From the Laboratory of Biochemistry, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892-8012 and the
¶ Laboratory of Clinical Science, NIMH, National Institutes of
Health, Bethesda, Maryland 29892-1264
Received for publication, September 26, 2002
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ABSTRACT |
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Human neuroblastoma cells, SH-SY5Y, contain
relatively low levels of thioredoxin (Trx); thus, they serve favorably
as a model for studying oxidative stress-induced apoptosis (Andoh, T.,
Chock, P. B., and Chiueh, C. C. (2001) J. Biol.
Chem. 277, 9655-9660). When these neurotrophic cells were
subjected to nonlethal 2-h serum deprivation, their neuronal
nitric oxide synthase and Trx were up-regulated, and the cells became
more tolerant of oxidative stress, indicating that NO may protect cells
from serum deprivation-induced apoptosis. Here, the mechanism by which
NO exerts its protective effects was investigated. Our results reveal
that in SH-SY5Y cells, NO inhibits apoptosis through its ability to
activate guanylate cyclase, which in turn activates the
cGMP-dependent protein kinase (PKG). The activated PKG is
required to protect cells from lipid peroxidation and apoptosis, to
inhibit caspase-9 and caspase-3 activation, and to elevate the levels
of Trx peroxidase-1 and Trx, which subsequently induces the expression
of Bcl-2. Furthermore, active PKG promotes the elevation of c-Jun,
phosphorylated MAPK/ERK1/2, and c-Myc, consistent with the notion that
PKG enhances the expression of Trx through its c-Myc-, AP-1-, and
PEA3-binding motifs. Elevation of Trx and Trx peroxidase-1 and
Mn(II)-superoxide dismutase would reduce H2O2
and O Reactive oxygen species have been implicated in the etiology
and/or progression of aging and disease (for review, see Refs. 1 and
2), particularly with respect to age-dependent neurological diseases (3-5). Serum deprivation has been shown to induce free radical generation and apoptotic cell death, particularly with human
SH-SY5Y neuroblastoma cells, which exhibit high sensitivity to
oxidative stress due to their low content of
Trx1 (6-8). Using this cell
line, we showed (6, 7) that both serum deprivation and
1-methyl-4-phenylpyridinium, a toxic metabolite of the
Parkinsonian-producing neurotoxin,
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, induces an elevation of
·OH, malondialdehyde, and 4-hydroxy-2-nonenal and causes the
cells to undergo mitochondria-mediated apoptosis. Furthermore, when the
cells were subjected to 2 h of nonlethal stress in serum-free medium 12 h prior to the 24-h lethal serum deprivation, these cells exhibited hormesis, a protective effect induced by the
preconditioning stress. This observed hormesis is associated with the
elevation of Trx, NO, and cGMP, and the biosynthesis of the neuronal
nitric oxide synthase. Furthermore, externally added Trx induced the synthesis of Bcl-2 and Mn(II)-superoxide dismutase and suppressed serum-free induced ·OH, lipid peroxidation, and apoptosis.
Nitric oxide is known to promote and protect cells from apoptosis
depending on conditions and cell types studied. At relatively high
concentrations, NO causes apoptotic cell death in macrophages (9),
whereas tumor necrosis factor- In this study, SH-SY5Y cells were used to investigate the mechanism by
which NO exerts its antiapoptotic protection during hormesis. Our
results indicate that NO, elevated by the preconditioning stress-induced synthesis of neuronal NOS, causes the elevation of cGMP,
which activates the PKG. The active PKG induces the synthesis of Bcl-2,
TPx-1, Trx, c-Jun, and Mn(II)-superoxide dismutase and the
phosphorylation of MAPK/Erk1/2 and c-Myc. Thus, with our system, the
antiapoptotic effect of NO is mediated via a
cGMP-dependent mechanism, whereas direct
S-nitrosylation of caspases appears not to be involved.
Materials--
Human neuroblastoma SH-SY5Y cells were kindly
provided by Dr. Carol Thiele (NCI, National Institutes of Health).
