From the UPRES-EA 1033, IFR 118, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq 59655, France
Received for publication, January 21, 2003
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ABSTRACT |
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The common neurotrophin receptor p75NTR
has been shown to initiate intracellular signaling that leads either to
cell survival or to apoptosis depending on the cell type examined;
however, the mechanism by which p75NTR initiates its
intracellular transduction remains unclear. We show here that the tumor
necrosis factor receptor-associated death domain protein (TRADD)
interacts with p75NTR upon nerve growth factor (NGF)
stimulation. TRADD could be immunodetected after p75NTR
immunoprecipitation from MCF-7 breast cancer cells stimulated by nerve
growth factor. In addition, confocal microscopy indicated that NGF
stimulation induced the plasma membrane localization of TRADD. Using a
dominant negative form of TRADD, we also show that interactions between
p75NTR and TRADD are dependent on the death domain of TRADD,
thus demonstrating its requirement for binding. Furthermore, the
p75NTR-mediated activation of NF- Nerve growth factor
(NGF)1 is the archetypal
member of the neurotrophin family of proteins (including brain-derived
neurotrophic factor, NT-3, and NT-4/5), which display a broad range of
survival and trophic activities for neuronal cells (1). In addition to
its neurotrophic function, other activities of NGF have been described.
For example, NGF can modulate gene expression in monocytes (2), is
chemotactic for melanocytes (3), and is motogenic for Schwann
cells (4). NGF also stimulates the proliferation of lymphocytes (5),
keratinocytes (6), and both prostate (7) and breast cancer (8) cells.
Cellular responses to NGF are elicited via two specific cell surface
receptors: TrkA tyrosine kinase receptors (p140TrkA) and the
common neurotrophin receptor, p75NTR, which belongs to the
tumor necrosis factor (TNF)-receptor gene family. Although NGF binding
to p140TrkA is known to activate its kinase domain, thus
triggering various downstream Ras signaling pathways such as the
mitogen-activated protein kinases, the function of p75NTR and
how it signals remain controversial. p75NTR has been shown to
regulate the activation of TrkA receptors but is also capable of
triggering cellular responses independent of them (9-11). In breast
cancer cells, the mitogenic activity of NGF is mediated through
activation of p140TrkA, whereas p75NTR is required for
its antiapoptotic effect, independent of p140TrkA (12). There
is evidence that NGF can both positively and negatively regulate cell
death and differentiation, depending on the cell type examined. In some
cases, p75NTR is an inducer of apoptosis, even without NGF
stimulation (13), whereas in other cases, the activation of
p75NTR results in a protection from cell death (14). The
mechanism by which p75NTR initiates such signaling is poorly
described, but the tumor necrosis factor receptor-associated factor
(TRAF) proteins, particularly TRAF2 and TRAF6, as well as the
receptor-interacting protein 2 have been shown to interact with
p75NTR and differentially modulate both the activation of the
transcription factor NF- The first protein recruited to TNF-receptor 1 is the TNF-R1-associated
death domain protein (TRADD), which serves as a platform to recruit
additional mediators such as the TRAF proteins (17). Although the
involvement of TRAF proteins in the p75NTR signaling complex
has been described, the requirement for TRADD has not been reported.
Here we show that TRADD functionally associates with
p75NTR in breast cancer cells. The interaction between
p75NTR and TRADD is required for activation of NF- Materials--
Cell culture reagents were provided by
Bio-Whittaker except insulin (Organon), transferrin (Sigma), and
fibronectin (Falcon-Biocoat). Recombinant NGF and other neurotrophins
(BDNF, NT-3, and NT-4) were purchased from R&D Systems. C2 ceramide
analogue (N-acetyl-D-sphingosine), Hoechst
33258, and electrophoresis reagents were from Sigma. The ExGen 500 transfectant and the Opti-MEM were from Euromedex. Protein A-agarose
was purchased from Transduction Laboratories (Lexington, KY). The mouse
monoclonal IgG1 anti-NGF receptor p75NTR antibody was from
Upstate Biotechnology, Inc. (Lake Placid, NY). The goat and rabbit
polyclonal antibodies (IgG) raised against TRADD and TRAF2,
respectively, were purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA), and the corresponding secondary antibodies were from Sigma.
The mouse monoclonal anti-Myc antibody was from Jackson Laboratories.
