From the Institute for Virus Research, Kyoto University, Kyoto 606, Japan
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ABSTRACT |
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We investigated the intracellular signaling of
OX40, a member of the tumor necrosis factor receptor family. Activation
of NF-B in OX40-transfected HSB-2 cells was detected by
electrophoretic mobility shift assay within 30 min after the binding of
the ligand gp34. In vitro binding experiments showed that
tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2,
TRAF3, and TRAF5 but not TRAF4 associated with glutathione
S-transferase-OX40 fusion protein. The cotransfection
experiments using human embryo kidney cell derived HEK 293T cells
showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in
vivo. Studies with OX40 deletion mutants demonstrated that the
cytoplasmic portion consisting of amino acid sequence 256-263
(GGSFRTPI) was required for the association with TRAFs and NF-
B
activation. The introduction of the dominant negative mutants of TRAF2
and TRAF5 into HSB-2-OX40 cells suppressed NF-
B activation in a
dose-dependent manner. In addition, the introduction of
TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5
further reduced NF-
B activation. These results indicate that the
NF-
B activation resulting from OX40 stimulation is mediated by both
TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3.
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INTRODUCTION |
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Human OX40 is a 50-kDa cell surface glycoprotein expressed
primarily on activated CD4+ T cells and some human T cell
leukemia virus type I
(HTLV-I)1-infected T cell
lines, but not on resting peripheral T cells, peripheral B cells, or
thymocytes. OX40 was originally described as a cell surface antigen on
the activated rat T cells (1). Molecular cloning of its cDNA (1-4)
revealed that OX40 is a member of the nerve growth factor
receptor/tumor necrosis factor receptor (NGF-R/TNF-R) superfamily which
is now known to include low affinity nerve growth factor receptor (p75
NGF-R), tumor necrosis factor receptors (p50/55 TNF-R1 and p75/80
TNF-R2), lymphotoxin- receptor, Fas antigen (CD95/APO-1), CD40,
CD30, CD27, and 4-1 BB (5, 6). All the members of this superfamily
share a characteristic repeating cysteine-rich motif in the
extracellular domain, which is believed to be related to their ability
to interact with the TNF-related ligands. The diverse cellular
responses such as cell growth, differentiation, and programmed cell
death (apoptosis) are triggered by the interaction between the members
of the NGF-R/TNF-R superfamily and their ligands.
The ligand for human OX40 was also cloned and identified as previously reported gp34, a cell surface protein expressed on HTLV-I-infected T cell lines and subsequently demonstrated to be induced by transactivator p40tax of HTLV-I (7-9). As expected, the deduced amino acid sequence of gp34 revealed that it is a member of the TNF family. Gp34 has been reported to be expressed on some HTLV-I-infected cell lines such as Hut 102 and MT-2 (10), human umbilical vein endothelial cells (11), and stimulated B lymphoblastoid cell line MSAB (12).
Since its first description, OX40 has been known to transmit
costimulatory signals to T cells. Recent studies with human T cells
have confirmed this finding and showed that the binding of gp34 to OX40
results in enhanced T cell proliferation and induction of interleukin-2
and -4 production in the presence of anti-CD3 or anti-T cell
receptor- antibody (7, 8). We recently reported that the
OX40/gp34 system directly mediates the adhesion of activated or
HTLV-I-transformed T cells to vascular endothelial cells (11, 13).
Furthermore, we examined the role of the OX40/gp34 system in the
development of angitis related diseases such as systemic lupus
erythematosus and erythema nodosum (14). Although these observations
have served to delineate OX40 as a multifunctional cell surface
molecule, its physiological as well as pathophysiological significance
in viral infection, inflammation (15), or malignant cell infiltration
has been poorly defined. In particular, intracellular signaling of OX40
has not been described to date.
