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B Activation, and c-Jun Kinase Activation1
Cytokine Research Section, Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030; and * Roche Biosciences, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B (NF-B) activation are mediated
through the p60 form of the endogenous receptor. What signals are
mediated through the p80 receptor is less clear. This study was an
effort to answer that question. HeLa cells, which express only p60
receptors, were transfected with p80 receptor cDNA and then examined
for apoptosis, NF-B activation, and c-Jun kinase activation induced
by TNF and by p60 or p80 receptor-specific muteins. The p80 mutein,
like TNF and the p60 mutein, induced apoptosis and activation of
NF-B and c-Jun kinase in cells overexpressing recombinant p80
receptor but had no effect on cells expressing a high level of
endogenous p80 receptor. The apoptosis mediated through the p60
receptor was also potentiated after overexpression of the p80 receptor,
suggesting a synergistic relationship between the two receptors.
Interestingly, Abs to the p80 receptor blocked apoptosis induced by all
ligands but by itself activated NF-B in the p80-transfected cells.
Overall, our results show that the p80 receptor, which lacks the death
domain, mediated apoptosis, NF-B activation, and c-Jun kinase
activation, but only when it was overexpressed, whereas endogenous p60
receptor mediated similar signals without overexpression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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29% homology in amino acid sequences)
but a dissimilar intracellular domain
(ICD)3 has led to the
proposal that these two TNF receptors must transduce distinct signals
(for references, see 1 . This hypothesis is further strengthened
by observations that the ICD of the p60 receptor contains an amino acid
sequence of approximately 80 amino acids, referred to as the death
domain (DD; due to its involvement in cell death), whereas the p80
receptor does not (2). Moreover, it has been shown that the ICD of the
p60 receptor recruits several signaling proteins distinct from that of
the p80 receptor (for references, see 3 , including TRADD, FADD,
FLICE, RIP, TRAF-2, ICH-1, MADD, TRIP, I-FLICE, sentrin, A-20, and
I-TRAF. In contrast, factors associated with
N-sphingomyelinase activation, PIP5K, TNF
receptor-associated protein-1, and TNF receptor-associated protein-2
bind to the juxtamembrane domain. The ICD of p80 without the DD
recruits TRAF-1, TRAF-2, IAP-1, IAP-2, and NF-B-inducing kinase (3, 4). Despite distinct ICD of the two receptors, TRAF-2 and IAP-1, which
bind to the p80 receptor, also bind through secondary interaction to
the p60 receptor, but most of the p60 receptor binding proteins do not
bind to the p80 receptor. In addition to these proteins, two distinct
putative serine/threonine protein kinases have been shown to be
recruited by the p60 and p80 receptors (5, 6). The roles of these
proteins in TNF signaling are not entirely clear, but it appears that
different proteins may mediate different TNF-induced signals. Even
though TRADD, FADD, FLICE, RIP, and ICH-1 have been implicated in cell
death (7, 8, 9, 10, 11, 12, 13), and RIP, TRADD, and TRAF-2 have been implicated in
TNF-induced activation of NF-B and c-Jun kinase (14, 15, 16, 17), the
mechanisms are unknown.
One of the most fundamental unanswered questions in TNF biology is thus
what types of signals are mediated through the p80 receptor compared
with the p60 receptor. In general, it has been shown that most of the
TNF signals are mediated through the p60 receptor. The p80 receptor has
been implicated in ligand passing, in cytotoxicity, and in potentiation
of p60 receptor-mediated cytotoxicity (18, 19, 20). Recent observations
using receptor-specific TNF muteins have indicated that the
cytotoxicity and NF-B activation are mediated entirely through the
endogenous p60 receptor (21).
In the present report we compared signaling through the endogenous
receptor with that through the recombinant p80 receptor. TNF muteins
that bind exclusively to either the p60 or the p80 receptor (22) were
employed for these studies. In p60 receptor-specific mutein, residue 32
was altered from arginine to tryptophan, and residue 86 was altered
from serine to threonine, whereas in p80 receptor-specific mutein, the
carboxyl-terminus at position 143 was mutated from aspartic acid to
asparagine and at position 145 from alanine to arginine. The p80
receptor-binding mutein induced apoptosis, activated NF-B, and
induced c-Jun kinase in cells overexpressing the recombinant receptor
but not in cells expressing endogenous receptor. Thus, our results
demonstrate that even though the ICD of the p80 receptor is distinct
from that of the p60 receptor and recruits several distinct proteins,
overexpressed p80 receptor can transduce signals similar to those of
the endogenous p60 receptor but distinct from those of the endogenous
p80 receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium, FCS, penicillin, streptomycin, and
trypsin-EDTA were obtained from Life Technologies (Grand Island, NY).
