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Am J Physiol Heart Circ Physiol 295: H2079-H2086, 2008. First published September 19, 2008; doi:10.1152/ajpheart.00092.2008
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Ser9 phosphorylation of mitochondrial GSK-3β is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis

Katsuhiko Ohori,1 Tetsuji Miura,1 Masaya Tanno,1,2 Takayuki Miki,1 Takahiro Sato,1 Satoko Ishikawa,1 Yoshiyuki Horio,2 and Kazuaki Shimamoto1

1Second Department of Internal Medicine and 2Department of Pharmacology, Sapporo Medical University School of Medicine, Sapporo, Japan

Submitted 28 January 2008 ; accepted in final form 11 September 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The aim of this study was to determine the role of GSK-3β in cardiomyocyte protection afforded by erythropoietin (EPO) against oxidant stress-induced apoptosis. Treatment with EPO (10 units/ml) induced Ser473 phosphorylation of Akt and Ser9 phosphorylation of GSK-3β and significantly reduced the proportion of apoptotic H9c2 cardiomyocytes after exposure to H2O2 from 38.3 ± 2.7% to 26.0 ± 2.9%. This protection was not detected in cells transfected with constitutively active GSK-3β (S9A), which lacks Ser9 for inhibitory phosphorylation. The antiapoptotic effect of EPO was mimicked completely by GSK-3β knockdown using small interfering RNA and partly by the transfection with kinase-deficient GSK-3β (K85R). The level of colocalization of intracellular GSK-3β with mitochondria assessed by enhanced green fluorescent protein-tagged GSK-3β or immunocytochemistry was not altered by EPO treatment. However, EPO increased the level of Ser9-phospho-GSK-3β colocalized with mitochondria by 50% in a phosphatidylinositol 3-kinase-dependent manner. Mitochondrial translocation of Bcl-2-associated X protein (BAX) after exposure to H2O2 was inhibited by EPO pretreatment and by GSK-3β knockdown. These results suggest that the suppression of GSK-3β activity by Akt-mediated Ser9 phosphorylation in the mitochondria affords cardiomyocytes tolerance against oxidant-induced apoptosis, possibly by inhibiting the access of BAX to the mitochondria.

oxidant stress; glycogen synthase kinase-3β

CYTOPROTECTION AFFORDED BY erythropoietin (EPO) receptor activation has been demonstrated against various types of injury (i.e., ischemia-reperfusion, trauma, and inflammation) in different tissues (6, 15, 20, 22, 23). In the heart, EPO and its derivatives have been shown to reduce myocardial necrosis after ischemia-reperfusion injury (15, 27, 28) and to suppress apoptosis and ventricular dysfunction after anthracyclin treatment (6, 17, 21). Although EPO receptor activation provokes the activation of multiple prosurvival signal pathways, recent studies, including ours, suggest that phosphatidylinositol 3-kinase (PI3K)-Akt-GSK-3β signaling plays a predominant role in protection by EPO against myocardial necrosis (14, 27, 28). GSK-3β is a putative regulatory factor of the threshold for opening of the mitochondrial permeability transition pore (mPTP), and several cytoprotective signals converge at this kinase (16). However, the role of GSK-3β in the protection of cardiomyocytes against apoptosis of cardiomyocytes remains unclear. Although inhibitors of PI3K have been shown to abolish both Ser9 phosphorylation in GSK-3β and EPO-induced myocardial protection against apoptosis induced by anthracyclin (17), whether Ser9-phospho-GSK-3β is necessary for the antiapoptotic effects of EPO has not been demonstrated. Furthermore, it also remains unclear whether EPO can afford cardioprotection against apoptosis induced by reactive oxygen species (ROS) that play important roles in ischemia-reperfusion (10) and chronic heart failure (24).

In the present study, we examined the hypothesis that EPO protects cardiomyocytes from oxidant stress-induced apoptosis by the inhibitory phosphorylation of GSK-3β at Ser9 and its translocation to the mitochondria. The rationale for this hypothesis is threefold. First, EPO treatment has been shown to elevate the threshold for opening of the mPTP (16), which triggers not only necrosis but also apoptosis (2, 9, 18, 30). Second, we recently found that GSK-3β phosphorylated by Akt and PKC translocates to the mitochondria and binds to an mPTP subunit protein after ischemia-reperfusion in rat hearts (29). Finally, GSK-3β activity has been shown to be a determinant of the apoptotic process, at least, in neuronal cells (13). To test the hypothesis, we used H9c2 cardiomyocytes and found that phosphorylation of Ser9 in GSK-3β is indeed a requisite for EPO to protect against apoptosis triggered by H2O2, a ROS mimetic. Although mitochondrial translocation of GSK-3β by EPO was not detected, the results suggest that EPO-induced GSK-3β phosphorylation occurs within the mitochondria by a PI3K/Akt-dependent mechanism.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. H9c2 cells (American Type Culture Collection) were cultured in DMEM (Sigma, St. Louis, MO) supplemented with 10% FBS at 37°C with 5% CO2. The cells were used for experiments when they were 70–80% confluent unless otherwise specified, and FBS was removed from the medium 24 h before the induction of apoptosis.

