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Journal of Virology, July 2000, p. 5810-5818, Vol. 74, No. 13
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Transforming Growth Factor Beta 1 Stimulates
Expression of the Epstein-Barr Virus BZLF1 Immediate-Early Gene
Product ZEBRA by an Indirect Mechanism Which Requires the MAPK
Kinase Pathway
Hassan
Fahmi,1,2
Chantal
Cochet,1
Zakariae
Hmama,2
Paule
Opolon,3 and
Irene
Joab1,*
Laboratory of Immunology, Faculty of Science,
Université Sidi Mohamed Ben Abdellah, Fès,
Morocco,2 and Laboratoire de
Pharmacologie Expérimentale et Clinique, INSERM EPI 99-32,
Institut de Génétique Moléculaire, 75010 Paris,1 and CNRS UMR 1582, Institut
Gustave Roussy, 94800 Villejuif,3 France
Received 9 December 1999/Accepted 4 April 2000
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ABSTRACT |
Disruption of Epstein-Barr virus (EBV) latency is mediated by
ZEBRA, the protein product of the immediate-early EBV gene, BZLF1. In
vitro, phorbol 12-myristate 13-acetate (PMA), a potent activator of
protein kinase C (PKC), induces reactivation of EBV. However, the
physiological stimuli responsible for the disruption of viral latency
are not well characterized. Transforming growth factor beta 1 (TGF-
1) has also been shown to trigger the reactivation of EBV in
Burkitt lymphoma cell lines; however, the effect of TGF-1 on ZEBRA
expression has not been reported. To further understand this
phenomenon, we have investigated the effect of TGF-1 on ZEBRA
expression. Our results indicate that the treatment of different EBV-positive Burkitt's lymphoma cell lines with TGF-1 induces a
time-dependent activation of BZLF1 transcription with a corresponding increase in the production of the protein ZEBRA. TGF-1 has been shown to exert its effects through a wide range of intracellular routes; in the present study, we have explored these pathways. Transient expression of Smad proteins on their own had no effect on
ZEBRA expression. A specific inhibitor of p38 mitogen-activated protein
kinase (MAPK), SB203580, did not affect TGF-1-induced ZEBRA
expression, whereas treatment with the MAPK/ERK kinase inhibitors, PD98059 and U0126, dramatically decreased this induction. This suggests
that TGF-1 effect on BZLF1 expression requires the MAPK pathway.
However, in Raji and B95-8 cells additional routes can be used, as (i)
the inhibition of ZEBRA induction by PD98059 or U0126 was incomplete,
whereas these inhibitors completely abolished PMA-induced ZEBRA
expression, (ii) TGF-1 induction of ZEBRA expression occurs in
PKC-depleted cells, (iii) in Raji and in B95-8 cells, the effect of
TGF-1 and PMA are additive. Transient transfection of the
EBV-negative B-cell line DG75 with a BZLF1 promoter-fusion construct
(Zp-CAT) showed that under conditions where the BZLF1 promoter is
activated by PMA treatment, TGF-1 had no significant effect on the
expression of the chloramphenicol acetyltransferase gene. Furthermore,
TGF-1 induction of BZLF1 transcripts is dependent on de novo protein
synthesis, which suggests that TGF-1 induces BZLF1 expression by an
indirect mechanism.
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INTRODUCTION |
Epstein-Barr virus (EBV), the
causative agent of infectious mononucleosis, is associated with a
growing number of malignant diseases, which include nasopharyngeal
carcinoma, African Burkitt lymphoma (BL), Hodgkin's disease,
non-Hodgkin's lymphoma in immunocompromised individuals
(48), and peripheral T-cell lymphoma (54). In vitro, EBV infection of human B lymphocytes results in the
immortalization of these cells with the virus maintained in a latent
state, expressing a minimum of six nuclear (EBNA 1, 2, 3A, 3B, 3C, and
EBNA LP) and three latent membrane (LMP1, LMP2A, and LMP2B) proteins.
EBV activation from latency is initiated by the expression of the BZLF1
gene product ZEBRA, also known as EB1 and Zta (11, 12).
ZEBRA shares partial amino acid homology to a region in the product of
the cellular proto-oncogene, c-fos. ZEBRA transactivates various EBV promoters through binding to AP-1-like sites and cyclic AMP
(cAMP)-responsive element consensus sequences (9, 21, 40,
58). The BZLF1 transcripts are derived from either one of two
promoters, Zp and Rp, as a 1-kb monocistronic or a 3-kb bicistronic
mRNA, respectively, (41). The more proximal promoter, Zp,
contains elements responsive to phorbol esters and anti-immunoglobulin G (anti-IgG) (7, 15, 22).
It has been reported that EBV can be reactivated in immunocompromised
hosts, e.g., organ-transplanted and AIDS patients (6, 62).