Recently, several types of intracellular signal transducer proteins
that bind to the members of the TNF-R family and initiate distinct
signal transduction have been identified. For example, Fas (CD95) and
TNF-R1 are reported to recruit FADD(MORT1)/RIP and
TRADD/FADD(MORT1)/RIP, respectively, to initiate signal transduction pathways leading to cell death (16-22). CD30, CD40, TNF-R1, TNF-R2, and lymphotoxin- receptor recruit several members of a second class
of signal transducer family called TRAFs (TNF-R-associated factors).
Some members of the TRAF family are responsible for the activation of
NF-
B (23-35) or c-Jun N-terminal kinase (36). However, the precise
mechanism of TRAF-mediated NF-
B activation (37, 38) or c-Jun
N-terminal kinase activation remains unclear.
In the present study, we examined the intracellular events of OX40
signaling using a unique coculture system of OX40-transfected T cells
and gp34-transfected adherent cells. We here demonstrate that the OX40
stimulation leads to TRAF-mediated NF-B activation. Based on the
experimental results, a possible physiological and pathophysiological
significance of the OX40 signaling in activated T cells is
discussed.
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MATERIALS AND METHODS |
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Preparation of Plasmid Constructs-- Based on the published cDNA sequence of human gp34, cDNA of the entire coding region of gp34 was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) method. The PCR products were ligated into an expression vector pMKIT Neo (a gift of Dr. K. Maruyama, Tokyo Medical and Dental University) to construct pMKIT Neo-gp34. The preparation of an expression vector, pMKIT Neo-OX40, was described previously (11). The constructs for OX40 cytoplasmic deletion mutants were generated by PCR method using TAG (stop codon)-tagged oligonucleotides as the primer and pMKIT Neo-OX40 as the template. The PCR products were ligated into pMKIT Neo to construct pMKIT Neo-OX40-del 1, -del 2, -del 3, -del 4, -del 5, and -del 6. The partial DNA sequences were determined for all the OX40 deletion mutants to confirm the constructs.
Based on the published cDNA sequences of murine TRAF1, TRAF2, TRAF5, and human TRAF3 and TRAF4, the cDNAs of TRAFs were obtained by RT-PCR. The oligonucleotides covering the entire coding regions of TRAFs and cDNAs from murine cell line DA-1 (for TRAF1, TRAF2, and TRAF5) or phytohemagglutinin-stimulated human peripheral blood mononuclear cells (for TRAF3 and TRAF4) were used as the primers and the templates, respectively. The PCR products were integrated into the expression vector pcDNA3 (Invitrogen, San Diego, CA) or HA-tagged expression vector pCMV4-3 HA+ (pCMV-HA, a gift of Dr. W. C. Greene, Gladstone Institute of Virology and Immunology, University of California, San Francisco) to construct pcDNA3-TRAFs or pCMV-HA-TRAFs. The cDNAs of truncated TRAF2 (TRAF2 DN) and truncated TRAF5 (TRAF5 DN) were generated by PCR with the primers covering the amino acid sequence (amino acids 256-501) of TRAF2 (21) and (amino acids 233-558) of TRAF5 (35), respectively, and then integrated into pcDNA3.Preparation of Soluble gp34-- A construct for soluble gp34 was designed by fusing the extracellular portion of gp34 (nucleotide sequence, 187-585) to the signal sequence of OX40 (nucleotide sequence, 6-116) at the SmaI site. The fused fragments were ligated into the expression vector pME18S (11) (a gift of Dr. K. Maruyama). Four µg of pME18S-soluble gp34 were transfected into COS-7 cells (1 × 107 cells) by the DEAE-dextran method (39). The transfected cells were cultured with Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal calf serum (Bio Whittaker, Verviers, Belgium) for 24 h and with 1% fetal calf serum for another 48 h. The culture supernatants were collected and concentrated 10-fold with Centriprep 10 (Amicon Inc., Beverly, MA) prior to the assay. The supernatant of COS-7 cells transfected with pME18S-soluble gp34 (soluble gp34-sup) was added to the cell culture at 25% v/v for each assay. The supernatant of COS-7 cells transfected with empty vector (mock-sup) was prepared by the same method as soluble gp34-sup and concentrated 10-fold prior to the assay.
