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INTRODUCTION |
Lymphotoxin-
receptor
(LT-
R)1 is a member of the
tumor necrosis factor receptor (TNFR) superfamily and is expressed on
the surface of most of cell types, including cells of epithelial and myeloid lineages but not on T and B lymphocytes (1, 2). LT-
R can
bind specifically to two ligands: the membrane form of lymphotoxin,
LT-
1/
2, (3, 4); and LIGHT, a recently identified member of TNF
superfamily (5, 6). LT-
R has been speculated to play an essential
role in the development of lymphoid organs. In LT-
knock-out mice,
the development of lymphoid organs is prevented (7). Studies involving
LT-
knock-out mice have shown impairment of lymph node development
and loss of splenic architecture (8). Similar results were observed
when the soluble LT-
receptor-immunoglobulin Fc chimera fusion
protein was introduced into the embryonic circulation by injecting
pregnant mice (9). Direct evidence to demonstrate the role of LT-
R
in lymphoid organ development comes from the fact that LT-
R
deficient mice lack Peyer's patches, colon-associated lymphoid
tissues, and all lymph nodes (10). Moreover, injection of the agonist
anti-LT-
R monoclonal antibody into the uteri of pregnant LT-
knock-out mice has been shown to result in the genesis of lymph nodes
in their progeny (11). In addition to its role in lymphoid organ
development, stimulation of LT-
R on certain cell lines by
LT-
1/
2 or anti-LT-
R antibodies can induce cell death (12),
chemokine secretion (13), and activation of nuclear factor
B
(NF-
B) (14). Thus, LT-
R may also have important biological
functions in the mature individuals.
The cytoplasmic domain of LT-
R, like other members of the TNF
receptor family, does not contain consensus sequences characteristic of
enzymatic activity. Therefore, signaling is thought to be mediated by
the proteins interacting with LT-
R. Two serine/threonine protein kinases, p50 and p80, have been shown to associate with LT-
R(CD) specifically (15), but the function of p50 and p80 in the LT-
R signaling pathway is still the subject of intensive study. Moreover, two members of the TNF receptor-associated factor (TRAF) family, TRAF3
and TRAF5, were found to associate with LT-
R (16, 17). Further study
has indicated that TRAF3 plays an important role in mediating
LT-
R-induced apoptosis (16, 18), whereas TRAF5 has been shown to be
involved in the activation of NF-
B (17). On the other hand, several
members of TNFR superfamily (such as TNFRI, Fas, DR3, DR4, and DR5)
contain a common motif, the death domain, in their cytoplasmic region
(19-24). These "death receptors" interact with other death
domain-containing proteins, such as TRADD (25), MORT1/FADD (26), RIP
(27), and RAIDD (28), via their death domains. MORT1/FADD and RAIDD can
in turn interact with MACH1/FLICE (caspase-8) and caspase-2,
respectively (28-30), and thus initiate the activation of caspase
cascades to execute apoptosis (31). LT-
R(CD) does not contain a
death domain, but signaling through LT-
R can also induce apoptosis
(12). It will be interesting to map the region of LT-
R responsible
for its cytotoxic effect and to determine whether LT-
R mediates
apoptosis via the activation of caspase cascades.
Both TNFRI and TNFRII can induce cell death when bound by TNF. It has
also been reported that clustering of TNF receptors due to interaction
either with trivalent TNF or with an agonist antibody is a crucial step
for subsequent intracellular signaling (32, 33). Because the
cytoplasmic domain of TNFRI can self-associate through its death
domain (34, 35), overexpression of TNFRI or of its cytoplasmic domain
alone can also induce receptor clustering resulting in the activation
of downstream signaling pathways (34, 36). In contrast, TNFRII has no
death domain and shows no tendency to self-associate, nor does a high
level of TNFRII expression result in spontaneous signaling (34, 37). In
this study, we have shown that LT-
R(CD) is capable of
self-association, despite the absence of a death domain. Moreover,
overexpression of LT-
R or LT-
R(CD) was sufficient to trigger
apoptosis without the need for ligand conjugation. The cytotoxic effect
mediated by LT-
R(CD) relies on the presence of its self-association
domain, suggesting that this domain is critical in the LT-
R
signaling pathway.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
HeLa cells were grown in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.), supplemented with 10% (v/v)
fetal bovine serum, in a 37 °C incubator containing 5% (v/v)
CO2.