Dulbecco's modified Eagle's medium and fetal bovine serum were
purchased from Invitrogen. Hoechst 33258 (bisbenzimide),
7-nitroindazole, LY-83,583, and DNCB were purchased from Sigma.
8-Br-cGMP, 8pCPT-cGMPS, and Rp-8-pCTP-cGMPS were obtained from
Calbiochem. The oligonucleotides of sense, antisense, and antisense
mutant for Trx mRNA were synthesized by Invitrogen. Antibodies
against TPx-1, TPx-II, TPx-III, and TPx-V were kindly provided by Dr.
S. G. Rhee (National Institutes of Health). Human Trx
antibody was obtained from MBL International (Watertown, MA).
Antibodies against Bcl-2, NF- Cell Cultures--
The SH-SY5Y cells were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum. These cells were incubated (37 °, 5% CO2) for
2-3 days before use.
Lipid Peroxidation Assay--
Cells were collected 24 h
after serum deprivation with or without treatment with either 8-Br-cGMP
or 8-pCPT-cGMPS. The extent of lipid peroxidation was quantitated by
monitoring the fluorescent adducts of malondialdehyde as described
previously (7). Results were presented as relative fluorescence
intensity per mg of protein.
Antisense and Sense Oligonucleotides for Trx mRNA--
The
nucleotide sequence for the antisense Trx mRNA is
5'-TCTGCTTCACCATCTTGGCTGCT-3', and the mutant antisense is
5'-TCGTTCTCACCATCTTGGTCCGT-3'; sites of mutation
are highlighted with bold letters. The corresponding nucleotide
sequence for the sense is 5'-AGCAGCCAAGATGGTGAAGCAGA-3'. These
phosphorthionate oligonucleotides (S-oligo) were designed to
be hybridized with human Trx mRNA (26). For transfection, 4 µM of each S-oligoprobe was mixed with 2 µl
of Tfx-50 (Promega, Madison, WI) in medium (800 µl) incubated with
cells for 2 days and then switched to serum-free media during the serum
deprivation experiments.
Staining of Nuclear DNA in Apoptotic Cells with Hoechst
33258--
Cells were harvested and fixed with 4% paraformaldehyde in
phosphate-buffered saline at 4 ° for 30 min. After rinsing with saline, nuclear DNA was stained with 1 µM Hoechst 33258 fluorescent dye for 5 min at room temperature (excitation/emission
wavelength: 365/420 nm) and imaged with a fluorescent microscope. The
percentage of apoptotic cells was quantitated using a fluorescent microscope.
Western Blotting--
Cells were homogenized in cell lysis
buffer containing 20 mM Hepes KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The protein concentration was determined using a Bio-Rad protein assay kit
(Bio-Rad). Cell proteins (5 or 20 µg) were separated by
electrophoresis using a 4-20% gradient SDS-polyacrylamide gel and
then transferred to polyvinylidene difluoride membrane (Millipore,
Bedford, MA). After blotting the membrane with a 5% skim milk
solution, it was then incubated with a 1:2000 dilution of antibody to
be tested at 4 ° overnight. It was subsequently incubated with a
horseradish peroxidase-linked antibody against mouse IgG (1:2000) for
1 h. Membrane-bound horseradish peroxidase-labeled protein bands
were reacted with a chemiluminescence detection solution (Amersham Biosciences). Chemiluminescent signals were detected using x-ray film
and analyzed using the NIH Image program.
Measurement of Caspase Activity--
Cells were homogenized in
cell lysis buffer and centrifuged at 10,000 × g for 5 min. After determining the protein concentration, one-half of the cell
lysates were used for measuring the activity of caspase-9 and -3 using LEHD-pNA and DEVD-pNA as substrate, respectively. The reaction
mixtures were incubated for 2 h at 37 °. Caspase activity was
monitored with the absorbance at 405 nm, reflecting chromophore pNA.
