Oxidative Stress Triggers STAT3 Tyrosine Phosphorylation and
Nuclear Translocation in Human Lymphocytes*
Modesto
Carballo
,
Manuel
Conde
,
Rajaa
El Bekay
,
Jose
Martín-Nieto§,
María Jesús
Camacho¶,
Javier
Monteseirín¶,
José
Conde¶,
Francisco
J.
Bedoya
, and
Francisco
Sobrino
¶
From the
Departamento de Bioquímica
Médica y Biología Molecular, Facultad de Medicina and the
¶ Departamento de Medicina, Servicio Regional de
Inmunología y Alergia, Hospital Universitario Virgen Macarena,
Universidad de Sevilla, 41009 Sevilla, and the
§ División de Genética, Universidad de Alicante,
Campus Universitario San Vicente, 03080 Alicante, Spain
 |
ABSTRACT |
Oxidizing agents are powerful activators of
factors responsible for the transcriptional activation of
cytokine-encoding genes involved in tissue injury. In this study we
show evidence that STAT3 is a transcription factor whose activity is
modulated by H2O2 in human lymphocytes,
in which endogenous catalase had previously been inhibited.
H2O2-induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by
immunoblotting of nuclear fractions and electrophoretic mobility shift
assays, and vanadate was found to strongly synergize with
H2O2. Moreover, anti-STAT3 antibodies
specifically precipitated a protein of 92 kDa that becomes
phosphorylated on tyrosine upon lymphocyte treatment with
H2O2. Phenylarsine oxide, a tyrosine
phosphatase inhibitor, and genistein, a tyrosine kinase inhibitor,
cooperated and cancelled, respectively, the
H2O2-promoted STAT3 nuclear translocation.
Evidence is also presented, using Fe2+/Cu2+
ions, that ·OH generated from H2O2
through Fenton reactions could be a candidate oxygen reactive species
to directly activate STAT3. Present data suggest that
H2O2 and vanadate are likely to inhibit the
activity of intracellular tyrosine phosphatase(s), leading to enhanced STAT3 tyrosine phosphorylation and hence its translocation to the
nucleus. These results demonstrate that the DNA binding activity of
STAT3 can be modulated by oxidizing agents and provide a framework to
understand the effects of oxidative stress on the JAK-STAT signaling pathway.
 |
INTRODUCTION |
Oxidative stress is characterized by high intracellular levels of
reactive oxygen intermediates
(ROI),1 which may function as
physiological mediators of a number of cellular responses by acting as
second messengers for specific signaling pathways (1-3). In addition,
ROI have been implicated in a variety of clinical conditions, including
rheumatoid arthritis and other autoimmune diseases (4, 5), and can also
play a role as tumor promoters (6). H2O2 as
well as superoxide anions (O
2) have traditionally been viewed
as potent microbicidal agents (7). H2O2 is a
small, diffusible, and ubiquitous molecule that can be rapidly
synthesized, as well as destroyed, in response to several stimuli. It
has been proposed that H2O2 is converted into
highly reactive ·OH radical via Fenton chemistry through its
reduction by ferrous/cuprous ions (8, 9). H2O2
fulfills the prerequisites for intracellular second messengers. In this
regard, recent studies have demonstrated that exposure of lymphocytes
to oxidants, such as H2O2 (10-12), diamide
(12, 13), or phenylarsine oxide (PAO) (14-16), results in an increased
tyrosine phosphorylation of intracellular proteins.
STATs (signal transducers and
activators of transcription) are a class of
transcription factors bearing SH2 domains that become activated upon
tyrosine phosphorylation. STATs are often activated by members of the
JAK family of protein-tyrosine kinases (PTKs) in response to cytokine
stimulation. This activation mechanism involves the SH2
domain-dependent recruitment of the STATs to tyrosine-phosphorylated cytokine receptors. The STATs then become phosphorylated by receptor-associated JAKs, which induces their dimerization via reciprocal SH2-phosphotyrosine interaction. STAT dimers then enter the nucleus and bind to specific DNA elements, thereby activating the transcription of a number of genes. The JAK-STAT
pathway has been the subject of many recent comprehensive reviews
(17-21). STAT3, a well characterized 92-kDa protein, has been shown to
become activated by both epidermal growth factor and interleukin-6 in
human A-431 cells (22). Because the ROI generated in response to
various external stimuli can play a role both as regulators of
transcription factors, including nuclear factors
B (2, 23) and AT
(24), and as inhibitors of protein-tyrosine phosphatases (PTPases)
(25-27), we have investigated whether H2O2 and
other oxidizing agents could modulate STAT3 function in human lymphocytes. Enhanced phosphotyrosine accumulation could then result
from the combined effects of increased phosphorylation and decreased
dephosphorylation. Moreover, the DNA binding activity of STATs is known
to depend primarily on tyrosine phosphorylation (19, 28-31), although
serine phosphorylation is also important in modulating the binding
affinity of STAT3 (32-34). Here we show for the first time that STAT3
is phosphorylated on tyrosine residue(s), translocated to the nucleus,
and elicited to bind to specific DNA elements upon lymphocyte treatment
with H2O2. An additive effect between
H2O2 and vanadate was also evidenced suggesting that inhibition of tyrosine phosphatase(s) by pervanadate could take
place. The effects of H2O2 were also enhanced
by the presence of Fe2+ and Cu2+ ions, which
indicates the participation of other chemical reactive species derived
from oxygen. These data collectively indicate that STAT3 is a major
component of the signaling pathways that become activated by oxidative
stress in human lymphocytes.
