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INTRODUCTION |
Epstein-Barr virus
(EBV)1 is a human herpesvirus
that is associated with several types of malignancy. EBV infects
resting B cells, stimulates their proliferation, and induces the
outgrowth of virus-transformed lymphoblastoid cell lines expressing the nuclear antigens EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA-LP and
the latent membrane proteins LMP1, LMP2A, and LMP2B (1).
Among the nuclear and membrane proteins expressed as a consequence of
EBV infection, the latent membrane protein 1 (LMP1) is of particular
interest because it induces the oncogenic transformation of rodent
fibroblast cell lines (2, 3). LMP1 expression is also essential for
EBV-mediated primary B cell transformation in vitro (1, 4)
and is associated with a number of human malignancies such as
Hodgkin's disease, undifferentiated nasopharyngeal carcinoma, and
EBV-related lymphoproliferative disease (1). Expression of this viral
oncogene in B cells can induce a plethora of activities including
up-regulation of cell surface markers such as CD23, CD40, and CD54
(intercellular adhesion molecule 1) and induction of anti-apoptotic
proteins such as A20 and members of the Bcl-2 family (5-7; for review,
see Ref. 8). In epithelial cells, LMP1 can also induce A20, CD40, and
CD54 expression and block differentiation, a property that may be
important in the pathogenesis of nasopharyngeal carcinoma (9-11). Of
particular interest is the reported ability of LMP1 to induce
production of cytokines such as interleukin-6 (IL-6) (12) and IL-10
(13), suggesting that this viral protein may activate inflammatory and immune-regulatory responses following EBV infection.
Structurally, LMP1 is a 63-kDa phosphoprotein comprising a short
23-amino acid NH2-terminal cytoplasmic domain, six
membrane-spanning domains of 162 amino acids, and a long 200-amino acid
COOH-terminal cytoplasmic tail. Mutational analysis has identified
COOH-terminal regions of the protein as being essential for
transformation and phenotypic changes. The 45 most membrane-proximal
residues of the LMP1 COOH terminus (amino acids 186-231) are critical
for EBV-mediated transformation of primary B cells, but recent studies suggest that the long term growth of these cells also requires the
distal COOH-terminal sequences (amino acids 352-386) (4, 14, 15).
Interestingly, these two functional domains of the LMP1 cytoplasmic
tail can also activate the transcription factor NF-
B, with the
extreme COOH-terminal activating region 2 (CTAR2, amino acids 351-386)
being the principal contributor to this effect in the majority of cell
lines (6, 16). This phenomenon can be attributed to the ability of
CTAR2 to associate with tumor necrosis factor (TNF) receptor-associated
death domain (TRADD) (15), a protein that mediates NF-
B
signaling from aggregated TNF receptor I (TNFRI) (17). The proximal
CTAR1 domain of LMP1 (amino acids 187-231) induces low levels of
NF-
B through its direct interaction with TNFR-associated factor 2 (TRAF2) (18, 19). TRAF3 has also been shown to bind CTAR1 (20).
Importantly, these CTAR1-TRAF interactions occur through a
P204xQ206xT208 TRAF binding motif,
common to the cytoplasmic tails of some TNFR family members such as
CD40 and CD30. In addition to NF-
B, LMP1 expression signals for
activation of a Ras/MAPK/ERK pathway (21) and of the JNK (c-Jun
NH2-terminal kinase, also known as the stress-activated protein kinase, SAPK) cascade (22-24), a phenomenon that is mediated through CTAR2 and results in the induction of the transcription factor
c-Jun/AP-1 (22, 24).
In this study we demonstrate that EBV-encoded LMP1 can also activate
the p38 MAPK pathway. This phenomenon occurs through both CTAR1 and
CTAR2 domains of the protein and appears to be mediated by the adaptor
protein TRAF2. p38 activation has been observed in response to a
variety of stimuli, including hyperosmotic shock, UV radiation,
lipopolysaccharide treatment, and stimulation by certain cytokines such
as IL-1 and TNF-
and requires phosphorylation of a closely spaced
tyrosine and threonine residue in the activation domain of the protein
(for review, see Refs. 25 and 26). Among the downstream targets of p38
are the heat-shock protein 27 (hsp27) and the transcription factors
ATF2 (27), Elk-1 (28), CHOP/GADD153 (29), and Max (30). In addition,
inhibition of p38 activity has been shown to interfere with
TNF-mediated NF-
B transactivation and to influence TNF-induced
apoptosis (31) and IL-6 production (32-34). In agreement with these
data we demonstrate that inhibition of p38 signaling impairs the
ability of LMP1 to induce IL-6 and IL-8 expression.
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MATERIALS AND METHODS |
DNA Constructs--
pSG5-based LMP1 and LMP1 deletion mutants
(332-386) and
(187-351) have been described previously (6).
pSG5-LMP1AxAxA was generated by site-directed mutagenesis
using the QuickChangeTM site-directed mutagenesis kit of
Stratagene and pSG5-LMP1 as substrate. The mutated oligonucleotide
primers used were: 5'-CCTCCCGCACGCTCAAGCAGCTGCCGATGA-3' and its
complementary. pSG5-LMP1AxAxA/378STOP was generated by a
similar approach using the mutated primers
5'-GATGACGACCCCCACTGACCAGTTCAGCTAAGC-3' and its complementary and
pSG5LMP1AxAxA as substrate. These mutations generate a STOP
codon at position 378 of the amino acid sequence of LMP1 and were
verified by sequencing. pSG5CD2.192-LMP1 has been described previously
(35). pIND-LMP1 was generated by excision of LMP1 coding sequences from
pSG5-LMP1 using EcoRI digestion and subsequent insertion
into the EcoRI site of the pIND vector (Invitrogen).
The 181-bp IL-8 promoter sequences (
135 to +46) were PCR amplified
from human genomic DNA using the following primers:
5'-GTGAGATCTGAAGTGTGATGACTCAGG-3' (IL8P-Forward), which contains an
artificial BglII site, and 5'-GTGAAGCTTGAAGCTTGTGTGCTCTGC-3' (IL8P-Reverse), which contains an artificial HindIII site.
The PCR product was then digested with
BglII/HindIII and inserted into the corresponding
restriction sites of the luciferase reporter plasmid pGL2-Basic
(Promega) to generate IL8-Luc. To generate the mutAP-1/IL8-Luc vector
that contains the same IL-8 promoter sequences but with a double
mutation that distorts the AP-1 consensus, the
5'-GTGAGATCTGAAGTGTGATATCTCAGG-3' (mut-IL8P-Forward) was used together
with IL8P-Reverse. The PCR product was again digested with
BglII/HindIII and ligated into pGL2-Basic.
The hemagglutinin (HA)-p46SAPK
-pcDNA3 and HA-p38 vectors were a
gift from James Woodgett (The Ontario Cancer Institute, Ontario, Canada), and the wild type and mutated TRAF2 expression vectors pcDNA3-TRAF2 and pcDNA3-TRAF2
(6-86), respectively, were
kindly provided by George Mosialos, Ken Kaye, and Eliott Kieff (Harvard Medical School, Boston). The CMV-driven I
B
[S32A/S36A] construct has been described previously (12). This double serine to alanine mutation inhibits degradation of I
B
, rendering it a
constitutively active molecule.