Polyclonal goat anti-rabbit IgG and donkey anti-goat IgG, both coupled
to Alexa 568, and polyclonal goat anti-mouse IgG coupled to Alexa 488 were purchased from Molecular Probes, Inc. (Eugene, OR). Full-length
TRADD cDNA or truncated cDNA lacking the sequence coding the
death domain (TRADD Cell Culture and Apoptosis Measurement--
The MCF-7 breast
cancer cell line was obtained from the American Type Culture Collection
and routinely grown as monolayers. Cells were maintained in minimal
essential medium (Earle's salts) supplemented with 20 mM
Hepes, 2 g/liter sodium bicarbonate, 2 mM
L-glutamine, 1% non-essential amino acids, 5 µg/ml
insulin, 10% fetal calf serum, 40 units/ml penicillin/streptomycin,
and 50 µg/ml gentamycin. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2.
Apoptosis of MCF-7 cells was induced by treatment with ceramide
analogue C2 (10 µM for 24 h), previously described
as a proapoptotic agent for human breast cancer cells (18, 19). For
determination of the proportion of cells in apoptosis, cells were fixed
with cold methanol ( Cell Transfection--
Transfection assays were carried out for
3 h in Opti-MEM transfection medium using ExGen 500 reagent, as
described by the manufacturer. To analyze the interaction of TRADD with
p75NTR by Western blotting, MCF-7 cells were transfected with
either 1 µg of PRK5 control vector or Myc-TRADD- or
Myc-TRADD Immunoprecipitation and Western Blot Analysis--
The
interaction of TRADD with p75NTR was assessed after NGF
stimulation by immunoprecipitation and Western blotting. Time course analysis was monitored at 5, 10, 15, and 30 min; dose dependence was
assessed for 5, 50, 100, and 200 ng/ml. The effect of other neurotrophins (BDNF, NT-3, and NT-4) was tested at concentrations of 50 and 100 ng/ml, after 10 min of stimulation. Cells were washed with PBS
and lysed in the following buffer: 150 mM NaCl, 50 mM Tris, pH 7.5, 1% Nonidet P-40, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium pyrophosphate, 10 µg/ml leupeptin and
aprotinin, and 1 µg/ml pepstatin. Cell lysate (1.5 mg) was precleared
with protein A-agarose (10 µl/250 µl, 60 min, 4 °C). After
centrifugation (10,000 × g, 2 min), the supernatant was incubated
with mouse monoclonal anti-p75NTR (20 µl/500 µl) at 4 °C
for 2 h. The immunocomplexes were precipitated with protein
A-agarose beads (10 µl, 1 h, 4 °C), which were then pelleted
by centrifugation (10,000 × g, 2 min). The pellet was rinsed three times with lysis buffer and boiled for 5 min in Laemmli buffer. After SDS-PAGE and electroblotting, nitrocellulose membranes (Schleicher & Schuell) were blocked with 3% bovine serum albumin. Membranes were then incubated with appropriate antibodies (1 µg/ml anti-p75NTR, 1:500 anti-TRADD) overnight at 4 °C, rinsed,
and incubated with appropriate secondary antibodies diluted at 1:500
for 2 h at room temperature. To assess the requirement of TRADD
death domain interactions with p75NTR, endogenous
p75NTR was immunoprecipitated from 1.5 mg of total protein
extracted from MCF-7 cells transfected with 1 µg of Myc-tagged-TRADD,
Myc-tagged-TRADD Immunofluorescent Labeling and Confocal
Microscopy--
Cells, cultured onto glass coverslips coated with
collagen, were treated for 15 min with NGF (200 ng/ml). Cells were
washed twice with PBS and then fixed with PBS containing 4%
paraformaldehyde for 30 min at room temperature. After washing with
ammonium chloride (10 min at room temperature), cells were
permeabilized with 0.05% saponin in PBS (10 min) and blocked for 10 min in permeabilizing buffer (PBS containing 2% BSA and 0.05%
saponin). Cells were then successively incubated for 1 h, at room
temperature, with primary antibodies (anti-p75 and then anti-TRADD).
After five washes with permeabilizing buffer, cells were incubated with
both the secondary antibodies (30 min at 37 °C at a 1:100 dilution).
Cells were washed twice with permeabilizing buffer, twice with PBS plus
2% BSA, and then with PBS alone. Slides were mounted using Vectashield mounting medium (Vector Laboratories).
Fluorescence-stained slides were examined under a Leica (TCS NT)
laser-scanning confocal microscope comprising a krypton/argon laser.
Simultaneous two-channel recording was performed. Frame scanning was
performed at ×1000 magnification, and a single optical section was
collected per field.
