(Received for publication, March 3, 1995)
From the
We have fused the cytoplasmic domain of the p55 tumor necrosis
factor (TNF) receptor to the extracellular and transmembrane domain of
the mouse platelet-derived growth factor (PDGF) receptor. Mouse mammary
gland epithelial (NMuMG) cells were stably transfected with the
PDGFR-TR55 chimeric receptor. These cells lack endogenous PDGF receptor
expression and do not respond to PDGF. In the PDGFR-TR55 transfectants,
PDGF elicited a cytotoxic response, which is indistinguishable from
that induced by the wild type p55 TNF receptor. In addition,
PDGF-induced activation of the PDGFR-TR55 chimeric receptor resulted in
nuclear translocation of NF-
Membrane receptors for TNF
Most cell surface receptors studied, e.g. tyrosine kinase receptors like the PDGF receptor, are activated by
ligand-induced dimerization of the receptor subunits (for reviews, see
Refs. 21 and 22). Unlike these receptors, TR55 (and probably most other
members of the TNF receptor superfamily) can form a trimeric structure
induced by binding of a trimeric
ligand(23, 24, 25) . However, the trimeric
structure of TR55 observed in x-ray diffraction studies may represent
only one of several possible conformations in intact
cells(26) .
To determine whether trimer formation of the p55
TNF receptor is absolutely required for signaling functions, the
extracellular and transmembrane domain of the TR55 was replaced by
domains of an unrelated molecule that is not activated by a trimeric
ligand. The PDGFR
Here,
we demonstrate that PDGF binds to the PDGFR-TR55 chimeric receptor
stably expressed in NMuMG cells and induces a cytotoxic response in
these transfectants as well as nuclear translocation of NF-
Figure 1:
Design of
the PDGFR-TR55 receptor chimera. The PDGFR provides the ligand binding
and transmembrane domain, while the cytoplasmic domain of TR55 provides
the interface to TNF-specific signal transduction components.
Nucleotide as well as amino acid sequences of the junction region are
indicated. The PDGFR and TR55 fragments were joined via Klenow
filled-in HindIII sites (outlined). Two new amino
acids (QA) were introduced as a result of the
fusion.
Stably
transfected NMuMG cells expressing the PDGFR-TR55 chimeric receptor
were obtained by cotransfection of pADB-PTR55 with BMGNeo (33) using electroporation at 960 microfarads/280 volts and
subsequent selection with 1000 µg/ml Geneticin (Life Technologies,
Inc.). Transfectants expressing vector alone without insert were
generated by cotransfecting pAD-CMV1 with BMGNeo.
For Scatchard
analyses, cells were incubated with serial dilutions of labeled ligand
(0.125-5 ng/well) in a total volume of 300 µl of
PBS/FCS/azide with or without unlabeled ligand. Cells were washed twice
in cold PBS/FCS/azide and analyzed in a
Nuclear extracts were prepared as
described(34) . Electrophoretic mobility shift assays were
performed by incubating 3 µg of nuclear extract with 4 µg of
poly(dI-dC) (Pharmacia Biotech Inc.) in binding buffer (5 mM HEPES, pH 7.8, 5 mM MgCl
The PDGFR-TR55 receptor was generated by fusing the
extracellular and transmembrane domain of the murine PDGF receptor to
amino acids 243-426 of the human p55 TNF receptor cytoplasmic
domain (see Fig. 1). The chimeric receptor was cloned into the
expression vector pAD-CMV1 under the transcriptional control of the
cytomegalovirus promoter and 5` to the simian virus 40 polyadenylation
site. The resulting plasmid was designated pADB-PTR55 (see
``Materials and Methods'').
A transient expression system
was initially utilized to monitor induction of NF-
Therefore, parental NMuMG
cells, which neither express detectable PDGF receptor mRNA nor PDGF
receptors at the cell surface, were stably transfected with plasmid
pADB-PTR55, and Geneticin-resistant colonies were cloned. A total of 32
clones was analyzed for cell surface expression of the PDGFR-TR55
chimeric receptor in binding assays for
Parental NMuMG cells, clones PAD-1,
PT55-1 to PT55-3, and the NMuMG transfectant line RI
expressing the full-length PDGF receptor were analyzed for expression
of endogenous murine p55 TNF receptors using human
Figure 2:
Expression of TR55, PDGFR, and PDGFR-TR55
by parental and transfected NMuMG cells. A, binding of
The expression of the PDGFR-TR55 chimeric
receptor by clones PT55-1 to PT55-3 was additionally
confirmed by fluorescence-activated cell sorting analysis using
antibody SM14 directed against the extracellular domain of the PDGF
receptor (data not shown).
