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INTRODUCTION |
Prostaglandins are important mediators in many physiological
processes including cell growth, ovulation, and immune functions. These
potent lipid molecules play key roles in the inflammatory response, and
inhibition of their synthesis is the target of most non-steroidal
anti-inflammatory drugs. Prostaglandin biosynthesis is
controlled by the cyclooxygenase
(COX)1 enzyme, which
catalyzes the initial conversion of arachidonic acid to prostaglandin
H2, a precursor common to all prostanoids. Two COX
isoenzymes are expressed in mammalian tissues, COX-1 and COX-2.
COX-1 is expressed constitutively in most tissues, whereas COX-2 is
induced by various pro-inflammatory cytokines and mitogenic agents in
different cell types and is thought to be responsible for the increased
production of prostaglandins in inflammatory disorders (1). COX-2
synthesis is regulated primarily at the transcriptional level via
distinct pathways in various cell types (including vascular endothelial
cells, epithelial cells, pancreatic cells, mast cells, and monocytes)
and involves transcription factors such as nuclear factor of
chain
in B cells (NF-
B), activator protein-1 (AP-1),
CCAAT/enhancer-binding protein (C/EBP), cAMP-responsive element-binding
protein (CREB), and nuclear factor of activated T cells (NFAT) (2-10).
COX-2 seems to act as an early T cell receptor (TCR)-responsive gene,
because TCR engagement results in COX-2 gene expression (11, 12)
through two NFAT motifs present in the COX-2 promoter region (3).
Moreover, inhibition of COX-1 or COX-2 activity by specific inhibitors
blocks T cell activation, and these two genes have been identified as
integral and sequential components of TCR signaling to p38
mitogen-activated protein kinase in T cell activation (12, 13).
One of the earliest events following TCR-mediated T cell activation is
an increase in tyrosine phosphorylation of specific proteins such as
cell surface receptors, adapter proteins, and kinases (14, 15). The
intracellular phosphotyrosine content is tightly regulated by a complex
balance between protein-tyrosine kinase (PTK) and protein-tyrosine
phosphatase (PTP) activities. The importance of PTP in T cell response
has been demonstrated by studies using specific PTP inhibitors that
reported an increase of T cell activation by these compounds (16). A
panoply of second messengers such as p56lck,
p59fyn, ZAP-70, and the mitogen-activated
protein kinase cascade are induced following treatment of lymphocytes
with PTP inhibitors such as pervanadate (17, 18). We have demonstrated
recently that treatment with potent PTP inhibitors bisperoxovanadium
(bpV) compounds (19) strongly activates both NF-
B and NFAT
transcription factors in T cell lines and peripheral blood mononuclear
cells (20, 21). These observations, along with the previously reported implication of NF-
B and NFAT in the control of COX-2 expression and
the pivotal function of COX-2 in T cell activation, led us to
scrutinize the effect of bpV molecules on the transcriptional induction
of COX-2 isoenzyme. In this study, we show that bpV compounds strongly
activate COX-2 promoter transcription in human T cells. The
bpV-mediated activating effect on COX-2 requires calcineurin and two
transcription factors, i.e. NFAT and AP-1.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
The lymphoid T cell lines used in this work
include Jurkat (clone E6.1), JCAM1.6, JCAM2, and J.RT3-T3.5. Jurkat is
considered a model cell line for the study of T cell signaling
machinery (22) whereas JCAM1.6 and JCAM2 are Jurkat derivatives that
are deficient in p56lck and
p36LAT expression, respectively (23, 24).
J.RT3-T3.5 is a TCR
chain-deficient mutant Jurkat cell line (25,
26). All cell lines were grown in RPMI containing 10% fetal calf serum
(Hyclone Laboratories) added with penicillin and streptomycin.
Plasmids and Antibodies--
The human COX-2 promoter constructs
and mutants were kind gifts from M. Fresno (University of Madrid,
Madrid, Spain). P2-1900, P2-274, P2-192, and P2-150 contain COX-2
promoter sequences from
1796,
170,
88, and
46, respectively, to
+104 cloned in the PXP2LUC reporter plasmid (3). P2-274
dNFAT-mut, P2-274 pNFAT-mut, and P2-192 CRE-mut bear mutations
destroying the distal NFAT (GTTAACAAT), the
proximal NFAT/AP-1 (CGTCTAGAAACAGCTG), or the
CRE motif (TTTGAGCTCA). The pCMV-LIP
(liver-enriched transcriptional inhibitory
protein), a generous gift from Dr. K. Kalame, encodes for a
truncated C/EBP protein (28). The expression vector for
p56lck, pEFneoLck-WT, and the
pEFneo-based empty vector have already been described (29) and were
kind gifts from C. Couture (Lady Davis Institute, Montreal,
Québec, Canada). The expression vector for
p36LAT, pCDNA3.1 LAT, was generously
provided by A. Weiss (University of California, San Francisco, CA)
(24). The vector encoding for SHP-1 and the control vector pSFFVneo
were provided by M. L. Thomas (Washington University School of
Medicine) (30). The expression vectors for TC-PTP and He-PTP, as
well as the control vector pEF/HA, were provided by T. Mustelin (La
Jolla Institute for Allergy and Immunology, San Diego, CA) (31). Rabbit
antisera raised against peptides from NFAT1 or all NFAT members (32) were obtained from N. Rice (NCI, National Institutes of Health, Frederick, MD). The polyclonal anti-NFATc (NFAT2) antibody was obtained
from Santa Cruz Biotechnology (Santa Cruz, CA).
