Acta
Physiologica Sinica,
June 25, 2003, 55(3):
311－316
Received
2002-08-15 Accepted 2002-11-18
This
project was supported by the National Natural Science Foundation of
Corresponding
author. Tel: +86-516-5748423;
Fax: +86-516-5748486;
E-mail: gyzhang@xzmc.edu.cn
Research Paper
Activation of STAT3 induced by
cerebral ischemia in rat
hippocampus and its possible mechanisms
LI Hong-Chun, ZHANG Guang-Yi*
Abstract: It has been demonstrated that signal transducer and activator of
transcription-3 (STAT3) is
activated after cerebral ischemia/reperfusion (I/R) in cortex and striatum. In
this study, we investigated whether STAT3 was rapidly activated in hippocampus
by cerebral ischemia without
reperfusion in four-vessel occlusion (4-VO) model of Sprague-Dawley (SD) rats.
The results showed that tyrosine phosphorylation and DNA binding activity of
STAT3 was rapidly increased by ischemia. The p-STAT3 level in cytoplasm
increased 5 min after occlusion and reached a peak at 10 min following ischemia
(1.7 folds vs sham) by means of immunoblotting (IB). P-STAT3 in nucleus was
gradually enhanced with its peak activity occurring at 30 min of ischemia (2.3 folds vs sham). Electrophoretic
mobility shift assay (EMSA) with STAT3 probe demonstrated that DNA binding
activity of STAT3 in nuclear extracts increased from 5 min and peaked at 30 min
of ischemia (3.2 folds vs sham). These changes were
prevented by genistein (a protein tyrosine kinase inhibitor) and antioxidant
N-acetyl-L-cysteine (NAC), but promoted by sodium orthovanadate (a protein
phosphatase inhibitor), which were administered to the SD rats 20 min before
ischemia. These results indicate that the activation of STAT3 following
cerebral ischemia may be modulated
by PTK/PTP, and that this pathway may be of benefit to the adaptation of the
hippocampal neurons to oxidative stress.
Key
words: STAT3;
cerebral ischemia; protein tyrosine kinase; protein tyrosine phosphatase;
oxidative stress
脑缺血大鼠海马信号转导与转录激活子-3的激活及其调控
李洪春, 张光毅*
徐州医学院生物化学与分子生物学研究中心, 徐州
221002
摘要: 以往的研究表明, 在脑缺血/再灌注的皮层和纹状体组织中信号转导与转录激活子-3
(STAT3)被激活。本实验旨在研究SD大鼠四动脉结扎诱导的全脑缺血是否引起海马组织STAT3的快速激活及其调控机制。结果表明, 脑缺血导致STAT3快速磷酸化激活及DNA结合活性增加。胞浆STAT3的磷酸化水平从缺血5
min起就显著增高, 10 min达高峰(增加约1.7倍), 然后开始下降。核内STAT3的磷酸化水平则逐渐增加, 缺血30 min时达高峰(增加约2.3倍)。电泳迁移率改变分析法显示,
STAT3的DNA结合活性从缺血5 min起就显著增加, 30 min达高峰(增加约3.2倍)。进一步的研究表明, 缺血前20 min腹腔注射给药, 然后缺血30
min, 发现蛋白酪氨酸激酶抑制剂染料木黄酮和抗氧化剂N-乙酰半胱氨酸能显著地抑制核内STAT3的磷酸化水平及DNA结合活性的增加(磷酸化水平从2.3和2.5倍分别降为1.2和1.4倍,
DNA结合活性则从2.8和3.7倍分别降为1.1和1.5倍), 而蛋白酪氨酸磷酸酶抑制剂矾酸钠则能明显地促进他们的增高(磷酸化水平从2.0倍增到3.4倍,
DNA结合活性从3.1倍增为5.1倍)。这些结果提示, 蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶可能共同参与了缺血诱导STAT3的激活调控, STAT3的激活可能有助于海马神经元适应氧化应激。
关键词: 神经生物学; 信号转导与转录激活子-3 (STAT3); 脑缺血; 蛋白酪氨酸激酶; 蛋白酪氨酸磷酸酶; 氧化应激
中图分类号: Q426; R743.31
Signal transducer and activator of transcription-3 (STAT3) belongs to a
family of transcription factors with unique dual functions that bear signals
from cell membrane to the nucleus. Phosphorylated-STAT3 allows dimerization,
and nuclear translocation. Once inside the nucleus, activated STAT3 can bind to
the serum inducible element in the c-fos gene promoter[1], to the jun-B
promoter[2], and interact with other transcriptional activators in the
regulation of gene expression involved in immune and stress response.
