Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment.

Abstract

A role for cellular inhibitors of apoptosis (IAPs [cIAPs]) in preventing CD95 death has been suspected but not previously explained mechanistically. In this study, we find that the loss of cIAPs leads to a dramatic sensitization to CD95 ligand (CD95L) killing. Surprisingly, this form of cell death can only be blocked by a combination of RIP1 (receptor-interacting protein 1) kinase and caspase inhibitors. Consistently, we detect a large increase in RIP1 levels in the CD95 death-inducing signaling complex (DISC) and in a secondary cytoplasmic complex (complex II) in the presence of IAP antagonists and loss of RIP1-protected cells from CD95L/IAP antagonist-induced death. Cells resistant to CD95L/IAP antagonist treatment could be sensitized by short hairpin RNA-mediated knockdown of cellular FLICE-inhibitory protein (cFLIP). However, only cFLIP(L) and not cFLIP(S) interfered with RIP1 recruitment to the DISC and complex II and protected cells from death. These results demonstrate a fundamental role for RIP1 in CD95 signaling and provide support for a physiological role of caspase-independent death receptor-mediated cell death.

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JCB: Article
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J. Cell Biol. Vol. 187 No. 7 1037–1054
www.jcb.org/cgi/doi/10.1083/jcb.200904158
1037
P. Geserick and M. Hupe contributed equally to this paper.
Correspondence to John Silke: j.silke@latrobe.edu.au; or Martin Leverkus:
leverkus@medizin.uni-magdeburg.de
Abbreviations used in this paper: 4-HT, 4-hydroxy-tamoxifen; CD95L, CD95
ligand; DD, death domain; DISC, death-inducing signaling complex; DKO,
double knockout; DL, death ligand; DR, death receptor; FADD, Fas-associated
protein with DD; FLIP, FLICE-inhibitory protein; IAP, inhibitor of apoptosis;
MEF, mouse embryonic fibroblast; PI, propidium iodide; RIP1K, RIP1 kinase;
SCC, squamous cell carcinoma; shRNA, short hairpin RNA; TRAIL, TNF-related
apoptosis-inducing ligand; vFLIP, viral FLIP; XIAP, X-linked IAP.
Introduction
The initiators of the extrinsic cell death pathway are a subclass
of TNF superfamily (TNFSF) receptors called death receptors
(DRs). A common feature of DR signaling is the formation of
a primary plasma membrane–associated death-inducing signal-
ing complex (DISC) and a secondary independent signaling
platform in the cytoplasm (complex II). Complex II was first
demonstrated for TNF-R1 (Micheau and Tschopp, 2003) but
subsequently was also shown for other DR pathways (Varfolomeev
et al., 2005; Lavrik et al., 2008). However, the mechanisms
leading to the formation of these secondary complexes and
their significance to signaling outcome are still unknown. DR
signaling pathways are controlled by inhibitors such as cellular
FLICE-inhibitory protein (FLIP [cFLIP]) or X-linked inhibitor
of apoptosis (IAP [XIAP]; for review see Meier and Vousden,
2007). The cFLIP gene can give rise to 11 distinct isoforms, but
in most cells, a long (cFLIPL) and a short isoform (cFLIPS) are
the only ones readily detected (for reviews see Kataoka, 2005;
Budd et al., 2006). cFLIPL has a caspase-like domain lacking the
critical catalytic residues present in caspase-8 in addition to
two death effector domains, whereas cFLIPS contains only two
death effector domains and is structurally related to viral FLIP
(vFLIP; Thurau et al., 2006). cFLIP isoforms interact with FADD
(Fas-associated protein with death domain [DD]) and caspase-8,
are recruited to the DISC, and interfere with caspase activation
within this signaling platform (Lavrik et al., 2005; Falschlehner
et al., 2007).
DRs can also cause nonapoptotic, caspase-independent cell
death and elicit nonapoptotic responses (for reviews see Wajant
et al., 2003; Kroemer et al., 2009). The significance of these caspase-
independent DR pathways is debated, and there is a need to pro-
vide additional examples in more physiological scenarios. RIP1
Cellular IAPs inhibit a cryptic CD95-induced cell
death by limiting RIP1 kinase recruitment
Peter Geserick,1,2 Mike Hupe,1 Maryline Moulin,3 W. Wei-Lynn Wong,3 Maria Feoktistova,1,2 Beate Kellert,1,2
Harald Gollnick,1 John Silke,3 and Martin Leverkus1,2
1Laboratory for Experimental Dermatology, Department of Dermatology and Venereology, Otto-von-Guericke University Magdeburg, 39120 Magdeburg, Germany
2Section of Molecular Dermatology, Department of Dermatology, Venereology, and Allergology, Medical Faculty of Mannheim, University of Heidelberg,
68167 Mannheim, Germany
3Department of Biochemistry, LaTrobe University, Melbourne 3086, Victoria, Australia
A
study, we find that the loss of cIAPs leads to a dramatic
sensitization to CD95 ligand (CD95L) killing. Surprisingly,
this form of cell death can only be blocked by a combina-
tion of RIP1 (receptor-interacting protein 1) kinase and cas-
pase inhibitors. Consistently, we detect a large increase in
RIP1 levels in the CD95 death-inducing signaling complex
(DISC) and in a secondary cytoplasmic complex (complex II)
© 2009 Geserick et al. This article is distributed under the terms of an Attribution–
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role for cellular inhibitors of apoptosis (IAPs [cIAPs])
in preventing CD95 death has been suspected but
not previously explained mechanistically. In this
in the presence of IAP antagonists and loss of RIP1-
protected cells from CD95L/IAP antagonist–induced death.
Cells resistant to CD95L/IAP antagonist treatment could
be sensitized by short hairpin RNA–mediated knockdown
of cellular FLICE-inhibitory protein (cFLIP). However, only
cFLIPL and not cFLIPS interfered with RIP1 recruitment to the
DISC and complex II and protected cells from death. These
results demonstrate a fundamental role for RIP1 in CD95
signaling and provide support for a physiological role of
caspase-independent death receptor–mediated cell death.
T H E J O U R N A L O F C E L L B I O L O G Y

