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Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Ghent, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation nor
granulocyte/macrophage-CSF secretion. Possibly, additional factors
different from TRAF2 are involved in TNF-mediated NF-B activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation (4, 5, 6, 10). Although the intracellular domains of
TNF-R55 and TNF-R75 are completely unrelated, they share certain
activities, such as induction of apoptosis and NF-B activation
(1, 11, 12). The role of TNF-R75 in cell death has long been controversial, and its activity was proposed to be independent of signal transduction (13, 14, 15, 16, 17, 18). The ligand-passing model reconciled many seemingly conflicting data and showed that, due to its higher affinity and rapid dissociation kinetics, TNF-R75 could regulate the rate of TNF association with TNF-R55 (17). However, in the past few years it has become clear that TNF-R75-specific triggering has a signaling role for apoptosis in PC60, KYM-1, HeLa, Colo205, and mature T cells (11, 15, 19, 20, 21, 22).
In recent years, the signaling events leading to TNF-R-mediated cell
death have been partially elucidated (1, 23, 24). Mainly by the use of
two-hybrid-based strategies, several intracellular TNF-R55-, TNF-R75-,
and Fas/Apo-1-signaling complex-constituting proteins were identified.
These molecules belong to four novel families of signal-transducing
effectors. First, the TNF-R-associated factors 1 and 2 (TRAF1 and
TRAF2) were cloned on the basis of their associating capacity with
TNF-R75 (25). TRAFs associate by means of a conserved C-terminal TRAF
domain with members of the TNF-R superfamily. A 78-residue C-terminal
region of TNF-R75, the TRAF-binding region, interacts with these signal
transducers. TRAF2 mediates NF-B activation by TNF-R55, TNF-R75, and
CD40 (26, 27). In general, TRAF family members seem to be involved in
cytokine-induced gene activation (26, 27, 28, 29, 30). Second, the TNF-R55- and
Fas-associated DD proteins TRADD, FADD/MORT1, and RIP, together with
the TNF-R family members TNF-R55, Fas, and the DR3–5, constitute a
family of molecules containing a DD motif, which is involved in
homotypic and heterotypic protein/protein interactions (7, 8, 9, 10, 31, 32, 33, 34).
Ligand-induced receptor aggregation targets these DD proteins to their
respective receptors, i.e., TRADD/FADD/RIP to TNF-R55 (although FADD
recruitment to the endogenous TNF-R55 has not been shown yet) and FADD
to Fas/Apo-1 (27, 35, 36, 37). Overexpression of these DD molecules leads
to cell death, probably by mimicking the formation of an intracellular
ligand-induced signaling complex. A FADD molecule lacking its
N-terminal region functions as a dominant negative (DN) mutant able to
inhibit TNF-R55-, Fas/Apo-1-, and DR3-induced apoptosis (5, 27, 37).
TRADD and RIP, besides their presumed role in cell death, are also
involved in NF-B activation by their ability to recruit TRAF2 (27, 35, 38). A third family of TNF-R complex-constituting proteins involves
the baculoviral IAP-related inhibitors of apoptosis c-IAP1 and c-IAP2
(39, 40). Although it is known that c-IAP1 and -2 participate in the
TNF-R75 complex and that c-IAP1 can be recruited to TNF-R55, in both
cases via their affinity for TRAF1 and/or TRAF2, their function in
signal transduction is still unclear. A fourth class of proteins links
the receptor-associating death-inducing FADD molecule to ICE/CED-3-like
cysteine proteases (caspases) involved in the process of apoptosis. The
caspase-8 molecule binds with its N-terminal part, by means of a
homologous death effector domain, to the N-terminally located death
effector domain of FADD (41, 42). Expression of caspase-8 mutants
lacking enzymatic activity block both TNF-R55- and Fas/Apo-1-mediated
cytotoxicity (41). The mechanism by which procaspase-8 becomes
activated after recruitment in the death-inducing signaling complex is
still unclear (43); but once activated, caspase-8 is able to activate
downstream caspases (44).
These findings indicate that members of the TNF-R family share various intracellular signaling proteins to activate similar transduction pathways. This sheds new light on the possible mechanism of TNF-R55 and TNF-R75 cooperation. Several recent reports have strongly suggested cooperation between TNF-R55 and TNF-R75, especially in the case of induction of cytotoxicity (11, 12, 21, 45, 46).
