From the Nelson Institute of Environmental Medicine,
New York University School of Medicine, New York, New York 10016, the
§ Health Effects Laboratory Division, NIOSH, National
Institutes of Health, Morgantown, West Virginia 26505,
The
Institute of Neuroscience, The Fourth Military Medical University,
Xi'an, 710032, People's Republic of China, and the ¶ Department
of Basic Pharmaceutical Science, West Virginia University, Morgantown,
West Virginia 26506
Received for publication, November 30, 2000, and in revised form, March 20, 2001
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ABSTRACT |
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The present study investigated the role of
reactive oxygen species (ROS) in activation of nuclear factor of
activated T cells (NFAT), a pivotal transcription factor responsible
for regulation of cytokines, by vanadium in mouse embryo fibroblast PW
cells or mouse epidermal Cl 41 cells. Exposure of cells to vanadium led
to the transactivation of NFAT in a time- and
dose-dependent manner. Scavenging of vanadium-induced
H2O2 with
N-acety-L-cyteine (a general antioxidant) or
catalase (a specific H2O2 inhibitor) or the
chelation of vanadate with deferoxamine, resulted in inhibition of NFAT
activation. In contrast, an increase in H2O2
generation by the addition of superoxide dismutase or NADPH enhanced
vanadium-induced NFAT activation. This vanadate-mediated
H2O2 generation was verified by both electron
spin resonance and fluorescence staining assay. These results
demonstrate that H2O2 plays an important role
in vanadium-induced NFAT transactivation in two different cell types. Furthermore, pretreatment of cells with nifedipine, a calcium channel
blocker, inhibited vanadium-induced NFAT activation, whereas A23187 and
ionomycin, two calcium ionophores, had synergistic effects with
vanadium for NFAT induction. Incubation of cells with cyclosporin A
(CsA), a pharmacological inhibitor of the phosphatase calcineurin,
blocked vanadium-induced NFAT activation. All data show that vanadium
induces NFAT activation not only through a calcium-dependent and CsA-sensitive pathway but also
involved H2O2 generation, suggesting that
H2O2 may be involved in activation of
calcium-calcineurin pathways for NFAT activation caused by vanadium exposure.
Vanadium is a transition metal widely distributed in the
environment. Occupational exposure to vanadium is common in oil-fired electrical generating plants and the petrochemical, steel, and mining
industries (1). The available data from the literature provides
evidence that in mammalian cell culture systems vanadium(V) is
biologically more active than vanadium(IV) in inducing toxic effects
(2). It has been reported that the cell can protect itself against
vanadate toxicity by reduced glutathione-mediated conversion of
vanadium(V) to vanadium(IV), whereas vanadate(IV) does not undergo any
change in valency state in BALB/3T3 cells (2). It has been found that
vanadium-containing compounds exert potent toxic and carcinogenic
effects, such as DNA damage and cell transformation (3-5). In animal
studies, vanadium compounds or vanadium-containing air pollution
particles have been shown to induce inflammation in the respiratory
tract (6, 7). It has been reported that vanadium associated with air
pollution particles, such as residual oil fly ash, can induce the
synthesis and expression of inflammatory cytokines, such as
IL-6,1 IL-8, and
TNF- The nuclear factor of activated T cells (NFAT) was originally described
as a transcriptional factor expressed in activated but not resting T
cells (11-14). The induction of NFAT in T cells required a
calcium-activated signaling pathway and was blocked by cyclosporin A
(CsA) and FK506 (15-21). Over the last decade, studies from
several laboratories have indicated that the pre-existing/cytoplasmic component of NFAT was a mixture of proteins belonging to a novel family
of transcription factors (22-24). The first member of this family
(NFATp, later renamed NFAT1) was purified from cytoplasmic extracts of a murine T cell cloned by affinity chromatography using the
distal NFAT site of murine IL-2 promoter (19, 25) and cloned from
murine (Ar-5) and human (Jurkat) T cell cDNA libraries (25, 26).
Other distinct proteins belonging to the same family, such as NFATc,
NFAT3, and NFAT4, were also isolated and cloned (27-30). There are three functional domains in NFAT family proteins: the Rel-similarity domain, which is responsible for DNA-binding activity and interaction with AP-1; the NFAT-homology region, which
regulates intracellular localization; and the transcriptional activation domain (31). The activation of NFAT in T cells includes dephosphorylation, nuclear translocation, and an increase in affinity for DNA binding (15). Stimuli that elicit calcium mobilization result
in rapid dephosphorylation of NFAT proteins and their translocation to
the nucleus. These dephosphorylated proteins show increased affinity
for DNA binding (15).