Statistical Analysis--
Data are presented as mean ± S.E. of n = 4-8 independent observations. Results
were analyzed by one-way analysis of variance, and p values
were assigned by using the Newman-Keuls test. Differences among means
were considered statistically significant when the p value
was less than 0.05.
A Coordinated Role for NOS1, Guanylyl Cyclase (GC), and PKG in the
Hormesis Effect--
We showed (7) that human neuroblastoma SH-SY5Y
cells, when subjected to a 24-h serum deprivation, underwent
apoptosis via an oxidative stress-induced, mitochondria-mediated
mechanism. However, if cells were preconditioned with a nonlethal 2-h
serum-free stress, a hormesis effect was observed. This protective
effect required the synthesis of both Trx and the active Trx reductase. In addition, Andoh et al. (6) showed that, during the 2-h
nonlethal serum deprivation, the mRNA for NOS1, NO, and cGMP were
elevated. Both cGMP-dependent (19-23) and -independent
(14-17, 25) pathways have been reported for the antiapoptotic effect
of NO. In this study, we attempted to elucidate the mechanism by which
hormesis protects SH-SY5Y cells from serum deprivation-induced
apoptosis. Using enzyme-specific inhibitors, we investigated the roles
of NOS1, GC, and PKG in the hormesis induced by nonlethal stress. When
SH-SY5Y cells were subjected to 2 h of serum
deprivation-preconditioning stress followed by a 10-h incubation in the
presence of serum and then subjected to 24 h of serum-free stress,
the amount of apoptotic cells was reduced from about 60 to 15% (Fig.
1). As shown in Fig. 1, this
pronounced hormesis effect was almost totally neutralized when the
activity of the NOS1, GC, or PKG was inhibited during the lethal 24-h
serum-free stress by 7-nitroindazole (30 µM), LY-83,583
(10 µM), or Rp-8-pCTP-cGMPS (10 µM),
respectively. This suggests that the protective effect is derived from
a sequential process in which NO is first elevated followed by the
increase of cGMP and PKG activation, required for downstream
cytoprotective processes. The fact that either the GC or PKG inhibitor
alone can eliminate the hormesis effect almost totally indicates that cytoprotection by S-nitrosylation of caspases can only play
a minor role in our system.
Cyclic GMP Prevents Serum Deprivation-induced Apoptotic Cell
Death--
Since NO is known to activate the activity of guanylyl
cyclase, the role of cGMP was investigated. Fig.
2A shows that using two
membrane-permeable cGMP analogs, 8-Br-cGMP (EC50, 300 µM) and 8-pCPT-cGMP (EC50, 5 µM), we observed that each of these cGMP analogs
protected SH-SY5Y cells from apoptosis induced by serum deprivation in
a concentration-dependent manner. Furthermore, their protective
efficiencies are correlated to their known EC50 values.
However, when cells were pretreated with the membrane-permeable PKG
inhibitor, Rp-8-pCTP-cGMPS, the cytoprotective effect of 8-Br-cGMP (1 mM) was diminished as a function of increasing
concentrations of the inhibitors (Fig. 2B). These results
and those shown in Fig. 1 indicate that PKG is expressed in SH-SY5Y
cells and that NO and cGMP exert their protective effects through a
PKG-mediated pathway. Fig. 3 shows that
both PKGI Reduced Trx Is Required for the cGMP- and Trx-mediated
Antiapoptotic and Antioxidative Effects--
Previously, we revealed
that, in addition to apoptosis, serum deprivation causes an elevation
of lipid peroxidation in SH-SY5Y cells and the hormesis involved the
elevation of the reduced Trx (7). Thus, the protective effect of cGMP
may be mediated via the Trx redox cycle. This point is illustrated
using the Trx reductase inhibitor, DNCB (28), in a concentration range
of 5-50 µM. It should be pointed out that 5-10
µM DNCB did not induce apoptosis. However, at 50 µM, it induced about 23% apoptosis in the
absence of serum (7, 29). Fig.