 |
MATERIALS AND METHODS |
Reagents--
Hydrogen peroxide (30%, v/v),
3-amino-1,2,4-triazole, Na3VO4, PAO, diamide,
genistein, fetal bovine serum, and 1,10-phenantroline were purchased
from Sigma Chemical (Madrid, Spain). Ficoll-Hypaque, phosphate-buffered
saline, and RPMI 1640 were obtained from Bio-Whittaker (Verviers,
Belgium). Phenylmethylsulfonyl fluoride (PMSF) and the double-stranded
SIE m67 oligonucleotide (5'-GTCGACATTTCCCGTAAATC-3') were obtained from
Roche Molecular Biochemicals (Barcelona, Spain). Interferon-
was
purchased from Schering-Plow (Brinny Innishannon, Ireland).
Antibodies--
Polyclonal antisera directed against STAT1 and
STAT3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Monoclonal antibodies (4G10) against phosphotyrosine (free or
conjugated to agarose beads) were purchased from Upstate Biotechnology,
Inc. (Lake Placid, NY). Polyclonal anti-STAT5B antibody was kindly provided by Dr. J. O'Shea (Bethesda, MD). Goat anti-rabbit and anti-mouse antibodies conjugated to horseradish peroxidase were purchased from Sigma Chemical.
Cell Culture and Treatment--
Human peripheral blood
lymphocytes (PBL) were prepared by Ficoll-Hypaque gradient
centrifugation from normal blood donors, following informed consent.
Normally, PBL were used immediately after they were obtained. In other
cases, PBL were cultured in RPMI 1640 medium supplemented with 10%
(v/v) fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin and maintained at
37 °C in an atmosphere of 5% CO2 and 95%
O2 for 24 h. For stimulation treatments, the cells
were washed with RPMI 1640, resuspended in fresh medium at a density of
10 × 106 cells/ml, and incubated in RPMI 1640 with
the reagents indicated in each case and for the specified times at
37 °C. Stimulation was terminated by centrifugation at 5,000 rpm for
30 s and washing once with ice-cold phosphate-buffered saline
supplemented with 400 µM Na3VO4
and 400 µM EDTA. In all the experiments in which H2O2 was used, the cells were previously
incubated with 25 mM aminotriazole for 30 min to inhibit
endogenous catalase activity, and the cells were not washed before
H2O2 addition.
Western Blotting Analysis--
Stimulated cells were pelleted
and lysed in 75 µl of ice-cold lysis buffer A, containing 20 mM Tris-HCl, pH 8.0, 1% (v/v) Nonidet P-40, 137 mM NaCl, 5 mM MgCl2, 10% glycerol,
and the following inhibitors of phosphatase and protease: 5 mM EDTA, 100 µM PAO, 1 mM
Na3VO4, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 1 mM PMSF. The lysates were kept on ice for
30 min with occasional mixing and then centrifuged at 10,000 × g for 10 min at 4 °C. The supernatants obtained
constituted the cytoplasmic fraction, and the nuclei-containing pellets
were washed three times with buffer A (35). Standard Laemmli sample
buffer was then added to cytosolic and nuclear fractions, and after
boiling for 5 min followed by centrifugation at 10,000 × g for 10 min, the supernatants were analyzed by SDS-PAGE on
7.5% polyacrylamide gels. The separated proteins were
electrophoretically transferred to nitrocellulose membranes, and after
blocking nonspecific interactions in TBST (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.1% Tween 20) containing 3% bovine
serum albumin for 1 h, incubation with anti-STAT or 4G10
antibodies (diluted 1:1000 in TBST) was carried out overnight at
4 °C. Next, the membranes were washed twice with TBST, and incubation with a 1:5000 dilution of goat anti-rabbit secondary antibody coupled to horseradish peroxidase was performed for 1 h
at room temperature. After washing three times with TBST,
immunoreactive bands were visualized by using the enhanced
chemiluminescence assay as described previously (36).
