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J. Biol. Chem., Vol. 277, Issue 46, 44155-44163, November 15, 2002
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From the Institute of Cell Biology and Immunology, University of
Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
Received for publication, July 23, 2002, and in revised form, September 3, 2002
Tumor necrosis factor (TNF) exists both as a
membrane-integrated type II precursor protein and a soluble cytokine
that have different bioactivities on TNFR2 (CD120b) but not on TNFR1
(CD120a). To identify the molecular basis of this disparity, we have
investigated receptor chimeras comprising the cytoplasmic part of Fas
(CD95) and the extracellular domains of the two TNF receptors. The
membrane form of TNF, but not its soluble form, was capable of inducing apoptosis as well as activation of c-Jun N-terminal kinase and NF- The majority of cell surface receptors initiate intracellular
signals after ligand-mediated homo- or heteromultimerization. Members
of the tumor necrosis factor
(TNF)1 ligand family
typically form stable homotrimers capable of multimerizing their
respective receptors (1). Essential for intracellular signal induction
is the subsequent recruitment of adaptor proteins (e.g.
members of the TNF receptor-associated factor (TRAF) family or death
domain-containing proteins like TNF receptor-associated death domain
protein (TRADD) or Fas-associated death domain protein (FADD)) (1).
These two protein groups define the two major signaling pathways
leading to either gene induction (TRAFs) or the activation of the
apoptotic program via autoproteolytic cleavage of initiator caspases by
induced proximity (1).
TNF binds to two membrane receptors, TNFR1 (CD120a; p55/60) and TNFR2
(CD120b; p75/80). Whereas TNFR1 seems to be constitutively expressed in
most tissues, TNFR2 expression is more restricted and can be found
especially in immune cells but also in endothelial and neuronal tissue
(2). TNFR2 is a typical member of the non-death domain-containing
subgroup of the TNF receptor family. It directly binds TRAF2, leading
to the activation of NF- Many aspects of the initial events during signal initiation of TNF,
however, are poorly understood. After ligand binding, TNF receptor
complexes are internalized or may be, alternatively, proteolytically
cleaved (5-7). Internalization has been shown to be important for
intracellular signal initiation by TNFR1 (8) but not for others like
Fas (CD95, APO-1) (8, 9). Further, in some cellular systems the
activation of the initiator caspase-8 was found to be necessary for Fas
cluster formation (9).
Remarkably, most ligands of the TNF family are expressed as type II
transmembrane proteins from which soluble ligands are formed by
proteolytic cleavage. In a recent publication, we have compared the
signaling capacity of the membrane-bound (memTNF) versus the
soluble form of TNF (sTNF) and have demonstrated that TNFR2, but not
TNFR1, differentially responds to these two ligand forms (10). It was
shown that memTNF, when acting on TNFR2, displays a superior capacity
to initiate various cellular responses in a positive cooperative manner
with TNFR1. Full TNFR2 activation can even cause a shift in the
phenotype of the respective cellular response to sTNF (10). In
addition, we have developed a TNFR2-specific monoclonal antibody,
termed 80M2, that mimics the bioactivity of memTNF when combined with
sTNF. Kinetic studies with iodinated TNF revealed that 80M2 stabilizes
ligand binding in terms of a prolonged receptor complex half-life (10).
These data raised the hypothesis that the kinetics of receptor ligand
complex formation and disintegration might at least in part determine
intracellular signaling strength. Accordingly, transient binding of
sTNF to TNFR2 would only allow formation of short living complexes that may inefficiently induce intracellular signals (10). On the other hand,
the bioactivity of 80M2 is dependent on its dimeric IgG1 structure,
since Fab fragments derived thereof are inactive (data not shown).
These data raised the possibility that secondary clustering of
TNF·TNFR2 complexes by 80M2 might reflect the underlying mechanism by which memTNF gains superior signaling capability on TNFR2.
Differences in the bioactivity of soluble and membrane-bound forms of
other members of the TNF ligand family have also been described
(e.g. for Fas ligand (FasL) (11), CD40L (12), and TRAIL
(13)). Soluble FasL can even exert antiapoptotic activity by serving as
an antagonist for the membrane-bound form of FasL (11).
In this study, we have analyzed receptor chimeras derived from the
extracellular domains of the two TNF receptors and the intracellular
domain of Fas. TNFR2-Fas chimera, comprising the extracellular domain
of TNFR2 and the cytoplasmic part of Fas, strongly induced apoptosis
only after treatment with memTNF-like stimuli but not with sTNF. In
contrast, both ligand forms induced a strong apoptotic signal in
TNFR1-Fas chimeras. These data show that the individual responsiveness
of TNFR1 and TNFR2 for soluble or membrane-bound TNF can be transferred
to a distinct intracellular signaling system, indicating that
responsiveness is dominantly controlled by the extracellular domains.
Cell Culture and Reagents--
HeLa cells stably transfected
with human TNFR2 (HeLa80) or TNFR2 plus Fas (HeLa80Fas) (14, 15)
and Chinese hamster ovary (CHO) cells expressing TNF Measurement of the Metabolic Activity by
Microphysiometry--
Description and operation of the Cytosensor
microphysiometer (Molecular Devices Corp., Sunnyvale, CA) have appeared
elsewhere (21, 22). HeLa80Fas (3 × 105 cells) were
seeded into chambers (Molecular Devices). The cell capsule was placed
into a flow- and temperature (37 °C)-regulated sensing chamber of
the microphysiometer, and pH changes were monitored. Cells were
perfused with low phosphate-buffered RPMI medium (Irvine Scientific,
Irvine, CA) in a cyclic manner. The pump cycle was 120 s,
comprising a flow-on period (100 µl/min for 80 s) followed by a
flow-off period (40 s). The extracellular acidification rate was
mathematically normalized to 100% at the data point just prior to
stimulation with TNF143N/145R (150 ng/ml) (basal acidification rate).
The addition of mAb 80M2 (2 µg/ml) was performed 30 min prior to
treatment with TNF mutants and extended to the whole TNF stimulation
time of 30 min.
Generation of Stably Transfected Cell Lines--
Expression
constructs encoding the fusion proteins TNFR1-Fas and TNFR2-Fas were
generated by PCR cloning. A KpnI site was introduced by
silent mutagenesis into the coding region of pBS-TNFR1 and pBS-TNFR2 at
bp 704 and 899, respectively, 3' of the potential transmembrane region.
