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INTRODUCTION |
The members of the tumor necrosis factor
(TNF)1 family are involved in
regulating diverse biological activities such as regulation of cell
proliferation, differentiation, cell survival, cell death, cytokine
production, lymphocyte co-stimulation, and isotype switching (1, 2).
Receptors in this family share a common structural motif in their
extracellular domains consisting of multiple cysteine-rich repeats of
approximately 30-40 amino acids (3). While TNFR1, CD95/Fas/APO-1,
DR3/TRAMP/APO-3, DR4/TRAIL-R1/APO-2, DR5/TRAIL-R2, and DR6 receptors
contain a conserved intracellular motif of ~80 amino acids called
death domain, associated with the activation of apoptotic signaling
pathways, other members, which contain a low sequence identity in the
cytoplasmic domains, stimulate the transcription factors NF-
B and
AP-1 (1-3).
Most TNF receptors contain a functional cytoplasmic domain. However,
some members of the TNFR superfamily do not have cytoplasmic domains
and are secreted, such as osteoprotegerin (OPG) (4), or linked to the
membrane through a glycophospholipid tail, such as TRID/DcR1/TRAIL-R3
(5, 6). Viral open reading frames encoding soluble TNFRs have also been
identified, such as SFV-T2 (7), Va53 (8), G4RG (9), and crmB
(3).
By searching an expressed sequence tag (EST) data base, a new member of
the TNFR superfamily was identified, named TR6, and was characterized
as a soluble cognate receptor for LIGHT and FasL/CD95L. LIGHT and FasL
mediate the apoptosis, which is the most common physiological form
of cell death and occurs during embryonic development, tissue
remodeling, immune regulation, and tumor regression.
LIGHT is highly induced in activated T lymphocytes and macrophages.
LIGHT was characterized as a cellular ligand for HVEM/TR2 and LT
R
(10). HVEM/TR2 is a receptor for herpes simplex virus type 1 (HSV-1)
entry into human T lymphoblasts. The soluble form of HVEM/TR2-Fc and
antibodies to HVEM/TR2 were shown to inhibit a mixed lymphocyte
reaction, suggesting a role for this receptor or its ligand in T
lymphocyte proliferation (10-12). The level of LT
R expression is
prominent on epithelial cells but is absent in T and B lymphocytes.
Signaling via LT
R triggers cell death in some adenocarcinomas (13).
LIGHT produced by activated lymphocytes could evoke immune modulation
from hematopoietic cells expressing only HVEM/TR2 and induce apoptosis
of tumor cells, which express both LT
R and HVEM/TR2 receptors (14,
15).
FasL is one of the major effectors of cytotoxic T lymphocytes and
natural killer cells. It is also involved in the establishment of
peripheral tolerance in the activation-induced cell death of lymphocytes. Moreover, expression of FasL in nonlymphoid and tumor cells contributes to the maintenance of immune privilege of tissues by
preventing the infiltration of Fas-sensitive lymphocytes (16). FasL is
also processed and shed from the surface of human cells (17).
Here we demonstrate that TR6 (DcR3), a new member of the TNFR
superfamily, binds LIGHT and FasL. Therefore TR6 may act as an
inhibitor in LIGHT-induced tumor cell death by blocking LIGHT interaction with its receptors.
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MATERIALS AND METHODS |
Identification and Cloning of New Members of the TNFR
Superfamily--
An EST cDNA data base, obtained from more than
600 different cDNA libraries, was screened for sequence homology
with the cysteine-rich motif of the TNFR superfamily, using the blastn
and tblastn algorithms. Three EST clones containing an identical open
reading frame, the amino acid sequence of which showed significant
homology to TNFR-II, were identified from cDNA libraries of human
normal prostate and pancreas tumor. A full-length TR6 cDNA clone
encoding an intact N-terminal signal peptide was obtained from a human
normal prostate library.
RT-PCR Analysis--
For RT-PCR analysis, total RNA was isolated
using TriZOL (Life Technologies, Inc.) from various human cell lines
before and after stimulation with phorbol 12-myristate
13-acetate/ionomycin or lipopolysaccharide. RNA was converted to
cDNA by reverse transcription and amplified for 35 cycles by PCR.