In such hosts, reactivation leads to increased susceptibility to
development of EBV-positive non-Hodgkin's-type B-cell lymphoma (27-29). In addition, the reactivation of EBV in
nasopharyngeal carcinoma has also been reported (42).
However, the factors responsible for the reactivation of the virus in
vivo are not known. To further our understanding of EBV reactivation,
it is essential to identify the physiological stimuli and to determine the mechanism(s) leading to this phenomenon. In vitro, reactivation of
the lytic cycle in latently infected B cells can be achieved by
treatment with various agents, such as phorbol 12-myristate 13-acetate
(PMA), Ca2+ ionophore, anti-IgG, human herpesvirus 6 infection, and transforming growth factor beta 1 (TGF-1) (5,
10, 17, 20, 56, 64).
TGF-1 regulates a wide range of physiological and pathological
cellular processes, including differentiation, immune response, inflammation, extracellular matrix synthesis, angiogenesis, and wound
healing in humans (39). Different studies have reported that
TGF-1 regulates the expression of various genes. However, the
signal-transducing mechanism of the cytokine is not completely understood. TGF-1 has been shown to exert its effects through a wide
range of intracellular routes. Recent studies from several laboratories
reported that Smads are intermediate effector proteins that transduce
the TGF-1 signal from the plasma membrane to the nucleus (14,
30, 43). TGF-1 can induce gene expression via c-Jun N-terminal
kinase (JNK) activation (3, 31, 60) or p38 mitogen-activated
protein kinase (MAPK) (1, 26). The role of MAPK/ERK in the
TGF-1 signaling pathway has also been described (4, 61).
Protein kinase C (PKC) has also been shown to be involved in PMA- and
anti-IgG-induced EBV reactivation (13, 15) and could also
play an important role in the signal transduction by TGF-1 (25,
50, 53).
This study was undertaken to elaborate on the role of TGF-1 in EBV
reactivation. As ZEBRA expression is a key step in the switch from
latency to lytic cycle, this study focused on the expression of this transactivator.
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MATERIALS AND METHODS |
Cell culture and reagents.
The EBV-positive B-cell lines
Daudi, P3HR1, Raji, B95-8, and Mutu I and the EBV-negative BL cell line
DG75 were maintained in RPMI 1640 supplemented with 100 UI of
penicillin/ml, 100 µg of streptomycin/ml, and 10% heat-inactivated
fetal calf serum (GIBCO BRL).
Purified recombinant TGF-1 was purchased from R&D Systems
(Minneapolis, Minn.), PMA, anisomycin, and cycloheximide were from Sigma Chemical Co. (St. Louis, Mo.),
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7),
bisindolylmaleimide (GF-109203X) [GFX], HA-1004, PD98059, and
SB203580 were from Alexis (San Diego, Calif.), and U0126 was from
Promega (Madison, Wis.).
Western blot analysis.
Cells were harvested, washed briefly
with phosphate-buffered saline, resuspended in a buffer composed of 100 mM Tris-Cl (pH 7.6), 50 mM NaCl, 2 mM EDTA, 0.5% NP-40,
phenylmethylsulfonyl fluoride (100 µg/ml), and 1 µg each of
leupeptin, pepstatin and aprotinin per ml, and sonicated; protein
concentrations were determined by the Bradford assay. Equal amounts of
protein in loading buffer, heated for 5 min at 100°C and separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on
10% gels were transferred by electroblotting to a nitrocellulose
membrane (Schleicher & Schuell, Ecquevilly, France). The membrane was
stained with Ponceau S sodium salt (Sigma) to verify that the same
amount of protein was deposited in each lane. Anti-ZEBRA monoclonal
antibodies (MAbs) Z125 and Z130 (E. Drouet, Faculté de Pharmacie,
Grenoble, France), were used as primary antibodies, and horseradish
peroxidase-conjugated IgG (Interchim, Montluçon, France) was used
as the secondary antibody; blots were developed by enhanced
chemiluminescence (Interchim).
Immunohistology.
Frozen cytospun slides were air dried,
fixed in acetone for 10 min, and air dried. Endogenous peroxidases were
quenched with H2O2 (0.3% in methanol) for 15 min. Slides were immersed in washing buffer (WB; BioGenex, San Ramon,
Calif.) and placed in coverplates (Shandon) filled with Power Block
universal blocking reagent (1/10 dilution; BioGenex) for 10 min.
Primary MAb Z130 was applied to the slides for 1 h. The slides
were washed twice with WB and incubated for 30 min at room temperature
with a biotinylated rabbit anti-mouse secondary antibody (1/200
dilution; Dako, Copenhagen, Denmark). After two 3-min washes with WB,
slides were incubated for 30 min with streptavidin peroxidase (1/20
dilution; Vector Laboratories). Following two 3-min washes, the
chromogen 3-amino-9-ethylcarbazole (Sigma) was added for 5 min. After
washing in WB, slides were counterstained with Mayer's hematoxylin and
mounted in aqueous medium (gel mount Microm).