Growing evidence indicates that NFAT is not only a T cell-specific
transcriptional factor but also is expressed in a variety of lymphoid
cells and in non-lymphoid tissue (15, 32, 33), NFAT involvement is
reasonably well established for the production of IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-18, TNF- Plasmids and Reagents--
CMV-neo vector plasmid and
NFAT-luciferase reporter plasmid were constructed as previously
reported (14, 33-36). Sodium metavanadate (vanadate) and vanadyl
sulfate trihydrate were purchased from Aldrich (Milwaukee, WI);
deferoxamine, N-acety-L-cyteine (NAC), Cell Culture--
The JB6 P+ mouse epidermal cell
line, Cl 41, and its transfectants, Cl 41 NFAT mass1 and
P+1-1, were established and cultured in monolayers at
37 °C, 5% CO2 using MEM containing 5% FBS, 2 mM L-glutamine, and 25 µg of gentamicin per
ml as described previously (33, 37, 38). Mouse embryo fibroblast PW
cells and its transfectants, PW NFAT mass1, were cultured
in DMEM with 10% FBS, 2 mM L-glutamine and 25 mg of gentamicin/ml.
Generation of Stable Co-transfectants--
PW cells were
cultured in a 6-well plate until they reached 85-90% confluence. 1 µg of CMV-neo vector, with or without 12 µg of NFAT-luciferase
reporter plasmid DNA and 15 µl of LipofectAMINE reagent, was used to
transfect each well in the absence of serum. After 10-12 h, the medium
was replaced with 5% FBS MEM. Approximately 30-36 h after the
beginning of the transfection, the cells were digested with 0.033%
trypsin and cell suspensions were plated onto 75-ml culture flasks and
cultured for 24-28 days with G418 selection (800 µg/ml). The stable
transfectants were identified by measuring basal level of luciferase
activity. Stable transfectant PW NFAT mass1 were
established and cultured in G418-free MEM for at least two passages
before each experiment.
Assay for NFAT Activity in Vitro--
Confluent monolayers of PW
NFAT mass1 or Cl 41 NFAT mass1 were
trypsinized, and 5 × 103 viable cells were suspended
in 100 µl of medium were added into each well of a 96-well plate.
Plates were incubated at 37 °C in a humidified atmosphere of 5%
CO2 and 95% air. The cells were then exposed to vanadium
for the indicated times and dosages. The cells were extracted with
lysis buffer, and luciferase activity was measured as previously
described (39). The results were expressed as NFAT activity relative to
controls or relative luciferase unit (RLU) (33).
Cellular Superoxide (O Electron Spin Resonance (ESR) Measurements--
ESR spin
trapping was used to detect short-lived free radical intermediates.
This technique involves the addition-type reaction of a short-lived
radical with a diamagnetic compound (spin trap) to form a relatively
long-lived free radical product, the so-called spin adduct, which can
be observed by conventional ESR. The intensity of the spin adduct
signal corresponds to the amount of short-lived radicals trapped, and
the hyperfine splittings of the spin adduct are generally
characteristic of the trapped radical. ESR measurements were carried
out using a Varian E9 ESR spectrometer and a flat cell assembly.
Hyperfine couplings were measured (0.1 G) directly from magnetic field
separation using potassium tetraperoxochromate (K3CrO8) and
1,1-diphenyl-2-picrylhydrazyl as reference standards. PW cells (1 × 106) were mixed with 100 mM DMPO, 100 µM NADPH, and 1 mM vanadate. The reaction
mixture was then transferred to a flat cell for ESR measurement as
described previously (2, 41).
Induction of NFAT Transactivation in PW Cells and Cl 41 Cells by
Vanadium--
To study the regulation of NFAT transcription activity
by vanadium in cells, we generated a NFAT-luciferase reporter stable transfectant by co-transfecting the NFAT-luciferase reporter plasmid and CMV-neo plasmid into mouse fibroblast cells, PW NFAT
mass1. The results observed from this stable transfectant
show that NFAT activities were markedly induced by exposure of PW cells
to either vanadium(V) (increasing RLU from 3837 ± 183 to
42838 ± 1920) or vanadium(IV) (increasing RLU from 3837 ± 183 to 38321 ± 2839) (p < 0.05) (Fig.