Cells and Culture Conditions-- Human T cell line HSB-2 and murine epithelial cell line MMCE were cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 60 mM tobramycin, and 2 mM L-glutamine. Human embryo kidney cell-derived cell line HEK 293T and COS-7 were cultured with Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.
The stable transfectants of OX40 (HSB-2-OX40 cells), or OX40 deletion mutants (HSB-2-OX40-del 1, -del 2, -del 3, -del 4, -del 5, or -del 6 cells) were prepared by introducing pMKIT Neo-OX40, or pMKIT Neo-OX40-del 1, -del 2, -del 3, -del 4, -del 5, or -del 6 into HSB-2 cells by the electroporation method. The stable transfectants of gp34 (MMCE-gp34 cells) were prepared by introducing pMKIT Neo-gp34 into MMCE cells by the electroporation method. The transfected cells were dispersed in 96-well flat-bottomed plates for clonal selection and cultured in RPMI 1640 medium containing 1 mg/ml G418 (Sigma) for 3 weeks. The expression of OX40 or gp34 on the transfected cells was examined with a FACScan (Becton Dickinson, San Jose, CA) prior to the assay. The empty vector was transfected into HSB-2 cells or MMCE cells to prepare HSB-2-mock cells or MMCE-mock cells. The empty vector transfected cells (mock cells) were cultured in 10-cm dishes with RPMI 1640 medium containing 1 mg/ml G418. The intracellular events in HSB-2-OX40 cells were examined after they were cultured on the monolayer of adhesive MMCE-gp34 cells. The contamination of MMCE-gp34 cells in the harvested cells was evaluated by RT-PCR (94 °C 1 min, 60 °C 1 min, 72 °C 2 min for 25 cycles) using murine specificElectrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts from HSB-2-OX40 cells or HSB-2-mock cells (2 × 106 cells) cocultured with either MMCE-gp34 cells or
MMCE-mock cells, or cultured with either soluble gp34-sup or mock-sup
for the indicated periods were prepared as described previously (40).
The nuclear extracts from HSB-2-OX40 cells preincubated (at 37 °C
for 30 min) with either anti-OX40 monoclonal antibody (50 µg/ml) (11)
or anti-interleukin-2 receptor chain antibody (anti-Tac, control antibody, 50 µg/ml) prior to coculture with MMCE-gp34 cells were also
prepared. Eight µg of nuclear extracts were mixed with
32P-labeled
B oligonucleotide containing a binding site
for NF-
B/c-Rel homodimeric and heterodimeric complexes
(5'-AGTTGAGGGGACTTTCCCAGGC-3') (Santa Cruz Biotechnology, Santa Cruz,
CA) or 32P-labeled mutant
B oligonucleotide
(5'-AGTTGAGGCGACTTTCCCAGGC-3') (Santa Cruz). The binding assay was
performed as described previously with a slight modification (41). The
composition of the induced NF-
B complex was examined by super shift
assay with anti-NF-
B p50 subunit antibody or anti-NF-
B p65
subunit antibody (Upstate Biotechnology Inc., Lake Placid, NY). Eight
µg of nuclear extracts in 10 µl of nuclear extract buffer (40) was
incubated with 100 ng of anti-NF-
B antibodies at room temperature
for 40 min prior to the binding assay.