Induction of apoptosis and EPO pretreatment. In preliminary experiments, we determined apoptosis of H9c2 cells at 2–6 h after treatment with 20–100 µM H2O2. Results of the experiments showed that apoptosis was induced in ~30% of cells either by treatment with 50 µM H2O2 for 6 h or by that with 100 µM H2O2 for 2 h. We primarily used the lower dose protocol, i.e., 6-h 50 µM H2O2, in the present study. In a series of experiments using small interfering (si)RNA, we used a 2-h 100 µM H2O2 incubation protocol to avoid the possibility that a long H2O2 incubation time modifies the effect of siRNA on GSK-3β expression during the experiments. To confirm that EPO affords protection against not only H2O2-induced apoptosis but also other forms of ROS-mediated apoptosis, we additionally examined the effects of EPO on angiotensin II-induced apoptosis. H9c2 cells were incubated with 100 or 150 µM angiotensin II or its vehicle for 24 h to induce apoptosis.

In EPO pretreatment groups, 10 units/ml of EPO were added to the culture medium 1 h before H2O2 or angiotensin II challenge. Vehicle was added to the medium in control groups. Both EPO (human recombinant EPO) and its vehicle were provided by Chugai Pharmaceutical (Tokyo, Japan). The present dose of EPO was selected based on previous findings from our laboratory that 1–5 units/ml of EPO significantly limited infarct size after ischemia-reperfusion in isolated buffer-perfused hearts (19, 27) and results of pilot experiments testing 1–30 units/ml in H9c2 cells. After exposure to H2O2 or angiotensin II, H9c2 cells were fixed with 4% paraformaldehyde and observed by confocal microscopy. Apoptosis of cells was defined as nuclear condensation revealed by nuclear staining with Hoechst 33342. In each time of the experiments, 500 cells were counted for each treatment group to determine a percentage of apoptotic cells.

Subcellular fractionation and immunoblot analysis. Whole cell lysates of H9c2 cells were obtained using CellLytic-M Mammalian Cell Lysis/Extraction agent (Sigma) before and after the treatment with EPO (10 units/ml). The samples were separated by electrophoresis in 10% SDS-polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). Proteins were visualized by use of antibodies against each protein of interest and LumiGLO (KPL, Gaithersburg, MD). Protein levels were determined by a lumino-image analyzer, LAS-1000 (Fujifilm, Tokyo, Japan). As primary antibodies for immunoblotting, antibodies against Akt, GSK-3β, Ser473-phospho-Akt, Ser9-phospho-GSK-3β (Cell Signaling Technology, Beverly, MA), common β-receptor subunit, EPO receptor (Santa Cruz Biotechnology, Santa Cruz, CA), prohibitin (Merck, Darmstadt, Germany), or Bcl-2-associated X protein (BAX) or Bcl-2 (BD Bioscience, San Jose, CA) were used. In some experiments, fractionation of the cell lysates into cytosolic and mitochondrial fractions was performed using a mitochondrial isolation kit (Pierce Biotechnology, Rockford, IL) before immunoblotting.

Plasmid construction and transfection. To construct the GSK-3β-enhanced green fluorescent protein (EGFP) fusion protein, the coding region of rat GSK-3β cDNA lacking its stop codon was cloned into the vector pEGFP-N3 (Clontech, Mountain View, CA) at the BglII and SalI sites and in frame with the EGFP coding region. To construct a constitutively active (S9A) and kinase-deficient (K85R) GSK-3β, site-directed mutagenesis was carried out using the QuickChange XL Mutagenesis kit (Stratagene, La Jolla, CA). Successful mutagenesis was confirmed by sequencing. H9c2 cells were transfected with each plasmid using the Cell Line Nucleofector Kit L (Amaxa, Gaithersburg, MD) and used for further analyses. Transfection efficiency was ~20% for wild-type, S9A, and K85R.

