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INTRODUCTION |
Sphingomyelinase
(SMase)1 activation and
ceramide generation have emerged as a lipid signaling pathway
transducing diverse biological effects of cytokine receptors like p55
tumor necrosis factor (TNF) receptor or CD95 (Fas, Apo-1) (1, 2).
Through binding to the 55-kDa TNF receptor (TNF-R55), TNF rapidly
activates two distinct forms of SMase, a membrane-associated neutral
(N-)SMase and an acid (A-)SMase (3) residing in caveolae (4) and in the
endosomal-lysosomal compartment. Each type of SMase hydrolyzes the
phosphodiester bond of sphingomyelin to yield the neutral lipid second
messenger ceramide and phosphorylcholine. Studies on TNF receptor
signaling suggested a model that clarifies how distinct SMases might
function through ceramide in diverse pathways (3, 5, 6). Using mutants
of the cytoplasmic domain of TNF-R55, we have shown previously that
specific receptor domains link to different sphingomyelinases. The
activation of N-SMase is signaled by a cytoplasmic portion of TNF-R55
containing a small motif of 9 amino acid residues at position 310-318
that is both necessary and sufficient for activation of N-SMase (5, 6). This region was termed NSD for neutral
sphingomyelinase activation domain.
The domain of TNF-R55 activating the A-SMase pathway strikingly
corresponds to the death domain signaling the cytotoxic effects of TNF
(1, 7). The molecular mode of action of the death domain has been
extensively investigated. The death domain of TNF-R55 binds an adaptor
protein, TRADD, that in turn recruits at least three further proteins,
TRAF2, FADD, and RIP (for review, see Ref. 8). The emerging picture
based on studies of many investigators indicates that TRAF2 mediates
the activation of the c-Jun N-terminal kinase (JNK) (9), RIP is
essentially involved in the NF-
B activation pathway (10), and FADD
signals apoptosis through activation of caspase 8 (FLICE/MACH)
(11-14). We have obtained evidence recently that neither TRAF2 nor RIP
affected A-SMase activation (15). In contrast, overexpression of TRADD
and FADD in 293 cells enhanced TNF-induced A-SMase activation without
changing the basal level of A-SMase enzymatic activity. Strikingly,
overexpression of the FADD-associated caspase 8 does not lead to
enhanced A-SMase activation after TNF treatment, indicating that the
apoptotic cascades initiated by caspase 8 segregate from the A-SMase
activation pathway. Enzyme kinetic analysis revealed that the observed
enhancement of A-SMase activation by TRADD and FADD is due to an
increased maximal velocity of substrate hydrolysis rather than a higher affinity for the substrate sphingomyelin.
Here we further delineate the role of FADD for TNF-induced activation
of A-SMase. Using embryonic fibroblasts from FADD-deficient mice, we
provide evidence for a stringent requirement of FADD for TNF-induced
activation of A-SMase.
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MATERIALS AND METHODS |
Cell Culture and Expression Vectors--
Primary embryonic
fibroblasts (EF) were derived from embryos at day 9.5 of gestation as
described previously (14). EF cells were cultured in high glucose
Dulbecco's modified Eagle's medium (ICN) supplemented with 10% fetal
calf serum, 10 mM glutamine, and 50 µg/ml each of
streptomycin and penicillin in a humidified incubator at 5%
CO2. Highly purified recombinant human TNF (3 × 107 units/mg) was kindly provided by Dr. G. Adolf, Bender,
Vienna, Austria.
Mammalian cell expression vectors encoding FADD cDNA were kindly
provided by Dr. V. Dixit, Genentech, San Francisco. The expression vector pRK-FADD was generated by inserting a
SalI-HindIII fragment of FADD cDNA into the
pRK5 vector. For transient expression experiments, 1.5 × 106 EF cells were seeded on 100-mm dishes (Falcon, 3803).
Cells were transfected the following day by the calcium phosphate
precipitation method (16). After 9 h of incubation, cells were
harvested, and enzymatic SMase assays were performed.
Assays for Neutral and Acid SMases--
The micellar SMase assay
using exogenous radiolabeled sphingomyelin was performed as described
previously (3). Briefly, cells were treated in triplicate in 0.5 ml of
medium with 100 ng/ml human recombinant TNF for the indicated periods
of time. To measure A-SMase, cells were homogenized in 0.2% Triton
X-100 lysis buffer. Radioactive phosphorylcholine produced from
[N-methyl-14C]sphingomyelin (labeled in the
choline moiety, 47 mCi/mmol, NEN Life Science Products) was determined
in the aqueous phase by liquid scintillation counting.