Cell Culture, Transfections, and Reporter Assays--
HEK 293 cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum and 2 mM glutamine. For transient transfections, 8 × 105 HEK 293 cells
were plated out on a 25-cm2 flask and the following day
were transfected using a standard calcium phosphate technique. HeLa and
Rat-1 cells, cultured in RPMI and 10% fetal calf serum supplemented
with 2 mM glutamine were transfected using the PrimeFector
lipofection kit (EquiBio Ltd, Kent, U. K.). The xanthogenate compound
D609 (tricyclodecan-9-yl-xanthogenate potassium) was purchased from
Calbiochem and dissolved in growth medium immediately before use. The
p38 inhibitor SB203580 was purchased from Calbiochem, dissolved in
dimethyl sulfoxide, and stored at
20 °C.
Luciferase reporter and
-galactosidase assays were performed as
described previously (12). 50 ng each of a CMV-driven
-galactosidase-expressing plasmid and of 3Enh.
B-ConALuc reporter,
which contains three tandem repeats of the NF-
B sites from the Ig
promoter, were used routinely to transfect 293 cells. Alternatively,
293 cells were transfected with 50 ng of IL8-Luc or mutAP-1/IL8-Luc and 50 ng of
-galactosidase, whereas HeLa cells were transfected with 1 µg each of these reporter constructs. Analysis of luciferase and
-galactosidase expression was performed at 36 h
post-transfection.
Oligomerization of CD2--
Oligomerization of CD2/LMP1 chimeric
protein was performed as described previously (35).
Immunoprecipitations, Kinase Assays, and Immunoblotting--
JNK
in vitro kinase assays were performed as described
previously (24). For phospho-p38 immunoblots or p38 kinase assays, cells were lysed in 300-500 µl of kinase lysis buffer (20 mM Tris, pH 7.6, 0.5% Triton X-100, 250 mM
NaCl, 3 mM EGTA, 3 mM EDTA, 2 mM
sodium vanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM DTT) for 20 min on ice. Cell debris were removed by
centrifugation, and the protein concentration was determined using a
commercially available Bio-Rad protein assay. p38 MAPK was
immunoprecipitated from 200-250 µg of total protein extracts using 1 µg of anti-HA antibody (Boehringer Mannheim) for 2 h and 25 µl
of protein G-Sepharose (Amersham Pharmacia Biotech) for an additional
1 h. After immunoprecipitation, beads were washed twice with
kinase lysis buffer and twice with assay buffer (25 mM
Tris, pH 7.5, 5 mM
-glycerophosphate, 10 mM
MgCl2, 2 mM DTT, 100 µM sodium
vanadate, and 1 µg/ml leupeptin). After the last wash, the beads were
drained using a fine gauge Hamilton syringe and resuspended in 50 µl
of assay buffer containing 2 µg of GST-ATF2(19-96) substrate (New
England Biolabs) and 200 µM ATP. Kinase reactions were
carried out at 30 °C for 30 min and stopped by the addition of 25 µl of 3 × Laemmli buffer and boiling for 5 min. Samples were
then analyzed on a 12.5% SDS-polyacrylamide gel, and ATF2
phosphorylation was detected by immunoblot using a phospho-specific
ATF2 antibody (New England Biolabs) which reacts with
Thr69/Thr71 doubly phosphorylated ATF2 followed
by densitometric analysis using a Bio-Rad GS-690 imaging densitometer.
Immunoblot analysis of anti-HA immunoprecipitates using a control p38
antibody (New England Biolabs) was also performed to demonstrate that
comparable amounts of HA-p38 were analyzed in cotransfection
experiments. Phosphorylation of p38 was determined by immunoblot
analysis of 50 µg of cell extracts using a phospho-specific p38 MAPK
(Thr180/Tyr182) antibody (New England Biolabs).
For LMP1 and TRAF2 immunoblotting, 25 µg of total cell lysates
isolated as described above was analyzed on a 10% gel, and LMP1 or
TRAF2 expression was detected with the anti-LMP1 mAbs CS.1-4 (36) or
the TRAF2(C-20) polyclonal antibody (Santa-Cruz) and ECL (Amersham
Pharmacia Biotech).
Electrophoretic Mobility Shift Assays (EMSAs)--
Cell nuclei
isolated by resuspending cells in a solution containing 10 mM HEPES, pH 7.9, 1.5 mM magnesium chloride, 10 mM potassium chloride, 0.5 mM DTT, and protease
inhibitors were subjected to lysis in a buffer constituting 20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM
magnesium chloride, 0.42 M sodium chloride, 0.2 mM EDTA, 0.5 mM DTT, and protease inhibitors.
The protein concentration of isolated nuclear extracts was determined
by the Bio-Rad protein assay, according to the manufacturer's
instructions. For EMSAs, a 29-bp HIV-
B probe
(5'-GATCAGGGACTTTCCGCTGGGGACTTTCC-3'), a 22-bp collagenase TRE probe
(5'-AGCTTGATGAGTCAGCCGGATC-3'), a 24-bp Jun2 TRE probe
(5'-AGCTAGCATTACCTCATCCCGATC-3'), and a 24-bp IL-8 probe containing the
AP-1 consensus (5'-GAAGTGTGATGACTCAGGTTTGCC-3') were made by annealing
complementary synthetic oligonucleotides and end labeling using
[32P]ATP (Amersham Pharmacia Biotech) and T4
polynucleotide kinase (Boehringer Mannheim) followed by spin-column
purification. Binding reactions containing 10 µg of nuclear protein
extract, 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech), 0.1 ng of
probe labeled to a specific activity of 2 × 108
cpm/µg, and binding buffer (4% glycerol, 1 mM EDTA, 5 mM DTT, 0.01 M Tris·HCl, pH 7.5, and 5 mM KCl) in a volume of 20 µl were incubated for 30 min at
room temperature and then resolved by electrophoresis through a 5%
polyacrylamide gel in a 0.55 × TBE buffer. Gels were then dried
and exposed to x-ray film for autoradiography (Kodak). In supershift
experiments, nuclear extracts were preincubated for 45 min with
antibodies directed against c-Fos (Santa Cruz, sc-052X) or c-Jun (Santa
Cruz, sc-045X) and then for a further 30 min with radiolabeled probe
before being subjected to EMSA as described above. For ATF2 depletion
experiments, 10-µg nuclear extracts were incubated with 1 µg of
anti-ATF2 or control c-Rel antibody (Santa Cruz, sc-242X or sc-272X,
respectively) for 2 h on ice. Depletion was achieved by the
addition of anti-rabbit IgG-coated agarose beads during the last 60 min
of the incubation. After a brief centrifugation at 12,000 × g, the extracts were subjected to EMSA as above. For EMSAs
using recombinant ATF2, 0.5 µg of full-length ATF2 protein (Santa
Cruz, sc-4007) was incubated for 30 min with collagenase TRE, Jun2 TRE,
or IL-8/AP-1 probe in the presence of poly(dI-dC) and binding buffer as
described above and was then analyzed on a 5% polyacrylamide gel electrophoresis.