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JCB • VOLUME 187 • NUMBER 7 • 2009 1038
MET1 cells but not A5RT3 were sensitized to TRAIL- or CD95
ligand (CD95L)–mediated cell death in a TNF-independent man-
ner in both short-term (Fig. 1 A) and clonogenic assays (Fig. 1 B).
DL-induced cell death correlated with phosphatidylserine exter-
nalization (Fig. 1 C and Fig. S4 A) and hypodiploidy (Fig. 1 D).
Consistent with an apoptotic cell death, we observed processing
of caspases and PARP (poly[ADP-ribose] polymerase) cleavage
within 4 h of stimulation with DLs in the presence of the IAP
antagonist (Fig. 1 E and not depicted). To determine whether IAP
antagonist–induced loss of cIAPs was important for this increased
sensitization, we tested SV40 large T immortalized mouse em-
bryonic fibroblasts (MEFs) lacking XIAP, cIAP1, cIAP2, or both
cIAP1 and -2 (double knockout [DKO]) for their sensitivity to
CD95L and TRAIL. DKO MEFs demonstrated an increased sen-
sitivity to DLs, and the IAP antagonist slightly decreased the
viability of these cells, suggesting that sensitivity was largely
regulated by both cIAP1 and -2 in MEFs (Fig. 2 A). Consistent
with this hypothesis, loss of XIAP did not sensitize MEFs to DLs
(Fig. 2 A). Inducible reconstitution of cIAP1 or -2 into DKO
MEFs (Fig. 2 B) increased the CD95L resistance of the DKOs
that was lost when cells were cotreated with the IAP antagonist
(Fig. 2 A, right). In contrast, increased expression of XIAP into
DKO MEFs did not substantially alter sensitivity of these MEFs
to the combination of CD95L and IAP antagonist (Fig. 2 C).
Similarly, knockdown of cIAP1 or both cIAPs sensitized
HaCaT cells to CD95L-induced death despite the fact that there
was a compensatory rise in the levels of cIAP2 (Fig. 2 D). This
rise in cIAP2 levels might be explained by posttranslational
regulation of cIAPs (Conze et al., 2005) or by increased NF-B
induced by loss of cIAP1 (Vince, J., personal communication). Spe-
cific knockdown of cIAP2 provides further evidence that it plays
a less important role than cIAP1 in these cells as reported previ-
ously (Diessenbacher et al., 2008), although the knockdown of
cIAP2 was weaker when compared with cIAP1 knockdown. We
also inducibly expressed cIAP1 or -2 in HaCaT. Overexpression
of cIAP2 conferred protection from CD95L-induced cell death in
the presence of the IAP antagonist (Fig. 2 E). In contrast, induc-
ible overexpression of cIAP1 did not alter sensitivity to CD95L
nor was it able to protect against the IAP antagonist–mediated
sensitization to CD95L death, presumably because of the effi-
cient degradation of endogenous and overexpressed cIAP1
(Fig. S4, C and D). These data argue that endogenous levels of
cIAPs in HaCaT are sufficient to confer resistance to CD95L-
induced cell death but that loss of both cIAPs sensitizes to CD95-
induced cell death. Our results demonstrate that cIAPs play an
important role in limiting DL toxicity in both human and mouse
cell lines.
IAP antagonist/DR-mediated cell death is
neither entirely caspase dependent nor
independent and requires RIP1K activity
DR-mediated apoptosis is initiated by DISC-activated caspase-8
(Peter and Krammer, 2003; Walczak and Haas, 2008). To in-
vestigate whether caspases caused DR-mediated cell death in
the presence of the IAP antagonist, we used the caspase inhibi-
tor zVAD-fmk. zVAD-fmk blocked cell death when cells were
stimulated with DLs for 24 h. However, DL-mediated cell death
(receptor-interacting protein 1) belongs to the RIP kinase family
but is the only family member with a C-terminal DD (Stanger
et al., 1995; for review see Festjens et al., 2007). RIP1 knockout
mice are born but die rapidly because of an increased sensitivity
to TNF (Kelliher et al., 1998). RIP1, and specifically its DD, was
reported to be critical for CD95-mediated necrosis independent
of NF-B–inducing activity or RIP1 kinase (RIP1K) activity
(Holler et al., 2000; Degterev et al., 2005). The development of
specific RIP1K inhibitors has facilitated experiments examining
the functional role of RIP1K in necrosis (Degterev et al., 2008),
but the precise role or potential targets of the kinase activity of
RIP1 are unknown (Hitomi et al., 2008).
A major goal of tumor therapies such as DR agonists is
to overcome transformation-induced apoptosis resistance
(Hanahan and Weinberg, 2000; Ashkenazi, 2008). However, un-
fortunately, resistant tumor cells are frequently selected during
treatment, exemplifying the need for novel treatments that can
further sensitize tumors to DR-mediated apoptosis. IAP antagonists
are synthetic compounds that were modeled on the N-terminal
IAP-binding motif of the mitochondrial protein Smac/DIABLO
(Wright and Duckett, 2005). The XIAP-interfering function of
Smac-mimetic compounds (IAP antagonists) is crucial for ther-
apeutic efficiency of TNF-related apoptosis-inducing ligand
(TRAIL) in xenograft tumor models (Vogler et al., 2008).
Recently, it has become apparent that compounds principally
designed to target XIAP also target cIAPs by rapid autoubiqui-
tylation and proteasomal degradation of cIAP1 and -2 (Gaither
et al., 2007; Petersen et al., 2007; Varfolomeev et al., 2007; Vince
et al., 2007; Bertrand et al., 2008).
Previous studies have shown that cIAPs can inhibit CD95-
and TRAIL-R–induced apoptosis (McEleny et al., 2004; Wang
et al., 2005). It is unlikely that their role will be as direct caspase
inhibitors because cIAPs are rather poor inhibitors of caspase
activity (Eckelman and Salvesen, 2006). Because cIAPs regulate
RIP1 in TNF-R1 and RIP1 plays a role in CD95 signaling, we
have investigated the mechanism of DR cell death in the context
of IAP inhibition. We show that cIAPs block DR-mediated cell
death and that in their absence, cell death proceeds in a caspase-
and RIP1K-dependent manner. Loss of cIAPs results in in-
creased RIP1 recruitment to the DISC and in increased formation
of complex II, which contains FADD, caspase-8, RIP1, and
cFLIP isoforms. Surprisingly, different cFLIP isoforms have
distinct signaling capabilities whenever cIAPs are repressed.
This function of cIAPs might be used as a target to overcome
apoptosis resistance in tumor therapy and might also be relevant
during virus infection or tumor immunity, in which the mode of
cell death is important (Lotze et al., 2007).
Results
The IAP antagonist sensitizes to death
ligand (DL)–mediated cell death
We characterized the sensitivity of different keratinocyte cell
lines and squamous cell carcinoma (SCC) cells with a recently
described IAP antagonist, compound A (Vince et al., 2007). A
rapid degradation of cIAP1 and, to a lesser extent, cIAP2 was
detected after IAP antagonist treatment (Fig. S1 A). HaCaT and

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1039cIAPs inhibit death receptor–mediated death • Geserick et al.
typical apoptotic cells demonstrating membrane blebbing, DNA
condensation, and fragmentation were detectable after CD95L
treatment and zVAD-fmk fully protected cell death and mem-
brane integrity (Fig. 3 B, left). Caspase inhibition only partly
protected from cell death in CD95L + IAP antagonist–treated
cells (Fig. 3 B, right). Dying cells under those conditions showed
in the presence of the IAP antagonist was only partially blocked
by zVAD-fmk (Fig. 3 A). One potential explanation for these
data is that DL treatment in the presence of the IAP antagonist
induced a caspase-independent form of cell death. To character-
ize the morphology of cell death in these cells, we performed
fluorescence microscopy experiments. Increased numbers of
Figure 1. The IAP antagonist sensitizes SCC and HaCaT to DL-mediated apoptosis independent of autocrine TNF secretion. (A) HaCaT, MET1, or A5RT3
cells were either pretreated with 100 nM of the IAP antagonist (Ant) alone or in combination with 10 µg/ml TNF-R2-Fc for 30 min and then stimulated with
TRAIL or CD95L. The viability of cells was analyzed by crystal violet assay after 18–24 h as indicated in Materials and methods. Mean and SEM of four
independent experiments are shown. (B) For clonogenic assays, HaCaT cells were prestimulated with 100 nM of the IAP antagonist for 30 min followed
by co-stimulation with 2.5 U/ml CD95L for 24 h. Colony formation was assayed as indicated in Materials and methods. One representative experiment
of a total of three independent experiments is shown. (C–E) HaCaT cells were either prestimulated with 100 nM of the IAP antagonist for 30 min alone
or stimulated/co-stimulated with 10 U/ml CD95L. (C) Cells were stained with annexin V–Cy5 and PI after 4 h and analyzed by FACS. (D) Cells were
incubated for 8 h and subsequently analyzed for hypodiploidy by FACS analysis (see Materials and methods). (E) Cells were treated with 100 nM of the
IAP antagonist, 2.5 U/ml CD95L, or the combination of both in the presence or absence of 10 µg/ml TNF-R2-Fc for the indicated time points. Western blot
analysis was performed for the expression of cIAP1 and -2, caspase-8 and -3, PARP-1, FADD, and RIP1. -Tubulin served as an internal loading control.
One of two representative experiments is shown. The asterisk marks an unspecific band. MM, molecular mass.