Transient Transfection and Stimulation of Cells--
Cells were
electroporated using a gene pulser I apparatus (Bio-Rad) (960 microfarad, 250 V) at room temperature. Cells were concentrated at
37.5 × 106 per ml of complete culture medium. Cells
(400-µl aliquots) were electroporated with either 5 µg of the
reporter construct DNA alone or, in the case of reconstitution
experiments, with 5 µg of reporter construct DNA and 0, 5, or 15 µg
of the expression plasmid. The total DNA amount for the reconstitution
experiments was maintained constant at 20 µg using the empty vector.
To minimize variations in plasmid transfection efficiencies, cells were
transfected in bulk and were separated at 36 h post-transfection into various treatments groups at a density of 105
cells/well (100 µl) in 96-well flat-bottom plates. For studies using
pharmacological inhibitors, cells were resuspended in fresh cell
culture medium (1 × 106 cells/ml) and were treated
for 60 min with subcytotoxic and subcytostatic concentrations of
cyclosporin A (1 to 100 ng/ml) (Fujisawa, Osaka, Japan), FK506 (1 to 10 ng/ml) (Sigma), TMB-8 (Sigma) (25 to 100 µM), or
piceatannol (Biomol) (0.1 to 50 µM) before addition of bpV(pic). Cells were either left untreated or were treated with phorbol
12-myristate 13-acetate (PMA) at 20 ng/ml (Sigma) alone or in
combination with ionomycin at 1 µM (Sigma) or with
various concentration of bpV(pic) (Alexis Biochemical), used either
alone or in combination with 10 µM forskolin (Cedarlane)
in a final volume of 200 µl. Next, cells were incubated at 37 °C
for 8 h unless otherwise specified. Luciferase activity was
determined following a previously described protocol (20). -Fold
induction was obtained by calculating the ratio between measured
relative light units of treated over untreated samples. Results shown
are the mean ± S.D. of four determinations and are representative of three different experiments.
Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assay--
Nuclear extracts were prepared according to a previously
described protocol (20). Briefly, untreated or treated cells (5 × 106) were first washed with phosphate-buffered saline.
Cells were then resuspended in 400 µl of hypotonic buffer (Buffer A)
(10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and
kept for 15 min on ice before lysis with 25 µl of Nonidet P-40 10%.
After brief vortexing and centrifugation, the supernatant was
discarded, and the pellet was resuspended with an hypertonic buffer
(Buffer B) (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride)
followed by gentle agitation for 15 min. The solution was then
centrifuged, and the supernatant was assayed for protein concentration
by BCA assay (Pierce) and stored at
85 °C until use.
Nuclear extracts (10 µg) were incubated for 20 min at room
temperature in 20 µl of 1× binding buffer (10 mM HEPES,
pH 7.9, 4% glycerol, 1% Ficoll, 25 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 25 mM NaCl, 2 µg of poly(dI-dC), and 10 µg of
nuclease-free bovine serum albumin fraction V) containing 0.8 ng of
[
-32P]-labeled double-stranded DNA oligonucleotide.
The following double-stranded DNA oligonucleotides were used as probes
and/or competitors: the distal NFAT binding site of the human COX-2
promoter (nucleotides
117 to
191),
5'-tcgaCAAGGGGAGAGGAGGGAAAAATTTGTGGC-3'; the NFAT/AP-1 proximal
site of the COX-2 promoter (nucleotides
82 to
58),
5'-tcgaCAAAAGGCGGAAAGAAACAGTCATTTC-3'; the distal NFAT binding site
from the murine interleukin-2 promoter,
5'-TCGAGCCCAAAGAGGAAAATTTGTTTCATG-3'; the consensus NF-
B binding
site, 5'-ATGTGAGGGGACTTTCCCAGGC-3'; and the consensus binding
site for Oct-2A (used for nonspecific competition). DNA-protein
complexes were resolved from free-labeled DNA by electrophoresis in
native 4% (w/v) polyacrylamide gels. The gels were subsequently dried
and autoradiographed on an Eastman Kodak Co. Biomax MR film at
85 °C. Cold competition assays were conducted by adding a 100-fold
molar excess of unlabeled double-stranded DNA oligonucleotide
simultaneously with the labeled probe. Supershift assays were performed
by preincubation of nuclear extracts with 1 µl of the appropriate
antibody in the presence of all of the components of the binding
reaction for 30 min on ice before addition of the labeled probe.
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RESULTS |
COX-2 Promoter Activity Is Strongly Activated by the PTP Inhibitor
bpV(pic)--
To assess whether PTP inhibitors can modulate the human
COX-2 gene promoter activity, we performed reporter assays using
various COX-2 promoter-based constructs. The P2-1900 construct, which comprises a region spanning from
1796 to +104 bp of the human COX-2
gene placed in front of the luciferase reporter gene (3), was
transiently transfected in Jurkat cells. As reported previously (3),
treatment of Jurkat cells with the PMA/ionomycin combination led to an
increase in COX-2 promoter-driven reporter gene activity (Fig.