NF-
B-Electrophoretic Mobility Shift Analysis--
EF cells
were transfected with 1.5 µg of expression constructs or vector
control. Cells were harvested the next day and left untreated or
stimulated with 10 ng/ml TNF for 20 min at 37 °C. Cells were washed
two times with ice-cold phosphate-buffered saline, and nuclear extracts
were prepared and analyzed by electrophoretic mobility shift assays as
described (17).
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RESULTS AND DISCUSSION |
Unresponsiveness of A-SMase in FADD
/
EF
Cells--
Primary EF cells obtained from FADD
/
,
FADD+/
, or FADD+/+ mice were stimulated with
TNF and assayed for A-SMase activity. As shown in Fig.
1, TNF did not enhance A-SMase activity
in FADD
/
EF cells within the time frame investigated.
These results suggest that FADD is indispensable for TNF-induced
A-SMase activation. In contrast, the A-SMase activation profile of
FADD+/
EF cells was unaltered when compared with
wild-type EF cells. Because the amplitude and kinetics of A-SMase
activation were not diminished in FADD+/
cells, we
conclude that FADD is not a rate-limiting factor for A-SMase activation
in wild-type fibroblasts.

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Fig. 1.
Lack of A-SMase responsiveness in
FADD / embryonic fibroblasts. EF cells were
stimulated with 100 ng/ml TNF. At indicated periods of time cellular
lysates were prepared and assayed for A-SMase activity. A-SMase
activity is expressed in percentage of control. The results are
representative of four independent experiments.
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TNF treatment of FADD-deficient EF cells resulted in activation of
N-SMase (Fig. 2A) and NF-
B
(Fig. 2B), which was indistinguishable from TNF-stimulated
wild-type EF cells. These findings indicate that FADD-independent TNF
signaling pathways are intact. Furthermore, like TNF, IL-1 readily
induced NF-
B in FADD
/
EF cells, which provides
further evidence that the status of FADD deficiency does not establish
a general unresponsiveness of EF cells.

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Fig. 2.
FADD-independent signaling in
FADD / EF cells. A, EF cells were
stimulated with 100 ng/ml TNF. At indicated times cellular lysates were
prepared and assayed for N-SMase activity. N-SMase activity is
expressed in percentage of control. B, EF cells were
stimulated for 20 min with 10 ng/ml TNF or 150 pg/ml IL-1. Nuclear
extracts were prepared, and NF- B was assessed by electrophoretic
mobility shift analysis.
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Specificity of FADD Action--
Unlike TNF, IL-1-induced
activation of A-SMase in FADD
/
EF cells, which was
similar in terms of amplitude and kinetics to that observed with
heterozygous FADD+/
or wild-type EF cells (Fig.
3). Thus, IL-1 stimulates activation of
A-SMase in a FADD-independent manner. Information about the molecular
mechanisms of IL-1-induced activation of A-SMase is sparse. Recently,
we reported that an accessory chain of the IL-1 receptor, IL-1RAcP, is
required for both IL-1 internalization and activation of A-SMase (18).
The functional domain of IL-1RAcP responsible for A-SMase activation
has not yet been mapped. Neither has a putative IL-1RAcP-associated
protein been identified that mediated A-SMase activation. At any rate,
FADD, in general, does not seem to play a role in IL-1 receptor
signaling. This is consistent with the unaltered A-SMase activation
observed in IL-1-treated FADD-deficient EF cells.

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Fig. 3.
IL-1-induced activation of A-SMase in
FADD / EF cells. EF cells were stimulated with 150 pg/ml IL-1. At indicated times, cellular lysates were prepared and
assayed for A-SMase activity.
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Restoration of A-SMase Responsiveness in FADD-deficient
Cells--
In order to provide further pertinent evidence for the
essentiality of FADD for the TNF-induced A-SMase activation pathway, FADD
/
EF cells were transiently transfected with a FADD
cDNA expression vector. As shown, Fig.
