From the Department of Microbiology and Immunology
and the Walther Oncology Center, Indiana University School of Medicine
and the Walther Cancer Institute, Indianapolis, Indiana 46202, § Human Genome Sciences, Rockville, Maryland 20850, and the
Department of Biological Sciences and the
Immunomodulation Research Center, University of Ulsan, Ulsan, Korea
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ABSTRACT |
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Among members of the tumor necrosis factor
receptor (TNFR) superfamily, 4-1BB, CD27, and glucocorticoid-induced
tumor necrosis factor receptor family-related gene (GITR) share a
striking homology in the cytoplasmic domain. Here we report the
identification of a new member, activation-inducible TNFR family member
(AITR), which belongs to this subfamily, and its ligand. The receptor is expressed in lymph node and peripheral blood leukocytes, and its
expression is up-regulated in human peripheral mononuclear cells mainly
after stimulation with anti-CD3/CD28 monoclonal antibodies or phorbol
12-myristate 13-acetate/ionomycin. AITR associates with TRAF1 (TNF
receptor-associated factor 1), TRAF2, and TRAF3, and induces nuclear
factor (NF)- Members of the TNFR1
superfamily share similar multiple cysteine-rich pseudorepeats of the
extracellular domain, each containing 30-45 amino acids with six
cysteines (1). Except for the death domain-containing family, which
includes TNFR1 (2), Fas (3-5), DR3 (6-9), DR4 (10-12), DR5 (13-17),
and decoy TNF-related apoptosis-inducing ligand (TRAIL) receptors
(18-21), no remarkable similarity is found within the intracellular
domain of these molecules. However, there is a striking homology in the
cytoplasmic domains of murine and human 4-1BB, CD27, and murine GITR
within TNFR superfamily members (22-24). Acidic amino acids are
especially highly conserved in the cytoplasmic domain of this
subfamily. Like other TNFR superfamily members (1), this subfamily is
implicated in diverse biological functions. First of all, 4-1BB and
CD27 molecules provide strong co-stimulatory signals for T cell
proliferation when ligated with their respective ligands or with
agonistic antibodies (1, 25). In addition to functioning as an
accessory molecule, CD27 induces apoptosis, which is mediated by a
death domain-containing molecule called Siva (26). Recently identified
murine GITR is shown to inhibit T cell receptor-induced apoptosis
(24).
Although the immunological functions of subfamily members have been
relatively well defined, insights into their signal transduction pathway have only recently been revealed (27-30). Two groups (27, 28)
have provided data indicating that association of 4-1BB with TRAF2
molecules initiates a signal cascade leading to activation of NF- Because the number of TNFR members is rapidly expanding, we expected
that even more numbers of the superfamily would exist. By searching an
EST (expressed sequence tag) data base, we identified a new member of
the TNFR and its ligand. Here we give an initial characterization of
the receptor and its ligand.
cDNA Cloning--
A data base containing more than 2 million
ESTs obtained from over 750 different cDNA libraries was generated
by Human Genome Sciences, Inc., using high throughput automated DNA
sequence analysis of randomly selected human cDNA clones. A
specific homology and motif search using the known amino acid sequence
and motif of TNFR members against this data base revealed several ESTs
with a translated sequence 35-55% homologous to that of the TNFR
family. Several clones were identified from cDNA libraries of
PHA-activated T cells, T helper cells, leukocytes, a healing abdomen
wound, primary dendritic cells, and adipose tissue. A full-length AITR cDNA clone encoding an intact N-terminal signal peptide was
obtained from a human activated T cell library and selected for further investigation. The complete cDNA sequence of both strands of this clone was determined, and its homology to TNFR members was confirmed. Similarly, TL6 (TNF ligand 6) was identified through a systematic comparison of sequence homology with TNF ligand family members. Partial
TL6 sequences, which were 25% homologous to that of TNF ligand family
members, were identified from endothelial, HUVEC (human umbilical vein
endothelial cell), brain, and fetal liver cDNA libraries. A
full-length cDNA clone was obtained from a human brain cDNA library.