Western blots using a monoclonal PDGF
receptor antibody demonstrated the presence of a reactive band of 78
kDa, the predicted size for the PDGFR-TR55 chimera (Fig. 2D). As expected, this 78-kDa protein was not
detected in NMuMG, RI, or PAD-1 cells. The full-length PDGF receptor in
RI cells was masked by the presence of a cross-reactive band
comigrating with the PDGF receptor. This band was observed in parental
NMuMG cells as well as in 70Z/3 pre-B-cells, which both are negative
for PDGF receptor expression (data not shown).
PDGF is a potent
mitogen for many cell types, while TNF exerts both proliferative and
cytotoxic effects depending on the target cell. To determine the type
of response the PDGFR-TR55 chimeric receptor would convey, we analyzed
proliferative and cytotoxic effects of PDGF and TNF on parental NMuMG
cells, PAD-1, RI, and PT55 transfectants.
First, cells were assayed
for PDGF- or TNF-mediated induction of DNA synthesis by
[
Figure 3:
Proliferative effects of TNF and PDGF on
NMuMG transfectants. Parental NMuMG cells, PAD-1 cells, RI cells
expressing the full-length PDGF receptor, and PT55 cells expressing the
PDGFR-TR55 chimera were kept in serum-free medium or stimulated with
TNF or PDGF or serum. [
Figure 4:
Cytotoxicity mediated through TR55 or
PDGFR-TR55. A, parental NMuMG cells, RI cells, PAD-1 cells (leftpanel), and PT55 clones 1-3 (rightpanel) were treated with serial dilutions of TNF. B, parallel analysis using serial dilutions of PDGF. Errorbars indicate the standard deviations obtained from six
parallel experiments.
As shown in Fig. 5(lanes1-6),
nuclear NF-
Figure 5:
PDGF induces NF-
The results of our study indicate that two key responses of
TNF signaling pathways, cytotoxicity and induction of NF-
Controversial data exist on the conformation of biologically active
TR55. Earlier studies reported that TNF acts as a trimer (38) that may induce the formation of both TR55 dimers and
trimers. It is widely acknowledged from x-ray diffraction studies (23, 24, 25) that TR55 can form trimers after
ligand binding. However, these studies have been performed on TR55
crystals and do not rule out the possibility that TR55 adopts other
conformations (e.g. dimers) at the cell surface when bound to
ligand. Active signaling of TR55 dimers might be deduced from
cross-linking experiments with agonistic
antibodies(39, 40) . However, antibody cross-linking
does not exclude the formation of trimeric or multimeric complexes.
Obviously, antibody-mediated receptor clustering does not always
correctly reflect results obtained by using physiologic ligands, which
is illustrated by experiments in which the cytoplasmic domains of TR55
or Fas were fused to the extracellular domain of CD40. Cross-linking
with agonistic CD40 antibodies did not activate the CD40-TR55 fusion
proteins(30) .
Indeed, not any physiologic ligand can
activate the TR55 cytoplasmic domain. A hybrid receptor consisting of
the TR55 cytoplasmic domain fused to extracellular sequences of the
CD44 hyaluronate receptor was incapable of inducing cell death after
ligand-mediated cross-linking, supporting the conclusion that TR55
requires a specific type of oligomerization different from that of
CD44(30) .
While oligomerization through CD44 is apparently
unable to activate TR55, PDGF-mediated cross-linking appears perfectly
sufficient to activate the TR55 cytoplasmic domain. PDGF represents a
classical dimeric ligand, that is, the plethora of data indicates that
PDGF activates its receptor by dimerizing two receptor
molecules(28, 29) . The same holds apparently true for
the PDGFR-TR55 chimeric receptor.
Since TR55 dimerization is
obviously sufficient to elicit cytotoxicity and induction of NF-
We thank H. Wagner for continuous support.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B. The data presented suggest that
cross-linking of the p55 TNF receptor cytoplasmic domain by a dimeric
ligand such as PDGF is sufficient to generate cellular responses that
do not differ from those observed with the trimeric ligand TNF.
(
)signal a
large number of cellular responses, including immunoregulatory
activities, anti-viral activity, cytotoxicity, and the transcriptional
regulation of many
genes(1, 2, 3, 4) . Two distinct
cell surface molecules of 55 kDa (TR55) and 75 kDa (TR75) apparent
molecular mass have been
cloned(5, 6, 7, 8, 9, 10) .