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JCB • VOLUME 187 • NUMBER 7 • 2009 1040
Figure 2. cIAPs specifically block CD95L-induced cell death of MEFs and human SCCs. (A) Transformed single knockout or DKO MEFs and respective
control wild-type (WT) MEFs were treated with 10 ng/ml CD95L or 500 ng/ml TRAIL for 24 h in the presence or absence of 500 nM of the IAP antagonist
(left). Cells were stained with PI and analyzed by flow cytometry. Four independent cIAP1 and -2 DKO MEFs were infected with inducible mouse cIAP1 or
Flag-cIAP2 and induced with 10 nM 4-HT for 24 h. Cells were then treated with CD95L and TRAIL for 24 h in the presence or absence of the IAP antagonist,

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1041cIAPs inhibit death receptor–mediated death • Geserick et al.
IAPs inhibit recruitment of RIP1 to the
DISC and suppress the formation of
complex II
To characterize how cIAPs negatively regulated CD95-mediated
cell death, we examined the DISC and the receptor-independent
complex II (Lavrik et al., 2008) in a cell line responsive to the IAP
antagonist (MET1) and compared it with cells resistant to the IAP
antagonist (A5RT3). We were readily able to detect recruitment of
cFLIP, caspase-8, and FADD after CD95L stimulation (Fig. 5 A,
left). Stimulation of CD95 led to SDS- and -mercaptoethanol–
insoluble CD95 complexes of higher molecular mass (Feig et al.,
2007), as seen in the CD95 Western blots of our DISC precipitates
(compare Fig. 5 with Fig. 7). We also detected small amounts of
RIP1 in the CD95 DISC in both cell types (Fig. 5 A, lanes 3 and 7).
Given the differential sensitivity, recruitment of FADD, FLIP,
and caspase-8 to the DISC was remarkably similar in MET1 and
A5RT3s, either in the presence or absence of the IAP antagonist.
In contrast, RIP1 recruitment was dramatically increased in the
CD95 DISC of MET1 in the absence of cIAPs (Fig. 5 A, lanes 3
and 4), whereas RIP1 recruitment was weaker in A5RT3 cells,
although still increased by the IAP antagonist (Fig. 5 A, lanes
7 and 8). When we examined complex II, we observed a simi-
lar stimulation-dependent interaction of FADD, cFLIP, and RIP1
with caspase-8. These experiments were performed in the pres-
ence of zVAD-fmk during stimulation because caspase inhibitors
stabilize complex II (Wang et al., 2008). As in the CD95 DISC,
there was a substantial increase in RIP1 recruitment to complex II
in the absence of cIAPs compared with CD95L-treated cells alone
(Fig. 5 A, lanes 19 and 20 and 23 and 24). Our data suggested that
loss of cIAPs increased the DISC recruitment of RIP1 or repressed
RIP1 degradation, which translated to an increased level of RIP1
in complex II.
zVAD-fmk stabilizes both the DISC and complex II
and allows for the easier detection of DISC and complex II
components (Micheau et al., 2002; Wang et al., 2008). Thus,
we tested whether zVAD-fmk affected DISC and complex
II composition. Reassuringly, the qualitative recruitment of
RIP1 was almost identical whether cells were treated with
zVAD-fmk or not, except that RIP1 was cleaved in the ab-
sence of caspase inhibitor as previously published (Fig. 5 B;
Kim et al., 2000; Martinon et al., 2000). Importantly, this ex-
periment showed that both DISC recruitment and complex II
formation increase over 1 h and then remain at steady levels
for the next hour, finally decreasing in abundance within 4 h.
It also demonstrated the effect of zVAD-fmk in increasing
the stability of both CD95 DISC and complex II, confirming
previous studies (Micheau et al., 2002; Wang et al., 2008).
a rounded shape, a lack of DNA condensation, and a later
disruption of cell membranes, which are indicative of caspase-
independent cell death (Fig. 3 B). These data suggested that cIAPs
inhibit a cryptic caspase-independent death pathway that ema-
nates from DRs. CD95 has the potential to activate a caspase-
independent form of cell death via RIP1 (for review see Festjens
et al., 2007). Because cIAPs are essential to ubiquitylate RIP1
in the TNF-R1 pathway (Bertrand et al., 2008), we hypothesized
that RIP1 was required for this form of cell death. Therefore, we
generated cell lines with decreased levels of RIP1 using stable
short hairpin RNA (shRNA) expression (Fig. 3 C) and tested for
sensitivity to CD95L/IAP antagonist–induced death. Interest-
ingly RIP1 knockdown cells were remarkably resistant to sen-
sitization to DL-mediated cell death by the IAP antagonist in
short-term viability (Fig. 3 D) or clonogenic assays (Fig. 3 E).
To rigorously test the requirement of RIP1 in our
CD95L/IAP antagonist–induced death, we tested RIP1 knock-
out MEFs. Consistent with our experiments in human cells, we
found that RIP1 knockout cells were not sensitized to DL-
mediated cell death in the absence of cIAPs (Fig. 4 A). How-
ever, the sensitivity of RIP1 knockout MEFs to CD95L was in-
creased when compared with wild-type cells, which is indicative
of a more complex role of RIP1. Our results imply that RIP1 is a
required component of a cryptic caspase-independent cell death
that is revealed when IAPs are antagonized. However, RIP1 also
blocks a cell death pathway in the presence of cIAPs. This dual
role is evident in TNF-R1 signaling where in the presence of
cycloheximide, RIP1 is protective, but in the presence of the IAP
antagonist, RIP1 is required for cell death (Kelliher et al., 1998;
Kreuz et al., 2004; Gaither et al., 2007; Petersen et al., 2007). To
determine whether the kinase activity of RIP1 was required for
this death, we treated cells with the RIP1K inhibitor Necrostatin-1
(Fig. 4 B). When added to DL- and IAP antagonist–treated cells,
Necrostatin-1 was unable to protect cells (Fig. 4 B). However,
coaddition of Necrostatin-1 and zVAD-fmk resulted in complete
protection from cell death (Fig. 4 B), annexin/propidium iodide
(PI) positivity (Fig. S4 A), or clonogenic survival of DKO MEFs
(Fig. 4 C). It has been suggested that the release of HMGB-1 (high
mobility group Box-1 protein) in the cellular supernatant repre-
sents a characteristic of necrotic cell death (Scaffidi et al., 2002).
When we investigated HMBG-1 release in IAP antagonist–treated
cells, zVAD-fmk failed to block HMGB-1 release (Fig. S4 B).
These data indicate that DLs activate a cell death pathway that
results in activated caspases that are, in most situations, sufficient
to kill cells. However, in the absence of cIAPs, a latent RIP1K-
dependent pathway is revealed. To fully block cell death, inhibi-
tion of both caspases and RIP1K is necessary.
stained with PI, and analyzed by flow cytometry. The mean + SEM is shown throughout. (B) DKO 1.5 MEFs, infected with inducible mouse cIAP1 and Flag-
cIAP2, were induced for 24 h with 4-HT. cIAP1 or Flag-cIAP2 expression was analyzed by Western blotting. -Actin served as a loading control. (C) DKO
2.7 and DKO 3.1 MEFs were infected with inducible mouse XIAP and induced with the indicated concentrations of 4-HT for 24 h. Induction of XIAP was
verified by Western blot analysis. Cells were treated with 20 ng/ml CD95L for 24 h in the presence or absence of 500 nM of the IAP antagonist, stained
with PI, and analyzed by FACS. The mean + SEM of a total of six independent experiments is shown. (D) HaCaT cells were transduced with control vector
or shRNA against cIAP1 or -2 or both. Western blot analysis shows expression of cIAP1 and -2. -Actin served as a loading control. Subsequently, cells
were analyzed for sensitivity to 2.5 U/ml CD95L in the presence or absence of 100 nM of the IAP antagonist 24 h later by crystal violet assay. (E) HaCaT
cells were transduced with lentiviral control vector or inducible cIAP2 as previously described (Diessenbacher et al., 2008). Cells were induced with 4-HT as
indicated and then treated with 2.5 U/ml CD95L for 24 h in the presence or absence of 100 nM of the IAP antagonist. Viability was examined by crystal
violet assay 24 h later. (D and E) Mean + SEM of three independent experiments is shown. MM, molecular mass.
 