Reverse Transcription PCR and ELISA--
cDNA synthesis from
total RNA and semiquantitative reverse transcription PCR were performed
as described previously (12). A 208-bp IL-8 cDNA fragment was
generated using the primers 5'-CAGTTTTGCCAAGGAGTGCTA-3' (IL8-Forward)
and 5'-AACTTCTCCACAACCCTCTGC-3' (IL8-Reverse) under the following
conditions: denaturation at 94 °C for 45 s, annealing at
52 °C for 45 s, and extension at 72 °C for 50 s for 24 cycles followed by a final extension step at 72 °C for 50 min. PCR
products were analyzed on a 1.5% agarose gel, transferred on Hybond
N+, and hybridized with the oligonucleotide probe
5'-TGATTGAGAGTGGACCACA-3' (IL8-Probe). Primers and amplification for
GAPDH have been described previously. IL-8 versus GAPDH
hybridization signals were quantified on a Molecular Dynamics PhosphorImager.
The presence of IL-6 or IL-8 protein in the supernatants of cultured
cells was determined by ELISAs (Pelikine human IL-6 or IL-8 ELISA, CLB,
Netherlands) as described (12). 12 h after transfection, cells
were washed with phosphate-buffered saline and plated on a 24-well
plate at a density of 40,000 cells/ml in 1 ml of complete medium in the
presence or absence of inhibitors. Supernatants were analyzed 24 h
later. Alternatively, 40,000 293EcR-LMP1 cells were pretreated for
1 h with 20 µM SB203580 and then induced to express
LMP1 by treatment with 10 µM ponasterone A; supernatants were analyzed 24 h later.
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RESULTS |
LMP1 Expression Induces Phosphorylation of p38 and ATF2--
To
determine the effects of LMP1 on the p38 MAPK pathway, an
ecdysone-inducible system was used to provide regulatable expression of
LMP1. This system is based on the binding of the steroid hormone ecdysone analog ponasterone A to a heterodimeric receptor comprising a
modified ecdysone receptor and the retinoid X receptor (RXR) which
allows the subsequent activation of an ecdysone-responsive promoter to
express the target gene (37). HEK 293 cells carrying the pVgRXR plasmid
(293EcR), which encodes the receptor subunits, were transfected with
pIND-LMP1, which contains the LMP1 coding sequences under the control
of ecdysone-response elements. Selected stable clones (293EcR-LMP1)
were then examined for ponasterone A-inducible gene expression. Results
from a representative clone (293EcR-LMP1/cl.4) are shown in Fig.
1A. Induction of LMP1 after the addition of 10 µM ponasterone A was observed as early
as 6 h and increased further at 12 h to remain stable for up
to 24 h of treatment. The levels of LMP1 achieved after treatment
with ponasterone A were comparable to those expressed in X50-7, an EBV-transformed B cell line (Fig. 1A, X50-7
lane). Control cultures, stably transfected with empty pIND
vector, showed no LMP1 expression (Fig. 1A). Cell lysates were also
analyzed for p38 phosphorylation by immunoblot using an antibody that
specifically recognizes the phosphorylated form of the protein (34,
38). These experiments demonstrated a transient increase in endogenous
p38 phosphorylation levels in parallel with the induction of LMP1
expression (Fig. 1B), and similar results were obtained with
an additional 293EcR-LMP1 clone (293EcR-LMP1/cl.5, data not shown). The
levels of p38 phosphorylation in vector control-transfected cultures
remained unaffected (Fig. 1B).

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Fig. 1.
Inducible LMP1 expression activates the p38
MAPK pathway. A, HEK 293 cells stably transfected with
an ecdysone-regulatable LMP1 expression system (293EcR-LMP1/cl.4) were
induced to express LMP1 by exposure to 10 µM ponasterone
A. Induction of LMP1 was observed as early as 6 h and increased
further at 12 h to remain stable for up to 24 h of treatment
(LMP1 lanes). X50-7, an EBV-transformed B cell line, is an
LMP1-positive control (25 µg, X50-7 lane). Untreated
293EcR-LMP1/cl.4 cells showed no LMP1 expression (first lane
from left). Control cultures, stably transfected with empty
pIND vector (vec lanes) remained unaffected. B,
LMP1 expression induces increased p38 phosphorylation. Cell lysates
were analyzed for p38 phosphorylation by immunoblot using an antibody
that specifically recognizes the phosphorylated form of the protein.
Data are representative of at least five independent experiments.
C, LMP1 induces p38 kinase activity. HEK 293EcR-LMP1/cl.4 or
control cells were transfected with HA-p38 and 24 h later were
stimulated with 10 µM ponasterone A for various time
intervals (0, 3, 6, 12, or 24 h). HA immunoprecipitates of cell
lysates were then analyzed for kinase activity using GST-ATF2(19-96)
fusion protein as substrate. ATF2 phosphorylation was determined by
immunoblot analysis using an antibody specific for the phosphorylated
form of ATF2. A maximum of an 8.1-fold increase in ATF2 phosphorylation
levels of 293EcR-LMP1 cells was observed at 12 h of treatment
compared to control cultures. As positive control, cells were treated
with sodium salicylate and analyzed for kinase activity as above
(first lane from left). Immunoblot analysis of
anti-HA immunoprecipitates using a control p38-specific antibody was
also performed to demonstrate that comparable amounts of HA-p38 were
analyzed in cotransfection experiments (lower panel). Three
independent experiments were performed and gave similar results.
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To demonstrate that the observed LMP1-mediated p38 phosphorylation is
functional, the ability of immunoprecipitated p38 to activate ATF2 was
examined. The transcription factor ATF2 is a known substrate for p38
MAPK, being phosphorylated primarily on threonine residues 69 and 71, and this event increases its transactivating properties. HEK
293EcR-LMP1/cl.4 or control cells were transiently transfected with
HA-tagged p38 expression vector and 24 h later were stimulated
with 10 µM ponasterone A for various time intervals (0, 3, 6, 12, or 24 h). Lysates from these cultures were isolated, immunoprecipitated with an HA-specific antibody, and assayed for kinase
activity using GST-ATF2(19-96) fusion protein as substrate. ATF2
phosphorylation was then determined by immunoblot analysis using an
antibody specific for the phosphorylated form of ATF2. As shown in Fig.
1C, no increase in p38 kinase activity was observed after a
3-h exposure to ponasterone A, in agreement with the lack of induction
of LMP1 expression and p38 phosphorylation at this time point. A
significant increase in ATF2 phosphorylation levels was, however, noted
at 6 h and peaked at 12 h of treatment, in parallel with p38
phosphorylation. A small decrease in p38 kinase activity was then
observed at 24 h of stimulation (Fig. 1C, upper panel). As a control for this experiment, HA-p38-transfected
293EcR-LMP1/cl.4 cells treated for 10 min with 20 mM sodium
salicylate, which has been shown to induce p38 activation (38), were
used (Fig. 1C, first lane). Immunoblot analysis
of HA immunoprecipitates using an anti-ATF2 control antibody verified
that comparable amounts of HA-p38 were analyzed (Fig. 1C,
lower panel). In similar immune complex kinase assays, the
anti-HA immunoprecipitate failed to phosphorylate GST substrate (data
not shown). Thus, inducible LMP1 expression triggers the
phosphorylation of p38 and ATF2.