Immunoprecipitation--
Stimulated cells were pelleted and
lysed in 75 µl of ice-cold lysis buffer B containing 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 300 mM
NaCl, 100 µM PAO, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 mM PMSF, 1 mM
Na3VO4, and 5 mM EDTA. The lysates
were centrifuged at 12,000 × g for 5 min. For
immunoprecipitation, incubation with anti-STAT3 specific antibodies
(0.5 µg/70 µl) was carried out with rotation for 2 h at
4 °C, followed by addition of 40 µl of a (50%) slurry of protein
A-Sepharose beads and further incubation for 2 h at 4 °C.
Alternatively, anti-phosphotyrosine antibodies covalently attached to
agarose (10 µg/70 µl) and a single incubation step performed for
2 h at 4 °C. The immune complexes were washed five times with
ice-cold lysis buffer B, and the proteins were extracted by boiling the
pellet in standard Laemmli sample buffer. After resolution by SDS-PAGE
(7.5% polyacrylamide), the proteins were electrotransferred to
nitrocellulose membranes and subjected to immunoblotting analysis as
indicated above.
Electrophoretic Mobility Shift Assay--
Stimulated cells were
lysed on ice for 10 min in hypotonic buffer C, which contained 20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM Na3VO4, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 20 mM NaF, 1 mM dithiothreitol, and 0.2% (v/v) Nonidet P-40. After centrifugation at 13,000 rpm for 1 min at 4 °C,
the supernatant obtained was collected as the cytoplasmic fraction.
Nuclear extracts were prepared by resuspension of the pellet in 25 µl
of high salt buffer (buffer C supplemented with 20% glycerol and 420 mM NaCl), followed by incubation on ice for 30 min with
occasional mixing. After centrifugation at 13,000 rpm for 10 min at
4 °C, the supernatants obtained constituted the nuclear extracts.
STAT3 DNA binding activity was assayed using the SIE m67 20-base pair
oligonucleotide as a probe (see sequence above) and the digoxigenin gel
shift assay kit from Roche Molecular Biochemicals. The reactions were
performed in 15 µl of a binding buffer containing 20 mM
Tris-HCl, pH 7.8, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% (v/v) Nonidet P-40, 6% glycerol,
and 10 µg of nuclear extract in the presence of 1 pmol of
digoxigenin-labeled oligonucleotide and allowed to proceed for 20 min
at room temperature. For competition assays, binding reactions were
performed in the presence of a 100-fold molar excess of unlabeled
oligonucleotide. The samples were finally electrophoresed on 5%
polyacrylamide native gels at 4 °C in 0.25× TBE buffer.
 |
RESULTS |
Hydrogen Peroxide Promotes Tyrosine Phosphorylation and Nuclear
Translocation of STAT3--
To investigate the changes on the STAT3
tyrosine phosphorylation status that take place under oxidative stress
conditions, PBL cells were treated with H2O2
for 2 min, and then STAT3 was immunoprecipitated using specific
antibodies. Immunoblotting analyses revealed that, whereas the amount
of STAT3 protein immunoprecipitated from
H2O2-treated and untreated cells was
comparable, the phosphotyrosine content of STAT3 was considerably
higher in lymphocytes treated with H2O2 (Fig.
1A). In other experiments,
cells treated with H2O2 and untreated control
cells were lysed and subjected to immunoprecipitation with
anti-phosphotyrosine antibodies. As shown in Fig. 1B, a high number of proteins underwent tyrosine phosphorylation upon
H2O2 treatment, as revealed by immunoblotting
using the same anti-phosphotyrosine antibodies. The presence of STAT3
as a 92-kDa protein among these phosphorylated polypeptides was further
evidenced by immunobloting with anti-STAT3 antibodies. A detectable
signal was only obtained in the immunoprecipitates from cells treated
with H2O2, which was virtually absent in
control untreated cells (Fig. 1B).

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Fig. 1.
H2O2 induces tyrosine
phosphorylation of STAT3 in human lymphocytes. A, PBL
were left untreated ( ) or were treated (+) with 2 mM
H2O2 for 2 min in RPMI 1640 medium at 37 °C.
The cells were then lysed in buffer B, and the supernatants obtained
after centrifugation were incubated with anti-STAT3 antibodies followed
by precipitation of the immune complexes with protein A-Sepharose
beads. The bound proteins were resolved by SDS-PAGE, transferred to
nitrocellulose membranes, and immunoblotted sequentially with
anti-phosphotyrosine antibodies (upper panel) or after
stripping with anti-STAT3 antibodies (lower panel).
