From the Department of Medicine, Tenovus Building, University of Wales College of Medicine, Cardiff CF14 4XX, United Kingdom
Received for publication, May 3, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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The latent membrane protein-1 (LMP1) of
Epstein-Barr virus induces gene transcription, phenotypic changes, and
oncogenic transformation. One cellular gene induced by LMP1 is that for
intercellular adhesion molecule-1 (ICAM-1), which participates in a
wide range of inflammatory and immune responses. ICAM-1 may enhance the
immune recognition of cells transformed by Epstein-Barr virus, and thus
combat development of malignancy. Despite growing understanding of the
various signaling functions of LMP1, the molecular mechanisms by which
LMP1 induces ICAM-1 are not understood. Here, we demonstrate that
transcriptional activation by LMP1 is absolutely dependent upon a
variant NF- The latent membrane protein-1
(LMP1)1 is considered to be
the major oncogene of Epstein-Barr virus (EBV), a persistent
herpesvirus that is associated with various malignant diseases (1).
LMP1 transforms rodent fibroblasts (2), induces lymphomas in transgenic mice (3), and is essential for EBV-induced immortalization of human
primary B-lymphocytes (4). The oncogenic properties of LMP1 are at
least in part due to the up-regulation of anti-apoptotic proteins such
as Bcl-2, A20, Mcl-1, and Bfl-1 (5-8). In addition, LMP1 up-regulates
components of the endogenous antigen processing pathway and some
intercellular adhesion molecules, such as ICAM-1 and LFA-3 (9, 10).
These latter functions ensure that EBV-transformed lymphocytes can be
recognized and regulated by cellular immune responses, which is an
important feature of EBV persistence in healthy individuals that
normally prevents the development of EBV-positive lymphomas (11).
Consistent with its diverse biological functions, LMP1 has been
reported to trigger a number of different signaling pathways, including
activation of NF- The intercellular adhesion molecule, ICAM-1 (CD54), is an inducible
cell surface glycoprotein and member of the immunoglobulin supergene
family (20, 21). ICAM-1 serves as a counter-receptor for a number of
cell surface molecules such as LFA-1 (CD11a) and MAC-1 (CD11b), and it
plays a central role in a wide range of inflammatory and immune
responses (22). ICAM-1 is constitutively expressed at low levels on
vascular endothelium and lymphocytes and at moderate levels on
monocytes. Induction of high levels of ICAM-1 occurs in response to
various inflammatory mediators, including bacterial lipopolysaccharide,
phorbol esters, oxidant stress and pro-inflammatory cytokines, such as
TNF The 5'-flanking region of the ICAM-1 gene contains numerous potential
regulatory elements that could be involved in the activation of the
promoter, some of which are tissue-specific and
cytokine-dependent (23). For example, The studies described here were designed to define the molecular
mechanisms involved in LMP1-induced ICAM-1 expression in lymphocytes.
We carried out promoter deletion analysis and luciferase reporter
assays to investigate the role of LMP1 at the transcriptional level and
to determine the activator site within the ICAM-1 promoter. Inhibition
of specific signaling pathways induced by LMP1 was achieved using
chemical inhibitors for the p38 and JAK3-pathways, and dominant
inhibitory molecules for the SEK and NF- Cell Lines--
Jurkat is a cell line derived from an
EBV-negative T cell lymphoma (33). Eli-BL is an EBV-positive B cell
line established from a Burkitt's lymphoma, and it displays a latency
I form of infection in which Epstein-Barr virus nuclear antigen 1 is
the only viral protein detected (34). DG75 is an EBV-negative
Burkitt's lymphoma B cell line, and the derived DG75-tTA-LMP1 line
contains a stable transfected tetracycline-regulated LMP1 expression
plasmid; this transfectant, together with the control DG75-tTA
transfectant, has been described previously (35). All the lymphoid cell
lines were grown in suspension in RPMI, 10% fetalm calf serum
supplemented with 2 mM glutamine and antibiotics (200 units/ml penicillin and 200 µg/ml streptomycin), and were maintained
at 37 °C in a humidified atmosphere with 5% CO2. The
DG75 transfectants were maintained in 1 µg/ml tetracycline and were
drug-selected with 0.8 mg/ml hygromycin B plus 2 mg/ml G418
(DG75-tTA-LMP1) or with 0.8 mg/ml hygromycin B only (DG75-tTA).
Plasmids and Inhibitors--
The ICAM-1 reporter constructs
containing 5' regions upstream of the ICAM-1 gene in front of a
luciferase gene were obtained from Harry C. Ledebur and have been
described elsewhere (30). The 3Enh-luc reporter plasmid, with three
A constitutively active I Gene Transfection--
For transient expression, 0.5 to 1 × 107 cells from a suspension culture were transfected by
electroporation using a Bio-Rad GenePulser II electroporator at 280 V
and 950 microfarads at room temperature in 500 µl of growth medium.
The cells were reseeded in 5 ml of fresh growth medium and were then
incubated under normal conditions. Transfection efficiency ranged from
10% to 20% for Eli-BL and from 40% to 50% for Jurkat, as assessed
by cotransfection with the EGFP-C1 expression vector and flow cytometry analysis.
Assay for Reporter Activity--
The activity of the different
reporter plasmids was measured at 18-24 h after transfection. Cells
were washed twice in phosphate-buffered saline and lysed in 150 µl of
lysis buffer containing 100 mM HEPES, pH 8.0, 2 mM magnesium chloride, 5 mM dithiothreitol, and
2% Triton X-100. Luciferase activity in 50 µl of clarified lysate
was analyzed in a Berthold LB9501 luminometer following injection of
100 µl of 0.5 mM luciferin (Amersham Pharmacia
Biotech) dissolved in luciferin assay reagent (30 mM
glycylglycine, pH 7.9, 1 mM MgCl2, 0.1 mM EDTA, 30 mM dithiothreitol, 0.3 mM coenzyme A, 0.5 mM ATP). Light release was
integrated over 10 s.
Assay for Cell Surface ICAM-1 Protein by Flow Cytometry--
The
induction of ICAM-1 protein in transfected cells was assayed by
immunofluorescence staining of viable cells, followed by flow cytometry
using a Becton Dickinson FACSCalibur analyzer as described previously
(19). Briefly, at 48 h after transfection, the cells were washed
and stained with a phycoerytherin-conjugated monoclonal antibody to
human CD54 (MCA675PE; Serotec) at 4 °C for 60 min. The transfected
population was marked by the expression of cotransfected EGFP-C1
plasmid, and this population was gated for analysis of ICAM-1 staining.
