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INTRODUCTION |
It is generally thought that oligosaccharides attached to
glycoproteins play crucial roles in the folding, stability, and sorting
of the protein (1). To clarify the function of each oligosaccharide
structure, many glycosyltransferases that catalyze the formation of
sugar chains have been identified, cloned, and characterized. However,
the precise physiological functions of each oligosaccharide structure
continue to remain elusive.
N-acetylglucosaminyltransferase
(GnT-III)1 is one such
glycosyltransferase. It catalyzes the attachment of a GlcNAc residue in
a
1, 4-linkage to the
1, 4-mannose residue in the core region of
N-glycans, producing a bisecting GlcNAc (2, 3). GnT-III suppresses the further processing and elongation of
N-glycans because the bisecting GlcNAc structure affects the
conformation of the sugar chains; as a result, other
glycosyltransferases such as GnT-II, GnT-IV, and GnT-V are no longer
able to act on the oligosaccharides containing the bisecting GlcNAc (4,
5). Many aspects of the biological significance of GnT-III have been examined. The metastatic potential of B16 mouse melanoma cells is
decreased by the introduction of the GnT-III gene
(6). This anti-metastatic effect of GnT-III is caused by an
increase in E-cadherin-mediated homotypic adhesion (7). The
overexpression of the GnT-III gene also affects nerve growth factor
signaling by suppressing the dimerization of TrkA in PC12 cells (8). Very recently, we reported that GnT-III gene induction resulted in
changes in N-glycan structures and enhanced epidermal growth factor (EGF)-induced Erk phosphorylation in HeLaS3 cells, and we
found that this up-regulation of Erk phosphorylation is due to the
enhancement of receptor internalization (9).
In the present study, we report on an examination of whether GnT-III
overexpression modulates signaling caused by
H2O2. It is known that
H2O2 is a major component of the reactive
oxygen species (ROS) in cells, which can trigger the activation of
multiple signaling pathways that influence the cytotoxicity. For
example, treatment of HeLa cells with H2O2
resulted in a time- and dose-dependent induction of
apoptosis accompanied by activation of various members of the
mitogen-activated protein kinase (MAPK) superfamily (10, 11). We
demonstrate that GnT-III overexpression in HeLaS3 cells suppresses
H2O2-induced apoptosis and the activity of two
stress-regulated MAPKs, JNK and p38, but not Erk 1/2. The results
indicate that the suppression of PKC
activity had an inhibitory
effect on JNK activity and apoptosis in GnT-III transfectants.
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EXPERIMENTAL PROCEDURES |
Cell Culture, Transfection, and Luciferase Assay--
A HeLaS3
cell line that stably expresses human GnT-III has been established
previously (9) and was maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum. To detect the pathway
activated by the overexpression of GnT-III, we utilized the Mercury
Pathway Profiling Systems (Clontech). Briefly,
HeLaS3 cells were cultured in 6-well plates, transiently transfected
with promoter-reporter plasmids using LipofectAMINE 2000 (Invitrogen)
according to the manufacturer's protocol. These plasmids contained a
luciferase gene downstream of several copies of the transcription
factor-binding sequences. Transfected cells were pre-cultured for
24 h before H2O2 (Nacalai Tesque,
Inc., Kyoto, Japan) was added to the media. After a 3-h
treatment with H2O2, the cells were harvested
and luciferase activities were measured using a luciferase reporter
gene assay kit (Roche Molecular Biochemicals) according to the
manufacturer's protocol.
Antibodies--
Anti-active MAPK and anti-active JNK antibodies
were obtained from Promega (Madison, WI). Anti-phosphorylated
Akt, p38-mapk, MKK4/SEK1, MKK3/MKK6, c-Jun, ATF-2, PKC
/
II
(Thr-638/641), PKC
(Thr-505), PKCµ (Ser-744/748), PKC
/
(Thr-410/413) antibodies and anti-p38-mapk, Akt, PKC
antibodies were
obtained from New England BioLabs. Anti-MAPK and anti-phosphotyrosine
(4G10) antibodies were obtained from Upstate Biotechnology (Lake
Placid, NY), and anti-JNK antibody was obtained from Santa Cruz (Santa
Cruz, CA).