Coimmunoprecipitation and in Vivo Binding Assay-- Two µg of pCMV-HA-TRAF1, -TRAF2, -TRAF3, -TRAF4, -TRAF5, or pCMV-HA were cotransfected with 2 µg of pME18S-OX40 or pME18S into HEK 293T (2 × 105 cells) by the calcium phosphate precipitation method (26). After 48 h of incubation, the cells were lysed in the Triton X lysing buffer (0.5% Triton X-100, 25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A). The cell lysates were immunoprecipitated with protein A-Sepharose 4FF (Pharmacia Biotech Inc., Uppsala, Sweden) and anti-OX40 monoclonal antibody (11). The immunoprecipitates were analyzed by SDS-PAGE with 7.5% gel (ATTO, Tokyo, Japan) and subjected to immunoblotting (11) with anti-HA monoclonal antibody, 12CA5 (Boehringer Mannheim). HA-tagged TRAFs were visualized by ECL detection system (Amersham Life Science, Arlington Heights, IL). The same membrane was reused for the detection of OX40 by immunoblotting.
Glutathione S-Transferase (GST) Fusion Protein Expression and in Vitro Binding Assay-- The cDNA of cytoplasmic portion of OX40 (corresponding to amino acids 241-277), OX40-del 3 (amino acids 241-249), OX40-del 4 (amino acids 241-255), OX40-del 5 (amino acids 241-263), or OX40-del 6 (amino acids 241-271) was inserted into the GST-fusion vector pGEX-5X-1 (Pharmacia). Glutathione-Sepharose 4B beads (Pharmacia) conjugated with GST-OX40, GST-OX40-del 3, GST-OX40-del 4, GST-OX40-del 5, GST-OX40-del 6, or GST were prepared as described previously (42). Four µg of pCMV-HA-TRAF1, -TRAF2, -TRAF3, -TRAF4, -TRAF5, or pCMV-HA was transfected into HEK 293T cells (2 × 105 cells) by the calcium phosphate precipitation method. After 48 h of incubation, the cell lysates were prepared by the same method as stated for the in vivo binding assay and mixed with either GST-OX40-, GST-OX40-del 3-, GST-OX40-del 4-, GST-OX40-del 5-, GST-OX40-del 6-, or GST-Sepharose 4B beads by rotating at 4 °C for 6 h. Protein bound to GST or GST-OX40 fusion protein was analyzed by SDS-PAGE with 7.5% gel (ATTO) and subjected to immunoblotting with anti-HA antibody 12CA5. Protein bound to GST-OX40-del 3, GST-OX40-del 4, GST-OX40-del 5, or GST-OX40-del 6 was analyzed by SDS-PAGE with 10.5% gel (ATTO) and subjected to immunoblotting with biotin-conjugated anti-HA-monoclonal antibody (Boehringer Mannheim). HA-tagged TRAFs were visualized by ECL detection system (Amersham).
Luciferase Assay--
In the studies of NF-B activation by
overexpression of TRAFs, 1.5 µg of pcDNA3-TRAF1, -TRAF2, -TRAF3,
-TRAF4, -TRAF5, -TRAF2 DN, or -TRAF5 DN were transfected into
HSB-2-OX40 cells (1 × 106 cells) together with 500 ng
of
B site integrated luciferase reporter gene
B-luc (43) (a gift
of Dr. W. Greene) and 250 ng of pCSK-LacZ (27) by the DEAE-dextran
method (44). The total amount of pcDNA3 constructs was adjusted to
3 µg by adding an empty vector. After 28 h of incubation the
cells were lysed in 250 µl of reporter lysis buffer (Toyo Ink Co.,
Tokyo Japan). Twenty µl of cell extract from each sample were
fractionated to measure the luciferase activity in accordance with the
manufacturer's protocol (Toyo Ink) using a luminometer (Bio-Orbit Oy,
Turku, Finland). Forty µl of cell extract were fractionated to
measure
-galactosidase activity as an internal control (27). The
luminescence values were normalized by the individual
-galactosidase
activity. In the experiments of TRAF2- and TRAF5-mediated NF-
B
activation, pcDNA3-TRAF2 DN, -TRAF5 DN, -TRAF3, or pcDNA3 was
cotransfected with 500 ng of
B-luc and 250 ng of pCSK-LacZ into
HSB-2-OX40 cells or HSB-2 mock cells (1 × 106 cells)
by the DEAE-dextran method. The total amount of pcDNA3 constructs
was adjusted to 3 µg by adding an empty vector. After 24 h of
incubation, the transfected cells were cocultured with either MMCE-gp34
cells or MMCE-mock cells for 24 h and then harvested to measure
luciferase activity and
-galactosidase activity as described
above.