We developed a thymoma system, based on the introduction of human TNF-R in the rat/mouse T cell hybridoma PC60, to investigate the role of TNF-R55 and TNF-R75 in TNF responses. Previously, we have shown that both receptors are signal-transducing in these cells (11, 12, 47). Remarkably, only PC60 cells expressing both of the receptor types were sensitive to TNF-induced apoptosis. Furthermore, we reported that the intracellular domain of TNF-R75 is required to mediate this receptor cooperation, thereby excluding a role for ligand passing (48). In the present report, we show the existence of a bidirectional TNF-R55 and TNF-R75 cooperation at the level of apoptosis induction and a unidirectional ligand-independent cooperation of TNF-R55 in TNF-R75-induced granulocyte/macrophage-CSF (GM-CSF) secretion. Our studies with TNF-R75 mutants suggest a role for the TNF-R75-associated TRAF1/TRAF2 complex in the observed TNF-R55 cooperation in apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purified E. coli-derived human TNF, the human TNF-R55-specific mutein R32WS86T, and the human TNF-R75-specific mutein D143F were prepared in our laboratory as described (11, 48). GM-CSF was quantified, using the murine GM-CSF preparation of the National Institute for Biological Standards (Potters Bar, U.K.) as a reference, in an FDCp1 proliferation assay (49).
Cells
The hybridoma PC60.21.14.4, provided by Dr. M. Nabholz (Swiss Institute of Experimental Cancer Research, Epalinges, Switzerland), was transfected with cDNAs coding for TNF-R55, TNF-R75, or both, as previously reported (11). The representative cell clones used for each type of transfectant were PC60 R55/8, PC60 R75/24, and PC60 R55R75/5, respectively.
Construction of mutant TNF-R75 and electroporation of PC60 cells
The starting plasmid for TNF-R75 mutagenesis, containing
wild-type (wt)-TNF-R75 under control of the SV40 early promoter, was
previously described (11). TNF-R75 M1 was generated by digestion of the
unique BstXI restriction site, removal of the protruding 3'
sticky end with T4 polymerase, and ligation to the XbaI
linker CCTCTAGAGG, generating an in-frame stop codon. Due to the
cloning procedure, an additional stretch of five irrelevant amino acids
was added. The M3 mutant (
304–345) resulted from the removal of the
sequence between two SacI restriction sites present in the
cytoplasmic domain. R75 M4 (288–340) was constructed by deleting an
XmaI fragment in the intracellular receptor part. M5
(403-end) was generated by introducing an in-frame stop codon at the
indicated position by PCR, thereby deleting the sequence coding for the
37 C-terminal amino acids. Mutant receptors were verified using dsDNA
sequencing. PC60 R55/8, PC60 R75/24, or PC60 R55R75/5 cells were
transfected by electroporation as reported (48). Briefly, cells were
washed with growth medium (RPMI 1640) supplemented with FCS. Wild-type
and mutant TNF-R expression plasmids (20 µg), together with 1 µg of
a selection plasmid pSV2Neo (G418 selection), or a murine TRAF2-DN
expression plasmid pRK5TRAF2-DN (20 µg), a generous gift of Dr.
D. V. Goeddel (25), together with the selection plasmid pUTSV1
(Cayla, Toulouse, France; phleomycin selection) were added to the
cells, and the mixture was exposed to a single electropulse (1500 µF,
300 V). Two days later, G418 (1500 µg; Life Technologies, Paisley,
U.K.) or phleomycin (200 µg; Cayla) was added, and cells were cloned
by limiting dilution. Cell lines derived from transfected PC60 R55/8 or
PC60 R75/24 were named PC60 R55NR75 and PC60 R75NR55, respectively.
Immunofluorescence and propidium iodide (PI) exclusion assays
TNF-R75 expression on transfected cells was analyzed by staining for 45 min with 1 µg utr-1/ml, a mAb against TNF-R75 (50), and subsequent incubation with a fluorescein-conjugated goat anti-mouse Ab, followed by fluorocytometric detection with a Coulter Epics 753 (Coulter, Hialeah, FL). The number of apoptotic cells in a given culture was measured by a PI exclusion assay as described (11). PI was added at a final concentration of 30 µM, and cells were analyzed on an Epics 753 fluorometer.