NF- Interaction of TRADD with p75NTR--
To investigate
possible interactions between p75NTR and TRADD, MCF-7 cell
lysates were prepared from NGF-treated cells followed by
immunoprecipitation with p75NTR antibody and Western blotting
with TRADD antibody. The results (Fig. 1)
indicate that TRADD co-immunoprecipitates with p75NTR when
cells have been stimulated with NGF. NGF concentration of 5-50 ng/ml
was sufficient to induce the interaction between p75NTR and
TRADD; a clear dose-dependent effect was observed (Fig.
1A). A study in which TRADD immunoprecipitates were Western
blotted with p75NTR, was performed at 100 ng/ml NGF. The time
course study (Fig. 1B) indicated that p75NTR/TRADD
interaction can be detected as soon as 5 min of NGF stimulation; the maximum effect was obtained after 10 min of stimulation. Other neurotrophins (BDNF, NT-3, NT-4) also mediate p75NTR/TRADD
interaction (Fig. 1C); 50 ng/ml was a sufficient dose for each tested neurotrophin. Confocal microscopy analysis revealed that
TRADD shifted to a plasma membrane localization upon NGF stimulation
(Fig. 1D). This co-localization was demonstrated by the
appearance of a merged fluorescence at the plasma membrane. The nuclear
translocation of TRADD has recently been reported (20), and in order to
investigate this possibility in the case of NGF stimulation, a
measurement of the intensity of staining for TRADD in both the
cytoplasm and the nucleus has been performed using the LSM-Image
Browser (Zeiss) software. Our results show that the ratio of nuclear to
cytoplasmic staining does not vary significantly upon NGF stimulation
(data not shown). Altogether, these results indicate a p75NTR
interaction with TRADD, which is regulated by NGF; in addition, no
interaction between TRADD and TrkA was ever detected (data not shown).
To further confirm the p75NTR/TRADD interaction, MCF-7 cells
were transfected with a vector coding Myc-TRADD or Myc-TRADD TRADD Is Required for p75NTR-induced Activation of
NF- TRADD Is Required for p75NTR-mediated Antiapoptotic Effect
of NGF--
We have previously shown that p75NTR-mediated
activation of NF- We have shown here for the first time that nerve growth factor
stimulation of the common neurotrophin receptor p75NTR results
in the recruitment of TRADD. It has previously been shown that
p75NTR interacts with TRAF protein family members (15, 21) and
that this interaction modulates p75NTR- induced cell death and
NF- Two intracellular domains have been identified within p75NTR.
The first one is homologous to the binding domain for TRAF proteins, and the second is homologous but distinct from the death domain of
TNFR1 (23). TRADD death domain has been reported to bind to the death
domain of TNFR1 (17). In our experiments, the death domain of TRADD was
required for interaction with p75NTR, suggesting that the death
domain of p75NTR is involved in TRADD binding. Therefore, the
activation of p75NTR and TNFR1 appears to involve a similar
mechanism of interaction with TRADD, further suggesting similar
downstream signaling pathways. Interestingly, it has been shown by NMR
spectroscopy and crystal structure determination that TRAF2 has a
higher affinity for TRADD than it has for TNFR1 itself (24, 25). TRAF2
has been shown to interact with p75NTR, an interaction we have
confirmed in breast cancer cells (data not shown); these data suggest
that TRADD linked to p75NTR might interact with TRAF2. However,
further structural analyses are required to define the precise
molecular mechanism involved in TRADD interaction with
p75NTR.
The role of NF- In conclusion, the findings reported here provide new insights into the
proximal elements of the p75NTR signaling pathway by
demonstrating the involvement of TRADD as a critical intermediary for
NF-B was inhibited by
transfection with a dominant negative TRADD, resulting in an inhibition
of NGF antiapoptotic activity. These results thus demonstrate that
TRADD is involved in the p75NTR-mediated antiapoptotic activity
of NGF in breast cancer cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and cell survival in response to NGF (15,
16).