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RESULTS |
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NF-B Is Activated by OX40 Stimulation--
Activation of
NF-
B in HSB-2-OX40 cells was detected by EMSA when the cells were
cocultured with MMCE-gp34 cells but not with MMCE-mock cells (Fig.
1A). The activation was
detected from 30 min up to 6 h (the end of culture period, data
not shown) after ligand stimulation and blocked clearly by
preincubation of HSB-2-OX40 cells with anti-OX40 antibody. The
supershift of the band with anti-NF-
B p50 subunit antibody or
anti-NF-
B p65 subunit antibody indicated the involvement of NF-
B,
consisting of p50 and p65 subunits in OX40-mediated activation. Similar
results were obtained when the HSB-2-OX40 cells were incubated with
soluble gp34-sup, but not with mock-sup (Fig. 1B).
Furthermore, the studies with HSB-2-OX40 deletion mutants demonstrated
that NF-
B activation was detected in HSB-2-OX40-del 5 (amino acids
1-263) cells and -OX40-del 6 (amino acids 1-271) cells, but not
HSB-2-OX40-del 4 (amino acids 1-255) cells after ligand stimulation.
These results indicated that the cytoplasmic portion of OX40 consisting
of the amino acid sequence 256-263 (GGSFRTPI) was required for the
activation of NF-
B (Fig. 1, C and D).
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TRAF 1, TRAF2, TRAF3, and TRAF5 but Not TRAF 4 Associate with OX40 in Vitro-- It has been reported that TRAFs associate with the receptors of several members of the TNF-R family and initiate the signal transduction upon the ligand stimulation. We examined the association of TRAFs with OX40 using HA-tagged-TRAFs and GST-OX40 fusion protein. As shown in Fig. 2A, HA-TRAF1, -TRAF2, -TRAF3, and -TRAF5 but not HA-TRAF4 associated with GST-OX40. HA-TRAF1, -TRAF2, -TRAF3, -TRAF4, and -TRAF5 were successfully expressed in HEK293 T cells and immunoprecipitated with anti-HA antibody (data not shown). Furthermore, the studies with GST-OX40 deletion mutants demonstrated that TRAF1, TRAF2, TRAF3, and TRAF5 associated with GST-OX40-del 5 (amino acids 241-263) and GST-OX40-del 6 (amino acids 241-271) (data not shown), but not with GST-OX40-del 3 (amino acids 241-249) (data not shown) or GST-OX40-del 4 (amino acids 241-255) in vitro (Fig. 2B). In other words, the cytoplasmic portion of OX40 consisting of the amino acid sequence 256-263 (GGSFRTPI) was required for the association with TRAF1, TRAF2, TRAF3, and TRAF5 in vitro.
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TRAF2, TRAF3, and TRAF5 Associate with OX40 in Vivo-- We next examined the association of TRAFs with OX40 in vivo. HA-TRAF2, HA-TRAF3, and HA-TRAF5 were coimmunoprecipitated with anti-OX40 antibody when coexpressed with OX40 in HEK 293T cells, indicating that these TRAFs can associate with OX40 in vivo (Fig. 3). HA-TRAF1 and HA-TRAF4 were successfully expressed in HEK 293T cells and immunoprecipitated with anti-HA antibody. However, the association of HA-TRAF1 or HA-TRAF4 with OX40 in vivo was not detected under this condition (data not shown).