Plasmid Construction--
A cDNA fragment encoding the
full-length LT-
R was amplified by reverse transcription-polymerase
chain reaction using a HepG2-derived cDNA template and the primers
5'-CGGGATCCATGCTCCTGCCTTGGGCCAC-3' (sense) and
5'-CGGGATCCTCAGTCATGGGTGATAAATTGG-3' (antisense). The DNA fragment
containing the human LT-
R cytoplasmic domain was amplified by
polymerase chain reaction using the primers
5'-GGAATTCCAAGAGCCACCCTTCTCTCTGC-3' (sense) and
5'-GGAATTCCTCAGTCATGGGTGATAAATTGG-3' (antisense). LT-
R(CD) were
subcloned into the pGEX vector as described previously (15). Deletion
mutants of LT-
R(CD) were generated by restriction enzyme digestion.
Expression of full-length LT-
R, LT-
R(CD), and its deletion
mutants in mammalian cells was achieved by subcloning the cDNAs
into the pFLAG-CMV2 vector (Eastman Kodak, Co.) in frame with the FLAG
tag at 5' end. The dominant negative mutant of TRAF3, TRAF3(367-568)
(16), was amplified by polymerase chain reaction and subcloned into the
pFLAG-CMV2 vector. For inducible expression of LT-
R(CD), the
cDNA encoding the LT-
R(CD) was subcloned into the
tetracycline-controlled expression vector, pTRE
(CLONTECH).
In Vitro Binding and Coprecipitation Assays--
For surface
biotinylation, HeLa cells were incubated in ice-cold phosphate-buffered
saline containing 0.5 mg/ml sulfo-NHS-LC-Biotin (Pierce) for 30 min at
4 °C. The cells were washed once with phosphate-buffered saline and
then incubated in Dulbecco's modified Eagle's medium for 15 min
before harvesting. 1 × 107 surface biotinylated cells
were resuspended in 1 ml of lysis buffer (20 mM Tris, pH
7.7, 0.5% (v/v) Nonidet P-40, 200 mM NaCl, 50 mM NaF, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotonin, and 0.1% (v/v) 2-mercaptoethanol) for 1 h at
4 °C, followed by centrifugation at 9000 × g for 10 min. The supernatant was precleared with 15 µg of GST bound to
glutathione-agarose beads for 2 h at 4 °C. The precleared
supernatant was mixed with 10 µg of GST-LT-
R(CD) attached to
glutathione-agarose beads for 2 h at 4 °C. Finally, the beads
were washed six times with lysis buffer. The sample was fractionated by
SDS-polyacrylamide gel electrophoresis (7.5% (w/v) acrylamide),
followed by Western blotting. The blot was probed with
peroxidase-conjugated avidin and then processed using ECL detection
reagents (Amersham International, Inc.).
Yeast Two-hybrid Interaction Analysis--
A cDNA encoding
the cytoplasmic domain of LT-
R was subcloned into the yeast vectors
pAS2-1 (GAL4 DNA-binding domain construct) and pACT2 (GAL4 activation
domain construct) (CLONTECH), respectively. For
protein-protein interaction assays, plasmids were used to transform
Saccharomyces cerevisiae strain Y190. Positive clones were
selected by prototrophy for histidine and tested by filter assays for
-galactosidase activity as described by the vender (CLONTECH).
Apoptosis Assay--
To identify the cells transfected with
LT-
R(CD), DNA constructs were cotransfected with a construct
designed to express
-galactosidase. Cells were then tested for
-galactosidase activity as described by Yang et al. (38).