4A depicts that pretreatment
with DNCB (IC50, 10 µM) prevented the cGMP
analog (1 mM)-induced protective effect against serum
deprivation-mediated apoptosis in a concentration-dependent manner. A protective effect was also observed when 1 µM
of membrane-permeable oxidized Trx was added extracellularly. The
oxidized Trx must be reduced intracellularly since addition of 50 µM DNCB completely eliminated the Trx protection
(Fig. 4B). Consistent with the notion that the antiapoptotic
effect of cGMP requires both PKG and reduced Trx, we observed that the
elevated activities of caspase-9 and caspase-3 caused by serum
deprivation was drastically reduced in cells pretreated with 8-Br-cGMP.
This cGMP analog-mediated effect was reversed, almost to its original
level, by the pretreatment with Rp-8-pCPT-cGMPS (10 µM)
or 50 µM of DNCB (data not shown).
Serum deprivation leads to ROS generation, including ·OH (7,
30). The ·OH can react with lipids to generate peroxy lipid
radicals that react with polyunsaturated fatty acids to form
malondialdehyde and 4-hydroxy-2-nonenal (31). Fig. 4C shows
that serum deprivation led to elevation of malondialdehyde, a marker of
lipid peroxidation, and this effect was drastically reduced by the
presence of 1 mM 8-Br-cGMP. The antioxidant effect of the
cGMP analog was blocked by pretreatment with either Rp-8-pCTP-cGMPS (10 µM) or DNCB (50 µM). This indicates that
the antioxidant activity exhibited by cGMP may be mediated by the redox
cycle of Trx. The reduced Trx can remove H2O2
when coupled with either the methionine and methionine sulfoxide
reductase (32) or Trx peroxidase systems (33). The latter is also
capable of removing organic peroxides (33, 34). Thus, elevation of Trx
would lead to reduction of ·OH and lipid peroxidation (7).
Furthermore, Trx can bind to the apoptosis signal-regulating kinase 1 and directly inhibit apoptosis (26). It is interesting to note from
Fig. 4A that the 8-Br-cGMP appears to be more effective in
protecting cells from apoptosis than the reduced Trx, particularly in
the presence of 50 µM DNCB. This suggests that the cGMP
analog may induce more pathways, relative to those due to Trx alone, in
its antiapoptotic mechanism.
cGMP-induced Expression of Thioredoxin Peroxidase
(TPx)--
To overcome the elevation of ROS induced by serum
deprivation, cGMP may regulate the synthesis of TPx (also known as
peroxiredoxin), which catalyzes the removal of
H2O2 and organic peroxide (33-35). This
hypothesis was verified by the results shown in Fig.
5A. Among the TPx isozymes
tested, only TPx-1 was elevated by about 2-fold due to 8-Br-cGMP (1 mM) treatment. The induction reached its maximal level
within 2 h and persisted for at least 24 h. Fig.
5B shows TPx-1 induction required active PKG since the
process was inhibited by Rp-8-pCPT-cGMPS. TPx-1 is the most abundant
cytosolic isozyme that can be regulated by phosphorylation catalyzed by cyclin-dependent kinase (35). Therefore, elevation of the
TPx-1 level makes sense since its activity, when necessary, can be
dampened by phosphorylation.
cGMP-induced Expression of Trx and Bcl-2--
SH-SY5Y cells are
highly sensitive to oxidative stress, which is, in part, due to
their low basal level of Trx. They contain about 1/5 to
1/7 of that detected in U-87MG, A549, and HeLa cells (7). We
showed that hormesis induced by 2 h of nonlethal preconditioning stress required the biosynthesis of Trx and that extracellularly added
Trx could cause elevation of Bcl-2 levels (7). Fig. 5C shows
that application of 1 mM 8-Br-cGMP induced a
time-dependent elevation of Trx that peaked at about 4 h. In addition, with a lag phase, the level of Bcl-2 also doubled and
reached its maximum at about 8 h. These data suggest that the
expression of Bcl-2 required Trx. The effect of 8-Br-cGMP on the
expression of Bad was also investigated, and no changes were found
(data not shown). However, if 8-Br-cGMP was added in the presence of 10 µM of the PKG inhibitor, no elevation of either Trx or
Bcl-2 was observed 4 h after the addition of cGMP analog (Fig.