Tris, glycine, NaCl, and BSA were obtained from Sigma Chemical (St.
Louis, MO). Hygromycin B was purchased from Calbiochem-Novabiochem
International (La Jolla, CA). Bacteria-derived recombinant human TNF
purified to homogeneity was provided by Genentech (South San Francisco,
CA). 32P-labeled 
-ATP with a sp. act. of 7000
Ci/mmol was obtained from Amersham Life Sciences (Arlington Heights,
IL). Recombinant human TNF muteins altered by site-specific mutagenesis
to bind to either the p60 or p80 receptor, expressed in bacteria, and
purified to homogeneity were supplied by F. Hoffmann-La Roche (Basel,
Switzerland). The isolation and characterization of these muteins has
been described previously (22). Rabbit anti-JNK polyclonal Ab was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse
anti-poly(A)DP ribose polymerase (anti-PARP) Ab was purchased
from PharMingen (San Diego, CA). Anti-mouse horseradish
peroxidase-conjugated secondary Ab was purchased from Transduction
Laboratories (Lexington, KY). Protein A/G-Sepharose beads were obtained
from Pierce Chemical (Rockford, IL). GST-Jun1–79 was
expressed in Escherichia coli and purified essentially as
previously described (5).
Cell lines
The human histiocytic cell line U-937, human myeloid ML-1a, and human rhabdomyosarcoma cell line KYM-1 were grown in RPMI 1640 medium supplemented with FBS (10%), penicillin (100 µg/ml), and streptomycin (100 µg/ml). Cells were seeded at a density of 1 x 105/ml in T75 flasks (Falcon 3013, Becton Dickinson Labware, Lincoln Park, NJ) containing 20 ml of medium and were grown at 37°C in an atmosphere of 95% air and 5% CO2. Cell cultures were split every third day. KYM-1 cells were trypsinized and seeded again every third day.
HeLa cells stably transfected with pCDM8 mammalian expression vector containing the full-length TNFRp80 cDNA, as previously described (18), were used for our studies. A plasmid carrying the hygromycin phosphotransferase gene was used as a selection marker and was cotransfected with the pCDM18 plasmid. Transfected cells were routinely grown in MEM containing 10% FBS, penicillin (100 µg/ml), streptomycin (100 µg/ml), and hygromycin (200 µg/ml).
Different clones were generated from the HeLa-p80 cells by two different methods. The first method involved sorting by FACS based on anti-p80 receptor Ab and anti-rabbit FITC-labeled secondary Ab. Depending on the level of p80 expression, four different populations of HeLa-p80 cells were obtained by this method. The second method involved isolation of different HeLa-p80 clones by limited dilution. Briefly, HeLa-p80 cells were diluted to 10 cells/ml and plated at 1 cell/100 µl/well into 96-well plates. The plates were incubated at 37°C for 2 wk, and thereafter, three different clones (no. 1–3) were isolated and further characterized.
The effect of p80 receptor transfection was also analyzed in another cell line, breast carcinoma MCF-7 (expresses only the p60 receptor), and by using a different vector. For this, full-length TNFRp80 cDNA (6) was cloned into pEC1214A(pTet), a modified tetracycline repressor system expression vector (a gift from Dr. Hong-Ji Xu, University of Texas M. D. Anderson Cancer Center, Houston, TX) containing a cloning site for the desired gene that is under the control of the tetracycline repressor, the constitutive expression of tetracycline repressor, and the neomycin resistance gene. MCF-7 cells were transfected with the pTet and pTet-p80 vector using Lipofectamine as described by the manufacturer (Life Technologies). A stable pool of MCF-7 cells harboring either pTet (MCF-7) or pTet-p80 (MCF-7 (p80)) were selected by growing the cells in complete medium containing tetracycline (1 µg/ml) and G418 (600 µg/ml). Induction of p80 receptor occurred after the withdrawal of tetracycline.
