From the Centre de Recherche en Infectiologie, § Centre de Recherche en Oncologie et Endocrinologie Moléculaire, Centre Hospitalier Universitaire de Québec, Pavillon CHUL and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec G1V 4G2, Canada
Received for publication, June 12, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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Intercellular adhesion molecule-1 (ICAM-1)
plays an important role in adhesion phenomena involved in the immune
response. The strength of adhesion has been shown to be modulated by
changes in ICAM-1 gene expression. In T cells, signaling
pathways are intimately regulated by an equilibrium between
protein-tyrosine kinases and protein tyrosine phosphatases (PTP). The
use of bis-peroxovanadium (bpV) compounds, a class of potent PTP
inhibitors, enabled us to investigate the involvement of phosphotyrosyl
phosphatases in the regulation of ICAM-1 gene expression in
human T cells. Here, we demonstrate for the first time that inhibition
of PTP results in an increase of ICAM-1 surface expression on both
human T lymphoid and primary mononuclear cells. The crucial role played by the NF- Intercellular adhesion molecule-1
(ICAM-1)1 is an inducible
cell surface glycoprotein belonging to the immunoglobulin supergene family that shows a molecular mass ranging from 76 to 114 kDa depending
on the degree of glycosylation. Its cognate ligands include the
membrane-bound integrin receptor LFA-1, Mac-1 on leukocytes, the
soluble molecule fibrinogen, rhinoviruses, and Plasmodium falciparum malaria-infected erythrocytes (1-5). Within the immune system, ICAM-1 is expressed on cells of the monocyte-macrophage lineage, B lymphocytes, plasma cells, and on both memory and activated T lymphocytes. The association between ICAM-1 and the activated form of
the LFA-1 counter-receptor has many important roles in adhesion
phenomena involved in the immune system. Its basic function is the
induction of a specific and reversible cell-cell adhesion that enables
intercellular communication, T cell-mediated defense mechanism, and
inflammatory response. In addition, ICAM-1 is also involved in
leukocyte-endothelial cell interaction, cell differentiation, and in
many pathological complications such as acquired immunodeficiency syndrome, malignancies of both myeloid and lymphoid origin, and allergic asthma (6-8).
In a normal immune response, the initial contact between T lymphocytes
and antigen presenting cells is made possible through an interaction
between adhesion molecules such as ICAM-1 and LFA-1 expressed on the
surface of both T lymphocytes and antigen presenting cells. This
interaction leads to the association between the T cell receptor-CD3
complex and antigenic peptides in the context of major
histocompatibility complex class I and II molecules. If the latter
interaction occurs, T cell receptor-CD3 receptors and major
histocompatibility complex molecules transmit activation signals in
both cell partners. One of the first reactions following such
activation is an increase of adhesion strength stabilizing the
association between T cells and antigen presenting cells. Additional
links occur between other molecules expressed on both cell surfaces
that are required for completing adhesion and cell signaling and
consequently determining the following response. It ensues that a
dysfunction in ICAM-1 gene expression results in an
immunological impairment or a physiopathological situation (7,
8).
The regulation of ICAM-1 gene expression occurs primarily at
the level of transcription and is cell type-specific. This phenomenon involves different signaling pathways and several enhancer elements such as palindromic interferon- In T cells, the expression of many genes is tightly regulated by an
equilibrium between two sets of enzymes with distinct properties, the
protein-tyrosine kinases and protein tyrosine phosphatases (PTP). The
role of protein-tyrosine kinases in T cell gene expression has been
well documented (29). Recently, some reports have described the role of
PTP in T cell signaling and T cell transduction (30-35), but the
involvement of PTP in the regulation of ICAM-1 gene
expression in T cells is unclear. Of interest is the observation that
the PTP inhibitor pervanadate can mimic IFN- The primary objective of the present work was to investigate the role
of PTP in the regulation of ICAM-1 gene expression in human
T cells. We show here that treatment of primary human peripheral blood
mononuclear cells and the human leukemic T cell lines Jurkat, HUT 78, and WE17/10 with the bis-peroxovanadium compound bpV[pic], a strong
inhibitor of PTP, results in the induction of ICAM-1 surface
expression. Further experiments revealed that NF- Reagents--
Phorbol 12-myristate 13-acetate (PMA) and
ionomycin (Iono) were purchased from Sigma and Calbiochem,
respectively. Sodium orthovanadate (Sigma) was freshly dissolved before
its use in 10 mM HEPES, pH 7.4. The bpV[pic] compound was
prepared as described previously (37). Briefly,
V2O5 was dissolved in an aqueous KOH solution
and then mixed with 30% H2O2 and an ancillary
ligand (picolinic acid anion in this study hence bpV[pic]) in
addition to the ethanol for optimal precipitation. Characterization of bpV[pic] was carried out by infrared 1H NMR and vanadium
51 (51V) NMR spectroscopy. Stock solutions of bpV[pic] (1 mM in phosphate-buffered saline, pH 7.4) were kept at
Cells and Culture Conditions--
The parental lymphoid T cell
line Jurkat (clone E6.1) was obtained from the American Type Culture
Collection (ATCC, Manassas, VA). Jurkat cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS)
(HyClone Laboratories, Logan, UT), 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.22%
NaHCO3, in a 5% CO2-humidified atmosphere. The
human IL-2-dependent T lymphoblastoid cell line WE17/10
(38) and the human cutaneous T lymphoma cell line HUT 78 (39) were provided by the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health (Bethesda), and were
maintained in complete culture medium in the presence of recombinant
human IL-2 (50 units/ml) for WE17/10. Peripheral blood mononuclear
cells (PBMCs) from healthy donors were isolated by Ficoll-Hypaque
density gradient centrifugation and were cultured in complete culture
medium in the presence of phytohemagglutinin (Sigma) (3 µg/ml) and
recombinant human IL-2 (30 units/ml) for 3 days at 37 °C. Such cells
were left untreated in complete culture medium containing 20%
heat-inactivated FBS for 3 days prior to treatment with either
bpV[pic] or PMA/Iono. The following reagent was obtained through the
AIDS Research and Reference Reagent Program: recombinant human
interleukin-2 from Maurice Gately, Hoffmann-La Roche (40).