Expression Vectors--
Full-length and HA (hemagglutinin
A epitope)-tagged AITR encoding the putative entire AITR protein (amino
acids 26-234) were amplified by PCR using sense
(5'-CTAGCTAGCTAGVVVAGCGCCCCACCGGGGGTCCC-3', and
5'-CTAGCTAGCTAGCTATCCATATGATGTTCCAGATTATGCTCAGCGCCCCACCGGGGGTCCC-3', respectively) and antisense (5'-AAGGAAAAAAGC
GGGCCGCTCACACCCACAGGTCTCCCAG-3') primers, cut with
NheI/NotI, and fused in-frame downstream of a CD5
leader sequence (28) into the pcDNA3.1 (pcDNA3.1/CD5L-AITR) and
pcDNA3 (pcDNA3/CD5L-AITR), respectively. Full-length TL6 was amplified by PCR (sense, 5'-AGACCCAAGCTTTT GAAAATGA TATGAGACGC-3'; antisense, 5'-AGACGGGATCCTCCTCCTATAG TAAGAAGGC-3'), cut with
HindIII/BamHI, and inserted into pcDNA3.1
(pcDNA3.1/TL6) and pCEP4 (Invitrogen, Carlsbad, CA; pCEP4/TL6).
pRK5-based expression vectors encoding Flag-tagged full-length TRAF1,
TRAF2, TRAF3, TRAF5, TRAF6, NIK, dominant negative TRAF2 (dnTRAF2), or
dnNIK have been described (28, 31-36). The
NF- Northern Blot and RT (Reverse Transcriptase)-PCR
Analysis--
For Northern blot analysis, cDNA probes were labeled
with 32P using the Rediprime DNA labeling system (Amersham
Pharmacia Biotech), according to the manufacturer's instructions.
Unincorporated nucleotide was removed from the labeled probe using
CHROMA SPIN-100 (CLONTECH). Two human multiple
tissue poly(A) RNA blots containing approximately 2 µg of poly(A) RNA
per lane from various human tissues were purchased from
CLONTECH. In addition, two cell line blots
containing 20 mg of total RNA from different cell lines were used.
Northern blotting was performed with the Expressed Hybridization
Solution (CLONTECH) according to the
manufacturer's manual. For RT-PCR analysis, total RNA was isolated
from human PBMC after stimulation with dexamethasone, PMA/ionomycin, or
anti-CD3/CD28 mAbs and from unstimulated or LPS-stimulated HUVEC cells.
RT-PCR was performed under standard conditions.
Interaction of AITR with TRAFs--
pcDNA3/CD5L-AITR-HA
plasmid (5 µg/10 cm-plate) was co-transfected into HEK293 EBNA cells
(2 × 106 cells/plate) by the standard calcium
phosphate precipitation method with pRK/TRAF1, -2, -3, -5, or -6-Flag
vector (5 µg/plate). Twenty four hours after transfection, cells were
lysed with 1 ml of lysis buffer (50 mM HEPES (pH 7.4), 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 10%
glycerol, and protease inhibitors). For immunoprecipitation, lysates
were incubated with anti-Flag M2 (Eastman Kodak Co.) or control murine
IgG1 mAb at 4 °C for 1 h, followed by incubation with 20 µl
of a 1:1 slurry of protein G-Sepharose (PharMingen, San Diego, CA) for
another hour. Precipitates were thoroughly washed with lysis buffer,
then fractionated on a 10% SDS-polyacrylamide gel before transfer to
polyvinylidene difluoride membrane (Millipore). Western blot analysis
was performed with anti-HA mAb coupled with horseradish peroxidase
(Boehringer Mannheim) and visualized using the enhanced
chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech).
