(Received for publication, December 1, 1995)
From the
CD95 (Fas/APO-1) and tumor necrosis factor receptor-1 (TNFR-1)
are related molecules that signal apoptosis. Recently, a number of
novel binding proteins have been proposed to mediate the signaling of
these death receptors. Here we report that an N-terminal truncation of
one of these candidate signal transducers, FADD/MORT1, abrogates
CD95-induced apoptosis, ceramide generation, and activation of the cell
death protease Yama/CPP32. In addition, this dominant-negative
derivative of FADD (FADD-DN) blocked TNF-induced apoptosis while not
affecting NF-B activation. FADD-DN bound both receptors, and in
the case of CD95, it disrupted the assembly of a signaling complex.
Taken together, our results functionally establish FADD as the
apoptotic trigger of CD95 and TNFR-1.
Recently, major advances have been made in understanding the
signal transduction of the tumor necrosis factor
(TNF)()/nerve growth factor receptor family. Activation of
these receptors is caused by aggregation mediated by the respective
ligands or agonist
antibodies(1, 2, 3, 4) . There are
no identifiable catalytic motifs (e.g. kinase or phosphatase)
in the cytoplasmic domains of these cell surface proteins. Instead, it
is becoming apparent that signal transduction is accomplished via
association with an emerging class of novel and diverse signaling
molecules. For example, a dominant-negative mutant of TRAF2 was shown
to block TNFR-2-mediated NF-
B activation(5) , while an
analogous mutant of CD40bp (also known as TRAF3, CRAF1, or
LAP1(6, 7, 8) ) was shown to inhibit
CD40-mediated up-regulation of CD23(7) . Numerous candidate
signal transducers have been identified for the two death receptors,
TNFR-1 and CD95, including FADD/MORT1, TRADD, RIP, FAP-1, FAF1, and
TRAP1/2(9, 10, 11, 12, 13) .
Both death receptors share a region of homology of about 80 amino acids
in their cytoplasmic domain required to signal
apoptosis(14, 15) . This shared ``death
domain'' suggests that both receptors engage a common component of
the apoptotic machinery. Here, we investigated the role of FADD in the
proximal signal transduction of CD95 and TNFR-1.
Overexpression of FADD causes apoptosis(9) , resulting in cleavage of the death substrate poly(ADP-ribose) polymerase (PARP) to signature apoptotic fragments (data not shown). A previously reported deletion mutant of FADD, NFD4 (hereon referred to as FADD-DN), was able to interact with CD95, but failed to initiate apoptosis (9) (Fig. 1A), suggesting that it may have a dominant negative effect on CD95 signaling. FADD-DN lacks 80 N-terminal amino acids, but contains the death domain responsible for association with the related death domain of CD95(9) . The B lymphoma cell line BJAB was transfected with either the expression vector pcDNA3 alone or as a FADD-DN expression construct. Stable transfectants were generated by neomycin (G418) selection and pooled populations assessed for FADD-DN expression and sensitivity to anti-Fas-induced apoptosis (BJAB-FADD-DN, Fig. 1B). Expression of FADD-DN in both a pooled population and in a mixture of selected clones (BJAB-sFADD-DN) dramatically abrogated CD95-induced cell death (Fig. 1B). The apoptotic nature of the cell death was confirmed by the TUNEL assay which detects 3`-OH DNA strand breaks. The FADD-DN expressing BJAB cells were not inherently resistant to apoptotic cell death, since the protein kinase inhibitor staurosporine and the calcium ionophore A23187 equally killed the three cell lines (data not shown). CD95 surface expression was equivalent in the vector and FADD-DN cell lines as assessed by flow cytometry (data not shown). The possibility that clonal variation in the stable lines was responsible for the observed resistance to CD95 killing was discounted by the observation that transient overexpression of the FADD derivative ablated CD95-induced cell death in BJAB and Jurkat cells (data not shown).