RNA isolation and Northern blot analysis.
Total RNA was
isolated on a Qiagen (Courtaboeuf, France) column according to the
manufacturer's instructions. Poly(A)+ RNA was isolated
using oligo(dT)-cellulose as instructed by the supplier (Pharmacia,
Courtaboeuf, France). Poly(A)+ RNA was separated by
electrophoresis through a 1% agarose-formaldehyde gel. The RNA was
transferred onto a nylon membrane (Hybond N; Amersham, Courtaboeuf,
France), and blots were prehybridized at 42°C for 3 h in 50%
formamide-5× SSPE (1× SSPE is 0.18 M NaCl, 10 mM
NaH2PO4, and 1 mM EDTA [pH 7.7])-5×
Denhardt's solution-0.5% SDS-100 µg of denatured salmon sperm
DNA/ml. The membranes were probed successively with random-primed
[32P]dCTP-labeled BZLF1 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNAs (106 cpm/ml) as recommended
by the manufacturer (Promega, Charbonnieres, France). Following
overnight incubation at 42°C, membranes were washed twice for 15 min
with 2× SSPE-0.1% SDS at room temperature, twice with 2×
SSPE-0.1% SDS at 65°C, and once with 0.5× SSPE-0.1% SDS at
65°C (10 min).
Plasmids, transfection, and CAT assays.
Plasmid 
221Zp-CAT,
generously provided by A. Sergeant (ENS, Lyon, France), contains BZLF1
promoter bp 221 to +12, relative to the transcription initiation
site, cloned upstream of the bacterial chloramphenicol
acetyltransferase (CAT) reporter gene. The Ia1 germ line
reporter construct, pAI-D-CAT, was kindly provided by P. Sideras, Umeå
University, Umeå Sweden (37). The latter plasmid was used
as a positive control of TGF-1 response. Plasmids containing Smad2,
Smad3, Smad4, and Smad7 coding sequences driven by the cytomegalovirus
promoter were kindly provided by Peter ten Dijke, Ludwig Institute for
Cancer Research, Uppsala, Sweden.
Plasmid DNA (10 µg) purified on two CsCl
2 density
gradients was mixed with 10
7 cells in 500 µl of RPMI
1640. The cells were exposed to a single
pulse at 230 V and 960 µF
(Bio-Rad, Richmond, Calif.). The transfected
cells were resuspended in
10 ml of complete culture medium, and
TGF-
1 or PMA was added
immediately following transfection. Cells
were harvested 48 h
later, washed with phosphate-buffered saline,
suspended in 100 µl of
25 mM Tris-HCl (pH 7.5), and frozen in
liquid nitrogen. Cells were
disrupted by three freeze-thaw cycles
in liquid nitrogen and 37°C.
Cell debris was removed by centrifugation
at 12,000 rpm for 10 min, and
protein concentration was determined
by the Bradford assay. For CAT
assays, equal amounts of protein
were incubated at 37°C with
[
14C]chloramphenicol in the presence of acetyl coenzyme A
as described
previously (
23) or in the presence of
N-butyryl coenzyme A (
52).
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RESULTS |
TGF-1 induces ZEBRA expression in EBV-infected BL cells.
To
determine whether TGF-1 treatment could induce ZEBRA expression,
EBV-positive B-cell lines B95-8, Raji, and Mutu I were cultured
overnight in absence or in presence of TGF-1 (1 or 5 ng/ml), and
ZEBRA expression was determined by immunoblotting. TGF-1 induced
ZEBRA expression in the cell lines tested (Raji, P3HR1, Daudi, B95-8,
and Mutu I) (Fig. 1). Similar results
were obtained whether cells were cultured in the presence or absence of
serum, and no expression of ZEBRA was observed in control cells treated
with the vehicle alone (data not shown).
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FIG. 1.
TGF-1-induced ZEBRA expression in EBV-infected BL
cells. Cells were incubated in absence or in presence of TGF-1 (5 ng/ml) for 18 h. Cells were lysed, and equal amounts of protein
were separated by SDS-PAGE and Western blotted with anti-ZEBRA
antibodies as described in Materials and Methods. The arrow indicates
the position of ZEBRA.
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Kinetics of BZLF1 mRNA and ZEBRA expression in TGF-1-stimulated
Raji cells.
Since TGF-1 induced the expression of ZEBRA
protein, we then looked at the appearance of the 1- and 3.0-kb mRNAs of
the BZLF1 gene. Raji cells were exposed to TGF-1 for various periods
of time and assayed for BZLF1 RNA expression by Northern blotting or
for ZEBRA expression by Western blotting.
RNA and proteins were extracted starting at 1 h and ending at
24 h poststimulation. The cDNA probe used for Northern analysis
recognizes both the monocistronic BZLF1 (1.0-kb) and the bicistronic
BZLF1-BRLF1 (3-kb) mRNAs transcribed from the Zp and Rp promoters,
respectively. The results (Fig.