Activation of the p38 MAPK pathway was also observed after transient
expression of LMP1. HeLa cervical carcinoma cells were transfected with
increasing doses (1 or 2.5 µg) of pSG5-LMP1, an SV40 early
promoter-driven LMP1 expression vector, in the presence of 0.5 µg of
HA-p38, and kinase activity was determined 36 h later by immune
complex kinase assays using GST-ATF2(19-96) as substrate. These
experiments demonstrated that increasing levels of LMP1 expression
correlated with increased p38 kinase activity (Fig. 2, A and B), and
similar results were obtained in HEK 293 cells (see Fig.
4C).

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Fig. 2.
Transient expression of LMP1 engages the p38
and JNK signaling pathways. HeLa cells were transiently
transfected with various amounts of pSG5LMP1 (0, 1, or 2.5 µg) in the
presence of HA-tagged p38 or JNK (SAPK) expression vectors, and 36 h later cell lysates were analyzed for LMP1 expression (A),
p38 kinase activity using GST-ATF2(19-96) fusion protein as substrate
(B), or JNK activity using GST-c-Jun(1-79) as substrate
(C). Immunoblot analysis of anti-HA immunoprecipitates using
a control p38 (B, lower panel) or JNK-specific
antibody (C, lower panel) was also performed to
demonstrate that comparable amounts of HA-p38 or
HA-p46SAPK -pcDNA3, respectively, were analyzed in cotransfection
experiments.
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Activation of the JNK pathway has recently been shown to occur upon
LMP1 expression (22-24). HeLa cells transiently transfected with
pSG5-LMP1 and HA-p46SAPK
-pcDNA3, a CMV-driven HA-tagged JNK
expression vector, showed increased JNK activity compared with control
cultures, as determined by immune complex kinase assays using
GST-c-Jun(1-79) as substrate (Fig. 2C). HA
immunoprecipitates were immunoblotted for JNK expression to verify that
comparable amounts of HA-p46SAPK
-pcDNA3 were analyzed in
cotransfection experiments (Fig. 2C, lower
panel).
LMP1-mediated p38 Activation Requires Oligomerization at the Plasma
Membrane and Can Be Primarily Dissociated from NF-
B
Activation--
Recent studies have demonstrated that LMP1-mediated
NF-
B activation requires oligomerization of the protein at the
plasma membrane (35, 39). Thus, chimeric molecules in which the
extracellular and transmembrane regions of CD2, CD4, or nerve growth
factor receptor have been linked to the cytoplasmic tail of LMP1 can activate NF-
B signaling only after antibody- or ligand-induced aggregation of the chimera.
To determine whether oligomerization is also important for
LMP1-mediated p38 activation, a CD2/LMP1 chimera comprising the extracellular and transmembrane domains of CD2 (amino acids 1-212) linked to the cytoplasmic COOH terminus of LMP1 (amino acids 192-386) (construct pSG5CD2.192-LMP1) (35) was used to transfect Rat-1 fibroblasts. CD2 and LMP1 expression levels in stable transfectants were verified using flow cytometry and immunoblot analysis,
respectively. Expression in a representative clone (Rat-1/CD2.192LMP1
cl.4) is shown in Fig. 3A. In
these assays LMP1 appeared as a broad band presumably caused by CD2
glycosylation (Fig. 3A, right panel). These
cultures were subsequently transfected with 2 µg of HA-p38 and
36 h later were either left untreated or stimulated for 3 h
with OX34 anti-CD2 mAb and cross-linking anti-mouse IgG, as described
(35, 39). Cell lysates were immunoprecipitated with a HA-specific
antibody and assayed for kinase activity using GST-ATF2(19-96) fusion
protein as substrate. As shown in Fig. 3B, untreated
Rat-1/CD2.192LMP1 cells demonstrated only basal levels of ATF2
phosphorylation (fifth lane); however, a significant
increase in p38 kinase activity was observed after aggregation of the
chimera (sixth lane), suggesting that oligomerization is
essential for activation of p38 by LMP1. ATF2 phosphorylation levels in
control cultures remained unaffected (Fig. 3B, first
two lanes).

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Fig. 3.
p38 activation by LMP1 requires protein
oligomerization on the cell membrane and can be dissociated from
LMP1-mediated NF- B induction.
A, Rat-1 fibroblasts were transfected with a CD2/LMP1
chimera comprising the extracellular and transmembrane domains of CD2
fused to the cytoplasmic COOH terminus of LMP1, and stable clones were
obtained. CD2 expression was determined by flow cytometry. Viable cell
suspensions of Rat-1/CD2.192LMP1 cl.4 or control cells were exposed to
the OX34 anti-CD2 mAb (left panel, CD2 squares)
or to control G28.5 anti-CD40 mAb (left panel, CD40
squares), then stained with fluorescein isothiocyanate-conjugated
goat anti-mouse IgG. The y axis represents cell number and
the x axis fluorescence intensity (logarithmic scale). Rat-1
control cells showed no CD2 expression (mean fluorescence (mf) 1.5, background mf 1.48), but Rat-1/CD2.192 cl.4 cells stained strongly (mf
59.5, background mf 1.9) with more than 98% of the population staining
above background. LMP1 expression in these cells was verified by
immunoblot (right panel). B, activation of p38
kinase activity by CD2 engagement in Rat-1/CD2.192LMP1 cl.4 cells is
inhibited by treatment with 20 µM SB203580 but not by
exposure to 25 µg/ml D609. Cells transiently transfected with HA-p38
were treated with appropriate concentrations of SB203580 (20 µM) or D609 (25 µg/ml) for 1 h, and they were then
stimulated with OX34/IgG for 3 h before being analyzed for kinase
activity using GST-ATF2(19-96) as substrate. CD2 ligation induced an
approximately 11-fold increase in ATF2 phosphorylation levels compared
with untreated cells. Treatment with OX34/IgG in the presence of
SB203580 induced only a 4.2-fold increase in p38 kinase activity.
Immunoblot analysis of anti-HA immunoprecipitates using a control p38
antibody (lower panel) was also performed to demonstrate
that comparable amounts of HA-p38 were analyzed in cotransfection
experiments. Data shown are representative of four independent
experiments. C, induction of NF- B binding activity after
CD2 engagement is inhibited in D609-treated but not SB203580-treated
Rat-1/CD2.192LMP1 cl.4 cells. Nuclear extracts isolated from parallel
cultures as described in B were subjected to EMSA using a
32P-labeled HIV long terminal repeat B probe. The
complex indicated by the arrow contains p50·p65
heterodimers.
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Because LMP1 also engages NF-
B signaling, experiments were carried
out to establish whether the p38 and NF-
B pathways are overlapping
or can be dissociated. For this purpose, we first examined whether
inhibition of LMP1-mediated p38 activation could influence NF-
B
signaling. Rat-1/CD2.192LMP1 cl.4 cells were stimulated for 3 h
with OX34/IgG in the presence of 20 µM SB203580, a
pyridinyl imidazole compound that has been shown previously to inhibit
p38 activity in response to a variety of stimuli (32, 40, 41). In
agreement with these reports, treatment with SB203580 significantly inhibited ATF2 phosphorylation induced by CD2 cross-linking (Fig. 3B, seventh lane). The requirement for high
concentrations of this compound to block p38 kinase activity compared
with amounts required in vitro could be attributed to the
ability of SB203580 to bind unactivated as well as activated p38 and to
compete with ATP in vivo (42); high p38 levels in
transfected cells would require increased amounts of inhibitor.