Briefly, HeLa cells were plated in 6-well (35 mm) plates at a density
of 2 × 105 cells/well and left overnight. The next
day, the cells were cotransfected with the LT-
R construct and
pCMV-LacZ (at a ratio of 10:1), using LipofectAMINETM (Life
Technologies, Inc.). After 24 h of incubation, cells were fixed in
1% glutaraldehyde in PBS at 4 °C for 5 min. Transfected cells
became blue after incubation with 1 mg/ml X-gal/5 mM
potassium ferricyanide/5 mM ferrocyanide/2 mM
MgCl2/0.02% Nonidet P-40/0.01% SDS in PBS at 37 °C for
4 h. The morphology of transfectants was observed using a phase
contrast microscope (Nikon), and the percentage of apoptotic cells was
calculated as the number of blue cells with apoptotic morphology
divided by the total number of blue cells. To inhibit the cytotoxic
effect, DEVD-FMK (Calbiochem, Inc.) or Z-VAD-FMK (KAMIYA Biomedical,
Co) was added to culture medium after transfection. At least 1000 blue
cells were counted for each sample.
Expression of LT-
R(CD) Using a Tet-off System--
HeLa cells
stably expressing the tetracycline-controlled transactivator
(CLONTECH) were transfected with pTRE-LT-
R(CD)
and pTK-Hyg (CLONTECH) simultaneously. Clones were
obtained from cells that were resistant to hygromycin (200 µg/ml).
Cells were grown in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.), supplemented with 10% (v/v) fetal bovine serum,
100 units/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml
neomycin, 100 µg/ml hygromycin, and 2 µg/ml tetracycline. To induce
the expression of LT-
R(CD), transfectants were incubated in the
above medium without tetracycline.
MTT Test--
The survival rate of cells was determined by an
MTT test. Briefly, cells were seeded in 96-well flat bottom plates at a
density of 3 × 103 cells/0.1 ml. After the indicated
time period, 10 µl of 5 mg/ml MTT/well was added, and the cells were
incubated at 37 °C for 4 h. The cells were then lysed by the
addition of 100 µl of 10% SDS in 10 mM HCl/well and
incubation at 37 °C for 24 h. The optical density of each
sample was determined by measuring the absorbance at 570 nm
versus 650 nm using an enzyme-linked immunosorbent assay reader (TECAN, RainBow).
Immunofluorescence Microscopy--
Cells were fixed with 1%
paraformaldehyde in PBS at room temperature for 20 min and then
permeabilized with acetone at
20 °C for 3 min. Cells were then
incubated with anti-FLAG monoclonal antibody (5 µg/ml) at room
temperature for 1 h, followed by incubation with fluorescein
isothiocyanate-conjugated goat anti-mouse IgG at room temperature for
1 h after washing with PBS three times. Cells were then examined
with a MRC600 scanning confocal microscope (Bio-Rad). All the
antibodies were diluted in 1% bovine serum albumin/PBS.
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RESULTS |
Mapping of the LT-
R Self-association Domain--
In a yeast
two-hybrid screen using LT-
R(CD) as a bait, Chen et al.
(39) found that most of the positive clones isolated from a human liver
cDNA library corresponded to LT-
R. This was indicative of a
tendency to self-associate. To confirm the ability of LT-
R to
self-associate, we tested whether a GST-LT-
R(CD) fusion protein is
capable of interacting with endogenous LT-
R from HeLa cells. HeLa
cells were surface biotinylated as described under "Experimental
Procedures." A GST-pull down assay was carried out, and precipitated
proteins were detected with avidin. We found that a 70-kDa protein was
coprecipitated with GST-LT-
R(CD) (Fig. 1, lane 3) but not with GST
under the same conditions (Fig. 1, lane 2). Furthermore, the
70-kDa protein associated with GST-LT-
R(CD) could be precleared from
HeLa cell extracts by anti-LT-
R antibodies (Fig. 1, lane
4). We are confident that the 70-kDa protein is endogenous LT-
R
for following reasons: (i) the 70-kDa protein is a surface protein
because it could be surface biotinylated and (ii) the molecular mass
(70 kDa) of the LT-
R(CD)-associated protein (Fig. 1, lane
3) is the same as that of endogenous LT-
R immunoprecipitated by anti-LT-
R antibodies (Fig. 1, lane
5). GST-LT-
R(CD) interacted less strongly with endogenous
LT-
R than did anti-LT-
R antibodies. This is probably due to the
lower affinity of the protein-protein association (Fig. 1,
lane 3) than that of the antigen-antibody
interaction (Fig. 1, lane 5). Based on the
observations above, we concluded that LT-
R(CD) can associate with
endogenous LT-
R and further confirmed the tendency of LT-
R to
self-associate.

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Fig. 1.