Detection of Proteins by Immunoblotting--
Cells were washed
in phosphate-buffered saline and lysed for 30 min on ice in luciferase
lysis buffer. The lysates were centrifuged for 5 min at 13,000 × g. An aliquot of the clarified lysate was added to an equal
volume of 2× gel sample buffer (0.1 M Tris buffer, pH 6.8, 0.2 M dithiothreitol, 4% sodium dodecyl sulfate, 20%
glycerol, 0.1% bromphenol blue) and boiled for 2 min. The solubilized
proteins were separated by SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride membrane for immunoblotting
using an alkaline phosphatase chemiluminescent detection protocol (41). LMP1 was detected by first incubating the membranes for 1 h with 1 µg/ml CS.1-4 (42) in I-BlockTM (Tropix Inc.) followed by incubating for 1 h with a 1/10,000 dilution of alkaline
phosphatase-conjugated goat anti-mouse IgG (Bio-Rad 170-6461). The
I Kinase Assays--
JNK and p38 in vitro kinase assays
were performed as described elsewhere (40). Briefly, at 48 h after
transfection with either HA-p46SAPKy-pcDNA3 or HA-p38, cells were
lysed in 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
dithiothreitol). An aliquot of 250 µg of protein extract was used for
immunoprecipitating HA-p38 or HA-JNK using 1 µg of anti-HA antibody
(Roche Molecular Biochemicals; 12CA5) on Sepharose beads. The washed
immunoprecipitates were then subjected to a kinase reaction, and
phosphorylation was determined by Western blot. In the JNK assay,
phosphorylation of Jun was determined by immunoblot analysis using
phospho-c-Jun (Ser63) antibody (New England Biolabs 9261),
and in the p38 assay phosphorylation of ATF2 was determined using
phospho-ATF2 (Thr71) antibody (New England Biolabs 9221).
RNase Protection Assay--
Total RNA was isolated from DG75
cells using Ultraspec solution (Biotecx) according to manufacturer's
recommendations. Quantification and purity of RNA was assessed by
A260/A280 absorption, and
an aliquot of 15 µg of RNA from each sample was used in the RNase protection assay.
The assay was performed using the Riboquant Multiprobe RNase protection
assay system as described by the manufacturer (PharMingen). The
customized Multiprobe ICAM1 template set and T7 RNA polymerase were
used to synthesize 32P-labeled antisense riboprobes, which
were hybridized to the RNA sample in solution at 54 °C for 16 h. After digestion of free probe and other single-stranded RNA with
RNases A and T1, the labeled "RNase-protected" fragments were
phenol:chloroform:isoamyl alcohol-extracted and ethanol-precipitated.
The fragments were than resolved on 6% denaturing polyacrylamide gels
and detected by autoradiography or using a phosphorimager (Bio-Rad).
Approximately 5000 cpm of "unprotected" labeled probe served as a
reference to identify the fragments.
To investigate ICAM1 mRNA levels, control plasmid and LMP1- or
MEKK1-expressing plasmids were transiently transfected together with
rCD2GFP into DG75 cells. After 48 h, transfections were pooled and
living positive transfected cells were stained with the rat CD2-specific antibody, Ox34 (American Tissue Culture Collection), and
sorted using MACS immunobeads (Miltenyi Biotec) according to
manufacturer's recommendations. Total RNA was extracted from cells and
analyzed for ICAM1 mRNA expression by RNase protection assay.
LMP1 Induces ICAM-1 Surface Expression and Promoter
Activation--
LMP1 up-regulates ICAM-1 protein expression in various
cell lines. This is illustrated in Fig.
1A, showing the results of flow cytometry analysis of ICAM-1 expression on Jurkat cells
transfected with a control vector or with the SG5-LMP1 expression
vector. The cells were cotransfected with a GFP expression vector so
that the transfected population could be identified and gated to allow analysis of ICAM-1 expression following staining of the cells with
phycoerytherin-conjugated CD54 antibodies. In this representative experiment, the vector control-transfected cells showed basal ICAM-1
surface expression with a mean fluorescence intensity of 29.4 arbitrary
units (Fig. 1A, upper histogram),
whereas cells transfected with 2 µg of SG5-LMP1 gave a mean
fluorescence intensity of 81.3 (Fig. 1A, lower
histogram). The LMP1-mediated induction of ICAM-1 protein in
Jurkat T lymphocytes (Fig. 1B) and Eli-BL B lymphocytes
(Fig. 1C) is dose-dependent. In the experiment
illustrated in Fig. 1B, induction of ICAM-1 in Jurkat cells
LMP1 caused a maximal 3-fold increase in ICAM1protein expression. In
separate independent experiments, the maximal induction of ICAM-1 by
LMP1 in Jurkat cells typically ranged between 2- and 6-fold at 48 h after transfection. In Eli-BL cells (Fig. 1C), a similar
magnitude of ICAM-1 induction by LMP1 was observed, but the
dose-response curve differed in that Eli-BL cells were responsive to as
little as 0.1 µg of SG5-LMP1 plasmid, whereas Jurkat cells required 5 to 10 times more plasmid to elicit a similar response. Nevertheless, the optimal induction of ICAM-1 surface expression in both cell lines
was achieved at a plasmid concentration of between 1 and 3 µg of
SG5-LMP1, which was used in subsequent experiments.
Since LMP1 is known to activate transcription factors, we investigated
the effects of LMP1 on the ICAM-1 promoter activation. The 5' region of
the ICAM-1 gene is well described, and the transactivator sites and
responsive regions for different members of the TNF
As a member of the TNFR superfamily, LMP1 might be predicted to
activate ICAM-1 using the TRE region within the ICAM-1 promoter. To
test this possibility, we used a series of luciferase reporters regulated by different regions of the ICAM-1 promoter (30). The results
of a representative experiment are shown (Fig. 2B) in which
the luciferase plasmids were cotransfected with SG5-LMP1 into Eli-BL
(black bars) and Jurkat (white
bars) cells, and the reporter activities measured at 24 h after transfection. LMP1 induced the ICAM-1 reporters with a similar
profile in both cell lines. The maximal luciferase induction of
5-6-fold was only obtained with the full-length 1.3ICAM1-luc
construct. Mutants deleted for 779 or 1126 5' bp in the regions
upstream of the TRE site (0.5-ICAM1 and TRE-ICAM1, respectively) showed
reduced activation levels but were still induced 3-4-fold by LMP1. In
contrast, neither the reporter mutant deleted for the TRE
(delTRE-ICAM1) nor the mutant deleted for 1248 5'-bases (0.1-ICAM1) was
induced by LMP1. It should be noted that the nonresponsive
0.1-ICAM1reporter contained the intact interferon response element.