2A)
indicate that as early as
90 min poststimulation, both the 1- and
3.0-kb RNAs were expressed.
Induction was maximal at 8 h, declined
by 12 h, and remained stable
thereafter. Western blot analysis
showed that ZEBRA expression
was detected at 4 h postinduction and
continued to increase throughout
the entire 24 h of culture (Fig.
2B).
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FIG. 2.
Kinetics of TGF-1-induced expression of BZLF1 RNA and
ZEBRA in Raji cells. (A) Raji cells were treated with TGF-1 (5 ng/ml) for the indicated time periods. Poly(A)+ RNA was
isolated and analyzed by Northern blot analysis. The blots were probed
with 32P-labeled BZLF1 cDNA. Equal loading was assessed by
rehybridization with 32P-labeled GAPDH cDNA. (B) Raji cells
treated with TGF-1 (5 ng/ml) for the indicated time periods were
lysed, and equal amounts of protein separated by SDS-PAGE were Western
blotted with anti-ZEBRA antibodies as described in Materials and
Methods. The position of ZEBRA is indicated by an arrow.
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ZEBRA expression is not affected by overexpression of Smad
proteins.
Smad proteins are known to trigger TGF-1 signaling.
It has been shown that cotransfection of Smad3 and Smad4 expression
vectors mimics the effect of the cytokine in TGF-1-responsive cells
and that Smad7 expression inhibits this effect (14, 59).
We cotransfected Mutu I or B95-8 cells with expression vectors for
Smad2, Smad3, and Smad4. The I
a1 germ line reporter
construct
pAI-D-CAT was included as positive control for the TGF-
1
effect.
When placed under the control of the pAI-D promoter, in Mutu I
cells, the coexpression of Smad2, Smad3, and Smad4 activated CAT
gene
expression to the same extent as TGF-
1 activation (Fig.
3C). However, coexpression of Smad2, -3, and -4 had no effect
on ZEBRA expression (Fig.
3A and B), whereas
TGF-
1 induces its
expression in the same cells. Similarly,
expression of Smad7 in
Mutu I reduces pAI-D-CAT expression brought
about by TGF-
1 induction
(Fig.
3), while no effect was observed on
TGF-
1 induction of
ZEBRA if Smad7 was expressed (Fig.
3A and B).
These findings suggest
that Smad proteins by themselves are not
sufficient for TGF-
1
signaling effects on ZEBRA induction.
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FIG. 3.
Effect of overexpression of Smad proteins on ZEBRA
expression. Mutu I cells (A) or B95-8 cells (B) were transfected by
electroporation with plasmids encoding Smad proteins, control plasmid
pCDNA3, or a plasmid containing the BZLF1 coding sequence under the
control of the cytomegalovirus promoter. Five hours after transfection,
the cells were treated with or without TGF-1 (2 ng/ml). After
24 h, cells were harvested and lysed, and equal amounts of protein
separated by SDS-PAGE were Western blotted with anti-ZEBRA antibodies
as described in Materials and Methods. The position of ZEBRA is
indicated by the arrow. (C) Mutu I cells were transfected by
electroporation with pAI-D-CAT in the absence or presence of plasmids
encoding Smad proteins. Five hours after transfection, the cells were
treated with or without TGF-1 (2 ng/ml). After 24 h, cells were
harvested and CAT activity was assayed in 50 µg of cell extract by
enzymatic butyrylation of radiolabeled chloramphenicol.
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Effect of PKC and PKA inhibitors on ZEBRA-induced expression by
TGF-1.
Since previous reports suggested that PKC may play an
important role in TGF-1 signal transduction (25, 50, 53),
we examined the effect of specific inhibitors of PKC and PKA on
TGF-1 BZLF1 induction. Raji cells were pretreated for 1 h with
the protein kinase inhibitors before stimulation by TGF-1 or PMA,
and induced ZEBRA expression was analyzed by Western blotting. As shown
in Fig. 4, GFX and H7, potent inhibitors
of PKC which interact with the catalytic subunit of the enzyme, inhibit
both PMA- and TGF-1-induced ZEBRA expression. These findings suggest
that in Raji cells, PKC not only mediates the PMA effect but also may
be involved in mediating the effect of TGF-1 on BZLF1 gene
expression. Conversely, in Mutu I cells, in which ZEBRA is not induced
by PMA treatment, GFX or H7 has no effect on TGF-1-mediated ZEBRA
induction (Fig. 5), suggesting that
diacylglycerol (DAG)-inducible PKC isoforms are not activated in these
cells.
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FIG. 4.