Analysis of NF- Recombinant Protein Production and Purification--
AITR-Fc
fusion protein was used for ligand screening and cell-binding
experiments. A fragment encoding the predicted extracellular domain of
AITR (amino acids 26-139) was amplified using a sense primer flanked
by an NheI site (5'-AGACCCAAGCTTGTGGGCTCTTGAAA CCCGGCATG-3')
and an antisense primer flanked by a BglII site (5'-GAAAGATCT GGGCTCTGCCGGCGGGGACCCTGGGAC-3'). The
amplified fragment was cut with NheI/BglII and
cloned into mammalian vector pCEP4, in-frame with CD5L at the 5' end
and with the Fc portion of human IgG1 at the 3' end
(pCEP4/CD5L-AITR-Fc). pCEP4/CD5L-AITR-Fc was transfected into HEK293
EBNA cells. AITR-Fc fusion protein was purified from
pCEP4/CD5L-AITR-Fc-transfected HEK293 EBNA cell supernatants using the
protein G column. To generate a Flag-tagged soluble form of TL6 protein
(amino acids 39-169), the Flag-tagged TL6 expression vector
(pCEP4/CD5L-TL6-Flag) was constructed by PCR amplification of TL6
coding sequences using sense (5'-CTAGCTAGCCCAGCGCCCCGACTACAAGGA CGACGATGACAAGGAGACT GCTAAGGAGCCC-3') and antisense (5'-CCGCTC GAGCTATAGTAAGAAGGCTCC-3') primers, digesting the product with NheI/XhoI, and cloning into pCEP4, in-frame with
the CD5L sequence. The construct was expressed in HEK293 EBNA cells.
Transfected cell supernatants containing secreted TL6-Flag were
harvested and used for binding assays. For some experiments, TL6-Flag
protein was purified from harvested supernatants, using anti-Flag gel (Sigma) according to the manufacturer's instructions.
Binding Assay--
Protein binding assays were done essentially
as described (12). For cell-binding assays, HEK293 EBNA cells were
transfected using pcDNA3.1/CD5L-AITR or pcDNA3.1, as described
above. Forty-eight hours after transfection, cells were harvested and
incubated consecutively with TL6-Flag-containing supernatant, anti-Flag
antibody, and FITC-conjugated anti-mouse IgG antibody (Southern
Biotechnology, Birmingham, AL). Flow cytometry analysis was performed
using the Becton Dickinson FACScan (San Jose, CA). Jurkat T cells were
stably transfected by electroporation using linearized
pcDNA3.1/CD5L-AITR and selected in the presence of Zeocin
(Invitrogen). A binding assay for this cell line was performed as
described above. To test the ability of AITR-Fc fusion protein to bind
membrane-bound TL6, pCEP4/TL6 was stably transfected into HEK293 EBNA
cells. After selection in the presence of hygromycin, TL6-expressing cells were harvested and incubated with AITR-Fc protein, followed by
FITC-conjugated anti-human IgG1 antibody (Southern Biotechnology). The
Becton Dickinson FACScan was used for flow cytometry analysis.
AITR was identified by searching an EST data base. A full-length
cDNA of a clone from a human activated T-cell cDNA library, which is tentatively named AITR (for activation-inducible TNFR family
member), encodes a 234-amino acid type I transmembrane protein with a
calculated molecular mass of 25 kDa (Fig.
1A). The receptor has a signal
peptide (the first 25 amino acids) and a single transmembrane region
(amino acids 140-158). When compared with the extracellular domain of
other TNFR family members, AITR displays three cysteine-rich
pseudorepeats corresponding to the second, third, and fourth TNFR
motif, respectively. The first cysteine pseudorepeat contains eight
cysteine residues and lacks C4. Therefore, it is unlikely that the
canonical pattern of C1-C2, C3-C5, and C4-C6 disulfide bridges exist in
this motif. The second pseudorepeat shows some features of the third
TNFR motif, but it is atypical in that C5 is not present even though it
contains 7 cysteine residues. The third pseudorepeat shows extensive
homologies with the fourth pseudorepeat of 4-1BB. The cytoplasmic
domain contains acidic amino acids that are highly conserved in the
cytoplasmic domains of 4-1BB, CD27, and GITR. Overall, AITR exhibits a
high homology (55% identity) to murine GITR (Fig. 1B), but
there is a mismatch in the first cysteine-rich pseudorepeat between
GITR and AITR, because the first pseudorepeat of GITR corresponds to the first TNFR cysteine-rich motif (24).