Primers used for amplification of the TR6 fragment are according to the
sequence of TR6.
-Actin was used as an internal control for RNA
integrity. PCR products were run on 2% agarose gel, stained with
ethidium bromide, and visualized by UV illumination.
Recombinant Protein Production and Purification--
The
recombinant TR6 protein was produced with hexahistidine at the C
terminus. TR6-(His) encoding the entire TR6 protein was amplified by
PCR. For correctly oriented cloning, a HindIII site on the 5' end of the forward primer
(5'-AGACCCAAGCTTCCTGCTCCAGCAAGGACCATG-3') and a
BamHI site on the 5' end of the reverse primer
(5'-AGACGGGATCCTTAGTGGTGGTGGTGGTGGTGCACAGGGAGGAAGCGCTC-3') were created. The amplified fragment was cut with
HindIII/BamHI and cloned into a mammalian
expression vector, pCEP4 (Invitrogen). The TR6-(His)/pCEP4 plasmid was
stably transfected into HEK 293 EBNA cells to generate recombinant
TR6-(His). Serum-free culture media from cells transfected
TR6-(His)/pCEP4 were passed through Ni-column (Novagen). The column
eluents were fractionated by SDS-PAGE, and TR6-(His) was detected by
Western blot analysis using the anti-poly(His)6 antibody (Sigma).
Production of HVEM/TR2-Fc, LT
R-Fc, and FLAG-tagged soluble LIGHT
(sLIGHT) fusion proteins were previously described (14). Fc fusion
protein-containing supernatants were filtered and trapped onto protein
G-Sepharose beads. FLAG-tagged sLIGHT proteins were purified with
anti-FLAG mAb affinity column.
Immunoprecipitation--
TR6-(His) was incubated overnight with
various FLAG-tagged ligands of the TNF superfamily and anti-FLAG
agarose in binding buffer (150 mM NaCl, 0.1% Nonidet P-40,
0.25% gelatin, 50 mM HEPES, pH 7.4) at 4 °C and then
precipitated. The bound proteins were resolved by 12.5% SDS-PAGE and
detected by Western blot with HRP-conjugated anti-poly(His)6 or anti-human IgG1 antibodies.
Cell-binding Assay--
For cell-binding assays, HEK 293 EBNA
cells were stably transfected using the calcium phosphate method with
pCEP4/full sequence of LIGHT cDNA or pCEP4 vector alone. After
selection with hygromycin B, cells were harvested with 1 mM
EDTA in phosphate-buffered saline and incubated with TR6-(His),
HVEM/TR2-Fc, or LT
R-Fc for 20 min on ice. For detecting Fc-fusion
protein, cells were stained with FITC-conjugated goat anti-human IgG.
To detect TR6 binding, cells were stained with
anti-poly(His)6 and FITC-conjugated goat anti-mouse IgG
consecutively. The cells were analyzed by FACScan (Becton Dickinson).
Cytotoxicity Assay--
Cytotoxicity assays using HT29 cells
were carried out as described previously (13). Briefly, 5000 HT29 cells
were seeded in 96-well plates with 1% fetal bovine serum, Dulbecco's
modified Eagle's medium, and treated with sLIGHT (10 ng/ml) and 10 units/ml human recombinant interferon-
(IFN-
) (supplied
from NIAID, NIH Repository). Serial dilutions of TR6-(His)
were added in quadruplicate to microtiter wells. Cells treated with
IFN-
and sLIGHT were incubated with various amounts of TR6-(His) for
4 days before the addition of [3H]thymidine for the last
6 h of culture. Cells were harvested, and thymidine incorporation
was determined using a liquid scintillation counter.