B, which
controls the antiapoptotic effect of nerve growth factor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DD:
195-312) were cloned into a PRK5-derived
vector containing a Myc tag. To obtain the Myc tag
(MASMEQKLISEEDLGIP), the oligonucleotides
5'-GGATGGCATCAATGGAGCAGAAGCTTATCTCAGAGGAAGATCTGGGAATACCGTGC-3' and
5'-GGCCGCACGGTATTCCCAGATCTTCCTCTGAGATAAGCTTCTGCTCCATTGATGCCATCCTGCA-3' were annealed and inserted in PstI/NotI-digested
PRK5 (Pharmingen). Human TRADD cDNA was obtained by PCR with the
oligonucleotides 5'-TGCGGCCGCATGGCAGCTGGGCAAA-3' and
5'-TGCGGCCGCTAGGCCAGGCCGCCATT-3' and was cloned in pCRII-TOPO
vector (Invitrogen). This insert was recovered after NotI
digestion and was cloned in PRK5 tag-Myc digested by NotI.
Truncated TRADD cDNA (TRADD
DD:
195-312) was cloned in PRK5
tag-Myc vector with the same strategy. The oligonucleotides used were
5'-TGCGGCCGCATGGCAGCTGGGCAAA-3' and
5'-GCGGCCGCGGCGGCGGCGGCTTCA-3'. A p75NTR dominant
negative, coding only the first 276 amino acids (corresponding to the
extracellular and plasma membrane region) of human p75NTR was
cloned into an episomic vector under CMV promoter control; the membrane
association has been assessed in Madin-Darby canine kidney cells using
immunocytochemistry. The activation of the NF-
B pathway was assessed
using a pNF-
B luciferase construct (Stratagene), which contains five
tandem repeats of consensus NF-
B binding site sequence. The control
was realized with pLuc-MCS from Stratagene. pCMV
-galactosidase
vector was from Clontech.
20 °C) for 10 min and washed twice with
phosphate-buffered saline (PBS) before staining with 1 µg/ml Hoechst
33258 for 10 min in the dark at room temperature. Cells were then
washed with PBS and mounted using Glycergel (Dako). The apoptotic cells
exhibiting condensed and fragmented nuclei were counted with an
Olympus-BH2 fluorescence microscope over randomly selected fields. A
minimum of 1000 cells were examined for each condition, and the results are expressed as a percentage of apoptotic cells observed of the total
number of cells counted.
DD-carrying vector. Cells were then grown for 24 h in
10% fetal calf serum minimal essential medium, serum-rinsed (2 h), and
then incubated with or without 200 ng/ml NGF for 24 h. To analyze
NF-
B activation, cells were co-transfected with 0.2 µg of
pCMV
-galactosidase, 1.2 µg of NF-
B-luciferase reporter, and 0.5 µg of expression plasmids encoding either the wild type TRADD, the
mutant TRADD
DD, or the mutant p75NTR. Cells were then grown
in fetal calf serum minimal essential medium, serum-rinsed, and
incubated in the presence of 10 µM C2 with or without 200 ng/ml NGF for another 24 h. For the study of the NGF antiapoptotic
effect, MCF-7 cells were co-transfected with 0.6 µg of green
fluorescent protein-carrying vector with either 0.8 µg of the PRK5
control vector or wild type TRADD- or dominant negative
TRADD
DD-carrying vector. Cells were fixed with paraformaldehyde 4%
(4 °C, 30 min), and the percentage of apoptosis in green fluorescent
protein-stained cells was determined as described above.
DD, or PRK5 empty vector. Immunoblotting was done as
described above with 1:500 anti-Myc antibody. Membranes were washed
extensively at room temperature, and the antibody complexes were
visualized using the ECL system (Amersham Biosciences) with Eastman
Kodak Co. X-Omat AR film.
B Activity Measurement--
After 24 h of NGF
treatment, transfected cells (TRADD, TRADD
DD, PRK5, NF-
B-Luc,
pLuc-MCS, and pCMV
-galactosidase) were harvested with a reporter
lysis buffer (Promega). NF-
B activity was determined using a
luciferase assay kit (Promega) and measured with a luminometer (Lumat
9501; Berthold). The induction of NF-
B was calculated by assessing
the luciferase expression by luminometer in cells expressing NF-
B
promoter element construct or control vector (pLuc-MCS from Stratagene)
lacking the NF-
B binding sequences. NF-
B activity was normalized
with the pCMV
control vector coding the
-galactosidase protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DD, the
latter a dominant negative form lacking the death domain (Fig.
2A), and the association was determined as above. The results (Fig. 2B)
demonstrated that the association between p75NTR and
TRADD requires the death domain of TRADD, since its deletion clearly
impairs the association.
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Fig. 1.
Interaction of TRADD with
p75NTR. A, dose dependence. Immunoblot
detection of TRADD associated with p75NTR is shown.
