 knockout MEFs show constitutive activation of noncanonical NF-κB (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200801010/DC1
; Grech et al., 2004
; Vince et al., 2007
), we hypothesized that TWEAK-mediated NF-κB signaling may be a direct result of the depletion of cIAP1 and TRAF2 and noncanonical in nature, despite the fact that degradation of cIAP1 after TWEAK signaling is never complete.
To measure TWEAK-induced NF-κB activation, we created stable cell lines containing an NF-κB reporter, where expression of EGFP is driven by a promoter containing four NF-κB binding elements. As expected, NIH 3T3 cells bearing the NF-κB reporter showed strong NF-κB induction when stimulated with TNFα (). TWEAK also induced a significant NF-κB response, although this was slower and not as large as the TNFα response (). TWEAK-induced NF-κB was not dependent upon autocrine-produced TNFα because induction of NF-κB could not be blocked by anti-TNFα (unpublished data). To investigate whether NF-κB was noncanonical, we examined processing of the NF-κB2 subunit from the p100 form to the activated, processed p52 form. In both OVCAR4 and KYM1 cell lines, processing of p100 to p52 became visible after 1 h of TWEAK stimulation () and correlated well with the TWEAK-induced loss of cIAP1–TRAF2 (). Also consistent with noncanonical activation of NF-κB, we observed that TWEAK treatment caused a remarkable stabilization of NIK (), which correlated with processing of p100 to p52, but observed no change in NF-κB1 p105 processing ().
Figure 3. TWEAK-induced cIAP1–TRAF2 loss activates noncanonical NF-κB. (A) An NIH 3T3 cell clone stably transformed with a lentiviral NF-κB reporter vector was stimulated with Fc-TNF or Fc-TWEAK for the indicated times. (B) Kym1 or OVCAR4 (more ...)
If TWEAK-induced loss of the cIAP1–TRAF2 complex is required to activate the noncanonical pathway, then genetic loss of either cIAP1 or TRAF2 might also result in constitutive activation of this pathway. Consistent with this model and our previous observations (Vince et al., 2007
), MEFs deleted for either cIAP1 or TRAF2 showed elevated p52 levels and an increase in p52 localization to a nucleus-containing fraction (Fig. S3 A). In contrast, p50 localization was unaffected by loss of these genes and was predominantly present in the unprocessed p105 form in the cytoplasm (Fig. S3 A).
If depletion of the cIAP1–TRAF2 complex is sufficient to activate NF-κB, then overexpression of these two proteins should inhibit TWEAK/FN14-induced NF-κB activity. To test this hypothesis, we used FN14-inducible NF-κB EGFP reporter cells in which maximal NF-κB activity was detected in cells that were simultaneously induced for FN14 expression and stimulated with TWEAK ligand and tested the effect of transiently transfecting cIAP1, TRAF2, or both () in this system. Individual expression of either TRAF2 or cIAP1 failed to block TWEAK/FN14-induced NF-κB activation (). However, the overexpression of both proteins together significantly reduced the amount of NF-κB activation (). Importantly, this was dependent on cIAP1 binding to TRAF2, because coexpression of TRAF2 with the ΔBIR1 cIAP1 mutant that is unable to bind TRAF2 (Fig. S1 E), was unable to inhibit FN14/TWEAK-induced activation of NF-κB (). NIK stabilization and p100 processing to p52 could be blocked by pretreatment of cells with NH4Cl but not by pretreatment with CA-074Me (). This suggests that relocalization to the lysosomal compartment is sufficient to trigger stabilization of NIK and subsequent processing of p100 rather than degradation in the lysosome per se.