These results suggest that LMP1 uses the same essential region as
TNF Comparison of the Effects of LMP1 and TNF
The results with the delTRE-luc reporter confirmed that the TRE is
essential for induction of ICAM-1 promoter activity both by LMP1 and by
TNF Activation of NF-
Having confirmed the effectiveness of EGFP-I Activation of NF- Other Signaling Pathways Known to Be Induced by LMP1 Are Not
Required to Induce ICAM-1 Protein--
In addition to NF- MEKK1 Cannot Induce ICAM-1-mRNA--
Since LMP1 and MEKK1
induce a similar subset of signaling pathways and MEKK1 very
efficiently induces ICAM-1 promoter but was not able to induce ICAM-1
protein, we wanted to investigate those differences in more detail. The
inability of MEKK1 to induce ICAM-1 surface expression suggests that
LMP1 regulates additional pathways. To further analyze the level at
which LMP1 and MEKK1 signal differed, we investigated the effects of
MEKK1 on ICAM-1 mRNA levels. DG75 cells were transfected with MEKK1
or LMP1 together with rat CD2 to identify the transfected cells. Cells
were incubated at 37 °C for 24 h to allow expression. The
transfected cells were stained with OX34 monoclonal antibody to rat
CD2, and were separated by immunomagnetic beads. This resulted in
greater than 90% purity of rat CD2-positive cells as assayed by
fluorescence-activated cell sorting (data not shown). Total RNA from
the purified cells was isolated and mRNA for ICAM-1 was assayed by
RNase protection using specific ICAM-1 probes. The results of these
experiments are shown in Fig. 8. The
upper panel shows a graphic representation of a
single experiment and clearly shows that LMP1 can up-regulate ICAM-1
mRNA (compare second lane with first
lane). However MEKK1 (third lane)
could not induce ICAM-1 mRNA. The lower panel
shows the average mRNA levels from the phosphorimager analysis of
three different experiments. Although LMP1 induced ICAM-1 mRNA
2-3-fold, MEKK1 had no effect on the ICAM-1 mRNA levels. These
data suggest that MEKK1 lacks a key event required for the effective
transactivation of the ICAM-1 gene.
CTAR1 and CTAR2 Provide Qualitative Different Signals to
ICAM-1--
LMP1 regulation of ICAM-1 clearly requires as yet
uncharacterized signals in addition to NF-
These data suggest that the deacetylation inhibitor, sodium butyrate,
can cooperate with CTAR2 and -3 to generate LMP1 wild type ICAM-1
expression. Therefore, we conclude that LMP1 induces a novel additional
signal or pathway to induce ICAM-1 protein, which can be mapped to the
CTAR1 region of LMP1.
Although it has been recognized for some time that the EBV-encoded
LMP1 is responsible for the up-regulation of ICAM-1 (10), the mechanism
of activation was unclear. The biological and signaling properties of
LMP1 share many features with the pro-inflammatory cytokines TNF A direct comparison of LMP1 and TNF Whatever the role of upstream elements, the TRE appears to be
critically involved in inducible regulation of ICAM-1 by various stimuli. Within the TRE, a critical feature is the variant NF- Despite the essential role for NF- The ability of NF- The major difference regarding MEKK1 and LMP1 lies in the ability to
induce mRNA, since LMP1 induces ICAM-1 mRNA 2-3-fold but
MEKK1-transfected cells have no increased ICAM-1 mRNA levels (Fig.
8). Thus, the LMP1-specific effect seems to act at the level of
transcription of the endogenous ICAM-1 gene, which is not completely reflected in the luciferase reporter assays. Signal-regulated acetylation events and their effects on gene transcription are recognized as another mechanism of gene activation (46, 56, 57). We
have shown (Fig. 9) that the deacetylation inhibitor, sodium butyrate,
can substitute for a function of LMP1, which locates to the CTAR-1
region. This identifies a new mechanism for activation of ICAM-1 by
LMP1. The precise nature of this activation process is not clear,
because sodium butyrate has other effects on cells, such as regulation
of proliferation (58), and induction of apoptosis (59), which may be
due to mechanisms in addition to its effects on histone deacetylation.
Furthermore, this new function of CTAR1, which can be substituted by
sodium butyrate, cannot alone explain our data. Thus, although
activation of NF- In summary, the present study demonstrates that NF-B motif within the tumor necrosis factor
(TNF
)
response element of the ICAM-1 promoter. Although the TNF
response
element is sufficient for TNF
induction of the ICAM-1 promoter, LMP1
also required the cooperation of additional upstream sequences for optimal induction. Inhibitor studies of known LMP1-induced signaling pathways ruled out the involvement of c-Jun N-terminal kinase (JNK),
p38 mitogen-activated protein kinase, and the Janus-activating tyrosine
kinase 3 (JAK3), and confirmed NF-
B as a critical factor for
induction of ICAM-1. However, although constitutive activation of
NF-
B efficiently induced promoter activity, it was not sufficient to
induce either ICAM-1 mRNA or ICAM-1 protein. Using signaling defective LMP1 mutants and deacetylation inhibitors, we showed that the
C-terminal activator region 1 of LMP1 delivers a new cooperating signal
to induce ICAM-1 mRNA.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B (5, 12), the mitogen-activated protein kinases,
JNK and p38, leading to activation of AP-1 and ATF-2 transcription
factors (13-15), and the JAK3/STAT1 pathway (16). LMP1 mimics a
ligand-independent, constitutive active receptor of the tumor necrosis
factor receptor (TNFR) superfamily by binding TNFR-associated factor
and TNFR-associated death domain (TRADD) to effect its signaling
functions (17, 18). The important regions of LMP1 are the so-called
C-terminal activator regions (CTAR-1, -2, and -3), which initiate the
signaling function of LMP1 by binding signaling molecules and which
need to cooperate together for optimal function (19). CTAR1 induces
NF-
B and is probably involved in the p38 pathway; CTAR2 signals
through NF-
B, the p38 pathway, and the JNK pathway; and CTAR3 binds
JAK3 (13, 16, 19).