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RESULTS AND DISCUSSION |
TR6 Is a New Member of the TNFR Superfamily--
TR6 was
identified by searching an EST data base. Three clones containing an
identical open reading frame were identified from cDNA libraries of
human normal prostate and pancreas tumor. A full-length TR6 cDNA
encoding an intact N-terminal signal peptide was obtained from a human
normal prostate library. The open reading frame of TR6 encodes 300 amino acids. To determine the N-terminal amino acid sequence of mature
TR6, hexahistidine-tagged TR6 was expressed in the mammalian cell
expression system, and the N-terminal amino acid sequences were
determined by peptide sequencing. The N-terminal sequence of the
processed mature TR6-(His) started from amino acid 30, indicating that
the first 29 amino acids constituted the signal sequence (Fig.
1A). Therefore, the mature
protein of TR6 was composed of 271 amino acids with no transmembrane
region. There was one potential N-linked glycosylation site
(Asn-173) in TR6. Like OPG (4), the predicted protein was a soluble, secreted protein, and the recombinant TR6 expressed in mammalian cells
was ~40 kDa as estimated on polyacrylamide gel. Fig. 1B shows the potential cysteine-rich motif aligned among TNFR-I, TNFR-II,
4-1BB, TR2/HVEM, LT
R, TR1/OPG, and TR6. TR6 contained two perfect
and two imperfect cysteine-rich motifs, and its amino acid sequence was
remarkably similar to the TR1/OPG amino acid sequence. TR6 shares
~30% sequence homology with OPG and TNFR-II.

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Fig. 1.
Sequence of TR6 and aligned amino acid
sequence of cysteine-rich motif. A, a deduced amino
acid sequence of TR6. The signal sequence is underlined. The
potential N-glycosylation site is underlined with
shadow. The N-terminal amino acid sequence of recombinant
TR6-(His) reads as VAETPT ... , which indicates that the first 29 amino acids constitute a signal sequence. B, aligned amino
acid sequence of the cysteine-rich motif of TR6 with other TNF receptor
family members. The amino acid sequence of TR6 was aligned with those
of TNFR-I, TNFR-II, 4-1BB, TR2 (HVEM), LT R, and TR1 (OPG) on the
basis of sequence homology and conserved cysteines.
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mRNA Expression--
We analyzed expression of TR6 mRNA in
human multiple tissue blots by Northern hybridization. Northern blot
analyses indicated that TR6 mRNA was ~1.3 kilobases in length and
was expressed predominantly in lung tissue and the colorectal
adenocarcinoma cell line SW480 (data not shown). RT-PCR analyses were
performed to determine the expression patterns of TR6 in various cell
lines. TR6 transcript was detected weakly in most hematopoietic cell
lines. The expression of TR6 was induced upon activation in Jurkat T
leukemia cells. Interestingly, TR6 mRNA was constitutively
expressed in endothelial cell line, HUVEC, at high level (Fig.
2).

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Fig. 2.
mRNA expression of TR6 in various human
cell lines. TR6 mRNA expression was detected with TR6-specific
RT-PCR. A low level of TR6 expression was detected in most hemopoietic
cells, and high level expression was found in stimulated Jurkat T
cells, monocytic THP-1 cells, and endothelial HUVEC cells.
Amplification of -actin was used as an internal control.
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Identification of the Ligand for TR6--
To identify the ligand
for TR6, several FLAG-tagged soluble proteins of TNF ligand family
members were screened for binding to recombinant TR6-(His) protein by
immunoprecipitation. TR6-(His) selectively bound LIGHT-FLAG and
FasL-FLAG among FLAG-tagged soluble TNF ligand members tested (Fig.
3). This result indicates that TR6 binds
at least two ligands, LIGHT and FasL. LIGHT exhibits significant
sequence homology with the C-terminal receptor-binding domain of FasL
(31%), but sLIGHT is unable to bind to Fas (10, 14). They may have a
similar binding epitope for TR6 binding.

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Fig. 3.
Biochemical identification of TR6
ligand. A, LIGHT and FasL are ligands for TR6.