Flow Cytometry--
Cell surface expression of ICAM-1 was
evaluated by flow cytometry as follows. Jurkat, HUT 78, WE17/10 cells,
and PBMCs (1 × 106) were washed once in
phosphate-buffered saline containing 2% FBS (PBSA). Cells were then
resuspended in 100 µl of PBSA to which was added 1 µg of monoclonal
anti-ICAM-1 antibody (clone RR1/1.1.1), vortexed gently, and incubated
for 30 min on ice. Cells were subsequently washed with PBS containing
2% FBS and resuspended in 100 µl of PBS containing
(R)-phycoerythrin-conjugated goat anti-mouse IgG (0.5 µg
total) and further incubated for 30 min on ice. Cells were finally
centrifuged and resuspended in 1% paraformaldehyde in PBS before being
analyzed by flow cytometry (EPICS XL, Coulter Corp., Miami, FL).
Plasmids and Antibodies--
Reporter plasmids of the ICAM-1
5'-regulatory element and mutants used in these experiments are cloned
upstream from the firefly luciferase gene. pGL1.3, pGL1.3 Transient Transfection by DEAE-Dextran--
Jurkat cells (5 × 106) were first washed once in TS buffer (137 mM NaCl, 25 mM Tris-HCl, pH 7.4, 5 mM KCl, 0.6 mM NaHPO4, 0.5 mM MgCl2, and 0.7 mM
CaCl2) and resuspended in 0.5 ml of TS containing 15 µg
of the indicated plasmids and 500 µg/ml DEAE-dextran (final concentration). The cell/TS/plasmid/DEAE-dextran mixture was incubated for 25 min at room temperature. Thereafter, cells were diluted at a
concentration of 1 × 106 per ml using complete
culture medium supplemented with 100 µM chloroquine
(Sigma). After 45 min of incubation at 37 °C, cells were
centrifuged, washed once, resuspended in complete culture medium, and
incubated at 37 °C for 24 h. Transiently transfected cells were
seeded at a density of 105 cells per well (100 µl) in
96-well flat-bottom plates. In most experiments, cells were left
untreated or were either treated with bpV[pic], sodium orthovanadate,
or PMA/Iono in a final volume of 200 µl for a period of 8 h for
bpV[pic] or 24 h for PMA/Iono and sodium orthovanadate. Cells
were then lysed, and luciferase activity was monitored with a
microplate luminometer (MLX; Dynex Technologies, Chantilly, VA).
Preparation of Nuclear Extracts--
Jurkat cells were either
left untreated or were incubated for different times at 37 °C with
bpV[pic] (10 µM) or PMA/Iono (20 ng/ml and 1 µM, respectively). Incubation of Jurkat cells with the
various stimulating agents was terminated by the addition of ice-cold
PBS, and nuclear extracts were prepared according to the microscale
preparation protocol (43). In brief, sedimented cells were resuspended
in 400 µl of cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride). After 15 min on ice, 25 µl of 10%
Nonidet P-40 was added. The lysate was vortexed for 10 s, and
samples were centrifuged for 30 s at 12,000 × g.
The supernatant fraction was discarded, and the cell pellet was
resuspended in 100 µl of cold buffer B (20 mM HEPES, pH
7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM
EGTA, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride). Cells were then rocked vigorously at
4 °C for 15 min. Cellular debris were removed by centrifugation at
12,000 × g for 5 min at 4 °C, and the supernatant
fraction was stored at Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assay was performed with 10 µg of nuclear extracts.
Protein concentrations were determined by the bicinchoninic assay with
a commercial protein assay reagent (Pierce). Nuclear extracts were
incubated for 30 min at room temperature in 15 µl of buffer C (100 mM HEPES, pH 7.9, 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM dithiothreitol, 5 mM
EDTA, 250 mM NaCl, 2 µg of poly(dI-dC), 10 µg of
nuclease-free bovine serum albumin (fraction V) containing 0.8 ng of
radiolabeled-labeled double-stranded DNA (dsDNA) oligonucleotide.
Double-stranded DNA (100 ng) was labeled with
[ ICAM-1 Expression Is Increased in Human T Lymphoid Cells and
Primary Cells by the PTP Inhibitor bpV[pic]--
Given that
intracellular tyrosine phosphorylation levels are crucial in the
regulation of numerous genes, we investigated the effect of the
PTP-specific inhibitor bpV[pic] on ICAM-1 protein expression in the
human leukemic T cell line Jurkat and also in primary cells
(i.e. PBMCs). In this set of experiments, cells were treated
either with the PMA/Iono combination (as a control) or bpV[pic]
compound, and the percentage of ICAM-1-expressing cells as well as the
mean fluorescence intensity (indicative of the number of molecules per
single cell shown on a logarithmic scale) were defined by flow
cytometry analyses with the use of an antibody specific for ICAM-1
(clone RR1/1.1.1). As depicted in Fig. 1,
A and D, ICAM-1 is constitutively expressed on
both Jurkat cells and PBMCs. PMA/Iono treatment resulted in a slight increase ICAM-1 expression on Jurkat cells, whereas a much greater induction of ICAM-1 protein was mediated by these stimuli in primary cells. Interestingly, treatment with the tyrosine phosphatase-specific inhibitor bpV[pic] resulted in a much greater up-regulation of ICAM-1
protein expression on Jurkat leukemic T cells than PMA/Iono. Inhibition
of PTP by the specific inhibitor bpV[pic] also leads to a marked
induction of ICAM-1 expression in PBMCs. Cell viability was not
affected by PMA/Iono and bpV[pic] treatments as monitored by
performing in parallel MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays (data not shown). These data represent the first demonstration that PTPs are implicated in ICAM-1 gene expression in human
T cells. It should be noted that we have made similar observations using HUT 78, another human T cell lymphoma line, and WE17/10, an
IL-2-dependent T cell receptor/CD4-expressing cell line
established from the blood cells of a patient with T cell lymphoma
(Fig. 1, B and C, respectively). This last series
of experiments indicate that the noticed bpV[pic]-mediated induction
of ICAM-1 gene expression is not an epiphenomenon since it
is observed in several human T cell lines.