Cytotoxicity assay
The cytotoxic effects of TNF and its muteins on adherent cells (KYM-1 and HeLa) were measured by the crystal violet dye uptake assay. Briefly, 10 x 103 cells were plated in 0.1 ml of medium in 96-well flat-bottom plates. After an overnight incubation at 37°C, the medium was removed, and different concentrations of TNF were layered in 0.1 ml of fresh medium. After 72 h of incubation at 37°C, the medium was removed, and the viability of cells was monitored by crystal violet staining according to a procedure described previously (23). The cytotoxic effects of TNF on nonadherent cells (U-937) were determined by the amount of [3H]thymidine incorporated by the cells as described previously (24). Briefly, cells were plated at 5000/well in 0.1 ml of medium in 96-well flat-bottom Falcon plates. Different concentrations of TNF were added in an additional 0.1 ml of medium and were incubated at 37°C for 72 h. During the last 6 h before harvesting, [3H]thymidine (5 mCi/mmol; Amersham) was added to each well (0.5 µCi/well), and then cells were harvested with the aid of Filtermate 196 harvester (Packard Instruments, Meriden, CT). Radioactivity bound to the filter was measured in a liquid scintillation counter (model 1600 TR, Packard Instruments).
Immunoblot analysis of PARP degradation
TNF-induced apoptosis was also examined by proteolytic cleavage of PARP (25). Briefly, cells (1 x 106/ml) were treated with TNF or the muteins for either 2 h (U-937 cells) or 4 h (KYM-1, HeLa and HeLa-p80 cells) in the presence of 1 µg/ml of cycloheximide. After treatment, cell extracts were prepared by incubating the cells for 30 min on ice in 0.05 ml of buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, and 1 mM DTT. The lysate was centrifuged, and the supernatant was collected. Cell extract protein (50 µg) was resolved on a 7.5% SDS-polyacrylamide gel, electrotransferred onto a nitrocellulose membrane, blotted first with mouse anti-PARP Ab and then with anti-mouse horseradish peroxidase-conjugated IgG as the secondary Ab, and then detected by chemiluminescence (ECL, Amersham). Apoptosis was represented by the cleavage of 116-kDa PARP into 85- and 41-kDa peptide products (25).
Electrophoretic mobility shift assays (EMSAs)
An EMSA to examine NF-B activation was conducted as described
previously (26, 27). Briefly, cells (2 x 106/ml)
were treated separately with different concentrations of TNF and its
muteins at 37°C for 30 min. Nuclear extracts were then prepared as
described previously (27). EMSA was performed by incubating 4 µg of
nuclear extracts with 16 fmol of 32P end-labeled 45-mer
double-stranded NF-B oligonucleotide from the HIV-1 long terminal
repeat, 5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3', in the
presence of 2 to 5 µg of poly(dI-dC) in a binding buffer (25 mM HEPES
(pH 7.9), 0.5 mM EDTA, 0.5 mM DTT, 1% Nonidet P-40, 5% glycerol, and
50 mM NaCl) for 15 min at 37°C. The DNA/protein complex formed was
separated from free oligonucleotide on a 7.5% native polyacrylamide
gel. A double-stranded mutant oligonucleotide was used as a control to
examine the specificity of binding of NF-B to the DNA. The
specificity of binding was also checked by competition with the
unlabeled oligonucleotide, by using mutant oligonucleotide, and by
supershift of the band with Abs against NF-B p50 and p65 proteins.
The radioactive bands from dried gels were visualized on a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and quantitated
using ImageQuant software.
Receptor binding assays
Receptor binding assays were performed as described previously (28). TNF was labeled with Na125I, using the Iodogen method, to an approximate sp. act. of 40 mCi/mg. Binding assays were performed in flexible 96-well plates. Cells (0.5 x 106/0.1 ml) were incubated in a binding medium (RPMI 1640 containing 10% FBS) with 125I-labeled TNF in the absence (total binding) or the presence of 100 nM unlabeled ligand (nonspecific binding) for 1 h at 4°C. Thereafter, cells were washed three times with ice-cold medium (PBS containing 0.1% BSA) at 4°C, and the cell-bound radioactivity was determined in a gamma counter (Cobra-AutoGamma, Packard Instrument). All determinations were performed in triplicate. Specific binding of TNF was calculated by subtraction of the amount (counts per minute) of nonspecific binding from the total binding.