Effect of protein kinase inhibitors on TGF-1- or
PMA-induced ZEBRA expression in Raji cells. Raji cells were pretreated
for 1 h, with or without the inhibitors listed, prior to
stimulation with TGF-1 (5 ng/ml) or PMA (20 ng/ml). The cells were
lysed, and equal amounts of protein separated by SDS-PAGE were analyzed
by Western blotting with anti-ZEBRA MAbs. The position of ZEBRA is
indicated by the arrow.
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FIG. 5.
Effect of PMA and protein kinase inhibitors on
TGF-1-induced ZEBRA expression in Mutu I cells. Mutu I cells were
pretreated for 1 h with or without the inhibitors listed, in the
absence or presence of TGF-1 (2 ng/ml) or PMA (20 ng/ml). The cells
were lysed, and equal amounts of protein separated by SDS-PAGE were
analyzed by Western blotting with anti-ZEBRA MAbs. The position of
ZEBRA is indicated by the arrow.
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The addition of HA-1004, an inhibitor of cAMP-dependent protein kinase,
to Raji cell cultures had no effect on the TGF-
1-mediated
ZEBRA
expression. PKA does not appear to be involved in this effect
of
TGF-
1.
TGF-1- and PMA-induced ZEBRA expression is additive.
A
dose-dependent TGF-1 (0 to 10 ng/ml) induction of ZEBRA
expression in Raji cells showed the protein induced with a dose as low
as 0.5 ng/ml, with a maximum effect observed at 5 ng/ml (Fig.
6A). The tumor-promoting phorbol ester
PMA was previously shown to induce BZLF1 expression (13,
38); we therefore compared the stimulating effect of this agent
to that of TGF-1 in Raji cells. As could be expected, PMA induced
ZEBRA expression in a dose-dependent manner, with maximum induction
attained at 20 ng/ml (Fig. 6B). Furthermore, the stimulation observed
with maximal concentrations of TGF-1 (10 ng/ml) and PMA (100 ng/ml)
was additive (Fig. 6C). This additive effect of PMA and TGF-1 on
ZEBRA expression was also seen in B95-8 cells (data not shown).
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FIG. 6.
Additive effect of TGF-1 and PMA on ZEBRA expression.
(A and B) Raji cells were treated with the indicated concentrations of
TGF-1 or PMA for 18 h. (C) Raji cells were treated with either
vehicle (C), TGF-1 (10 ng/ml; T), PMA (100 ng/ml; P), or TGF-1
(10 ng/ml) plus PMA (100 ng/ml) (T+P) for 18 h. Cells were lysed,
and equal amounts of protein were separated by SDS-PAGE and analyzed by
Western blotting with anti-ZEBRA antibodies as described in Materials
and Methods. The position of ZEBRA protein is indicated by the arrow.
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We compared the fraction of cells producing ZEBRA in response to the
various inducing agents. Figure
7 shows
that treatment
of Raji cells with TGF-
1, PMA, or both TGF-
1 and
PMA induces
ZEBRA expression in 74 of 1,645 (4.45%), 88 of 1,566 (5.6%), and
74 of 1,322 (5.6%) cells, respectively. A chi-square test
showed
no statistical difference (
P = 0.27). This
result shows that the
additive effect of PMA and TGF-
1 on ZEBRA
expression was not
due to a higher number of cells responding to the
combined treatment.
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FIG. 7.
Percentage of Raji cells producing ZEBRA. Raji cells
were treated for 18 h with or without TGF-1 (5 ng/ml), PMA (20 ng/ml) or TGF-1 (5 ng/ml) plus PMA (20 ng/ml). Detection of ZEBRA
expression was performed by immunochemistry as described in Materials
and Methods. ZEBRA-positive and -negative cells were counted on
low-magnification photographs. More than 1,300 total cells were counted
for each treatment, and a chi-square test was performed for statistical
analysis.
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These findings provide evidence that activation of ZEBRA expression by
TGF-
1 is mediated by a pathway distinct from that
used for the
stimulation by
PMA.
TGF-1 induction of ZEBRA expression requires a non-PMA-inducible
protein kinase.
Different isoforms of PKC (
, , and 
) are
down-regulated by chronically treating cell culture with PMA. This
procedure depletes these isoforms and desensitizes the enzymes to
subsequent activation by PMA (34, 55, 57). We exposed Raji
cells to PMA (300 ng/ml) or vehicle (dimethyl sulfoxide [DMSO]) for
48 h, and mRNA expression was evaluated following the addition of
either TGF-1 or PMA. As shown in Fig.
8, PMA pretreatment caused a marked
decrease in the induction of BZLF1 expression by PMA compared to that
seen in controls pretreated with the vehicle alone. In contrast,
TGF-1 promoted a strong response which was even higher than that
seen in cells which were not pretreated with PMA.
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FIG. 8.
Effect of PMA-sensitive PKC down-regulation on TGF-1-
or PMA-induced BZLF1 expression. Raji cells were pretreated with PMA
(300 ng/ml) or control vehicle (DMSO) for 48 h prior to
stimulation with TGF-1 (10 ng/ml) or PMA (20 ng/ml) for 4 h.