Figure 1:
FADD
mediates CD95 signal transduction. A, schematic representation
of FADD and FADD-DN (NFD-4). Amino acid residues are given for
selected junctures. B, BJAB cells expressing FADD-DN are
resistant to CD95-induced apoptosis. The indicated cell lines were
incubated for 16 h with various concentrations of anti-Fas IgM and cell
death assessed by nuclear morphology. At least 250 cells were counted
in three independent experiments (mean ± S.D.). Expression of
FADD-DN is shown in the photographic insets. FADD-DN migrates
as a doublet around 18 kDa due to post-translational modification(23).
The TUNEL assay is shown in the graphical inset, and at least
250 cells were counted in three independent experiments (mean ±
S.D.). C, anti-CD95-induced ceramide generation is abrogated
by FADD-DN. The indicated BJAB cell lines were treated with anti-Fas
IgM (1 µg/ml) for the various times listed and ceramide levels
subsequently assessed (mean ± S.D.; n = 3).
Significant levels of ceramide could not be detected at 5-, 10-, 30-
and 60-min time points (inset). D,
Cceramide (C2), but not
C
-dihydroceramide (DHC2), can bypass the dominant
negative effect of the FADD derivative. The cells characterized include
BJAB vector, BJAB-FADD-DN, and BJAB-sFADD-DN. As a control, cells were
also exposed to the structurally related inactive analog,
C
-dihydroceramide (33) . Viabilities were not
decreased significantly, thus validating the specificity of the
cytotoxic effect of C
-ceramide. The x axis refers
to the concentration of synthetic ceramide used and the y axis
refers to viability as assessed by 3-(4,5-dimethyl
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide conversion. Viability is
expressed as percentage of vehicle-treated control ± S.E.
Results are representative of three independent experiments. E, CD95-induced activation of the apoptotic protease
Yama/CPP32 is blocked by FADD-DN. BJAB vector and BJAB-sFADD-DN were
left untreated or treated with 100 ng/ml anti-Fas IgM for 18 h. Lysates
were then run on a 15% gel and immunoblotted with polyclonal antibodies
directed against the p17 and p12 subunits of Yama (upper
panel). Cleavage of the death substrate poly(ADP-ribose)
polymerase was also assessed (lower
panel).
The sphingolipid ceramide has been
implicated as a signaling intermediate in the CD95
pathway(20, 24, 25, 26) . To
determine whether this signaling event was blocked by the FADD
derivative, BJAB cells expressing FADD-DN were treated with anti-Fas
IgM and, subsequently, ceramide levels assessed. Consistent with a
proximal role of FADD in CD95 signaling, FADD-DN inhibited
CD95-mediated ceramide generation (Fig. 1C).
Additionally, vector and FADD-DN transfected BJAB cells were equally
susceptible to cell death induced by the cell-permeable, active
ceramide analogue, C-ceramide, confirming that the block in
the death pathway was prior to ceramide generation (Fig. 1D).
Mammalian homologs of the Caenorhabditis elegans cell death protease CED-3 are thought to be distal effectors of the CD95 cell death pathway. Here we show that the apoptotic protease Yama/CPP32 (17, 27, 28) is activated by CD95 engagement (Fig. 1E). Endogenous Yama is expressed as a 32-kDa pro-enzyme and upon activation is proteolytically processed into active p17 and p12 subunits(17, 27) . One of the proposed substrates of Yama is the nuclear enzyme PARP(17, 27) . As expected, CD95-mediated activation of Yama and resulting PARP cleavage was blocked by FADD-DN (Fig. 1E).
In the yeast two-hybrid assay, FADD had a
weak but specific interaction with TNFR-1(9) . To determine
whether FADD has a role in TNFR-1-induced cell death, the FADD
derivative was transfected into TNF-sensitive MCF7 breast carcinoma
cells and stable cell lines generated. Interestingly, FADD-DN
expressing MCF7 cells were equally resistant to both CD95- and
TNF-induced cell death (Fig. 2A, Table 1),
suggesting a proximal convergence of the cytokine-mediated cell death
pathway. Additionally, 293T cells transiently overexpressing TNFR-1
were protected from cell death by co-transfecting FADD-DN (data not
shown). As with the BJAB cell lines, the FADD-DN MCF7 cell lines were
not resistant to staurosporine-induced apoptosis (Table 1).