Cell Viability Assay--
Approximately 4 × 104 cells were plated onto a 96-well plate and allowed to
grow for 24 h, after which they were treated with various
concentrations of H2O2 in triplicate for
18 h. Ten µl of
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
reagent (Wako, Tokyo, Japan) was then added, and the mixture was
incubated for an additional 6 h. The resulting dark crystals were
dissolved in 50% isopropanol and 0.02 N HCl, and the absorbance at 550 nm was measured.
DNA Analysis and Detection of Apoptotic Cells--
For DNA
analysis of apoptotic cells, cells were treated with or without
H2O2 for 18 h and incubated for 10 min in
500 µl of buffer containing 20 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.2% Triton X-100 on ice, and then centrifuged at
10,000 × g for 10 min. The supernatant was incubated
overnight at 50 °C with 100 µg/ml Proteinase K. After
phenol/chloroform extraction, DNA was precipitated with ethanol and
resolved by 1.5% agarose gel electrophoresis (12). For detection of
apoptotic cells, cells were treated with or without 300 µM H2O2 for 18 h and then
washed once with methanol containing 1 µg/ml
4,6-diamidino-2-phenylindole (DAPI). Cells were then stained with 1 µg/ml DAPI-methanol solution for 15 min. The nuclear morphology of
the cells was examined by fluorescence microscopy.
Electrophoretic Mobility Shift Assay--
Nuclear extracts for
an electrophoretic mobility shift assay were prepared by resuspending
phosphate-buffered saline-washed cell pellets in a high salt buffer
containing Nonidet P-40 (13). After 30 min on ice, debris was removed
by centrifugation for 5 min at 15,000 × g. Equal
amounts of protein were reacted with 32P-end-labeled
double-stranded oligonucleotide. The 32P-labeled
probe was prepared by annealing the oligonucleotide CTAGCAAGGGACTTTCCGCTA with the oligonucleotide
CTAGTAGCGGAAAGTCCCTTG and labeling it with Klenow
fragments and [32P]dCTP (Amersham Biosciences).
DNA binding proteins were resolved by electrophoresis on native 4%
polyacrylamide gels, and the dried gels were visualized by autoradiography.
Western Blot Analysis--
HeLaS3 cells (80% confluent) were
washed with ice-cold phosphate-buffered saline and solubilized in lysis
buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% (w/v) Nonidet P-40, 10% (w/v) glycerol, 5 mM sodium pyrophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 10 mM
-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 5 µg/ml leupeptin, and 1 mM
dithiothreitol and then centrifuged at 15,000 × g for
15 min. The supernatants were collected, and the protein concentrations
were determined using a protein assay CBB kit (Nacalai Tesque). Equal
amounts of proteins were fractionated by 12.5% SDS-PAGE and then
transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel,
Germany). The membrane was incubated with primary and secondary
antibodies for 1 h each, and detection was performed using an ECL
kit (Amersham Biosciences) according to the manufacturer's instructions.
Immunoprecipitation--
Cells were washed with ice-cold
phosphate-buffered saline, lysed in lysis buffer, and centrifuged at
15,000 × g for 15 min. The supernatants were collected
and incubated with 4 µg of anti-phosphotyrosine antibody (4G10) for
1 h and then with 15 µl of Protein G-Sepharose 4 Fast Flow
(Amersham Biosciences) for 1 h at 4 °C. The immune complex was
washed three times with lysis buffer and subjected to Western Blot analysis.