Transcriptional Regulation of the ICAM-1 Gene by bpV[pic]
Compound--
It is well documented that ICAM-1 gene
expression is primarily regulated at the transcriptional level. In an
attempt to study the effect of bpV[pic] on ICAM-1 transcription, a
dose-response experiment was initially carried out using increasing
concentrations of this PTP inhibitor. To this end, Jurkat cells were
transiently transfected with a reporter construct made of the
luciferase gene placed downstream of the entire ICAM-1
promoter (i.e. pGL1.3). Next, cells harboring the
ICAM-1-luciferase vector were either left untreated or were treated for
8 h with the indicated bpV[pic] concentrations (Fig.
2A). A
dose-dependent increase of ICAM-1 promoter
activity in Jurkat cells transiently transfected with pGL1.3 was
observed when using concentrations of bpV[pic] ranging from 1 to 10 µM (1.2-42.9-fold increase). A slight decrease of ICAM-1-driven luciferase activity was detected at the highest concentration tested (i.e. 20 µM) (34.0-fold
increase), which could be due to cell toxicity. Subsequent experiments
were thus conducted using bpV[pic] at a maximal concentration of 10 µM. Sodium orthovanadate
(Na3VO4), a commonly used PTP inhibitor, was
similarly tested in this series of investigations. As shown in Fig.
2B, a weak increase in ICAM-1-driven luciferase activity was
obtained with concentrations of Na3VO4 ranging
from 12.5 to 50 µM (1.1-1.5-fold induction). Therefore,
these data suggest that bpV[pic] is a much more potent activator of
ICAM-1 promoter transcription than the other PTP inhibitor
tested, i.e. sodium orthovanadate. Kinetic analyses were
also performed to define the appropriate incubation time to reach
optimal bpV[pic]- and PMA/Iono-mediated activation of ICAM-1
transcription. As shown in Fig. 2C, bpV[pic] was found to
be markedly more potent than PMA/Iono combination with respect to
activation of ICAM-1 transcription. Moreover, maximal activation of
ICAM-1-dependent luciferase activity was reached after
8 h following bpV[pic] treatment (22.6-fold increase), whereas
the highest induction of ICAM-1 transcription was seen following
24 h of treatment with PMA/Iono (6.7-fold increase). These time
points were thus used for the following series of investigations.
Identification of bpV[pic]-responsive Elements in the ICAM-1
Promoter in Human T Cells--
By having demonstrated that bpV[pic]
compound acts as a potent inducer of ICAM-1 transcription in human T
cells, we next characterized the cis-regulatory element(s)
located within the 5'-flanking sequences of the ICAM-1
promoter that confers responsiveness to this tyrosine phosphatase-specific inhibitor. This goal was achieved using a series
of ICAM-1 reporter constructs carrying either deletions or point
mutations in the 5' region of the promoter and
trans-dominant negative mutants of some specific
transcription factors. Each of these molecular constructs was
transiently transfected into Jurkat cells, and the luciferase
activities of control, PMA/Iono, and bpV[pic]-treated cells were determined.
We initially tested the involvement of the mammalian ubiquitous
transcription factor NF-
Previous observations suggest that NF-
The ICAM-1 promoter contains also a more distal consensus
NF-
Our previous results indicated that a point mutation in the proximal
NF-
The proximity of pI
It has been previously demonstrated that IFN- ICAM-1 gene expression is regulated by numerous
inducing (e.g. TNF- Our results first demonstrated that bpV[pic] was effective in
inducing ICAM-1 surface expression. Flow cytometry analyses showed that
bpV[pic] compound was able to increase both the number of
ICAM-1-expressing cells and the mean fluorescence intensity. The
physiological significance of the current work was provided by the
observation that bpV[pic] treatment leads to the induction of ICAM-1
expression not only in established T cell lines (i.e. Jurkat, HUT 78, and WE17/10) but also in primary human PBMCs. Further
experiments revealed that the up-regulatory effects of bpV[pic] on
ICAM-1 transcriptional activity are far superior than those of sodium
orthovanadate (Na3VO4), another described
powerful PTP inhibitor. Interestingly, such results are consistent with a previous report showing that bpV molecules are more potent inducers of human immunodeficiency virus promoter activity than
Na3VO4 (49). These observations suggest that
the inhibitory effects of bpV[pic] on intracellular PTP may be
distinct from those of sodium orthovanadate, in terms of potency and/or
substrate specificity. Further studies are needed to shed light on this matter.
Data from several reports have clearly indicated that the transcription
factor NF- The nuclear phosphoproteins Ets have been reported to be involved in
activation of the ICAM-1 promoter in various cell types including rabbit kidney carcinoma, human choriocarcinoma, and endothelial cell lines (26, 27). To evaluate the implication of Ets
transcriptional factors in bpV-mediated activation of ICAM-1, we
transiently transfected Jurkat cells either with pGLE, pGLE In addition to binding sites for NF- In conclusion, our findings indicate that ICAM-1 gene
expression in human T cells is under the control of constitutive PTP activity, which serves to maintain ICAM-1 expression at a basal level.
A more detailed understanding of the transcription factors involved in
bpV[pic]-mediated activation of ICAM-1 expression will be needed to
determine how the various cooperative protein-protein interactions
regulate the transcriptional activation of the ICAM-1 gene
in human T cells.
B-, Ets-, and pI
RE-binding sites in bpV[pic]-mediated activation of ICAM-1 was demonstrated using various 5' deletion and
site-specific mutants of the ICAM-1 gene promoter driving the luciferase reporter gene. Co-transfection experiments with trans-dominant mutants and electrophoretic mobility shift
assays confirmed the importance of constitutive and inducible
transcription factors that bind to specific responsive elements in
bpV-dependent up-regulation of ICAM-1 surface expression.