B, PBL were treated and lysed as in A, and
immunoprecipitation was carried out with anti-phosphotyrosine
antibodies cross-linked to protein A-agarose. Tyrosine phosphorylation
was detected by immunoblotting with anti-phosphotyrosine antibodies,
and after blot stripping, STAT3 protein levels were analyzed by
immunoblotting with anti-STAT3 antibodies. The sizes of molecular mass
markers, expressed in kDa, are indicated at the left of
panel B. Arrows in A and B
show the position of migration of STAT3. IP,
immunoprecipitation; PY, phosphotyrosine.
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To test whether the H2O2-promoted increase in
tyrosine phosphorylation of STAT3 was accompanied by its translocation
from the cytosol to the nucleus, PBL cells treated with
H2O2 for different times were lysed, and the
cytoplasmic and nuclear fractions were subject to immunoblotting
analysis using anti-STAT3 antibodies. As illustrated in Fig.
2A, STAT3 was clearly
detectable in the nucleus after 10 min of treatment with
H2O2, whereas in the cytosol the STAT3 signal
at this time was significantly diminished. Nuclear translocation of
STAT3 appeared to be completed after 30 min, because no cytosolic STAT3
was detected at this point. In some untreated cell preparations, a
certain amount of STAT3 was found in the nuclei, attributable to basal
levels of activation in the absence of stimulus.

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Fig. 2.
H2O2 and PHA induce
STAT3 translocation from the cytosol to the nucleus in human
lymphocytes. PBL were treated with 2 mM
H2O2 (A) or 10 µg/ml PHA
(B) for the indicated times under culture conditions. The
cells were then lysed in buffer A, and the cytoplasmic and nuclear
fractions were obtained as described under "Materials and Methods."
The proteins were resolved by SDS-PAGE and subject to immunoblotting
analysis with anti-STAT3 antibodies. C, PBL were treated or
not with 10 µg/ml PHA for 24 h and then were treated in the
presence or absence of 2 mM H2O2
for 15 min, as indicated by the minus and plus
symbols. The cells were then lysed, and cytoplasmic and nuclear
fractions were analyzed by immunoblotting with anti-STAT3 antibodies.
The sizes of molecular mass markers, expressed in kDa, are indicated at
the left of the three panels. Arrows indicate the
position of migration of STAT3.
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The effect on STAT3 nuclear translocation of phytohemagglutinin (PHA),
a well known promoter of lymphocyte proliferation, was also analyzed
alone or in combination with H2O2. As shown, PHA by itself was able to slightly induce STAT3 nuclear translocation (Fig. 2B, lane 8). This effect was, however,
considerably enhanced when PHA acted in combination with
H2O2, conditions under which a synergistic
effect was found (Fig. 2C). Recently, it has been documented
that the treatment of T lymphocytes with interleukin-2 results in an
increase of STAT3 tyrosine phosphorylation, although no such effect was
observed to occur in cells treated with PHA for 3 days (37). Present
results suggest that PHA enhances the transduction of signals that are
required for optimal H2O2 effect.
Hydrogen Peroxide and Vanadate Act Synergistically to Induce
Nuclear Translocation of STAT3--
It has been previously proposed
that the combination of H2O2 and orthovanadate
potently inhibits PTPase activity (25, 26) and/or synergistically
stimulates insulin-dependent PTK activity (38) in T
lymphocytes. Also, the mixture of H2O2 and
sodium orthovanadate promotes tyrosine phosphorylation of phospholipase C
1 and activates p56lck and p59fyn tyrosine
kinases (23). These facts have been explained in the light of
inhibition of protein-tyrosine phosphatase(s) by pervanadate (38).
Further experiments were therefore used to analyze whether a decrease
in intracellular PTPase activity could be implicated in the
H2O2-induced STAT3 nuclear translocation
effect. Toward this end, human PBL were treated with orthovanadate,
alone or in combination with H2O2, and the
nuclear levels of STAT3 were analyzed. Vanadate alone at 100 µM (the maximal dose tested) promoted a slight
accumulation of STAT3 in the nucleus (Fig.
3A, lane 5), and
H2O2 alone (Fig. 3A, lanes
1-4) induced this STAT3 translocation detectable only at the
highest tested concentration (2 mM). However, the
combination of vanadate and H2O2 markedly
stimulated in a synergistic fashion the nuclear translocation of STAT3,
enhancing about 6-fold the effect obtained with 2 mM
H2O2 alone. As shown in Fig. 3A, at
100 µM vanadate combined with a low dose of
H2O2, a nuclear translocation of STAT3 took
place (lane 6), whereas at higher concentrations of
H2O2 an intense nuclear STAT3 signal was
observed (lanes 7 and 8).