Immunocytochemistry. H9c2 cells cultured on a collagen-coated glass dish were stained with Mito-Tracker Red (0.2 µM) for 15 min and fixed with 4% paraformaldehyde. The cells were then washed with PBS, blocked with 3% BSA in PBS for 30 min, and incubated overnight in PBS containing 3% BSA and anti-GSK-3β or anti-phospho-GSK-3β antibodies. The bound antibodies were labeled using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes).

Double fluorescence for red (Mito-Tracker Red) and green (EGFP or Alexa Fluor 488) channels was imaged by a confocal microscopy (Bio-Rad, Hercules, CA) using a x100 objective lens with excitation of an argon-krypton laser at the wavelength of 543 and 488 nm. The acquired images were merged, and quantitative analysis was performed by use of image analysis software (Adobe Photoshop version 7). The degree of colocalization of transfected EGFP-GSK-3β, Alexa Fluor 488-labeled GSK-3β, or Alexa Fluor 488-labeled Ser9-phospho-GSK-3β with mitochondria was determined as green pixels (GSK-3β) overlapped with red pixels (mitochondria) as a percentage of entire red pixels.

Transfection of siRNA. GSK-3β-siRNA, GSK-3{alpha}-siRNA, and EPO receptor-siRNA were synthesized by B-Bridge. A cocktail of three siRNAs targeted to the gene was used to efficiently knock down the gene. The sequences of sense and anti-sense siRNAs in the cocktail were GGUCAUUUGGUGUGGUAUATT (sense) and UAUACCACAC CAAAUGACCTT (antisense), GUUCAGAAGUCUAGCCUAUTT (sense) and AUAGGCUAGACUUCUGAACTT (antisense), and CCAACAAGGGAGCAAAUUATT (sense) and UAAUUUGCUCCCUUGUUGGTT (antisense), respectively, for GSK-3β-siRNA and CGAGGGAACUGGUGGCCAUTT (sense) and AUGGCCACC AGUUCCCUCGTT (antisense), GCUCUAGCCUGCUGGAGUATT (sense) and UACUCCAGCAGGCUAGAGCTT (antisense), and GGGCAGAGGUAAAUGAACUTT (sense) and AGUUCAUUUACCUCUGCCCTT (antisense), respectively, for GSK-3{alpha}-siRNA. The sequences of EPO receptor-siRNAs in the cocktail were CUUACCAGCUCGAAGGUGATT (sense) and UCACCUUCGAGCUGGUAAGTT (antisense), CUGAGUGUGUCCUGAGCAATT (sense) and UUGCUCAGGACACACUCAGTT (antisense), and GACUAUGGAUGAAGGUUCATT (sense) and UGAACCUUCAUCCAUAGUCTT (antisense). H9c2 cells were transfected with 100 nM of the cocktail using the Nucleofector Kit L (Amaxa) according to the manufacturer's instructions. Forty-eight hours after transfection, the cells were used for further analyses.

Statistical analysis. Results are presented as means ± SE. Differences between groups were tested by one-way or two-way ANOVA, and the Student-Newman-Keuls post hoc test was used for multiple comparisons when ANOVA indicated significant differences. Differences were considered significant when P was <0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Receptors for EPO in H9c2 cells. Since not only the EPO receptor but also common β-receptor subunit, which putatively forms a heterodimer receptor together with EPO receptor monomer, has been reported to transmit prosurvival signals (4), we immunoblotted for these proteins in H9c2 cells without any pretreatments. As shown in Fig. 1A, both the EPO receptor protein and common β-receptor subunit protein were detected in H9c2 cells under baseline conditions.


Figure 1
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  		Fig. 1. Time courses of Akt- and GSK-3β phosphorylation by erythropoietin (EPO) receptor (EPO-R) activation. A: representative immunoblottings for EPO-R and common β-receptor subunit (βcR) protein in 10 or 20 µg of protein obtained from H9c2 cell lysates. B: representative immunoblottings for total and phosphorylated Akt and GSK-3β proteins and summary of phospho-GSK-3β-to-total GSK-3β ratio and phospho-Akt-to-total Akt ratio for each time point. Columns and bars stand for means and SE, respectively. Each time course experiment was repeated 5 times. AU, arbitrary unit. *P < 0.05 vs. baseline (i.e., time = 0).