Interaction of
LT- R(CD) with endogenous
LT- R in vitro. 1 × 107 HeLa cells were surface biotinylated and then
precleared with pre-immune serum (lanes 2, 3, and
5) or anti-LT- R antibodies (lane 4). The
precleared HeLa cell extracts were then incubated with GST-adsorbed
(lane 2) or GST-LT- R(CD)-adsorbed (lanes 3 and
4) glutathione-agarose beads or with anti-LT- R antibodies
bound to protein A beads (lane 5). All these samples and
HeLa cell extracts (from 2 × 104 cells) (lane
1) were fractionated on 7.5% (w/v) SDS-polyacrylamide gels, and
Western blot analysis was carried out using peroxidase-conjugated
avidin to detect biotinylated proteins. Molecular mass standards (in
kDa) are marked on the left, and the position of full-length
LT- R is indicated by an arrow.
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To identify the self-association domain, LT-
R(CD) and its deletion
mutants were subcloned into the vectors pAS2-1 (containing the GAL4
DNA binding domain) and pACT2 (containing the GAL4 activation domain),
and the resultant constructs were used to carry out yeast two-hybrid tests. As shown in Table I,
LT-
R(CD), LT-
R(CD)(aa234-377), and LT-
R(CD)(aa324-377)
clones exhibited
-galactosidase activity in filter assays when
cotransformed with LT-
R(CD), indicating that self-association had
occurred. In contrast, the deletion mutants LT-
R(CD)(aa234-324) and
LT-
R(CD)(
324-377) did not give rise to
-galactosidase
activity, suggesting that the absence of amino acids 324-377 resulted
in a loss of self-association. These results show that amino acids
324-377 of LT-
R(CD) are necessary and sufficient for LT-
R
self-association.
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Table I
Mapping of LT- R self-association domain by yeast two-hybrid assay
The cDNAs encoding LT- R(CD) and its deletion mutants were
subcloned into the yeast pAS2-1 (GAL4 DNA-binding domain construct)
and pACT2 (GAL4 activation domain construct) vectors as shown. The
yeast strain Y190 was transformed with various combinations of these
expression constructs. Clones phototrophic for histidine were subjected
to filter assays. Blue color indicated the presence of
-galactosidase activity.
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Expression of LT-
R(CD) Triggers Cell Death--
Recombinant
LT-
1/
2 or anti-LT-
R antibody has been shown to trigger
apoptosis in certain cell lines, including the human cervical carcinoma
cell line HeLa (12, 39). Here, we asked whether the overexpression of
LT-
R or LT-
R(CD) has a similar effect. Full-length LT-
R and
LT-
R(CD) were subcloned into FLAG-tagged expression vectors and
cotransfected with pCMV-LacZ (which expresses
-galactosidase) into
HeLa cells. Of the transfected cells that expressed
-galactosidase
activity, only those expressing either full-length LT-
R (Fig.
2B) or LT-
R(CD) (Fig.
2C) became rounded and condensed, which are the typical
morphological alterations of adherent cells undergoing apoptosis. In
addition, nuclear condensation was observed when the cells were stained
with Hoechest 33342 (data not shown). In contrast, cells transfected
with the pFLAG vector alone did not undergo apoptosis (Fig.
2A). The proportions of cells expressing LT-
R or
LT-
R(CD) that underwent apoptosis were about 76 and 74%,
respectively, whereas this occurred in only ~8% of control cells
(Fig. 2D). From this observation, it is clear that
overexpression of LT-
R or LT-
R(CD) can result in cell death, without the need for ligand binding or receptor cross-linking by an
agonist antibody. Similar results were obtained in the human adenocarcinoma cell line HT29 overexpressing LT-
R or LT-
R(CD) (data not shown).

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Fig. 2.
Ligand-independent apoptosis induced by
LT- R or
LT- R(CD). A-C, FLAG vector,
FLAG-LT- R, and FLAG-LT- R(CD) plasmids were transiently
cotransfected into HeLa cells with -galactosidase cDNA. Cells
were stained with X-gal 24 h after transfection, followed by
examination under a phase contrast microscope. D, the
percentage of apoptotic cells was calculated as the number of blue
cells with apoptotic morphology divided by the total number of blue
cells. At least 1000 blue cells were counted for each sample. The data
shown here are the averages ± S.D. of triplicate
experiments.