5B). Thus, the enhanced synthesis of both Trx and Bcl-2 in
response to preconditioning stress is regulated by a PKG-mediated
pathway. It should be pointed out that NO (20) and cGMP (6) have been
shown to elevate Bcl-2 and prevent apoptosis of splenic B lymphocytes
and SH-SY5Y cells, respectively, by an unknown mechanism.
To verify that the elevated expression of Bcl-2 induced by the cGMP
analog required the synthesis of Trx, we investigated the effects of
inhibiting Trx synthesis by transfecting the cells with the Trx
antisense mRNA. Fig. 5D reveals that 8-Br-cGMP induced the expression of both Trx (determined after 4 h incubation) and Bcl-2 (measured after 24 h of stress). However, when the synthesis of Trx was inhibited by antisense S-oligo transfection, the
level of Bcl-2 was also reduced drastically, implying that Trx is
needed for Bcl-2 expression. Note that the 8-Br-cGMP-induced Bcl-2
level was not suppressed when the cells were treated with sense and antisense mutants. In addition, controlled experiments showed that the
antisense S-oligo probe for Trx mRNA did not bind to the
Bcl-2 mRNA (data not shown). Furthermore, the antiapoptotic effect
of 8-Br-cGMP was generally compromised only when cells were transfected
with the antisense S-oligo probe for Trx mRNA but not
those with the sense or antisense mutant (Fig. 5E).
cGMP Induces c-Jun Expression, MAPK-Erk1/2, and c-Myc
Phosphorylation--
To elucidate how PKG enhances Trx expression, we
investigated the effect of PKG on transcription factors known to
regulate Trx expression. The promoter region of the human Trx-encoding gene was shown (36) to contain many possible regulatory elements that
are compatible with both a basal constitutive expression and an
inducible transcriptional regulator. The binding sites of the inducible
transcriptional factors include AP-1, AP-2, NF- Proposed Reaction Scheme for Serum Free-induced Apoptosis and
NO-mediated Hormesis in SH-SY5Y Cells--
Together, the
current results and those reported earlier (6, 7) implicate
the mechanistic scheme shown in Fig. 7
for the serum deprivation-induced apoptosis and hormesis. Prolonged, e.g. 24-h, serum-free treatment causes an elevation of
·OH, malondialdehyde, and 4-hydroxy-2-nonenal, most likely due to increased accumulation of H2O2 and organic
peroxides, and leads to mitochondria-mediated apoptosis. The hormesis
resulting from 2 h of nonlethal serum-free treatment leads to the
increased expression of NOS1 mRNA and enzyme, which leads to
elevation of NO and cGMP (6). In addition, the protective effect
required the biosynthesis of Trx. The elevated level of Trx leads to
increased biosynthesis of Bcl-2 and Mn(II)-superoxide dismutase, a
reduction in cytochrome c released from mitochondria, and
the inhibition of procaspase-9 and procaspase-3 activation (7). Our
current data reveal that elevation of NO leads to activation of PKG,
whose activity is required for the enhanced biosynthesis of TPx-1 and
c-Jun and the elevation of c-Myc and MAPK/Erk1/2 phosphorylation. The
elevated TPx-1 and Trx enhances the elimination of
H2O2 and a number of organic peroxides (35).
The elevated level of Trx also induces the biosynthesis of Bcl-2 (Fig.
7) and Mn(II)-superoxide dismutase (7) known to inhibit
mitochondria-mediated apoptosis (42) and to protect cells from
peroxynitrite-mediated cellular toxicity (43-45), respectively.