In Vitro Kinase Assay--
For measurement of JNK1 activity
in vitro, HeLaS3 cells were lysed in lysis buffer. The
lysates were clarified by centrifugation, and the JNK1 was
immunoprecipitated with anti-JNK1 antibody C-17 (Santa Cruz). The
immune complex was recovered with Protein A-Sepharose 4 Fast Flow and
washed twice with lysis buffer and twice with reaction buffer (25 mM HEPES, pH 7.4, 20 mM MgCl2, 20 mM
-glycerophosphate, 0.5 mM EGTA, 0.5 mM NaF, 0.5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride). The reaction was
initiated by adding 20 µl of kinase reaction mixture (reaction buffer
plus 5 µCi of [
-32P]ATP (Amersham Biosciences), 20 µM unlabeled ATP, 1 mM dithiothreitol, and 1 µg of c-Jun (Santa Cruz) as a substrate). After 20 min of incubation
at 37 °C, the reactions were terminated by the addition of 5×
sample buffer and boiled for 5 min. Samples were subjected to SDS-PAGE
and analyzed by phosphorimaging (BAS-2000, Fuji Film).
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RESULTS |
GnT-III Overexpression Inhibits
H2O2-induced Apoptosis in HeLaS3 Cells--
To
investigate the effect of GnT-III transfection on cell survival, we
examined H2O2-induced cell death in GnT-III
transfectants. GnT-III transfectants and control cells were treated
with various concentrations of H2O2 for 18 h, and their viability was analyzed by means of an MTT assay. Fig.
1A shows that
H2O2-induced cell death was suppressed in
GnT-III transfectants; about 60% of control cells died when the cells
were treated with 300 µM H2O2,
whereas less than 30% of GnT-III transfectants were found to be dead
at the same concentration. A similar result on cell viability was obtained from human mammary carcinoma MCF7 cells following
H2O2 exposure (data not shown). Nuclear DNAs
were next examined in the same system, in order to distinguish between
apoptosis and necrosis. DNA fragmentation was observed in control cells
treated with 200-600 µM H2O2 for
18 h but not in the GnT-III transfectants (Fig. 1B).
The degree of DNA fragmentation was apparently reduced at
concentrations higher than 600 µM of
H2O2 in both cells. In this condition, almost
all the cells showed signs of necrotic cell death with focal rupture of
cell membranes or surface blebbing (data not shown), and no difference
in cell viability was seen between control cells and GnT-III
transfectants (Fig. 1A). When cells were treated with 300 µM H2O2 for 18 h, the nuclei
consistently appeared condensed and fragmented by DAPI staining in
control cells (Fig. 1C, panel b), and chromatin
condensation was strongly suppressed in the GnT-III
transfectants (Fig. 1C, panel d). Next, when
cells were pretreated with caspase3-specific inhibitor, DEVD-CHO, to
prevent apoptotic cell death, 300 µM
H2O2-induced cell death in control cells was
markedly reduced to the same level as in GnT-III transfectants
(Fig. 1D). These results suggest that GnT-III overexpression
down-regulates H2O2-induced apoptosis.

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Fig. 1.
GnT-III overexpression inhibits
H2O2-induced apoptosis in HeLaS3 cells.
A, HeLaS3 cells (2 × 104 cells per well of
96-well plates) were incubated with the indicated concentrations of
H2O2 for 18 h, and cell viability was then
assessed by MTT assay. Data from three independent experiments with
triplicate samples, each of which was averaged and expressed as the
mean ± S.D., are shown. B, soluble DNA was extracted
from HeLaS3 cells treated with the indicated concentrations of
H2O2 for 18 h and resolved by 1.5%
agarose gel electrophoresis as described under "Experimental
Procedures." C, control cells (upper panel) or
GnT-III transfectants (lower panel) were treated with 300 µM H2O2 for 18 h
(panel b and d) or left untreated as control
(panels a and c). Apoptotic cells were detected
by DAPI staining. The arrowheads indicate typical apoptotic
nuclei with chromatin condensation. Scale bar, 20 µm.
D, cells were pretreated with or without 10 nM
DEVD-CHO for 30 min before incubation with 300 µM
H2O2 for 18 h; cell viability was then
assessed by MTT assay. Data from three independent experiments with
triplicate samples, each of which was averaged and expressed as the
mean ± S.D., are shown.
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GnT-III Overexpression Does not Affect NF-
B Activation and Akt
Phosphorylation--
The activation of NF-
B in GnT-III
transfectants was examined because it has been demonstrated that
activation of the transcription factor NF-
B has an essential role in
protecting cells from apoptosis, as mediated by a variety of stressful
stimuli, including oxidative stress such as
H2O2 (14-16). Fig.