The cells were then harvested, mRNA was isolated, and the Northern blot
was probed with the 32P-labeled BamHI Z fragment
cDNA. Equal loading was assessed by rehybridization with
32P-labeled GAPDH cDNA.
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Taken together, these results strongly suggest that PMA activates BZLF1
expression through DAG-sensitive PKC, whereas GFX-sensitive
protein
kinase(s) (other than DAG-sensitive PKCs, or other isoforms
of PKC)
mediate the TGF-
1
effect.
Effect of MAPK inhibitors on TGF-1 induction of ZEBRA
expression.
The role of MAPK kinase (4, 61) as well as
that of p38 MAPK in the TGF-1 signaling pathway has been described
elsewhere (1, 26). Treatment of Mutu I, Raji, and B95-8
cells with SB203580 (120 nM) had no effect on the rate of ZEBRA
production through TGF-1 induction in any of these cell lines (data
not shown). This result demonstrate that the p38 MAPK is not required in TGF-1 signaling pathway for ZEBRA induction.
Transfection of a dominant negative form of JNK coding sequences
(
24) in Mutu I, Raji, and B95-8 cells had no effect on
TGF-
1-mediated ZEBRA induction, indicating that JNK is not involved
in TGF-
1 signaling for ZEBRA expression. The dominant negative
form
of JNK is efficiently expressed in Mutu I, Raji, and B95-8
cells, as
measured by its activity on transfected TRE-CAT plasmid
(not
shown).
We investigated the effect MAPK/ERK kinase inhibitors on the induction
of ZEBRA expression by TGF-
1 or PMA using PD098059,
which prevents
the MEK1,2 activation by Raf and U0126, which inhibits
both active and
inactive MEK1,2 (
18). Mutu I, Raji, and B95-8
cells were
pretreated for 2 h with 100 µM PD98059, and TGF-
1
(2 or 5 ng/ml) or PMA (20 ng/ml) was then added for 18 h. The
results are
presented in Fig.
9A. TGF-
1-induced
production of
ZEBRA in Mutu I, Raji, and B95-8 cells is reduced by
pretreatment
with PD98059. In B95-8 cells, this inhibition affects only
the
induced and not the basal ZEBRA production. Experiments performed
with U0126 gave similar results. These results show that MAPK/ERK
kinase pathway is required for TGF-
1 induction of ZEBRA expression.
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FIG. 9.
Effect of protein kinase inhibitors on TGF-1- or
PMA-induced BZLF1 expression. (A) Mutu I, Raji, and B95-8 cells were
pretreated for 2 h with PD98059 (100 µM), U0126 (50 µM), or
vehicle (DMSO) before the addition of TGF-1 (2 ng/ml) or PMA (20 ng/ml). Fifteen hours later, cells were harvested and resuspended in
Laemmli sample buffer. Equal amounts of protein were separated by
SDS-PAGE and analyzed by Western blotting with anti-ZEBRA antibodies as
described in Materials and Methods. (B) Mutu I cells pretreated for
2 h with PD98059 (100 µM), U0126 (50 µM), or vehicle before
the addition of TGF-1 (2 ng/ml). Four hours later, cells were
harvested, and total RNA was extracted and analyzed on Northern blots
probed with 32P-labeled BZLF1 cDNA.
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The partial effects on ZEBRA expression observed with PD98050 and U0126
in Raji and B95-8 cells suggest that these cells use
an additional
route(s) to mediate the TGF-
1 effect on ZEBRA
expression.
Conversely, PD98050 and U0126 completely inhibited PMA-induced ZEBRA
expression in Raji and B95-8 cells. Thus, the signal-transducing
pathway of PMA appears to implicate solely that of MAPK/ERK kinase.
In
Mutu I cells, while PD98059 exhibits partial TGF-
1-mediated
ZEBRA
induction, U0126 completely inhibits this induction. Since
U0126
inhibits the MAPK/ERK signaling pathway at the level of
both Raf and
MEK activation, an additional pathway leading to
MEK activation could
occur through TGF-
1 signaling. Nevertheless,
as inhibition of
TGF-
1 induction by U0126 is complete in Mutu
I cells, it would
appear that no pathway other than the one leading
to MAPK/ERK kinase is
used.
These results were confirmed by Northern blot analysis. Figure
9B shows
that the induction of the 1- and 3-kb BZLF1 transcripts
by TGF-
1 is
partially inhibited by PD98059 in Mutu I cells, while
U0126 completely
inhibits this induction. These results show that
both the induction and
the inhibition of BZLF1 expression occur
at the transcriptional
level.
The Zp BZLF1 promoter does not respond to TGF-1 in the
EBV-negative cell line DG75.