To determine the levels of p60 and p80 forms of TNF receptors, affinity-purified Abs specific for each type of receptor were employed (21). Cells (0.5 x 106/0.1 ml) were preincubated with the Ab (5 µg/ml) for 1 h at 4°C and then examined for specific binding of labeled TNF as described above. TNF muteins were also used to compete for TNF binding sites. Specific binding of TNF observed on competition with TNF (p60) or on cells pretreated with anti-p60 Ab would be due to the p80 receptor and vice versa.
c-Jun kinase assay
The c-jun kinase assay was performed using a modified method as
described previously (29). Briefly, after treatment of cells (3 x
106/ml) with TNF for 15 min, cell extracts were
prepared by lysing cells in buffer containing 20 mM HEPES (pH 7.4), 2
mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2
µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, and 1 mM DTT.
Cell extracts (150–250 µg) were subjected to immunoprecipitation
with 0.03 µg of anti-JNK Ab for 30 min at 4°C. Immune complexes
were collected by incubation with protein A/G-Sepharose beads for 30
min at 4°C. The beads were collected by centrifugation and washed
extensively with lysis buffer (four times, 400 µl each time) and
kinase buffer (twice, 400 µl each time; 20 mM HEPES (pH 7.4), 1 mM
DTT, and 25 mM NaCl). Kinase assays were performed for 15 min at 30°C
with 2 µg GST-Jun1–79 in 20 µl containing 20 mM HEPES
(pH 7.4), 10 mM MgCl2, 1 mM DTT, and 10 µCi
[-32P]ATP. Reactions were stopped with 15 µl of
SDS-sample buffer, boiled for 5 min, and subjected to SDS-PAGE.
GST-Jun1–79 was visualized by staining with Coomassie
Blue, and the dried gel was analyzed by a PhosphorImager (Molecular
Dynamics) and quantitated by ImageQuant Software (Molecular
Dynamics).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation, and c-Jun kinase activation. To
distinguish the signaling transduced by the p80 receptor from that of
p60, we used genetically engineered TNF muteins that bind to either the
p60 or the p80 receptor. The results were confirmed by the use of
affinity-purified Abs specific to each receptor. We also compared the
signaling of the recombinant p80 receptor with that of the endogenous
p80 receptor. Characterization of endogenous vs recombinant p80 receptors
We used U-937 and KYM-1 cells, which have been shown to express a
high density of high affinity (5000 binding sites with a
Kd of 0.1 nM) TNF receptors (30). Furthermore,
the level of p80 receptors on these cells, determined using
receptor-specific Abs, was approximately 3000 sites/cell (30, 31). In
the present report we further examined the levels of p60 and p80
receptors by using TNF muteins specific to each receptor as a
competitor. TNF (p60) inhibited TNF binding on U-937 cells by
approximately 46%, and TNF (p80) inhibited it by 56% (Fig. 1
, upper panel),
suggesting that they expressed almost equal numbers of p60 and p80
receptors. Like U-937 cells, KYM-1 (Fig. 1, lower
panel) cells expressed a high level of TNF receptors, and
their binding was equally inhibited by p60- and p80-specific muteins.
Compared with p60, U-937 cells expressed slightly lower p80, and KYM-1
cells expressed slightly higher p80 receptor levels.
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A) indicate that
nontransfected HeLa cells expressed p60 receptor, as TNF binding could
be blocked by anti-p60 Ab only (upper
right) and inhibited by TNF (p60) mutein and not by TNF
(p80) mutein (upper left). HeLa cells, when
transfected with p80 receptor cDNA, bound 20 to 30 times more labeled
TNF, and this binding could be blocked primarily by anti-p80 Abs
(lower right) and inhibited by TNF (p80)
mutein. Anti-p60 Ab or TNF (p60) had a minimal effect on binding of TNF
to transfected cells. These results clearly indicate that transfected
HeLa cells overexpressed p80 receptors. Scatchard analysis revealed
that transfected HeLa cells express approximately 190,000 binding
sites/cell (18).