B activation via TRAF2. The ligand for AITR (AITRL) was
found to be an undescribed member of the TNF family, which is expressed
in endothelial cells. Thus, AITR and AITRL seem to be important for
interactions between activated T lymphocytes and endothelial cells.
INTRODUCTION
Top
Abstract
Introduction
References
B.
In the CD27 signaling pathway, both TRAF2 and TRAF5 mediate NF-
B and
SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase)
activation, and NIK (NF-
B-inducing kinase) is a common downstream
kinase of TRAF2 and TRAF5 (30).
EXPERIMENTAL PROCEDURES
B-dependent E-selectin-luciferase reporter gene
(pELAM-Luc) and pRSV-
galactosidase (pRSV-
-gal) plasmids were
also described elsewhere (31, 37).
B by Reporter Assay--
Approximately
0.5 × 106 HEK293 EBNA cells/well were seeded on
6-well plates. After 24 h, cells were transfected by the standard calcium phosphate precipitation method using various combinations of
pcDNA3.1/CD5L-AITR plus pRK5 plasmids encoding TRAFs, dnTRAF2, NIK,
or dnNIK. The total amount of plasmid was adjusted to 2.0 µg by
adding empty vector. Twenty-four hours after transfection, cells were
lysed in 200 µl of reporter lysis buffer (Promega, Madison, WI).
Luciferase activity was measured using 20 µl of cell extract. 5 µl
of cell extract was used to assay
-galactosidase activity as an
internal control, and luminescence values were normalized by individual
-galactosidase activity.
RESULTS AND DISCUSSION
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Fig. 1.
Sequence and expression analysis of
AITR. A, deduced amino acid sequence of AITR. The
potential signal sequence is underlined. The putative
glycosylation site is indicated as bold
characters, and the transmembrane region is indicated in
boxes. B, comparison of the amino acid sequence
of AITR with murine GITR. Bold letter
cs within three cysteine pseudorepeat motifs indicate
conserved cysteine residues found in the extracellular domain of TNFR
superfamily members. Conserved acidic amino acid clusters of the
cytoplasmic domain are indicated in boxes. C,
expression of AITR: Northern blot analysis in various tissues and
cancer cell lines (left panels) and RT-PCR
analysis in human PBMC (right panels). Northern
blots of poly(A) RNA from human tissues and cancer cell lines were
analyzed by hybridization to a 32P-labeled cDNA probe
containing the entire AITR coding regions. For RT-PCR, 2.0 × 106 human PBMC were activated by treatment with different
stimuli (dexamethasone (Dex), PMA/ionomycin
(P/I), or CD3/
CD28 mAbs) for 24 and 48 h. Total
RNA was extracted using TriZOL. 1 µg of total RNA was used for
reverse transcriptase and PCR reaction.
-Actin was amplified as an
internal control.
We investigated expression of AITR mRNA in multiple human tissues by Northern blot hybridization (Fig. 1C). 1.25-kilobase mRNA was detected in lymph node, peripheral blood leukocytes, and, weakly, in spleen. We also tested a variety of tumor cell lines for expression of AITR mRNA (Fig. 1C). A 1.25-kilobase message was detected only in the colorectal adenocarcinoma cell line, SW480, among the cell lines tested. The expression of virtually all members of the TNFR superfamily is enhanced by antigen stimulation/lymphocyte activation (1). Consistent with this idea, AITR expression was up-regulated in PBMC after stimulation. No AITR message was detectable in unstimulated PBMC when we used a sensitive RT-PCR method (Fig. 1C). AITR expression was clearly induced within 24 h by typical PBMC stimulation such as treatment with PMA plus ionomycin or soluble anti-CD3 plus anti-CD28 mAbs. FACS analysis for AITR expression, however, showed that a small population of activated PBMC expressed AITR on the cell surface at 48 h after stimulation, suggesting that a prolonged period of stimulation is required for maximum expression of AITR.2 Expression of AITR was not induced by treatment with dexamethasone. This property was different from that of GITR (24).