Furthermore, the fact that inhibition of any one of the enzymes in the
sequence, NOS1, GC, and PKG, is capable of eliminating the hormesis
effect (Fig. 1) indicates that the neuroprotective effect of NO in
SH-SY5Y cells proceeds via the PKG-mediated mechanism and that its
capacity to inhibit caspases via S-nitrosylation plays only
a minimal role, if any at all.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and lipopolysaccharide induce NO
synthesis in rat PC12 cells and apoptosis (10). However, at
physiologically relevant concentrations, NO protects cells from
apoptosis (6, 11-13). This protective mechanism has been shown to
proceed via either a cGMP-dependent or -independent
pathway. NO can also directly S-nitrosylate the active
site cysteine of caspases and inhibit apoptosis (14-17).
S-Nitrosylation of the active site cysteine of the
caspase-activating enzyme has also been reported (18). In addition,
caspase-3 zymogens were found to be S-nitrosylated at their
active site cysteine, and Fas induced its denitrosylation (16). NO
donors have also been shown to induce S-nitrosylation of
overexpressed caspase-3 in vivo (17). With respect to the
cGMP-dependent signaling pathway, NO is known to activate
soluble guanylyl cyclase. The cGMP-dependent protective pathway is normally augmented by membrane-permeable cGMP analogs and
inhibited by guanylyl cyclase and PKG inhibitors (18-23). In addition,
NO could protect cells by up-regulating antiapoptotic proteins, such as
heme oxygenase-1 (24), ferritin (12), and Bcl-2 (25). The elevation of
Bcl-2 was accomplished by the inhibition of Bcl-2 degradation catalyzed
by caspase-3 (25).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B, Oct-1, PEA-3, c-Myc, phosphorylated
c-Myc, PKGI
, PKGI
, and PKGII were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Antibodies phosphorylated and
nonphosphorylated c-Jun, MAPK/Erk1/2, MEK1/2, I
B, and SAPK/JNK were
obtained from Cell Signaling Technology, Inc. (Beverly, MA). A
horseradish peroxidase-linked antibody against IgG was obtained from
Amersham Biosciences.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Effects of NOS1, GC, and
PKG on hormesis against apoptosis induced by serum deprivation.
SH-SY5Y cells were treated with or without ( ) a nonlethal 2-h
prestress at 12 h prior to lethal 24-h serum deprivation stress.
The NOS1 inhibitor (NOS1i; 7 nitroindazole, 30 µM), GC inhibitor (GCi; LY-83,583, 10 µM), and PKG inhibitor (PKGi; Rp-8-pCPT-cGMPS,
10 µM) was presented, as indicated, during the 24-h
stress. The dashed line shows the data obtained in
serum-containing medium without any other treatment. n = 6.
and PKGII (27) are present in SH-SY5Y cells, based on the
Western blotting analysis.
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Fig. 2.
Effects of cGMP analogs and PKG inhibitor on
serum deprivation-induced apoptosis of SH-SY5Y cells. As shown in
A, 8-Br-cGMP and 8-pCPT-cGMP protect SH-SY5Y cells from
serum deprivation-induced apoptosis. Cells were subjected to 24-h
serum-free stress in the presence of the indicated concentration of
8-Br-cGMP ( ) or 8-pCPT-cGMP (
). The apoptotic cell nuclei were
stained with fluorescent DNA dye (Hoechst 33258) and quantitated as
described under "Experimental Procedures." In the absence of the
cGMP analog, 67 ± 5% apoptotic cells were observed.
B, effects of the PKG inhibitor on the antiapoptotic effect
of 8-Br-cGMP. Cells were pretreated with the indicated concentration of
the PKG inhibitor, Rp-8-pCTP-cGMPS, for 10 min before adding 1 mM of 8-Br-cGMP and then subjected to serum-free stress for
24 h. The apoptotic cells were quantitated as described above. The
open column shows the data from the serum deprivation group
in the absence of 8-Br-cGMP and Rp-8-pCPT-cGMPS. The dashed
line represents the level of apoptotic cells in the non-treated
serum control group. n = 6.