Immunoprecipitation and immunoblotting
PC60 cells (107) expressing different TNF-R combinations were washed twice with ice-cold PBS A and lysed (15 min) in 1 ml lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 100 µg/ml PMSF, 0.27 trypsin-inhibitory U/ml aprotinin, 10 µg/ml leupeptin, and 2 mM pefabloc) (27). After centrifugation at 14,000 x g (10 min), 20 µg of the TNF-R75-specific Ab utr-4 (50) was added to the supernatant and left for 1 h at 4°C, then mixed with 40 µl of a 1:1 slurry of protein A-Sepharose for another 1.5 h. Beads were washed twice with lysis buffer, twice with high salt (1 M) lysis buffer, and again twice with low salt lysis buffer. Proteins were eluted with Laemmli gel-loading buffer containing 4.2% 2-ME, loaded on a 10% SDS-polyacrylamide gel, and after electrophoresis electroblotted on a nitrocellulose membrane. TNF-R75 proteins were revealed using a rabbit polyclonal anti-TNF-R75 antiserum (a gift of Dr. W. A. Buurman, University of Maortricht) and enhanced chemiluminescence (ECL; Amersham Life Science, Amersham, U.K.). For coimmunoprecipitations, the parental PC60 R55R75/5 and PC60 R55R75T2DN cells (108) were washed with warm growth medium (37°C) and incubated for different time intervals in the presence or absence of TNF (1 µg/ml) in a 10-ml volume on a rotating wheel, then lysed with 4 ml lysis buffer (150 mM NaCl) further supplemented with 30 mM NaF and 2 mM Na-pyrophosphate. The lysate was further incubated with a combination of utr-4 Ab (50) and protein A-Trisacryl beads or with protein A-Trisacryl beads alone and further treated as above (27). Immunoblots were developed with anti-murine TRAF2 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-TNF-R75 antisera using ECL.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we have shown that PC60 cells transfected with both
human TNF-R were sensitive to TNF-mediated apoptosis, in contrast to
cells expressing only one TNF-R type. Both TNF-R55 and TNF-R75 were
able to elicit intermediate apoptosis levels in the double
transfectants when separately triggered by agonistic Abs or
receptor-specific TNF muteins (11). The percentage of PI-positive cells
was taken as a measure for apoptotic death in these cultures. To
further confirm these results, we transfected TNF-R55+
and TNF-R75+ single transfectant PC60 cells with TNF-R75 or
TNF-R55 expression plasmids, respectively. Cell lines expressing both
TNF-R were selected by immunostaining, and subsequent cytofluorometric
analysis in the case of TNF-R75, or by treating cells with the
TNF-R55-specific TNF mutein R32WS86T and scoring the GM-CSF levels
induced in the case of TNF-R55 (data not shown). PC60 R55NR75 and PC60
R75NR55 cells were the new double transfectants so obtained. When
treated with TNF, both cell clones responded in an apoptotic way (Fig. 1
A). This response was
in contrast to cells transfected only with the selection plasmid (PC60
R55/8 Neo and PC60 R75/24 Neo, respectively).
Separate clustering of receptors by the TNF muteins R32WS86T (TNF-R55)
or D143F (TNF-R75) resulted in intermediate levels of apoptosis in
R55R75+ cells (Fig. 1B), while
simultaneous activation of TNF-R55 and TNF-R75 was as cytotoxic as
stimulation with wt-TNF. Specific stimulation of TNF-R75 in PC60 R75
cells induced consistently low, but reproducible GM-CSF levels (Ref.
47; Fig. 2). After introduction of
TNF-R55 in these cells, treatment with the TNF-R75-specific agent D143F
resulted in considerably enhanced GM-CSF production (Fig. 2). Since
single-transfected TNF-R55+ cells already produced elevated
levels of GM-CSF after specific receptor stimulation and since the
levels of induced cytokine varied between different clones, we were not
able to conclude unambiguously whether, reciprocally, the presence of
TNF-R75 facilitated the R55 GM-CSF-inducing capacity (see below and
data not shown). These results confirm and extend our previous
observations indicating cooperation by even an unliganded second
receptor in PC60 cells (11). Our data show a bidirectional cooperation
between both TNF-R in inducing apoptosis and a clear help of the
TNF-R55 presence in TNF-R75-signaling to GM-CSF secretion.