1A). Interestingly, treatment
with the PTP inhibitor bpV(pic) at 20 µM induced an even
stronger activation of COX-2 promoter. A greater bpV-mediated induction
of COX-2 activity (i.e. 70-fold increase) was observed with
P2-274 (Fig. 1B), a molecular construct that comprises the
promoter region spanning from
170 to +104. This suggests that the
upstream region contains negative regulation elements. Dose-response
and time course experiments indicated that the highest transcriptional
induction was achieved following an 8-h incubation time period with 20 µM bpV(pic) (Fig. 1, B and C).
Altogether these data suggest that inhibition of PTP activity triggers
COX-2 promoter activity in human T cells.

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Fig. 1.
COX-2 transcriptional regulation by
bpV(pic). Jurkat T cells were transiently transfected with the
COX-2 promoter constructs P2-1900 (panel A) or P2-274
(panels B and C). A, cells were next
either left untreated (NT) or were treated for 8 h with
PMA (10 ng/ml), ionomycin (iono; 1 µM),
PMA/ionomycin combination (PMA/iono), or bpV(pic) (20 µM) before being assayed for luciferase activity.
B, cells were treated with the indicated concentrations of
bpV(pic) for 8 h. C, cells were treated with bpV(pic)
(20 µM) for the indicated time periods before assessment
of luciferase activity. Results are expressed as -fold increase in
luciferase activity in treated over untreated samples and represent the
mean ± S.D. of quadruplicate samples. Data shown are
representative of three different experiments.
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The Calcineurin-mediated Signaling Pathway Is Involved in COX-2
Transcriptional Activation by bpV(pic)--
We have demonstrated
previously (20, 21) that bpV compounds can induce both
calcineurin-dependent and -independent signaling pathways in T cells. To elucidate the signal transduction
pathway(s) involved in bpV-mediated COX-2 transcriptional activation,
we used the immunosuppressive drugs FK506 and CsA. Jurkat cells were first transiently transfected with P2-274 and next pretreated with
increasing amounts of FK506 or CsA before activation by bpV(pic). As
depicted in Fig. 2, treatment with both
calcineurin inhibitors resulted in an almost complete inhibition of
COX-2 transcriptional activation. Similar observations were made when
using the P2-1900 COX-2 promoter construct (data not shown). No
detectable cell toxicity was observed upon cell incubation with these
inhibitors at the tested concentrations (data not shown). These results
demonstrate that COX-2 transcriptional activation by bpV occurs through
a pathway that relies on calcineurin activity.

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Fig. 2.
bpV-mediated induction of COX-2 promoter
activity is abolished by calcineurin inhibitors. Jurkat cells
transiently transfected with P2-274 were pretreated for 60 min with
FK506 (panel A) or CsA (panel B) at the indicated
concentrations before stimulation with bpV(pic) (20 µM).
Results are expressed as -fold increase in luciferase activity in
treated over untreated samples and represent the mean ± S.D. of
quadruplicate samples. Data shown are representative of three different
experiments.
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The two NFAT Binding Sites in the COX-2 Promoter Are Involved in
Transcriptional Activation by bpV(pic)--
We next wanted to identify
the precise cis-acting regions required for the inducibility of the
COX-2 promoter region to the bpV(pic) compound. To this end, we first
used several 5' deletion constructs that were created from the P2-274
vector (Fig. 3A). A deletion
of 83 bp (i.e. P2-192 vector) resulted in a 60% decrease in the inducibility by bpV(pic) (Fig. 3B). A supplementary
deletion of 42 bp (i.e. P2-150 vector) decreased
transcriptional activity by an additional 50%, leaving only a 20%
residual activation. These results suggest that regions spanning from
170 to
88 and
88 to
46 bp relative to the transcription start
site of the human COX-2 gene include sequences responsible for the
bpV-mediated positive effect on COX-2 gene. These regions contain a
binding site for C/EBP, a CRE, and two NFAT binding sites, one of which contains an adjacent AP-1 motif (i.e. pNFAT) (Fig.
3A). Given that the two NFAT elements have been shown to be
crucial for COX-2 transcriptional activation by PMA and ionomycin (3),
and treatment of human T cells with bpV molecules resulted in induction
of NFAT (21), we next tested the importance of NFAT binding sites in the noticed bpV-induced COX-2 activation. To this end, we used constructs bearing mutations destroying proximal (pNFAT-mut) or distal
(dNFAT-mut) NFAT sites. Transfection of Jurkat cells with the
P2-274 vector bearing either COX-2 dNFAT or pNFAT mutated sites resulted in a 40 to 50% diminution in the responsiveness to
bpV(pic) (Fig. 3C). Moreover, data obtained with dNFAT-mut were comparable with results seen when using the P2-192 deletion mutant, which lacks the entire dNFAT binding site. The inducibility by
bpV(pic) was even further diminished when cells were transiently transfected with a P2-192-based construct carrying mutations in the
remaining pNFAT site (i.e. P2-192 pNFAT-mut vector). These data support the idea that NFAT plays a crucial role in bpV-mediated transcriptional induction of the human COX-2 gene in human T cells.

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Fig. 3.
Implication of NFAT binding sites in
bpV-dependent induction of COX-2 promoter activity.
A, COX-2 promoter-based vectors used in this work.
cis-acting sequences are represented by boxes.
LUC, luciferase gene. B and C, Jurkat
cells were transiently transfected with the indicated constructs and
were either left untreated or were treated for 8 h with bpV(pic)
(20 µM) before assessment of reporter gene activity.