 
Phosphorylation of Akt and GSK-3β by EPO in H9c2 cells. Time courses of Akt and GSK-3β phosphorylation after EPO receptor activation were assessed in H9c2 cells. As shown in Fig. 1B, increases in Ser473-phospho-Akt and Ser9-phospho-GSK-3β started to appear at 30 min after the addition of EPO (10 units/ml) to the culture medium and reached significant levels at 60 min. In previous studies from our laboratory (19, 27), significant phosphorylation of Akt and GSK-3β was detected at 15 min after EPO receptor activation in buffer-perfused beating hearts. Differences in cell subtypes (H9c2 cells vs. adult cardiomyocytes) and/or conditions of loading and contraction may be responsible for the difference between the time courses of Akt/GSK-3β phosphorylation.

In H9c2 cells transfected with the EPO receptor siRNAs, EPO failed to induce phosphorylation of Akt and GSK-3β (data not shown). Significant suppression of EPO receptor expression in the tranfected cells was confirmed by RT-PCR of EPO receptor mRNA.

Protection of H9c2 cells from apoptosis by EPO. Cytoprotective effects of EPO against oxidant stress-induced apoptosis were examined by use of H2O2 and angiotensin II. Based on the results shown in Fig. 1B, we treated H9c2 cells with EPO (10 units/ml) or a vehicle for 1 h before the induction of apoptosis by H2O2 or angiotensin II. The proportion of apoptotic cells at 6 h after the addition of 50 µM H2O2 was reduced from 38.3 ± 2.7% to 26.0 ± 2.9% by EPO (Fig. 2A). Significant protection by EPO against apoptosis was also observed at 24 h after incubation with angiotensin II, whereas EPO alone did not significantly affect the number of apoptotic cells during the 24-h control period (Fig. 2B).


Figure 2
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  		Fig. 2. Effects of pretreatment with EPO on apoptosis in H9c2 cells. A, top: percentage of apoptotic cells at 6 h after addition of 50 µM H2O2 to the culture medium. In the EPO-treated groups, EPO (10 units/ml) was added to the culture medium 1 h before addition of H2O2. A, bottom: representative images of apoptotic cells in each group. B, top: percentage of apoptotic cells at 24 h after addition of indicated dose of angiotensin II (ANG II) to the medium. B, bottom: representative images of apoptosis induced by 150 µM of ANG II with or without EPO pretreatment. Each experiment was repeated 6 times.

 
Effects of GSK-3β modification on EPO-induced cytoprotection. To examine the causal relationship between inhibitory Ser9 phosphorylation of GSK-3β and protection afforded by EPO, we assessed changes in antiapoptotic tolerance of H9c2 cells by the reduction of GSK-3β protein expression and by the elimination of Ser9-mediated regulation of GSK-3β. As shown in Fig. 3, the reduction of GSK-3β protein level by siRNA significantly suppressed H2O2-induced apoptosis of H9c2 cells, whereas knocking down the {alpha}-isoform of GSK-3 did not affect tolerance against apoptosis.


Figure 3
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  		Fig. 3. Effects of suppression of GSK-3 proteins on tolerance against apoptosis. A: representative immunoblotting for GSK-3{alpha} and GSK-3β in the control-, GSK-3{alpha}-, and GSK-3β small interfering (si)RNA-treated groups 48 h after transfection. Reblotting for GAPDH is shown as loading controls. B: percentages of cells with apoptotic phenotypes at 6 h after addition of 50 µM H2O2 to the medium in each treatment group. The cells were transfected with siRNAs 48 h before the H2O2 challenge. Each experiment was repeated 5 times. NS, not significant.

 
In H9c2 cells transfected with wild-type GSK-3β, pretreatment with EPO suppressed apoptosis induced by H2O2 from 34.1 ± 1.4% to 23.2 ± 1.8% (Fig. 4). However, transfection with S9A alone significantly enhanced apoptosis in response to H2O2 challenge and also abolished protective effects of EPO (Fig. 4). In contrast, in H9c2 cells transfected with K85R, a dominant-negative GSK-3β, apoptosis tended to be reduced compared with that in wild-type GSK-3β-transfected controls, and EPO failed to further suppress apoptosis.


Figure 4
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  		Fig. 4. Effects of mutations in GSK-3β on EPO-induced protection against apoptosis. H9c2 cells were transfected with wild-type GSK-3β (WT), S9A, or K85R, which were tagged with enhanced green fluorescent protein (EGFP). Percentage of transfected cells showing apoptotic phenotype at 2 h after addition of 100 µM H2O2 to the medium is shown for each group. Cells were pretreated with a vehicle (control) or 10 units/ml of EPO for 1 h before exposure to H2O2. *P < 0.05 vs. control; {dagger}P < 0.05 vs. WT control. Each experiment was repeated 7 times.