Reactive oxygen species (ROS) can
activate JAK-STAT pathway[3－7]. An extensive body of work has demonstrated that
STAT3 pathway plays a crucial role in the expression of stress-responsive genes
in a variety of cells and organs[8,9]. Experimental evidence has indicated that
STAT3 was rapidly induced in response to H2O2 and inhibited by
antioxidants[7,10]. In the nervous system, a variety of pathogenetic stimuli
can activate STAT3, including ischemic insults[11,12] and cytokines, etc. Upon
stimulation, STAT3 becomes activated by phosphorylation on a specific tyrosine
residue (Tyr705) in reponse to cytokines and growth factors via their
subsequent receptors in a janus kinase (JAK) dependent manner or through
intrinsic receptor tyrosine kinase domains or by other tyrosine kinase, such as
Src family protein tyrosine kinase (PTK)[13]. On the other hand, the state of
tyrosine phosphorylation of STAT3 is also controlled by protein tyrosine
phosphatases (PTP).
Hippocampus is one of the brain regions most vulnerable to ischemic damage. The purpose of the
present study was to determine the mechanism by which the state of the tyrosine
phosphorylation and DNA binding activity of STAT3 are regulated during cerebral
ischemia in rat hippocampus.LI Hong-Chun
et al: STAT3 Activation and
Regulation in Ischemic Insult Acta Physiol. Sin., June 25, 2003, 55(3): 311－316
1 MATERIALS AND METHODS
1.1 Materials. Mouse monoclonal
anti-p-STAT3 (sc-8059), rabbit
polyclonal anti-STAT3 (sc-482) antibodies, and STAT3 consensus probe (sc-2571)
were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa
Cruz, CA). Genistein, NAC, vanadate, alkalline phosphatase conjugated goat
anti-rabbit IgG and goat anti-mouse IgG were from Sigma. [γ-32]ATP was from
Yahui Biological and Medical Engineering Co, Beijing. T4 polynucleotide kinase,
BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro blue tetrazolium)
were from Promega (Madison, WI, USA).
All other chemicals were from Sigma unless indicated otherwise.
1.2 Animals
and induction of ischemia. Adult
male Sprague-Dawley (SD) rats (purchased from Sippr-BK Experimental Animal Ltd
Co, Shanghai, Grade Ⅱ, Certificate No D52, n=45) weighing 250－300 g were
used. Cerebral ischemia was induced
by 4-VO as described before[14]. Briefly, under anesthesia with chloral hydrate
(350 mg/kg, i.p.), vertebral arteries were electrocauterized and common carotid
arteries were exposed. On the next day, both carotid arteries were occluded
with aneurysm clips for 5, 10, 15 or 30 min without reperfusion, respectively.
Then the whole brains were removed immediately for dissections, and the
hippocampi were frozen in liquid nitrogen until use. During ischemia, animals
were required to meet the following criteria: (1) completely flat EEG during
occlusion; (2) maintenance of dilated pupils and absence of a cornea reflex due
to strong light stimulation, and (3) rectal temperature was maintained at 37－37.5℃.
Animals not meeting the criteria were excluded. Sham control animals received
the same surgical procedures except those carotid arteries were not occluded.
When necessary, N-acetyl-L-cysteine (NAC), genistein (GN), sodium orthovanadate
(SV) or dissolvents (0.9% NaCl or Me2SO) were administered to the rats by
intraperitoneal injection (i.p.) 20 min before 30 min of ischemia.