In addition, the cytoplasmic region of Fas was amplified by PCR
introducing 5' and 3' appropriate restriction sites for TNFR ligation
(pBS TNFR1-Fas and pBS TNFR2-Fas). TNFR1-Fas and TNFR2-Fas were
subcloned into the expression vector pEF PGKpuro (23), using
BamHI and EcoRV, generating pEFpuroTNFR1-Fas and pEFpuroTNFR2-Fas. All constructs generated by PCR were verified by
sequencing. Immortalized mouse fibroblasts (4 × 105
cells) from TNFR1 and TNFR2 double knockout cells were transfected with
pEFpuroTNFR1-Fas or pEFpuroTNFR2-Fas using LipofectAMINE Plus
(Invitrogen) according to the manufacturer's recommendations. The day
after, cells were selected for stably expressing cells by 1-5 µg/ml
puromycin A, and 2 weeks later they were sorted for TNFR1-Fas- and
TNFR2-Fas-positive cells by flow cytometry using a FACStar+
(Becton Dickinson, San Jose, CA). Briefly, 5 × 105
cells were harvested and resuspended in PBA (0.025% bovine serum albumin, 0.02% NaN3 in PBS) containing mouse 5 µg/ml
anti-huTNFR1 (Htr 9) or mouse anti-TNFR2 antibodies (MR2-1). After
incubation for 1 h at 4 °C, cells were washed once with PBA,
resuspended in PBA containing secondary fluorescein
isothiocyanate-labeled goat anti-mouse IgG plus IgM antibodies
(Dianova, Germany), and incubated at least for 30 min at 4 °C. Cells
were washed again and subjected to fluorescence-activated cell sorting.
10,000-30,000 positive cells were collected and grown in cell culture
medium containing 1 µg/ml puromycin A. TNFR1-Fas and TNFR2-Fas
expressing mouse fibroblasts (MF-R1-Fas and MF-R2-Fas cells,
respectively) were stable for at least 3 weeks.
Cell Death Assays--
Mouse fibroblasts (1.5 × 104 cells/well) were grown in 96-well plates overnight.
Cells were then treated as indicated and cultivated overnight. The next
day, cells were washed three times with PBS followed by crystal violet
staining (20% methanol, 0.5% crystal violet) for 15 min. The wells
were washed with H2O and air-dried. The dye was resolved
with methanol for 15 min, and optical density at 550 nm was determined
with an enzyme-linked immunosorbent assay plate reader (SPECTRAmax
340PC; Molecular Devices).
For coculture experiments, CHO cells (1.5 × 105) were
seeded into a 12-well plate. The next day, MF-R2-Fas cells (1.5 × 105) were given on top and cultivated at 37 °C. Cells
were visualized by light microscopy, and pictures were taken 1 h
after seeding of the mouse fibroblasts.
Coimmunoprecipitation and Western Blotting--
HeLa80Fas cells
(5 × 106 cells) were pretreated where indicated with
antagonistic TNFR1-Fab fragment (H398-Fab) for 30 min followed by
stimulation with sTNF (100 ng/ml) or Cys-TNF (116 ng/ml). After
incubation at 37 °C for the indicated times, cells were washed with
ice-cold PBS and scraped off the plate in PBS. Cells were pelleted and
lysed in 300 µl of lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 30 mM NaF, 0.5 mM phenylmethylsulfonyl fluoride,
protease inhibitor mix (Roche Molecular Biosciences). Proteins were
extracted by vortexing three times for 40 s, and TNFR2 complexes
were immunoprecipitated for 3 h at 4 °C while tumbling. Immune
complexes were washed in lysis buffer and assessed by Western blotting
using TRAF2-specific antibodies. Proteins were visualized by
chemiluminescence (Super Signal; Pierce).
Electrophoretic Mobility Shift Assays of NF- Immunocomplex JNK Assay--
JNK assays were performed basically
as described (25). Briefly, following stimulation, cells (5 × 106 cells) were lysed in kinase lysis buffer (20 mM Tris, pH 7.4, 5 mM MgCl2, 1%
Triton X-100, 150 mM NaCl, 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM NaVO4, and
1 mM NaF) by sonification. JNK was immunoprecipitated with
0.6 µg of JNK-specific antiserum (Santa Cruz Biotechnology) and
subjected to kinase assays using GST-Jun-(5-89) (0.5 µg/assay) and 5 µCi of [32P]ATP as substrate. The
reactions were carried out in assay buffer (20 mM MOPS, pH
7.2, 10 mM EGTA, 2 mM MgCl2, 0.1%
Triton X-100, and 1 mM dithiothreitol) at 37 °C for 20 min.
Transient Transfections and Confocal Microscopy--
Cells were
harvested and transiently transfected with 10 µg of expression
plasmids of pFADD-EGFP and pTRAF2-EGFP, respectively, or with 4 µg of
pCaspase-8(C369S)-EGFP plus 4 µg of murine pFADD by electroporation.
Cells (800 µl; 1.25 × 106 cells/ml) were
electroporated at 1800 microfarad and 250 V in a 0.4-cm cuvette (Peqbio
Easyject Plus; Peqlab). After electroporation, cells were immediately
transferred into cell culture medium, and 3 × 105
cells/dish (35 mm; Maltek) were grown for 18 h before analysis. Electroporation with pCaspase-8(C369S)-EGFP required the addition of 20 µM z-VAD-fmk. For live imaging TNF, mAb 80M2 and
Cys-TNF143N/145R were coupled with AlexaFluor-546 (Molecular Probes,
Inc., Eugene, OR) according to the manufacturer's instructions. Cells
were washed in PBS and, where indicated, preincubated with
AlexaFluor-546-coupled 80M2 (80M2(red)) (6 µg/ml) for 4 min at room temperature or 1.7 µg/ml AlexaFluor-546-coupled sTNF
(sTNF(red)) on ice followed by two washings with cell
culture medium without phenol red. Cells were placed into a chamber
held constantly at 37 °C and 5% CO2 and treated where
indicated with 300 ng/ml TNFR2-specific TNF mutant (Cys-TNF143N/145R),
and pictures were taken at the indicated time points. Alternatively,
105 cells were seeded onto coverslips in a 24-well plate
and treated as above. Instead of performing live imaging of the cells,
cells were washed with ice-cold PBS, fixed with 3.5% paraformaldehyde in PBS for 15 min at 37 °C at various time points, mounted with Flouromount-G (Biozol, Germany) onto glass slides, and examined using a
Leica DM IRBE confocal immunofluorescence microscope. Pictures were
taken at a resolution of 1024 × 1024 pixels with a magnification
of ×630 for live imaging and ×1000 for fixed cells.