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JCB • VOLUME 187 • NUMBER 7 • 2009 1042
Figure 3. DR-mediated cell death in the presence of the IAP antagonist uses caspases and caspase-independent signaling pathways. (A) HaCaT cells were
pre- or co-stimulated with 10 µM zVAD-fmk for 1 h and 100 nM of the IAP antagonist for 30 min. Subsequently, cells were stimulated with the indicated
concentration of TRAIL or CD95L. The viability of cells was analyzed by crystal violet assay 18–24 h later as indicated in Materials and methods. Mean +
SEM for three (TRAIL) or six (CD95L) independent experiments is shown. (B) HaCaT cells were either pretreated with 10 µM zVAD-fmk for 1 h or 100 nM
of the IAP antagonist for 30 min. Cells were subsequently stimulated with 5 U/ml CD95L for 4 or 24 h. 5 µg/ml Hoechst 33342 and 5 pM SYTOX green
were added for 15 min at 37°C, immediately followed by transmission (left) or fluorescence (right) microscopy. One of two independent experiments is
representatively shown. (C) Stable knockdown of RIP1 in HaCaT cells was performed as indicated in Materials and methods and controlled by Western
blot analysis for RIP1. Reprobing of the membrane with -tubulin antibodies served as a control for protein loading. MM, molecular mass. (D) Transduced
HaCaT cells as shown in C were prestimulated for 30 min with 100 nM of the IAP antagonist or diluent alone and subsequently stimulated with the indicated
concentrations of TRAIL or CD95L for 18–24 h followed by crystal violet assay. Mean ± SEM of three (TRAIL) or four (CD95L) independent experiments is
shown. (E) Transduced HaCaT cells, as described in C, were preincubated with 100 nM of the IAP antagonist (Ant) for 30 min and then stimulated with
0.5 U/ml CD95L. After 24 h, colony formation was assayed as indicated in Materials and methods. One of four representative independent experiments
is shown. Bar, 10 µm.
Collectively, our results demonstrate that the IAP antagonist
leads to a consistent increase in recruitment of RIP1 to the
DISC and does not merely delay or block the transition of
RIP1 to complex II.
Cells resistant to the sensitizing effect of the IAP antago-
nist contained cIAP2 within both the DISC and complex II de-
spite treatment with IAP antagonist, opening up the possibility
that increased levels of cIAP2 in A5RT3 could account for the

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1043cIAPs inhibit death receptor–mediated death • Geserick et al.
Figure 4. RIP1 is an important regulator of DL-mediated cell death in the absence of cIAPs. (A) RIP1 knockout (KO) or wild type (WT) MEFs were stimulated
with 10 ng/ml CD95L or 10 ng/ml CD95L and 500 nM of the IAP antagonist for 24 h and then assayed for cell viability using PI and flow cytometry. The
mean + SEM of a minimum of three independent experiments is shown. (B) The combination of caspase inhibitor zVAD-fmk and RIP1K inhibitor Necrostatin-1
completely protects HaCaT cells from DL-mediated cell death in the presence of the IAP antagonist. HaCaT cells were separately prestimulated with 10 µM
zVAD-fmk for 1 h, 50 µM Necrostatin-1 for 1 h, and 100 nM of the IAP antagonist for 30 min, followed by stimulation with 50 ng/ml TRAIL or 2.5 U/ml
CD95L for 18–24 h and subsequent crystal violet assay. Mean + SEM of three (TRAIL) or six (CD95L) independent experiments is shown. (C) Transformed
cIAP1 and -2 DKO 2.3 and 2.7 MEFs were treated with 10 ng/ml CD95L for 24 h in the presence or absence of 10 µM QVD and/or 50 µM Necrostatin-1
(Necro). Respective control wild-type MEFs were treated as the DKO MEFs but in the presence or absence of the IAP antagonist (Ant). Subsequently, cells
were harvested, and an aliquot was replated in 6-well plates and cultured for another 4 d followed by crystal violet staining of colonies.

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JCB • VOLUME 187 • NUMBER 7 • 2009 1044
Figure 5. Induction of ligand-induced receptor-bound CD95 complex (DISC) or intracellular caspase-8–containing complex (complex II) in the presence or
absence of the IAP antagonist. (A) The CD95 DISC was precipitated from MET1 or A5RT3 cells preincubated with 10 µM zVAD-fmk and 100 nM of the IAP
antagonist for 1 h and subsequently treated with CD95L-Fc for 2 h. CD95L DISC (left) was precipitated as detailed in Materials and methods. Precipitation of
receptor complexes after lysis () served as internal specificity control when compared with ligand affinity precipitates (IP; +). Equal amounts of DISC (CD95L IP)

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1045
cIAPs inhibit death receptor–mediated death • Geserick et al.
resistance of these cells to CD95L/IAP antagonist–induced
death. However, when we performed knockdown of cIAP2,
A5RT3 cells were not sensitized to CD95L-induced cell death
by the IAP antagonist (Fig. S2 C). Moreover, overexpression of
TRAF2 (Fig. S2 B), which is expressed at substantially lower
levels in A5RT3 cells than in MET1 cells (Fig. S2 A), did not
render A5RT3 cells capable of being sensitized by the IAP
antagonist, largely excluding TRAF2 as a critical candidate
explaining the difference between IAP antagonist–sensitive
and –resistant cells.
cFLIP isoforms differentially contribute to
resistance to DL-mediated cell death in the
absence of IAPs
There are several conflicting results with respect to signaling
capabilities of different cFLIP isoforms (for review see Yu and
Shi, 2008). Although cFLIP recruitment appeared very similar
in the three human cell lines tested, we wished to test whether
cFLIP isoforms could confer resistance to IAP antagonist–
mediated sensitization to DLs. Knockdown of both expressed
isoforms of cFLIP sensitized A5RT3 to IAP antagonist–DL
cell death (Fig. S3). Therefore, we generated HaCaT (Fig. 6 C)
and MET1 (Fig. S5 A) cell lines expressing different cFLIP
isoforms. We observed that sensitivity to the IAP antagonist
alone was increased in cells overexpressing cFLIPS but not
cFLIPL (Fig. 6, A and B; and Fig. S5, B and C). Furthermore,
overexpression of cFLIPS was unable to protect from DR-
mediated cell death in the presence of the IAP antagonist
(Fig. 6 B and Fig. S5 B, panels 7 and 8) even if zVAD-fmk
was added and despite the fact that it was perfectly competent
at protecting cells from CD95L treatment alone (Fig. 6 B and
Fig. S5 B, panels 2 and 3). Intriguingly, Necrostatin-1 pre-
vented cell death in short-term assays under those conditions
(Fig. 6 B and Fig. S5 B, panel 9). In contrast, cFLIPL was
very effective in blocking CD95L/IAP antagonist–induced
cell death (Fig. 6, A and D; and Fig. S5 C). These experiments
show that cFLIPS and cFLIPL differentially regulate cell death
pathways in the absence of cIAPs in a previously unsus-
pected manner.
cFLIP isoforms differentially influence
CD95-induced recruitment of RIP1 to
the DISC and complex II
To elucidate the molecular mechanism of this cFLIP isoform
phenomenon, we precipitated the CD95 DISC and com-
plex II in cells expressing cFLIPL or cFLIPS. This experi-
ment was performed in the absence of zVAD-fmk to allow
detection of differences in caspase activity. We observed
a dramatic increase in RIP1 levels in the DISC of control
cells treated with the IAP antagonist despite the fact that the
majority of RIP1 was now cleaved within the DISC (Fig. 7 A,
lanes 1–4). Consistent with previous studies for the TRAIL
DISC (Harper et al., 2001; Wachter et al., 2004), cFLIPL
repressed the recruitment of RIP1 to the CD95 DISC, and
caspase-8 and cFLIP were recruited and cleaved as previously
published (Krueger et al., 2001; Geserick et al., 2008). In com-
plex II, an increased amount of RIP1, FADD, and cFLIPL (pro
form as well as p43) was detected in control cells (Fig. 7 A,
lanes 18–21). In contrast, cFLIPL and, to a substantially lesser
extent, cFLIPS blocked the formation of complex II (Fig. 7 A,
lanes 22–29). Interestingly, complex II formation in cFLIPS-
expressing cells was detected at low levels in the absence of
DL stimulation (Fig. 7 A, lane 27). Inhibition of RIP1K activ-
ity with Necrostatin-1 blocked RIP1–caspase-8 interaction in
complex II, suggesting that RIP1K activity may facilitate tran-
sition from the DISC to complex II, which is important for cas-
pase-independent cell death (Fig. 7 A, lanes 13–16 and 30–33).
To analyze this particular aspect also in parental HaCaT cells,
we tested DISC and complex II formation in the presence of
zVAD-fmk, the IAP antagonist, Necrostatin-1, and the combi-
nation thereof (Fig. 7 B). Because of the known stabilization of
the DISC and complex II by zVAD-fmk, comparisons should
be made between similarly treated samples. Comparable levels
of RIP1 were retained in the DISC in the presence or absence
of Necrostatin-1 (Fig. 7 B, compare lane 12 with lane 16 for
the CD95 IP, RIP1 vs. FADD). Comparison of lane 8 with lane
14 for the CD95 DISC and lane 25 with lane 31 for complex
II indicates that Necrostatin-1 did not detectably change RIP1
association with the CD95 DISC, if the DISC-associated cleav-
age of RIP1 is taken into account (Fig. 7 B). In contrast, the
level of RIP1 in complex II was decreased by Necrostatin-1
(Fig. 7 B, compare lane 29 with 33, low exposure RIP1). Thus,
RIP1K activity contributes to the translocation of a complex
II in the parental HaCaT cells, which is in line with our over-
expression data. Thus, cIAPs normally limit RIP1 recruitment
to the DISC and maturation of a RIP1-containing DISC into a
RIP1-containing complex II. In the absence of cIAPs, over-
expressed cFLIPL is able to block this increased recruitment and
prevent cell death, whereas overexpressed cFLIPS is not. Thus,
cFLIPS is unable to block increased RIP1 recruitment even
though it is able to completely block caspase-8 activation and
caspase activity in the DISC. However, cFLIPS is nevertheless
insufficient to block CD95-induced cell death.
TWEAK sensitizes to CD95L-induced cell
death comparable with the IAP antagonist
in cells lacking XIAP
To further understand the physiological role of cIAPs for
CD95-induced cell death, we studied the treatment of cells
with TWEAK, which was recently shown to induce cIAP
or complex II (caspase-8 IP) were subsequently analyzed by Western blotting for the indicated molecules. Equal amounts of total cellular lysates (TL) were
loaded on the same gels to allow comparison of signal strength between CD95L-IP, complex II, and total cellular lysates. (B) Kinetics of DISC (left) or complex
II (right) in the presence or absence of the IAP antagonist. The CD95 DISC was precipitated from parental HaCaT cells either prestimulated with 100 nM
of the IAP antagonist and 10 µM zVAD-fmk for 1 h alone or the combination of both and subsequently stimulated with 250 U/ml CD95L for the indicated
times. CD95L DISC (left) or complex II (right) was precipitated as detailed in Materials and methods and specified for A. MM, molecular mass.
 