Results are expressed as -fold increase in luciferase activity in
treated over untreated samples and represent the mean ± S.D. of
quadruplicate samples. Data shown are representative of three different
experiments.
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bpV Induces NFAT and AP-1 Binding Activities onto COX-2
Promoter--
The COX-2 pNFAT element contains an adjacent AP-1-like
binding site that is destroyed in pNFAT mutant. Thus, to establish the
possible implication of the AP-1 transcription factor and to further
incriminate the participation of NFAT in the observed phenomenon, we
performed electromobility shift assays with nuclear extracts of Jurkat
cells using DNA probes specific for the distal or the proximal NFAT
elements. Two specific retarded complexes were induced with different
kinetics when using the proximal NFAT probe (Fig.
4A). The intensity of complex
I was maximal at 270 min whereas complex II reached a peak after only
30 min of bpV treatment. A single retarded complex, i.e.
complex II, was seen when using the distal NFAT probe. Both complexes
were efficiently competed away with 100-fold molar excess of the
unlabeled specific probe but not by a nonspecific oligonucleotide
(i.e. Oct-2A). The intense, faster migrating band was
considered as nonspecific, because it was not consistently competed
away by an excess of specific oligonucleotide. Migrating complexes were
further identified using specific competitions and supershift assays.
Complex I was specifically competed away by an NFAT-specific
oligonucleotide and also by an AP-1-specific sequence, but not by a
control NF-
B-specific oligonucleotide (Fig. 4B,
lanes 1-4), suggesting that this complex contains both NFAT
and AP-1 bound to their specific sites. Complex II was also
specifically competed away by a NFAT-specific oligonucleotide, but not
by either NF-
B- or AP-1-specific sequences, suggesting that NFAT is
responsible for this complex (Fig. 4B, lanes
1-4). However, because this band was not completely competed away
either by the consensus NFAT or by the distal NFAT sequences
(lanes 2 and 5) but only by the pNFAT
oligonucleotide, it is likely that the complex II represents a
superimposition of a major NFAT complex and a minor AP-1 complex.
Supershift assays indicated that NFAT1, but not NFAT2, was present in
both the NFAT and NFAT·AP-1 complexes (lanes 8 and
9). Incubation with a pan-NFAT antiserum prevented the
formation of both the NFAT and the NFAT·AP-1 complexes (lane 10). Similar observations were made when the distal NFAT sequence was used as a probe (lanes 11-20), thus confirming that
treatment of Jurkat cells with bpV molecules induces the binding of
NFAT1 on both NFAT elements of the COX-2 promoter.

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Fig. 4.
Treatment of Jurkat cells with bpV(pic) leads
to binding of NFAT- and AP-1-specific complexes onto the COX-2
promoter. A, nuclear extracts from Jurkat cells treated
with bpV(pic) (20 µM) for the indicated time periods were
incubated with [ -32P]-labeled probes corresponding to
the proximal (i.e. pNFAT) or the distal NFAT binding site
(i.e. dNFAT). The complexes were resolved on a native 4%
polyacrylamide gel. Competitions were performed with a 100-fold molar
excess of specific (S) or nonspecific (NS) Oct-2A
oligonucleotides. B, nuclear extracts from Jurkat cells
stimulated with bpV(pic) for 270 min were incubated with probes
corresponding to the proximal (lanes 1-10) or distal
(lanes 11-20) NFAT binding site. Competition experiments
were performed with a 100-fold molar excess of cold oligonucleotides
representing the consensus NFAT (lanes 2 and 12),
NF- B (lanes 3 and 13), AP-1 (lanes
4 and 14), COX-2 distal NFAT (lanes 5 and
15), COX-2 proximal NFAT (lanes 6 and
16) sequences, or a nonspecific oligonucleotide (lanes
7 and 17). Supershift assays were performed with
polyclonal antisera to NFAT1 (lanes 8 and 18),
NFAT2 (lanes 9 and 19), or a pan-NFAT
antiserum (lanes 10 and 20). SS,
supershifted complex. NS, nonspecific complex.
Arrows indicate bpV-induced migrating complexes.
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Transcriptional Activation of COX-2 by bpV(pic) Is Independent of
C/EBP Transcription Factors--
Given that the COX-2
promoter contains a C/EBP binding site, we also assessed the importance
of C/EBP transcription factors in bpV-dependent induction
of the COX-2 promoter. The implication of the C/EBP family of
transcription factors was assessed by using pCMV-LIP, a construct that
encodes a truncated C/EBP protein that has only the DNA binding and
leucine zipper domains and possesses a dominant negative function (28).
Co-transfection of this expression vector, along with P2-274, had no
effect on bpV-induced reporter gene expression (Fig.
5), thus ruling out any implication of
C/EBP in COX-2 transcriptional regulation by the PTP inhibitor
bpV(pic).

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Fig. 5.
C/EBP transcription factors are not essential
for COX-2 promoter activation by bpV(pic). Jurkat cells were
co-transfected with the C/EBP dominant negative expression vector
pCMV-LIP or the empty control vector, along with P2-274. Such cells
were next either left untreated or were treated for 8 h with
bpV(pic) (20 µM) before determination of luciferase
activity. Results are expressed in relative luciferase activity
(RLU) and represent the mean ± S.D. of quadruplicate
samples. Data shown are representative of three different
experiments.