Because TWEAK has been reported to activate the canonical pathway, we also examined the effects of TWEAK and cIAP1 or TRAF2 loss on canonical signaling markers. Consistent with previous observations (Saitoh et al., 2003
), we observed TWEAK-induced rapid phosphorylation of IκB and p65. Loss of either cIAP1 or TRAF2 resulted in almost identical responses, with higher basal phosphorylation of IκB and p65 and a significantly delayed TWEAK-induced increase (). This highlights that the cIAP1–TRAF2 complex plays an important role in both NF-κB pathways induced by TWEAK.
TWEAK induces cell death through NF-κB–dependent induction of TNFα
Tumor cell lines sensitive to synthetic IAP antagonists are killed through NF-κB–dependent autocrine production of TNFα (Vince et al., 2007
). Moreover, it has been described that TWEAK can kill Kym1 cells in a TNFα-dependent manner (Schneider et al., 1999
), although how TWEAK stimulated TNFα in Kym1 cells remains unknown. We therefore asked whether TWEAK acted in a similar manner to synthetic IAP antagonists by causing an increase in the abundance of TNFα driven through the activation of NF-κB.
We observed that the levels of TNFα in the cell lysate of TWEAK-treated cell lines increased significantly in all three cell types that are killed by TWEAK treatment alone (; and Fig 5 A) with a concomitant increase of TNFα released into the media supernatant (). In contrast, cell lines that are not killed by TWEAK treatment alone did not produce TNFα when TWEAK was added (unpublished data), suggesting that induction of TNFα is necessary for TWEAK to cause apoptosis.
Figure 4. TWEAK-mediated NF-κB activation induces TNFα in TWEAK-sensitive lines. Kym1 (A) or SKOV3 and OVCAR4 (B) cells were treated with Fc-TWEAK for 8 or 24 h, respectively and the amount of TNFα in cell lysates was measured by ELISA. (more ...)
To test whether activation of NF-κB by TWEAK/FN14 was required for the enhanced TNFα production observed in TWEAK-sensitive cell lines, we created stable inducible nondegradable IκBSR (IκB superrepressor) SKOV3 and OVCAR4 cell lines. Induction of IκBSR inhibited TWEAK-induced NF-κB activity (Fig. S3 A) and significantly reduced the TWEAK-dependent increase in levels of cellular and secreted TNFα in both SKOV3 and OVCAR4 cells ().
Inhibition of TNFα signaling or caspase 8 blocks TWEAK/FN14 cell death
Previous work with synthetic IAP antagonists (Gaither et al., 2007
; Varfolomeev et al., 2007
; Vince et al., 2007
) and the data presented here with TWEAK demonstrate that either treatment results in an increase in TNFα, which is driven by NF-κB. Remarkably, tumor cell lines that are killed by treatment with a synthetic IAP antagonist alone, such as OVCAR4, SKOV3, and Kym1 cells, (Vince et al., 2007
) are also killed by TWEAK.
TWEAK killing of sensitive cell lines was prevented by TNFα-blocking antibodies but not by TRAIL- or Fas ligand–neutralizing antibodies in both short-term (), and long-term clonogenic survival assays (Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200801010/DC1
), which is consistent with a conserved mechanism of cell death between synthetic IAP antagonist compounds and TWEAK. In addition, expression of the extracellular domain of TNF-R2 fused to a GPI-anchor (dnTNFR2), which is able to sequester and hence neutralize TNFα (Vince et al., 2007
), significantly inhibited cell death caused by TWEAK (). In contrast, neither dnCD27 nor dnTRAIL-R2 had any protective effect ().
Figure 5. TWEAK-induced cell death is mediated by TNFα. (A) TWEAK-induced death is blocked by neutralizing TNFα antibodies. Kym1, SKOV3, and OVCAR4 cells were incubated with Fc-TWEAK or Fc-CD27 (control) for 24 (Kym1) or 48 (SKOV3 and OVCAR4) h (more ...)
Caspase 8 activity was necessary for TWEAK to induce apoptosis because Kym1 and SKOV3 cell lines inducibly expressing the caspase 8 inhibitor crmA were significantly resistant to TWEAK killing in both short-term () and long-term clonogenic survival assays with Kym1 cells (Fig. S4 B).