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JCB • VOLUME 187 • NUMBER 7 • 2009 1046
in keratinocytes (Leverkus et al., 2003b), which is similar
to the role of XIAP in type II cells (Jost et al., 2009).
TWEAK-induced sensitization similarly uncovered a caspase-
independent, RIP1-dependent form of cell death (Fig. 8 C).
Most interestingly, TWEAK-induced sensitization was blocked
by cFLIPL but not cFLIPS (Fig. 8 D), indicating that the find-
ings for the IAP antagonist depicted in Figs. 6 and 7 and
Fig. S5 are also relevant in a physiological setting. Of par-
ticular interest, cells expressing cFLIPS were fully protected by
Necrostatin-1 alone, indicating that TWEAK-induced degra-
dation of cIAPs may be sufficient to allow a CD95-induced
degradation and sensitize cells to TNF-mediated cell death
(Vince et al., 2008; Wicovsky et al., 2009). TWEAK treat-
ment of HaCaT cells caused down-regulation of cIAP1 and
-2 similarly to the IAP antagonist (Fig. 8 B). HaCaT cells
were sensitized to CD95 killing by TWEAK to the same
extent as HaCaT cells treated with the IAP antagonist and
were similarly protected by the combination of zVAD-fmk
and Necrostatin-1 (Fig. 8 A). MET1 cells that expressed high
levels of XIAP (Fig. S2 A) were substantially less sensitized
when comparing TWEAK with the IAP antagonist (Fig. 8 A).
These data confirm that XIAP can contribute to DL resistance
Figure 6. cFLIP is an important regulator of DL-mediated cell death in the absence of cIAPs. (A and B) HaCaT cells were transduced with cFLIPL (A) or cFLIPS (B)
or control vector. Total cellular lysates were analyzed for cFLIP and caspase-8. -Tubulin served as an internal control for protein loading. The mean + SEM
of six independent experiments is shown. (C) Cells were prestimulated with 10 µM zVAD-fmk for 1 h, 50 µM Necrostatin-1 for 1 h, and 100 nM of the
IAP antagonist for 30 min or diluent alone. Subsequently, cells were stimulated with 2.5 U/ml CD95L. The viability of cells was analyzed by crystal violet
assay after 18–24 h. MM, molecular mass. (D) Transduced HaCaT cells were prestimulated with 100 nM of the IAP antagonist (Ant) for 30 min followed
by co-stimulation with 2.5 U/ml CD95L. 24 h later, colony formation assay was performed as described in Materials and methods. One representative
experiment of a total of three independent experiments is shown.

Page 11

1047cIAPs inhibit death receptor–mediated death • Geserick et al.
sensitivity to caspase-independent cell death once cIAPs
are inactivated in a given cell irrespective of the mode of
their degradation.
caspase-independent signaling pathway to be unblocked.
Moreover, these data suggest that the stoichiometry of
cFLIPL and cFLIPS might be important to determine the
Figure 7. cFLIPL but not cFLIPS blocks the for-
mation of complex II. (A) DISC or complex II
formation in the presence or absence of the
IAP antagonist. To allow for caspase activity,
these experiments were performed without
zVAD-fmk. HaCaT cells were stimulated with
CD95L-Fc for 2 h. Subsequently, the CD95L
DISC (left) was precipitated using ligand affin-
ity precipitation as detailed in Materials and
methods. Precipitation of receptor complexes
after lysis () served as an internal specificity
control when compared with ligand affinity
precipitates (IP; +). Equal amounts of DISC
(CD95L IP) or complex II (caspase-8 IP) were
subsequently analyzed by Western blotting for
the indicated molecules. Equal amounts of total
cellular lysates (TL) were loaded on the same
gels to allow comparison of signal strength be-
tween IP and total cellular lysates. (B) DISC or
complex II formation in parental HaCaT. Cells
were either pre- or co-stimulated with 10 µM
zVAD-fmk, 50 µM Necrostatin-1, and 100 nM
of the IAP antagonist for 1 h and subsequently
stimulated with 250 U/ml CD95L for 2 h. The
CD95L DISC (left) or complex II (right) was pre-
cipitated as detailed in Materials and methods
and specified for A. Equal amounts of DISC
(CD95L IP) or caspase-8–interacting proteins
(complex II) were subsequently analyzed by
Western blotting for the indicated molecules.
MM, molecular mass.