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To further dissect the mechanism of TNF-R cooperation in PC60
cells, we characterized the intracellular part of TNF-R75 responsible
for cooperation with TNF-R55. Therefore, several TNF-R75 deletion
mutants were constructed and expressed in PC60 R55+
cells. Comparison of the human and mouse TNF-R75 intracellular
sequences revealed two major boxes of homology: a conserved domain
containing a sequence of six consecutive serine residues and the
TRAF-binding region (Fig. 3A). We decided to make
deletion mutants in which the serine-rich region (mutants M3
(304–345) and M4 (288–340)) or the TRAF-binding domain (M1,
313-end) were removed (Fig. 3B). Rothe et al.
previously reported that deletion of the 37 C-terminal amino acids
resulted in an inactive receptor due to the inability to bind the
TRAF1/TRAF2 complex (25). This deletion mutant was also included in
our structure-function studies (M5, 403-end).
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C). Since the levels of transfected TNF-R55 in PC60
cells are extremely low (11), TNF-R55 presence could be evaluated only
on the basis of functionality, viz the induction of GM-CSF by
R55-specific stimulation (data not shown, see further). To demonstrate
that the transfected cell lines expressed the mutant TNF-R75 gene
products, the receptors were immunoprecipitated from the respective
cell lines using the mAb utr-4 (50), then further analyzed by
immunoblotting (Fig. 3D). Deletion of the serine box-containing domain has no influence on the functionality of TNF-R75
PC60 R55NR75 M3 (304–345) and PC60 R55NR75 M4 (288–340)
cells expressing TNF-R75 lacking the Ser box were stimulated with
TNF-R-specific muteins or TNF, and the levels of GM-CSF induced were
quantified. From Figure 4A it
was clear that all cell lines responded to TNF-R55-specific triggering
(R32WS86T), thereby showing the presence of TNF-R55 on the transfected
PC60 cells. D143F stimulation of the mutant TNF-R75, M3 and M4,
stimulated the secretion of similar amounts of GM-CSF as activation of
wt-TNF-R75 in PC60 R55R75/5 cells. Simultaneous activation of both
TNF-R55 and TNF-R75 resulted in a synergistic production of GM-CSF in
wt as well as in R75 M3- and R75 M4-coexpressing cells. This indicated
that the gene-inducing capacity of TNF-R75 was not affected by removal
of the conserved Ser box. When these cultures were analyzed for the
percentage of apoptotic cells induced by TNF-R triggering, it was clear
that R75 M3 and R75 M4 were still able to cooperate with R55 to render
PC60 cells susceptible to TNF-R-mediated cell death, as did wt-R75
(Fig. 4B). Additionally, M3 and M4 R75 mutants
induced apoptosis after specific stimulation with D143F. Parental PC60
R55/8 cells and control PC60 R55/8 Neo transfectants showed
no apoptotic cells after mutant TNF treatment. Hence, we concluded that
the TNF-R75 Ser box region was not involved in the process of receptor
cooperation, nor did deletion of this R75 region influence its
apoptosis- or gene-inducing capability.
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Complete deletion of the TNF-R75 TRAF-binding domain (M1,
313-end) resulted in loss of receptor functionality.
TNF-R75-specific triggering of PC60 R55NR75 M1 cells was not able to
induce any GM-CSF secretion neither on its own, nor in synergy with
TNF-R55 stimulation (Fig. 5A). Moreover, this
mutated receptor was no longer capable of transducing a signal required
for receptor cooperation in inducing apoptosis by TNF-R55 (Fig. 5B). Removal of the 37 C-terminal amino acids of the
intracellular TNF-R75 domain (403-end), which disturbs the TRAF
complex binding to this receptor (25), had identical effects. PC60
R55NR75 M5 cells stimulated with D143F did not produce GM-CSF and did
not show an apoptotic response even when stimulated with TNF (Fig. 5).
These results led us to postulate an involvement of the TRAF-binding
domain, and hence the TRAF1/TRAF2 complex, not only in R75-mediated
GM-CSF induction but also in the cooperation that allows TNF-R55 to
induce apoptosis.