Flag-tagged soluble members of the TNF ligand superfamily, TRANCE,
LIGHT, FasL, 4-1BBL and unpublished novel protein, TL3, TL6, and TL7,
in 1 ml of 150 mM NaCl, 0.1% Nonidet P-40, 0.25% gelatin,
50 mM HEPES (pH 7.4) buffer was mixed with poly(His)-tagged
TR6, incubated, and then precipitated with anti-FLAG agarose. The bound
TR6-(His) was resolved by SDS-PAGE (12.5%) and detected by immunoblot
with anti-poly(His) antibody. B, TR6 competitively inhibits
the HVEM/TR2-Fc-LIGHT-Flag interaction. The same concentrations of
HVEM-Fc (20 nM) and TR6-(His) (20 nM) were
incubated with cultured supernatant containing LIGHT-FLAG protein and
then precipitated with anti-FLAG agarose. The bound protein was
resolved by SDS-PAGE and detected HVEM-Fc with HRP-conjugated
anti-human IgG antibody (top), and stripped membrane was
reused to detect TR6-(His) with HRP-conjugated anti-poly(His) antibody.
C, TR6 inhibits the HVEM/TR2-Fc-LIGHT-FLAG binding and
LT R-Fc-LIGHT-FLAG binding. HVEM/TR2-Fc (6 nM) and
LT R-Fc (6 nM) with or without TR6-(His) (20 nM) were mixed with LIGHT-FLAG and then precipitated with
anti-FLAG antibody and resolved on 12.5% SDS-PAGE. The bound
HVEM/TR2-Fc and LT R-Fc were detected with anti-human IgG
antibody.
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Previously, Zhai et al. (14) and Harrop et al.
(15) reported the biological functions of LIGHT and its possible
mechanisms of action as a ligand for HVEM/TR2 and/or LT
R. LIGHT is
expressed in activated T cells. LIGHT, in conjunction with serum
starvation or addition of IFN-
, inhibits the cell proliferation in
tumor cells, MDA-MB-231 and HT29.
To determine whether TR6 might act as an inhibitor to LIGHT
interactions with HVEM/TR2 or LT
R, TR6-(His) was used as a
competitive inhibitor in LIGHT-HVEM/TR2 interaction. When LIGHT was
immunoprecipitated with HVEM/TR2-Fc in the presence of TR6-(His),
HVEM/TR2-Fc binding to LIGHT was decreased competitively by TR6-(His),
but TR6-(His) binding to LIGHT was not changed by HVEM/TR2-Fc (Fig.
3B). Furthermore, the binding of HVEM/TR2-Fc (6 nM) or LT
R (6 nM) was completely inhibited
by 20 nM TR6-(His) protein in immunoprecipitation assays (Fig. 3C). These results support the notion that TR6 may act
as a strong inhibitor of LIGHT function through HVEM/TR2 and
LT
R.
Binding of TR6-(His) to LIGHT-transfected Cells--
To determine
whether TR6 binds to LIGHT expressed on the cell surface, we performed
a binding assay using LIGHT-transfected HEK 293 EBNA cells by flow
cytometry. LIGHT-transfected HEK 293 EBNA cells were stained
significantly by TR6-(His) as well as by HVEM/TR2-Fc and LT
R-Fc. No
binding was detected by HVEM/TR2-Fc or LT
R-Fc on pCEP4
vector-transfected HEK 293 EBNA cells. (Fig. 4). Furthermore, control isotype did not
bind to LIGHT-transfected HEK 293 EBNA cells, and none of the above
fusion proteins bound to vector-transfected cells, confirming the
specificity of these bindings. These bindings indicate that TR6 can
bind to both soluble and membrane-bound forms of LIGHT.

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Fig. 4.
Identification of the membrane-bound TR6
ligand. HEK293 EBNA cells were transfected with pCEP4 control
vector (shaded area) or with pCEP4/encoding full-length
LIGHT cDNA (solid line). Cells were incubated with
HVEM/TR2-Fc (0.34 µg) (A), LT R-Fc (0.34 µg)
(B), TR6-(His) (0.34 µg) or buffer control (same as
vector) (C). Cells were stained with anti-hIgG-FITC for
detecting HVEM/TR2 and LT R binding. For detecting TR6 binding, cells
were stained with anti-poly(His) and anti-mIgG-FITC. They were analyzed
for binding by FACS.