Binding Kinetics--
Receptor-ligand studies were performed as
described (26). Briefly, TNF was labeled with 125I by the
chloramine-T method. Murine fibroblasts were incubated with 0.2 nM 125I-TNF in the presence or absence of mAb
80M2 (2 µg/ml) for 1 h on ice. Cells were incubated for several
time periods at 37 °C, and cell-bound 125I-TNF was
determined after centrifugation of the cells through a phthalate oil mixture.
Construction of Fibroblasts Expressing Chimeric TNF
Receptor/Fas Proteins--
To get a better understanding
for the molecular basis of the differential signaling capacity of sTNF
and memTNF, we constructed chimeras of Fas and TNFR1 and TNFR2,
respectively. These fusion proteins consist of the extracellular and
transmembrane region of the respective human TNFRs fused to the
cytoplasmic domain of human Fas (Fig.
1A). Hybrid constructs
TNFR1-Fas and TNFR2-Fas were stably expressed in large T
antigen-immortalized fibroblasts derived from TNFR1/TNFR2 double
knockout animals, obtaining a cellular system devoid of any TNF
background responsiveness. Fluorescence analyses of the TNFR1-Fas- and
TNFR2-Fas-expressing mouse fibroblast cells, MF-R1-Fas and MF-R2-Fas,
respectively, are shown in Fig. 1B. Equilibrium binding
studies using iodinated TNF revealed ligand binding sites of 15,000 for
MF-R1-Fas cells and 45,000 for MF-R2-Fas cells per cell (data not
shown).
Induction of Apoptosis in MF-R1-Fas and MF-R2-Fas Cells--
As
expected, we could not detect any TNF responsiveness in the parental
mouse fibroblasts devoid of both mouse TNF receptors (data not shown).
MF-R1-Fas cells, however, developed a strong cytotoxic response after
treatment with serial dilutions of sTNF (Fig.
2A). Development of cell death
was nearly maximum at TNF concentrations as low as 1 ng/ml, and the
majority of the cells showed typical blebbing already after 3 h of
sTNF treatment (data not shown). Further, cell death could be blocked
by the inhibitor z-VAD-fmk, demonstrating the involvement of caspases
(data not shown). Wild type TNFR1 is equally well activated by sTNF and memTNF (10). We confirmed this for TNFR1-Fas using the TNF mutein Cys-TNF32W/86T, derived from a TNFR1-specific mutant of sTNF (27), which allows additional receptor cross-linking due to the
formation of cysteine-linked multimers (Fig. 2A). The
respective TNFR2-specific mutein of TNF, Cys-TNF143N/145R, did not
induce apoptosis in MF-R1-Fas cells (Fig. 2A).
In a parallel set of experiments, we investigated the chimeric receptor
TNFR2-Fas. MF-R2-Fas cells were treated with sTNF up to concentrations
of 300 ng/ml, but no significant cytotoxic response could be observed
after overnight culture (Fig. 2B). In the presence of the
antibody 80M2, however, a strong cytotoxic response was observed with a
half-maximum effect at a TNF concentration of about 100 pg/ml, whereas
80M2 on its own was not toxic (Fig. 2B). Cell death
developed rapidly; most cells showed massive signs of disintegration
after 1 h of memTNF-like stimulation (data not shown). Again,
induction of cytotoxicity could be efficiently blocked with the pan
caspase inhibitor z-VAD-fmk (data not shown). The TNFR2-selective
derivative Cys-TNF143N/145R also induced a significant apoptotic
response, although with reduced efficacy when compared with sTNF plus
80M2 (Fig. 2B). In contrast, the TNFR1-specific mutein
Cys-TNF32W/86T was ineffective (Fig. 2B). To confirm that
the transmembrane form of TNF is also able to induce apoptosis in
MF-R2-Fas cells, these were cocultured for 1 h with CHO cells
expressing a mutant form of memTNF that cannot be processed by the
tumor necrosis factor Gene Induction Pathways Initiated by TNFR-Fas
Chimeras--
Although Fas represents the prototype of a death
receptor, it is also known to activate gene expression (e.g.
via the activation of the transcription factor NF- Recruitment of FADD to TNF Receptor/Fas
Chimeras--
We then asked whether the observed differences in the
signaling strength of the various ligand/receptor chimera combinations are mirrored at the level of RISC formation. One of the first intracellular reactions after ligand-induced oligomerization of Fas is
the recruitment of FADD, leading to caspase-8 activation. We therefore
monitored transiently expressed human FADD-EGFP in mouse fibroblast
cell lines by confocal microscopy after stimulation with
AlexaFluor-546-labeled TNF receptor agonists. Fig.
3 shows the overlays of the green and the
red fluorescence observed. As expected, MF-R1-Fas cells transiently
transfected with FADD-EGFP showed a green cytosolic fluorescence (Fig.
3A). These cells had been preincubated with
Alexa-546-labeled sTNF, detectable as a weak red cell surface staining.
No prominent colocalization with FADD-EGFP at the cell surface can be
observed. After incubation of the cells for 15 min at 37 °C,
however, the majority of the TNFR1-Fas-bound sTNF had formed clusters
mostly colocalized with FADD-EGFP (Fig. 3B). These data
demonstrate that sTNF is able to recruit significant amounts of
FADD-EGFP to TNFR1-Fas molecules, leading to the formation of large
RISC aggregates.
In contrast, the respective sTNF treatment of MF-R2-Fas cells did not
reveal a significant colocalization of sTNF and FADD-EGFP, and no signs
of cell surface located cluster formation could be observed (Fig. 3,
C and D). Stimulation for only 2-8 min with a
memTNF-like agent, however, consisting of sTNF and the red fluorescent antibody 80M2, induced rapid formation of cell surface-associated clusters of colocalized TNFR2-Fas and FADD-EGFP (data not shown; see
Fig. 3, E and F). Similar results were obtained
using MF-R2-Fas cells transiently transfected with
pCaspase-8(C360S)-EGFP, expressing an catalytic inactive caspase-8, and
murine FADD expression constructs in the presence of z-VAD-fmk. Again,
sTNF was able to induce recruitment of caspase-8 to TNFR1-Fas but not
to TNFR2-Fas chimeras (data not shown). In contrast, memTNF-like
stimuli were efficient in both cellular systems (data not shown; Fig.