Parallel cultures were analyzed for NF-
B activation by EMSAs using a
32P-labeled HIV-
B probe. It was found that treatment of
Rat-1/CD2.192LMP1 cells with SB203580 did not impair the ability of
LMP1 to activate NF-
B after CD2 engagement (Fig. 3C,
sixth and seventh lanes). In addition, no
inhibition in antibody-induced JNK activation was observed at this
SB203580 concentration (data not shown). We then examined the effects
of the metabolic inhibitor D609 on p38 and NF-
B activation. This
compound has been shown previously to prevent nuclear translocation of
NF-
B and subsequent NF-
B transactivation in response to a variety
of stimuli, including LMP1 expression (24, 43). In agreement with these
reports, exposure of OX34/IgG-stimulated Rat-1/CD2.192LMP1 cl.4 cells
to 25 µg/ml D609 significantly inhibited LMP1-mediated NF-
B
binding activity (Fig. 3C, far right lane).
Lysates from parallel cultures, cotransfected with HA-p38 expression
vector, were analyzed for p38 kinase activity. It was found that far
from reducing kinase activity, D609 increased ATF2 phosphorylation in
response to LMP1 signaling. This is in agreement with previous reports
showing that antioxidants and potent NF-
B inhibitors increase p38
kinase activity in response to various stimuli, such as TNF-
and
phorbol 12-myristate 13-acetate (41). Our data demonstrate that
compounds that efficiently block LMP1-mediated NF-
B activation in
Rat-1 cells do not impair its ability to signal on the p38 axis, and conversely, inhibition of p38 activity does not influence NF-
B binding, indicating divergence of signals.
Both CTAR1 and CTAR2 Domains of LMP1 Mediate p38
Activation--
Previous studies have shown that LMP1 activates
signaling on the NF-
B axis through two distinct regions in its
cytoplasmic COOH terminus, namely CTAR1 (amino acids 187-231) and
CTAR2 (amino acids 351-386). To determine whether these LMP1 domains
are also implicated in p38 signaling, LMP1 deletion and point mutants
with inactivated CTAR1 or CTAR2 were used (Fig.
4A).

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Fig. 4.
Both CTAR1 and CTAR2 domains of LMP1
contribute to p38 MAPK activation. A, schematic
representation of the LMP1 protein and the deleted LMP1 gene sequences
used in this study. Solid black lines represent wild type
(wt), and dotted lines denote deleted LMP1
sequences. CTAR1 is located at residues 194-232 and CTAR2 at residues
351-386. The asterisks represent a triple
P204xQ206xT208 AxAxA mutation.
B, induction of NF- B-dependent
transcriptional activity by LMP1 and LMP1 deletion mutants. HEK 293 cells were transfected with 1 µg of pSG5-based constructs in the
presence of 50 ng of NF- B-regulated luciferase reporter plasmid
BConA-Luc and 50 ng of -galactosidase expression vector. Relative
luciferase values (RLV), which represent the luciferase
values normalized on the basis of -galactosidase expression, were
determined at 36 h post-transfection. Data shown represent fold
increase in RLV relative to vector control, which was given
the arbitrary value of 1 and are the mean (±S.D.) of at least three
independent experiments. C, induction of p38 kinase activity
by LMP1 and LMP1 mutants. HEK 293 cells were transfected with 0.5 µg
of HA-p38 and 1 µg of pSG5 or pSG5-based LMP1 expression vectors; p38
kinase activity was assessed using immune complex kinase assays and
GST-ATF2(19-96) as substrate. Densitometric analysis using a Bio-Rad
GS-690 imaging densitometer showed the following increases in ATF2
phosphorylation levels compared with control vector, which was given
the arbitrary value of 1: LMP1, 7.5; LMP1 (332-386), 1.9;
LMP1 (187-351), 5.5; LMP1AxAxA, 4.7;
LMP1AxAxA/378STOP, 0.9. Three independent experiments were
performed and gave similar results.
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These mutated LMP1 constructs were first analyzed for their effects on
NF-
B activation using luciferase reporter assays in transiently
transfected HEK 293 cells. It was found that deletion of CTAR2
(construct pSG5-LMP1
(332-386)) significantly reduced NF-
B
activity to approximately 20% of wild type LMP1 (Fig. 4B). Deletion of CTAR1 (construct pSG5-LMP1((187-351)) induced only a small
reduction in NF-
B levels to approximately 75% of the wild type
molecule. Comparable levels of NF-
B activation were observed after
expression of pSG5-LMP1AxAxA, which contains a
P204xQ206xT208
AxAxA mutation
and therefore acts as a CTAR2 effector. Indeed, this triple mutation
has been shown previously to block CTAR1-mediated NF-
B by abrogating
TRAF binding to this LMP1 domain (12, 19). Because the extreme COOH
terminus of LMP1 is important for association with TRADD and CTAR2
signaling (15, 58), we have introduced a stop codon at amino acid
position 378 of pSG5-LMP1AxAxA (construct pSG5-
LMP1AxAxA/378STOP, Fig. 4A) and examined the
ability of this mutant to engage NF-
B signaling. Removal of the last
8 amino acids from LMP1AxAxA completely abolished the
ability of this EBV protein to signal on the NF-
B axis (Fig.
4B). These data verify that CTAR2 is the major
NF-
B-activating domain and demonstrate that the LMP1 constructs used
are functional.
To determine whether CTAR1 and CTAR2 also contribute to p38 activation,
HEK 293 cells were transfected with 1 µg of pSG5 or pSG5-based LMP1
constructs together with 0.5 µg of HA-tagged p38 expression vector.
Lysates from transfected cells were subjected to immune complex kinase
assays using GST-ATF2(19-96) fusion protein as substrate. As shown in
Fig. 4C, expression of LMP1
(187-351) or
LMP1AxAxA induced significant p38 kinase activity, albeit
lower than wild type LMP1, whereas transfection of the CTAR1-expressing
pSG5-LMP1
(332-386) construct had only a small effect on ATF2
phosphorylation. Removal of the last 8 amino acids from
LMP1AxAxA abolished LMP1-mediated p38 signaling, in
agreement with the loss of its functional CTAR1 and CTAR2 domains (Fig.
4C, middle panel). Immunoblot analysis using the
CS.1-4 mAbs (36) was also performed to confirm LMP1 expression from
these plasmids (Fig. 4C, top panel). As has been
documented previously (6), the CTAR1-deleted LMP1
(187-351)
construct was not detectable by immunoblotting, but expression was
confirmed by immunofluorescence staining with the CS.1 mAb (data not
shown). Thus, both the TRAF-interacting CTAR1 and TRADD-binding CTAR2
domains of LMP1 can activate p38 signaling with CTAR2 being the major contributor.