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TR6 Inhibits LIGHT-induced Cytotoxicity in HT29
Cells--
Browning et al. (13) have shown that Fas
activation leads to rapid cell death (12-24 h) whereas LT
R takes
2-3 days in induction of apoptosis for the colorectal adenocarcinoma
cell line, HT29. Zhai et al. (14) also reported that LIGHT
leads to the death of the cells expressing both LT
R and HVEM/TR2 but
not the cells expressing only the LT
R or HVEM/TR2 receptor. Both
HVEM/TR2 and LT
R are involved cooperatively in LIGHT-mediated
killing of HT29 cells (14).
To determine whether binding of TR6 inhibits LIGHT-mediated
cytotoxicity, HT29 cells were incubated with 10 ng/ml sLIGHT and IFN-
(10 units/ml) in the presence of 200 ng/ml LT
R-Fc or
TR6-(His). As shown in Fig.
5A, TR6-(His) blocked
significantly the LIGHT-mediated cell killing. Cells were also
incubated with sLIGHT and/or IFN-
in the presence of varying
concentrations of TR6-(His). TR6-(His) blocked sLIGHT-induced cell
death in a dose-dependent manner (Fig. 5B).
Taken together, TR6 appears to act as a natural inhibitor of
LIGHT-induced tumor cell killing. The data also suggest that TR6
contributes to immune evasion of tumors.

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Fig. 5.
TR6 inhibits LIGHT-induced cell death in HT29
cells. A, HT29 cells were incubated in 96-well plates
with control medium, 10 units/ml IFN- alone, purified sLIGHT protein
(10 ng/ml) in the absence or presence of IFN- (10 units/ml),
purified sLT R-Fc (200 ng/ml), or TR6-(His) (200 ng/ml) in the
presence of IFN- (10 units/ml) and sLIGHT (10 ng/ml). B,
cells were incubated with various doses of TR6-(His) and IFN- (10 units/ml) with (open circle) or without (filled
circle) sLIGHT (10 ng/ml). In all assays, cells were cultured for
4 days, and proliferation was detected during the last 6 h of
culture by the addition of 1 µCi of [3H]thymidine.
Cells were harvested, and thymidine incorporation was determined using
a liquid scintillation counter.
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LIGHT interaction with HVEM/TR2 and/or LT
R may trigger the distinct
biological events, such as T cell proliferation, blocking of
HVEM-dependent HSV1 infection, and anti-tumor activity (10, 14, 15). TR6 may act as an inhibitor of LIGHT interaction and may play
diverse roles in different cell types. Indeed, while this paper was in
preparation, another group reported an identical cDNA to TR6, which
they called decoy receptor (DcR) 3 and showed that it bound to FasL and
might contribute to immune evasion by certain tumor (18). TR6 may act
as a decoy receptor and contribute to immune evasion both in slow and
rapid tumor cell death, which is mediated by LIGHT or the FasL-mediated
apoptosis pathway.
TR6 may function as a cytokine to trigger membrane-bound FasL or LIGHT
and transduce signals through FasL or LIGHT. Recently the Desbarats and
Suzuki groups (19, 20) reported that FasL could itself transduce
signals, leading to cell cycle arrest and cell death in
CD4+ T cells but cell proliferation in CD8+ T
cells. Therefore, TR6 may be involved in signaling through FasL and LIGHT.
HUVEC cells constitutively expressed TR6 in RT-PCR analysis. LIGHT and
FasL have been known to be expressed in activated T cells. Therefore it
is speculated that TR6 and its ligands are important for interactions
between activated T lymphocytes and endothelium. TR6 may be involved in
activated T cell trafficking as well as endothelial cell survival.
In this paper we have identified a novel soluble member of the TNFR
superfamily, TR6, which is constitutively expressed in lung tissue,
tumor cells, and in endothelial cells. We have also identified the
ligands for TR6, LIGHT, and FasL, which are involved in the cell death
pathway. TR6 bound specifically to LIGHT and FasL and inhibited their
activities. Like DcR1, DcR2, and another soluble member of the TNFR
superfamily, OPG, TR6 may act as an inhibitor of signaling through TNF
family members, FasL and LIGHT. Hence, TR6 may have important roles in
the inhibition of apoptosis and tumor modulation.