To provide a nongenetic test that TWEAK-driven NF-κB was sufficient to kill cells, we used Geldanamycin because it completely blocked TWEAK-induced NF-κB (Fig. S4 C). As has been shown before (Wang et al., 2006
), inhibiting the IKK1/2 complex with Geldanamycin is sufficient to sensitize OVCAR4 and wild-type MEFs to TNFα (Fig. S4 E), presumably by blocking NF-κB–induced transcription of prosurvival genes (Wang et al., 2006
). Remarkably, however, Geldanamycin was able to block TWEAK-induced NF-κB (Fig. S4, C and D) and TWEAK-induced cell death of Kym1 and OVCAR4 cells (Fig. S4 E). Moreover, although Geldanamycin-treated Kym1 cells showed reduced survival in long-term clonogenic growth assays, cells treated with TWEAK and Geldanamycin still showed clonogenic protection when compared with TWEAK treatment alone (Fig. S4 A).
TWEAK/FN14 signaling sensitizes cells to exogenously supplied TNFα
Although TWEAK kills OVCAR4 and SKOV3 cells through induction of autocrine TNFα, it is known that, like most other cell types, these cells are resistant to TNFα treatment alone (). It has been recently shown that removal of cIAP1 by either synthetic IAP antagonists or in gene knockout MEFs sensitizes these cells to TNFα killing (Li et al., 2004
; Gaither et al., 2007
; Vince et al., 2007
). Therefore, we hypothesized that TWEAK not only induces TNFα but also sensitizes cells to TNFα-induced cell death through degradation of the cIAP1–TRAF2 complex in a similar manner to synthetic IAP antagonists, which sensitize tumor cells to TNFα killing by depleting cIAP1, albeit in a mechanistically distinct fashion.
Figure 6. TWEAK-induced cIAP1–TRAF2 loss sensitizes transformed cells to TNFα-induced death. (A) The indicated cell lines were treated with 100 ng/ml Fc-TWEAK and/or 100 ng/ml Fc-TNFα for 24 h, followed by propidium iodide staining and flow (more ...)
To test this hypothesis, exogenous TNFα was applied to TWEAK-sensitive (OVCAR4) and -resistant (D645 and MEF) cell lines alone or in combination with TWEAK for 24 h. Consistent with an additional sensitizing role for TWEAK, OVCAR4 cells were killed by TWEAK/TNFα treatment far more efficiently and rapidly than with TWEAK alone. Even more significantly, D645 and MEF cells (among many other cell types; not depicted) were resistant to treatment with TWEAK or TNFα alone but were extremely sensitive to combined TWEAK/TNFα treatment (). Even a subset (2/12) of primary human tumor lines was significantly sensitized to TNFα by TWEAK treatment ().
TWEAK sensitization to TNFα killing was examined further by Western blot on the TWEAK (and TNFα)-resistant D645 glioma cell line. As in TWEAK-sensitive cell lines, TWEAK treatment reduced cIAP1 and TRAF2 levels, whereas TNFα treatment alone had no effect (). Consistent with the lack of cell death (), the individual treatments of TWEAK or TNFα did not alter caspase 8 cleavage (). In contrast, upon cotreatment of TWEAK with TNFα, processing of caspase 8 into the p43/p41 forms and the active p18 subunit was observed within 3 h () and correlated with the loss of cIAP1–TRAF2 and the rapid death of these cells (). To allow a direct comparison with our synthetic IAP antagonist, we also incubated D645 cells with compound A alone or compound A and TNFα. Treatment with compound A alone resulted in the rapid loss of cIAP1 but did not affect either TRAF2 or caspase 8 levels (). Significantly, cIAP1 loss alone was sufficient to sensitize D645 cells to TNF to a similar level as that of TWEAK-induced depletion of the cIAP1–TRAF2 complex ().