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TRAF2 and TRAF5 Mediate NF-B Activation in OX40 Signaling, while
TRAF3 Negatively Modulates NF-
B Activation--
To evaluate the
ability of various TRAFs to mediate NF-
B activation in HSB-2-OX40
cells, pcDNA3-TRAFs were transfected with the luciferase reporter
plasmid
B-luc into HSB-2-OX40 cells. The luciferase assay of the
transfected cell lysates demonstrated that TRAF2 and TRAF5 but not
TRAF1, TRAF3, or TRAF4 were able to mediate NF-
B activation when
overexpressed in HSB-2-OX40 cells (Fig.
4A). Since TRAF2, TRAF3, and
TRAF5 were found to associate with OX40 in vivo, we examined
the effects of TRAF3 on NF-
B activation mediated by TRAF2 or TRAF5
in HSB-2-OX40 cells. As shown in Fig. 4A, the introduction
of TRAF3 reduced the levels of NF-
B activation by overexpressed
TRAF2 or TRAF5. Based on this experiment, we further examined the roles
of TRAF2, TRAF5, and TRAF3 in NF-
B activation resulting from OX40
stimulation by introducing the dominant negative forms of TRAF2 (TRAF2
DN), TRAF5 (TRAF5 DN), or wild type TRAF3 into HSB-2-OX40 cells. The
transfected HSB-2-OX40 cells were stimulated by MMCE-gp34 cells. As
shown in Fig. 4B, the introduction of TRAF2 DN or TRAF5 DN
suppressed the luciferase activity in a dose dependent manner.
Furthermore, the introduction of both TRAF2 DN and TRAF5 DN suppressed
the luciferase activity markedly to the lowest level. The introduction
of TRAF3 together with TRAF2 DN or TRAF5 DN reduced NF-
B activation
further, which suggests that TRAF3 modulates NF-
B activation
negatively in OX40 signaling.
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DISCUSSION |
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In the present study, we demonstrated that TRAF2- and
TRAF5-mediated NF-B activation was induced by OX40 stimulation. We have had difficulties in the study of human OX40 signaling, since neither of our two anti-OX40 monoclonal antibodies (11) could trigger
OX40 signaling even when cross-linked with the second antibody. We,
therefore, employed a unique coculture system of OX40-transfected HSB-2
cells and human gp34-transfected MMCE cells. The separation of
HSB-2-OX40 cells from MMCE-gp34 cells was easy and the contamination of
MMCE-gp34 cells in the harvested cells was estimated to be less than
0.5% by RT-PCR method using murine specific primers. We also employed
the culture supernatants of COS-7 cells transfected with the soluble
gp34-construct in most of the assays to confirm the data obtained from
the coculture system. Although the signals triggered by soluble gp34
were somewhat weaker than those by membrane-bound gp34, the experiments
with soluble gp34 gave essentially the same results.
The studies with OX40 deletion mutants demonstrated that the
cytoplasmic portion of OX40 consisting of the amino acid sequence 256-263 (GGSFRTPI) was required for association with TRAFs and NF-B
activation. It is notable that the potential phosphorylation site for
protein kinase C is included in this portion of 8 amino acid residues
(4). In addition, our preliminary studies showed the induction of c-jun
mRNA by OX40 stimulation, for which the same intracytoplasmic
portion GGSFRTPI was required (data not shown). Taken together, we
consider that this portion of 8 amino acid residues constitutes a part
of the crucial domain that initiates multiple signal transductions upon
OX40 stimulation.
Previous studies demonstrated that several members of TRAFs associate
with the members of the TNF-R family and initiate signal transduction.
For example, TNF-R2 is associated with TRAF1, TRAF2, or TRAF3 (23-26).