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B activation in PC60 R55R75 cells
Since our results with the TNF-R75 M5 mutant pointed to a role for
the TRAF1/TRAF2 complex in receptor cooperation in PC60 R55R75 cells,
we transfected these cells with a TRAF2-DN molecule lacking its
RING-finger domain. This mutant TRAF2 was previously shown to be able
to inhibit TNF-R55-, TNF-R75-, and CD40-mediated NF-B activation
after transient overexpression (26, 27). The function of TRAF1 is still
unresolved. PC60 R55R75T2DN clones expressing high levels of the
TRAF2-DN protein were selected (Fig. 6A). Functional
analysis of TRAF2-DN-expressing PC60 R55R75 cells, however, revealed
that there was no influence on the induction of apoptosis by TNF-R55
and/or TNF-R75 (Fig. 6B). PC60 R55R75/5 and PC60
R55R75T2DN cells were both equally sensitive to different
concentrations of TNF (data not shown). Surprisingly, we were also
unable to show any inhibitory effect on the stimulation of GM-CSF
secretion or NF-B activation (Fig. 6C and data not
shown), although a strong involvement of NF-B in GM-CSF promoter
activity has been reported (51). This lack of effect was not because we
had for some reason been working with an inappropriate TRAF2-DN
molecule, since DNA sequence analysis revealed the correct sequence
(data not shown) and transient overexpression in HEK293 cells could
block TNF-R55-mediated activation of NF-B determined by a luciferase
reporter gene construct (data not shown), as reported previously
(27).
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B
activation by TRAF2-DN overexpression, immunoprecipitations of
TNF-R75 were conducted to examine whether TRAF2-DN indeed interacted
with this receptor in PC60 R55R75 cells. In PC60 R55R75/5 cells, we
could show a TNF-dependent recruitment of endogenous wt TRAF2 (Fig. 6D). This recruitment seemed to be an early event in
signal transduction, since TRAF2 could already be detected in the
TNF-R75 immunoprecipitates after 1 min of TNF treatment. A maximum
TRAF2 recruitment was observed after 15 min of TNF treatment. In
contrast, PC60 R55R75T2DN cells showed no enodogenous wt TRAF2
recruitment to TNF-R75 upon TNF triggering. Instead, the overexpressed
TRAF2-DN molecule was recruited to TNF-R75 in a TNF-dependent way. We
could even detect a small amount of TRAF-2DN in untreated cells,
probably due to an excess of overexpressed TRAF2-DN molecules, which
are recruited with low efficiency to TNF-R75 dimerized by means of the
immunoprecipitating utr-4 mAb. Immunoprecipitations with beads alone
did not reveal TRAF2 or TRAF2-DN molecules. Taken together, these
observations show that the level of TRAF2-DN expression in PC60
R55R75T2DN cells was sufficient to completely inhibit the recruitment
of functional endogenous TRAF2 to the TNF-R75 signaling complex, in
this way blocking TRAF2-dependent signaling pathways. Therefore, it is
conceivable that PC60 R55R75 cells might use other receptor-associated
molecules leading to NF-B activation. Considering the clear
association of TRAF2-DN with TNF-R75 in PC60 R55R75T2DN cells, we
conclude that in PC60 cells no known TRAF2-mediated activities are
implicated in receptor cooperation leading to apoptosis and activation
of NF-B. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
activation, as measured by gel shift assay, was similar in PC60 cells
carrying one or both TNF-R types (G.D., unpublished observation). The
molecular basis for this cooperation is still unknown. Most cases of
receptor cooperation are a result of simultaneous receptor triggering,
although the observation of unliganded receptor cooperation is not
restricted to PC60 R55R75 cells. Indeed, it was reported that selective
loss of TNF-R75 expression can result in resistance to TNF-mediated
cytotoxicity in KYM-1-derived cell lines (45). In this study, a role
for ligand passing was excluded by making use of TNF-R-specific
Abs.
Our structure/function studies with TNF-R75 deletion mutants focused on
two conserved regions in the intracellular part of the receptor, namely
a conserved region upstream of the TRAF complex-binding domain
containing a Ser box of six consecutive serine residues, and the
TRAF1/TRAF2-binding domain itself. No role for the Ser box region in
receptor cooperation was found. These data are consistent with the
earlier observation that this region is not involved in
TNF-R75-mediated NF-B activation (25). Disruption of the TNF-R75
TRAF complex-binding domain (amino acids 346–423) resulted in loss of
receptor functionality. Complete deletion of this region (PC60 R55NR75
M1 cells, 313-end) or removal of the TNF-R75 37 C-terminal amino
acids (PC60 R55NR75 M5) abrogated TNF-R75-mediated induction of GM-CSF
and apoptosis, in addition to TNF-R55-induced apoptosis. Rothe et al.