1a). The activation of NFAT by
vanadium appears to be time- and dose-dependent manner
(Fig. 1, c and d). The maximum induction of NFAT
activity by vanadium occurred between 36 and 48 h after cells were
exposed to vanadium (Fig. 1d). To test whether vanadium-induced NFAT activation is cell-type-specific, we also exposed
the Cl 41 NFAT mass1 to vanadium. The results show that exposure of Cl 41 cells to vanadium resulted in significant activation of NFAT activity at 25 and 100 µM
(p < 0.05) (Fig. 1b). These results
demonstrate that vanadium is a stimulus for NFAT transactivation. This
activation is not limited to a single cell type but appears to be a
more generalized reaction to vanadium treatment.
It is well known that, in T cells, transactivation of NFAT is regulated
tightly in response to elevations of both intracellular calcium ion
(Ca2+) and diacylglycerol following activation of
phospholipase C (15). Increased intracellular calcium stimulates the
activation of calmodulin (15). It is believed that binding of
calmodulin to a region near the C terminus of calcineurin displaces the
auto-inhibiting domain and exposes the calcineurin active site (15).
Activated calcineurin subsequently dephosphorylates the cytoplasmic
NFAT proteins, leading to NFAT nuclear translocation (16, 48). NFAT
then forms a heteromeric transcriptional co-activator complex with AP-1
that co-induces NFAT-dependent transactivation (15). Because our previous data indicated that vanadium exposure also led to
transactivation of AP-1 (3), the data shown above can not rule out the
involvement of AP-1 in vanadium-induced NFAT transactivation even
though the NFAT-luciferase reporter only contains the firefly
luciferase under the control of three NFAT binding sites. To test this,
we used a chemical inhibitor, SB202190, that can inhibit AP-1
activation by inhibition of AP-1 upstream kinase, p38 kinase. We find
that pretreatment of cells with SB202190 does not show any inhibitory
effects on vanadate-induced NFAT activation (Fig.
2A), whereas it markedly
inhibits AP-1 activation induced by vanadate (Fig. 2B).
These results suggest that vanadium-induced NFAT transactivation in PW
cells is only dependent on NFAT, but not AP-1.
Reactive Oxygen Species Are Involved in NFAT Activation by
Vanadium--
Our previous studies have indicated that reactive oxygen
species (ROS) are involved in vanadate-induced biological activities (42). If NFAT activation is responsible for some of the biological effects caused by vanadium, ROS generation may play a role in vanadium-induced NFAT activation. To test this possibility, ROS generation was determined in PW cells exposed to vanadium by both dye
staining and ESR. The results show that PW cells alone did not generate
any detectable amount of free radicals (Fig.
3A), whereas PW cells exposed
to vanadate generated a strong ESR signal (Fig. 3B). The
spectrum consists of a 1:2:2:1 quartet with hyperfine splittings of
aH = aN = 14.9 G, where aN and
aH denote hyperfine splittings of the nitroxyl nitrogens
and Synergistic Effect of A23187 and Ionomycin on Vanadium-induced NFAT
Activation--
Ionomycin reportedly has a synergistic effect with TPA
for NFAT transactivation in T and B cells, whereas ionomycin or TPA alone does not induce NFAT transactivation (14). Our previous studies
have demonstrated that A23187 exhibited a synergistic effect with UV
radiation for NFAT induction (33). To test the effect of
Ca2+ ionophore on vanadium-induced NFAT activation in PW
cells, we incubated PW NFAT mass1 with either A23187 or
ionomycin. The results show that both A23187 and ionomycin exhibited a
high synergistic augmentation of vanadium-induced NFAT activation
(p < 0.05) (Fig. 7,
a and b), whereas administration of A23187 or
ionomycin alone did not show induction of NFAT activity in PW cells
(Fig. 7, a and b). These synergistic effects
appear to be dose-dependent (Fig. 7, c and
d). These data suggest that elevation of intracellular
calcium in PW cells plays a role in the activation of NFAT in response
to vanadium.
Vanadium-induced NFAT Activation Is Dependent on Calcium
Mobilization in PW Cells--
To determine the role of
calcium-dependent pathways in vanadium-induced NFAT
activation, we investigated the effect of nifedipine, a calcium channel
blocker, on vanadium-induced NFAT activation. Blocking calcium channel
activity by pretreatment of PW cells with nifedipine resulted in
inhibition of vanadium-induced NFAT activation (p < 0.05) (Fig. 8). These data suggest that
activation of NFAT by vanadate and the synergistic effect of A23187 and ionomycin on vanadium-induced activation of NFAT both occur through calcium-dependent pathways.