, interleukin-1
(IL-1
), and
-interferon (
-IFN) (23,
24). Furthermore, certain viruses (e.g. rhinovirus,
respiratory syncytial virus, and EBV) are also known to up-regulate
ICAM-1 surface expression (11, 25, 26). With regard to EBV, the LMP1
protein is the major effector of ICAM-1 up-regulation (27). The
mechanism by which LMP1 effects up-regulation of ICAM-1 is poorly
understood, but studies on the other stimuli of ICAM-1 suggest that all
of the transcription factors activated by LMP1 are potential regulators of ICAM-1 transcription.
-IFN and TNF
have been shown to mediate ICAM-1 induction at the level of
transcription, using different signal transduction pathways and
specific activator sites (28-30). The interferon response element maps
to
76 bp to
66 bp, whereas the TNF
-responsive element (TRE)
ranges from
227 bp to
177 bp. The TRE was shown to be both
necessary and sufficient to induce ICAM-1 promoter activation by
TNF
, and it critically required the binding of the NF-
B family of
transcription factors, specifically p65 homodimers, to a variant
B-site (30). However, flanking sequences surrounding this
B
binding site are also required for transcription factor binding and
transactivation in TNF
-mediated induction of ICAM-1 (31). The ICAM-1
TRE also contains Sp-1 and C/EBP binding sites located upstream of the
modified NF-
B site, and binding of C/EBP homo- or heterodimers to
the C/EBP site was reported to be necessary for maximal induction of
ICAM-1 by TNF
(32).
B pathways. The importance
of these individual pathways for LMP1-induced ICAM-1 promoter and
protein expression at the cell surface was thus determined and revealed
a hitherto unrecognized function of LMP1 to activate ICAM-1 surface
expression. Finally, we will show that this novel function cooperates
with one of the C-terminal activator regions of LMP1 and is essential
for optimal ICAM-1 induction.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B elements upstream of a minimal conalbumin promoter driving the
expression of the firefly luciferase gene (36), and the
p(IL-6
B)3 plasmid, containing three copies of the IL-6
promoter
B site in front of a luciferase gene (37), were used to
assay NF-
B activity. Plasmid pSG5-LMP1 expresses wild-type LMP1
cloned from the B95.8 strain of EBV and has been described
previously (38). The LMP1 mutant CTAR1
plasmids replace
amino acids Pro208, Gln210, and
Thr212 with alanines, and the CTAR2
mutant
replaces Tyr384 with glycine; these mutants were described
elsewhere (14, 39). The green fluorescent protein expression plasmid,
pEGFP-C1, was purchased from CLONTECH, and the
pMEKK1-expressing plasmid was from BioLabs.
B
/GFP fusion protein vector was
generated by amplifying the I
B
gene from the pCMV I
B
N
plasmid (kindly provided from Dean W. Ballard, Howard Hughes Medical
Institute, Nashville, TN) using a forward 5'-primer, which binds to the
base pairs after the codon 36, and a 3'-primer lacking the stop codon of the protein. The purified PCR fragment was then cloned into the
BglII site of the pEGFP-C1 plasmid to generate plasmid
EGFP-I
B
DN. The hemagglutinin-tagged kinase vectors,
HA-p46SAPKy-pcDNA3 and HA-p38, and the dominant inhibitor SEKDN
vector (40) were provided by Aristides Eliopoulos (CRC Institute,
Birmingham, United Kingdom). The pyridinyl imidazole SB20380
(Calbiochem), a specific inhibitor of the p38 MAPK pathway, was
prepared as a 20 mM stock solution in dimethyl sulfoxide
and a JAK3 inhibitor (Calbiochem) was also prepared in dimethyl
sulfoxide as a 25 µg/ml stock solution. Both inhibitors were added to
the cultures at a dilution of 1/1000. The deacetylation inhibitor
sodium butyrate (Sigma) was used in a final concentration of 1 mM.
B and EGFP-I
BDN proteins were detected with 1 µg/ml rabbit
polyclonal antibodies to I
B
(Santa Cruz sc371) followed by an
alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad
170-6518). Specific antibody-protein complexes were detected using
CDP-StarTM (Tropix Inc.) development reagent.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Induction of cell surface ICAM-1 protein
expression by LMP1 in lymphocytes. A, Jurkat cells were
transfected with 2 µg of control vector (SG5) or an LMP1-expressing
vector (SG5-LMP1), together with pEGFP-C to mark the transfected cell
population. At 48 h after transfection, the ICAM-1 surface
expression in GFP-positive cells was measured by staining with
phycoerytherin-conjugated antibodies to CD54 and analyzing by flow
cytometry. The upper histogram shows the basal
ICAM-1 cell surface expression of vector control-transfected Jurkat
cells, and the lower histogram shows the induced
ICAM1 surface expression in Jurkat cells transfected with SG5-LMP1. The
mean fluorescence intensity (m.f.i.) is indicated in each
case. B, dose response of LMP1-induced up-regulation of
ICAM-1 in the Jurkat T cell line. Increasing amounts of SG5-LMP1
plasmid were transfected into Jurkat cells and the ICAM-1 expression
measured as in A. Data shown represent the mean values (± S.D.) of at least three independent experiments. C, dose
response of LMP1-induced up-regulation of ICAM-1 in the Eli-BL B cell
line, analyzed as for Jurkat in B. The results shown are the
mean (± S.D.) of at least three independent experiments.
receptor family
have been identified (30, 43). Only 1381 base pairs of the 5'-ICAM-1
gene region are required for the ICAM-1 induction by the
proinflammatory cytokines TNF
, Il-1
, and
-IFN. Fig.
2A shows a schematic structure
of the promoter region with the potential transcription factor binding
sites, and the characterized TNF
response region (TRE). The nuclear
transcription factors involved in the regulation of ICAM-1 promoter
include: Ap1, NF-
B, C/EBP, Ets, STAT, and Sp1. The locations of
their binding sites in the ICAM-1 5'-regulatory region are shown,
together with the mapped AP2 and AP3 sites, and the translational start
sites (CAT/TATA boxes).
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Fig. 2.
LMP1 activates the ICAM-1 promoter using the
TNF response region. A,
structure of the ICAM-1 gene full-length promoter ranging from the ATG
site in the ICAM-1 gene to
1353 bp of the 5'-region. The schematic
shows the individual transcription factor binding sites: AP1 (
277 and
1244), AP2 (
264 and
41), AP3 (
364), SP1 (
203 and
53), C/EBP
(
197), Ets (
153), NF-
B (
183 and
498), and STAT (
75). The
box indicates the TRE region (
227 to
178,
boxed) containing Sp1, C/EBP, and NF-
B sites.