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Fig. 3.
Expression of cGMP-dependent
protein kinase subtypes in SH-SY5Y cells. The levels of
cGMP-dependent protein kinase type I
(PKGI
), I
(PKGI
), and II
(PKGII) in 20 and 10 µg of protein from SH-SY5Y cells were
determined by Western blotting method using anti-PKGI
, -PKGI
, and
-PKGII antibodies.
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Fig. 4.
Role of Trx cycling in cytoprotection
mediated by 8-bromo-cGMP. A, effects of DNCB on
cytoprotection mediated by 8-Br-cGMP. Cells were pretreated with the
Trx reductase inhibitor, DNCB, 10 min prior to the extracellular
administration of 8-Br-cGMP (1 mM). They were then
subjected to serum deprivation for 24 h. The apoptotic cells were
quantitated as described in the legend for Fig. 2. n = 6. B, effects of DNCB on the antiapoptotic action of Trx.
Oxidized Trx (1 µM) was applied to cells at the beginning
of the 24-h serum deprivation stress. In one group, cells were
pretreated with DNCB (50 µM) for 10 min prior to the
administration of oxidized Trx. After being subjected to oxidative
stress under serum-free conditions for 24 h, the apoptotic cells
were quantitated. n = 6. C, effects of
8-Br-cGMP on lipid peroxidation induced by serum deprivation. Lipid
peroxidation was measured by detecting the formation of fluorescent
adducts of malondialdehyde as described under "Experimental
Procedures." The relative fluorescent units are depicted as
relative fluorescence units/mg of protein. The open column
shows the control data from the serum-deprived group without the
treatments. The PKG inhibitor (PKGi) group was pretreated
with the PKG inhibitor, Rp-8-pCPT-cGMPS (10 µM), for 10 min before adding 8-Br-cGMP. The DNCB group was pretreated with DNCB
(50 µM). n = 6. The dashed
line shows the results of the non-treated serum control group.
RFU, relative fluorescence unit.
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Fig. 5.
Cyclic GMP-induced TPx, Trx, and Bcl-2
expression in SH-SY5Y cells. A, time course of the
expression of TPx subtypes induced by 8-Br-cGMP. 8-Br-cGMP (1 mM) was applied to SH-SY5Y cells. The expression of TPx was
detected by Western blotting with anti-TPx antibodies (TPx-I ( ), II
(
), III (
), and V (
)). The data were quantitated using
the NIH Image program and presented as % of the control level.
n = 3. B, effect of the PKG inhibitor on
cGMP-induced Tpx-I, Trx, and Bcl-2 expression. The PKG inhibitor
(PKGi, 10 µM) was applied 10 min prior to the
application of 8-Br-cGMP (1 mM). Cellular protein (20 µg)
was collected after 2 h of incubation for TPx-1, 4 h of
incubation for Trx, and 24 h of incubation for Bcl-2 measurements
using Western blot analysis. n = 3. CTL
(control) shows the results of the non-treated group. C,
time course of the expression of Trx and Bcl-2 induced by
8-Br-cGMP. 8-Br-cGMP (1 mM)-treated cells were collected at
indicated time points. The protein levels, expressed as % of control,
of Trx (
) and Bcl-2 (
) in 20 µg of protein extracts were
detected by Western blotting. As shown in D, cyclic
GMP-induced expression of Bcl-2 required the biosynthesis of Trx. Cells
were transfected with sense (S), antisense
(Anti-S), or antisense mutant (Anti-S mutant)
S-oligo probes for Trx mRNA as described under
"Experimental Procedures" and subjected to 24 h in serum-free
media with or without 8-Br-cGMP treatment. These stressed cells were
harvested at 4 and 24 h after 8-Br-cGMP treatment for detecting
the expression of Trx and Bcl-2 proteins, respectively. Human Trx and
Bcl-2 antibodies were used in Western blot analysis.
n = 6. E, effects of the antisense
oligonucleotide probe on the antiapoptotic effects induced by
8-Br-cGMP. S-Oligo probes for Trx mRNA were applied as described in
above. After transfection and 24 h of serum deprivation stress,
the apoptotic nuclei were quantitated. The open column shows
the data from the serum-deprived group without the pretreatment with
S-oligo probes. n = 6. The dashed
line represents the results of the non-treated serum control
group.