TRAF2 Is a Mediator of LMP1-activated p38 Signaling--
The
inability of LMP1AxAxA/378STOP mutated construct, which
lacks both the TRAF- and TRADD-interacting domains of LMP1 to induce p38 kinase activity, suggests that these adaptor proteins are important
for p38 signaling. TRADD and TRAF2 in particular are known to regulate
TNFR, CD40, and LMP1-mediated JNK and NF-
B signaling (44-47, 58).
We therefore examined the ability of TRAF2 to regulate p38 activation
by LMP1. For this purpose, HEK 293 cells were cotransfected with 1 µg
of pSG5LMP1 and HA-p38 in the presence or absence of 1 µg of a
CMV-driven TRAF2
(6-86) (18). This NH2-terminal deleted
TRAF2-expressing construct has been shown to act as a dominant negative
mutant of TRAF2 activities because it blocks JNK and NF-
B signals
from CD40, TNFRII, and LMP1 (12, 18, 19, 44-47, 58). TRAF2 associates
with TRADD, and dominant negative TRAF2 can also inhibit TNFRI- and
TRADD-mediated signals (44, 48).
Expression of LMP1 and TRAF2 in transiently transfected 293 cells was
verified by immunoblot analysis (Fig. 5,
A and B). Immune complex kinase assays
demonstrated that although TRAF2
(6-86) alone had no effect on ATF2
phosphorylation, expression of this mutated TRAF2 protein reduced
LMP1-mediated p38 kinase activity (Fig. 5C, first four
lanes). This observation, coupled with the ability of TRAF2
overexpression to activate p38 MAPK (Fig. 5C, far
right lane) suggests that TRAF2 is a mediator of LMP1-activated p38 signaling. Overexpression of TRADD also induced ATF2
phosphorylation in the presence of the anti-apoptotic cowpox virus crmA
protein; but TRAF3, another LMP1-associated protein, had no significant effect (data not shown).

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Fig. 5.
TRAF2 mediates LMP1-induced p38
activation. HEK 293 cells were cotransfected with 0.5 µg of
HA-p38 and 1 µg of pSG5 or pSG5LMP1 in the presence or absence of 1 µg of TRAF2 (6-86) (first four lanes). The effects of
transient expression of TRAF2 were also determined (far right
lane). Transfected DNA was normalized to 2.5 µg using empty
vector. 36 h later, lysates were analyzed for LMP1 (A)
or TRAF2 expression (B) or used to assess p38 kinase
activity (C). Although TRAF2 (6-86) alone had no effect
on p38 activity, expression of this mutated TRAF2 molecule inhibited
LMP1-induced ATF2 phosphorylation by 60%. Transfection of TRAF2
potently activated p38 MAPK, inducing an 8.1-fold increase in ATF2
phosphorylation. Results shown are representative of three independent
experiments.
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The p38 Pathway Regulates IL-6 and IL-8 Secretion in Response to
LMP1 Expression--
To identify the physiological consequences of p38
activation by LMP1, we have examined the effects of p38 inhibition on
interleukin production. Previous data have demonstrated the importance
of p38 signaling in TNF-mediated IL-6 synthesis (32-34), and we have shown recently that LMP1 expression induces IL-6 production via a
pathway involving TRAFs (12).
HeLa cells were transiently transfected with pSG5-LMP1 or control
vector, and condition supernatants were analyzed 36 h later using
an IL-6-specific ELISA. These assays demonstrated that LMP1 induced a
significant, 130-fold increase in IL-6 production compared with
vector-transfected cells. However, in the presence of 20 µM SB203580, a dramatic decrease in LMP1-induced IL-6
secretion was observed (Fig.
6A). Immunofluorescence
staining for the detection of LMP1 expression in transfected cultures
showed that more than 35% of the cells were positive for LMP1 in every
experiment performed (data not shown).

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Fig. 6.
LMP1 induces IL-6 and IL-8 secretion via a
p38-regulated mechanism. A, the p38 MAPK-specific
inhibitor SB203580 impairs the ability of LMP1 to induce IL-6
secretion. Conditioned supernatant collected from HeLa cells
transiently transfected with pSG5 or pSG5LMP1 in the presence or
absence of 20 µM SB203580 was analyzed for IL-6 levels
using an IL-6-specific ELISA. Results shown are the mean values
(±S.D.) from three independent experiments with triplicate
determinations for each experiment performed. B, p38
inhibition induces a significant reduction in LMP1-mediated IL-8
secretion. The same supernatants described in A were
analyzed for IL-8 levels using an IL-8-specific ELISA. C,
inducible LMP1-activated IL-8 secretion is inhibited by treatment with
SB203580. HEK 293EcR-LMP1/cl.4 cells or control cultures were treated
with 20 µM SB203580 for 1 h before the addition of
10 µM ponasterone A. After a 24-h incubation in the
presence of both agents, cell culture supernatants were collected and
analyzed for IL-8 levels using an IL-8-specific ELISA. Results from
triplicate determinations of a representative experiment are
shown.
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The same supernatants were also analyzed for IL-8 production.
Interestingly, we have found that LMP1 potently enhanced IL-8 protein
synthesis, inducing an approximately 100-fold increase in IL-8
secretion, and this effect was inhibited in the presence of nontoxic
concentrations of SB203580 (Fig. 6B). To examine the generality of the observed phenomenon, 293EcR-LMP1/cl.4 cells were
induced to express LMP1 in the presence or absence of 20 µM SB203580. Whereas untreated cells produced very little
IL-8, treatment with ponasterone A dramatically increased IL-8
synthesis in 293EcR-LMP1 but not in control cultures (Fig.
6C). However, in the presence of p38 inhibitor,
LMP1-mediated IL-8 secretion was reduced. The observed differences in
the levels of IL-8 secreted from HEK 293 and HeLa cells is a cell
line-dependent phenomenon rather than the result of
differential LMP1 expression, as transient transfection of pSG5-LMP1 in
293 cells induces IL-8 production at levels comparable to
ponasterone-treated cells (data not shown). Overall, these data suggest
that the p38 pathway may influence the ability of LMP1 to induce both
IL-6 and IL-8 production.
Involvement of p38 in the Transcriptional Control of IL-8
Synthesis--
Because the ability of LMP1 to induce IL-8 production
is novel, we have examined this phenomenon in more detail. We first determined whether LMP1 activates IL-8 at the transcriptional level.
For this purpose, RNA isolated from HeLa cells transiently transfected
with LMP1 or control vector was subjected to semiquantitative reverse
transcription PCR using primers specific for IL-8 and using GAPDH as
internal control. These experiments showed that IL-8 mRNA levels
increased approximately 12-fold in response to LMP1 expression (Fig.
7A). Interestingly, treatment
with 20 µM SB203580 dramatically reduced IL-8 RNA levels
in LMP1-transfected HeLa cells, suggesting that the p38 MAPK pathway
modulates the ability of LMP1 to induce IL-8 synthesis at the
transcriptional level.