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B). U-937, ML-1a, and KYM-1 expressed variable
amounts of endogenous p80 receptor protein, with the highest being
expressed by KYM-1. As expected, nontransfected HeLa cells expressed no
detectable p80 receptor, whereas after transfection these cells
expressed a high level. A small amount of a low m.w. band was also
found in transfected cells. The origin of this band is unclear at
present, but may represent the degraded form of the p80 receptor. Overexpression of p80 receptor mediates apoptosis
To examine signaling through the endogenous p80 receptor, we
examined the cytotoxic responses to TNF, TNF (p60), and TNF (p80). Both
TNF and TNF (p60) were cytotoxic to U-937 cells (Fig. 3A, upper
panel) and to KYM-1 cells (Fig. 3A, lower
panel) in a dose-dependent manner. As little as 0.01 ng/ml
of the ligand showed a significant effect. TNF (p80) mutein, however,
had no effect on these cells, even when used at 200 ng/ml, suggesting
that the p80 receptor is inert in these cells. As will be shown later,
the lack of this effect could not be attributed to the possibility
that TNF (p80) mutein was not biologically active.
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B, upper
panel). These results imply that the p80 receptor, when
overexpressed, could also mediate TNF-induced cell killing. To
determine whether TNF could kill the cells without the assistance
of the endogenous p60 receptor, we used TNF muteins. Interestingly, we
found that p80 receptor transfection also enhanced the activity of the
mutein specific to the p60 receptor (Fig. 3B, middle
panel), suggesting a synergistic effect in the signaling
mediated through the p60 and p80 receptors. TNF (p80) mutein, which was
inactive on cells expressing endogenous receptor, was also inactive on
nontransfected HeLa cells (which express only the p60 receptor; Fig. 3B, bottom panel). However, when examined
on cells transfected to overexpress the p80 receptor, TNF (p80) mutein
was as active as TNF, indicating that unlike the p60 receptor, the p80
receptor can mediate cytotoxicity only when overexpressed. These
results also indicate that p80 not only can mediate cytotoxicity but
can also enhance p60-mediated cytotoxicity.
Besides cytotoxicity, which required 72-h treatment to be expressed, we
also examined TNF-induced cleavage of PARP, another early
characteristic feature of apoptosis (25). After treatment of KYM-1
cells for 4 h, both TNF and the TNF (p60) mutein induced cleavage
of PARP; the TNF (p80) mutein, however, had no effect (Fig. 4). Similarly, in U-937 cells the TNF
(p60) mutein induced PARP cleavage within 2 h, whereas the TNF
(p80) mutein did not (Fig. 4). These results clearly indicate that
apoptosis is mediated through the endogenous p60 receptor but not the
endogenous p80 receptor. We also found that the TNF (p80) mutein
induced PARP cleavage in p80-transfected HeLa cells but not in cells
expressing endogenous p60 receptor. Thus, as was the case for
cytotoxicity, PARP cleavage was mediated through the recombinant p80
receptor but not through the endogenous receptor.
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B activation
Both TNF and TNF (p60) activated NF-B in U-937, ML-1a, and
KYM-1 cells, but the TNF (p80) mutein had no effect on any of these
cells (Fig. 5A). Even
TNF (p80) at 10 nM (100-fold higher) failed to activate NF-B in
KYM-1 cells (Fig. 5B). Thus, endogenous p80 receptor
appears to be inactive for induction of both apoptosis and NF-B
activation. Both TNF and TNF (p60) activated NF-B within 30 min in a
dose-dependent manner in HeLa cells, whereas TNF (p80) was inactive
even when tested at a 100-fold excess (Fig. 5C, upper
panel). In contrast, on p80-transfected cells, TNF (p80)
mutein was as active as TNF or TNF (p60) for this cellular response
(Fig. 5C, middle panel). To determine the
specificity of the TNF-mediated response, we used anti-p80 Abs,
which block endogenously expressed p80-mediated signaling.
Serendipitously, we found that when these Abs to the p80 receptor were
used to treat cells that overexpress recombinant p80 receptor, NF-B
was activated through the p80 receptor (Fig. 5C, lower
panel). Thus, the Ab acted as a TNF agonist. These results
indicate that in contrast to endogenous p80 receptor, recombinant p80
receptor can also activate NF-B.
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Recently, it has been reported that overexpression of TRAF2, which
associates with the p80 receptor, can activate c-Jun kinase (13, 15).