Overexpression of interleukin-1 converting enzyme (ICE) could
``bypass'' the dominant-negative effect of the FADD
derivative, suggesting that the death pathway was being blocked
upstream of the ICE-like proteases implicated in the apoptotic pathway.
Figure 2:
FADD mediates TNF-induced cell death, but
not TNF-induced NF-B activation. A, the stable cell lines
utilized include MCF7-sFADD-DN, which represents a pool of nine
resistant clones and a corresponding MCF7 vector control. Expression of
FADD-DN is shown in the left panels. FADD-DN migrates as a
doublet around 18 kDa, possibly due to post-translational modification.
The indicated cell lines were either left untreated (UnRx) or
treated with anti-Fas IgM (250 ng/ml) plus cycloheximide (CHX,
10 µg/ml) or 100 ng/ml TNF for 24 h and stained with propidium
iodide. Similar results were obtained using anti-APO-1 antibody plus
soluble protein A in the absence of cycloheximide (data not shown).
Phase contrast micrographs are shown with corresponding confocal
micrographs (insets) depicting nuclear morphology. B,
MCF7 vector or MCF7-sFADD-DN cells were transfected with an
NF-
B-dependent E-selectin-luciferase reporter construct (5) and were either untreated or treated with TNF for 9 h.
Luciferase activities were assessed as described
previously(5) , and values shown are mean ± S.D. of
three independent experiments.
While the main activity of CD95 is to trigger apoptosis, TNFR-1 can
signal an array of diverse pro-inflammatory and immunoregulatory
activities(29) . Distinct from CD95, TNFR-1 is an inducer of
nuclear factor B (NF-
B)(30) . We therefore
investigated whether expression of FADD-DN modulated TNF-induced
NF-
B activation. MCF7 vector and MCF7-sFADD-DN cells were
transfected with an NF-
B-dependent reporter gene (5) and
relative NF-
B activity assessed (Fig. 2B). In both
cell lines, NF-
B was activated equally well, suggesting that
TNFR-1 utilizes FADD to transduce the death signal and activates
NF-
B by a different mechanism.
To determine the mechanism by which the FADD derivative exerts its dominant negative effect, co-immunoprecipitation of FADD and FADD-DN with CD95 or TNFR-1 was assessed. 293T cells were co-transfected with AU1 epitope-tagged FADD constructs and FLAG epitope-tagged CD95, FLAG-TNFR-1, or FLAG-B94(31) . Cell lysates were immunoprecipitated with anti-FADD antibody and subsequently immunoblotted with anti-FLAG antibody. TNFR-1 and CD95, but not a control cytoplasmic protein, B94, co-immunoprecipitated with FADD-DN (Fig. 3A). The association of FADD and FADD-DN with CD95 was 10-fold greater than with TNFR-1, correlating with the relative apoptotic potential of the two death receptors(32) . Thus, our data suggest that FADD-DN exerts its inhibitory action by directly or indirectly forming a complex with the death receptors, preventing recruitment of endogenous FADD. It remains formally possible, however, that FADD-DN functions by binding and sequestering other death signaling molecules.