Inhibition of Vanadium-induced NFAT Transactivation by Cyclosporin
A (CsA)--
Previous studies demonstrated that in T cells the major
NFAT activation pathway appears to involve a
calcium/calmodulin-dependent phosphatase, calcineurin (15,
16). To test the role of calcineurin in vanadate-induced
NFAT-dependent transcription activity in PW cells and Cl 41 cells, cyclosporin A (CsA), a widely used pharmacological inhibitor of
the phosphatase calcineurin, was used. Pretreatment of cells with CsA
resulted in a dramatic inhibition of NFAT transactivation induced by
vanadium (p < 0.05) in both PW (Fig.
9) and Cl 41 cells (date not shown).
These data suggest that activation of calcineurin is required for
vanadium-induced NFAT activation and that vanadium activates the NFAT
transcription activity in mouse embryo fibroblasts and epidermal cells
through the pathway that is similar to that in T cells.
The antigen-regulated, cyclosporin A-sensitive nuclear factor of
activated T cells (NFAT) is not only expressed in lymphoid cells as
once thought but also expressed in other cells and organs, such as the
skin and lung (15, 32). The stimuli and regulation of NFAT in cells
other than lymphoid cells, however, remains unknown. In this report, we
investigated the possible involvement of H2O2 in NFAT activation of fibroblasts and epidermal cells in response to
vanadium. Exposure of cells to vanadium caused marked increases in
NFAT-dependent transcription in a time- and
dose-dependent manner. Co-stimulation studies show that
A23187 or ionomycin augments the NFAT-mediated transcription
synergistically in response to vanadium. Pretreatment of cells with
nifedipine or CsA results in impairment of NFAT transactivation induced
by vanadium. These results strongly suggest that vanadium is able to
induce NFAT activation. Furthermore, scavenging of vanadium-induced
H2O2 by NAC or catalase, or the chelation of
vanadate by deferoxamine resulted in inhibition of NFAT activation,
whereas an increase in H2O2 generation in
response to SOD or NADPH enhanced vanadium-induced NFAT activation.
These results suggest that vanadium induces NFAT transactivation is
dependent on H2O2 generation caused by vanadium.
NFAT isoforms are expressed in different tissues. It has been reported
that NFAT1 and NFAT2 mRNAs have been
detected in brain, heart, skeletal muscle, testis, placenta, pancreas,
small intestine, prostate, colon, skin tumors, as well as in lung (15,
32). NFAT expression or NFAT-derived transactivation has also been described in several types of nonlymphoid cells, including mast (43),
endothetial (44), neuronal (45), vascular smooth muscle (46), and
liver-derived Chang cells (47). In this study, we have found that
exposure of either mouse embryo fibroblasts (PW cells) or mouse
epidermal cells (Cl 41) resulted in marked activation of
NFAT-dependent transcription. These results are not only
consistent with previous findings that NFAT mRNA is detectable in
skin (32) but also for the first time provide the evidence that NFAT is expressed in embryo fibroblasts, suggesting that NFAT may play some
role in embryonic development. The activation of NFAT includes dephosphorylation, nuclear translocation, and an increase in affinity for DNA binding (15). Stimuli that elicit calcium mobilization result in rapid dephosphorylation of NFAT proteins and their
translocation to the nucleus. NFAT then binds to the promoter region of
its target genes and initiates the transcription of these target genes, in turn leading to translation (15). Therefore, the
translocation of NFAT occurs within 3 h (33), whereas its target
gene proteins could be detected between 24 and 48 h after cells
exposed to stimulus (15). The system used in this study is
NFAT-luciferase reporter cDNA, which includes the firefly
luciferase cDNA under the control of NFAT regulatory element
(promoter region of mouse IL-2, a typical target gene of NFAT). Thus,
the NFAT activation by vanadium appears at 12 h and reaches peak
around 36-48 h after cells exposed to vanadium.