Altogether, these observations suggest that expression of ICAM-1 in
human T cells is regulated by phosphotyrosyl phosphatase activity
through NF-
B-, Ets-, and STAT-1-dependent signaling pathways.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-responsive element (pI
RE),
NF-
B, Ets, C/EBP, AP-1-like, Sp1, and retinoic acid response
elements (7-9). These numerous enhancer elements contained in the
ICAM-1 promoter suggest a complex regulation that is still
ill-defined in human T cells. In various cell types, signal transducers
and activators of transcription (STAT) factors, and more specifically STAT-1 and STAT-3, can bind the ICAM-1 promoter pI
RE and
are strongly involved in ICAM-1 gene expression (10-19).
NF-
B has also been reported to play a pivotal role in
ICAM-1 gene regulation where RelA (p65)/RelA, RelA/c-Rel,
and RelA/NF-
B1 (p50) dimers can potently induce ICAM-1 expression in
several cell types (20-25). Both JAK/STAT and NF-
B pathways have
been shown to be modulated by phosphorylation events that lead to their
translocation into the nucleus. In addition to JAK/STAT and NF-
B,
the Ets gene family of transcriptional factors is also involved in the
regulation of ICAM-1 expression (26, 27). The control of highly diverse sets of genes by Ets proteins involves their own regulation at different levels which include, among others, specific phosphorylation events mediated by the Ras-MAPK pathway in response to extracellular signals (28).
-mediated induction of
ICAM-1 expression via nuclear translocation of STAT-1 proteins in human
keratinocytes (17). Moreover, calyculin A and okadaic acid, two
phosphoseryl/threonyl phosphatase inhibitors, induce an
ICAM-1/LFA-1-dependant homotypic aggregation of both Jurkat and U937
cells (36). However, the mechanisms leading to this ICAM-1/LFA-1
aggregation have not been defined. Altogether, these reports suggest
that both PTP and phosphoseryl/threonyl phosphatases are involved in
ICAM-1 expression.
B, Ets, and
pI
RE-binding sites are important sequence motifs in
bpV[pic]-mediated up-regulation of ICAM-1 expression. These results
suggest that ICAM-1 is regulated in human T cells by PTP activity.
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ABSTRACT
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DISCUSSION
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85 °C into small aliquots until used.
Bmut, pGL
HindIII, and pGL HindIII IRE mut were provided by
Dr. T. P. Parks (Boehringer Ingelheim, Ridgefiel, CN), and pGLE
WT, pGLE
138mut, pGLE
158mut, and pGLE
138/
158mut were kindly
supplied by Dr. Y. de Launoit (Institut Pasteur, Lille, France).
Anti-STAT-1, anti-STAT-3, and anti-p50 antibodies were purchased from
Santa Cruz Biotechnology. Dr. N. Rice (NCI, Frederick, MD) kindly
provided the polyclonal anti-p65 antibodies. Dr. Rothlein
(Boehringer Ingelheim, Ridgefield, CN) provided the anti-ICAM-1
antibody RR1/1.1.1 (anti-CD54) (41). The dominant negative
I
B
-expressing vector pCMV-I
B
S32A/36A has been described
previously (42) (a kind gift from Dr. W. C. Greene, The Gladstone
Institutes, San Francisco). The DNA filler pCMV-EcoRV/SmaI was constructed from the
expressing vector pCMV-I
B
S32A/36A by deletion of the cDNA
encoding for I
B
S32A/36A with EcoRV/SmaI digestion.
70 °C until used.
-32P]ATP and T4 polynucleotide kinase in a kinase
buffer (New England Biolabs, Beverly, MA). This mixture was incubated
for 20 min at room temperature, and the reaction was stopped with 5 µl of 0.2 M EDTA. The labeled oligonucleotide was
extracted with phenol/chloroform and passed through a G-50 spin column.
The dsDNA oligonucleotides, which were used as probes or as
competitors, contained either the nonspecific probe Oct-2A
(5'-GGAGTATCCAGCTCCGTAGCATGCAAATCCTCTGG-3'), the proximal
NF-
B-binding site (5'-GATTGCTTTAGCTTGGAAATTCCGGAGCTG-3'), the distal
NF-
B-binding site (5'-AGGGAGCCCGGGGAGGATTCCTGGGCC-3'), the pI
RE
(5'-AAGGCGGAGGTTTCCGGGAAAGCAGCACC-3'), the wild-type
138/
158
Ets-binding sites (5'-CTGTCAGTCCGGAAATAACTGCAGCATTTGTTCCGGAGGGGAAG-3'), or the
138/
158-mutated Ets-binding sites
(5'-CTGTCAGTCCCCAAATAACTGCAGCATTTGTTGGGGAGGGGAAG-3') of the ICAM-1 5'-regulatory element. DNA-probe complexes were resolved
from free labeled DNA by electrophoresis in native 4% (w/v)
polyacrylamide gels containing 50 mM Tris-HCl, pH 8.5, 200 mM glycine, and 1 mM EDTA. The gels were
subsequently dried and autoradiographed. Cold competitor assays were
carried out by adding a 100-fold molar excess of homologous unlabeled
dsDNA proximal or distal NF-
B, pI
RE, or Ets oligonucleotides
simultaneously with the labeled probe. Supershift assays were performed
by preincubation of nuclear extracts with 1 µl of specific
antibodies in the presence of all the components of the binding
reaction described above for 30 min at 4 °C.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Cytometric analyses of PMA/Iono- and
bpV[pic]-dependent modulation of ICAM-1 surface
expression on Jurkat, HUT 78, WE17/10 cells, and PBMCs. Cells were
incubated with an antibody specific for ICAM-1 (clone RR1/1.1.1) in
combination with a (R)-phycoerythrin-conjugated goat
anti-mouse IgG (dotted lines). Controls consisted of cells
incubated with an isotype-matched irrelevant monoclonal antibody
(solid lines).
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Fig. 2.