1.3 Tissue
preparation and nuclear extracts．
Nuclear extracts of hippocampus were carried out with a modification of
a previously described procedure[15]. Briefly, tissue samples were homogenized
in ice cold buffer A (mmol/L: HEPES 10,
NaF 50, DTT 1, Na3VO4
1, pH 7.9) containing enzyme inhibitors: 0.5 mmol/L PMSF, 10 mg/L each
of aprotinin, leupeptin, and pepstatin A. The homogenates were allowed to swell
on ice for 10 min, after which the last concentration of 0.6% solution of NP-40
was added and the tubes were vigorously vortexed for 30 s and centrifuged at
800×g for 10 min at 4℃. The supernatants containing the cytoplasmic fraction
were collected and stored at －80℃
until use. The nuclei-containing pellets were washed three times with buffer A,
then resuspended in ice-cold buffer B (mmol/L: HEPES 20, NaCl 400, DTT 1, Na3VO4 1, 20% glycerine, pH 7.9) containing enzyme inhibitors
mentioned above. Then the tubes were vigorously rocked at 4℃ for 30 min on a
shaking platform. After centrifugation at 12000×g for 15 min at 4℃, the nuclear
extracts were aliquoted and frozen in liquid nitrogen, then stored at －80℃
until use. Protein concentration was determined using Lowry method[14].
1.4 Immunoblotting
(IB). IB of p-STAT3 was
performed as described before[14]. Briefly, samples were mixed with loading
buffer and boiled for 5 min. Proteins (100 μg) were fractionated by 7.5%
SDS/PAGE. Gels were electroblotted onto nitrocellulose membrane and probed with
monoclonal anti-p-STAT3 antibody (1∶200) and visualized by using the image
analyzer.
1.5 Electrophoretic
mobility shift assays (EMSA). EMSA
were performed as described by Shen et al.[16] with some modification. Briefly,
a double-stranded 24-mer DNA probe: 5'-GAT CCT TCT GGG AAT TCC TAG ATC-3'
containing the SIE consensus sequence (in boldface) and the reverse complement
3'-CTA GGA AGA CCC TTA AGG ATC TAG-5' were labeled with [γ-32]ATP by T4
polynucleotide kinase. For binding assays, 10 μg of nuclear proteins extracts from
hippocampus and 1 μg of [ploy (dI·dC)(dI·dC)] (Amersham Pharmacia) were incubated for
10 min prior to addition of labeled probe. Reaction was allowed to take place
for 20 min at room temperature. Samples were mixed with loading buffer and
loaded on 4% nondenaturing polyacrylamide gel in 0.5×Tris/borate/Ethylene
diamine tetraacetic acid (EDTA). Autoradiograms were developed by exposing
vacuum-dried gels to x-ray film at －80℃ with intensifying screens for 20－48 h.
Competition experiment was carried out using 100-fold excess non-radioactive
STAT3 probe. Specificity control was carried out by using 100-fold excess
non-radioactive irrelated probe that was specific for SP-1. Supershift of STAT3
was incubated with anti-STAT3 antibody.
1.6 Statistical
analysis． Values were expressed as
mean±SD from three independent animals. Statistical software package (Stata,
version 4.0) was used for data analysis. Statistical analysis of the results
was carried out by one-way analysis of variance (ANOVA) followed by the
Duncan's new multiple range method or Newman-Keuls test and P<0.05 was
considered significant.
2 RESULTS
2.1 Rapid
activation of STAT3 in rat hippocampus during cerebral ischemia
To evaluate whether a rapid activation
of STAT3 was induced after cerebral ischemia by 4-VO, the p-STAT3 and DNA
binding activity were systematically examined at serial ischemia time points
(5, 10, 15 and 30 min) without reperfusion in rat hippocampus. Levels of
p-STAT3 in cytoplasmic fractions increased rapidly from 5 min after occlusion
and reached its peak level at 10 min of ischemia (1.7 folds vs sham), then decreased, and
had a marked reduction at 30 min (Fig.1A, C). IB analysis of nuclear extracts
with anti-p-STAT3 antibody demonstrated that sequence of ischemia by 4-VO
resulted in a markedly rapid increase of p-STAT3 at 10 min after occlusion and
reached peak activity at 30 min of ischemia (2.3 folds vs sham; Fig.1A, C).
EMSA with STAT3 probe showed that DNA binding activity of STAT3 had a similar
fashion to that of p-STAT3 by IB in nucleus extracts [3.2 folds vs sham at ischemia 30 min (I 30 min) Fig.1B, C], but starting from 5 min of
ischemia.