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Fig. 3.
Orthovanadate and
H2O2 cooperatively induce STAT3 nuclear
translocation. A, PBL were treated with several doses
of H2O2 in the presence or absence of 100 µM sodium orthovanadate for 15 min at 37 °C. The cells
were then lysed in buffer A, and nuclear fractions obtained as
described under "Materials and Methods" were resolved by SDS-PAGE
and analyzed by immunoblotting with anti-STAT3 antibodies. The
arrow indicates the STAT3 band. The sizes of molecular mass
markers, expressed in kDa, are indicated at the left of
panel A. B, STAT3 nuclear levels obtained from
densitometric analysis of panel A are plotted
versus the concentration of
H2O2.
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Effects of Phenylarsine Oxide, Diamide, and Genistein on
H2O2-induced Nuclear Translocation of
STAT3--
Because the above effects of pervanadate could be due to
the inhibition of intracellular tyrosine phosphatase(s), experiments were attempted to analyze whether other reagents known to alter the
tyrosine phosphorylation status of proteins could influence the nuclear
translocation of STAT3. Because the effect of PAO as a PTPase inhibitor
in T lymphocytes is well known (39), we decided to test its effect on
STAT3 nuclear translocation in human PBL. As shown in Fig.
4A, PAO induced a
dose-dependent enhancement of STAT3 nuclear translocation,
as monitored in nuclear fractions from cells treated with different
concentrations of PAO, upon immunoblotting analysis with anti-STAT3
antibodies. It could also be observed that the nuclear levels of this
factor reached in the presence of 2 mM
H2O2 could be mimicked by 50 µM
PAO. Furthermore, PAO and H2O2 were found to
act in a synergistic fashion (Fig. 4B, compare lanes
2 and 4 with lane 5), which suggests that
both molecules could be cooperatively acting as inhibitors of
intracellular tyrosine phosphatases. Diamide, a compound with oxidative
properties (12, 13), was also shown to stimulate the nuclear
translocation of STAT3 (Fig. 4B, lane 3). This
drug was reported to induce a marked enhancement of the tyrosine
phosphorylation status of several cellular proteins (13, 40), possibly
mediated by inhibition of tyrosine phosphatase(s) (13, 37). In an
opposite fashion, genistein, an inhibitor of protein-tyrosine kinases,
produced a partial inhibition of the STAT3 nuclear translocation
elicited by H2O2 (Fig. 4B,
lane 6) or diamide (lane 7). However, genistein was unable to counteract the stimulating action of PAO (lane
8). These results clearly suggest that the nuclear levels of STAT3 are balanced by the opposite actions of intracellular PTKs and PTPases.

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Fig. 4.
PAO induces nuclear translocation of
STAT3. A, PBL were left untreated (lane 1)
or were treated for 15 min with 2 mM
H2O2 (lane 2), with 1 µl of Me2SO (lane 3), or with the indicated
doses of PAO (lanes 4-8). The cells were then lysed in
buffer A, and nuclear fractions were resolved by SDS-PAGE and analyzed
by immunoblotting with anti-STAT3 antibodies. B, PBL were
subjected to the indicated treatments. PBL were left untreated
(lanes 1-5) or treated (lanes 6-9) with
genistein (100 µM) for 30 min, followed by 2 mM H2O2 (lanes 2 and
6), 1 mM diamide (lanes 3 and
7), or 100 µM PAO (lanes 4 and
8) for 15 min. In lanes 5 and 9,
H2O2 and PAO were added together at the same
time. Nuclear fractions were analyzed by immunoblotting with anti-STAT3
antibodies. The arrows indicate the STAT3 band, and the
sizes of molecular mass markers, expressed in kDa, are indicated at the
left of both panels.
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Experiments were also conducted to study the specificity of
oxidant-induced STAT nuclear translocation. Immunoblotting analyses of
PBL nuclear extracts reveals that H2O2
treatment lightly induced the nuclear translocation of STAT1 and
STAT5B, although to a lesser extent than that observed for STAT3 (Fig.
5). The distinct behavior displayed by
these STAT proteins upon cell challenge with
H2O2 agrees with recent observations regarding
their activation by different cytokines and growth factors (41). In
this regard, G-CSF induces tyrosine phosphorylation of STAT1, STAT3,
and STAT5B; however, STAT5B can be activated by mutant receptors that
lack tyrosine residues (41, 42). Moreover efficient STAT3 activation requires additional receptor sequences that include Tyr704
(42).

