Down-regulation of Hydrogen Peroxide-induced PKCdelta Activation in N-Acetylglucosaminyltransferase III-transfected HeLaS3 Cells*

Yukinao Shibukawa, Motoko Takahashi, Isabelle Laffont, Koichi Honke, and Naoyuki TaniguchiDagger

From the Department of Biochemistry, Osaka University Graduate School of Medicine, B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

Received for publication, August 2, 2002, and in revised form, October 30, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

N-acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue. Our previous studies have shown that modification of N-glycans by GnT-III affects a number of intracellular signaling pathways. In this study, the effects of GnT-III on the cellular response to reactive oxygen species (ROS) were examined. We found that an overexpression of GnT-III suppresses H2O2-induced apoptosis in HeLaS3 cells. In the case of GnT-III transfectants, activation of Jun N-terminal kinase (JNK) following H2O2 treatment was markedly reduced compared with control cells. Either the depletion of protein kinase C (PKC) by prolonged treatment with phorbol 12-myristate 13-acetate or the inhibition of PKC by the specific inhibitor H7 attenuated the H2O2-induced activation of JNK1 and apoptosis in control cells but not in the GnT-III transfectants. Furthermore, we found that H2O2-induced phosphorylation of PKCdelta was markedly suppressed in GnT-III transfectants. Rottlerin, a specific inhibitor of PKCdelta , significantly inhibited H2O2-induced activation of JNK1 in control cells, indicating that PKCdelta is involved in the pathway. These findings suggest that the overexpression of GnT-III suppresses H2O2-induced activation of PKCdelta -JNK1 pathway, resulting in inhibition of apoptosis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 1, 4-linkage to the beta 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 PKCdelta activity had an inhibitory effect on JNK activity and apoptosis in GnT-III transfectants.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, PKCalpha /beta II (Thr-638/641), PKCdelta (Thr-505), PKCµ (Ser-744/748), PKCzeta /lambda (Thr-410/413) antibodies and anti-p38-mapk, Akt, PKCdelta 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 beta -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 beta -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 [gamma -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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

GnT-III Overexpression Does not Affect NF-kappa B Activation and Akt Phosphorylation-- The activation of NF-kappa B in GnT-III transfectants was examined because it has been demonstrated that activation of the transcription factor NF-kappa 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-kappa B activation following H2O2 exposure. A luciferase assay using a plasmid containing the NF-kappa 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-kappa B and Akt signaling pathway. A, HeLaS3 cells were treated with H2O2 for 3 h or 20 ng/ml TNFalpha , used as a positive control, for 1 h. After incubation with H2O2 or TNFalpha , 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-kappa B binding site-containing plasmids using LipofectAMINE 2000 as described under "Experimental Procedures." Transfected cells were preincubated for 24 h before H2O2 or TNFalpha 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.

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-kappa 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.

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.

GnT-III Overexpression Inhibits H2O2-induced JNK1 Activation through Suppression of PKCdelta 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 PKCalpha , beta II in the conventional family, PKCzeta , lambda  in the atypical family, and PKCdelta , µ in the novel family. As shown in Fig. 5, H2O2- or PMA-induced phosphorylation on threonine residue in PKCdelta was markedly decreased in the GnT-III transfectants. No difference in the protein expression level of PKCdelta between control cells and GnT-III transfectants was detected (data not shown). It has also been suggested that PKCdelta was phosphorylated on multiple tyrosine residues by H2O2 exposure and that tyrosine phosphorylation of PKCdelta increases its kinase activity (23-25). Fig. 6A showed that the tyrosine phosphorylation of PKCdelta 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 PKCdelta is involved in the down-regulation of JNK1 in GnT-III transfectants, we used the PKCdelta -selective inhibitor, rottlerin. The results showed that rottlerin inhibited H2O2-induced PKCdelta 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 PKCdelta .


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Fig. 5.   GnT-III overexpression inhibits H2O2-induced PKCdelta 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 PKCdelta 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 PKCdelta 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PKCdelta 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-kappa B and Akt (16, 18, 19, 26); several groups have reported that H2O2 induces NF-kappa B activation (14-16). Therefore, we first examined NF-kappa B activation in our system, but the findings show that there is little or no NF-kappa 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-kappa 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 PKCdelta . PKCdelta 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-alpha , -beta I, -beta II, -gamma ) are calcium- and phospholipid-dependent and are activated by phosphatidylserine and diacylglycerol (DAG). Novel PKC isoforms (nPKC-delta , -epsilon , -eta , -theta , -µ) can be activated by DAG but not by calcium. Atypical PKC isoforms (aPKC-iota , -lambda , -zeta ,) are calcium- and phospholipid-independent. The different PKC isotypes may have specific roles in signal transduction. Recent reports have suggested that PKCdelta 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 PKCdelta increases its kinase activity (23-25). In this study, it was shown that H2O2-induced threonine and tyrosine phosphorylation of PKCdelta was markedly suppressed in the GnT-III transfectants, resulting in down-regulation of JNK1 activation. Our results are consistent with the report that PKCdelta is a pro-apoptotic molecule that activates JNK and is required for mitochondrial-dependent apoptosis (20, 34-37). It has been also demonstrated that PKCdelta is involved in cancer metastasis. Kiley et al. (37) reported that PKCdelta phosphorylates cytoskeletal substrate proteins, such as adducin, and plays an important role in migration and attachment. Suppression of the activity of PKCdelta 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 PKCdelta in response to ROS and how GnT-III overexpression suppresses H2O2-induced PKCdelta activation. One possible mechanism would involve some modification by GnT-III on some molecule(s) or some domain in PKCdelta 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 PKCdelta -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.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed. Tel.: 816-6879-3420; Fax: 816-6879-3429; E-mail: proftani@biochem.med.osaka-u.ac.jp.

Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M207870200

    ABBREVIATIONS

The abbreviations used are: GnT-III, N-acetylglucosaminyltransferase III; ROS, reactive oxygen species; MTT, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide; JNK, Jun N-terminal kinase; SEK1, stress-activated protein kinase/extracellular signal-regulated kinase kinase 1; ERK, extracellular signal-regulated protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; MAPK, mitogen-activated protein kinase; DAPI, 4,6-diamidino-2-phenylindole; EGF, epidermal growth factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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