B, the structure of the different ICAM-1 promoter luciferase
constructs and the luciferase-expressing vector (basic) are shown on
the left panel; the numbers given to
some constructs represent the approximate nucleotide position of the
5'-end relative to the translation initiation codon of the gene. The
TRE construct is deleted for 5' bases upstream of the TRE region (
227
bp), and the del TRE construct consists of the full-length promoter
deleted for the TRE region only. The lymphocyte cell lines Eli-BL
(black columns) and Jurkat (white
columns) were transfected with the ICAM-1 promoter
luciferase constructs together with SG5-LMP1 or the SG5 control vector.
The luciferase activity was measured at 24 h after transfection,
and is expressed as -fold induction relative to the SG5 vector control.
The results shown are representative of seven independent
experiments.
to activate the ICAM-1 promoter.
on the ICAM-1
Promoter--
Since the TRE has been shown to be essential and
sufficient for TNF
-induced up-regulation of ICAM-1 (30), we tested
mutant reporters with or without this region to investigate the
similarities between LMP1 and TNF
. We analyzed the luciferase
activity of 1.3ICAM1, TRE-ICAM1, and delTRE-ICAM1 (see Fig.
2A) together with a full-length reporter in which the
variant
B-site in the TRE had been inactivated by point mutation
(NF-
Bneg-ICAM1). In one representative experiment shown in Fig.
3, cells transfected with each of these
reporters were cotransfected either with SG5-LMP1 or with SG5 vector;
the SG5 vector transfectants were then treated with TNF
at 12 h
after transfection and for 6 h prior to harvesting. The results in
Fig. 3 show that LMP1 induced the TRE-ICAM1-luc reporter only to 58%
of the full-length promoter activity, whereas TNF
stimulated the
TRE-reporter to 140% of the full-length promoter activity. The reduced
inducibility of the TRE-ICAM-1 reporter by LMP1 suggests that, in
contrast to TNF
stimulation, the TRE region is not sufficient for
optimal induction of ICAM-1 by LMP1.
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Fig. 3.
Comparison LMP1- and
TNF -induced ICAM-1 promoter activity.
Jurkat cells were transfected either with the basic luciferase
reporter, with a reporter containing the full-length ICAM-1 promoter
(1.3ICAM-1-luc), or with reporters affecting the TRE region (TRE-luc,
delTRE-luc, and
Bneg-ICAM-1-luc). One set of transfectants was
cotransfected with SG5-LMP1 (left panel), whereas
a parallel set was cotransfected with SG5 vector and was stimulated
with 100 units/ml TNF
(right panel), which was
added to the cultures at 12 h after transfection and for a time
period of 6 h. The cells were analyzed for luciferase activity at
18 h after transfection. The results are expressed relative to the
activity of the full-length promoter reporter, which was designated as
100%. The results are representative of three separate
experiments.
. Within this region, the variant NF-
B motif has been reported
to be critical for transcriptional activation of ICAM-1 by TNF
(30),
and we therefore tested the importance of this site for LMP1-induced
ICAM-1 promoter activation. Using the
B-negative mutant ICAM1
reporter, we always observed an increased background promoter activity
of about 2-fold above the basic luciferase reporter activity, which
suggests that this functional
B site regulates basal ICAM-1 levels
as well as the inducibility. However, the
B-negative reporter was
not induced further either by LMP1 or by TNF
(Fig. 3). Taken
together, the results in Fig. 3 indicate that LMP1 differs from TNF
in utilizing regions upstream of the TRE to maximize the inducibility
achieved from the TRE, and that within the TRE the variant
B site is
critical for induction both by LMP1 and by TNF
.
B Is the Major Event in LMP1-induced
Up-regulation of ICAM-1--
We wanted to further investigate the
importance of the LMP1-induced NF-
B pathway in regulating ICAM-1.
Therefore, we examined the effect of the NF-
B inhibitor protein,
I
B
, upon LMP1-induced activation of the ICAM-1 promoter reporter
and up-regulation of cell surface ICAM-1 protein. To enable endogenous
and transfected I
B
to be distinguished, we designed a
constitutive active I
B
that was deleted for the first 36 amino
acids (thus removing the two regulatory phosphorylation sites) and
fused to GFP (EGFP-I
B
DN; see Fig.
4A). Expression of this
construct following transfection into Jurkat cells was determined by
immunoblotting with a rabbit anti-I
B
antibody (Fig.
4B). In Jurkat cell extracts, this antibody always detected
endogenous I
B
as low molecular mass bands, between 39 and 44 kDa,
and the transfected EGFP-I
B
DN as a higher molecular mass band,
between 68 and 70 kDa, of similar intensity to the endogenous I
B
.
The inhibitory function of EGFP-I
B
DN on LMP1-induced NF-
B
signaling was analyzed in reporter assays using the
B-dependent luciferase reporters 3Enh-luc and
IL-6(
B)3-luc, which contain triple repeats of
B
response elements from the Ig
promoter and the IL-6 promoter,
respectively. EGFP-I
B
DN was fully functional, being able to
inhibit LMP1-induced activation of the 3Enh-luc reporter by 93%, and
the IL-6(
B)3-luc reporter by 94% (Fig.
4C).
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Fig. 4.
Design and functional analysis of a dominant
negative form of
I B
.
A, schematic representation of the structure of the I
B
wild type gene (1-317 amino acids) with the two phosphorylation sites
at amino acid positions 32 and 36 and the typical ankyrin repeats
marked as black boxes. The dominant active form
of I
B
was created by deletion of the first 36 amino acids of the
gene and ligating to the C terminus of the EGFP gene to create the
fusion protein, EGFP-I
B
DN. B, Western blot showing
expression of the EGFP-I
B
DN fusion protein in Jurkat cells.