Dose-dependent and kinetic
analyses of bpV[pic]-, PMA/Iono-, and sodium orthovanadate-mediated
effect on ICAM-1 gene expression in human T
cells. Jurkat cells were transiently transfected with pGL1.3 and
were next stimulated for 8 h with increasing doses of bpV[pic]
(1, 2.5, 5, 10, and 20 µM) (A) or for 24 h with Na3VO4 (12.5, 25, and 50 µM) (B). Cells were then lysed, and luciferase
activity was monitored with a microplate luminometer. Transiently
transfected Jurkat cells were either treated with bpV[pic] (10 µM) or PMA/Iono (20 ng/ml and 1 µM,
respectively) for different times (2, 4, 6, 8, 24, and 48 h) prior
to monitoring luciferase activity in cell lysates (C).
Results shown are the means ± S.D. of four determinations. These
results are representative of three independent experiments.
B in bpV-induced activation of
ICAM-1 promoter transcription. NF-
B is a pleiotropic
transcription factor complex that mediates the regulated expression of
multiple immunomodulatory genes bearing cis-acting
B
enhancer elements, including the
light chain of immunoglobulins,
cytokines, as well as known genes for some cell adhesion molecules
including ICAM-1 (44). NF-
B has been postulated to play a key role
in the cell type- and stimulus-specific regulation of ICAM-1 (7).
Considering that the proximal NF-
B-binding site located about 200 bp
upstream of the translation initiation site has been demonstrated to be
particularly important for the induction of ICAM-1 transcription (45,
46), we used pGL1.3 and a luciferase-encoding vector constituted of the
full-length ICAM-1 promoter bearing a point mutation in the
most proximal NF-
B-binding site (i.e. pGL1.3
Bmut).
Cells were then either left untreated or were treated with bpV[pic]
for 8 h and PMA/Iono for 24 h. Again, high levels of ICAM-1
induction were observed with the reporter construct containing the
complete ICAM-1 promoter (Fig.
3A). However, mutation of the
proximal NF-
B-binding site resulted in a significant decrease in the
induction ratio in response to both bpV[pic] (compare 20.3- and
4.4-fold induction) and PMA/Iono treatment (compare 8.9- and 5.1-fold
increase). Therefore, it can be concluded that the proximal
NF-
B-binding site is a critical DNA-regulatory element responsible
for ICAM-1 induction in T cells which is seen upon treatment with the
PTP-specific inhibitor bpV[pic].
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Fig. 3.
Involvement of NF- B
in bpV[pic]-mediated induction of ICAM-1 transcription.
A, Jurkat cells were transiently transfected with pGL1.3 or
pGL1.3
Bmut and were next either left untreated or were treated with
PMA/Iono (20 ng/ml and 1 µM, respectively) or bpV[pic]
(10 µM). B, Jurkat cells were transiently
co-transfected with pGL1.3 and either
pCMV-EcoRV/SmaI (empty vector control) or
pCMV-I
B
S32A/36A. Next, cells were left untreated or were treated
with PMA/Iono (20 ng/ml and 1 µM, respectively) or
bpV[pic] (10 µM). The cells were lysed, and luciferase
activity was monitored with a microplate luminometer. Results shown are
the means ± S.D. of four determinations. These results are
representative of three independent experiments.
B complexes can interact with
other transcription factors that are recognized to bind to the
ICAM-1 promoter (47, 48). Thus, to address more completely the relative contribution of NF-
B in bpV-dependent
activation of ICAM-1 transcription, Jurkat cells were co-transfected
with pGL1.3 and a construct encoding for a dominant negative version of
I
B
mutated to alanine on both serine 32 and 36 residues
(i.e. pCMV-I
B
S32A/36A). The I
B
repressor
protein encoded by pCMV-I
B
S32A/36A is sequestered in the
cytoplasm and renders the NF-
B complex unable to translocate to the
nucleus. The capacity of the used I
B
repressor to abolish nuclear
translocation and activation of NF-
B was initially tested by
transient transfection of Jurkat cells with pCMV-I
B
S32A/36A and
pNF-
B-LUC, a vector made of five consensus binding sites for NF-
B
(data not shown). Transfection of the empty promoter vector
(i.e. pCMV-EcoRV/SmaI) had no effect on bpV[pic]-mediated reporter gene activity (Fig. 3B).
However, when the mutated version of the repressor was instead used,
incubation of the cells with bpV[pic] compound showed a marked
reduction in ICAM-1 promoter activity (compare 35.0- and
16.0-fold increase). A more dramatic diminution of ICAM-1-driven
reporter gene activity was seen following treatment with PMA/Iono
combination (compare 7.8- and 1.0-fold induction). These data further
confirmed the essential role played by NF-
B in bpV[pic]-induced
ICAM-1 promoter activity.
B-binding site. In an attempt to assess the putative implication of both domains in the noticed up-regulation of ICAM-1 transcription by
bpV[pic] compound, the proximal and distal NF-
B-binding sites of
the ICAM-1 promoter were labeled and used as probes for DNA mobility shift assays. Nuclear extracts from untreated, PMA/Iono-, and
bpV[pic]-treated Jurkat cells were used in these experiments. Nuclear
proteins were extracted after 60 min of treatment with either PMA/Iono
or bpV[pic] since maximal NF-
B nuclear translocation was seen at
this time (data not shown), an observation that is consistent with a
previous report (49). As illustrated in Fig. 4, a complex was formed following
treatment with both the tyrosine phosphatase-specific inhibitor
bpV[pic] (Fig. 4A, compare lanes 2 and
1) and PMA/Iono combination (Fig. 4B, compare
lanes 2 and 1) only when using the proximal
NF-
B-binding site as a probe. Indeed, no such complex could be
induced by either PMA/Iono or bpV[pic] treatment when the distal
NF-
B-binding site was used as a probe. The complex was competed away
by the addition of a 100-fold molar excess of cold NF-
B
oligonucleotide (lanes 5). However, complex formation was
not affected by a nonspecific oligonucleotide (Oct-2A) (lanes
6), demonstrating the specificity of the signal. To identify the
NF-
B proteins involved in complex formation, antibodies against two
of the most prominent NF-
B isoforms (i.e. p50 and p65)
were incubated with nuclear extracts from PMA/Iono- and
bpV[pic]-treated Jurkat cells before the addition of labeled probes.