Therefore, we examined the role of endogenous p80 receptor in mediating
c-Jun kinase activation by using TNF muteins. In U-937 cells, both TNF
and TNF (p60) activated c-Jun kinase, whereas TNF (p80) had no effect
(Fig. 6). Both TNF and TNF (p60)
activated JNK on control HeLa cells and transfected cells, whereas TNF
(p80) activated JNK only on transfected cells (Fig. 6). These results
demonstrate that the p80 receptor can mediate c-Jun kinase activation
only when overexpressed. Our results to date suggest that endogenous
p80 receptor lacks the ability to mediate apoptosis, NF-B
activation, and c-Jun kinase activation.
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Why there are two distinct TNF receptors and whether there is any
communication between the two receptors with respect to signaling have
not been established. Since both receptors appeared to respond
independently of each other in our system, we examined possible
cross-talk between the two receptors. Given that our earlier results
indicated that these receptors may function in a synergistic manner
(Fig. 3B, middle panel), we examined the
effect of anti-p80 Ab on TNF-, TNF (p60)-, and TNF (p80)-mediated
cytotoxicity in p80-transfected cells. The results show that
anti-p80 blocked not only TNF- and TNF (p80)-mediated cytotoxicity,
but also the TNF (p60)-mediated effect (Fig. 7, upper panel).
Inhibition of TNF (p60)-induced cytotoxicity by anti-p80 occurred
in a dose-dependent manner (Fig. 7, lower panel). The
results with PARP cleavage also showed that the TNF (p60)-mediated
effect was blocked by anti-p80 (Fig. 4). These results provide
further evidence in HeLa-p80 cells that to mediate its effects, TNF
(p60) mutein requires the p80 receptor in addition to the p60
receptor.
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B
activation (Fig. 5, lower panel) and for c-Jun kinase
activation (Fig. 6, lower panel) showed antagonistic
activity for cytotoxicity (Fig. 7). Different clones of HeLa-p80 cells mediate TNF-induced signaling through the p80 receptor
All the results shown above were conducted with a single clone of
p80-transfected HeLa cells. To ascertain that signaling through p80
receptor is not clone specific, we generated different clones of
HeLa-p80 both by FACS sorting and by single cell cloning. HeLa-p80
cells were sorted into different populations based on the level of p80
receptor expression by FACS analysis. The different populations were
designated A (untransfected), B (very low), C (low), D (medium), E
(high), and F (unsorted). The level of p80 receptor expression on these
cells was evaluated by labeled TNF binding (Fig. 8A) and by Western blot
(Fig. 8B). In cells with medium and high levels of
p80 expression (populations D and E), TNF (p80) mutein could induce
cytotoxicity (Fig. 8C), activation of NF-B (Fig. 8D), and activation of c-Jun kinase (Fig. 8E). Thus, these data suggest that there is a direct
correlation between increasing expression of the p80 receptor in HeLa
cells and its ability to transduce various signals. Similarly, when
different clones were isolated by single cell cloning, the level of
expression of p80 (Fig. 9A) again correlated
with NF-B activation (Fig. 9B) and cytotoxicity
(Fig. 9C) induced by TNF (p80) mutein.
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B activation
To ascertain that signaling through the recombinant p80 receptor
is not restricted to HeLa cells, we also investigated the effect of p80
receptor transfection in MCF-7 cells, which normally express only p60
receptor. For this, MCF-7 cells were stably transfected with an
inducible expression vector containing the p80 receptor (MCF-7 (p80)).
The p80 receptor expression was induced as evaluated by Western blot
(Fig. 10A) and by
labeled TNF binding (Fig. 10B). While MCF-7 cells
were completely resistant to increasing doses of the TNF (p80) mutein,
expression of the p80 receptor in MCF-7 (p80) cells caused cytotoxicity
in a dose-dependent manner when exposed to TNF (p80) mutein (Fig. 10C). The expression of the p80 receptor in MCF-7
(p80) also led to activation of NF-B by the TNF (p80) mutein (Fig. 10D). These results suggest that overexpression of
the p80 receptor leads to signaling not only in HeLa cells, but also in
MCF-7 cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and of c-Jun kinase, all previously known to be
mediated through the p60 receptor.