(25) showed that this TNF-R75 mutant could no longer signal for
proliferation and NF-B activation in CT6 T cells, presumably due to
the absence of TRAF1/TRAF2 binding. This TNF-R75 M5 mutation deletes
part of a putative TRAF interaction site (53). Hence, our results with
PC60 R55NR75 M1 and R55NR75 M5 cells strongly point to a role for the
TRAF1/TRAF2 complex in apoptosis, both by induction of apoptosis by
TNF-R75 itself and by receptor cooperation in TNF-R55. The function of
TRAF1 is still unclear, while TRAF2, a molecule involved in both
TNF-R55 and TNF-R75 signal transduction (26, 27), might be a likely
candidate for the molecule involved in receptor cooperation in PC60
R55R75 cells. Furthermore, recent data suggest a link between TRAF
molecules and death signaling. First, CD40, which binds directly to
TRAF2, TRAF3, and TRAF5 (26, 28, 29, 54), but presumably not to the
DD-containing proteins TRADD, FADD, or RIP (29), is also capable of
inducing apoptosis in transformed cells (26, 28, 29, 54, 55). Second,
signals mediated by CD30 in combination with signals transduced by the
TCR induce Fas-independent cell death in T cell hybridomas (56).
Deletion analysis shows that the 66 C-terminal amino acids of the CD30
cytoplasmic domain, necessary for binding TRAF1 and TRAF2, are required
to induce cell death. Third, overexpression of an N-terminal truncated
TRAF3 molecule abrogated LTß-R-induced cell death, but not activation
of NF-B, in HT29 cells (57). Fourth, transient transfection of a
TRAF2-DN mutant in HeLa cells renders these cells more susceptible to
TNF-mediated apoptosis, pointing to a protective role of TRAF2 in
apoptosis (58). Fifth, the TNF-R75 TRAF2 association site seems to be
required for TNF-R75-dependent enhancement of TNF-R55-mediated
cytotoxicity in HeLa cells (46).
To test the possible involvement of TRAF2 in receptor cooperation in
PC60 R55R75 cells, we transfected this cell line stably with an
expression vector coding for a murine TRAF2(87–501) mutant, which
having lost its biologic functions acts as a DN molecule. This mutant
is still capable of binding the TRAF domain of R75, while it
blocks NF-B, JNK, and p38 MAPK activation (25, 26, 58). Although a
clear inhibition of the TNF-dependent recruitment of functional,
endogenous wt TRAF2 to the TNF-R75 signaling complex by overexpression
of a TRAF2-DN mutant could be shown, PC60 R55R75T2DN cells were neither
impaired in TNF-R75-mediated apoptosis nor in cooperation involved in
TNF-R55-dependent apoptosis. The observation that PC60 R55R75T2DN cells
were as sensitive to TNF as PC60 R55R75/5 cells indicated that TRAF2
was not involved in providing anti-apoptotic signals in these
cells. The kinetics of TRAF2 recruitment to the TNF-R75 were comparable
with these of TRADD recruitment to the TNF-R55 signaling complex (27).
Additionally, the rapid ligand-dependent association of TRAF2 with
TNF-R75 within 1 min following TNF stimulation is also seen in the case
of FADD or TRAF3 targeting to the trimerized Fas receptor or LTß-R,
respectively (36, 57). The low level of TNF-independent TRAF2-DN
recruitment to TNF-R75 in PC60 R55R75T2DN cells is probably caused by
the bivalent immunoprecipitating Ab and indicates that TRAF2 also can
bind to dimerized TNF-R75. This phenomenon is also observed in
experiments showing ligand-dependent TRAF3 association with the LTß-R
(57). However, endogenous TRAF2 recruitment in PC60 R55R75/5 control
immunoprecipitations could not be observed, even after overexposition
of the ECL Western blot. Previously, TRAF2 was postulated as an
essential molecule in TNF-R-mediated NF-B activation by means of
transient overexpression experiments (26, 27). Recently, reports on the
basis of TRAF2-DN transgene and TRAF2-/- mice indicated
the existence of TRAF2-independent pathways leading to the activation
of NF-B (59, 60). Our results confirm these observations. Hence,
TNF-R complex-associated proteins that overrule the need for TRAF2 in
NF-B activation remain to be identified. RIP, a signaling molecule
that interacts with TRADD and TRAF2 (35), seems to be a good candidate
to fulfill this function, since a RIP-deficient Jurkat cell line is
defective in TNF-induced NF-B activation (38). This model can be
true for TNF-R55-induced NF-B activation, since TRADD can recruit
RIP to this signaling complex. On the other hand, we were unable to
show RIP association to the TNF-R75 by its affinity for TRAF1 and/or
TRAF2 in overexpression experiments (W.D., unpublished observation).