3, G and H). Together, these data demonstrate
that the different signaling capacities of the two TNF forms are
directly reflected at the level of RISC formation.
Enhanced Recruitment of TRAF2 to Wild-type TNFR2 by memTNF--
We
next investigated whether a lack of significant adaptor protein
recruitment to TNFR2-Fas by sTNF can also be observed in wild type
TNFR2-positive cells. In HeLa cells stably overexpressing TNFR2
(HeLa80), transiently expressed TRAF2-EGFP was also primarily located
in the cytosol (Fig. 4, upper
panel). The addition of the TNFR2-selective sTNF mutant
sTNF143N/145R, marked with a red fluorescing dye, revealed a staining
of the plasma membrane but did not result in any visible recruitment of
TRAF2-EGFP to the cell membrane (Fig. 4A, upper
panel). Cellular stimulation with sTNF143N/145R in the
presence of the antibody 80M2, however, resulted in the strong
formation of membrane-associated TRAF2-EGFP aggregates showing
colocalization with TNFR2 (Fig. 4A, lower
panel). Significant TRAF2-EGFP recruitment could also be
induced using the secondary cross-linked TNFR2-selective TNF mutein
Cys-TNF143N/154R (data not shown). Similar data were obtained using
mouse fibroblasts transfected with wild type TNFR2 and TRAF2-EGFP (data
not shown).
To study TRAF2 recruitment to TNFR2 after memTNF-like stimulation also
at physiological TRAF2 levels, we assessed coimmunoprecipitation studies with endogenously expressed TRAF2. TNFR2 was immunoprecipitated from HeLa cells, stably expressing TNFR2, and the precipitates were
investigated for TRAF2 by Western blotting. 15 and 30 min after
stimulation of the cells with sTNF, only slightly enhanced TRAF2
amounts were detectable in the immunoprecipitates as compared with
unstimulated cells (Fig. 4B). TRAF2 coimmunoprecipitation could not be blocked with a TNFR1-specific antagonistic Fab fragment (
We verified that enhanced adaptor recruitment to wild type TNFR2, as
depicted in Fig. 4, A and B, is in fact
paralleled by an enhanced cellular response. To this, HeLa80 cells and
KYM-1 cells, known to activate NF- Ligand Association and Dissociation Studies--
In a previous
publication, we showed that the enhanced signaling capacity of sTNF in
the presence of 80M2, representing a memTNF-like stimulus, correlates
with the stabilization of ligand-receptor complexes due to a strongly
reduced dissociation rate (10). Using MF-R1-Fas and MF-R2-Fas cells,
respectively, association and dissociation studies with iodinated sTNF
at 37 °C were performed. The association kinetics of iodinated sTNF
to both receptors were rapid and similar, with half-maximum binding
after about 2 min for TNFR1-Fas and about 1 min for TNFR2-Fas at a
ligand concentration of 0.3 nM (data not shown). The
presence of the antibody 80M2 did not significantly change ligand
association kinetics of TNFR2 (kon(sTNF) = 3.0·109 M In contrast to TNFR1, which is equally well activated by both sTNF
and memTNF, TNFR2 can only be efficiently activated by memTNF.
To address the molecular mechanisms underlying these different bioactivities, we have constructed receptor chimeras containing the
extracellular domains of the TNF receptors fused to the intracellular part of Fas. In the present study, we demonstrate that these chimeric receptors exhibit identical activation requirements regarding the two
TNF forms as the wild type TNF receptors. Moreover, our data suggest
that the half-life of TNF·TNFR complexes becomes translated into the
efficiency of intracellular adaptor recruitment, thus controlling the
intensity of the transmitted signal.
Experimental systems quantitatively assessing the signal capacity of
cell surface expressed memTNF are difficult to handle. We therefore
used available tools that mimic memTNF signaling. We have recently
described the TNFR2-specific monoclonal antibody 80M2 that, in
combination with sTNF, induces intracellular signals comparable with
the transmembrane form of TNF (10). Furthermore, we have utilized
muteins of sTNF that form intermolecular disulfide bonds via an
N-terminal cysteine residue (Cys-TNF and the receptor-specific derivatives Cys-TNF32W/86T and Cys-TNF143N/145R), resulting in the
formation of secondary cross-linked TNF trimers. These TNF derivatives
also show an enhanced signaling capacity via TNFR2 as compared with
sTNF (Fig. 2B).2
To obtain a suitable molecular system for investigation of sTNF and
memTNF action, we transferred the extracellular domains of the two TNF
receptors to the cytoplasmic part of Fas, another TNF receptor family
member. A simple exchange of the intracellular TNF receptor domains
turned out to be inappropriate due to the constitutive cytotoxic
activity of the intracellular region of TNFR1 (data not shown). To omit
problems with endogenous TNF responsiveness, receptor chimeras were
expressed in a fibroblast-derived cell line from TNFR1/TNFR2 double
knockout mice. As expected, both chimeric receptors, TNFR1-Fas and
TNFR2-Fas, could be expressed in quite high numbers in these cells,
with about 15,000 and 45,000 TNF binding sites for MF-R1-Fas and
-R2-Fas cells, respectively (data not shown). In the absence of any TNF
receptor-specific stimulus, both cell lines proliferate well without
indications of spontaneous apoptosis (data not shown).
In various studies on TNFR1- and Fas-expressing cells, conflicting
results have been obtained regarding the involvement of membrane rafts
or the requirement of internalization for signal initiation. Recently,
the possible arrangement of Fas in lipid rafts was shown (31, 32),
which may be true for type II but not for type I cells (9, 33).
Furthermore, Fas signaling has been reported to be independent
of receptor complex internalization (8, 9), whereas TNFR1 was not (8).