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Fig. 7.
p38 coregulates LMP1-induced IL-8 synthesis
at the transcriptional level. A, transient expression
of LMP1 induces IL-8 RNA synthesis that is inhibited by treatment with
SB203580. HeLa cells transiently transfected with pSG5LMP1 (third and
fourth lanes) or control vector (first two lanes)
were exposed to 20 µM SB203580 (second and
fourth lanes), and RNA isolated 24 h later was
subjected to reverse transcription PCR using primers specific for IL-8
(top panel) and GAPDH (middle panel). The
arrows show hybridization signals of the PCR products. The
relative IL-8 RNA expression levels are depicted in histogram form
(bottom panel). B, LMP1 activates the IL-8
promoter. A reporter construct (IL8-Luc) containing 181 bp of the IL-8
promoter sequences linked to the luciferase gene was transfected into
HeLa or HEK 293 cells together with wild type or mutated LMP1
expression vectors, and relative reporter activity (ratio of luciferase
versus -gal activity; RLV) was assessed. Data shown are
the mean (±S.D.) of at least four independent experiments.
C, the p38-specific inhibitor SB203580 influences IL-8
promoter activity. HeLa or HEK 293 cells were transfected with IL8-Luc
or a mutated AP-1 IL8-Luc construct (mAP-1/IL8-Luc) in the presence of
1 µg of pSG5LMP1 or control vector. After treatment with 20 µM SB203580, IL-8 promoter luciferase and
-galactosidase activities were determined. Relative luciferase
values were then assessed; data shown represent mean values (±S.D.)
relative to vector control of four independent experiments.
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To analyze further the effects of LMP1 on IL-8 transcription, direct
studies on the IL-8 promoter were performed. A reporter plasmid
(IL8-Luc) containing 181 bp of the IL-8 promoter sequences linked to
the luciferase gene was constructed and transfected in HeLa and HEK 293 cells together with wild type or mutated LMP1 expression vectors. LMP1
expression was found to induce IL-8 promoter activity significantly in
both HeLa (6.0 ±1.4-fold increase) and 293 cells (12.6 ±3.2-fold
increase, Fig. 7B). Transfection of the CTAR1-expressing
LMP1
(332-386) had only a marginal effect whereas expression of
CTAR2 (constructs LMP1
(187-351) and LMP1AxAxA)
significantly induced IL-8 promoter activity in both cell lines, in
agreement with the function of CTAR2 as the predominant contributor of
LMP1 signals. Transfection of HeLa or HEK 293 cells with
LMP1AxAxA/378STOP, which is inactive for both NF-
B and
p38, failed to induce IL-8 promoter activity above background levels
(Fig. 7B).
Nucleotide sequence analysis of the 181-bp IL-8 promoter has revealed
the existence of three cis-acting elements that have been
shown to be functional in cell lines: a TGACTCA AP-1 binding motif 100 bp upstream the TATA box, an AGTTGCAAAT C/EBP (NF-IL6) binding site 64 bp upstream of the TATA box, and a GGGAATTTCC NF-
B binding site 52 bp upstream of TATA box (49). To examine the contribution of the AP-1
site to LMP1-mediated IL-8 promoter activity, a 2-nucleotide mutation
within the AP-1 motif (TATCTCA), which abolishes
transcription factor binding (49), was introduced in IL8-Luc. This
mutated construct (mut AP-1/IL8-Luc) was cotransfected with pSG5-LMP1
or control vector into HeLa or HEK 293 cells and examined for
luciferase activity. It was found that mutation of the AP-1 site from
the IL-8 promoter significantly inhibited its activity in response to
LMP1 expression by 50-60% (Fig. 7C). These results suggest
that AP-1 is a positive regulator of IL-8 promoter activation by LMP1.
Unlike AP-1, mutations that abrogate C/EBP binding on IL-8 promoter had
no effect on its activation after LMP1 expression (data not shown). A
role for NF-
B in IL-8 promoter regulation was demonstrated by the
ability of a constitutively active I
B
mutant
(I
B
[S32A/S36A]), which has been shown previously to interfere
with LMP1-mediated NF-
B (24), to abolish LMP1-induced IL8/Luc and
mutAP-1/IL8-Luc reporter activity (Fig.
8). Thus, NF-
B confers a potent
positive regulatory role in IL-8 promoter activity.

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Fig. 8.
Suppression of LMP1-mediated
NF- B activation by constitutively active
I B [S32A/S36A]
abolishes IL-8 and mutated AP-1 IL-8 promoter activity. HEK 293 cells were cotransfected with mAP-1/IL8-Luc and -gal constructs and
1 µg of pSG5LMP1 or control vector in the presence or absence of 1 µg of a CMV-driven I B [S32A/S36A]. This I B expression
construct has been shown previously to inhibit LMP1-mediated NF- B
activation (24). Relative luciferase values (RLV) were determined at
36 h post-transfection. Data shown represent fold increase
(mean ± S.D.) in luciferase value relative to vector control,
which was given the arbitrary value of 1.
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To gain more insight into the composition of the protein complex bound
to the IL-8 AP-1 consensus, we have performed EMSAs using nuclear
extracts from vector or LMP1-transfected HEK 293 cells. As shown in
Fig. 9A, LMP1 expression
dramatically increased binding activity on the IL-8 AP-1 motif. The
addition of anti-Fos antibody in nuclear extracts did not influence the
mobility or intensity of binding, whereas the addition of anti-Jun/AP-1
antibody supershifted the complex. Exposure of nuclear extracts from
LMP1-transfected HEK 293 cells to anti-ATF2 antibody and subsequent
precipitation of the formed immune complex reduced the intensity of
protein-DNA interaction by approximately 30-40%, suggesting that ATF2
is a component of the complex bound to the IL-8 AP-1 site (Fig.
9A). Parallel depletion experiments using a control
anti-c-rel antibody failed to induce any significant change in AP-1
binding (data not shown). To verify that ATF2 binds the IL-8 AP-1
consensus, EMSAs were performed using recombinant ATF2 protein.
Previous studies have shown that ATF2 binds the Jun2 TRE sequence from the c-Jun promoter but not the collagenase promoter TRE (50, 51). In
agreement with these reports, recombinant ATF2 strongly interacted with
a 32P-labeled Jun2 TRE oligonucleotide (Fig. 9B,
right lane) but did not bind the collagenase AP-1 sequence
(Fig. 9B, left lane). Interestingly, recombinant
ATF2 was found to interact with the IL-8 promoter AP-1 motif, although
to less extent than with Jun2 TRE (Fig. 9B, center
lane).

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Fig. 9.
The transcription factors c-Jun and ATF2 bind
the AP-1 motif of IL-8 promoter. A, nuclear extracts
from vector (first lane) or LMP1-transfected 293 cells
(second through fifth lanes) were subjected to
EMSA using a 32P-labeled IL-8 AP-1 probe. LMP1 induces
binding activity (second lane). The composition of the IL-8
AP-1 consensus-bound complex was examined by supershift analysis using
antibodies raised against c-Fos or c-Jun (third and
fourth lanes) or by depletion experiments using an anti-ATF2
antibody that recognizes the DNA binding and dimerization domain of the
protein (fifth lane). B, recombinant ATF2 protein
binds the AP-1 motif of IL-8 promoter (second lane). As a
negative control, ATF2 failed to interact with a collagenase TRE
oligonucleotide (first lane), whereas Jun2 TRE strongly
bound recombinant ATF2 protein (third lane).
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To determine whether the p38 MAPK pathway is involved in IL-8 promoter
regulation, the p38 inhibitor SB203580 was used to treat HeLa or HEK
293 cells transfected with pSG5-LMP1 or control vector and IL-8
promoter constructs. It was found that p38 inhibition significantly
reduced LMP1-mediated IL-8 promoter activity by 45-60% in both cell
lines (Fig. 7C).