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Cyclic AMP-mediated Signal Transduction Pathways Potentiate
bpV-induced COX-2 Activation--
Because the CRE presents in the
COX-2 promoter has been shown to be involved in COX-2 regulation in
various cell types such as monocytes, macrophages, and endothelial and
epithelial cells (6-8, 33), we investigated the importance of
cAMP-related effectors in induction of COX-2 upon treatment of T cells
with bpV(pic). When Jurkat cells were transfected with COX-2 promoter
constructs and were treated with the cAMP-elevating agent forskolin
(FSK), a very modest increase in transcriptional activity was observed (never more than 2-fold) (Fig. 6,
panels A and B). However, when FSK was used with
bpV(pic), this combination resulted in an induction of COX-2 activity
that was superior to the one seen when bpV(pic) was used alone. This
up-regulating effect was observed with both COX-2 molecular constructs,
i.e. P2-1900 and P2-274. Cyclic AMP is a ubiquitous
intracellular second messenger that transmits information to several
proteins including protein kinase A and a family of cAMP-responsive
nuclear factors that bind to the CRE consensus sequence.
Phosphorylation of these CREBs by the C subunit of protein kinase A
modulates their overall activity. To assess the importance of CREB
factors in COX-2 transcriptional activation, we used a dominant
negative form of CREB named killer CREB (KCREB), which is unable to
bind to CRE DNA sequence and blocks the binding of wild type CREB when
present as a KCREB·CREB heterodimer. Jurkat cells were
co-transfected with increasing amounts of the KCREB expression vector,
along with P2-274, and were next treated with bpV(pic) and/or FSK. The
dominant negative CREB was found to severely decrease COX-2
transcriptional activation that is seen following treatment with
bpV(pic) alone (Fig. 6C). The enhancing effect of FSK was
completely abolished in the presence of high concentrations of KCREB,
therefore suggesting that the transactivating potential showed by FSK
is mediated by CREB transcription factors. Surprisingly, mutation of
the CRE binding site in the COX-2 promoter had no effect on induction
by both bpV(pic) and the combination of bpV(pic) and FSK (Fig.
6D). These observations suggest that CREB transcription factors are not acting directly on COX-2 promoter but are activating the expression of other transcription factors that are able to regulate
the COX-2 promoter. In an attempt to define whether
bpV-dependent induction of intracellular cAMP could lead to
an increased expression of AP-1 via an effect on CRE elements present
in Fos and Jun promoters, we performed electrophoretic mobility shift
assay using as a probe the COX-2 proximal NFAT sequence. Treatment of
Jurkat cells with FSK did not induce any specific binding activities,
but treatment with bpV(pic) and FSK resulted in a much stronger
NFAT·AP-1 complex when compared with treatment with bpV(pic) alone
(Fig. 6E).

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Fig. 6.
Cyclic AMP-dependent signaling
events further increase COX-2 activity that is seen upon bpV(pic)
treatment. Jurkat T cells were transiently transfected P2-1900
(panel A) or P2-274 (panel B). C,
cells were co-transfected with 0, 10, and 20 µg of the CREB dominant
negative expression vector KCREB, along with P2-274. D,
cells were transiently transfected with the COX-2 promoter construct
P2-192 bearing either wild type or mutated CRE site. Cells were next
either left untreated or were treated for 8 h with bpV(pic) (10 µM), FSK (10 µM), or a combination of both
agents, before determination of luciferase activity. Results are
expressed as -fold increase in luciferase activity in treated over
untreated samples and represent the mean ± S.D. of quadruplicate
samples. Data shown are representative of two different experiments.
E, nuclear extracts from Jurkat cells that had been treated
with bpV(pic) (10 µM), FSK (10 µM), or a
combination of both agents for 4 h were incubated with a
[ -32P]-labeled probe corresponding to the proximal
NFAT/AP-1 site (i.e. pNFAT). The complexes were resolved on
a native 4% polyacrylamide gel.
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COX-2 Transcriptional Activation by bpV(pic) Is Dependent on
TCR-mediated Signaling Cascade Events--
We next wanted to
characterize the bpV-induced signaling pathway(s) leading to COX-2
transcriptional activation. We reported previously (21) that induction
of NFAT by bpV treatment in T cells is a process that relies on
TCR-dependent biochemical events. Because NFAT is directly
involved in the inducibility by bpV(pic) of the COX-2 promoter, we
assessed the implication of different TCR-related signal transducers in
this process. We used Jurkat-derived cell lines that are TCR-negative
(i.e. J.RT3-T3.5) or deficient for either the PTK
p56lck (i.e. JCAM1.6) or the adapter
protein p36LAT (i.e. JCAM2). When
such cells were transiently transfected with P2-274, the TCR-negative
J.RT3-T3.5 cell line was found to be fully responsive to bpV(pic)
stimulation, thus ruling out an involvement of the TCR in COX-2
activation by bpV compounds (Fig.
7A). In contrast, both JCAM1.6
and JCAM2 cell lines were unresponsive to bpV stimulation (Fig.