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JCB • VOLUME 187 • NUMBER 7 • 2009 1048
Figure 8. TWEAK sensitizes to CD95L-induced cell death in a RIP1-dependent manner and is negatively regulated by cFLIPL but not cFLIPS. (A) HaCaT and
MET1 cells were either prestimulated with 0.5 ng/ml TWEAK for 2 h or 100 nM of the IAP antagonist (Ant) for 30 min and then treated with 2.5 U/ml
CD95L. In the same experiments, HaCaT cells were treated with 10 µM zVAD-fmk for 1 h, 50 µM Necrostatin-1 for 1 h, and 0.5 ng/ml TWEAK for 2 h.
Subsequently, cells were stimulated with 2.5 U/ml CD95L. Viability was analyzed by crystal violet assay after 18–24 h. (B) TWEAK leads to rapid down-
regulation of cIAP1 and -2 expression in HaCaT. Cells were stimulated with 0.5 ng/ml TWEAK for the indicated time. Subsequently, total cellular lysates
were analyzed for expression of cIAP1 or -2 by Western blotting. -Tubulin served as a loading control. MM, molecular mass. (C) Stable knockdown of
RIP1 protects HaCaT cells from CD95L-induced cell death in the presence of TWEAK. Transduced HaCaT cells as shown in Fig. 3 C were prestimulated for
2 h with 0.5 ng/ml TWEAK or diluent alone, subsequently stimulated with CD95L for 18–24 h, and assayed by crystal violet assay. The mean ± SEM of
three independent experiments is shown. (D) cFLIPL but not cFLIPS protects HaCaT cells from CD95L-induced cell death in the presence of TWEAK. cFLIPL,
cFLIPS, and vector-transduced control HaCaT as specified in Fig. 6 C were prestimulated with 10 µM zVAD-fmk for 1 h, 50 µM Necrostatin-1 for 1 h, and
0.5 ng/ml TWEAK for 2 h or diluent alone. Cells were then stimulated with 2.5 U/ml CD95L, and viability of cells was analyzed by crystal violet assay
after 18–24 h. (A and D) The mean + SEM of three independent experiments is shown.
Discussion
Our study contributes several important findings for the under-
standing of signaling pathways activated by DRs. We found that
IAP antagonists, which sensitize cells to TRAIL-induced cell
death as shown previously (Fulda et al., 2002), also dramati-
cally alter sensitivity of cells to CD95L. We studied these aspects
in human SCC cells treated with a pharmacological inhibitor
that induces degradation of IAPs within minutes and cIAP-
deficient MEFs. Our data demonstrate that DL-induced signaling

Page 13

1049cIAPs inhibit death receptor–mediated death • Geserick et al.
several studies have shown that RIP1 is a direct target of the
E3 ligase activity of cIAPs, this decrease in RIP1 modification is
likely to be caused by a reduction in RIP1 ubiquitylation (Park
et al., 2004; Bertrand et al., 2008; Varfolomeev et al., 2008).
However, our experiments cannot distinguish whether cIAPs
limit RIP1 recruitment into the DISC or whether they limit the
accumulation of RIP1 within the DISC by K48 ubiquitylating
RIP1, leading to its proteasomal degradation. Future studies
using ubiquitin-specific antibodies will be able to address this
issue (Newton et al., 2008). It is tempting to speculate that the in-
creased amount of RIP1 in the DISC and complex II subsequently
leads to autoactivation of the kinase within the complex (Fig. 9),
which is consistent with the activation mechanism of other
kinases (for review see Eswarakumar et al., 2005). Identifying
targets of RIP1K will undoubtedly promote greater insight into
this caspase-independent cell death program (Hitomi et al., 2008).
While this manuscript was under revision, RIP3, a kinase in-
volved in the apoptosis/necrosis shift in TNF-mediated cell death,
was reported to interact with RIP1 (Cho et al., 2009; He et al.,
2009) and autophosphorylate in a RIP1K-dependent manner. In
turn, activated RIP3 may regulate metabolic enzymes that could
promote the necrotic phenotype (Zhang et al., 2009).
The precise physiological relevance of complex II forma-
tion after DL stimulation remains unresolved to date (Varfolomeev
et al., 2005; Lavrik et al., 2008). We have now studied this com-
plex under conditions in which cIAPs are absent and find that
complex II formation is increased whenever cIAPs are down-
regulated and contain high amounts of RIP1 in association with
caspase-8. Remarkably, less RIP1 is detected in complex II in
cells insensitive to IAP antagonists, which suggests that it is
a shift of the stoichiometric balance of complex II–associated
proteins that is relevant for the outcome of apoptotic/necrotic
signaling. RIP1 is not only ubiquitylated in the DISC but is also
cleaved by caspases. This fact raises a question concerning the
impact of caspase activation on RIP1-dependent cell death. Our
kinetic analysis of DISC and complex II formation showed a
marked increase of full-length RIP1 in the CD95 DISC and
complex II in the presence of zVAD-fmk when compared with
control cells or in cleaved form in the absence of zVAD-fmk. A
large body of evidence about alternative DR-induced cell death
pathways for CD95 (Vercammen et al., 1998; Holler et al.,
2000) or TNF (Chan et al., 2003) has been generated by studies
using chemical caspase inhibitors (e.g., zVAD-fmk). Based on
our data, increased RIP1 recruitment to the DISC and/or com-
plex II in the absence of cIAPs may explain the increase in DR-
induced cell death in the presence of zVAD-fmk. However,
zVAD-fmk is unable to fully block DISC-associated activity of
caspase-8 in the DISC (Wachter et al., 2004) and does not block
the enzymatic activity of the pro form of caspase-8 (Boatright
et al., 2004), which makes it difficult to draw further mechanistic
conclusions from such experiments. Thus, we attempted to clarify
the role of caspases by investigating the impact of cFLIP isoforms
as potent caspase-8 inhibitors that can block DL-mediated cell
death. However, surprisingly, we showed that cFLIPS is insuffi-
cient to prevent the alternative RIP1 cell death pathway that
proceeds independent of inhibition of caspases by zVAD-
fmk. In contrast, cFLIPL, with its equivocal caspase inhibitory
pathways are profoundly regulated by cIAPs. XIAP appeared to
play only a minor role because even overexpression of XIAP in
cIAP1- and cIAP2-deficient MEFs failed to increase resistance
to CD95L. Furthermore, TWEAK-FN14 signaling, which leaves
XIAP levels unaffected (Vince et al., 2008; Wicovsky et al.,
2009), duplicates the effects of the IAP antagonist. Although
XIAP undoubtedly plays a role in regulating CD95 signaling in
type II cells (Jost et al., 2009), CD95 signaling in type II cells
requires amplification via the mitochondrial pathway to effec-
tively kill cells, and therefore, XIAP inhibits caspases at a late
point in the signaling pathway. In our cells, we show that cIAPs
regulate death signaling at the level of the DISC and complex II
and suggest that this alters the cell death signal strength or charac-
ter such that it can no longer be effectively inhibited by XIAP.
CD95L induces apoptosis in many cell types. Our data
show that the presence of cIAPs favors the apoptotic pathway and
therefore, in the presence of cIAPs, CD95 killing can be blocked
by caspase inhibitors such as zVAD-fmk, cFLIPS, and cFLIPL.
However, in the absence of cIAPs, a cryptic alternative pathway
is revealed. This type of death shows hallmarks of apoptosis, in-
cluding phosphatidylserine exposure and cleavage of substrates
such as PARP by caspases. However, it also shows features of a
necrotic type of cell death, including HMGB-1 release and other
morphological characteristics. This CD95L/IAP antagonist–
induced death cannot be blocked by either zVAD-fmk or
Necrostatin-1 alone. Therefore, the fact that the specific RIP1
inhibitor Necrostatin-1 (Degterev et al., 2008) in combination
with zVAD-fmk inhibits CD95L/IAP antagonist killing indicates
that RIP1 plays a pivotal role in this death. Support for this concept
comes from a previous study by Holler et al. (2000) that showed
DISC recruitment of RIP1 in the complete absence of FADD.
Several other studies have shown that blocking the CD95 or TNF
apoptotic pathway, often with chemical caspase inhibitors, induces
an alternative cell death pathway variously called programmed
necrosis or necroptosis (Vercammen et al., 1998; Matsumura et al.,
2000), and in the absence of FADD or caspase-8, a caspase-
independent cell death is operative (Bell et al., 2008; Ch’en et al.,
2008). In line with our findings, a recent study has identified
extracellular pH as a possibility to switch DR-induced apoptotic
cell death to RIP1-dependent necrotic cell death in the absence of
pharmacological caspase inhibitors such as zVAD-fmk (Meurette
et al., 2007). However, despite these insights, the molecular
mechanisms that regulate each response under physiological con-
ditions are unresolved (for review see Festjens et al., 2007).
Using a combined genetic and biochemical approach, we
demonstrate that cIAPs inhibit a caspase-independent cell death
and thereby facilitate an apoptotic death. In the absence of cIAPs,
a cryptic alternative death pathway is revealed. Using RIP1/
MEFs and a specific RIP1K inhibitor, Necrostatin-1, we show
that this alternative death pathway is dependent on RIP1K activ-
ity. The recruitment of RIP1 to the CD95 membrane-bound com-
plex (CD95 DISC) is decreased by cIAPs, whereas caspase-8
recruitment and processing is unaltered, thus suggesting
a molecular mechanism for how cIAPs inhibit this RIP1K-
dependent cell death pathway. Not only is the total amount of
RIP1 increased in the CD95 DISC and complex II in the absence
of cIAPs, the degree of RIP1 modification is also less. Because