Antibody against p50 significantly decreased the complex formation and
a supershift (lanes 3), whereas anti-p65 antibody caused a
partial supershift (lanes 4). It is thus clear that the protein complex bound to the proximal NF-
B-binding site of the ICAM-1 promoter is composed of both p50 and p65 subunits.
These results clearly indicate that bpV[pic] compound is mediating
nuclear translocation of NF-
B, and such a finding is perfectly in
line with our previous transcriptional studies (Figs. 2 and 3).
View larger version (56K):
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Fig. 4.
bpV[pic] induces
NF- B complexes that bind to the proximal
NF-
B-binding site of the ICAM-1
promoter. Labeled proximal (prox) and distal
(dist) NF-
B-binding sites of the ICAM-1
promoter were incubated with nuclear extracts from Jurkat cells either
left untreated (lanes 1 and 7) or treated for 60 min with bpV[pic] (A, lanes 2-6 and 8-12) or
PMA/Iono combination (B, lanes 2-6 and
8-12). Binding specificity was tested by adding a 100-fold
molar excess of either cognate NF-
B oligonucleotide (lanes
5 and 11) or a nonspecific (non-spec) probe
(Oct-2A) (lanes 6 and 12). For gel supershift
assays, nuclear extracts were also incubated with antibody against p50
and p65 isoforms. These results are representative of three independent
experiments.
B-binding site or the use of a dominant negative form of the
I
B
repressor did not completely eliminate the ability of the
full-length ICAM-1 promoter to respond to bpV[pic] (Fig. 3, A and B). Thus, it suggests that
NF-
B-independent signal transduction pathway(s) might be involved as
well. Previous studies have identified two functional Ets-binding sites
in the ICAM-1 proximal promoter (26, 27). It should be
emphasized that the role of these two transcription regulatory elements
in human T cells with respect to the transcriptional regulation of the
ICAM-1 gene remains to be defined. Cells were then
transfected in a transient fashion with a luciferase-encoding vector
driven by the first 176 nucleotides of the ICAM-1 promoter
region (pGLE WT). This region of the ICAM-1 promoter harbors
two Ets-binding sites (positions
138 and
158 relative to the start
of transcription) in addition to the pI
RE, Sp1- and AP-2-binding
sites, and a TATA box. Moreover, cells were also transfected with
molecular constructs bearing point mutations either at each (pGLE
138mut and pGLE
158mut) or both (pGLE
138/
158mut) Ets-binding
sites. Jurkat cells were next either left untreated or were treated
with bpV[pic] for 8 h and PMA/Iono for 24 h. A comparable
level of bpV[pic]- and PMA/Iono-mediated ICAM-1 induction was
observed when using wild-type and mutated versions of the ICAM-1 promoter carrying single point mutation in either
138 or
158 Ets-binding site (Fig. 5).
Interestingly, when both Ets-binding sites were mutated, the induction
by bpV[pic] was severely reduced (31.2-fold versus
14.2-fold increase). It should be noted that PMA/Iono-mediated
induction of ICAM-1 transcription is not markedly affected by such
mutations. To determine whether these two sequence motifs can function
as protein-binding sites, the responsive region composed of both
138
and
158 wild-type Ets-binding sites was labeled and used as a probe
for mobility shift assays. First, we noticed that there was a
constitutive nuclear translocation of Ets in Jurkat cells that was not
modulated by a treatment with bpV[pic] or PMA/Iono (Fig.
6, compare lanes 1 and
2). The Ets complex was competed away by addition of a
100-fold molar excess of cold Ets-binding sites (lane
3) and not by a nonspecific oligonucleotide (Oct-2A) (lane
4). No signal was detected when using a probe containing mutations
at the two Ets-binding sites (lanes 5-8). Altogether results from transient transfection experiments and mobility shift assays demonstrated the importance of Ets-binding sites in
bpV[pic]-mediated ICAM-1 gene expression, although the
formed migrating complex with the Ets probe is not modulated by
bpV[pic] treatment.
View larger version (16K):
[in a new window]
Fig. 5.
bpV[pic]-mediated up-regulation of
ICAM-1 transcription necessitates Ets-binding sites. Jurkat
cells were transiently transfected with pGLE WT, pGLE 138mut, pGLE
158mut, and pGLE
138/
158 mut before being either left untreated
or treated with PMA/Iono (20 ng/ml and 1 µM,
respectively) or bpV[pic] (10 µM). Next, cells were
then lysed, and luciferase activity was monitored with a
microplate luminometer. Results shown are the means ± S.D. of
four determinations. These results are representative of three
independent experiments.
View larger version (56K):
[in a new window]
Fig. 6.
Constitutive expression of transcription
factors that bind to the Ets-binding sites in the ICAM-1
promoter. Labeled Ets-binding sites of the ICAM-1
promoter were incubated with nuclear extracts from Jurkat cells either
left untreated (lanes 1 and 5) or treated for 60 min with bpV[pic] (lanes 2 and 6) or PMA/Iono
combination (lanes 2 and 6). Binding specificity
was tested by adding a 100-fold molar excess of a probe constituted of
either the cognate 138/
158 WT Ets oligonucleotide (lanes
3 and 7) or a nonspecific (non-spec) probe
(Oct-2A) (lanes 4 and 8). These results are
representative of three independent experiments.
RE (i.e. about 100 bp upstream of the
translation initiation site) to the Ets-binding sites prompted us to
investigate the functional importance of the ICAM-1 pI
RE palindromic STAT-binding site in the transcriptional regulation of the
ICAM-1 gene by the potent tyrosine phosphatase inhibitor
bpV[pic]. Cells were transiently transfected with the ICAM-1 WT
reporter construct (pGL1.3), a 5' deletion mutant of pGL1.3 that
contains 277 bp of the ICAM-1 promoter (pGL1.3
HindIII) or a vector carrying a site-specific mutation in
pI
RE (pGL1.3 HindIII IRE mut). It is clear that the first
277-bp region of the ICAM-1 5'-flanking sequence is as efficient as the
full-length ICAM-1 promoter to drive the expression of a
reporter gene in response to bpV[pic] and PMA/Iono treatment (Fig.