2A shows that GnT-III
transfection had no effect on NF-
B activation following
H2O2 exposure. A luciferase assay using a
plasmid containing the NF-
B-binding sequence also showed similar
results (Fig. 2B).

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Fig. 2.
GnT-III overexpression does not affect the
H2O2-induced activation of the
NF- B and Akt signaling pathway.
A, HeLaS3 cells were treated with
H2O2 for 3 h or 20 ng/ml TNF , used as a
positive control, for 1 h. After incubation with
H2O2 or TNF , cells were harvested and
subjected to electrophoretic mobility shift assay with
32P-labeled probe as described under "Experimental
Procedures." The experiment was performed three times with similar
results. The protein-DNA complex is indicated by an
arrowhead. B, HeLaS3 cells were transiently
transfected with an NF- B binding site-containing plasmids using
LipofectAMINE 2000 as described under "Experimental Procedures."
Transfected cells were preincubated for 24 h before
H2O2 or TNF was added to the medium for
3 h and harvested for luciferase assays. The experiment was
performed in triplicate three times with similar results. The mean ± S.D. values are shown. C, HeLaS3 cells were treated with
300 µM H2O2 or 50 ng/ml epidermal
growth factor, used as a positive control, for the indicated periods.
Cell lysates were subjected to SDS-PAGE and analyzed by Western
blotting using an anti-phospho Akt antibody (upper panel)
and an anti-Akt antibody (lower panel). D, cells
were pretreated with or without 100 nM wortmannin for 30 min before incubation with 300 µM
H2O2 for 18 h; cell viability was then
assessed by MTT assay. Data from three independent experiments with
triplicate samples, each of which was averaged and expressed as the
mean ± S.D., are shown.
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The possibility that GnT-III suppresses
H2O2-induced apoptosis through PI3K/Akt
up-regulation was also examined because previous reports have suggested
that H2O2 activates the PI3K/Akt signaling pathway that protects cells from ROS-mediated apoptosis (17-19). As
shown in Fig. 2C, H2O2 enhanced Akt
phosphorylation in both control cells and GnT-III transfectants, and
there was no difference between them. There was also no difference in
cell viability in the presence or absence of wortmannin, a specific
inhibitor of PI3K (Fig. 2D). These results indicate that the
inhibition of H2O2-induced apoptosis in GnT-III
transfectants does not occur via NF-
B or PI3K/Akt activation.
GnT-III Overexpression Inhibits
H2O2-induced JNK and p38-mapk Activation, but
not Erk1/2--
To elucidate how GnT-III suppresses
H2O2-induced apoptosis, we examined MAPK
activation in GnT-III transfectants because various members of the MAPK
family have been implicated in apoptosis in response to oxidative
stress (10, 11). MAPKs activation in the GnT-III transfectants and
control cells following H2O2 exposure is shown
in Fig. 3. H2O2
stimulated a striking increase in the level of Erk 1/2 phosphorylation
in both control cells and GnT-III transfectants in a time- and
dose-dependent manner (Fig. 3, A and
B, upper panels), and no difference between
control cells and GnT-III transfectants was observed. On the other
hand, the phosphorylation of JNK 1/2 and p38-mapk was also enhanced by
H2O2 treatment in both a time- and
dose-dependent manner in control cells. However, it was
suppressed in the GnT-III transfectants (Fig. 3, A and
B). To investigate the activation of JNK and p38-mapk, phosphorylation of transcription-factor c-Jun and ATF-2, which are
phosphorylated by JNK and p38-mapk, respectively, was examined. As
expected, H2O2-induced c-Jun and ATF-2
phosphorylation was significantly suppressed in the GnT-III
transfectants (Fig. 3C). Furthermore, phosphorylation of
MAPKKs, MKK4/SEK1, and MKK3/MKK6, which are able to activate JNK and
p38-mapk, was examined. As shown in Fig. 3D,
H2O2-induced MKK4/SEK1 and MKK3/MKK6
phosphorylation was also suppressed in the GnT-III transfectants. These
results indicate that the phosphorylation of JNK and p38-mapk by
H2O2 is markedly inhibited in the GnT-III
transfectants and that the inhibitory effects exist both upstream and
downstream of JNK and p38-mapk.

