(Received for publication, March 25, 1997)
From the Department of Molecular Oncology, Genentech, Inc., South
San Francisco, California 94080-4918 and the
Department of Molecular Biology, Tularik, Inc.,
South San Francisco, California 94080
The mammalian tumor necrosis factor receptor
(TNFR) family consists of 10 cell-surface proteins that regulate
development and homeostasis of the immune system. Based on an expressed
sequence tag, we have cloned a cDNA encoding a novel member of the
human TNFR family. A closely related protein, designated HVEM (for
herpesvirus entry mediator), was identified independently by another
group as a mediator of herpesvirus entry into mammalian cells
(Montgomery, R., Warner, M., Lum, B., and Spear, P. (1996)
Cell 87, 427-436). HVEM differed from our clone by two
amino acid residues, suggesting that the two proteins represent
polymorphism of a single HVEM gene. We detected HVEM mRNA
expression in several human fetal and adult tissues, although the
predominant sites of expression were lymphocyte-rich tissues such as
adult spleen and peripheral blood leukocytes. The cytoplasmic region of
HVEM bound to several members of the TNFR-associated factor (TRAF)
family, namely TRAF1, TRAF2, TRAF3, and TRAF5, but not to TRAF6.
Transient transfection of HVEM into human 293 cells caused marked
activation of nuclear factor-B (NF-
B), a transcriptional
regulator of multiple immunomodulatory and inflammatory genes. HVEM
transfection induced also marked activation of Jun N-terminal kinase,
and of the Jun-containing transcription factor AP-1, a regulator of
cellular stress-response genes. These results suggest that HVEM is
linked via TRAFs to signal transduction pathways that activate the
immune response.
Members of the TNFR1 family play a key
role in regulating the immune response to infection (1). For example,
TNFR1, TNFR2, and CD40 modulate the expression of proinflammatory and
costimulatory cytokines, cytokine receptors, and cell adhesion
molecules through activation of the transcription factor NF-B (2).
Some TNFR family members regulate also the AP-1 transcription factor,
whose target genes are less well defined (3). NF-
B is the prototype of a family of dimeric transcription factors whose subunits contain conserved Rel regions (4). In its latent form, NF-
B is complexed with members of the I
B inhibitor family; upon inactivation of I
B
in response to certain stimuli, released NF-
B translocates to the
nucleus, where it binds to specific DNA sequences and activates gene
transcription (4). AP-1 represents a family of dimeric complexes
composed of members of the Jun and Fos protein families (3). AP-1
activation is mediated in part through phosphorylation of Jun proteins
by Jun N-terminal kinases (JNKs), also known as stress-activated
protein kinases (SAPKs) (3, 5).
Transcriptional regulation by TNFR family members is mediated by a
family of signal transducers known as TNFR-associated factors (TRAFs)
(6). For example, TRAF2 associates directly with TNFR2, CD40, and CD30
and plays a key role in NF-B activation by these receptors (6-8).
TRAF2 also mediates TNFR1 activation of NF-
B and of JNK, although it
associates with TNFR1 indirectly, via the TRADD adaptor protein; TRADD
signals apoptosis activation by TNFR1 as well (9, 10). TRAF1 associates
with TNFR2 indirectly, by interaction with TRAF2 (6), while
TRAF3/CD40bp/CRAF1 associates directly with and mediates signaling by
CD40 and the lymphotoxin
receptor (LT
R) (7, 11-13), as does
TRAF5 (14, 15). TRAF6 binds to CD40 and contributes to CD40 signaling
(16), as well as to signaling by interleukin-1 (17).
By using an expressed sequence tag (EST) approach, we have identified a
member of the TNFR family that proved to be closely related to the
recently isolated HVEM (18). In the present study, we provide evidence
suggesting that HVEM interacts with several members of the TRAF family
and modulates activation of NF-B, JNK, and AP-1.
Human fetal and adult tissue poly(A) RNA blots (CLONTECH) were analyzed by Northern hybridization using a 32P-labeled cDNA probe containing the entire HVEM coding region. In addition, poly(A) RNA was prepared from purified human peripheral blood T cells (19) and from Jurkat T cells (ATCC) using a FastTrack kit (Invitrogen), and 50 ng of RNA were analyzed for HVEM expression by reverse transcriptase-polymerase chain reaction (PCR) using oligonucleotide primers that amplify a 240-base pair sequence in the putative extracellular region of HVEM.