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JCB • VOLUME 187 • NUMBER 7 • 2009 1050
experiments, may be of pathophysiological relevance. To
test whether the phenotype we described is unique to HaCaT
cells, we generated cFLIP-expressing MET1 cells that are p53
wild type and express XIAP. Reassuringly, overexpression of
cFLIPL or cFLIPS resulted in very similar results in respect to
the isoform-specific effects of cFLIP, indicating a more general
phenomenon. One intriguing observation is that cFLIPL p43 re-
covered in the DISC in HaCaT cells was only found at very low
levels in cells expressing cFLIPS. Thus, one added hypothesis
explaining the functional difference of both cFLIP isoforms in
the absence of cIAPs may be that high levels of cFLIPS inhibit
incorporation of cFLIPL into the DISC, and this may result in
increased RIP1 in the DISC. Further experiments are required
to test this hypothesis. With these considerations in mind, we
believe our experiments suggest a remarkable and previously
unsuspected specificity concerning the mechanism of death in-
hibition by cFLIP isoforms and open up interesting questions
for future studies.
cFLIPL was reported to mediate binding to proteins such
as TRAFs, RIP1, or others (for review see Kataoka, 2005).
TRAF2 is a binding partner of cIAPs and RIP1, and in some
cells, cFLIPL overexpression results in increased TRAF2 re-
cruitment to the CD95 DISC (Siegmund et al., 2007). We were
consistently unable to specifically detect TRAF2 in the DISC or
complex II using either CD95L-Fc– or antibody-mediated pre-
cipitation, and because TRAF2 overexpression did not alter the
sensitivity of A5RT3 cells to IAP antagonist CD95L death in
our cells, TRAF2 is unlikely to be the critical molecule mediat-
ing the different effects of cFLIPL and cFLIPS. In summary, our
data point to novel and differential functions of cFLIP isoforms
in the absence of cIAPs. Intriguingly, we detect an increased
spontaneous complex II in IAP antagonist–treated cells as well
capability, effectively blocks RIP1K-dependent death, and this
inhibitory potential is associated with its ability to repress the
efficient formation (or maintenance) of RIP1 in association with
caspase-8 within the DISC and complex II. In particular, the
interaction of RIP1 with caspase-8 in complex II is repressed by
cFLIPL but not cFLIPS. These data point to a critical role of the
caspase-like domain of cFLIPL that may allow cleavage of RIP1
within the DISC, thereby limiting its ability to form complex II.
Alternatively, cFLIPL might simply be better than cFLIPS at in-
hibiting RIP1 recruitment because of steric hindrance. Finally,
it is also possible that cFLIP isoforms act independently of the
DR complex, as previously suggested in lymphoid cell (Golks
et al., 2006). For example, it is possible that nonubiquitylated
forms of RIP1 bind to FADD independently of DISC formation,
subsequently leading to DR-independent complex II formation
and thereby facilitating necrotic cell death.
Our study using isoforms of cFLIP has the obvious limita-
tion of ectopic overexpression of both isoforms. HaCaT cells ex-
press low levels of endogenous cFLIP as compared with primary
keratinocytes (Leverkus et al., 2000) and are therefore ideally
suited for these experiments (Wachter et al., 2004). However,
overexpression experiments can result in nonphysiological re-
sponses, and in the case of cFLIP, a lack of any clear consensus
about the physiological relevance of different isoforms makes it
challenging to interpret such experiments. Even in the case of
vFLIP isoforms, which are highly similar to cFLIP isoforms, it
is unknown what the intracellular protein levels of vFLIP are
during a virus infection or how endogenous cFLIP could affect
vFLIP effects. Of note, vFLIP of HHV8 dramatically increases its
expression during late stages of HHV8-induced Kaposi sarcomas
(Stürzl et al., 1999). Thus, a major shift of the ratio of cFLIPL
to cFLIPS, such as that which occurs in our overexpression
Figure 9. The role of cIAPs during DR-mediated
cell death. cIAPs block recruitment to or degrada-
tion of RIP1 in the DISC. This signaling platform
induces cell death in a caspase-dependent as
well as -independent manner. A secondary com-
plex II, which is critical for necrotic cell death,
also contains the initiator caspase-8 and FADD.
In the presence of high levels of RIP1, RIP1 might
be autoactivated and induce necrotic cell death
or may require additional binding partners such
as RIP3 (Cho et al., 2009; He et al., 2009;
Zhang et al., 2009). cFLIPL but not cFLIPS is able
to block complex II formation and the pro-
necrotic activity of RIP1 in complex II. In contrast,
both isoforms block apoptotic cell death initiated
by caspase-8 at the DISC. Caspase-8–mediated
cleavage of RIP1 is one hypothetical mecha-
nism of down-regulation of RIP1 within the com-
plexes. Alternatively, RIP1 is only recruited to the
DISC when ubiquitylated. cIAPs transfer ubiqui-
tin chains of currently debated specificity (e.g.,
Lys63, -48, or -11) to its substrate RIP1 (Park
et al., 2004; Bertrand et al., 2008; Varfolomeev
et al., 2008; Blankenship et al., 2009).