Numerous studies have demonstrated that the majority of TNF activities
is mediated by TR55(11, 12, 13) . Signal
transduction through TR55 involves at least two independent pathways,
one initiated by a neutral sphingomyelinase, which results in
subsequent activation of a ceramide-activated protein kinase,
phospholipase A
, as well as the protein kinase
Raf(14, 15, 16) . The other pathway involves
activation of a phosphatidylcholine-specific phospholipase C, which
regulates stimulation of protein kinase C and an acidic
sphingomyelinase, which in turn mediates induction of the transcription
factor NF-
B(15, 17) . NF-
B controls
expression of a variety of TNF-responsive genes (18, 19, 20) and, among other molecules,
represents an important mediator of the proinflammatory responses
caused by TNF.
was chosen because it represents one of the
best characterized receptor tyrosine kinases(22, 27) .
In addition, it has clearly been demonstrated that the homo-dimeric
structure of the receptor ligand (in this case PDGF-BB) activates the
receptor by dimerizing two subunits(28, 29) .
B.
These results support the concept that dimerization of TR55 is
sufficient to mediate TNF-specific responses.
Plasmids and Reagents
The expression
vectors pADB-TR55, containing the full-length human p55 TNF receptor
cDNA, pAD-CMV1, and pSVRCD4 have been
described(6, 11, 31) . Murine and human
recombinant TNF- were provided by Dr. G. Adolf (Boehringer
Ingelheim, Vienna), and human recombinant BB-chain PDGF was obtained
from Drs. B. Ratzkin and L. Souza (Amgen, Thousand Oaks). Antibody SM14
was kindly provided by Dr. J. B. Bolen (Bristol-Myers Squibb,
Princeton). Anti-PDGF
receptor monoclonal antibodies were
purchased from Affinity Research Products (Nottingham).
Construction of the PDGFR-TR55
Molecule
The extracellular domain and parts of the
cytoplasmic domain of TR55 were removed from the expression vector
pADB-TR55 by digestion with SalI/HindIII. The
extracellular and transmembrane domain of the murine PDGF receptor was
recovered from the expression vector pSVRCD4 as an EcoRI-HindIII DNA fragment and ligated into the SalI-HindIII site of pADB-TR55 after treatment with
Klenow polymerase to restore the reading frame. The resulting plasmid,
pADB-PTR55, was sequenced to ensure that the fusion had occurred in
frame (see Fig. 1). In vitro transcription-translation
revealed that the chimeric gene was translated into a protein of the
appropriate size.(
)
Cell Culture and
Transfections
NMuMG cells (32) were obtained from
ATCC. NMuMG cells stably transfected with the full-length murine PDGF
receptor cDNA (cell line RI) were a generous gift from Dr. J. Escobedo
(University of California, San Francisco). Cells were grown in DMEM
supplemented with 10% bovine calf serum, 50 µg/ml penicillin, and
50 µg/ml streptomycin (Life Technologies, Inc.).
Radioligand Binding Assays and Scatchard
Analyses
Cells were detached by incubation in PBS, 2 mM EDTA at 37 °C for 15 min, washed, and resuspended in cold PBS
with 0.2% FCS and 0.02% sodium azide. Assays were set up in triplicate
as follows: 1 ng of I-radiolabeled human recombinant
BB-chain PDGF (DuPont NEN; specific activity 1480 kBq/µg) or human
recombinant TNF-
(DuPont NEN; specific activity 1630 kBq/µg)
was added to 3
10
cells in a total volume of 300
µl of PBS/FCS/azide with or without a 1000-fold excess of unlabeled
ligand. Cells were incubated at 0 °C for 2 h and washed twice in
cold PBS/FCS/azide, and total as well as nonspecific binding was
analyzed in a
-counter (Canberra Packard).
-counter (Canberra
Packard).
Immunoblots
Cells were lysed in TNE
buffer (50 mM Tris, pH 8.0, 1% Nonidet P-40, 2 mM EDTA) containing 10 µg/ml aprotinin/leupeptin. The cell
lysates were denatured in sample buffer and boiled, and 25 µg of
cell protein per lane were resolved by electrophoresis on 7%
SDS-polyacrylamide gels. After electrophoretic transfer to
nitrocellulose, reactive proteins were detected using anti-PDGF
receptor monoclonal antibodies (Affinity Research Products) and the ECL
detection kit (Amersham Corp.).
Proliferation Assays
2 10
cells/well were seeded in 24-well plates and kept in DMEM with 5%
FCS for 3 days. After further incubation in quiescing medium (DMEM with
0.1% bovine serum albumin, 5 µg/ml transferrin) for 1 day, cells
were stimulated by adding 2 nM human recombinant BB-chain PDGF
or 0.6 nM murine recombinant TNF-
for 16 h, pulsed with
18.5 kBq [methyl-
H]thymidine (DuPont
NEN) for 1 h, washed in PBS three times, lysed in 0.1% SDS, and counted
in a scintillation counter (Canberra Packard).