Lane 1 contains protein from 2 × 105 mock
Jurkat transfectants, and lane 2 contains protein
from Jurkat cells transfected with 1 µg of EGFP-I
B
DN plasmid;
protein extracts were prepared at 24 h after transfection. Western
blots were probed with a rabbit polyclonal antibody to the C terminus
of I
B
. The endogenous I
B
was detected in both samples,
whereas lane 2 also shows the expression of
transfected EGFP-I
B
DN fusion protein with an expected molecular
mass of ~69 kDa. C, Jurkat cells were transfected with 3 µg of SG5-LMP without (black columns) or with
(gray columns) 0.5 µg of EGFP-I
B
DN, and
with 3 µg of one of two different NF-
B-dependent
luciferase reporters, 3Enh-luc or Il6(-
B)3-luc. Cells
were assayed for luciferase activity at 24 h after transfection.
The results of reporter induction are expressed as -fold induction
relative to vector control. The numbers over the
gray columns denote the inhibition caused by
EGFP-I
B
DN for each reporter. Results shown are the mean of six
separate experiments.
B
DN as an inhibitor
of the LMP1-induced NF-
B pathway, we investigated its effect on
LMP1-induced ICAM-1 up-regulation. Increasing amounts of LMP1 with or
without EGFP-I
B
DN were transfected into Jurkat lymphocytes, and
the effects upon luciferase activity of the full-length 1.3ICAM-1 promoter reporter (Fig. 5A) as
well as ICAM-1 protein expression (Fig. 5B) were measured.
The results show that LMP1-induced ICAM-1 promoter activity was
completely inhibited by EGFP-I
B
DN at low doses of SG5-LMP1 (
1
µg), and was inhibited by about 80% at the highest input doses (2 and 4 µg) of SG5-LMP1. It should be noted that the constitutive SV40
promoter of the SG5-LMP1 plasmid itself is not completely unaffected by
EGFP-I
B
DN. However, although EGFP-I
B
DN reduced LMP1
expression by up to 50% at the lowest doses of SG5-LMP1, at the higher
doses, there was no significant effect on LMP1 expression (data not
shown). The flow cytometry analysis of the cell surface ICAM-1 protein
expression in the same transfected cell population revealed that LMP1
induction of ICAM-1 protein is completely abolished in the presence of
the NF-
B inhibitor. These experiments were also performed with the Eli-BL B cell line and showed similar results (data not shown).
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Fig. 5.
Effect of
EGFP-I B
DN upon
LMP1-induced ICAM-1 promoter activity and protein expression.
Jurkat cells were transfected with 3 µg of the full-length ICAM-1
promoter luciferase reporter (1.3ICAM-1) and increasing amounts of
SG5-LMP1 DNA without (filled symbols) and with
(hollow symbols) 0.5 µg of EGFP-I
B
DN
plasmid. Aliquots of cells were assayed for luciferase activity at
24 h and for cell surface ICAM-1 protein expression at 48 h
after transfection. The upper graph
(A) shows the relative luciferase activity, and the
lower graph (B) shows the relative
number of ICAM-1-positive cells. The data shown are the mean (± S.D.)
of three independent experiments.
B Is Essential but Not Sufficient for ICAM-1
Induction--
For confirmation that NF-
B is the major activator of
ICAM-1 expression, we tested the effects of a constitutive activated form of MEKK1, a potent inducer of the NF-
B pathway. The active kinase expression plasmid, when transfected into Jurkat cells, was
shown to induce the 3Enh-luc NF-
B dependent luciferase reporter by
110-fold (data not shown). Therefore, we transfected MEKK1 with and
without SG5-LMP1 into Jurkat cells and measured its effect on the
ICAM-1 reporter activation as well as on the expression of cell surface
ICAM-1 protein. In the representative experiment shown in Fig.
6, MEKK1 induced the ICAM-1 full-length
promoter (1.3ICAM-1) 465-fold over background level, which is 63 times higher than the LMP1-induced reporter luciferase activity (Fig. 6A). In contrast, flow cytometry analysis of cell surface
ICAM1 protein expression (Fig. 6B) revealed that, although
the cells transfected with LMP1 showed a substantial 10-fold increase
in ICAM-1 mean fluorescence intensity, the cells transfected with MEKK1
alone showed no induction of ICAM-1 expression. Cotransfection of LMP1
and MEKK1 showed that the MEKK1did not interfere with the ability of
LMP1 to up-regulate ICAM-1 protein (Fig. 6B). These results
show that NF-
B activation alone is sufficient for activation of the
ICAM-1 reporter, but is not sufficient to effect up-regulation of
ICAM-1 protein. This suggests that other signaling pathways of LMP1, in
addition to NF-
B, are required to up-regulate ICAM-1 protein
expression.
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Fig. 6.
Effects of MEKK1-induced
NF- B on ICAM-1 promoter activity and protein
expression. Jurkat cells were transiently transfected with 3 µg
of control vector (SG5) or SG5-LMP1 plasmid, with and without 1 µg of
MEKK1 plasmid DNA. After 24 and 48 h, aliquots of cells were
assayed for luciferase activity (A) and ICAM-1 surface
expression (B), respectively. Luciferase activity is
expressed as -fold induction relative to control values. ICAM-1 protein
expression was measured by flow cytometry, and the data shown are the
-fold increase of mean fluorescence intensity over control. Results
shown are the mean values (± S.D.) of three independent
experiments.
B, the
known LMP1-induced signaling pathways include the JNK pathway, which
leads to activation of the c-Jun transcription activator; the p38
pathway, resulting in ATF2 translocation; and a JAK3/STAT pathway. The
importance of these individual pathways in LMP1-mediated up-regulation
of ICAM-1 was analyzed using specific inhibitors. To inhibit the JNK
pathway, we used SEKDN, a dominant negative form of JNK kinase that is deleted for the phosphorylation site and is therefore unable to phosphorylate the Jun-activating kinase, JNK (13). To inhibit the p38
pathway, we used a chemical inhibitor SB203580, which specifically
blocks p38 phosphorylation and translocation without affecting other
signaling pathways (44). A chemical inhibitor was also used to
specifically inhibit JAK3 (45). These inhibitors were all shown to
affect their respective targets without affecting the expression of
LMP1 from the SG5-LMP1 vector (Fig.
7A and data not shown). The
inhibition of LMP1-mediated JNK activation with SEKDN was less
efficient than was the inhibition of other pathways (Fig.
7A), but the 60-70% inhibition was similar to that
previously reported by other workers (40). Having confirmed the
functionality of the inhibitors, we analyzed their effects on
LMP1-induced ICAM-1 protein (Fig. 7B). As a positive control
for inhibition of ICAM-1 induction, we cotransfected EGFP-I
B
DN
together with SG5-LMP1 in Jurkat cells. The results shown demonstrate
that, although LMP1-induced up-regulation of ICAM-1 protein was
completely inhibited by EGFP-I
B
DN, the other inhibitors tested
did not affect the up-regulation of ICAM-1 protein.