Further evidence supporting the observation that TWEAK/TNFα kill in a death receptor–dependent pathway was obtained using FADD−/− MEFs, as these were completely resistant to TWEAK/TNFα-induced death (). In contrast, TWEAK/TNFα killing was independent of the Bax/Bak-dependent apoptotic pathway, as Bax/Bak double knockout MEFs showed a similar TWEAK/TNFα sensitivity to wild-type MEFs ().
As expected, cIAP1−/− (Vince et al., 2007
), TRAF2−/−, and TRAF2/TRAF5−/− double knockout or compound A–treated MEFs were all extremely sensitive to killing by TNFα alone (; Tada et al., 2001
), supporting the hypothesis that TWEAK-induced loss of the cIAP1–TRAF2 complex is sufficient to sensitize MEFs to TNFα killing. Surprisingly, cIAP2−/− MEFs were not sensitive to TNFα-mediated cell death (), making it unlikely that cIAP2 has a role in TWEAK-mediated sensitization to TNFα.
Pretreating wild-type MEFs with TWEAK for 8 h before addition of TNFα caused a reduction in the total canonical response. However simultaneous treatment with TWEAK/TNFα resulted in an augmented canonical response (Fig. S5 A, available at http://www.jcb.org/cgi/content/full/jcb.200801010/DC1
), making it unlikely that a reduction in prosurvival NF-kB signal from TNFα is the reason for TWEAK-induced sensitization to TNFα when the two cytokines are added simultaneously. Consistent with this data, pretreating wild-type MEFs with either TNFα or TWEAK alone for 24 h before cotreatment with TWEAK/TNFα or compound A/TNFα did not change the amount of cell death observed when cells were cotreated for the same time period (Fig. S5 B). This suggests that the prosurvival signals elicited by TNFα, such as NF-κB–induced gene transcription, are not sufficient to counteract TWEAK/TNFα killing.
TWEAK/TNFα treatment distinguishes between normal and transformed cells
Genetic knockout cIAP1 mice display no obvious phenotypic defects in apoptotic signaling (Conze et al., 2005
; unpublished data), raising the possibility that primary cells may be less sensitive to TWEAK/TNFα-induced death. Consistent with this possibility, primary MEFs showed only a twofold increase in death after TWEAK/TNFα stimulation, whereas a 14-fold increase was observed in SV40 large T immortalized MEFs (). TWEAK-induced loss of cIAP1–TRAF2 was observed in both primary MEFs and transformed MEFs (), as was activation of noncanonical NF-κB (). Although similar levels of FN14 were initially present in both MEF lines, these increased dramatically after TWEAK stimulation (), implying that FN14 expression is regulated by TWEAK.
Figure 7. TWEAK/TNFα treatment distinguishes between normal and transformed cells, and TWEAK/TNFα killing is suppressed by elevated cIAP1–TRAF2 levels. (A) Primary MEFs are resistant to TWEAK/TNFα-induced death. Three primary MEF (more ...)
Because TWEAK-mediated loss of the cIAP1–TRAF2 complex is sufficient to sensitize tumor cells to TNFα-induced death, we tested whether a liver progenitor tumor cell line, PIL2, which expresses high levels of the cIAP1–TRAF2 complex (), was resistant to TWEAK/TNFα killing relative to a liver progenitor cell line, PIL4, with lower levels (). Treatment of these cells with TWEAK/TNFα killed >90% of PIL4 cells, whereas only 35% of PIL2 (cIAP1 high) cells were killed (). Western blot analysis showed that PIL2 and PIL4 cells expressed equal levels of FN14 (). Significantly, TWEAK-induced degradation of cIAP1–TRAF2, and increased FN14 levels, were attenuated in the PIL2 cells, implying that enhanced expression of cIAP1–TRAF2 inhibits FN14 signaling and counters TWEAK-induced sensitivity to TNFα-induced death.