MCF-7 cells were cultured in 10% fetal calf serum-containing medium
before serum starvation for 24 h. Cells were then stimulated or
not with 5, 50, 100, and 200 ng/ml NGF for 10 min. Total proteins were
extracted, 1.5 mg was immunoprecipitated with anti-p75NTR (or
anti-TRADD) and immunoblotted (Western blotting (WB))
against anti-p75NTR and anti-TRADD. NGF stimulation enhanced
the association of endogenous TRADD with endogenous p75NTR
receptor. Similar results were obtained in three separate experiments.
B, time course of p75NTR/TRADD interaction.
Immunoprecipitation with anti- p75NTR followed by immunoblot
against anti-p75NTR and anti-TRADD were performed after 5, 10, 15, and 30 min of stimulation by 100 ng/ml NGF. C,
stimulation of p75NTR/TRADD interaction by BDNF, NT-3,
and NT-4. Immunoprecipitations and Western blotting were
realized as described above. D, fluorescence confocal
micrographs showing paraformaldehyde-fixed MCF-7 cells treated with or
without NGF. Cellular localization of p75NTR and TRADD was
assessed using secondary antibodies directed against
anti-p75NTR and anti-TRADD coupled to Alexa 488 (green) and Alexa 568 (red), respectively. NGF
induced a shift of TRADD to the plasma membrane.
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Fig. 2.
p75NTR/TRADD
interaction requires the death domain of TRADD. Subconfluent MCF-7
cells were transiently transfected with 1 µg of expression plasmid
coding several constructs (A): Myc-tagged TRADD, Myc-tagged
TRADD DD, or control vector PRK5. Transfection was followed by
stimulation with NGF (200 ng/ml) for 24 h before
immunoprecipitation of endogenous p75NTR from 1.5 mg of cell
lysate and immunoblot against anti-Myc (B). Control to
assess the levels of expression of the transfected products was
performed with anti-Myc immunoblot of total cell lysate.
B--
Stimulation of p75NTR is known to activate
the transcription factor NF-
B in breast cancer cells so that it
translocates to the nucleus (12). Since TRADD is known to be a mediator
of TNFR1-induced NF-
B activation (17), it was important to determine
whether TRADD was also involved in the activation of NF-
B mediated
through p75NTR. The results (Fig.
3) demonstrated that TRADD wild type
control vector strongly enhanced the NGF-mediated induction of NF-
B. Furthermore, this induction was inhibited by p75NTR dominant
negative (lacking the cytoplasmic tail) or by TRADD
DD dominant
negative, thus demonstrating the involvement of TRADD in the activation
of NF-
B in breast cancer cells.
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Fig. 3.
TRADD is required for
NF- B activation by NGF. MCF-7 cells were
co-transfected with NF-
B-luciferase,
-galactosidase reporters,
and 0.5 µg of expression plasmids encoding either wild type TRADD or
the mutant TRADD
DD lacking the death domain, the dominant negative
p75NTR (m p75NTR), or the
corresponding empty vector. A control luciferase vector (pLuc-MCS)
lacking the NF-
B binding site was also used. After 24 h, cells
were serum-starved and treated with or without 200 ng/ml NGF, in the
presence of 10 µM ceramide C2, for 24 h. The effects
of ectopically expressed proteins are assessed as induced
NF-
B-luciferase normalized for
-galactosidase activity. The
induction of NF-
B was calculated by assessing the luciferase
expression in cells expressing NF-
B promoter element construct or
control vector (pLuc-MCS) lacking the NF-
B binding sequences.
NF-
B activity was normalized with the pCMV
-galactosidase control
vector. Results are expressed as the means ± S.D. of five separate
experiments.
B results in the survival of breast cancer cells
(12). We show here that transfection with TRADD
DD prevents the
antiapoptotic effect of NGF in MCF-7 breast cancer cells (Fig.
4A). However, the
antiapoptotic activity of NGF was not totally inhibited with TRADD
DD, since transfection with a vector coding a
mutated form of I
B suggests that TRADD is not the only intermediate
associating with p75NTR to activate NF-
B. In contrast,
transfection with TRADD enhances the antiapoptotic effect of NGF,
confirming the involvement of TRADD in p75NTR-mediated survival
activity. The morphology of cell nuclei observed after Hoescht staining
is shown in Fig. 4B.
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Fig. 4.