In T cells, transactivation of NFAT is regulated tightly in response to
elevations of both intracellular calcium ion (Ca2+) and
diacylglycerol following activation of phospholipase C (15). Increased
intracellular calcium stimulates the activation of calmodulin (15). It
is believed that binding of calmodulin to a region near the C terminus
of calcineurin displaces the auto-inhibiting domain and exposes the
calcineurin active site (15). Activated calcineurin subsequently
dephosphorylates the cytoplasmic NFAT proteins, leading to NFAT nuclear
translocation (15, 16, 48). NFAT forms a heteromeric transcriptional
co-activator complex with AP-1 that co-induces
NFAT-dependent transactivation (15). It has also been
reported that phosphorylation of NFAT is regulated by several protein
kinases, including GSK3 and JNK2 (15, 49-51). Other than lymphocytes, NFAT activation has been demonstrated in only a
few cell types, such as vascular smooth muscle cells following
induction with platelet-derived growth factor and liver-derived Chang cells following exposure to hepatitis B virus × protein (46, 47). The results obtained from the present investigation show that
vanadium alone could induce an increase of NFAT activity of more than
10-fold. It appears that vanadium alone is the strongest NFAT inducer
reported thus far. Recently, we reported (33) that UV irradiation
resulted in activation of NFAT-mediated transcription through a
calcium-dependent pathway in mouse epidermal JB6 cells as
well as in mouse skin. The present study found that A23187 or ionomycin
augmented the NFAT-mediated transcription synergistically in
combination with vanadium. Pretreatment of cells with nifedipine or CsA
resulted in impairment of NFAT transactivation induced by vanadium.
Therefore, the vanadium-induced NFAT transactivation appears to require
an increase in free intracellular calcium and calcineurin activation.
Vanadium-containing compounds exert potent toxic and carcinogenic
effects, such as cell transformation (3-5). It has been demonstrated
that vanadium-mediated generation of reactive oxygen species (ROS) is
responsible for most biological effects caused by vanadium (41, 42). It
is well accepted that extracellular stimuli trigger signals through a
cascade of protein-protein interactions (52-54). It is generally
believed that these extracellular stimuli generate and/or require
reactive free radicals or derived oxidant species to successfully
transmit their signals to the nucleus (53, 55). In addition to inducing
cellular injury, such as DNA damage and lipid peroxidation, ROS also
function as intracellular messengers (2, 53, 56). Accumulating data
suggest a vital role of ROS in mediating cellular responses by various
extracellular stimuli (2, 40-42, 53, 56). It has been reported that
generation of H2O2 is required for
platelet-derived growth factor signal transduction (57). Involvement of
ROS in NFAT activation in T lymphocytes was first demonstrated by the
observation that treatment of T cells with dithiocarbamate resulted in
inhibition of NFAT activation and NFAT-mediated cytokine gene
expression (58, 59). The results presented here demonstrate that
increased intracellular H2O2 levels are
associated with induction of NFAT activity by vanadium. The following
experimental observations support this conclusion: (a) the
ESR spectrum and dye staining show that vanadium induces ROS generation
in cells; (b) catalase, a scavenger of H2O2, inhibited NFAT activation induced by
vanadium; (c) sodium formate, an ·OH radical
scavenger, did not affect vanadium-induced NFAT activation; (d) SOD, which scavenges O
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(6-9). Vanadium exposure in vitro was also
found to cause the production of IL-1, TNF-
, and prostaglandin E2
(6, 10).
, interferon
,
and granulocyte macrophage-colony-stimulating factor in a variety of
cell types (15). In addition, previous studies from our laboratory and
other groups have shown that TNF-
, IL-6, and IL-8 are involved in
vanadium-induced inflammation (6-10). Therefore, the objective of the
present study was to determine if activation of NFAT occurred in
response to vanadium and if so to investigate the molecular mechanism,
by which lead to NFAT activation.
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MATERIALS AND METHODS
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-nicotinamide adenine dinucleotide phosphate (NADPH), superoxide dismutase (SOD), and sodium formate were purchased from Sigma Chemical
Co. (St. Louis, MO); luciferase assay substrate was obtained from
Promega (Madison); fetal bovine serum (FBS), Eagle's minimal essential
medium (MEM), and Dulbecco's modified Eagle's medium (DMEM) were from
BioWhittaker (Walkersville, MD). LipofectAMINE was from Life
Technologies, Inc.; A23187, ionomycin, as well as cyclosporin A (CsA)
were purchased from Alexis Biochemicals (San Diego, CA);
dichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (HE)
were purchased from Molecular Probe (Eugene, OR).
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Fig. 1.