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Fig. 7.
Effects of inhibitors of LMP1-induced
signaling. A, kinase assays were carried out to confirm
the functionality of the Jun and p38 pathway inhibitors, SEKDN and
PB203580. Jurkat cells were transfected with control vector (SG5) and
SG5-LMP1 in the presence and absence of the inhibitors. At 48 h
after transfection, immunocomplex kinase assays were carried out to
measure kinase activity using GST-Jun-(1-79) (left
panel) or GST-ATF2-(19-96) (right
panel) as substrates. Three independent experiments were
performed and gave similar results. B, Jurkat cells were
transfected with EGFP-C1 and different amounts of SG5-LMP1 DNA with and
without dominant negative SEK (SEKDN) or EGFP-I B
DN plasmids or
were treated with 20 µM p38 inhibitor SB203580 or 25 µg/ml JAK3 (Calbiochem) inhibitor as described under "Materials and
Methods." After 48 h, GFP-positive transfected cells were
assayed by flow cytometry for ICAM-1 surface expression. The results
are shown as ICAM-1 mean fluorescence intensity and are representative
of three independent experiments.
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Fig. 8.
RPA analysis of ICAM-1 mRNA levels in
transfected DG75 cells. DG75 cells were transfected with rCD2GFP
and either control vector or LMP1- or MEKK1-expressing plasmids. After
48 h, the cells were immunomagnetically sorted for CD2 expression.
The RPA was performed on total RNA samples prepared from those positive
sorted cells. The upper panel shows a
representative RPA autoradiogram (8-h exposure) of ICAM-1 mRNA
levels in the individual samples indicated above each
track. Levels of ICAM-1 mRNA were quantitated on a
phosphorimager. The data shown in the lower panel
represent ICAM-1 mRNA levels relative to the housekeeping gene
L32 mRNA levels. Three independent experiments were
performed and gave similar results (S.D. for control mRNA levels
was less than 10%).
B. The full nature of
these signal remains elusive. In the case of LMP1 signaling, the
cooperation of the C-terminal activator regions CTAR1 and CTAR2 has
been shown to be essential for LMP1 function (19). Furthermore, histone acetylation of genes and their promoters was identified to be involved
in coordinate regulation of transcription (46). We wanted to test if
these mechanisms could also be important for LMP1-induced ICAM-1
up-regulation. Therefore, we investigated two point mutants of LMP1,
defective for either one (CTAR1
) or the other
(CTAR2
) C-terminal activator region and MEKK1 in the
presence of a chemical compound that has been shown to inhibit histone
deacetylation and thus prevent gene silencing. Eli-BL cells were
transfected with control or LMP1-, CTAR1
-,
CTAR2
-, and MEKK1-expressing plasmid together with
EGFP-C1 to control for transfection. After 24 h the cells were
stimulated with sodium butyrate, and, after an additional 12-h
incubation, the ICAM-1 surface expression was analyzed using flow
cytometry. The ICAM-1 surface expression of these transfected cells is
shown in Fig. 9 as mean values of three
independent experiments. LMP1 induces ICAM-1 expression as seen before
(column 2 compared with column 1). The CTAR1
mutant (column
3), which has intact CTAR2 and CTAR3 domains, induced only
half of the ICAM-1 protein compared with LMP1, and the
CTAR2
mutant with intact CTAR1 and CTAR3 induced even
less (column 5). However, after incubation with
the deacetylation inhibitor sodium butyrate, only the
CTAR1
-transfected cells showed ICAM-1 expression
comparable to wild type LMP1 levels (column 4 compared with column 2), whereas the ICAM-1
levels of the CTAR2
transfected cells remained low. In
the case of the MEKK1-transfected cells, the ICAM-1 expression levels
showed no significant difference between cells treated with or without
the deacetylation inhibitor. The ICAM-1 surface expression in
MEKK1-transfected cells remained at basal levels (columns
7 and 8).
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Fig. 9.
Effects of deacetylase inhibitor on induced
ICAM-1 protein expression. Analysis of ICAM-1 surface expression
in Eli-BL cells transfected with EGFP-C1 marker plasmid and either
control plasmid, LMP1, LMP1 mutants (CTAR1 mutated at
amino acids 204, 206, and 208; CTAR2
construct mutated at
amino acid 384), or MEKK1-expressing plasmid. After 24 h cells
were treated with sodium butyrate (NaB, 1 mM).
ICAM-1 protein expression in GFP-positive cells was assayed by flow
cytometry after an additional 12-h incubation. Bars
represent the average (± S.D.) of the ICAM-1 mean fluorescence
intensity (×1000) in three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
-IFN (9, 16-18), both of which transcriptionally regulate ICAM-1
expression through distinct regulatory elements in the ICAM-1 promoter.
Our results show that LMP1 acts primarily through the TRE located at
178 bp to
227 bp, but not the interferon response element located
at
76 bp to
66 bp. However, LMP1 and TNF
are subtly different in
their regulation of ICAM-1 (Fig. 3), which highlights the complex range
of signaling pathways and transcription factors involved in ICAM-1
activation. Consistent with this observation, although a similar range
of signal transduction factors are known to associate with LMP1 and
TNFR1, and an overlapping spectrum of signaling pathways are activated,
there are important differences. For example, the death domain at the C
terminus of TRADD is involved in TNFR1/TRADD interaction, whereas LMP1
recruits TRADD via its N-terminal domain, and the different topology of TRADD binding affects the mechanisms by which LMP1 and TNFR1 activate NF-
B and JNK (47, 48).
signaling (Fig. 3) showed that
the TRE is both essential and sufficient for TNF
-induced ICAM-1, as
reported previously (30); in contrast, this site was essential but not
sufficient for optimal induction by LMP1 (Figs. 2 and 3). Taken
together, these data suggest that LMP1 might differ from TNF
by
targeting additional transcription activation site(s) located upstream
of the TRE between
574 bp and
1381 bp. In this respect, potential
sites include an AP1 binding site at position
1284 and a second
NF-
B site at position
531. Other upstream regulatory enhancer
elements have also been described, which are thought to be more
important for constitutive ICAM-1 expression rather than for inducible
ICAM-1 (29), although it is possible that they have a role in LMP1
induction of ICAM-1.