TRADD is required for p75NTR-mediated
antiapoptotic effect of NGF. A, apoptosis was assessed in
MCF-7 cells transiently cotransfected with green fluorescent
protein-carrying vector along with either expression plasmid for wild
type TRADD or dominant negative TRAD DD. 24 h after
transfection, cells were serum-starved and treated with 10 µM of C2 with or without 200 ng/ml NGF for another 24-h
period. Control consists of MCF-7 cells transfected with PRK5 control
vector. B, Hoechst staining of cell nuclei in control, C2,
C2 + NGF-treated MCF-7, and C2 + NGF-treated TRAD
DD-transfected
cells. Cells were serum-starved and treated with C2 + NGF as described
above. Apoptosis was monitored 24 h after transfection, using the
Hoechst 33258 staining as described under "Experimental
Procedures." Results are expressed as the means ± S.D. of five
separate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation with contrasting effects (15). TRAF4 inhibited the
NF-
B response, whereas TRAF2 and TRAF6 enhanced
p75NTR-induced NF-
B activation. However, these experiments
were carried out in immortalized human epithelial kidney HEK 293T cells
in which p75NTR stimulation by NGF resulted in cell death
induction. In contrast, for breast cancer cells, p75NTR
stimulation by NGF has been shown to be antiapoptotic and mediated by
the nuclear translocation of NF-
B (12). TRADD is a multifunctional intracellular signaling adaptor protein that is recruited by TNFR1, leading to IKK activation and so to NF-
B nuclear translocation (22).
Our results indicate that, in breast cancer cells, TRADD is also
recruited by p75NTR, resulting in an activation of NF-
B,
similar to the signaling initiated by TNFR1. Cell survival induced by
NGF was observed from 50 ng/ml, and the maximum effect was obtained at
200 ng/ml, providing a coherence between the p75NTR/TRADD
interaction and the biological effect of NGF in breast cancer cells. In
addition, it should be emphasized that our experiments were performed
without transfection of p75NTR or TRADD, strengthening the
physiological relevance of the p75NTR/TRADD interaction in
breast cancer cells.
B activation is well established for the survival of
breast cancer cells, and its involvement in the antiapoptotic effect of
NGF is clearly related to p75NTR signaling (12). TRADD is a
potent activator of the I
B kinase, IKK, which leads to the
ubiquitination/degradation of I
B and nuclear translocation of the
free NF-
B (17). We have shown that transfection of breast cancer
cells with a dominant negative form of TRADD resulted in an inhibition
of the NGF-mediated antiapoptotic effect, demonstrating the involvement
of TRADD in the NF-
B-mediated survival pathway. The trophic function
of NGF for breast cancer cells, involving both mitogenic and
antiapoptotic activities, has recently been shown (8, 12). The
prognostic value of both p75NTR and TrkA receptors has also
secondarily been reported (26), as has the possibility of targeting
p140TrkA-mediated mitogenic signaling with tamoxifen, a drug
commonly used in breast cancer therapy (27). Our present data also
suggest that targeting the p75NTR/TRADD/NF-
B signaling
pathway may be a promising avenue for future treatment of breast cancer.
B activation. In immortalized striatal neurons, p75NTR
was found to induce apoptosis without the participation of TRADD as an
adaptor protein (28). Whether or not the interaction of TRADD with
p75NTR provides a more specific pathway for NGF-induced
antiapoptotic activity remains to be determined, but TRADD should now
be considered as a significant influence in the balance between
proapoptotic and antiapoptotic signaling pathways initiated by
p75NTR.
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ACKNOWLEDGEMENTS |
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We thank L. Brunet and G. Courtand for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Ligue Nationale Contre le Cancer (Comité du Pas-de-Calais), the French Ministry of Research and Education, and the Genopole of Lille.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.
Supported by a fellowship from the "Région Nord
Pas-de-Calais" and the "Ligue Nationale Contre le Cancer,
comité départemental."
§ Recipient of an Association pour la Recherche sur le Cancer (ARC) fellowship. Present address: UMR-CNRS 6061, Université de Rennes 1, 34043 Rennes Cedex, France.
¶ To whom correspondence should be addressed: UPRES-EA 1033, Bâtiment SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France. Tel.: 33-3-20-43-40-97; Fax: 33-3-20-43-40-38; E-mail: hubert.hondermarck@univ-lille1.fr.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M300631200
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ABBREVIATIONS |
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The abbreviations used are: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; p75NTR, p75-neurotrophin receptor; TNF, tumor necrosis factor; TRAF, tumor necrosis factor receptor-associated factor; TRADD, TNF receptor-associated death domain protein; CMV, cytomegalovirus; PBS, phosphate-buffered saline.
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