In the present report, we demonstrate that the endogenous p80 receptor
is incapable of inducing these three signals. However, p80 receptor
can, when overexpressed, mediate cytotoxicity, NF-B activation, and
induction of c-Jun kinase in a ligand-dependent manner. In addition, we
found that overexpression of p80 receptor potentiates p60
receptor-mediated cytotoxicity in a synergistic manner, and p60
receptor-mediated cytotoxicity was blocked by Abs to the p80 receptor.
The p80 Abs, which was antagonistic on endogenous receptor acted as an
agonist on the cells overexpressing p80 receptor for NF-B
activation.
Although several reports indicate that the endogenous p60 receptor can
mediate cytotoxicity, NF-B activation, and induction of c-Jun
kinase, very few indicate that endogenous p80 receptor can mediate
similar responses (31, 32, 33, 34, 35, 36, 37). This has led to suggestions of an alternate
role of p80 receptor including ligand passing to the p60 receptor, the
receptor for transmembrane form of TNF, and the receptor required for
immunomodulation by TNF (19, 32, 33). Only when overexpressed could the
p80 receptor signal for all the cellular responses examined. Induction
of apoptosis by overexpression of p80 is consistent with the findings
of a previous report (18).
Why overexpression of p80 receptor is needed for its signaling, whereas the p60 receptor can signal without overexpression is not clear. It has been shown that receptor clustering is needed for cytokine signaling, so it is possible that receptor clustering is more efficient for the p60 receptor than for the p80 receptor. That the ICD of the p60 receptor contains a DD that itself has a strong tendency to self-associate, whereas the p80 receptor lacks this domain, may explain the differential behavior of the two receptors. This may also explain why overexpression of the p60 receptor leads to ligand-independent signaling, whereas overexpression of the p80 receptor, as noted in our studies, still requires activation by the ligand to signal.
The DD of the p60 receptor has been shown to recruit several proteins
that are involved in TNF-mediated cytotoxicity, NF-B activation, and
induction of c-Jun kinase. How p80 receptor signals without the DD
binding proteins is not known. TRAF-2 and cellular IAP-1 have been
shown to bind to both receptors (38, 39, 40). The potential role of TRAF-2
in NF-B activation and induction of JNK has been demonstrated
(13, 14, 15, 16, 17). However, TRAF-2 plays no role in TNF-induced apoptosis. Thus,
how p80 receptor can mediate cytotoxicity independent of the p60
receptor is not clear. Our results also showed that p60
receptor-mediated cytotoxicity is potentiated by p80 receptor (see Fig. 3B) and that p60 receptor-mediated cytotoxicity is
blocked by anti-p80 (Fig. 7). How p80 receptor potentiates the p60
receptor-mediated signaling is not clear. Our results, however, are in
agreement with those of a recent report that showed enhancement of p60
receptor-mediated cytotoxicity by p80 receptor (20). The ability of p80
receptor to cooperate with the activation of p60 receptor was found to
be due to TRAF-2 (20). Overexpression of TRAF-2 alone, however, does
not induce cytotoxicity. The role of TRADD in p60 receptor-mediated
cytotoxicity has been demonstrated (14). It is possible that the p80
receptor recruits proteins similar to TRADD that can also mediate
cytotoxicity. Alternatively, it is also possible that both TRADD and
TRAF2 are needed for cell killing by TNF. Ligand-induced formation of a
heterocomplex between p60 and p80 has recently been demonstrated (41).
In any case, our results provide evidence of cross-talk not only at the
receptor level, but also for downstream signaling.
We showed that when U-937 and KYM-1 cells, which express endogenous p80
receptor, are stimulated with TNF (p80), they do not show activation of
NF-B. However, when the p80 receptor was overexpressed, the TNF
(p80) mutein activated NF-B. This is consistent with the report that
overexpression and clustering of TRAF2 can by itself activate NF-B
(13). The p80-mediated activation of NF-B seen in HeLa-p80 cells was
perhaps due to such a clustering of the TRAF2 molecules. Besides TNF
(p80), anti-p80 receptor Ab, which was antagonistic to induction of
apoptosis (see Fig. 7), activated NF-B in HeLa-p80 cells (Fig. 5C, bottom panel). How an antagonistic Ab
can become an agonist is not clear. This may be because differential
receptor clustering is needed for different signals.