Cytotoxicity Assays
10 cells
were seeded in flat-bottom 96-well plates and incubated overnight in
100 µl of DMEM, 10% FCS to allow cells to adhere. The cells were
then preincubated in medium with 100 ng/ml actinomycin D for 2 h before
serial dilutions of murine recombinant TNF-
or human recombinant
BB-chain PDGF were added. After 18 h, 20 µl of MTT (Sigma, 2.5
mg/ml in PBS) were added, and incubation was continued for an
additional 2 h to allow metabolization of MTT to
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylformazan, which was
solubilized with isopropanol-HCl (24:1) and colorimetrically determined
at 570 nm in a microplate reader (MWG-Biotech).
Stimulation of Cells, Preparation of Nuclear
Extracts, and Electrophoretic Mobility Shift Assays for
NF-
3 B
10
cells/10-cm dish were
stimulated by adding human recombinant BB-chain PDGF or murine
recombinant TNF-
to a final concentration of 10 nM (PDGF)
or 0.6 nM (TNF). The cells were incubated at 37 °C for 20
min or for the indicated time, washed twice in ice-cold PBS, and
harvested with a cell scraper.
, 50 mM KCl,
0.5 mM dithiothreitol, 10% glycerol in a total volume of 20
µl for 20 min at room temperature. Then, end-labeled
double-stranded oligonucleotide probe (NF-
B-specific
oligonucleotide containing two tandemly arranged NF-
B binding
sites of the HIV-1 long terminal repeat enhancer
(5`-ATCAGGGACTTTCCGCTGGGGACTTTCCG-3`), 1
10
to 5
10
cpm) was added, and the reaction mixture was
incubated for 7 min. The samples were separated on native 6%
polyacrylamide gels in low ionic strength buffer (0.25
Tris
borate-EDTA). For competition experiments, unlabeled or a mutated
oligonucleotide containing altered
B sites
(5`-GATCACTCACTTTCCGCTTGCTCACTTTCCAG-3`) was added before the initial
20-min incubation.
B in NMuMG cells
transfected with pADB-PTR55 and a 4
immunoglobulin/HIV-
B
chloramphenicol acetyltransferase reporter plasmid(35) . Due to
low transfection efficiencies, however, this system was not suitable to
conduct detailed studies of the potential signal transduction through
the PDGFR-TR55 chimera (data not shown).
I-PDGF. Three
positive clones (designated PT55-1 to PT55-3) were
characterized further. As a control, transfectants were selected that
contain the expression vector pAD-CMV1 without insert. Of those, clone
PAD-1, together with parental NMuMG cells, served as a negative control
in the subsequent experiments.
I-TNF.
All cell lines examined specifically bound
I-TNF,
confirming endogenous TR55 expression (Fig. 2A). When
analyzed for the binding of
I-PDGF, both NMuMG and PAD-1
cells proved negative, while RI cells demonstrated significant binding.
Clones PT55-1 to PT55-3 did also prove positive for binding
of
I-PDGF, though at a lower level than that seen with
the full-length PDGF receptor in RI cells (Fig. 2B).
I-labeled TNF-
to endogenous p55 TNF receptors of
parental NMuMG cells, cells transfected with pAD-CMV1 without insert
(PAD-1), cells expressing the full-length PDGF
receptor (RI), or
transfectants expressing the PDGFR-TR55 chimeric receptor (PT55-1
to PT55-3). B, binding of
I-labeled
PDGF-BB to the full-length PDGF receptor or the PDGFR-TR55 receptor
chimera. The filledcolumns indicate total binding (TB), and the opencolumns indicate
nonspecific binding in the presence of excess unlabeled ligand (NSB). Errorbars represent the standard
deviations from three experiments. C, Scatchard plot of
specific
I-PDGF binding to the PDGFR-TR55 receptor
chimera on PT55 cells or to the full-length PDGF receptor on RI cells.
The lines were plotted by linear regression analysis (PT55-1: r = -0.96; PT55-2: r =
-0.94; PT55-3: r = -0.98; RI: r = -0.93). Each value represents the mean of
triplicate experiments. Receptor numbers per cell and the K value for each cell line are indicated. D, Western blot
analysis of the PDGFR-TR55 chimeric receptor in NMuMG transfectants.
Protein extracts from parental NMuMG cells, PAD-1 cells, RI cells, and
clones PT55-1 to PT55-3 were separated on 7%
SDS-polyacrylamide gels and immunoblotted using a monoclonal PDGFR
antibody (Affinity Research Products). The arrow indicates the predicted size for the receptor
chimera.