TNF-R1 is associated with TRAF2 through TRADD (21). CD40 is associated
with TRAF2 (26, 27) or TRAF3 (23, 28-30). CD30 is associated with
TRAF1, TRAF2, TRAF3, or TRAF5 (31-34). Lymphotoxin- receptor is
associated with TRAF3 (23) or TRAF5 (35). In some of these reports, two
independent motifs in the receptors, EEEGKE and PXQE, have been
described as "TRAFs-binding motifs" (31, 32, 45). While the EEEGKE
motif that interacts with TRAF1 and TRAF2, but not with TRAF3, is not
found in OX40, the PXQE motif that interacts with TRAF2 and TARF3 is
conserved among CD30 (amino acids 561-564), CD40 (amino acids
250-253), and OX40 (amino acids 262-265). However, the OX40 deletion
mutant HSB-2-OX40 del 5 (amino acids 1-263) cells were able to trigger intracellular signaling leading to NF-
B activation upon ligand stimulation and GST-OX40 del 5 (amino acids 241-263) was able to
associate with TRAFs in vitro. We, therefore, need further evaluation to determine whether all four amino acid residues (PXQE) are
indispensable for the association with TRAFs and NF-
B activation. Several reports indicate that TRAFs are recruited to the receptor by
forming the complex with the receptor-associated molecules such as
TRADD (21) and/or TRAF-associated molecules such as c-IAP (24) and TRIP
(46). It is, therefore, possible that the binding site and the binding
affinity between TRAFs and the receptors are affected by such
interacting molecules.
In most of the members of the TNF-R family, TRAF2 and/or TRAF5 are
responsible for the activation of NF-B, while the function of TRAF1
(31), TRAF3, or TRAF4 (47) has not been clearly understood. We
demonstrated that both TRAF2 and TRAF5 mediated NF-
B activation in
OX40 signaling, whereas TRAF3 exerted suppressive effects on NF-
B
activation as previously reported in CD30 signaling (31). Further
studies will be needed to elucidate the precise role of TRAF3 in OX40
signaling.
TRAF-mediated NF-B activation, indeed, has furnished a clue to the
understanding of the signaling in the several members of the TNF-R
family. However, studies of the downstream events after NF-
B
activation as well as other signaling pathways would be one of the key
issues to be addressed to give a satisfactory explanation of the
diverse cellular responses triggered by the stimulation of the members
of the TNF-R family. Recently several groups reported that the
activation of NF-
B blocked apoptosis (48-50), which may help to
understand the role of the OX40/gp34 system in vivo. The
conspicuous feature of the OX40/gp34 system among the TNF-R/TNF family
is its ability to mediate adhesion between activated or
HTLV-I-transformed T cells and endothelial cells. It is, therefore,
possible that the OX40 signaling in T cells is triggered by the
interaction with endothelial cells of the tissues where activated T
cells expressing OX40 are infiltrating. OX40-mediated NF-
B
activation in T cells may serve to protect them from apoptosis, which
results in the amplification and prolongation of the immune responses,
or in the case of adult T cell leukemia, prolonged survival of leukemic
cells in the infiltrated tissues.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. K. Maruyama for
providing pMKIT Neo and pME18S expression vectors, and Dr. W. C. Greene for the luciferase reporter plasmid construct B-luc and
HA-tagged vector pCMV-HA. We also thank K. Fukunaga for excellent
technical assistance and useful discussions.
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FOOTNOTES |
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* This study was supported in part by grants from the Japanese Ministry of Education, Science, Sports and Culture.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.
To whom correspondence should be addressed: Institute for Virus
Research, Kyoto University, 53 Shogoin-kawaracho, Sakyo-ku, Kyoto 606 Japan. Tel.: 81-75-751-4048; Fax: 81-75-751-4049.
1 The abbreviations used are: HTLV-I, human T cell leukemia virus type I; NGF, nerve growth factor; TNF, tumor necrosis factor; R, receptor; TRAF, tumor necrosis factor receptor-associated factor; RT, reverse transcriptase; PCR, polymerase chain reaction; HA, hemagglutinin; luc, luciferase; CMV, cytomegalovirus; HEK, human embryo kidney; EMSA, electromobility shift assay; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
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REFERENCES |
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