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Fig. 5.
H2O2 induces nuclear
translocation of STAT3 but not of STAT1 or STAT5B. PBL were left
untreated ( ) or were treated (+) with 2 mM
H2O2 for 15 min, and nuclear fraction samples
were resolved by SDS-PAGE and analyzed by immunoblotting with
anti-STAT3, anti-STAT1, or anti-STAT5B antibodies, as indicated.
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Effect of Fe2+/Cu2+ Ions on
H2O2-promoted STAT3 Nuclear
Translocation--
After passive diffusion through the plasma
membrane, H2O2 can be converted into other
active oxygen species, such as O
2 and ·OH (23). The
most likely mode of intracellular ·OH radical production is via
Fenton chemistry, which involves the reduction of
H2O2 by ferrous ions according to the reaction: H2O2 + Fe2+
·OH + OH
+ Fe3+ (8). Also, cuprous ions are
elemental producers of ·OH radicals via Fenton reactions (9). We
have analyzed whether the chemical species generated from
H2O2 through Fenton reactions could affect
STAT3 nuclear translocation. Fig. 6
illustrates that the prior addition of Fe2+ and
Cu2+ ions notably enhanced the
H2O2-induced nuclear translocation of STAT3
(lane 3). By contrast, the pretreatment of cells with 1,10-phenantroline, a chelator of copper and iron ions resulted in
cancellation of the STAT3 nuclear accumulation induced by
H2O2 (lane 4). These results
indicate that the intracellular reduction of
H2O2 to yield more reactive oxygen species,
such as ·OH, could constitute a more efficient mechanism for the
positive action of H2O2 on STAT3.

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Fig. 6.
Pretreatment with
FeSO4/CuSO4 or 1,10-phenantroline modulates
STAT3 nuclear translocation induced by
H2O2. PBL were left untreated (lane
1), treated with 2 mM H2O2
(lane 2) for 15 min, or pretreated for 15 min with 100 µM FeSO4/100 µM
CuSO4 (lane 3) or 20 µM
1,10-phenantroline (1,10-Phe, lane 4) and then
exposed to 2 mM H2O2 for a further
15 min. Nuclear fractions were resolved by SDS-PAGE and analyzed by
immunoblotting with anti-STAT3 antibodies.
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Hydrogen Peroxide Enhances STAT3 DNA Binding Activity--
The
enhancement of the ability of nuclear STATs to bind to specific DNA
elements in PBL cells upon cell stimulation by oxidizing agents was
assessed through gel mobility shift assays using a double-stranded
oligonucleotide probe containing a high affinity target sequence for
STAT factors. This binding sequence, named m67, was derived from the
sis-inducible element (SIE) of the human c-fos
promoter (43, 44). Nuclear extracts were thus prepared from PBL
challenged with orthovanadate, H2O2, or both
and incubated with the digoxigenin-labeled SIE m67 oligonucleotide
probe. Fig. 7A illustrates
that H2O2 induced a rapid assembly of complexes able to bind to the SIE m67 probe within PBL nuclei (lane
3). The same effect was observed upon vanadate treatment
(lane 2), and a cooperative effect was found to take place
between vanadate and H2O2 (lane 4).
As a positive control, we also analyzed the effect of interferon-
(18), with the same results (lane 5). The shifted band could
be competed by an excess of unlabeled SIE m67 oligonucleotide; thus, it
indicates that nuclear factor binding was specific (lane 6).
However, quite intriguingly, we found that treatment with PAO, despite
this compound enhanced STAT3 nuclear translocation, cancelled
completely nuclear factor binding to the STAT specific sequence, even
below basal levels of binding seen in the absence of
H2O2 (data not shown). We cannot offer any
explanation for this fact, although it could be related to the
hyperphosphorylation of proteins (e.g. STAT3) found to occur after PAO treatment (38, 45, 46). Fig. 7B illustrates that the presence of Fe2+/Cu2+ in combination with
H2O2 greatly enhanced the capacity of nuclear factors to bind to the SIE m67 oligonucleotide (Fig. 7B,
lane 3) and that the addition of 1,10-phenantroline reduced
the H2O2-induced nuclear factor binding. These
results show a clear correlation with the observations described in
Fig. 6 regarding the nuclear translocation of STAT3.

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Fig. 7.