Interaction of HVEM with TRAFsThe cytoplasmic region of HVEM was cloned into the pGEX-2TK vector (Pharmacia Biotech Inc.), expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), and subsequently purified by glutathione-agarose affinity chromatography (20). HEK293 cells (ATCC) were transiently transfected by the calcium phosphate precipitation method with pRK5-based expression vectors encoding Flag epitope-tagged TRAFs 1, 2, 3, 5, or 6 (5 µg/10-cm dish). Total cell lysates were prepared 36 h later (7). Aliquots from each lysate were incubated for 2 h at 4 °C with ~1 µg of GST-HVEM fusion protein. Bound complexes were precipitated with glutathione-agarose beads, resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by immunoblot with anti-Flag monoclonal antibody (Eastman Kodak Co.) using the enhanced chemiluminescence Western blotting detection system (Amersham). The interaction of GST-HVEM with endogenous TRAFs in nontransfected HEK293 cells was determined using a similar approach, except that the TRAFs were detected by sequential immunoblot with polyclonal anti-human TRAF1 or TRAF2 antibodies (21), 4E7 anti-human TRAF3 monoclonal antibody (BIOS), or polyclonal anti-murine TRAF5 antibody (Zymed), which cross-reacts with human TRAF5.2
Electrophoretic Mobility Shift Assay (EMSA)The activation
of NF-B and AP-1 was analyzed by EMSA using oligonucleotide probes
specific for each transcription factor. Nuclear extracts were prepared
24 h after transient transfection (22) of HEK293 cells by HVEM or
control expression plasmids (10 µg/10-cm dish). Aliquots from each
extract (1 µg of total protein) were reacted (22) with a
32P-labeled oligonucleotide probe. For NF-
B, we used as
a control a radioprobe based on a mutated NF-
B target sequence (plus
strand sequence 5
-AGTTGAGGCGACTTTCCCAGGC-3
) (23), or a
radioprobe based on a wild type NF-
B target sequence (plus strand
sequence 5
-AGTTGAGGGGACTTTCCCAGGC-3
) (23). For AP-1 we used a control radioprobe based on a mutated AP-1 target sequence (plus strand sequence 5
-CGCTTGATGACTTGGCCGGAA-3
) (24), or a radioprobe based on a wild type AP-1 target sequence (plus strand sequence 5
-CGCTTGATGACTCAGCCGGAA-3
) (24). The reactions were subjected to PAGE
and visualized by phosphorimager analysis (22).
JNK activation was determined with a SAPK/JNK assay kit (New England Biolabs). Cell lysates were prepared 24 h after transient transfection of HEK293 cells by HVEM or control expression plasmids. JNK was precipitated with a GST-c-Jun fusion protein bound to glutathione-Sepharose beads. After washing, the kinase reaction was allowed to proceed in the presence of ATP, and was resolved by SDS-PAGE. Phospho-c-Jun was detected by immunoblot with antibody specific for c-Jun phosphorylated on Ser-63, a site important for transcriptional activity (5), using chemiluminescence.
TNFR family members share sequence homology primarily in their extracellular region, which contains three to six characteristic cysteine-rich pseudorepeats (1, 25). To identify new members of the TNFR family, we searched the DNA data bases for ESTs that exhibit homology to individual TNFR family members. We identified a human retinal EST (GenBank locus AA021617), which upon translation showed homology to the cysteine-rich regions of several TNFR family proteins. To isolate the full-length cDNA, we screened a human retinal cDNA bacteriophage library (CLONTECH) by hybridization to a 60-base pair oligonucleotide probe based on a region of consensus between AA021617 and several related ESTs. We identified five independent positive clones containing cDNA inserts of 1.8-1.9 kb. Three of the inserts were subcloned into the pRK5 plasmid, and sequenced on both strands. The cDNA sequences were identical (with the exception of an intron found in one of the clones), and encoded a putative 283-amino acid transmembrane protein that showed significant extracellular sequence homology to the TNFR family (data not shown). Subsequently, a protein designated HVEM was reported independently (18), and turned out to be closely related to the protein we had identified. HVEM was isolated from a human HeLa cell cDNA library as a mediator of herpesvirus entry into mammalian cells; the cloning strategy was based on a functional screen of transfected cDNA clones for ability to confer herpesvirus sensitivity upon resistant cells (18). The protein we identified and HVEM (18) differ, respectively, in two extracellular amino acid residues: codon 108 encodes a serine or a threonine, and codon 140 encodes an alanine or an arginine. These differences may be due to polymorphism in the HVEM gene; hence, we refer to both proteins here as HVEM.
We investigated expression of the HVEM mRNA in human tissues by
Northern blot hybridization (Fig. 1, A-C).
We detected a major transcript of about 1.8 kb, which is similar to the
size of our cDNA clones, in multiple fetal and adult tissues; this
transcript was most highly expressed in adult spleen and peripheral
blood leukocytes. A second transcript of about 3.8 kb, as well as some larger transcripts also were detected. In addition, we analyzed the
expression of HVEM mRNA in peripheral T cells purified from human
blood and in the human Jurkat T cell line by reverse transcriptase-PCR (Fig. 1D). Both primary T cells and the T cell line
expressed HVEM mRNA. The data of Fig. 1B are consistent
with the previous observation that HVEM mRNA is expressed in
several non-lymphoid tissues (18). However, our results demonstrate
that in fact the predominant sites of HVEM mRNA expression are
lymphocyte-rich tissues, i.e. spleen and peripheral blood
leukocytes (Fig. 1C), and show further that HVEM mRNA is
expressed in purified T cells (Fig. 1D) and in several fetal
tissues (Fig. 1A).