Induction of NFAT-dependent
transcription by vanadium in PW cells and Cl 41 cells. 8 × 103 cells of PW NFAT mass1 (a,
c, and d) or Cl 41 NFAT mass1
(b) were seeded into each well of 96-well plates. After
being cultured at 37 °C overnight, the cells were treated with:
a, vanadium (100 µM) for 24 h;
b and c, for a dose-response study, the cells
were exposed to different concentrations of vanadium as indicated for
24 h; d, for a time course study, the cells were
exposed to 100 µM vanadium for various times as
indicated. Then, the luciferase activity was measured. Each
bar indicates the mean and standard deviation of four repeat
assay wells. The results are presented as NFAT-dependent
transcription activity relative to control. The asterisk
indicates a significant increase from control (p < 0.05).
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Fig. 2.
Pretreatment of cells with SB202190 caused
inhibition of vanadate-induced activation of AP-1, but NFAT.
8 × 103 cells of Cl 41 NFAT mass1
(A) or P+1-1 (B) were seeded into
each well of 96-well plates. After being cultured at 37 °C
overnight, the cells were pretreated with 4 µM SB202190
for 30 min. The cells were then treated with vanadate (100 µM) for 24 h, and the luciferase activity was
measured. Each bar indicates the mean and standard deviation
of four repeat assay wells. The results are presented as relative
luciferase unit (RLU). The asterisk indicates a significant
decrease from vanadate control (p < 0.05).
-hydrogen, respectively. Based on these splittings and the
1:2:2:1 line shape, the spectrum was assigned to the DMPO-OH adduct,
which is evidence of ·OH radical generation. Time course studies
show that maximum generation of free radical is between 6.3 and 11.2 min after addition of vanadate (Fig. 4).
Addition of catalase, a scavenger of H2O2, inhibited ·OH radical generation (Fig. 3C),
indicating that H2O2 was produced in the
vanadate-treated cells and involved in ·OH generation. Addition
of sodium formate, an ·OH radical scavenger, decreased the
signal intensity (Fig. 3D), further supporting the
·OH radical generation. Incubation of the mixture with
deferoxamine, a metal chelator, dramatically decreased the
signal intensity (Fig. 3E). Measurements using HE, a
specific fluorescent dye for O
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Fig. 3.
Measurement of vanadate-induced ROS
generation in PW cells by ESR. ESR spectra were recorded 6 min
after mixing 1 × 106 PW cells, 100 mM
DMPO, 1 mM sodium vanadate, and 100 µM NADPH
with or without different ROS scavengers as indicated. The final
concentrations of these scavengers were: catalase, 2000 units/ml; or
sodium formate, 100 mM. Deferoxamine, 2 mM, was
used as a metal chelator.
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Fig. 4.
Time course of vanadate-induced ROS
generation in PW cells. ESR spectra were recorded at different
time points after mixing 1 × 106 PW cells, 100 mM DMPO, 1 mM sodium vanadate, and 100 µM NADPH.
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Fig. 5.
Determination of O
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Fig. 6.
Effects of free radical modifiers on NFAT
activation by vanadium. PW NFAT mass1 cells suspended
in 10% FBS DMEM were added to each well of 96-well plates and cultured
overnight. The cells were pretreated with different free radical
modifiers at the concentrations indicated. The cells were then exposed
to vanadium (100 µM) for 24 h. The NFAT
activity was determined by the luciferase activity assay. The results
are presented as relative NFAT activity. Each column and
bar indicates the mean and S.D. from triplicate assays. The
asterisk indicates a significant increase (c and
d) and a significant decrease (e) from vanadium
alone (p < 0.05).
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Fig. 7.
Synergistic effect of A23187 and ionomycin on
vanadium-induced NFAT activity. 8 × 103 PW NFAT
mass1 cells suspended in 10% FBS DMEM medium were added to
each well of 96-well plates. After being cultured at 37 °C
overnight, the cells were treated with (a) vanadium (100 µM), A23187 (1 µM), or vanadium (100 µM) + A23187 (1 µM), or (b)
vanadium (100 µM), ionomycin (1 µM), or
vanadium (100 µM) + ionomycin (1 µM). For
the dose-response study, the cells were exposed to vanadium (100 µM) plus different concentrations of A23187
(c) or ionomycin (d). The cells were harvested
after being cultured for 48 h. The NFAT activity was measured by
luciferase activity assay. The results are presented as relative
NFAT-dependent transcription activity. Each bar
indicates the mean and standard deviation of assays from triplicate
wells. The asterisk indicates a significant increase from
vanadium alone (p < 0.05).