Recently it has been shown that 4-1BB molecules associate with TRAF1,
TRAF2, and TRAF3 (27-29). Because the cytoplasmic domain of AITR is
similar to that of 4-1BB, we tested its ability to co-precipitate five
of the six known TRAFs that were overexpressed in HEK293 EBNA cells. We
observed an interaction of AITR with TRAF1, TRAF2, and TRAF3 but not
with TRAF5 and TRAF6 (Fig.
2A). The association of AITR
with TRAF2 suggested that, like other members of the TNFR superfamily
(27, 28, 30, 31, 39-41), AITR might mediate NF-B activation through
TRAF2. To test this possibility, we used an NF-
B reporter system in
HEK293 EBNA cells (31). Co-transfection with the AITR expression vector
typically induced greater than 3-fold higher luciferase activity when
compared with the vector transfection control (Fig. 2, B and
C). When co-expressed with TRAF2, AITR induced greater
luciferase activity than did TRAF2 alone (Fig. 2, B and
C). More importantly, overexpression of dominant-negative
TRAF2, which lacked the RING and zinc finger motifs (31), abrogated the
luciferase activity induced by AITR (Fig. 2B). This
indicates that TRAF2 is an important mediator of NF-
B activation for
AITR. A similar observation was made when we blocked the activity of
NIK, which was thought to lie downstream of TRAF2 in the NF-
B
signaling pathway, by overexpression of the dominant-negative NIK (36),
which lacked the two lysine residues of the catalytic domain (Fig.
2B). Taken together, these data indicate that AITR mediates
NF-
B activation through the TRAF2/NIK pathway. Since TRAF1 and TRAF3
were found to associate with AITR in HEK293 EBNA cells, we examined the
effects of TRAF1 and TRAF3 on NF-
B activation induced by AITR. As
shown in Fig. 2C, introduction of TRAF3 nearly abolished the
luciferase activity induced by AITR overexpression. To a lesser extent,
TRAF1 overexpression diminished AITR-induced NF-
B activation. These
data suggest that TRAF1 and especially TRAF3 down-regulate AITR-induced
NF-
B activation.
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To identify AITRL, we screened a panel of Flag-tagged candidate TNF
ligand proteins for binding to AITR-Fc fusion protein by
immunoprecipitation. AITR-Fc selectively bound TL6-Flag among Flag-tagged TNF ligand proteins tested (Fig.
3A). In our experimental conditions, 4-1BB and TR2 (HVEM) bound their cognate ligands, 4-1BBL
and LIGHT (38), respectively (data not shown). Furthermore, our data
clearly showed that TL6-Flag protein bound AITR transiently expressed
on the cell surface of HEK293 EBNA cells and AITR constitutively expressed on the cell surface of Jurkat cell (Fig. 3B,
upper and middle panels, respectively). Since TL6
is a transmembrane protein (see below), we used flow cytometry to
determine whether AITR-Fc fusion protein was able to bind HEK293 EBNA
cells that were stably transfected with full-length TL6. We found that
AITR-Fc protein was capable of binding TL6 expressed on HEK293 EBNA
cells (Fig. 3B, lower panel). Next, we tested
whether interactions between AITR and TL6 would result in NF-B
activation. In an NF-
B reporter assay, ligand-dependent
NF-
B activation was demonstrated by co-transfecting transmembrane
TL6 with AITR (data not shown) or transfecting TL6-expressing HEK293
EBNA cells (Fig. 3C). In addition, when AITR was transiently transfected into HEK293 EBNA cells, which constitutively secreted soluble TL6 protein, NF-
B activation markedly increased as compared with empty vector-tansfected HEK293 EBNA cells (Fig. 3C).