H2O2 enhances STAT3
DNA binding activity. A, PBL were left untreated
(lane 1) or were treated with 100 µM
Na3VO4 (lane 2), 2 mM
H2O2 (lane 3), both
H2O2 and Na3VO4 added
at the same time (lane 4), or 1000 units/ml interferon-
(lane 5) for 10 min. The cells were then lysed in buffer C,
and nuclear extracts were prepared as described under "Materials and
Methods." DNA binding to the digoxigenin-labeled SIE m67
oligonucleotide probe was assessed by gel mobility shift assays. The
retarded band is indicated by an arrow. Where indicated by
an asterisk (lane 6), binding reactions were
performed in the presence of a 100-fold molar excess of unlabeled
oligonucleotide in the reaction performed with nuclear extracts from
H2O2-treated human lymphocytes. B,
PBL were left untreated (lane 1) or treated with 2 mM H2O2, alone (lane 2)
or in combination with 100 µM FeSO4/100
µM CuSO4 (lane 3) or 20 µM 1,10-phenantroline (1,10-Phe, lane
4) for 15 min. Nuclear extracts were used for gel mobility shift
assays as indicated for panel A.
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 |
DISCUSSION |
The intracellular processes that become triggered upon oxidative
stress constitute nowadays a focus of extensive research. Very
recently, we have described that H2O2 and other
peroxides are able to inhibit calcineurin activity and to inactivate
nuclear factor
B DNA binding in human neutrophils (36). In contrast, Schreck et al. (2) have provided evidence that peroxides
activate nuclear factor
B in lymphocytes through an undetermined
mechanism involving the synthesis of ROI. Also,
H2O2 has been implicated in the positive
modulation of the activity of a number of PTKs whose function is
critical for lymphocyte activation (11, 47). An involvement of STAT
factors in mediating leukocyte activation elicited by interleukin-2 has
been recently discovered (33, 37, 48). However, the potential role of
STAT factors as mediators of peroxide-triggered lymphocyte activation,
by acting as target molecules of oxidative processes, has not been
previously explored. In this paper, we provide the first evidence that
H2O2 promotes tyrosine phosphorylation of STAT3
in peripheral blood human lymphocytes, which is followed by its
translocation to the nucleus and binding to a specific DNA element.
It is well established that the activation of STAT factors requires
their phosphorylation on tyrosine residue(s) (34). To explain the
mechanism whereby they become activated by oxidants, two possibilities
can be contemplated. First, the alteration of their redox status could
directly alter STAT conformation, in such a way that their interaction
with cytosolic proteins responsible for nuclear targeting would be
enabled. Because the structures of STAT3 and especially its presumptive
redox centers are not completely understood, this hypothesis requires
further investigation. The current state of knowledge thus makes more
likely a second explanation based on the ability of oxidants to act as
inhibitors of tyrosine phosphatases. According to this possibility,
STAT3 nuclear translocation would be facilitated or promoted by its increased status of tyrosine phosphorylation, this being the
consequence of inhibition by H2O2 of the
activity of intracellular PTPases (25, 39). In this context,
exogenously added H2O2 has been shown to
increase protein-tyrosine phosphorylation in several cell types, such
as lung epithelial and FAO cells (49, 50). Also,
H2O2 can directly inhibit PTPase activity
in vitro, and this inhibition can be completely reversed by
dithiothreitol (25). All PTPases are known to contain an essential
sulfhydryl group at their active site, which is susceptible to
oxidation because of its unusually low pKa (<5)
(51). These observations thus suggest that PTPases may constitute
targets for intracellularly generated H2O2 and
that their inhibition by oxidants may well lead to activation of number
of signaling molecules, such as STAT3, by virtue of their increased
phosphotyrosine content. It can be presumed that because the specific
activities of PTPases in vitro are 10-1000 times higher
than those of PTKs (52), in most cell types the activation of a
receptor tyrosine kinase upon ligand binding may not be sufficient to
increase the steady-state level of protein-tyrosine phosphorylation, so
that concurrent inhibition of PTPases by H2O2
and/or derived ROI could additionally be necessary.
The treatment of lymphocytes with vanadium derivatives induces a strong
tyrosine phosphorylation of protein kinases of the Erk2 and Syk
families, leading to their activation (45). Also, it has been reported
that H2O2 treatment of blood T cells elicits tyrosine phosphorylation of Lck kinases and that a much stronger response on tyrosine phosphorylation of multiple cellular proteins is
observed following treatment with the sulfhydryl oxidizing agent
diamide (12). Furthermore, the Src kinases, such as p56lck and
p59fyn in T cells, were transiently activated by
H2O2 in combination with vanadate (23). It is
interesting to note that vanadate by itself exerts a limited effect on
the tyrosine phosphorylation of STAT3, as shown in this work. However,
upon the simultaneous addition of H2O2 and
vanadate, the resulting pervanadate enhanced severalfold the effects
exerted separately by these two compounds. It is of interest that
pervanadate administered as such induces the tyrosine phosphorylation
and nuclear translocation of a number of STAT proteins in liver cells,
including STAT3 (53). Also, pervanadate has been shown to induce T cell
activation and transcription of c-fos (54), the activation
of interferon-
- and prolactin-dependent transcription
factors, and the activation of JAK kinases in epithelia cells (55, 56).