Fig.1.Rapid
activation of STAT3 after cerebral
ischemia without reperfusion by 4-VO. A:
IB analysis of hippocampal cytoplasm and nucleus derived from rats at
various ischemic time points with anti-p-STAT3 antibody. B: DNA- binding
activity to the STAT3 probe was assessed by EMSA. C: Bands corresponding to p-STAT3 and STAT3
binding complex were scanned and the intensities were represented as folds
vs sham control. Data are expressed
as mean±SD from three independent animals (n=3). aP<0.05 vs sham， bP<0.05 vs I 10 min. I, ischemia; OD, optical
density.
Fig.2Inhibitory
effect of NAC on tyrosine phosphorylation and DNA-binding activity of STAT3
induced by ischemia in hippocampus. Nuclear extracts were prepared from rats
subjected to 4-VO that had been treated with NAC or with vehicle (0.9% NaCl).
A: IB was used to analyze the
levels of p-STAT3 with anti-p-STAT3 antibody. B: DNA-binding activity was
evaluated using STAT3 specific probe by EMSA. The competition assay was
performed in the absence (a) or presence of 100-folds unlabeled STAT3 consensus
probe (d), or 100-folds excess of an unrelated SP1 probe (b). The supershift
assay was carried in the presence of anti-STAT3 antibody (c). C: Bands corresponding to p-STAT3 and STAT3
binding complex were scanned and the intensities were represented as folds vs.
sham control. Data are expressed as mean±SD from three independent animals
(n=3). aP<0.05 vs sham control, bP<0.05 vs I 30 min +NaCl. NAC, N-acetyl-L-cysteine
(100 mg/kg) was administered to the rats 20 min before 30 min of ischemia.
2.2 Inhibitory
effect of NAC on tyrosine phosphorylation and DNA binding activity of STAT3
induced by cerebral ischemia
To demonstrate the effect of ROS on
activation of STAT3 induced by
cerebral ischemia in hippocampus neurons, the antioxidant NAC (100
mg/kg) and an equal volume of vehicle (0.9% NaCl) were intraperitoneally
injected into 4-VO rats at 20 min before 30 min of ischemia. The ischemia-induced
increase of tyrosine phosphorylation and DNA-binding activity of STAT3 in
nucleus was significantly eliminated by pretreatment with NAC (Fig.2, the level
of p-STAT3 was from 2.8 folds of vehicle control to 1.5 folds of NAC vs sham;
DNA-binding activity of STAT3 from 3.7 folds to 1.6 folds vs sham).
2.3 Effects
of genistein and sodium orthovanadate on tyrosine phosphorylation and DNA
binding activity of STAT3 during cerebral ischemia
Genistein (30 mg/kg), an inhibitor of
PTK, or sodium orthovanadate (15 mg/kg), a PTP inhibitor, was administered to
the rats by i.p. 20 min before 30 min of ischemia. Control rats received
intraperitoneal injections of an equal volume of dissolvent (Me2SO) or vehicle
(0.9% NaCl), respectively. Samples from drug-treated or vehicle-treated groups were immunoblotted using anti-p-STAT3
antibody and were initiated with STAT3 probe by EMSA. Figure 3 shows a
significant inhibition in
activation of STAT3 in the presence of genistein (the level of p-STAT3
was from 2.7 folds of vehicle control to 1.5 folds of genistein vs sham;
DNA-binding activity of STAT3 from 3.6 folds to 1.7 folds vs sham) and a
promotion by pretreatment with sodium orthovanadate (the level of p-STAT3 was
from 2.5 folds of vehicle control to 3.8 folds of sodium orthovanadate vs sham;
DNA-binding activity of STAT3 from 3.4 folds to 4.5 folds vs sham).
Fig.3.Effects
of genistein and vanadate on tyrosine phosphorylation and DNA-binding activity
of STAT3 in hippocampus after cerebral ischemia. Nuclear extracts were prepared
from rats subjected to 4-VO that had
been treated with GN or SV or with vehicles (Me2SO or 0.9% NaCl,
respectively). A: p-STAT3 was
analyzed by IB with anti-p-STAT3 antibody. B: EMSA was carried out to analyze DNA
binding activity. C: Bands corresponding to p-STAT3 and STAT3
binding activity were scanned and the intensities were represented as folds vs.
sham control. Data are expressed as mean±SD from three independent animals
(n=3). aP<0.05 vs sham control,
bP<0.05 vs I 30 min +
Me2SO. cP<0.05 vs I 30 min+NaCl.