Page 15

1051cIAPs inhibit death receptor–mediated death • Geserick et al.
(Cancer Research UK, London, England, UK). Cell lines were cultured
exactly as previously described (Boukamp et al., 1988; Proby et al., 2000;
Mueller et al., 2001).
Inducible lentiviral reconstitution of murine or human IAPs
The generation of MEFs and lentiviral particles have previously been de-
scribed (Vince et al., 2007, 2008). In brief, MEFs were generated from
embryos in accordance with standard procedures and were infected
with SV40 large T antigen–expressing lentivirus. In vivo DKO cIAP1 and
-2 MEFs were obtained from LoxP/LoxP cIAP1 and FRT/FRT cIAP2 mouse
crossed first with Cre transgenic mouse followed by Flp transgenic mouse.
DKO MEFs were generated at embryonic day (E) 10 instead of E15. To
generate DKO MEFs expressing mouse cIAP1 or Flag-cIAP2 or mouse
XIAP, 293T cells were transfected with packaging construct pCMV ðR8.2,
VSVg, and the relevant lentiviral plasmid. DKO MEFs were infected with
packaged lentivirus, and polyclonal MEFs were obtained after puromycin
(2–5 µg/ml: pF 5xUAS selection) and hygromycin selection (100–500 µg/ml:
GEV16 selection). HaCaT cells were transduced to express GFP or Flag-
tagged cIAP1 or -2 in an inducible manner (Diessenbacher et al., 2008).
Cells were subsequently tested for expression of the respective proteins after
24 h of induction with 10 or 100 nM 4-hydroxy-tamoxifen (4-HT).
Retroviral infection
For infection of HaCaT cells, the pCFG5-IEGZ retroviral vector containing
cDNA inserts of cFLIPL, cFLIPS, or TRAF2 (provided by H. Wajant, Univer-
sity of Würzburg, Würzburg, Germany) was used as previously described
(Leverkus et al., 2003a; Geserick et al., 2008). In brief, the amphotropic
producer cell line NX was transfected with 10 µg of the retroviral vec-
tors by Ca phosphate precipitation. Cell culture supernatants containing
viral particles were generated by incubation of producer cells with HaCaT
medium (DME containing 10% FCS) overnight. After filtration (45-µm filter;
Schleicher & Schuell), culture supernatant was added to HaCaT cells seeded
in 6-well plates 24 h earlier in the presence of 1 µg/ml polybrene. After
centrifugation for 3 h at 30°C, viral particle–containing supernatants were
replaced by fresh medium. After 10–14-d Zeocin selection of bulk-infected
cultures, FACS analysis for GFP expression (always >90%) and Western blot
analysis were performed on polyclonal cells to confirm ectopic expression
of the respective molecules. The empty retroviral vector served as control.
Aliquots of cells were used for the experiments between passages 2 and 6
after initial characterization for all subsequent experiments.
Stable siRNA expression
We used stable expression of siRNA against cFLIP or cIAP1 or -2 as recently
published (Diessenbacher et al., 2008; Schmidt et al., 2009). RIP1 siRNA as
well as a hyper random sequence not matched by any gene in the National
Center for Biotechnology Information database (HRS; Vogler et al., 2007)
were used. The HRS construct was provided by S. Fulda (Ulm University, Ulm,
Germany). For generation of the constructs, cDNA 64-mer oligomers con-
taining RIP1-targeting sequence (nt start position +193) were cloned into the
pSuper.retro retroviral vector (pRS) using HindIII and BglII restriction sites.
The resulting vectors or control vector containing a sequence not found in the
human genome were transfected into the amphotropic producer cell line
exactly as outlined in the previous section. The retrovirus-containing superna-
tant was then used to infect A5RT3 and MET1 cells with HRS or cFLIP shRNA,
respectively. HaCaT cells were infected either with HRS or RIP1 or cIAP1 or -2
shRNA, and infected cells were selected with 1 µg/ml puromycin (Sigma-
Aldrich) for 3 d to obtain puromycin-resistant bulk-infected cultures for further
analysis. The respective control constructs served as internal control. FACS
analysis of GFP expression (always >90%) and Western blot analysis were
performed on polyclonal cells to confirm ectopic expression of the respective
molecules. Aliquots of cells were used for cytotoxicity assays and biochemical
characterization between passage 2 and 6 after the antibiotic selection.
FACScan analysis
For surface staining of TRAIL receptors (TRAIL-R1 and -R2) and CD95, cells
were trypsinized, and 4 × 105 cells were incubated with monoclonal anti-
bodies against TRAIL-R1 or -R2, CD95, or isotype-matched control IgG for
60 min followed by incubation with biotinylated goat anti–mouse secondary
antibodies (BD) and Cy5-phycoerythrin–labeled streptavidin (Invitrogen) as
described previously (Wachter et al., 2004). For all experiments, 2 × 104
cells were analyzed by FACScan (BD).
Western blot analysis
After stimulation as indicated, cells were washed twice with ice-cold PBS
and lysed for 30 min on ice by the addition of lysis buffer (30 mM Tris-HCl,
as increased DL-induced formation of complex II in cFLIPS-
expressing cells. Because cFLIPL can block these events, it
suggests that the caspase-like domain of cFLIPL is involved in
negatively regulating complex II formation, but whether and
which cFLIPL interacting proteins are required for this effect
are unclear.
Our discoveries concerning the role of RIP1 in an alterna-
tive RIP1-dependent death emanating from the CD95 receptor
were made initially with IAP antagonist drugs that rapidly de-
plete cIAP levels and antagonize XIAP. Although IAP antago-
nists have attracted great interest as cancer therapeutics, it could
be questioned whether these findings have any physiological rel-
evance. TWEAK is able to promote the rapid degradation of
cIAPs in an analogous manner to IAP antagonists (Vince et al.,
2008; Wicovsky et al., 2009). TWEAK and other ligands such as
TRAIL, CD95L, or TNF are likely to be present in the same
physiological scenario (Vince and Silke, 2006). Thus, stimulation
of FN14, as shown in this manuscript, or possibly other receptors
such as CD30 or -40 able to recruit cIAPs based on their protein
structure (for example DR6, TRAIL-R2 or -R4, CD27, or EDAR)
could deviate DR-mediated apoptotic to necrotic cell death with
major physiological and pathophysiological consequences dur-
ing tumorigenesis or the inflammatory response in multicellular
organisms (Leverkus et al., 2008; Kerstan et al., 2009).
Materials and methods
Materials
The following antibodies were used for Western blot analysis: antibodies to
caspase-8 (C-15 [provided by P.H. Krammer, German Cancer Research
Center, Heidelberg, Germany] and C-20 [Santa Cruz Biotechnology, Inc.]);
cFLIP (NF-6; Enzo Life Sciences, Inc.); XIAP, FADD, and RIP1 (BD); caspase-3
(CPP32; provided by H. Mehmet, Merck Frosst, Kirkland, Quebec, Can-
ada); PARP-1 (clone C-2-10; Enzo Life Sciences, Inc.); rat antibodies to
cIAP1 (Silke et al., 2005) and cIAP2 (Vince et al., 2009); and -tubulin
(clone 2.1; Sigma-Aldrich). Polyclonal antibodies to HMGB-1 were pur-
chased from Abcam, and TRAF2 (C-20) and CD95 antibodies (C-20) were
purchased from Santa Cruz Biotechnology, Inc. -Actin antibodies were
purchased from Sigma-Aldrich, and HRP-coupled monoclonal antibody to
GFP was purchased from Clontech. His-Flag-TRAIL (HF-TRAIL) was produced
as previously described (expression construct provided by H. Walczak, Im-
perial College London, London, England, UK; Diessenbacher et al., 2008).
For expression of Fc-CD95L or Fc-TWEAK, we used constructs previously
published (Bossen et al., 2006), which were provided by P. Schneider (Uni-
versity of Lausanne, Epalinges, Switzerland). 1 U of Fc-CD95L was deter-
mined as a 1:500 dilution of the stock Fc-CD95L supernatant, and 1 U/ml
of Fc-CD95L supernatant was sufficient to kill 50% (LD50) of A375 mela-
noma cells, as previously described (Geserick et al., 2008). Ligand-
mediated cell death was completely blocked by the addition of either
soluble TRAIL-R2-Fc protein or CD95-Fc protein. HRP-conjugated goat anti–
rabbit, goat anti–rat IgG, and goat anti–mouse IgG antibodies and HRP-
conjugated goat anti–mouse IgG1, IgG2a, IgG2b, and IgG1- were
obtained from SouthernBiotech. TRAIL-R1 (HS 101) and TRAIL-R2 (HS 201)
monoclonal antibodies for FACScan analysis of surface receptor expression
were used as previously described (Leverkus et al., 2003b) and are avail-
able from Enzo Life Sciences, Inc. CD95 antibodies (Apo-1 IgG1 and
IgG3a) were provided by P.H. Krammer. Cy5-conjugated annexin V was
purchased from BD. The IAP antagonist (compound A) was provided by
TetraLogic Corp.
Cell culture
The generation of MEFs has previously been described (Vince et al., 2007,
2008). RIP knockout mice were provided by M. Kelliher (University of Mas-
sachusetts Medical School, Worcester, MA). The spontaneously trans-
formed keratinocyte line HaCaT and the derived metastatic clone A5RT3
(Mueller et al., 2001) were provided by P. Boukamp (German Cancer Re-
search Center). MET1 cells (Popp et al., 2000) were provided by I. Leigh