B, Oct-1, PEA3, and
c-Myc. A series of transcriptional factors was monitored for their
responses due to PKG activation. Fig. 6
shows that 8-Br-cGMP induced an elevation of c-Jun but not of c-Jun phosphorylation and elevations of the phosphorylated MAPK/Erk1/2 and
c-Myc but not of their protein levels. The kinetic study revealed that
phosphorylation of MAPK/Erk1/2 preceded that of c-Myc (data not shown).
Furthermore, the PKG inhibitor, Rp-8-pCPT-cGMPS (10 µM),
eliminated the elevation of c-Jun and the phosphorylation of
MAPK/Erk1/2 and c-Myc induced by the cGMP analog. The inhibitor of PKG
exerted no effect on the levels of phosphorylated c-Jun and the
protein levels of MAPK/Erk1/2 and c-Myc. These results suggest that
cGMP is capable of inducing Trx transcription via its c-Myc-, AP-1-,
and/or PEA3-binding sites since the cGMP analog elevates the levels of
P-c-Myc, c-Jun, and P-MAPK/Erk1/2, respectively. The
phosphorylation-activated MAPK/Erk1/2 has been shown to regulate the
transcriptional factor PEA3 (37) and to phosphorylate c-Myc at
T-58/S-62 (38) for its transcriptional activation (39). It is
interesting to note that PEA3 and AP-1 often exhibit strong synergistic
effects (40, 41).
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Fig. 6.
PKG induces c-Jun expression and
phosphorylation of MAPK/Erk1/2 and c-Myc in SH-SY5Y cells. The PKG
inhibitor (PKGi; Rp-8-pCPT-cGMPS, 10 µM) was
applied 10 min before the application of 8-Br-cGMP (1 mM).
Cells were collected, and the protein was extracted 30, 60, and 120 min
after 8-Br-cGMP treatment for Western blot analysis of c-Jun and
P-c-Jun, MAPK/Erk1/2, and phosphorylated MAPK/ERK1/2
(P-MAPK/Erk1/2), and c-Myc and phospho-c-Myc
(P-c-Myc), respectively. n = 3. The relative
quantitative data obtained from the three experiments were presented on
the right side. CTL, control.
View larger version (21K):
[in a new window]
Fig. 7.
Schematic representation of a proposed serum
deprivation-induced apoptosis and NO-mediated hormesis in SH-SY5Y
cells. indicates increase, and LOOH represents
organic peroxides.
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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.
§ Present address: Dept. of Applied Pharmacology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyoam 930-0194, Japan.
To whom correspondence should be addressed: LB/NHLBI/National
Institutes of Health, Bldg. 50, Rm. 2134, 50 South Dr., MSC-8012, Bethesda, MD 20892-8012. Tel.: 301-496-2073; Fax: 301-496-0599; E-mail:
pbc@helix.nih.gov.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M209914200
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ABBREVIATIONS |
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The abbreviations used are: Trx, thioredoxin; PKG, cGMP-dependent protein kinase; NOS, nitric oxide synthase; NOS1, neuronal NOS; DNCB, 1-choloro-2-dinitrobenzene; TPx-1, thioredoxin peroxidase-1; S-oligo, phosphorthionate oligonucleotides; 8-pCPT-cGMP, 8-(4-chlorophenylthio)-cGMP; Rp-8-pCPT-cGMPS (PKGi), 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, Rp-isomer; GC, guanylyl cyclase; ROS, reactive oxygen species; SAPK, stress-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PEA3, polyomavirus enhancer A-binding protein-3.
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