In the present study, both TNFR-Fas chimeras have been expressed in
reasonable receptor numbers in mouse fibroblasts and are therefore
supposed to induce a very strong initial signal upon appropriate
stimulation. This is in accordance with the rapid TNF-induced
morphological changes, typical for apoptosis, within 1 (TNFR2-Fas) to
3 h (TNFR1-Fas). In addition, pretreatment of MF-R2-Fas cells with
methyl- The Studies Performed with the TNFR-Fas Chimeras Allow Two Direct
Conclusions--
First, the differential response pattern of the two
TNF receptors to sTNF and memTNF could be fully transferred to the Fas signaling system (i.e. in the case of TNFR2 from a gene
inductory pathway, acting via TRAF2 binding, to the apoptotic pathway
of Fas, acting via FADD mediated caspase-8 activation). Identical patterns were found for three different cellular responses
(i.e. the induction of apoptosis, activation of
NF-
Second, exogenously initiated cross-linking of ligand-receptor
complexes is not necessary for induction of full signaling and receptor
cluster formation. Cross-linking reagents like antibodies are commonly
used for the efficient signal induction of Fas, TNFR2, or CD40 (11, 12,
16). Due to their multivalent nature, the treatment of cells with
antibodies is paralleled by the formation of large receptor clusters
(32, 35). However, the functional role of these clusters for signal
initiation and strength has not been fully elucidated. In our studies,
the efficient recruitment of FADD and TRAF2, respectively, is also
paralleled by the formation of large receptor clusters, visible by
colocalization of receptors with EGFP-tagged adaptor proteins (Figs. 3
(B, F, and H) and 4A) and
in coimmunoprecipitation studies (Fig. 4B). This cluster
formation always correlated with the particular signaling strength. In
the case of TNFR2 and the TNFR2-derived Fas chimera, exogenously
initiated cross-linking of ligand-receptor complexes was necessary for
induction of full signaling and receptor cluster formation (Figs. 3
(F and H) and 4A). However, sTNF on
its own is sufficient to initiate a strong intracellular signal via
TNFR1-Fas, whereas in parallel triggering the formation of large
receptor clusters (Figs. 2A and 3B). Since there
are no observable differences in the efficiency of sTNF and memTNF upon
TNFR1 stimulation (10),3 the
results argue against a mandatory role of an external, additional cross-linking agent for full activation of a given receptor. Recent observations by the group of Peter, demonstrating that formation of
large Fas clusters is not directly dependent on the cross-linking properties of the stimulating agent (9), are consistent with these observations.
As discussed above, cluster formation occurs in parallel with enhanced
signaling and is independent of the cytoplasmic part of the TNF
receptors. It is therefore likely that the extracellular parts of
ligand-bound receptors mediate cluster formation via additional
interactions possibly involving supplementary molecules. Since the
TNFR-Fas chimeras used in our studies contain the transmembrane parts
of the respective TNF receptors, the possibility cannot be excluded
that these domains are involved in receptor aggregate formation. When
chimeric fusion proteins comprising the extracellular domain of the
erythropoietin receptor and the intracellular part from TNFR2 were
investigated, an exchange of the respective transmembrane domains had
moderate effects on the signaling capacity (36). However, the present
structural data of ligand trimerized receptors indicate that the
transmembrane domains are unlikely to directly interact with each other
between individual complexes (37, 38). More likely, a direct
interaction of the ligated extracellular receptor domains might occur.
Based on the dimeric crystal structure of the extracellular part of
TNFR1, such a cluster formation has been already proposed by Naismith
et al. (39). A possible candidate for receptor/receptor
interaction is the recently defined preligand assembly domain,
identified in both TNF receptors and in Fas, which is most likely
present also in other members of the TNF receptor family (40).
What mechanisms beside additional cross-linking of receptor complexes
could determine the enhanced signaling capability of memTNF? We propose
that the stability of individual receptor-ligand complexes could be the
important factor. Interactions of sTNF with TNFR1 and TNFR2 differ
strongly in this regard. Whereas sTNF forms very stable complexes with
TNFR1 (half-life = 33 min) (10) and TNFR1-Fas (half-life = 32 min) (Fig. 6A), ligation of TNFR2 (half-life = 1.1 min)
(10) and TNFR2-Fas (half-life = 1.2 min) (Fig. 6B)
occurs only very transiently. These differences are most likely
inherent properties of the TNF/TNF receptor interactions rather than
caused by different stoichiometries of complexes or subsequent steps
like receptor cluster formation. This is evident from studies with TNF
receptor-derived IgG fusion proteins that revealed very similar results
(41). The exchange rates of radiolabeled sTNF complexed with TNFR-IgG
fusion proteins showed a half-life of about 7 min for TNFR2-derived
complexes, whereas TNFR1 complexes were extremely stable
(half-life = 8 h) (41). Accordingly, the stability of
individual ligand receptor complexes, most likely comprising three
receptor molecules, would control subsequent steps leading to the
formation of large, stable clusters, capable of effectively recruiting
adaptors and initiating a strong intracellular signal. Certain
conditions must be met, however. First, the initial sTNF binding must
be rapid as compared with the subsequent steps. This holds true, since
sTNF binding occurs very fast (26) and is mainly controlled by
diffusion.3 Second, receptor cluster formation should be
dependent on preformed ligand-receptor complexes and should not occur
spontaneously with unligated receptors. Most likely, this also holds
true, since we observe no receptor patches directly after ligand
binding on ice in our microscopy studies (Figs. 3 and 4). Third,
formation of large receptor clusters must stabilize ligand binding in
TNFR2 and TNFR2-Fas molecules. This also seems very reasonable, since integration of single receptor-ligand complexes into a lattice-like structure should inhibit dissociation of individual ligand-receptor complexes. We therefore suggest a causal relationship between the
stability of individual receptor complexes and the efficiency of
adaptor protein recruitment, the latter being directly translated into
signaling strength. This principle might hold true for many, if not
all, members of the TNF receptor family but is not necessarily restricted to these molecules.
We thank I.-W. von Broen (Knoll AG,
Ludwigshafen, Germany) for recombinant TNF, D. Männel for
murine fibroblasts, M. Lenardo for pCaspase-8-EGFP and pFADD-EGFP, H. Wajant for pTRAF2-EGFP, and A. Strasser (The Walter and Eliza Hall
Institute, Melbourne, Australia) for pEF PGKpuro. We also thank K. Pfizenmaier and H. Wajant for helpful discussion.