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Fig. 3.
GnT-III overexpression suppressed
H2O2-induced JNK and p38 activation, but not
Erk1/2 activation. A, HeLaS3 cells were stimulated with
300 µM H2O2 for the indicated
periods. Lysates were prepared and analyzed by Western blotting using
anti-phospho-MAPKs antibodies or anti-MAPKs antibodies. B,
HeLa cells were treated with the indicated concentrations of
H2O2 for 30 min. Western blotting was then
performed using the same antibodies as shown in panel A. C and D, HeLaS3 cells were treated with 300 µM H2O2 for the indicated times.
Cell lysates were analyzed by Western blotting using
anti-phosphorylated c-Jun, ATF2, MKK4/SEK1, and MKK3/MKK6 antibodies as
indicated.
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PKC-JNK1 Signaling Pathway Is Involved in
H2O2-induced Apoptosis in HeLaS3 Cells and Is
Suppressed in GnT-III Transfectants--
We next investigated whether
protein kinase C (PKC) is involved in the suppression of
H2O2-induced JNK activation and apoptosis in
GnT-III transfectants, because PKC has been reported to be involved in
JNK activation and apoptosis induced by UV or ionizing radiation (20,
21). To deplete PKC, prolonged PMA treatment was performed. In the
control cells, H2O2-induced phosphorylation of
JNK2 was not affected by PKC depletion, whereas phosphorylation of JNK1
was reduced to the same levels as that of the GnT-III transfectants
(Fig. 4A), indicating that the
phosphorylation of JNK1 by H2O2 treatment is
mediated by PKC in control cells. The fact that JNK1 activity has been
reported to be required for bcl-2 phosphorylation and apoptosis (22)
suggests that the PKC-JNK1 signaling pathway is involved in
H2O2-induced apoptosis in HeLaS3 cells. We
further investigated the effects of GnT-III overexpression in PKC and
JNK1 activation. H2O2-induced JNK1 activity was
significantly reduced in control cells by PKC depletion by prolonged
PMA treatment but remained unchanged in GnT-III transfectants (Fig.
4B). Similar results were obtained using a PKC-specific
inhibitor, H7 (Fig. 4C). In addition, PKC depletion by
prolonged PMA treatment was effective in blocking
H2O2-induced cell death in control cells, but
little or no effect was detected in the GnT-III transfectants (Fig.
4D). Similar results were obtained when the PKC-specific inhibitors, H7 and sangivamycin, were used (data not shown). Thus, it
was suggested that GnT-III overexpression inhibits JNK1 activation and
apoptosis in a PKC-dependent manner.

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Fig. 4.
PKC-JNK1 signaling pathway is involved in
H2O2-induced apoptosis in HeLaS3 cells and is
suppressed in GnT-III transfectants. A, cells were
preincubated with or without 1 µM PMA for 48 h and
treated with 300 µM H2O2 for the
indicated times. Cell lysates were analyzed by Western blotting using
an anti-phosphorylated JNK antibody as indicated. B and
C, cells were pretreated with or without 1 µM
PMA for 48 h (B) or 5 µM H7 for 30 min
(C) before incubation with 600 µM
H2O2 for the indicated times. JNK1 activity was
analyzed by an in vitro kinase assay using c-Jun as a
substrate and visualized by autoradiography and quantified as described
under "Experimental Procedures." The results are representative of
at least three independent experiments. D, cells were
preincubated with 1 µM PMA for 48 h and treated with
the indicated concentrations of H2O2 for
18 h; cell viability was then assessed by an MTT assay. Data from
three independent experiments with triplicate samples, each of which
was averaged and expressed as the mean ± S.D., are shown.