Alternatively, TNF may activate sphingomyelinases, leading to ceramide
generation and subsequent NF-B activation (61), although acidic
sphingomyelinases do not seem to be involved in TNF-induced NF-B
activation (62). In addition, stable overexpression of mutant TRAF
family members TRAF3 and TRAF5 block CD40-mediated induction of CD23
expression (28, 29), although experiments with
TRAF3-/-cells could not confirm a role for TRAF3 in CD40
signaling (63).
The observation that both TNF-R55 and TNF-R75 share the same intracellular signaling molecules brings us back to the question of why the presence of both TNF-R types is required for induction of apoptosis in PC60 cells. A possible explanation could be the formation of heteromeric receptor complexes, which need to contain both TNF-R55 and TNF-R75 to be fully active. Since we were able to induce apoptosis in PC60 R55R75/5 cells making use of TNF-R-specific muteins or TNF-R-specific Abs (11), such a receptor complex could not be formed by simultaneous interaction of both TNF-R with the same ligand molecule. Possibly, mixed TNF-R complex formation might be due to bridging between associated molecules. However, ligand-induced conformational changes, enabling TNF-Rs to interact with each other, cannot be excluded. Alternatively, a low level of spontaneous homomeric TNF-R aggregation resulting in low level signal transduction sufficient for receptor cooperation in PC60 cells is another possible explanation. This synergizing process would be abrogated if one of the receptors was nonfunctional. We should stress that the necessity for both TNF-R types to allow TNF-R75-induced apoptosis seems not to be a general mechanism, since TNF-R75 has been shown able to induce apoptosis in a subset of T cells in TNF-R55-/- mice (22). However, a similar mechanism of receptor cooperation probably exists in HeLa cells (46).
In summary, our data indicate the existence of bidirectional TNF-R55
and TNF-R75 cooperation in PC60 cells in the case of apoptosis. We were
also able to demonstrate ligand-independent TNF-R55-mediated
cooperation in TNF-R75-induced GM-CSF secretion, but not the reverse.
Mutational analysis of the TNF-R75 intracellular domain revealed the
involvement of the TRAF1/TRAF2-binding region in TNF-R75-mediated
receptor cooperation in apoptosis. However, (over)expression of a
TRAF2-DN molecule did not block receptor cooperation; nor did it result
in inhibition of TNF-R55- or TNF-R75-mediated apoptosis, GM-CSF
induction, or NF-B activation, despite the fact that TNF-dependent
recruitment of endogenous wt TRAF2 was prevented by the TRAF2-DN
mutant.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. P. Vandenabeele, Laboratory of Molecular Biology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium. E-mail address:
3 Abbreviations used in this paper: DD, death domain; DN, dominant negative; PI, propidium iodide; TRAF, TNF-R-associated factor; wt, wild-type; GM-CSF, granulocyte/macrophage-CSF; ECL, enhanced chemiluminescence; TRADD, TNF-R1-associated DD protein; FADD, Fas-associating protein with DD; ICE/CED, IL-1D-converting enzyme/Caenorhabditis elegans death gene; C-IAP, cellular inhibitor of apoptosis; RIP, receptor-interacting protein.
Received for publication August 22, 1997. Accepted for publication February 20, 1998.
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References |
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B. Curr. Biol. 6:1669.[Medline]
B activation. Cell 81:495.[Medline]
B by TNF receptor 2 and CD40. Science 269:1424.B but not Fas/APO-1-initiated apoptosis. EMBO J. 15:6189.[Medline]
) in KYM-1 cells involves both TNF receptors. Cytokine 5:556.[Medline]
and IL-1ß and partial amino acid sequence of rat GM-CSF. Lymphokine Res. 9:381.[Medline]
B activation and binding specificity. Proc. Natl. Acad. Sci. USA 93:9699.B. Proc. Natl. Acad. Sci. 94:2460.B activation prevents cell death. Cell 87:565.[Medline]
B activation and regulates lymphocyte proliferation and survival. Immunity 7:703.[Medline]
B activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7:715.[Medline]
B- degradation in a cell-free system. J. Biol. Chem. 269:13760. activates NF-B in acid sphingomyelinase-deficient mouse embryonic fibroblasts. J. Biol. Chem. 272:10904.This article has been cited by other articles:
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