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant GR 1307/3-3 and Sonderforschungsbereich 495, project A4.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, September 4, 2002, DOI 10.1074/jbc.M207399200
2
A. B. Hammer, J. Gerspach, P. Scheurich,
and K. Pfizenmaier, manuscript in preparation.
3
A. Krippner-Heidenreich, F. Tübing, S. Bryde, S. Willi, G. Zimmermann, and P. Scheurich, unpublished data.
The abbreviations used are:
TNF, tumor necrosis
factor;
TNFR, TNF receptor;
EGFP, enhanced green fluorescent protein;
FADD, Fas-associating protein with death domain;
JNK, c-Jun
amino-terminal kinase;
mAb, monoclonal antibody;
MF, mouse fibroblast;
RISC, receptor-induced signaling complex;
TRAF, TNF receptor-associated
factor;
z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp
fluoromethyl ketone;
TRADD, TNF receptor-associated death
domain protein;
memTNF, membrane-bound TNF;
sTNF, soluble TNF;
CHO, Chinese hamster ovary;
PBS, phosphate-buffered saline;
MOPS, 4-morpholinepropanesulfonic acid.
Control of Receptor-induced Signaling Complex
Formation by the Kinetics of Ligand/Receptor Interaction*
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B
via the TNFR2-derived chimera. In contrast, the TNFR1-Fas chimera
displayed strong responsiveness to both TNF forms. This pattern of
responsiveness is identical to that of wild type TNF receptors,
demonstrating that the underlying mechanisms are independent of the
particular type of the intracellular signaling machinery and rather are
controlled upstream of the intracellular domain. We further demonstrate
that the signaling strength induced by a given ligand/receptor
interaction is regulated at the level of adaptor protein recruitment,
as shown for FADD, caspase-8, and TRAF2. Since both incidents, strong
signaling and robust adapter protein recruitment, are paralleled by a
high stability of individual ligand-receptor complexes, we
propose that half-lives of individual ligand-receptor complexes control
signaling at the level of adaptor protein recruitment.
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B and the c-Jun N-terminal kinase (JNK).
TNFR1 carries a death domain in its cytoplasmic part and therefore
represents a direct activator of apoptotic caspases after recruitment
of TRADD and FADD. In parallel to its cytotoxic activity (and this
seems to be unique within the TNF receptor family), TNFR1 is a strong
activator of gene induction. Receptor-bound TRADD serves as an assembly
platform also for recruitment of TRAF2 and receptor-interacting protein
(3), which act together in the activation of the inhibitor of B
kinases, leading to the activation of NF-B (4).
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1-12
(CHOTNF
1-12) (10) have been described elsewhere. Simian
virus 40 large T-immortalized murine fibroblasts have been generated
from TNFR1 and TNFR2 double knockout mice and were generously supplied
by Daniela Männel (University of Regensburg, Regensburg,
Germany). HeLa cells, CHO cells, and immortalized mouse fibroblasts
were grown in RPMI 1640 medium supplemented with 5% (v/v)
heat-inactivated fetal calf serum and 2 mM
L-glutamine. 1 µg/ml puromycin A was added once a week to
TNFR1-Fas and TNFR2-Fas expressing mouse fibroblasts routinely. Kym-1
cells were grown in Clicks-RPMI medium containing 10% fetal calf serum
and 2 mM L-glutamine (10). Recombinant human
TNF (2 × 107 units/mg) was provided by Knoll AG
(Ludwigshafen, Germany). Cys-TNF and mutants derived thereof
(Cys-TNF143N/145R and Cys-TNF32W/86T) have been generated in
Escherichia coli and purified to homogeneity using a
nickel-chelate column.2
The monoclonal mouse antibody 80M2 (16), H398 (17), and Htr9 (18) have been described. The TNFR2-specific monoclonal antibody MR2-1 was kindly provided by W. Buurman (University of Limburg, Maastricht, The Netherlands). Additional antibodies specific
for TNFR2 were purchased from R&D (goat anti-huTNFR2), TRAF2-specific antibodies (mouse anti-huTRAF2, clone C90-481) were from Pharmingen, and antibodies specific for JNK (rabbit anti-huJNK; C-17) were from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). z-VAD-fmk was
purchased from Bachem. The expression plasmids pFADD-EGFP and
pCaspase-8-EGFP were kind gifts from Michael Lenardo (National Institutes of Health, Bethesda, MD), and pTRAF2-EGFP was from Harald
Wajant (University of Stuttgart, Stuttgart, Germany), and they have
been described elsewhere (19, 20).
B
Activation--
5 × 105 cells/well HeLa80Fas cells
or mouse fibroblasts were seeded in six-well plates and grown
overnight. Where indicated, cells were pretreated with 2 µg/ml mAb
80M2 for 30 min. The cells were then stimulated for various times with
the indicated reagents. Nuclear extracts were prepared as described
(24), and samples were adjusted for identical protein levels. As probe,
[32P]ATP-end-labeled NF-B-specific oligonucleotides
(5'-AGTTGAGGGGACTTTCCCAGGC-3') were used.
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Fig. 1.
Stable expression of chimeric receptors in
mouse fibroblasts. A, schematic representation of
TNFR1-Fas and TNFR2-Fas chimeric proteins. The cytoplasmic domain of
Fas, amino acids 191-335, was fused to the C terminus of the potential
transmembrane region of TNFR1 (amino acids 236) or TNFR2 (amino acids
301). Amino acids in the fused region are indicated. B,
immortalized mouse fibroblasts were stably transfected with TNFR1-Fas
or TNFR2-Fas expression plasmids. Expression of the chimeras was
analyzed by flow cytometry using TNFR1-specific (Htr9) and
TNFR2-specific (MR2-1) antibodies. The percentage of TNFR-Fas-positive
cells is indicated.
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Fig. 2.
Response pattern of TNFR1-Fas and TNFR2-Fas
expressing cells to sTNF and memTNF. A and
B, MF-R1-Fas (A) and MF-R2-Fas (B)
cells were treated with serial dilutions of sTNF or TNFR1-specific
(Cys-TNF32W/86T) and TNFR2-specific (Cys-TNF143N/145R) TNF mutants,
respectively. For costimulation with the monoclonal antibody 80M2,
cells had been preincubated with 2 µg/ml 80M2 for 30 min at 37 °C
before sTNF treatment. Cell viability was determined by crystal violet
staining the next day. All experimental groups shown were performed in
parallel, one representative experiment out of three is shown.