7). A significant decrease in the
induction ratio with bpV[pic] was seen with the site-specific mutant
plasmid pGL1.3 HindIII IRE mut as compared with the 5'
deletion mutant pGL1.3 HindIII (compare 25.3- and 5.4-fold
induction). We next defined by EMSA whether treatment of Jurkat cells
with bpV[pic] induced the specific binding of transcription factors
to the ICAM-1 pI
RE site. Results from Fig.
8 indicate that bpV[pic] (compare
lane 3 with lane 1) but not PMA/Iono (compare
lanes 9 and 10 with 8) treatment leads
to a DNA-protein complex of increased intensity. The complex formation
was competed away by the addition of a 100-fold molar excess of
an unlabeled pI
RE probe (lane 6) but not by a nonspecific
oligonucleotide (Oct-2A) (lane 7).
View larger version (12K):
[in a new window]
Fig. 7.
pI RE is important in
the ICAM-1 promoter to confer responsiveness to
bpV[pic] treatment. Jurkat cells were transiently transfected
with pGL1.3, pGL 1.3 HindIII, or pGL HindIII IRE
mut. Next, cells were either left untreated or were treated with
PMA/Iono (20 ng/ml and 1 µM, respectively) or bpV[pic]
(10 µM) and lysed, and luciferase activity was monitored
with a microplate luminometer. Results shown are the means ± S.D.
of four determinations. These results are representative of three
independent experiments.
View larger version (99K):
[in a new window]
Fig. 8.
Treatment with bpV[pic] leads to nuclear
translocation of STAT-1 that binds to the pI RE
element in the ICAM-1 promoter. Labeled pI
RE
oligonucleotide was incubated with nuclear extracts from Jurkat cells
either left untreated (lanes 1 and 8) or treated
for 60 and 120 min with bpV[pic] (lanes 2 and
3) or PMA/Iono combination (lanes 9 and
10). Binding specificity was tested by adding a 100-fold
molar excess of a probe composed of either the cognate pI
RE
oligonucleotide (lanes 6 and 13) or a nonspecific
(non-spec) probe (Oct-2A) (lanes 7 and
14). For gel supershift assays, nuclear extracts were also
incubated with antibody specific either for STAT-1 (lanes 4 and 11) or STAT-3 (lanes 5 and 12).
These results are representative of three independent
experiments.
induces STAT-1,
whereas IL-6 mediates binding of both STAT-1 and STAT-3 transcriptional factors to the pI
RE element in the ICAM-1 promoter (17,
50). Thus, we defined whether the bpV[pic]-induced complex is
constituted of either STAT-1, STAT-3, or both by performing supershift
analyses. To this end, nuclear proteins were extracted from untreated
and bpV[pic]-treated Jurkat cells (60 and 120 min) before incubation with anti-STAT-1 or anti-STAT-3 antibody and the radiolabeled ICAM-1
pI
RE. Antibody against STAT-1 diminished the binding and caused a
partial supershift of the bpV[pic]-induced complex (compare lane 4 and 3), whereas anti-STAT-3 antibody did
not affect the complex formation mediated by bpV[pic] treatment
(compare lanes 5 and 3). These results suggest
that bpV[pic] results in the formation of a DNA-protein complex
constituted of ICAM-1 pI
RE site and STAT-1 transcription factor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, IFN-
, PMA, and IL-1) or
inhibitory factors (e.g. IL-4, IL-10, and glucocorticoids).
The architecture of the ICAM-1 promoter is complex and is
thus regulated by an interplay between different transcription factors
such as STAT, NF-
B, Ets, C/EBP, and Sp1 as demonstrated in various
cell types including acute myeloid leukemia blast cells, B lymphocytes,
endothelial cells, and epithelial cells (7, 8). Previous studies
reported that protein phosphorylation could be involved in
ICAM-1 gene expression. In human monocytic U937 and human
lymphocytic Jurkat cell lines, okadaic acid and calyculin A (Ser/Thr
phosphatases inhibitors) promote ICAM-1 and LFA-1-mediated homotypic
aggregation (36). In addition, phenylarsine oxide, pervanadate, and
diamide (PTP inhibitors) have been shown to block the TNF-induced
endothelial cell surface adhesion molecules (ICAM-1, VCAM-1, and
ECAM-1) (45). Another study reported that pervanadate mimics the
IFN-
induction of ICAM-1 gene expression (17). Although
central to many different pathways in T cells, tyrosine phosphorylation
events per se have not been directly investigated in the
regulation of ICAM-1 gene expression. In this study, we have
thus analyzed the role of tyrosine phosphorylation events in
transcriptional regulation of the ICAM-1 promoter in human T
cells. The bpV[pic] compound, previously characterized as one of the
most potent PTP inhibitors, was used in this study to break the
equilibrium between PTP and protein-tyrosine kinases and therefore to
increase intracellular phosphotyrosine levels.
B is playing a central role in the induction of ICAM-1
(25, 46-48) that is seen following treatment with PMA, TNF-
, IL-1,
and lipopolysaccharide (20, 24, 51). This family of transcriptional
factors has the ability to interact with other transcriptional factors
such as Fos/Jun, C/EBP, and Sp1 (52-54). Our results are perfectly in
line with these previous observations since we defined that NF-
B is
a second messenger actively participating in bpV[pic]-mediated
expression of ICAM-1 in human T cells. Although Imbert and colleagues
(55) have reported that the PTP inhibitor pervanadate can activate
NF-
B via tyrosine phosphorylation of I
B
with no concomitant
proteolytic degradation of I
B
, our experiments performed with a
dominant negative form of I
B
lead us to propose that
bpV[pic]-dependent nuclear translocation and activation
of NF-
B is most likely associated with phosphorylation on both
serine residues 32 and 36 of I
B
. This series of events is known
to be necessary to ensure dissociation of I
B
from NF-
B through ubiquitination of I
B
and its final degradation by the proteasome. Our data indicate also that bpV[pic]-mediated activation of ICAM-1 gene expression is dependent on both p50 and p65
NF-
B/Rel family members. These findings are consistent with previous
reports indicating that the ICAM-1
B site binds p50 and p65 (20,
48). Although we provide conclusive evidence of the intimate interplay between the phosphotyrosine level and NF-
B-dependent
induction of ICAM-1 expression in T cells, the various putative
protein-protein interactions between NF-
B complexes and other
transcription factors binding to the ICAM-1 promoter
(e.g. AP-1 and C/EBP) remain to be explored.