B binding site, which is essential for transcriptional up-regulation of
ICAM-1 mediated by LMP1 (Fig. 3), TNF
(30), and other cytokines (24). The critical role for NF-
B in LMP1-mediated up-regulation of
ICAM-1, suggested by our analysis with mutant reporters, was supported
by complementary experiments showing that a constitutively active
I
B
efficiently abolished LMP1-mediated up-regulation both of the
full-length 1.3ICAM-1 reporter and expression of ICAM-1 protein (Fig.
5). Our results shed new light on an area of confusion since two
previous studies have reported the use of I
B
to inhibit the
ability of LMP1 to up-regulate ICAM-1 protein expression, but there was
disagreement about the efficiency of this effect (49, 50). Our data, in
line with those of Devergne and colleagues (50), show that efficient
blocking of NF-
B activation can completely abrogate the ability of
LMP1 to up-regulate ICAM-1 protein.
B, it is clear that activation of
this transcription factor alone is unable to induce ICAM-1 protein
expression. Thus, we have observed previously that transfection and
overexpression of p50 and p65 NF-
B species has no effect upon ICAM-1
protein expression in lymphoid
cells.2 We now show that
transfection of active MEKK1, whose effects include activation of
NF-
B besides AP-1 and p38 activation (51, 52), is able to activate
ICAM-1 transcription more efficiently than does LMP1 but does not
affect ICAM-1 protein expression (Fig. 6). These results implicate
other LMP1-activated signal pathways in up-regulating ICAM-1 protein.
However, our experiments with specific inhibitors mitigate against a
role for the Jun/AP-1, p38/ATF-2, and JAK3/STAT pathways (Fig. 7). It
could be argued that, because the inhibition of the JNK/AP-1 pathway by
the dominant-negative SEKDN molecule was not as efficient as the other
inhibitors used in this study (Fig. 7), it is possible that the
residual LMP1-induced AP-1 activation is sufficient to cooperate with
NF-
B to up-regulate ICAM-1 protein. However, this is contradicted by
the observations (i) that the SEKDN inhibitor did not even partially
inhibit up-regulation of ICAM-1 (Fig. 7), and (ii) that MEKK1, which
activates both AP-1 and NF-
B, was unable to up-regulate ICAM-1
protein. With regard to the lack of affect of SB203580 inhibition of
p38 on LMP1-induced ICAM-1, it is noteworthy that this inhibitor has been shown to abrogate two other biological functions of LMP1, the
induction of IL-6 and IL-8 (40). During the preparation of this report,
the activation of Cdc42 (a small GTPase) in fibroblasts was identified
as yet another signaling function for LMP1 (53). However, this function
was reported to be mediated by the transmembrane domains of LMP1 (53),
whereas activation of ICAM-1 requires only domains on the C-terminal
cytosolic regions of LMP1 (19). Therefore, we are drawn to conclude
that another, as yet uncharacterized, signaling pathway(s) cooperates
with NF-
B to control ICAM-1 regulation by LMP1.
B to activate the ICAM-1 reporter but not ICAM-1
protein expression (Fig. 6) is intriguing. There are a number of
possible factors that could be involved, including mRNA stabilization, promoter accessibility regulated by nucleosomes, and
various post-transcriptional mechanisms. With regard to mRNA stability, both phorbol esters and
-IFN have been shown to stabilize the otherwise labile ICAM-1 mRNA in murine fibroblast and monocytic cell lines (54, 55). Furthermore, stabilization of mRNA has been
shown to be a feature of LMP1-mediated up-regulation of Bfl-1 in
lymphocytes by as yet unknown mechanisms (8). However, using the same
human lymphoid cell model (BJAB cells transfected with a
tetracycline-regulated LMP1 expression vector) that was used to
demonstrate Bfl-1 mRNA stabilization, we found that LMP1 does not
affect the stability of ICAM-1 mRNA (data not shown).
B was the only known signaling function of LMP1
that was shown to be critical for induction of ICAM-1 protein by LMP1
(Fig. 7), sodium butyrate was unable to cooperate with the NF-
B
activated by the LMP1-CTAR2
mutant or by MEKK1 (Fig. 9).
There are several possible explanations for this; one being that there
is yet another unknown signaling function of LMP1. The precise analysis
of these new features of LMP1 signaling is ongoing.
B is a key
signaling pathway stimulated by LMP1 in the transcriptional regulation
of ICAM-1, and that the other known signaling pathways activated by
LMP1 appear not to be critical for up-regulation of ICAM-1. However,
NF-
B activation is not sufficient by itself to up-regulate ICAM-1
protein. We described a new cooperating signaling mechanism induced by
LMP1, which may act at the level of promoter accessibility and maps to
the CTAR1 domain of LMP1. Further characterization of this pathway and
the detailed analysis of the signal-regulated acetylation event and
gene transcription are the next critical steps toward our understanding
of LMP1 function.
![]() |
FOOTNOTES |
---|
* This work was supported by EC BioMed-2 Program Grant BMH4-97-2567, by Leukaemia Research Fund (London) Grants 9533 and 9842, and by a grant from the Wellcome Trust.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.
Present address: Dept. of Biochemistry, Astra-Zeneca, Loughborough
LE11 5RH, United Kingdom.
§ To whom correspondence should be addressed. Tel.: 44-2920-742579; Fax: 44-2920-743868; E-mail: rowem@cf.ac.uk.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M003758200
2 M. Rowe, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
LMP1, latent
membrane protein-1;
EBV, Epstein-Barr virus;
CTAR, C-terminal activator
region;
ICAM-1, intercellular adhesion molecule-1;
NF-B, nuclear factor
B;
TNF
, tumor necrosis factor
;
I
B, inhibitor of NF-
B;
JNK, Jun N-terminal kinase;
JAK3, Janus-activating tyrosine kinase 3;
STAT1, signal transducing and
transcription factor-1;
TNFR, tumor necrosis factor receptor;
TRADD, TNFR-associated death domain;
IL, interleukin;
-IFN,
-interferon;
TRE, TNF
-responsive element;
EGFP, enhanced green fluorescent
protein;
HA, hemagglutinin-tagged;
SAPK, stress-activated protein
kinase;
MEKK, mitogen-activated protein/extracellular signal-regulated
kinase-activating kinase kinase;
bp, base pair(s);
GFP, green
fluorescent protein;
GST, glutathione S-transferase;
C/EBP, CAAT/enhancer-binding protein.
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REFERENCES |
---|
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