C, wild type Chinese hamster ovary (CHOwt) cells
(left) or CHO cells stably expressing a noncleavable form of
memTNF (CHOTNF 1-12) (right) were grown
overnight. MF-R2-Fas cells were seeded on top, and induction of
apoptosis was followed by light microscopy. Pictures were taken after
1 h of coculture. The percentage of apoptotic mouse fibroblasts
was calculated after counting about 200 cells. Bars, 75 µm. D and E, MF-R1-Fas (D) and MF-R2-Fas
(E) cells were treated with TNF (100 ng/ml), CysTNF (112 ng/ml), without or after pretreatment with mAb 80M2 (2 µg/ml) for 30 min as indicated. Cell lysates were prepared directly before (
) or
after the indicated time points of TNF stimulation, and JNK activity
was measured by immunocomplex kinase assay with GST-c-Jun-(5-89) as a
substrate (upper panels), or the translocation of
NF-B was investigated from isolated nuclei by gel shift analysis
(lower panels). The positive control (pos.
c) corresponds to HeLa cells stimulated with TNF (100 ng/ml) for
30 min. The specificity was controlled by the addition of a 100-fold
excess of unlabeled oligonucleotides (100×
cold).
-converting enzyme (10). These cells (Fig.
2C, right panel), but not control CHO cells (Fig. 2C, left panel), induced a
strong cytotoxic response in TNFR2-Fas-expressing mouse fibroblasts.
When apoptotic cells were counted after 3 h of coculture,
<5% of apoptotic MF-R2-Fas cells were determined in cocultures with
control CHO cells, whereas between 68 and 79% (n = 3)
of MF-R2-Fas cells showed an apoptotic phenotype in cocultures with
memTNF-expressing CHO cells (data not shown). These values are in good
agreement with the percentage of the MF-R2-Fas cells expressing high
numbers of chimeric receptors as estimated from flow cytometry data
(Fig. 1B). As expected, memTNF-expressing CHO cells were
also toxic for MF-R1-Fas cells (data not shown). In summary, these
results show that the divergent responsiveness of TNFR1 and TNFR2 to
sTNF is independent of the cytoplasmic domain of the receptors, since
it is transferable to the intracellular part of Fas.
B and activation
of mitogen-activated protein kinases) (25, 28). To investigate whether
the differential responsiveness of TNFR2-Fas to sTNF and memTNF also
holds true for noncytotoxic Fas responses, we analyzed the activation
of NF-B and JNK in both MF-R1-Fas and MF-R2-Fas cells after
treatment with sTNF and memTNF-like stimuli. The activations of NF-B
and JNK were investigated by electromobility shift assays and
immunocomplex kinase assays, respectively, revealing an identical
response pattern for both cellular responses (Fig. 2, D and
E) as compared with the induction of apoptosis (Fig. 2,
A and B). These results strongly suggest that the
molecular basis of the difference in signal initiation by memTNF and
sTNF is at or upstream of the formation of the receptor-induced signaling complex (RISC). In accordance with literature data, JNK
activation induced by TNFR-Fas chimera could be blocked by z-VAD-fmk (25, 29, 30), implicating the dependence on the activation of caspases, whereas nuclear translocation of NF-B was
rather augmented by this caspase inhibitor (data not shown).
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Fig. 3.
Differential RISC formation of TNFR-Fas
chimeras. MF-R1-Fas (A and B) or MF-R2-Fas
(C-H) cells were transiently transfected with constructs
expressing human FADD-EGFP (A-F) or human
caspase-8(C360S)-EGFP plus murine FADD in the presence of 20 µM z-VAD-fmk (G and H). The day
after, cells were pretreated with Alexa 546-labeled 80M2
(80M2(red); 6 µg/ml) (E-H) or sTNF
(TNF(red); 1.7 µg/ml) (A-D) for 4 min on ice
followed by washing with PBS. Subsequently, unlabeled sTNF (40 ng/ml)
was added to the 80M2(red)-treated cells, and cells were
examined by live imaging (A-F) or fixed at the indicated
time points (G and H) and examined using a
confocal laser-scanning microscope.
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Fig. 4.
Recruitment of TRAF2 by stimulation of TNFR2
with a memTNF-like agent but not with sTNF. A, HeLa
cells, stably expressing TNFR2 (HeLa80), were transiently transfected
with constructs expressing huTRAF2-EGFP. The day after, cells were
pretreated with Alexa 546-labeled mAb 80M2 (6 µg/ml) or
TNFR2-specific TNF (sTNF143N/145R; 1.7 µg/ml) for 4 min on ice
followed by washing with PBS. Subsequently unlabeled membrane-like TNF
(Cys-TNF143N/145R) was added to the mAb 80M2-treated cells. Live
imaging was performed with a confocal microscope, and pictures were
taken at the indicated times. B, HeLa cells stably
expressing TNFR2 plus Fas (HeLa80Fas) were treated with sTNF (100 ng/ml) or Cys-TNF143N/145R (116 ng/ml) for the indicated times,
followed by cell lysis. Where indicated, cells had been pretreated with
antagonistic TNFR1-Fab fragments ( TNFR1-Fab; 14 µg/ml) to prevent
binding of sTNF to TNFR1 and subsequent TRAF2 recruitment. Lysates were
subjected to coimmunoprecipitation with TNFR2-specific antibodies, and
Western blot analysis was performed using TRAF2-specific antibodies. As
controls, TNFR2-specific antibodies (control IgG) and lysates from
cells overexpressing huTRAF2 (TRAF2) were used.
TNFR1-Fab), capable of inhibiting TNF binding to TNFR1 (Fig. 4B). In parallel experiments, we used TNFR2-specific
Cys-TNF143N/145R for a memTNF-like stimulation of TNFR2.
Immmunoprecipitates from these cells contained significantly larger
amounts of TRAF2 as compared with that obtained from sTNF-treated cells
(Fig. 4B), confirming the data obtained by confocal
microscopy (Fig. 4A). Together, these results show that also
in wild type TNFR2, the enhanced signaling capacity of memTNF-like
stimuli, like Cys-TNF143N/145R, is linked to an enhanced recruitment of
adaptor proteins and not dependent on overexpression of these
intracellular signaling molecules.