GN, genistein (30 mg/kg); SV,
sodium orthovanadate (15 mg/kg) was administered to the rats 20 min before 30
min of ischemia.
3 DISCUSSION
The results of our studies
demonstrate that a rapid activation of STAT3 took place in rat hippocampus during cerebral ischemia induced by 4-VO. In
cytoplasm, STAT3 was rapidly activated, starting from 5 min after occlusion, peaking at 10 min of
ischemia, and then decreasing
again. But the p-STAT3 in nucleus extracts increased gradually and reached its
peak at 30 min of ischemia. The presence of activated STAT3 in the nucleus was
confirmed by EMSA. All these indicated that rapid tyrosine phosphorylation of
STAT3 occured shortly after
ischemia, resulting in an increase in p-STAT3 and DNA binding activity in the
nucleus. These results suggest that ischemic cerebral injury induces a rapid
activation of STAT3 protein in the ischemic area and that p-STAT3 may be
involved in the cytokine regulation of immediate early gene[17,18]. Our study
also reveals that nucleus DNA binding activity of STAT3 is rapidly induced as
early as 5 min of ischemia when compared with the sham control rats. The
possible explanation for more significant
increase in DNA binding activity than tyrosine phosphorylation of STAT3
in nucleus extracts is that serine phosphorylation of STAT3 (Ser 727) induced
by mitogen-activated protein kinase (MAPK) increases the transcription activity
of STAT3[19,20]. The precise mechanism will be studied in our later work.
Rapid activation of different JAKs or
STATs in response to H2O2 has been reported and is inhibited by
antioxidants[5,10]. But the direct effect of antioxidants on activation of
STAT3 induced by cerebral ischemia is still unclear. To delineate the relationship
between STAT3 activation and ROS-induced neuronal injury in vivo, we choose
antioxidant NAC to determine whether it would modify the activation of STAT3 in
response to cerebral ischemia insults. Our studies indicated that the tyrosine
phosphorylation and DNA binding activity of STAT3 induced by ischemia were
significantly attenuat-ed in the presence of NAC. These data indirectly support
the view that ROS induced after ischemia would activate STAT3 and that
antioxidant could eliminate these effects. But the exact mechanism of STAT3
activation by ROS is still unclear. Dose
it act indirectly through JAKs, Src or directly on STAT3, or both? All
these questions are worthwhile studying further. The findings suggest that ROS
may be a second messenger for the activation of STAT3 and this pathway may play
a key role in the adaptive response to oxidative stress.
When rapid changes in protein activity in
response to stimuli are required, phosphorylation and dephosphorylation is an
important fashion of the regulation for the signalling transduction. Although
several studies have demonstrated the effects of genistein and sodium
orthovanadate on the activation of STAT3 in some models[4,5,21], the precise
regulation process in ischemic cerebral injury remains to be further
elucidated. In this report, we showed that sodium orthovanadate dramati-cally
promoted the tyrosine phosphorylation and DNA binding activity of STAT3 in
hippocampus induced by ischemia, whereas genistein obviously counteracted these
increases. Previous report has shown that brief periods of cerebral ischemia resulted in an
increase in total PTK activity in hippocampus, but PTP activity did not change
in the same fashion[22]. Our results could be interpreted from two aspects.
First, the reduction of STAT3 activation might be induced by genistein through
inhibition of the upstream tyrosine kinases of STAT3, such as JAKs and Src, or through direct inhibition of the
tyrosine phosphorylation of STAT3[5,6]. The Second possibility is that the
effect of sodium orthovanadate on the tyrosine phosphorylation and DNA binding
activity of STAT3 could be ascribed to the inhibition of basal protein tyrosine
phosphatases or in coordination with ROS, leading to STAT3 activation[8]. Taken
together, the results suggest that the activation of STAT3 induced by cerebral
ischemia is controlled by PTK and PTP.
In conclusions, the present studies
identify that the activation of STAT3 is induced rapidly by cerebral ischemia in hippocampus and
eliminated in the presence of antioxidant and modulated by PTK and PTP. These
observations impel us to further investigate the precise role and mechanism of
variation of STAT3 in ischemic brain injury.
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