Figure 3:
A mechanism for the inhibitory action of
FADD-DN. A, 293T cells were co-transfected with AU1 epitope
tagged FADD constructs and FLAG-tagged constructs encoding CD95,
TNFR-1, and B94. The cells were lysed and FADD, or FADD-DN was
immunoprecipitated with anti-FADD polyclonal antibody, run on a 15%
SDS-polyacrylamide gel, and subsequently transferred to a
nitrocellulose membrane. Co-precipitating FLAG-Fas and FLAG-TNFR-1 were
identified by immunoblotting with anti-FLAG antibody. B94, a 73-kDa
protein(31) , did not co-precipitate with FADD, verifying the
specificity of the protein-protein interaction. All transfected
components were assessed for expression by immunoblotting cell lysates
(data not shown). B, a truncated derivative of FADD exerts a
dominant-negative effect by displacing endogenous FADD from activated
CD95 and thereby inhibits DISC formation. BJAB vector (upper
panels) and BJAB-sFADD-DN cells (middle panels) were
metabolically labeled (with [S]cysteine and
[
S]methionine), lysed with Triton X-100,
immunoprecipitated with anti-APO-1/PA-Sepharose, and subsequently
analyzed by two-dimensional isoelectric focusing, 12%
SDS-polyacrylamide gel electrophoresis. As expected, the four CAPs, as
well as FADD-DN, failed to associate with the unactivated (not
oligomerized) APO-1. However, cells stimulated with anti-APO-1 for 5
min and then lysed show association of the four CAP proteins
with the activated receptor in vector transfected cells. By contrast,
in FADD-DN-expressing cells, FADD-DN associated with the oligomerized
APO-1, while the CAPs did not. The lower left panel is a
schematic illustration of the migration positions of APO-1, CAPs, and
FADD-DN. Large open arrowhead, migration positions of
endogenous FADD (CAP1, CAP2). Large closed arrowhead, FADD-DN. Small open arrowhead, CAP3 (26 kDa) and CAP4 (55 kDa). APO-1
runs as an array of spots around 54 kDa on the basic half of the gel.
FADD-DN was identified in activated sFADD-DN cells by immunoblotting
using anti-AU1 antibody (lower right panel). C, a
model for the role of FADD in the CD95 and TNFR-1 death
pathways.
In the
case of CD95, the endogenous signaling machinery was studied. Four
proteins, termed CAPs (for cytotoxicity-dependent APO-1-associated
proteins), associate with CD95 in a ligand-dependent
fashion(23) . CAP1 and CAP2 were shown to be FADD, while CAP3
and CAP4 remain unidentified(23) . The oligomerized receptor,
along with the associated CAPs, has been designated the DISC (23) . CAP3 or CAP4 are not the recently described candidate
signaling molecules FAP-1 or RIP, as antipeptide antibodies capable of
detecting endogenous FAP-1 or RIP, respectively, were unable to detect
either protein associated with activated or unactivated CD95. ()As expected, in vector transfected BJAB cells, the DISC
formed upon anti-APO-1 treatment (Fig. 3B). By
contrast, anti-APO-1-stimulated sFADD-DN cells did not form a complete
DISC. Instead, FADD-DN complexed with CD95 in a ligand-dependent
fashion and inhibited the recruitment of FADD (CAP1 and CAP2), CAP3 and
CAP4, thereby disrupting the DISC (Fig. 3B). Similar
results were obtained using FADD-DN expressing MCF7 cells (data not
shown). Thus, our results suggest that the N terminus of FADD, which is
missing in FADD-DN, is required for the recruitment and assembly of the
downstream DISC components, CAP3 and CAP4. Studies are underway to
identify an analagous TNFR-1 DISC.
In conclusion, this is the first
report to functionally establish FADD, one of numerous candidate
signaling molecules, in the proximal signal transduction of CD95. Many
of the contenders, including RIP and FAP1, were not found associated
with the active or inactive receptor, nor are dominant negative
inhibitors likely to exist. Of paramount importance is the fact that
the dominant-negative version of FADD potently abrogates TNF-induced
apoptosis but not TNF-induced NF-B activation. This suggests that
FADD is the common conduit of the cytokine-mediated death signal and
also demonstrates that the signaling pathways for TNF-induced apoptosis
and NF-
B activation are distinct (Fig. 3C). Taken
together, our results demonstrate that FADD is both a necessary and
sufficient mediator of CD95 and TNFR-1-induced apoptosis as
overexpression of FADD engages the cell death machinery (9) ,
while a truncated derivative acts as a potent dominant-negative
regulator. Future studies will hopefully elucidate downstream
components of the pathway, thus linking FADD to the apoptotic proteases
of the ICE/ced-3 family.