Cyclic GMP-dependent Protein Kinase Regulates the Expression of Thioredoxin and Thioredoxin Peroxidase-1 during Hormesis in Response to Oxidative Stress-induced Apoptosis*

Tsugunobu AndohDagger §, Chuang Chin Chiueh, and P. Boon ChockDagger ||

From the Dagger  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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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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, respectively. Thus, the cytoprotective effect of NO in SH-SY5Y cells appears to proceed via the PKG-mediated pathway, and S-nitrosylation of caspases plays a minimal role.

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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-alpha 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).

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.

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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-kappa B, Oct-1, PEA-3, c-Myc, phosphorylated c-Myc, PKGIalpha , PKGIbeta , and PKGII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies phosphorylated and nonphosphorylated c-Jun, MAPK/Erk1/2, MEK1/2, Ikappa 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.

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.

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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.


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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.

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 PKGIalpha 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 (black-triangle). 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 Ialpha (PKGIalpha ), Ibeta (PKGIbeta ), and II (PKGII) in 20 and 10 µg of protein from SH-SY5Y cells were determined by Western blotting method using anti-PKGIalpha , -PKGIbeta , and -PKGII antibodies.

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).


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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.

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.


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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 (open circle ), III (triangle ), 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 (open circle ) 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.

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-kappa 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.

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.


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Fig. 7.   Schematic representation of a proposed serum deprivation-induced apoptosis and NO-mediated hormesis in SH-SY5Y cells. up-arrow  indicates increase, and LOOH represents organic peroxides.


    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.

§ 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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