7A). To further establish the importance of
p56lck, a member of the Src family of
non-receptor PTK, we cotransfected JCAM1.6 cells with increasing
amounts of the p56lck-encoding vector
pEFneoLckWT, along with P2-274. The bpV-induced COX-2 transcriptional
activation was partially restored (Fig. 7B), confirming the
implication of p56lck in the noticed
up-regulation of COX-2 promoter activity. Similarly, co-transfection of
a p36LAT expression vector also partially
restored the inducibility of the JCAM2 cell line, demonstrating that
p36LAT is also part of the bpV-induced signaling
pathway (Fig. 7C). The incomplete restoration of
inducibility to bpV treatment is most likely because of the low
transfection efficiency of JCAM1.6 and JCAM2 cell lines. The
implication of ZAP-70 was investigated using the Syk family kinase
inhibitor piceatannol (34). Pretreatment of Jurkat cells transfected
with P2-274 with increasing concentrations of piceatannol induced a
dose-dependent inhibition of the COX-2 transcriptional
activation by bpV(pic) (Fig. 7D).

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Fig. 7.
Implication of TCR-dependent
signaling effectors and calcium in COX-2 transcriptional activation
that is seen upon bpV treatment. A, Jurkat, J.RT3-T3.5,
JCAM1.6, and JCAM2 cells were transiently transfected with P2-274 and
were then either left untreated or were treated with bpV(pic).
B, JCAM1.6 cells were co-transfected with P2-274 (5 µg)
and different concentrations of the
p56lck-encoding vector pEFneoLckWT.
C, JCAM2 cells were co-transfected with P2-274 (5 µg) and
different concentrations of the p36LAT-encoding
vector pCDNA3.1 LAT. DNA amounts were kept constant by addition of
the empty vector. Transfected cells were either left untreated or were
treated for 8 h with bpV(pic) before measuring luciferase
activity. D, Jurkat cells transiently transfected with
P2-274 were pretreated for 60 min with the inhibitor of Syk kinases
piceatannol at the indicated concentrations. Cells were then either
left untreated or were treated for 8 h with bpV(pic) before
measuring luciferase activity. E, Jurkat cells transiently
transfected with P2-274 were pretreated for 60 min with the inhibitor
of calcium mobilization TMB-8 at the indicated concentrations. Cells
were then either left untreated or were treated for 8 h with
bpV(pic) before measuring luciferase activity. Results are expressed as
-fold increase in luciferase activity in treated over untreated samples
and represent the mean ± S.D. of quadruplicate samples. Data
shown are representative of three different experiments.
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More downstream effectors of the TCR signaling cascade
include the release of intracellular calcium, an event that is known to
be crucial for NFAT activation. To monitor the importance of calcium,
we used the modulator of intracellular calcium homeostasis TMB-8 that
blocks endogenous calcium release. Pretreatment of Jurkat cells
transiently transfected with P2-274 with increasing concentrations of
TMB-8 resulted in a marked diminution of COX-2 promoter-driven
luciferase activity upon bpV(pic) stimulation (Fig. 7E). No
loss of cell viability was observed with the tested doses of TMB-8
(data not shown).
Possible Involvement of SHP-1 and TC-PTP in COX-2 Transcriptional
Activation by bpV(pic)--
We next seeked to identify the tyrosine
phosphatase(s) that, once inhibited by bpV(pic), is resulting in an
increased phosphorylation of kinases such as
p56lck and ZAP-70 and, ultimately, in COX-2
transcriptional activation. We focused our efforts on CD45, SHP-1,
He-PTP, and TC-PTP based on the notion that these PTPs are expressed at
high levels in Jurkat cells (35). The implication of the transmembrane
PTP CD45 was ruled out by the observation that bpV-mediated activation of COX-2 promoter in the CD45-negative cell line J45.01 is comparable with that seen in parental CD45-expressing Jurkat cells (data not
shown). However, overexpression of SHP-1 and TC-PTP, but not He-PTP,
caused a marked reduction in bpV-induced COX-2 transcriptional activation (Fig. 8). The noticed
inhibition of bpV-mediated increase in COX-2 activity with wild type
SHP-1 and TC-PTP could not be compared, because the two proteins are
expressed from different promoters.

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Fig. 8.
bpV-mediated increase in
COX-2 transcriptional induction is diminished by SHP-1 and
TC-PTP overexpression. Jurkat cells were transiently transfected
with P2-1900 (5 µg), along with 5 or 15 µg of a vector encoding
for wild type SHP-1, TC-PTP, or He-PTP. The total amount of DNA was
kept constant in each transfection by the addition of an empty control
vector. Cells were then either left untreated or were treated for
8 h with bpV(pic) before measuring luciferase activity. Results
are expressed as percentage of the activation observed with P2-1900
alone, which was arbitrarily fixed at 100.
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DISCUSSION |
Although it has been shown that COX-2 gene expression in T cells
is regulated by various stimuli, the exact intracellular signaling
events involved in this regulation have not yet been elucidated. In
this study we analyzed the effect of the PTP inhibitor bpV(pic) on
regulation of COX-2 transcriptional activity. We demonstrate here that
PTP inhibition by means of a treatment with bpV(pic) results in a
significant increase in COX-2 promoter activity through induction of
both NFAT and AP-1 transcription factors. These results confirm
previous observations demonstrating the importance of NFAT and AP-1
binding sites in the transcriptional induction of COX-2 isoenzyme that
is seen upon T cell activation (3). Both the COX-2 distal NFAT and
proximal NFAT/AP-1 binding sites were found to be involved in
bpV-dependent enhancing effect on COX-2 promoter activity,
because selective mutation of each site resulted in a diminution of the
promoter inducibility (Fig. 3). Other cis-acting elements were
described previously to be important for COX-2 gene regulation in non-T
cells. For example, a C/EBP element appears to be crucial for COX-2
transcriptional regulation in macrophages, as well as in fibroblasts
and osteoblasts, with the involvement of different members of the C/EBP
family in each cell type (2, 4, 5, 9, 10). A cyclic AMP-responsive
element is also involved in COX-2 regulation in various cell types
including endothelial and epithelial cells (6-8) and
monocytes/macrophages (33). We did not observe any implication of these
two motifs in COX-2 transcriptional activation induced by bpV(pic) (see
Fig. 5 and Fig. 6D). Indeed, the COX-2 gene appears to be
regulated by a particular set of transcription factors for each cell
type and stimulus, a situation that could be related to the various
physiological roles played by the COX-2 protein in various cell types,
for example inflammation, tissue damage, tumorigenesis, or pain transmission.