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Fig. 8.
Inhibition of vanadium-induced NFAT activity
by pretreatment of cells with nifedipine. 8 × 103 PW NFAT mass1 cells suspended in 10% FBS
DMEM were added to each well of 96-well plates. After being cultured at
37 °C overnight, the cells were first treated with 40 µM nifedipine for 30 min and sequentially were exposed to
vanadium (100 µM). After being cultured for 48 h,
the cells were harvested, and the NFAT activity was measured by
luciferase activity assay. The results are presented as relative
NFAT-dependent transcription activity. Each bar
indicates the mean and standard deviation of assays from triplicate
wells. The asterisk indicates a significant decrease from
vanadium alone (p < 0.05).
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Fig. 9.
Blocking vanadium-induced NFAT
transactivation by cyclosporin A. PW NFAT mass1 were
seeded to each well of 96-well plates and cultured until 90%
confluent. The cells were then treated with cyclosporin A at
(a) 0.5 µM or (b) at the
concentration as indicated for 30 min and sequentially were exposed to
100 µM vanadium. After being cultured for 48 h, the
cells were harvested and the NFAT activity was measured by luciferase
activity assay. Each bar indicates the mean and standard
deviation of assays from triplicate wells. The asterisk
indicates a significant decrease from vanadium alone (p < 0.05).
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Fig. 10.
The model of signal transduction pathway for
NFAT activation induced by vanadium.
NFAT is a transcription factor, which has been reported to play an
essential role in IL-2 gene expression (14). Binding sites for NFAT
have also been found within the promoter regions of cytokines,
including granulocyte macrophage-colony-stimulating factor, TNF-,
IL-1, IL-3, IL-4, IL-5, and IL-8 (15, 47, 50). Previous studies have
indicated that expression of IL-8, TNF-
, and other cytokines is
associated with the initiation and control of effective immune and
inflammatory responses and may be related to cancer development (47,
50). Therefore, we suggest that NFAT is involved in vanadium-induced
inflammation and may subsequently be involved in the carcinogenic
effects of vanadium. This idea is supported by previous findings that
CsA and FK506, the two most commonly used inhibitors for NFAT
activation, exhibit strong anti-tumor promotion activity by
specifically only targeting and blocking the activation of a
Ca2+-dependent phosphatase, calcineurin (61,
62).
In conclusion, we demonstrate that NFAT is expressed in
embryonic cells and vanadium is a strong inducer of NFAT activation. Vanadium induces NFAT activation through CsA-sensitive and
calcium-dependent signal transduction pathways, which
required H2O2 generation caused by vanadium.
Transactivation of NFAT in cells other than lymphocytes supports the
hypothesis that NFAT activation may play an important role in
vanadium-induced biological effects, such as inflammation and tumor
promotion, possibly through NFAT-mediated expression of inflammatory
cytokines, such as IL-1, IL-2, IL-6, IL-8, and TNF-.
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FOOTNOTES |
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* 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.
** To whom correspondence should be addressed: Health Effects Laboratory Division, NIOSH, National Institutes of Health, Morgantown, WV 26505. Tel.: 304-285-6158; Fax: 304-285-5938; E-mail: xas0@cdc.gov.
Published, JBC Papers in Press, April 5, 2001, DOI 10.1074/jbc.M010828200
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ABBREVIATIONS |
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The abbreviations used are:
IL, interleukin;
NFAT, nuclear factor of activated T cells;
CsA, cyclosporin A;
AP-1, activated protein-1;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
NAC, N-acety-L-cyteine;
NADPH, -nicotinamide
adenine dinucleotide phosphate;
SOD, superoxide dismutase;
DCFH-DA, dichlorofluorescein diacetate;
HE, dihydroethidium;
FBS, fetal bovine
serum;
MEM, Eagle's minimal essential medium;
DMEM, Dulbecco's
modified Eagle's medium;
ROS, reactive oxygen species;
CMV, cytomegalovirus;
PBS, phosphate-buffered saline;
ESR, electron spin
resonance;
RLU, relative light unit(s);
DMPO, 5,5-dimethy-1-pyrroline
N-oxide;
GSK3, glycogen synthase kinase-3;
JNK, c-Jun N-terminal kinase.
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