B after appropriate stimulation
via TNFR2 (16, 20), were examined for the activation of NF-B by
electrophoretic mobility shift assay. Nuclear translocation and DNA
binding of NF-B was determined after 30 min of receptor stimulation
(i.e. within the same time range also investigated in the
confocal microscopy experiments). These experiments revealed a similar
stimulatory capacity of TNFR1 and TNFR2 for both cell lines (Fig.
5A). The TNFR2-selective
sTNF143N/145R on its own showed only a marginal, if any, capacity to
activate NF-B, whereas the TNFR2-selective Cys-TNF143N/145R
possessed an intermediate stimulatory capacity. This confirms that in
HeLa and KYM-1 cells a memTNF-like activity is mandatory for full
activation of TNFR2, whereas sTNF is sufficient to fully activate
TNFR1. Finally, in TNFR2-expressing HeLa cells the overall metabolic
response to a selective stimulation of TNFR2 by sTNF in the absence and
presence of 80M2 was determined using a microphysiometer. The results
confirm a strong cellular (i.e. metabolic) response
initiated by TNFR2 only when stimulated with sTNF143N/145R in the
presence of 80M2 (Fig. 5B).
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Fig. 5.
Enhanced NF- B
activation and metabolic activity induced by memTNF-like stimuli.
A, KYM-1 cells (top) and HeLa cells expressing
TNFR2 (HeLa80, bottom) were treated with various sTNF-like
stimuli (sTNF, 30 ng/ml; TNFR1-specific sTNF32W/86T, 30 ng/ml;
TNFR2-specific sTNF143N/145R, 300 ng/ml) or memTNF-like stimuli
(Cys-TNF, 30 ng/ml; sTNF/80M2, 30 ng/ml/2 µg/ml; TNFR1-specific
Cys-TNF32W/86T, 30 ng/ml; TNFR2-specific Cys-TNF143N/145R, 300 ng/ml)
for 30 min. An agonistic TNFR2-specific serum (M80, 1:200) and a rabbit
control serum (1:200) were also included. Nuclear extracts were
prepared, and 10 µg of protein was subjected to gel shift analysis
using 32P-labeled NF-B-specific oligonucleotides.
B, the metabolic activity of HeLa80Fas cells was analyzed
using a microphysiometer. HeLa80Fas cells (3 × 105)
had been pretreated with mAb 80M2 for 30 min at 37 °C where
indicated. Cells were then stimulated with the TNFR2-specific TNF
mutein sTNF143N/145R in the presence or absence of mAb 80M2 for 30 min
(arrow). The change in the metabolic activity was followed
over time and is expressed as percentage acidification.
1 min1
and kon(sTNF + 80M2) = 2.5·109 M1 min1).
To study ligand dissociation, untreated MF-R1-Fas and MF-R2-Fas cells,
as well as 80M2-pretreated MF-R2-Fas cells, were incubated with 0.2 nM iodinated sTNF for 1 h on ice to allow receptor
complex formation. The temperature was then shifted to 37 °C, and in
the presence of a 200-fold excess of unlabeled sTNF, the release of the
radiolabeled ligand was followed. Fig.
6A shows that sTNF dissociates
only slowly from MF-R1-Fas cells (half-life = 32 min). In
contrast, sTNF·TNFR2-Fas complexes have a low half-life of about 1.2 min, whereas 80M2 pretreatment stabilizes ligand·TNFR2-Fas complexes
more than 10-fold (half-life = 14.5 min). These association and
dissociation data are in good agreement with the results obtained with
wild type TNF receptors (10). Accordingly, also the dissociation constants (Kd values) at 37 °C, as calculated for
the chimeric receptors, are very similar to those values determined for
the wild type TNF receptors (29 pM for TNFR1-Fas, 19 pM for TNFR1; 286 pM for TNFR2-Fas, 420 pM for TNFR2) (26). In summary, we show that the stability
of ligand-receptor complexes correlates with the formation of the RISC
and signal capacities of TNFR1-Fas and TNFR2-Fas chimeras.
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Fig. 6.
Time course of TNF dissociation at 37 °C.
MF-R1-Fas (A) and MF-R2-Fas (B) cells were
incubated for 1 h with 10 ng/ml 125I-TNF at 4 °C in
the presence or absence of mAb 80M2 (2 µg/ml). Dissociation of the
radiolabeled ligand was measured at 37 °C in the presence of 2 µg/ml unlabeled sTNF. Nonspecific binding determined in the
presence of a 200-fold excess of unlabeled sTNF was less than 5% of
total binding and has been subtracted. Half-lives of the TNF-receptor
complexes were calculated from exponential decay curves.
DISCUSSION
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DISCUSSION
REFERENCES
-cyclodextrine, which disrupts lipid rafts by cholesterol
depletion (34), or monodansylcadaverine, which blocks receptor
internalization (8), did not affect cell death kinetics of MF-R2-Fas
cells treated with sTNF plus 80M2 in a 3-h assay (data not shown). In
agreement, using radioiodinated sTNF, we did not find significant
internalization of ligand-receptor complexes within 30 min of
incubation at 37 °C (<5% of TNFR1-Fas when stimulated with sTNF;
<10% of TNFR2-Fas when stimulated with sTNF with or without 80M2;
data not shown). Together, all of these data suggest that our cellular
model displays a very rapid apoptotic response after appropriate TNF
treatment that is largely independent of secondary processes following
RISC formation and/or cofactors.
B, and activation of the mitogen-activated protein kinase JNK)
(Fig. 2). These results clearly show that the responsiveness of the TNF
receptors to the soluble versus the membrane bound form of
TNF is independent of the particular intracellular signaling machinery.
A direct consequence is that the decisive process, able to distinguish
between sTNF and memTNF in the case of TNFR2 and TNFR2-Fas, is located
upstream of the recruitment of cytoplasmic factors. This strongly
argues for the existence of a general principle able to control the
signaling strength of a given receptor within the TNF receptor family,
which is not determined solely by the affinity of ligand/receptor
interaction, since sTNF acting at saturating concentrations on
TNFR2-Fas elicits only weak, if any, responses (Fig. 2, B
and E).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 49-711-685-6987;
Fax: 49-711-685-7484.
ABBREVIATIONS
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ABSTRACT
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DISCUSSION
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