To investigate whether HVEM interacts with TRAFs, we generated a GST
fusion protein based on the cytoplasmic region of HVEM and tested its
ability to co-precipitate five of the six known TRAFs upon their
overexpression in HEK293 cells (Fig. 2, A and B). We observed strong association of HVEM with TRAF2;
weaker association with TRAF5, TRAF3, and TRAF1; and no association
with TRAF6. Consistent with these results, HVEM bound also to
endogenous TRAFs in HEK293 cells, with a similar rank order (Fig.
2C). LTR recognized the same set of endogenous TRAFs in
HEK293 cells as did HVEM, while TNFR2 interacted mainly with TRAF2 and
TRAF1 (Fig. 2C). These results are intriguing, because the
58-amino acid cytoplasmic region of HVEM is related to the cytoplasmic
domain of LT
R (11 identities), but shows no homology to the
cytoplasmic region of TNFR2. CD40, another TNFR family member that
shows significant cytoplasmic region homology to HVEM (12 identities),
also interacts with TRAF2, TRAF3, and TRAF5 (7, 11, 12), but unlike
HVEM, it recognizes TRAF6 as well (16).
To explore possible HVEM-regulated signaling pathways, we investigated
whether transfection of HEK293 cells by HVEM affects NF-B activity
(Fig. 3). Cells transfected by a pRK5-based HVEM expression plasmid showed significant NF-
B activation relative to
cells transfected by pRK5 alone (Fig. 3A). For comparison, we tested two other TNFR family members that have been shown previously to activate NF-
B, namely TNFR1 and Apo-3/DR-3/WSL-1 (2, 26-28); the
level of NF-
B activation by these receptors was similar to the level
of activation by HVEM (Fig. 3A). A specific antibody to the
p65/RelA subunit of NF-
B, but not preimmune serum, inhibited the
mobility of the NF-
B probe in the case of each receptor (Fig. 3B). Hence, the NF-
B complexes activated by HVEM, TNFR1,
and Apo-3 in HEK293 cells appear to contain the p65/RelA protein (Fig. 3B). These results indicate that HVEM is linked to an
NF-
B activation pathway. It is possible that interaction of HVEM
with TRAF2 and/or TRAF5 (Fig. 2) mediates the NF-
B activation, since
these TRAFs are key to NF-
B activation by other related TNFR family
members (see above).
Next, we investigated whether transfection of HEK293 cells by HVEM
activates JNK, as well as the transcription factor AP-1, which is
activated through JNK-mediated phosphorylation of Jun proteins (Fig.
4). HVEM-transfected cells showed marked JNK activation as compared with cells transfected by pRK5 alone (Fig. 4A);
the level of activation was comparable to the level induced by TNFR1 transfection. Consistent with JNK activation, HVEM-transfected cells
showed AP-1 activation as well (Fig. 4B). Anti-Jun D
antibody, but not preimmune serum, inhibited the migration of the
AP-1-specific probe (Fig. 4C), suggesting that Jun D
participates in the AP-1 complexes activated by HVEM in HEK293 cells.
These results indicate that HVEM is coupled to the JNK/AP-1 signaling
pathway. The interaction of HVEM with TRAF2 (Fig. 2) may contribute to
JNK/AP-1 activation by HVEM, since TRAF2 plays a key role in mediating
JNK activation by TNFR1 (10). However, it remains to be established
which TRAFs signal NF-B and JNK/AP-1 activation by HVEM.
Using an EST-based approach, we have identified a novel member of
the human TNFR family. Montgomery et al. (18) isolated a
closely related protein, HVEM, as an entry receptor for herpesvirus. There are only two amino acid differences between the two receptors, suggesting that these proteins may be encoded by a single, polymorphic HVEM gene. Our results provide an important step toward understanding the physiological function of HVEM. HVEM associates with members of the
TRAF family and activates JNK/SAPK, as well as the transcription factors NF-B and AP-1, which control expression of multiple immune, inflammatory, and acute phase action genes in response to infection or
cellular stress (3-5). The induction of NF-
B and AP-1 by HVEM,
together with the relatively abundant expression of HVEM mRNA in T
cells and in lymphocyte-rich tissues, suggests that HVEM is involved in
regulating lymphocyte activation.
We thank C. Clark for comments on the manuscript; W. Wood for EST searches; M. Vasser, P. Ng, and P. Jhurani for oligonucleotide synthesis; and M. Hamner for help with DNA sequencing.