Scatchard analyses of clones PT55-1, PT55-2, and
PT55-3 demonstrated high affinity binding of radiolabeled PDGF-BB
to the PDGFR-TR55 receptor (Fig. 2C), comparable to the
affinity described for PDGF binding to the wild type PDGF receptor on
murine 3T3 cells(36) . In addition, Scatchard analyses of RI
cells demonstrated an almost identical binding affinity for the
transfected wild type PDGF receptor (Fig. 2C),
indicating that the reduced levels of I-PDGF binding
observed with PT55 cells result from reduced numbers of the PDGFR-TR55
molecule rather than from an impaired binding capability of the
receptor chimera. Reduced expression of PDGFR-TR55 is probably due to
toxic effects of a highly expressed TR55 cytoplasmic
domain(37) .
H]thymidine incorporation assays (Fig. 3). Engagement of the endogenous TR55 by TNF did not
induce any significant proliferation of either cell line. PDGF induced
DNA synthesis in RI cells expressing the full-length PDGF receptor. In
contrast, PDGF did not stimulate the proliferation of parental NMuMG
cells, PAD-1 cells, or of clones PT55-1 to PT55-3
expressing the PDGFR-TR55 chimera. These results indicate that neither
endogenous TR55 nor the PDGFR-TR55 receptor are able to mediate a
proliferative response in these cells.
H]Thymidine incorporation
into cellular DNA is shown relative to serum-stimulated cells (100%). Errorbars indicate the standard deviations derived
from triplicate determinations. The actual cpm values obtained in these
experiments for serum-stimulated cells were: NMuMG cells, 40618
± 2773; PAD-1 cells, 16801 ± 3304; RI cells, 21749
± 3055; PT55-1 cells, 27444 ± 625; PT55-2
cells, 9059 ± 1972; PT55-3 cells, 10003 ± 2066. The
data shown are representative of a total of three independent
experiments.
TNF treatment of parental and
transfected NMuMG cells resulted in cytotoxic effects (Fig. 4A), which is likely mediated through endogenous
TR55. When PT55 clones expressing the PDGFR-TR55 chimera were treated
with PDGF, a pronounced cytotoxic effect was elicited. In contrast,
parental NMuMG, PAD-1, or RI cells did not show a cytotoxic response to
PDGF (Fig. 4B).
We finally examined whether the
PDGFR-TR55 chimera was capable to mediate the activation of the
transcription factor NF-B, an important mediator of TNF responses.
B activity in parental NMuMG and PAD-1 cells was
markedly increased after stimulation of cells with TNF but not after
PDGF treatment. In PT55 cells, NF-
B activation could be induced by
both TNF and PDGF (Fig. 5, lanes7-24),
and the kinetics of NF-
B activation were similar in any instance
(data not shown). Competition experiments using oligonucleotides
containing wild type or mutated NF-
B sites confirmed NF-
B
binding activity induced by PDGF (Fig. 5, lanes17 and 18).
B in PT55 cells.
Parental NMuMG cells and PAD-1 cells were left unstimulated (lanes1 and 4) or treated with either TNF (lanes2 and 5) or PDGF (lanes3 and 6) for 20 min as described under ``Materials and
Methods.'' PT55-1 cells were left untreated (lane7) or stimulated with TNF for 20 min (lane8, positive control). Alternatively, PT55-1 cells
were stimulated with PDGF for the indicated times (lanes9-16) or for 20 min with addition of wild type (lane17) or mutated (lane18)
NF-
B oligonucleotides for competition. Lanes19-24, same experiment as in lanes1-6, using cell lines PT55-2 and
PT55-3.
B, can be
mediated solely by the TR55 cytoplasmic domain without requirement for
extracellular or transmembrane sequences. Furthermore, dimerization of
the TR55 cytoplasmic domain by a bivalent ligand, PDGF, is sufficient
to activate the intrinsic functions of the receptor. This mode of
activation ranges TR55 among the group of other receptors that are
dimerized prior to activation, e.g. the epidermal growth
factor receptor, the PDGFR, and many more(21) .
B,
one might speculate that signaling through a dimeric p55 TNF receptor
employs the same pathways as signaling through the TR55 trimer. More
knowledge is needed about the signaling events that take place
following TR55 dimerization, e.g. which proteins associate
with TR55, and if those proteins represent the same set that binds to
an activated TR55 trimer or if different sets of associated proteins
exist for different states of TR55 clustering. The identification and
cloning of those proteins will provide essential information about the
p55 TNF receptor signal transduction pathways.
ler, and M. Krönke,
unpublished observation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.