TGF-1 stimulation results in the
induction of BZLF1 mRNA and protein expression. BZLF1 gene induction is
initiated by activation of its promoter by lytic cycle inducers. The
EBV-negative BL cell line DG75 was transfected with a construct
containing BZLF1 promoter nucleotides 221 to +12 linked to the
bacterial CAT reporter 221Zp-CAT. The cells were then induced with
TGF-1 (10 ng/ml) and/or PMA (20 ng/ml). The Ia1 germ
line reporter construct pAI-D-CAT was included as the positive control.
An increase in CAT activity was observed following 221Zp-CAT
stimulation with PMA, whereas no significant activation was detected in
response to TGF-1 (Fig. 10). Since
TGF-1 activated the pAI-D promoter, the lack of a response for
Zp-CAT could not be attributed to failure of TGF-1 signal transduction in DG75 cells.
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|
FIG. 10.
Response to TGF-1 and PMA of a transiently
transfected BZLF1 promoter-driven CAT reporter plasmid in DG75 cells.
The BZLF1 promoter reporter construct 221Zp-CAT and the pAI-D-CAT
construct were transfected into DG75 cells by electroporation.
Immediately following transfection, cells were treated with TGF-1
(10 ng/ml) and/or PMA (20 ng/ml). After 48 h, cells were harvested
and the level of CAT activity was determined by quantification of
acetylated chloramphenicol species with a PhosphorImager (Molecular
Dynamics).
|
|
The presence of a positive TGF-
1 response element(s) outside the
221-bp region, or negative regulatory element(s) in the
221-bp
region, was assayed with constructs containing extensions
of the Zp
promoter 5' region to
500 bp and various deletions
in the
221-bp
Zp, respectively. These insertions or deletions
had no effect on
TGF-
1 inductivity of Zp (data not shown). A
similar negative result
was obtained with the promoter of the
3-kb
transcript.
To determine if TGF-
1 induction of BZLF1 transcripts is dependent on
de novo protein synthesis, Mutu I cells were treated
with TGF-
1 (2 ng/ml) in the presence or absence of either one
or both of two
inhibitors of protein synthesis (anisomycin or
cycloheximide). Cells
were harvested 2 or 4 h after addition of
TGF-
1, and RNA
prepared from these cells was analyzed for the
presence of BZLF1
transcripts by Northern blotting. As seen in
Fig.
11, each inhibitor (or both together
[data not shown]) completely
abolished the appearance of both the 1- and 3-kb BZLF1 transcripts,
which suggests that induction of the
lytic cycle is dependent
on de novo protein synthesis.
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|
FIG. 11.
Effect of protein synthesis inhibitors on
TGF-1-induced BZLF1 expression. Mutu I cells were treated (or not)
with TGF-1 (1 ng/ml) for indicated time periods, with or without 10 µM anisomycin or 40 µM cycloheximide. Total RNA was isolated and
probed with 32P-labeled BZLF1 cDNA on Northern blots. Equal
loading was assessed by rehybridization with [32P]GAPDH
cDNA.
|
|
| |
DISCUSSION |
It was reported that TGF-1 induces EA expression in EBV
latently infected B cells (10, 16); however, the molecular
mechanisms involved in the effect of TGF-1 are unknown. The present
study was aimed at defining the molecular mechanisms by which EBV is reactivated following exposure to TGF-1. Since the expression of
ZEBRA is an early event which precedes EA production, we focused our study on the activation of ZEBRA by this cytokine. Our results indicate that exposure of different EBV genome-positive B cells to
TGF-1 results in the expression of the immediate-early EBV protein,
ZEBRA. This supports earlier observations that low levels of TGF-1
can induce the lytic cycle in latently infected B cells (16).
Northern blot analysis showed that stimulation by TGF-1 in Raji
cells involves the simultaneous expression of both the 1- and 3.0-kb
RNAs, suggesting that both promoters could be activated by changes in
the activity of cellular factors associated with TGF-1 treatment.
Although the BZLF1 transcripts are produced in a short period of time
following TGF-1 induction, de novo protein synthesis is required to
produce this effect.
Depending on the cell type, TGF-1 signal transduction has implicated
virtually every second messenger pathway including cAMP, inositol
phosphate hydrolysis, calcium influx, DAG, immediate-early genes
c-jun and c-fos, p21ras,
p38, JNK, and PKC (25, 35, 36, 44-46). More recently, a pathway involving Smad proteins has been documented (43).
However, overexpression of Smad proteins is not able to mimic the
TGF-1 effect on ZEBRA expression, suggesting that on their own,
Smads are not sufficient in mediating TGF-1 induction of ZEBRA.