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To further confirm this observation, we used the Tet-off-inducible
system to allow the expression of LT-
R(CD) to be turned on when
tetracycline was removed from culture medium. In Tet-off HeLa cells
stably transfected with pTRE-LT-
R(CD), LT-
R(CD) protein became
detectable at day 3 after induction (data not shown). Significant cytotoxicity was observed when tetracycline was removed (Fig. 3A, right panel),
whereas cells did not undergo apoptosis in the presence of tetracycline
(Fig. 3A, left panel). By staining the surviving
cells with MTT, we found that cell death became apparent at day 4 after
removal of tetracycline (Fig. 3B). Based on the observations
above, we concluded that expression of LT-
R(CD) alone is sufficient
to induce apoptosis.

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Fig. 3.
Viability of Tet-off HeLa cells transfected
with LT- R(CD). The
tetracycline-controlled construct encoding the LT- R(CD) was stably
transfected into Tet-off HeLa cells that express the
tetracycline-controlled transactivator constitutively
(CLONTECH). A, cells were examined under
a phase contrast microscope in the presence and absence of
tetracycline. Photographs were taken at day 6 after tetracycline was
removed. B, the viability of cells was determined by MTT
tests at the times indicated. The percentage of survival cells in the
absence of tetracycline was compared with the cell population in the
presence of tetracycline at various times.
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Characterization of the Apoptotic Pathway Initiated by
LT-
R(CD)--
It has been shown that the dominant negative TRAF3
mutant, TRAF3(367-568), can inhibit cell death triggered by
LT-
1/
2 or anti-LT-
R antibodies (16). Therefore, we asked
whether the TRAF3 mutant can also affect LT-
R(CD)-induced apoptosis.
TRAF3(367-568) was cotransfected with LT-
R(CD) into HeLa cells. We
found that TRAF3(367-568) provided partial protection from the
cytotoxic effect of LT-
R(CD) (Fig.
4A), suggesting that TRAF3 is
involved in LT-
R(CD)-induced apoptosis.

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Fig. 4.
Effects of a TRAF3 dominant negative mutant
and caspase inhibitors on LT- R(CD)-induced
apoptosis. A, HeLa cells were cotransfected with
pFLAG-LT- R(CD) and pCMV-lacZ, in conjunction with TRAF3(367-568) in
a ratio of 7:1:7. The amounts of total transfected DNA were equalized
with control vector. Apoptosis assays were performed as described in
the legend to Fig. 2. B, HeLa cells were cotransfected with
pFLAG-LT- R(CD) and pCMV-lacZ at a ratio of 7:1 or with
pFLAG-LT- R(CD), pCMV-lacZ, and CrmA at a ratio of 7:1:5. The cells
cotransfected with pFLAG-LT- R(CD) and pCMV-lacZ were incubated
either with the caspase peptide inhibitors Z-VAD-FMK (20 µM) and
DEVD-FMK (150 µM) or with dimethyl sulfoxide
(DMSO) (0.1%) as a control.
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Signaling through TNFRI and Fas can initiate caspase cascades to
execute apoptosis (31). To examine whether LT-
R(CD) can also
activate caspases, the effects of Z-VAD-FMK (a broad spectrum caspase
inhibitor), DEVD-FMK (a CPP32 inhibitor), and the CrmA of cowpox virus
on LT-
R(CD)-induced apoptosis were tested. As shown in Fig.
4B, the cytotoxicity induced by LT-
R(CD) was blocked in
cells coexpressing CrmA. In addition, LT-
R(CD)-induced apoptosis was
almost completely inhibited by Z-VAD-FMK but only partially by
DEVD-FMK. This observation suggests that caspase(s) may participate in
the apoptotic pathway activated by LT-
R(CD). The partial inhibition of DEVD-FMK might result from the lower membrane permeability of HeLa
cells for this species, because similar results were obtained when
looking at TNF-induced cytotoxicity under the same conditions (data not shown).