4A, FADD-transfected cells
responded to TNF with approximately half-maximal A-SMase activation
when compared with FADD-deficient cells stimulated with IL-1 for
control. This partial A-SMase response corresponded well with the
transfection efficiency, which ranged from 35 to 45% as judged from
parallel transfection with a GFP expression plasmid (not shown). In
TNF-stimulated FADD-deficient EF cells, transfected with an empty
vector for control, the changes of A-SMase activity were not
significant as inferred from the respective S.E. In addition, results
obtained from different independent experiments did not reveal a
significant increment of A-SMase activity (Fig. 4B and data
not shown). When different amounts of FADD expression vector were
transfected into FADD
/
EF cells, a
dose-dependent increase of A-SMase activation was observed
(Fig. 4B). Thus, the A-SMase responsiveness to TNF in FADD-deficient EF cells can be reconstituted by transfection with FADD
expression plasmids. Together, the results of this study indicate that
FADD is an essential member of the TNF-induced A-SMase activation
pathway.

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Fig. 4.
Overexpression of FADD restores TNF
responsiveness of A-SMase in FADD / EF cells.
A, FADD / EF cells were left untreated or
transfected with either control vector or 3 µg of expression
construct for FADD. Cells were either left untreated (white
bars) or stimulated with 100 ng/ml TNF or 150 pg/ml IL-1,
respectively, for 3 min (solid bars) or 4 min (hatched
bars). Cellular lysates were prepared and assayed for A-SMase
activity. B, FADD / EF cells were left
untreated or transfected with the indicated amounts of FADD expression
construct. EF cells were stimulated with 100 ng/ml TNF for 3 min and
analyzed for A-SMase activity.
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The question then arises how FADD signals A-SMase activation. More
specifically, how does the FADD-derived signal cross the membrane
bilayer to target the endosomal A-SMase. Caspase 8 does not appear to
mediate FADD activation of A-SMase (15), and A-SMase is not a target
for caspase 8 proteolytic cleavage. Rather, recent evidence suggests
that internalization of TNF-R55 is required for A-SMase activation
(19). While the formation of TNF-R55-containing endosomes brings
together TNF-R55 and A-SMase at the same subcellular compartment, TRADD
and FADD will stay at the cytoplasmic face of the endosomal membrane,
which does not allow any direct physical interaction between A-SMase
and TRADD or FADD. Thus, TRADD and FADD may either activate a cytosolic
enzyme that generates a lipid messenger able to cross the endosomal
membrane. Alternatively, TRADD and FADD may simply serve to stabilize a
TNF-R55 multimer to allow activation of A-SMase through the
extracellular domains of TNF-R55 located in the endosomal lumen.
Further studies will be necessary to address in detail the molecular
mechanisms of FADD-mediated A-SMase activation.
It is well established that FADD can interact through a C-terminal
death domain with DD-containing proteins like CD95, TRADD, and death
receptor 3 (DR3). This interaction unmasks a N-terminal death effector
domain of FADD resulting in the recruitment and activation of caspase 8 triggering an apoptotic caspase cascade (20). Using FADD-deficient EF
cells, Yeh et al. (14) demonstrated recently that FADD is
essential for signaling TNF-R55-, CD95-, or DR3-induced apoptosis.
Remarkably, FADD was also found to be essential for mouse embryo
development in particular for the ventricular myocardium. The lethality
of FADD-deficient mice contrasts with the phenotypically normal
embryonic development and viability of CD95- and TNF-R55-deficient mice
(21, 22). This led to the conclusion that FADD, besides its signaling
function for death receptors, may also be used by other receptors
regulating embryonic development. In addition, the early lethality of
FADD-deficient embryos points to physiologic functions of FADD other
than caspase 8 activation. In the present study we identify A-SMase as
a further FADD-dependent enzyme, indicating that FADD,
indeed, may couple to signaling systems distinct from caspase 8. Although A-SMase has been implicated in numerous cellular responses
including apoptosis (for review, see Ref. 1), A-SMase can be viewed an
"orphan" enzyme, whose precise role in signaling transduction has
become elusive. Notably, A-SMase-deficient mice do not show early
embryonic lethality (23, 24). Thus the effects of FADD deficiency on embryogenesis cannot be explained by a lack of A-SMase activation. Further investigations are needed to clarify the role of FADD and
A-SMase in nonapoptotic signaling.