The DAG-inducible PKC could be a step in this TGF-1 signaling
pathway, as the PKC inhibitors H7 and GFX are able to inhibit TGF-1-mediated ZEBRA induction. Activation of PKC leads to the MAPK/ERK pathway (13, 19). Furthermore, several lines of
evidence suggest that the MAPK/ERK pathway is involved in TGF-1
induction of ZEBRA in B95-8 and in Raji cells since incubation with
PD98059 or U0126, potent inhibitors of this pathway, inhibit the
TGF-1 ZEBRA induction. It remains to be determined if ras
activation of raf is involved in this pathway. It has been
shown that the MAPK/ERK pathway is involved in anti-Ig activation of
ZEBRA expression in Akata cells (51). We now provide
evidence for the use of this pathway in triggering the TGF-1
induction of ZEBRA. Nevertheless, in B95-8 and in Raji cells an
additional route (or routes) could also be used since (i) the
inhibition by PD98059 or U0126 was incomplete (inhibitors which
completely abolished PMA-induced ZEBRA expression in the same cell
lines), (ii) TGF-1 induction of ZEBRA expression occurred in
PKC-depleted cells, and (iii) the effects of TGF-1 and PMA are
additive in Raji and in B95-8 cells. This model is represented in Fig.
12A. The proposed additional pathway
does not involve the p38 MAPK or JNK because TGF-1 ZEBRA induction
is not affected by incubation with the p38 MAPK inhibitor SB203580 or
by transient expression of a dominant negative JNK mutant.
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|
FIG. 12.
Proposed scheme for a second messenger pathway
triggered by TGF-1 leading to the induction of ZEBRA. (A) In Raji
and B95-8 cells, TGF-1 causes activation of PKC and/or initiation of
the Raf-MEK-ERK MAPK cascade. TGF-1 also used an activity determined
by the method described by Gorman et al. (23) as an
additional signaling pathway to mediate the induction of ZEBRA
expression. (B) In Mutu I cells, the Raf-MEK-ERK MAPK cascade is used
as signal transducer. Moreover a cross talk between pathways will lead
to a Raf-independent MEK1,2 activation. Thick arrows represent proposed
routes. PD98059 acts by inhibiting the activation of MEK1,2 by Raf
kinase, U0126 acts both on inactive and active forms of MEK1,2.
GF-109203X is a potent inhibitor of PKC.
|
|
In Mutu I cells, U0126 completely abolished TGF-1-induced ZEBRA
expression, showing that only the MAPK/ERK pathway is used. As PD98059
only partially inhibits the TGF-1 effect, a pathway leading to
activation of MEK1,2 could be used. This activation might be due to
MEKK1,3, as shown by Yujiri et al. (63). The model is
represented in Fig. 12B.
The MAPK pathway is also activated by the EBV latent membrane protein
LMP1 (19), which plays a critical role in the regulation of
cell growth and differentiation. This might suggest that the lytic
cycle requires a some step of differentiation.
We observed that the Zp BZLF1 promoter does not respond to TGF-1 in
transfected cells and that TGF-1 induction of BZLF1 transcripts is
dependent on de novo protein synthesis. These data are in agreement
with the two-step induction model proposed by Flemington and Speck
(21), in which induction of the EBV lytic cycle requires an
initial activation signal of sufficient magnitude to allow expression
of enough ZEBRA to autoactivate Zp.
TGF-1, a potent immunosuppressive cytokine which suppresses T-cell
responses and deactivates macrophage effector functions, is produced by
a wide variety of cells (39). In addition, B-lymphoma cells
and Hodgkin's Reed-Sternberg cells have been shown to produce TGF-1
(33, 47). Therefore, in vivo, TGF-1-mediated EBV
reactivation may occur through a paracrine or an autocrine mode.
Furthermore, EBV binding (2) and ZEBRA (8) have
been shown to induce TGF-1 expression. Thus, a vicious cycle may be
initiated, whereby replication of EBV and production of TGF-1
amplify one another. In immunodeficient individuals, this may lead to
an increase in the number of EBV-infected cells and thus favor the
development of EBV-associated diseases. Moreover, high levels of the
cytokine may perpetuate the immunosuppressive circuits.
| |
ACKNOWLEDGMENTS |
We thank A. Alberga for critically reading the manuscript and
Azzedine Atfi for helpful discussion. We thank E. Drouet for the
anti-ZEBRA monoclonal antibodies, A. Sergeant for the Zp-CAT plasmid,
P. Sideras for pAI-D-CAT, Peter ten Dijke for plasmids containing
sequences encoding Smad2, Smad3, Smad4, and Smad7 proteins, Roger Davis
for the plasmid with the sequence of the dominant negative form of JNK,
and E. Connault for technical assistance.
This project was supported by ARC (9474) and by the Ligue Nationale
contre le Cancer (SF-98).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Pharmacologie Expérimentale et Clinique, INSERM EPI 99-32, Institut de Génétique Moléculaire, 27 rue Juliette
Dodu, 75010 Paris, France. Phone: 33 (1) 42 49 92 68. Fax: 33 (1) 42 49 48 38. E-mail: i.joab{at}chu-stlouis.fr.
| |
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Journal of Virology, July 2000, p. 5810-5818, Vol. 74, No. 13
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