Our results show that p80 receptor can transduce similar signals as the p60 receptor but require a higher level of expression. It is possible that the normal level of p80 receptors present in the cells does signal, but the level is too low to be detectable. Alternatively, a normal level of p80 receptor expression is responsible for long term effects, but is too low to trigger short term effects. Overall, our results indicate that both p60 and p80 receptors can mediate identical signals, but the p60 receptor is more efficient than the p80 receptor. Our results also provide evidence for cross-talk between the two receptors.
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2 Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Section, Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. E-mail address:
3 Abbreviations used in this paper: ICD, intracellular domain; DD, death domain; FADD, Fas-associated death domain; FLICE, Fas-associated death domain-like IL-1-converting enzyme; RIP, receptor-interacting protein; TRAF, tumor necrosis factor receptor-associated factor; IAP, inhibitor of apoptosis; NF-B, nuclear factor-B; JNK, c-Jun kinase; PARP, poly(A)DP ribose polymerase; GST, glutathione-S-transferase; EMSA, electrophoretic mobility shift assay. TRADD, TNF-receptor associated death domain; ICH-1, ICE and ced-3 homolog-1; MADD; Map kinase-activated death domain protein; TRIP, TRAF-interacting protein; I-FLICE, Inhibitor of FLICE; I-TRAF-Inhibitor of TRAF.
Received for publication July 9, 1997. Accepted for publication December 3, 1997.
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S. K. Manna and B. B. Aggarwal Interleukin-4 Down-regulates Both Forms of Tumor Necrosis Factor Receptor and Receptor-mediated Apoptosis, NF-kappa B, AP-1, and c-Jun N-Terminal Kinase. COMPARISON WITH INTERLEUKIN-13 J. Biol. Chem., December 11, 1998; 273(50): 33333 - 33341. [Abstract] [Full Text] [PDF]
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S. K. Manna and B. B. Aggarwal IL-13 Suppresses TNF-Induced Activation of Nuclear Factor-{kappa}B, Activation Protein-1, and Apoptosis J. Immunol., September 15, 1998; 161(6): 2863 - 2872. [Abstract] [Full Text] [PDF]
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B. G. Darnay, V. Haridas, J. Ni, P. A. Moore, and B. B. Aggarwal Characterization of the Intracellular Domain of Receptor Activator of NF-kappa B (RANK). INTERACTION WITH TUMOR NECROSIS FACTOR RECEPTOR-ASSOCIATED FACTORS AND ACTIVATION OF NF-kappa B AND c-JUN N-TERMINAL KINASE J. Biol. Chem., August 7, 1998; 273(32): 20551 - 20555. [Abstract] [Full Text] [PDF]
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V. Haridas, J. Ni, A. Meager, J. Su, G.-L. Yu, Y. Zhai, H. Kyaw, K. T. Akama, J. Hu, L. J. Van Eldik, et al. Cutting Edge: TRANK, a Novel Cytokine That Activates NF-{kappa}B and c-Jun N-Terminal Kinase J. Immunol., July 1, 1998; 161(1): 1 - 6. [Abstract] [Full Text] [PDF]
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S. K. Manna, H. J. Zhang, T. Yan, L. W. Oberley, and B. B. Aggarwal Overexpression of Manganese Superoxide Dismutase Suppresses Tumor Necrosis Factor-induced Apoptosis and Activation of Nuclear Transcription Factor-kappa B and Activated Protein-1 J. Biol. Chem., May 22, 1998; 273(21): 13245 - 13254. [Abstract] [Full Text] [PDF]
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C. Seitz, P. Muller, R. C. Krieg, D. N. Mannel, and T. Hehlgans A Novel p75TNF Receptor Isoform Mediating NFkappa B Activation J. Biol. Chem., May 25, 2001; 276(22): 19390 - 19395. [Abstract] [Full Text] [PDF]
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A. Mukhopadhyay, J. Suttles, R. D. Stout, and B. B. Aggarwal Genetic Deletion of the Tumor Necrosis Factor Receptor p60 or p80 Abrogates Ligand-mediated Activation of Nuclear Factor-kappa B and of Mitogen-activated Protein Kinases in Macrophages J. Biol. Chem., August 17, 2001; 276(34): 31906 - 31912. [Abstract] [Full Text] [PDF]
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