Mapping of the Apoptotic Domain of LT-
R--
Because there is
no death domain in the cytoplasmic region of LT-
R, we wished to
identify the sequence responsible for LT-
R(CD)-induced apoptosis. As
shown in Fig. 5, apoptosis was observed
in cells transfected with either LT-
R(CD) or the deletion mutant
LT-
R(CD)(aa234-377). In contrast, the deletion mutants
LT-
R(CD)(aa234-324) and LT-
R(CD)(
324-377) had no effect on
cell viability. These results provided direct evidence that amino acids
324-377 are essential for LT-
R(CD)-induced apoptosis. This is also
the region required for LT-
R self-association (Table I).

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Fig. 5.
Mapping of the apoptotic domain of
LT- R. LT- R(CD) and the deletion
mutants shown on the left of the figure were cloned into the
pFLAG-CMV2 vector. HeLa cells were transfected with each of the
pFLAG-LT- R(CD) mutants and were subjected to apoptosis assays
24 h after transfection.
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Localization of the LT-
R Deletion Mutants--
Loss of the
extracellular and transmembrane domains would be expected to result in
LT-
R(CD) in the cytoplasm of cells. However, LT-
R(CD) was shown
to associate with endogenous full-length LT-
R, which is a
transmembrane protein (Fig. 1). Therefore, we wished to clarify the
localization of the LT-
R(CD). HeLa cells transiently transfected
with LT-
R(CD) or its deletion mutants were processed for
immunostaining (Fig. 6). Interestingly,
we found that LT-
R(CD) and LT-
R(CD)(aa234-377) were localized in
the proximity of plasma membrane (Fig. 6, A and
B), whereas deletion mutants lacking the self-association
domain (LT-
R(CD)(aa234-324) and LT-
R(CD)(
324-377)) were
predominantly localized in the cytoplasm (Fig. 6, C and
D). The membrane juxtaposition of LT-
R(CD) was also
observed in Tet-off HeLa cells stably transfected with LT-
R(CD)
(data not shown). Considering that LT-
R(CD) and
LT-
R(CD)(aa234-377) contain the self-association domain and have
the ability to self-associate (Fig. 1 and Table I), it is conceivable
that they might be directed to the inner side of plasma membrane
through their interactions with endogenous LT-
R. This observation
suggests that the self-association domains are able to interact
in vivo.

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Fig. 6.
Localization of
LT- R(CD) and deletion mutants by confocal
immunofluorescence microscopy. HeLa cells were transfected with
each of the pFLAG-LT- R(CD) mutants shown in Fig. 5 and then
processed for immunostaining as described under "Experimental
Procedures." The anti-FLAG monoclonal antibody was used to detect
FLAG-tagged proteins, and this was recognized by fluorescein
isothiocyanate-labeled goat anti-mouse IgG. Images were obtained by
confocal microscopy. A, LT- R(CD). B,
LT- R(CD)(aa234-377). C, LT- R(CD)(aa234-324).
D, LT- R(CD)( 324-377).
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DISCUSSION |
The cytoplasmic domains of TNFRI and Fas have been shown to be
able to self-associate via their death domains. This self-association can prompt signaling events, giving rise to TNF and Fas effects (34,
35). In contrast, the cytoplasmic region of TNFRII has no death domain
and shows no tendency to self-associate, nor does overexpression of
this receptor induce cell death (34, 37). Like TNFRII, LT-
R does not
contain a death domain. However, studies using the yeast two-hybrid and
GST-pull down systems have clearly demonstrated the self-association
tendency of LT-
R(CD) (Ref. 39 and the present report). In addition,
we have observed that overexpression of LT-
R or LT-
R(CD) is
sufficient to trigger apoptosis, without the need to cross-link LT-
R
with ligand or an agonist antibody. It has been shown that a dominant
negative mutant of TRAF3, TRAF3(367-568), can inhibit apoptosis
induced by LT-
1/
2 or anti-LT-
R antibodies (16). Likewise, our
results showed the dominant negative effect of TRAF3(367-568) on
LT-
R(CD)-induced apoptosis. This result suggests that apoptotic
signaling mediated by LT-
R(CD) may share the same pathway as that
triggered by ligand conjugation. Furthermore, activation of NF-
B was
also observed in cells overexpressing
LT-
R(CD).2 This is in
accordance with previous reports that stimulation of LT-
R by
recombinant LT-
1/
2 or anti-LT-
R antibodies can transduce
signals not only for apoptosis (12) but also for NF-
B activation
(14). Based on the observations above, overexpression of LT-
R(CD)
seems to be able to activate downstream signaling events equivalent to
those induced by LT-
1/
2 or anti-LT-
R
antibodies.