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GnT-III Overexpression Inhibits
H2O2-induced JNK1 Activation through
Suppression of PKC
Activation--
It is known that the PKC family
consists of three groups: conventional, novel, and atypical PKC
isoforms. Neither Gö-6976 nor Ro-32-0432, both of which are
conventional PKC-specific inhibitors, attenuated the
H2O2-induced activation of JNK1 in control
cells (data not shown), indicating that novel or atypical PKC mediates H2O2-induced JNK1 activation in HeLaS3 cells
and that GnT-III overexpression might affect the activation of those
PKCs. We further investigated the specific type of PKC that is affected
in the GnT-III transfectants. We examined
H2O2-induced phosphorylation of PKC
,
II
in the conventional family, PKC
,
in the atypical family, and
PKC
, µ in the novel family. As shown in Fig.
5, H2O2- or
PMA-induced phosphorylation on threonine residue in PKC
was markedly
decreased in the GnT-III transfectants. No difference in the protein
expression level of PKC
between control cells and GnT-III
transfectants was detected (data not shown). It has also been suggested
that PKC
was phosphorylated on multiple tyrosine residues by
H2O2 exposure and that tyrosine phosphorylation
of PKC
increases its kinase activity (23-25). Fig.
6A showed that the tyrosine
phosphorylation of PKC
was enhanced by H2O2
treatment in control cells but it was suppressed in the GnT-III
transfectants (Fig. 6A, upper panel). To further
examine the issue of whether the suppression of PKC
is involved in
the down-regulation of JNK1 in GnT-III transfectants, we used the
PKC
-selective inhibitor, rottlerin. The results showed that
rottlerin inhibited H2O2-induced PKC
activation as expected (Fig. 6B, upper panel),
and H2O2-induced JNK1 phosphorylation was
significantly suppressed in control cells but was not changed in
GnT-III transfectants (Fig. 6B, lower panel). Thus, these results suggest that GnT-III overexpression inhibits H2O2-induced JNK1 activation and apoptosis by
suppressing the activation of PKC
.

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Fig. 5.
GnT-III overexpression inhibits
H2O2-induced PKC
activation. HeLaS3 cells were treated with 300 µM H2O2 for the indicated periods
or 1 µM PMA for 15 min. Cell lysates were subjected to
SDS-PAGE and analyzed by Western blotting using specific antibodies as
indicated. In the upper panel, quantification of each PKC
phosphorylation state was determined by densitometry. cPKC,
conventional PKC. aPKC, atypical PKC. nPKC, novel
PKC.
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Fig. 6.
GnT-III overexpression inhibits
H2O2-induced JNK1 activation through
suppression of PKC activation.
A, cells were treated with or without 600 µM
H2O2 for the indicated times. Cell lysates were
subjected to immunoprecipitation with anti-phospho-tyrosine antibody
(4G10). The lysates and immunoprecipitants were subjected to
SDS-PAGE and analyzed by Western blotting using PKC antibody.
B, cells were preincubated with or without 50 µM rottlerin for 30 min and treated with 300 µM H2O2 for 30 min. Cell lysates
were analyzed by Western blotting, using specific antibodies as
indicated.
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DISCUSSION |
The effects of GnT-III overexpression on
H2O2 signaling in HeLaS3 cells were examined.
We found that overexpression of GnT-III markedly suppresses
H2O2-induced apoptosis by down-regulating JNK1
activation. We propose that H2O2-induced
activation of PKC
is suppressed in GnT-III transfectants, which is
responsible for inhibition of JNK1 activation.
One of the major signaling pathways for influencing the survival of
cells subjected to oxidative stress is mediated by NF-
B and Akt (16,
18, 19, 26); several groups have reported that
H2O2 induces NF-
B activation (14-16).
Therefore, we first examined NF-
B activation in our system, but the
findings show that there is little or no NF-
B activation either in
control HeLaS3 cells or GnT-III transfectants (Fig. 3). Similar results have been reported by using myocytes (27). Thus, we have concluded that
H2O2-induced NF-
B activation might be highly
cell type-dependent. There was also no difference in Akt
activation between control and GnT-III transfectants.
Our results support the contention that MAPKs play a critical role in
H2O2-induced apoptosis in HeLaS3 cells (10).