Similarly, higher NF-
B activation was induced by treating with
soluble TL6 protein HEK293 cells that were transiently transfected with
AITR (data not shown). This indicates that TL6 is able to trigger
AITR-specific activation of NF-
B. It appears that higher induction
of NF-
B by TL6 is correlated with a stronger association of AITR
with TRAF2 in HEK293 EBNA cells, since stronger association of AITR with TRAF2 was observed in cells that were co-transfected with TL6 than
in cells that were transfected with AITR alone.2
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TL6 was one of the TNF ligand proteins initially identified by an EST data base search. Hydrophilicity analysis of a full-length TL6 clone from a brain cDNA library predicts a single hydrophobic transmembrane domain and the absence of a signal sequence (Fig. 4A). TL6 contains two potential glycosylation sites in the C-terminal region. These features suggest that TL6 is a type II membrane protein with the C-terminal region extracellular. Northern blot analysis of human tissue RNAs revealed expression of a single 2.4-kilobase TL6 mRNA in pancreas (Fig. 4B). Various human cell lines and PBMC were also examined for TL6 expression. No message was detectable in either unstimulated or stimulated T-cell lines (CEM-6 and Jurkat), B-cell lines (Priess and Frev), promyelocytic cell line (HL-60), monocytic cell line (THP-1), and PBMC by RT-PCR (data not shown). In contrast, HUVEC cells constitutively expressed TL6, and its expression was up-regulated after stimulation with LPS (Fig. 4B). Therefore, it is speculated that AITR and its ligand are important for interactions between activated T lymphocytes and blood vessels.
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AITR has 55% identity with murine GITR at the amino acid level. The high sequence conservation between human and mouse provides evidence that AITR is the human homologue of murine GITR. At this point, however, the possibility remains that these two receptors may serve distinct functions from one another, based on the following facts. 1) There is a mismatch in the first cysteine-rich pseudorepeat between GITR and AITR; 2) in contrast to GITR, AITR is not inducible by dexamethasone.
In summary, we have identified a novel protein of the TNFR superfamily,
AITR, which activates NF-B through a TRAF2-mediated mechanism.
Expression of AITR is activation-inducible. The ligand for AITR is a
member of the TNF ligand family and is constitutively expressed in an
endothelial cell line. This indicates that AITR and its ligand may be
involved in activated T cell trafficking.
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ACKNOWLEDGEMENTS |
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We thank Sister Mary Etta Kiefer and Pat Mantel for editing the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI28125 and DE12156 and Molecular Medicine Program 98-mm-02-01-A-04 from the Ministry of Science and Technology, Korea.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF117297 and AF117713.
¶ Present address: Mendel Biotechnology, Inc., Hayward, CA 94545.
To whom correspondence should be addressed: Dept. of
Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202. Tel.: 317-274-3950; Fax: 317-274-4090; E-mail: kkwon{at}sunflower.bio.indiana.edu.
** These two authors contributed equally to this work.
2 B. Kwon, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
TNFR, tumor necrosis
factor receptor;
AITR, activation-inducible TNFR family member;
GITR, glucocorticoid-induced TNFR family-related gene;
PMBC, peripheral
mononuclear cells;
TRAF, TNF receptor-associated factor;
mAb, monoclonal antibody;
PHA, phytohemagglutinin;
HA, hemagglutinin;
EST, expressed sequence tag;
PMA, phorbol 12-myristate 13-acetate;
HUVEC, human umbilical vein endothelial cell;
RT, reverse transcriptase;
PCR, polymerase chain reaction;
LPS, lipopolysaccharide(s);
NIK, NF-B-inducing kinase;
dn, dominant negative;
FITC, fluorescein
isothiocyanate.
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REFERENCES |
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