Our results indicate that both NFAT and AP-1 binding activities are
induced following bpV(pic) stimulation in Jurkat cells. NFAT1 is the
most prevalent NFAT family member that is translocated to the nucleus
(Fig. 4B). This is consistent with earlier reports demonstrating the predominance of NFAT1 in activated T cells (32), which is in line with the previous observation indicating that bpV
stimulation of T cells leads to the binding of NFAT1 onto the
regulatory elements of human immunodeficiency virus (21). Another study
suggests that NFAT2 could also be implicated in COX-2 transcriptional
regulation (3); however, this observation was made following
overexpression of NFAT2 by transient transfection. We also identified
by mobility shift assays an NFAT·AP-1 complex that is strongly
induced upon bpV treatment. Such highly stable ternary complexes are
known to act in synergy and to regulate expression of various genes
(36-38). NFAT alone is able to bind to the dual NFAT/AP-1 site
(complex II in Fig. 4). However very weak, if any, binding
of AP-1 alone was observed. This suggests that NFAT is the
transcription factor that is crucial for COX-2 activation by bpV(pic).
This is exemplified by the observation that bpV(pic)-mediated induction
of COX-2 promoter activity is abolished by a pretreatment with the
calcineurin inhibitors FK506 and CsA (Fig. 2). Activation of
calcineurin is necessary to dephosphorylate NFAT proteins and to allow
their translocation to the nucleus; however, no such role was described
for AP-1. Although a calcineurin-dependent regulation of
Jun proteins has been shown in activated T cells (39), induction of
AP-1 was found to be inhibited by CsA in primary cells (40) but not in
Jurkat cells (41). Consequently, the observed inhibitory effect of
FK506 and CsA is linked with a blockade of nuclear translocation of
NFAT.
Differences in the kinetics of NFAT and AP-1 activation following bpV
treatment were detected when performing mobility shift assays. Indeed,
nuclear translocation of NFAT was already maximal at 30 min whereas the
intensity of the NFAT·AP-1 complex reached a peak at 270 min after
bpV treatment (Fig. 4A). Such a delayed translocation
suggests that AP-1 activation by bpV compounds requires transcription
and translation of Fos and/or Jun.
Additional experiments revealed that the PTKs
p56lck and ZAP-70, the adapter protein
p36LAT, and calcineurin are all implicated in
bpV-dependent up-regulation of COX-2 promoter activity in
human T cells. These observations suggest that the bpV(pic) PTP
inhibitor affects the most proximal membrane events of TCR signaling.
The TCR complex itself is not required for the triggering of COX-2
activity by bpV(pic) (Fig. 7A). Interestingly, ZAP-70 is
able to interact with an immunoreceptor tyrosine-based activation motif
distinct from the TCR/CD3 complex (42). The observed similarity between
signaling pathways induced upon TCR engagement and bpV treatment is not
surprising if one considers that addition of bpV to T cells treated
with phytohemagglutinin or anti-TCR/CD28 antibodies results in
activation of the interleukin-2 promoter through a concomitant
induction of NF-
B, NFAT, and AP-1 transcription factors (21). In
this study, the working model to explain activation of NFAT by bpV
molecules proposed that inhibition of PTPs led to
p56lck activation, which, via phosphorylation of
ZAP-70, induced intracellular calcium increase and calmodulin
activation (21).
Interestingly enough, CREB is also crucial for COX-2 gene activation by
bpV(pic), with no implication of the CRE site present in the COX-2
promoter (Fig. 6, panels C and D). This
observation suggests that PTP inhibition by bpV(pic) induces, via an
increase in intracellular calcium, activation of calcium-calmodulin
kinase IV, a kinase that is known to phosphorylate CREB serine 133 (43, 44). This phosphorylated CREB does not directly transactivate the COX-2
promoter but rather acts indirectly by increasing c-Fos and/or c-Jun
expression. Indeed, both fos and jun regulatory
regions contain consensus sites for CREB (45). This indirect effect accounts for the delayed apparition of the NFAT·AP-1 complex
(compared with the NFAT complex) observed in the electrophoretic
mobility shift assay experiments. The residual CREB-independent
transactivating potential could be explained by a signaling cascade
initiated by ZAP-70 and p36LAT. ZAP-70 could
also activate the Ras/Raf pathway, leading to transcriptional activation of c-fos via the extracellular signal-regulated
kinase pathway and phosphorylation of c-Jun by the c-Jun N-terminal
kinase (JNK) (46, 47). The implication of p36LAT
is also possible, because this adapter protein recruits phospholipase C
to the membrane, leading to the activation of protein kinase C via
formation of diacylglycerol. Protein kinase C is involved in
c-fos transcriptional activation by the Elk-1 and serum
response factor (SRF) transcription factors via a
p21ras-dependent cascade (48), as well as in
the direct activation of c-Fos by c-Fos kinase (49) and in c-Jun
activation by JNK (50).