The possibility that the H2O2 and pervanadate effects on nuclear translocation of STAT3 could be ascribed to inhibition of tyrosine phosphatases is reinforced by experiments using
PAO and diamide, two compounds able to act as PTPase inhibitors. As
shown in this work, these drugs elicited an enhancement of the
H2O2 action, whereas genistein, an inhibitor of
protein-tyrosine kinases, antagonized the H2O2
and diamide effects. Genistein did not, however, counteract the
increase in STAT3 phosphorylation promoted by PAO, possibly due to an
imbalance between the phosphorylation and dephosphorylation of STAT3 at
the study doses of both agents. Taken together, present results are in
line with previous reports, demonstrating that
H2O2 acts an inhibitor of tyrosine
phosphatase(s) (10-12), leading in turn to enhanced STAT3 tyrosine
phosphorylation. The complexity of this phenomenon is evidenced,
however, by the recent demonstration that phosphorylation of STAT3 on
the residue Ser727 is also required for competent STAT3
transcriptional activation (33, 34). It may thus be speculated that
H2O2 could as well increase a serine kinase
activity necessary for the activation of STAT3.
It is noteworthy that the enhancing effect of
H2O2 on STAT3 nuclear translocation seems
rather specific, because neither STAT1 nor STAT5B underwent migration
to the nucleus under oxidative conditions, as shown in this paper.
However, which is the active oxygen species directly responsible for
the observed H2O2 effects on STAT3 activation
is currently unknown. Exogenously H2O2 added is
rapidly converted into other chemical species, and in this context we
present evidence, using a combination of Fe2+ and
Cu2+ ions, that ·OH radicals generated through
Fenton reactions could fulfill such a role. In fact, in the presence of
these metal ions, an enhanced STAT3 nuclear translocation and DNA
binding was found in cells exposed to H2O2.
Accordingly, it is clear that H2O2 is not the sole species implicated in the nuclear translocation of STAT3. In line
with a recent paper, showing that 1,10-phenantroline, a chelator of
iron and copper ions, completely prevented DNA strand breakage induced
by H2O2 (57), we have found that this compound was able to cancel STAT3 nuclear translocation induced by
H2O2. These results speak in favor of the
possibility that intracellular Fe2+/Cu2+ ions
could play a role as intermediates under
H2O2-induced oxidative stress conditions.
In summary, present observations support a role for oxidants in the
regulation of STAT3 transcription factor activity in vivo in
human lymphocytes. In keeping with extensive work done elsewhere, our
results favor the hypothesis that H2O2 by
itself and/or derived oxidative species generated through Fenton
reactions can modulate the STAT3 tyrosine phosphorylation status and
hence promote its translocation to the nucleus and the formation of
bound complexes at specific DNA promoter elements.
 |
ACKNOWLEDGEMENT |
We thank Dr. O'Shea for kindly providing the
anti-STAT5B antiserum.
 |
FOOTNOTES |
*
This work was supported by Fondo Investigaciones Sanitarias
Grants 94/1484 and 97/1289 (to F. S.) and 97/207 (to J. C.),
a grant from the Foundation of Sociedad Española Alergia
Immunologia Clinica of Spain (to J. M.), Grant SAF 96/0205 from
the Dirección General Investigación Científica
Técnica (to F. J. B.), a grant from Consejería
Salud (Junta de Andalucía) (to F. S.), and a grant from
Innogenetis Diagnostica y Terapeutica S.A. from Spain.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: Dept. de
Bioquímica Médica y Biología Molecular. Facultad
de Medicina. Avda. Sánchez Pizjuán 4, 41009 Sevilla Spain.
Fax: 34-95-4907041; E-mail: fsobrino{at}cica.es.
 |
ABBREVIATIONS |
The abbreviations used are:
ROI, reactive oxygen
intermediates;
PAO, phenylarsine oxide;
JAK, Janus kinase;
PTK, protein-tyrosine kinases;
PTPase, protein-tyrosine phosphatases;
PMSF, phenylmethylsulfonyl fluoride;
PHA, phytohemagglutinin;
PBL, peripheral
blood lymphocytes;
PAGE, polyacrylamide gel electrophoresis;
SIE, sis-inducible element.
 |
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