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DISCUSSION |
The EBV-encoded LMP1 has recently attracted much attention as it
appears to function as a constitutively activated TNF family receptor.
Indeed, LMP1 has been shown to interact with TRAFs and TRADD and to
mimic many of the phenotypic consequences of CD40 or TNFR activation.
Expression of this viral protein induces a plethora of activities in
target cells. These include the oncogenic transformation of rodent
fibroblast cell lines, up-regulation of anti-apoptotic proteins and
cell surface markers, cytokine production, and differentiation blockade
in epithelial cells. Furthermore, LMP1 expression is essential for
EBV-induced B cell immortalization in vitro. The signaling
pathways that mediate these phenomena are a subject of intense
investigation (for review, see Ref. 52). Previous studies have
demonstrated that LMP1 expression leads to the rapid activation of the
transcription factor NF-
B, an effect mediated independently by two
domains in the cytoplasmic COOH terminus of the protein: CTAR1 (amino
acids 187-231) and CTAR2 (amino acids 351-386) (6, 16). More recent
reports indicate that LMP1 also mediates activation of a
Ras/MAPK-dependent pathway (21) as well as the JNK/AP-1
cascade (22-24).
In this study we provide evidence of another signaling pathway
activated by LMP1. Thus, we have shown that inducible LMP1 expression
leads to activation of the p38 MAPK, as evidenced by the ability of
LMP1 to induce p38 phosphorylation. Furthermore, p38 immunoprecipitated
from LMP1-transfected cells induced phosphorylation of the
transcription factor ATF2, one of the downstream targets of p38 MAPK.
This phenomenon depends on oligomerization of LMP1 on the cell membrane
because a CD2/LMP1 chimera, comprising the extracellular and
transmembrane domain of CD2 and the cytoplasmic tail of LMP1, can
induce p38 kinase activity only after antibody-induced aggregation of
the chimera. Mutational analysis of the LMP1 cytoplasmic COOH terminus
identified both TRAF-interacting CTAR1 and TRADD-binding CTAR2 domains
contributing to p38 signaling. To exclude the possibility that
induction of an autocrine loop is responsible for p38 activation, conditioned supernatants from vector or LMP1-transfected HEK 293 cells
were used to treat untransfected HEK 293 cultures. These experiments
showed that none of the conditioned media was able to induce ATF2
phosphorylation above background levels (data not shown), suggesting
that LMP1 activates the p38 MAPK cascade directly.
Because LMP1 also engages NF-
B signaling, experiments were performed
to determine whether these two pathways run on the same axis or in
parallel. We have found that inhibition of p38 MAPK by treatment with
the highly specific inhibitor SB203580 did not affect LMP1-induced
NF-
B binding activity. Conversely, although the metabolic inhibitor
D609 impaired LMP1-mediated NF-
B activation, it did not inhibit
signaling on the p38 axis, suggesting that these two LMP1-activated
pathways can be primarily dissociated. This is in agreement with our
previous work demonstrating NF-
B and JNK signal divergence in
response to LMP1 expression (24). However, we cannot exclude the
possibility of LMP1-mediated NF-
B transactivation being a target for
p38. Indeed, a role for p38 MAPK in TNF- or CD40-induced NF-
B
transactivation in the absence of a direct effect on NF-
B binding
activity has been reported recently (33, 41, 53), and our preliminary
results using a luciferase reporter plasmid containing
B elements
from the Ig
enhancer (12) suggest that SB203580 induces a small
inhibition in LMP1-mediated NF-
B transcriptional activity.
Our data demonstrate the involvement of TRAF2 in LMP1-mediated p38
activation. The ability of TRAF2 also to engage signaling on the
NF-
B axis suggests that bifurcation of NF-
B and p38 signals occurs downstream of this adaptor protein. Indeed, in this study we
have shown that transient overexpression of TRAF2 significantly induced
p38 kinase activity and that a dominant negative TRAF2
(6-86) molecule that has been shown previously to interfere with LMP1-mediated NF-
B partially inhibited p38 activation.
The functional implications of LMP1-mediated p38 activation have been
demonstrated by the ability of SB203580 to down-regulate LMP1-activated
IL-6 and IL-8 production. These cytokines play an important role in
initiation and maintenance of acute inflammatory responses. IL-8 is a
potent chemotactic agent and may contribute to the accumulation of a T
cell infiltrate in EBV-positive malignancies such as nasopharyngeal
carcinoma and Hodgkin's disease, where LMP1 is expressed. In addition,
IL-8 has been shown to induce angiogenesis and haptotactic migration
and to enhance metastatic potential in melanoma cells (54, 55). These
data coupled with the recently reported ability of LMP1 to induce
up-regulation of matrix metalloproteinases (56) suggest that in
addition to its transforming potential, LMP1 may contribute to
metastasis of EBV-associated tumors. The effects of p38 MAPK on
LMP1-mediated IL-8 production were found to occur at the
transcriptional level as treatment of HeLa cells with SB203580
significantly inhibited up-regulation of IL-8 RNA. This is in agreement
with the reported ability of SB203580 to block TNF-mediated IL-6 RNA
and protein synthesis (32, 40).
To gain more insight into this novel LMP1-mediated phenomenon, we have
examined the effects of LMP1 expression on IL-8 promoter and found that
transfection of HeLa or HEK 293 cells with this viral protein
significantly enhanced promoter activity. Elements important for IL-8
promoter regulation are localized within the first 135 bp of the
5'-flank and include an AP-1 and a NF-
B binding site (49). LMP1 is
known to engage signaling on the NF-
B axis, and inhibition of this
pathway by coexpression of a constitutively active
I
B
[S32A/S36A] mutant abolished wild type or AP-1-mutated IL-8
promoter activity. Thus, LMP1-mediated NF-
B activation can positively regulate IL-8 activity.
Furthermore, we have found that LMP1 expression induced nuclear protein
binding on the AP-1 element of IL-8 promoter, whereas mutations within
this element significantly impaired LMP1-mediated IL-8 transcriptional
induction. Taken together, these data support a positive regulatory
role for LMP1-activated AP-1-bound proteins in IL-8 synthesis. Using
EMSAs, we have identified c-Jun and ATF2 proteins as components of the
bound complex. Binding sites for c-Jun/ATF2 have been found in several
promoters including those of c-Jun,
-interferon, E-selectin, and the
human urokinase enhancer (50, 51, 57). A role for p38 in IL-8 promoter
regulation is demonstrated by the inhibitory effect of SB203580 on
LMP1-induced IL-8 transcriptional activity. However, the ability of
LMP1 to induce c-Jun phosphorylation through activation of JNK suggests that this kinase pathway may also contribute to modulation of IL-8
expression. A similar complex regulation of the E-selectin promoter by
JNK, p38, and NF-
B has been reported recently (57).
Overall, in this study we demonstrate the ability of a viral protein,
namely EBV-encoded LMP1, to activate the p38 MAPK pathway. This
phenomenon appears to be important for IL-6 and IL-8 production and may
play a significant cooperative role in regulating additional LMP1 activities.