Another pathway leading to COX-2 activation following TCR triggering
involves Cot kinase, a mitogen-activated kinase kinase kinase. This
kinase was shown to activate NFAT-mediated transactivation of the human
COX-2 promoter in a CsA-independent manner and to synergize with the
calcineurin-dependant pathway (51). We showed that bpV-induced COX-2
transcriptional activation is inhibited by both CsA and FK506, which
rules out any implication of Cot kinase in our findings.
We also demonstrate that cAMP-mediated signaling events cooperate with
bpV(pic) to induce a more powerful activation of COX-2 (Fig. 6,
panels A and B). This effect is mediated by the
CREB family of transcription factors (Fig. 6C) and, most
likely, via CREB phosphorylation on its serine 133 by protein kinase A. However, the CRE element located in the COX-2 promoter is not necessary for this transactivating potential (Fig. 6D), thus
suggesting an indirect effect of CREB on COX-2 transcriptional
activity, most likely via an increase in Fos and/or Jun expression.
This hypothesis in strengthened by the observation that FSK treatment induced a marked increase of the NFAT·AP1 complex that is seen upon
bpV(pic) treatment (Fig. 6E). The inability of cAMP-mediated signals to fully transactivate COX-2 gene expression can then be
explained by the fact that AP-1 can activate transcription only when
complexed with NFAT, which is not induced upon FSK treatment. This is
paralleled by the very inefficient transcriptional activation induced
by PMA or ionomycin alone as compared with the PMA/ionomycin combination (Fig. 1A). Another possible effect of cAMP would
involve serine phosphorylation of He-PTP by protein kinase A, which, in turn, would inhibit binding of He-PTP to MAP kinases, leading to their
activation (52). This effect would be sufficient to induce
transcription from the c-fos promoter and could then
contribute to the increase in AP-1 binding to the COX-2 promoter that
is observed upon FSK treatment.
It can be proposed that bpV molecules act by inhibiting one or several
PTP(s) that normally attenuate a constitutive kinase activity. Several
PTP are most likely targeted by bpV(pic), leading to the activation of
multiple kinases and to COX-2 transcriptional activation. However, our
results show that the PTK p56lck and ZAP-70 are
both critical to the bpV-induced activation, suggesting that the
signaling cascade is initiated by the inhibition of a PTP that normally
dephosphorylates p56lck and/or ZAP-70. Signaling
events transduced through the TCR complex following treatment with the
PTP inhibitor pervanadate was shown to be mediated by inhibition of
CD45 (18). The fact that bpV-mediated activation of COX-2 promoter was
still detected in the CD45-negative cell line J45.01 is an indication
that this T cell membrane PTP is not participating to this process
(data not shown). The Src homology-containing PTP SHP-1 represents an
ideal candidate, because it has been proposed as a negative regulator
of T cell activation by decreasing the activity of several molecules
involved in TCR signaling machinery, including
p56lck, phospholipase C
, ZAP-70, and Syk (25,
30, 53-55). Our data showing an inhibition of bpV-induced
transcriptional activation when SHP-1 is overexpressed further
strengthen this hypothesis. Overexpression of SHP-1 may counteract the
inactivation caused by bpV(pic) and restore the inhibition of kinase
activity. Our results are in contradiction with a previous report
stating that a PTP distinct from CD45, SHP-1, and SHP-2, present in T
cell membranes, is responsible for the
p56lck-dependent tyrosine
phosphorylation events that are induced by PTP inhibitors (56). It
should be mentioned that PTP inhibitors used in this study were
hydrogen peroxide and vanadate. It is thus possible that bpV molecules
target PTPs that are different from the ones affected by hydrogen
peroxide and vanadate.
Our data do not suggest an implication of He-PTP, although this
cytosolic PTP was shown to inhibit the TCR-induced activation of
NFAT/AP-1 (31). This discrepancy is probably related to some differences in the signaling pathway induced by bpV compared with TCR
stimulation. On the other hand, we observed an inhibition of
bpV-induced activation following overexpression of TC-PTP. Localization
of TC-PTP in the nucleus or endoplasmic reticulum suggests that this
PTP is not playing an active role in the most proximal TCR signaling
events (35). The inhibitory effect that is seen following
overexpression of wild type TC-PTP is then most likely related with
dephosphorylation of some transcription factors in the nucleus, leading
to a decrease in COX-2 transcriptional activity. Last, we cannot
totally exclude the implication of the Pro-Glu-Ser-Thr (PEST)-enriched
phosphatase that acts as a potent negative regulator of T cell
activation through an association with Csk and a possible direct
dephosphorylation of Src family PTK (27).
In summary, we have demonstrated that COX-2 transcriptional activity is
augmented following treatment by the PTP inhibitor bpV(pic), in a
manner very similar to the stimulation induced by TCR triggering. These
findings render bpV compounds very attractive for the restoration of
immune functions in immunosuppressed individuals, especially knowing
that COX-2 is an integral component of the TCR signaling cascade and is
crucial for T cell activation.