H2O2 is known to activate both Erk and JNK (10,
11). The general hypothesis is that the dynamic balance between Erk and
JNK pathways is important in determining whether a cell survives or
undergoes apoptosis (28). Wang et al. (10) reported that
H2O2-induced apoptosis was markedly enhanced
when ERK was inhibited by PD098059 and it decreased when JNK activation
was inhibited by expression of a dominant negative mutant form of MEK4.
Recent studies suggest that the JNK activity is necessary for the
stress-induced activation of the cytochrome c-mediated death
pathway via bcl-2/bcl-xL phosphorylation (22, 29, 30). Our
results show that GnT-III overexpression suppresses JNK1 activation but
does not affect the Erk pathways, suggesting that this inhibition of
JNK1 results in cell survival. p38-mapk is also activated by various
stressful stimuli and is involved in the apoptosis signal (31, 32).
However, a p38-mapk-specific inhibitor, SB20358, had no effect on cell
viability in our system (data not shown), indicating that p38-mapk is
not involved in the prevention of H2O2-induced
apoptosis in GnT-III transfectants.
In this report, we have shown that PKC-JNK1 pathway is involved in
H2O2-induced apoptosis in HeLaS3 cells.
Although it has been reported that PKC is involved in JNK activation
and apoptosis by UV or ionizing radiation, the signaling pathway of JNK
activation induced by H2O2 has not been well
defined. As indicated in Fig. 4, PKC inhibition by pretreatment with
PMA- or PKC-specific inhibitors was effective in blocking
H2O2-induced JNK1 activation and apoptosis in
control cells.
We have shown that GnT-III overexpression suppressed both PMA- and
H2O2-induced activation of PKC
. PKC
belongs to the PKC family, which consists of at least 12 members and
which can be classified into three groups based on their biochemical
properties and sequence homologies. Conventional PKC isoforms
(cPKC-
, -
I, -
II, -
) are calcium- and
phospholipid-dependent and are activated by
phosphatidylserine and diacylglycerol (DAG). Novel PKC isoforms (nPKC-
, -
, -
, -
, -µ) can be activated by DAG but not by
calcium. Atypical PKC isoforms (aPKC-
, -
, -
,) are calcium- and
phospholipid-independent. The different PKC isotypes may have specific
roles in signal transduction. Recent reports have suggested that PKC
was phosphorylated on multiple tyrosine residues by various tyrosine
kinases, such as Fyn, Src, Lyn, Abl, and growth factor receptors and
that the tyrosine phosphorylation of PKC
increases its kinase
activity (23-25). In this study, it was shown that
H2O2-induced threonine and tyrosine phosphorylation of PKC
was markedly suppressed in the GnT-III transfectants, resulting in down-regulation of JNK1 activation. Our
results are consistent with the report that PKC
is a pro-apoptotic molecule that activates JNK and is required for
mitochondrial-dependent apoptosis (20, 34-37). It has been
also demonstrated that PKC
is involved in cancer metastasis. Kiley
et al. (37) reported that PKC
phosphorylates
cytoskeletal substrate proteins, such as adducin, and plays an
important role in migration and attachment. Suppression of the activity
of PKC
in GnT-III transfectants might be involved in decreased
metastatic property of GnT-III transfectants observed in B16 mouse
melanoma cells (6).
Further studies will be required to clarify the activation mechanisms
of PKC
in response to ROS and how GnT-III overexpression suppresses
H2O2-induced PKC
activation. One possible
mechanism would involve some modification by GnT-III on some
molecule(s) or some domain in PKC
itself, as we suggested for the
intracellular domain of epidermal growth factor receptor in GnT-III
transfectants (9).
In summary, we have provided evidence that PKC-JNK1 pathway is involved
in H2O2-induced apoptosis in HeLaS3 cells and
that the overexpression of GnT-III results in the suppression of
H2O2-induced apoptosis through inhibition of
PKC
-JNK1 pathway. The role of N-glycans in signaling
pathway has started to be examined from many aspects. It is possible
that the suppression of PKC activity by GnT-III overexpression that is
observed here shares the same mechanisms underlying other phenomena
observed in GnT-III transfectants.