138mut,
pGLE
158mut, or pGLE
138/
158mut. Our results demonstrated that no
significant change occurred following a point mutation in either the
138 (pGLE
138mut) or
158 (pGLE
158mut) Ets-binding sites when
compared with a vector carrying the wild-type Ets-binding sites (pGLE
WT). However, when both Ets-binding sites were mutated, the
bpV[pic]-induced up-regulation of ICAM-1 promoter-driven
luciferase activity was significantly decreased. Our results are
contrasting with previous data indicating that a point mutation in the
158 Ets-binding site strongly diminished the ICAM-1
promoter activity (27). In this report, it was demonstrated that
expression vectors encoding for Ets-2 and ERM significantly
up-regulate ICAM-1 transcription in rabbit kidney carcinoma RK13 cells,
and the ERM-mediated activation of ICAM-1 transcription was strongly
diminished when using a mutated
158 Ets-binding site. In addition, a
mutated version of the
138 Ets-binding site did not change the
ERM-mediated induction of ICAM-1 gene expression. There are
at least two possibilities that could explain such a discrepancy.
First, experiments conducted by de Launoit and co-workers (27) were
performed using established cell lines different from the one used in
the current study. Second, in contrast to their work that is based on
overexpression of Ets-2 and ERM, we could not detect any positive
modulation of Ets nuclear translocation upon bpV[pic] treatment. The
observation that both Ets-binding sites in the ICAM-1
promoter are important for bpV[pic]-mediated induction of ICAM-1
transcription despite an absence of Ets activation is suggestive of a
functional interaction between binding sites for Ets and another
transcription factor. This possibility is supported by a previous study
demonstrating that the H2O2-responsive element
in the ICAM-1 promoter is constituted of the Ets- and AP-1-binding sites (26). Similarly, a synergic activation of ICAM-1 by
retinoic acid and TNF-
is due to a functional cooperation between
retinoic acid response elements and NF-
B-binding sites (56). Further
studies are warranted to identify the binding site(s) in the
ICAM-1 promoter that functionally cooperate with Ets-binding
sites to form the bpV[pic]-responsive elements.
B and Ets transcription factors,
our results indicate that the JAK/STAT signaling pathway is also
implicated in the induction of ICAM-1 expression in human T cells that
is seen upon treatment with the potent PTP inhibitor bpV compound.
Indeed, a point mutation in the IFN-
-responsive element
(i.e. pI
RE) markedly reduced bpV[pic]-mediated
up-regulation of ICAM-1 promoter activity. We also
demonstrated that bpV[pic] treatment was resulting in nuclear
translocation of STAT-1. Our results are consistent with a previous
report demonstrating that the PTP inhibitor pervanadate activates the
protein-tyrosine kinases JAKs which will in turn induce tyrosine
phosphorylation of STAT-1, STAT-3, and STAT-6 (57). Of interest for the
present study, STAT-1 has been recently shown to be inactivated by a
nuclear PTP (58). Therefore, it can be proposed that the PTP inhibitor bpV[pic] could result in phosphorylation of STAT-1 in the cytoplasm, translocation to the nucleus, and then the maintenance for a longer period of a high level of STAT-1 in an active phosphorylated state.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Benoit Barbeau for critical
reading of the manuscript; Dr. M. Dufour for technical assistance in
flow cytometry studies; Drs. T. P. Parks and Y. de Launoit for the
reporter vectors of the ICAM-1 promoter (i.e.
pGL1.3, pGL1.3 Bmut, pGL HindIII, pGL HindIII
IRE mut, pGLE, pGLE
138mut, pGLE
158mut, and pGLE
138/
158mut);
Dr. W. C. Greene for pCMV-I
B
S32A/S36A; Dr. R. Rothlein for
RR1/1.1.1 antibody (anti-ICAM-1); and Dr. N. Rice for antisera against
NF-
B proteins.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant HOP-15575 from the Canadian Institutes of Health Research HIV/AIDS Research Program (to M. J. T.).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.
Submitted in partial fulfillment for the M.Sc. degree from the
Microbiology-Immunology Program, Faculty of Medicine, Laval University.
¶ Recipient of a Chercheur National award from the Fonds de la Recherche en Santé du Québec.
Holds a Canada Research Chair in Human Immuno-Retrovirology
and recipient of Canadian Institutes of Health Research Investigator award. To whom correspondence should be addressed: Laboratoire d'Immuno-Rétrovirologie Humaine, Centre de Recherche en
Infectiologie, RC709, Centre Hospitalier Universitaire de Québec,
Pavillon CHUL, 2705 Blvd. Laurier, Ste-Foy, Québec G1V
4G2, Canada. Tel.: 418-654-2705; Fax: 418-654-2212; E-mail:
Michel.J.Tremblay@crchul.ulaval.ca.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M005067200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ICAM-1, intercellular adhesion molecule-1;
PTP, protein tyrosine phosphatases;
bpV, bis-peroxovanadium;
bpV[pic], bis-peroxovanadium compound
carrying the picolinic acid as an auxillary ligand;
pIRE, palindromic interferon-
-responsive element;
STAT, signal transducers
and activators of transcription;
PMA, phorbol 12-myristate 13-acetate;
Iono, ionomycin;
FBS, fetal bovine serum;
PBMCs, peripheral blood
mononuclear cells;
PBS, phosphate-buffered saline;
WT, wild type;
dsDNA, double-stranded DNA;
mut, mutant;
IL, interleukin;
IFN, interferon;
TNF-
, tumor necrosis factor-
;
bp, base pair.
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