It has been shown that apoptotic signaling mediated by TNFRI or Fas
occurs via interaction with other death domain-containing proteins,
which can initiate caspase cascades to execute apoptosis (31). Here, we
demonstrated that LT-
R(CD)-induced apoptosis was inhibited by
several caspase inhibitors: CrmA, Z-VAD-FMK, and DEVD-FMK, suggesting
that the activation of CPP32 or ICE-related caspases might be involved
in the apoptotic signaling of LT-
R. Because LT-
R does not contain
a death domain, it will be intriguing to discover how LT-
R initiates
the caspase pathway. This question might be answered by examining
whether TRAF3 or other LT-
R(CD)-associated proteins could recruit
and activate caspase(s).
LT-
1/
2 or anti-LT-
R antibodies cannot trigger apoptosis
without the presence of IFN-
(12). However, LT-
R(CD)-induced apoptosis does not require the presence of IFN-
, nor did IFN-
result in a synergistic enhancement of LT-
R(CD) cytotoxicity (data
not shown). The mechanism of IFN-
action in apoptosis induced by
LT-
R is still unclear. Recently, IFN-
was found to modulate cell
death by inducing several apoptosis-related genes, including the TNFR
family members, TNFRI and Fas; a bcl-2 family member, bak; and the
caspase family members, ICE, CPP32, and FLICE (40). Nevertheless,
IFN-
does not up-regulate LT-
R expression on HT-29 (12) and HeLa
(data not shown) cell lines. It is possible that IFN-
might modulate
LT-
R-induced cell death by regulating caspases involved in the
cytotoxic effect. However, the amounts of LT-
R(CD) expressed by
transfected cells might be sufficient to initiate caspase cascades to
execute apoptosis, such that IFN-
does not further enhance the
cytotoxic effect. This speculation is supported by the fact that
LT-
R(CD)-induced apoptosis can be inhibited by several caspase
inhibitors, suggesting that the activation of caspases is important in
this apoptotic pathway.
Because there are no consensus sequences for membrane anchorage in the
FLAG tag or LT-
R(CD), the FLAG-LT-
R(CD) fusion protein would be
expected to express as a cytoplasmic protein. However, the
FLAG-LT-
R(CD) was shown to be localized in the proximity of plasma
membrane. Interestingly, we also observed that membrane juxtaposition
of LT-
R(CD) and its deletion mutants correlates with their ability
to self-associate and to induce apoptosis. Because the self-association
domain is required for targeting of LT-
R(CD) to the plasma membrane
(Fig. 6), we speculate that membrane juxtaposition of LT-
R(CD) might
be due to its association with endogenous LT-
R (Fig. 1), which may
therefore induce receptor clustering to activate apoptotic signaling.
Nevertheless, our result does not rule out the possibility that
LT-
R(CD) can be linked to the membrane by other proteins, either
located on the inner side of cell membrane or associated with
endogenous LT-
R.
Previous studies have shown that the death domain of TNFRI has a strong
tendency to self-associate and interact with other death
domain-containing proteins and plays an obligatory role in signaling
cell death. In the present report, self-association of LT-
R(CD) was
mapped to amino acids 324-377, a region that is also required for
apoptotic signaling. Interestingly, the self-association domain (amino
acids 324-377) is the same region as that required for the
interactions between LT-
R and its associated kinases, p50 and p80
(15). Moreover, the self-association domain of LT-
R almost overlaps
with a region of LT-
R, which has been shown to interact with the
core antigen of hepatitis C virus (39, 41). Nevertheless, the
self-association domain of LT-
R does not show significant sequence
similarity with the death domains of TNFRI and Fas; therefore, the
self-association domain of LT-
R represents a novel motif that is
essential for receptor self-association, as well as interaction with
both cytoplasmic and viral proteins, and that might regulate the
receptor signaling for cell death. The identification of proteins
capable of interacting with amino acids 324-377 of LT-
R will be
crucial to understand LT-
R signaling.