 
Effects of EPO on intracellular distribution of GSK-3β. First, we determined effects of EPO on the intracellular localization of GSK-3β by transfection of EGFP-tagged GSK-3β. The percentage of mitochondria overlapped with EGFP-tagged GSK-3β was 28.7 ± 4.0% under baseline conditions and 30.0 ± 5.4% 1 h after treatment with EPO, which were not statistically different (Fig. 5). To rule out the possibility that tagging GSK-3β with EGFP modified the capacity of the GSK-3β molecule to translocate within the cell, we examined GSK-3β distribution also by immunocytochemistry. As shown in Fig. 6, A and C, immunostaining using anti-GSK-3β antibody showed no significant change in GSK-3β-mitochondria colocalization induced by EPO. Interestingly, however, colocalization of Ser9-phospho-GSK-3β with mitochondria was significantly increased from 17.1 ± 1.2% to 25.4 ± 1.7% after EPO treatment (Fig. 6, B and D). Furthermore, LY-294002, a PI3K inhibitor, prevented the EPO-induced increase in the level of phospho-GSK-3β-mitochondria colocalization (18.7 ± 1.7%), although LY-294002 alone did not modify. These findings suggest that the activation of the EPO receptor induces Ser9 phosphorylation of GSK-3β in the mitochondria via PI3K/Akt activation without the recruitment of this kinase to the mitochondria compartment.


Figure 5
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  		Fig. 5. Colocalization of EGFP-GSK-3β with mitochondria. Mito-Tracker-stained mitochondrial area (Mito), which overlapped with EGFP-GSK-3β signal, was expressed as a percentage of total mitochondrial area. Representative images (A) and summarized data (B) from 40 cells counted for each treatment are shown. EPO is treatment with 10 units/ml of EPO for 1 h. Each experiment was repeated 5 times.

 

Figure 6
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  		Fig. 6. Effects of EPO on colocalization of total GSK-3β and phospho-GSK-3β with mitochondria. A and B: representative images of GSK-3β and Ser9-phospho-GSK-3β, respectively. Mito-Tracker-stained mitochondrial area (Mito), which overlapped with Alexa Fluor 488 signal from total GSK-3β (A and C) or Ser9-phospho-GSK-3β (B and D), is expressed as a percentage of total mitochondrial area and summarized for each treatment group in C and D. GSK-3β(+)-mito is mitochondrial area overlapped with GSK-3β; phospho-GSK-3β(+)-mito is mitochondrial area overlapped with phospho-GSK-3β; EPO is treatment with 10 units/ml of EPO for 1 h; LY-294002 (LY; 30 µM alone) + EPO is combined treatment with LY-294002 (30 µM) and EPO; LY-294002 was added to the medium 1 h before the addition of EPO. *P < 0.05 vs. vehicle; {dagger}P < 0.05 vs. EPO. Each experiment was repeated 6–8 times.

 
To confirm the elevation of phospho-GSK-3β level in the mitochondria by EPO by immunoblotting, mitochondria and cytosolic fractions were prepared from H9c2 cells with or without 1-h treatment with EPO. In contrast with the findings from total cell lysates (Fig. 1), immunoblotting for phospho-GSK-3β using subcellular fractions of EPO-treated cells showed a very slight increase of phospho-GSK-3β in the mitochondria (data not shown). Thus we repeated experiments using IGF-I, which is a more potent ligand than EPO for the induction of GSK-3β phosphorylation. Treatment with IGF-I (10 nM) for 1 h mimicked the effects of EPO on GSK-3β colocalization with mitochondria. There was no change in total GSK-3β colocalized with mitochondria (Fig. 7, A and C), but phospho-GSK-3β colocalized with the mitochondria was significantly increased (Fig. 7, B and D). Immunoblotting experiments also showed that IGF-I induced a clear elevation of the phospho-GSK-3β level in the mitochondrial fractions and a modest increase in cytosolic phospho-GSK-3β (Fig. 7E). Taken together, these results support the notion that phosphorylation of GSK-3β at Ser9 can be induced in the mitochondria without the recruitment of GSK-3β from extramitochondrial compartments.


Figure 7
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  		Fig. 7. Effects of IGF-I on colocalization of total GSK-3β and phospho-GSK-3β with mitochondria. A and B: representative images of GSK-3β and Ser9-phospho-GSK-3β, respectively. Levels of total GSK-3β (C) and phospho-GSK-3β (D) colocalized with mitochondria (Mito) are expressed as in Fig. 6. IGF-I is treatment with IGF-I (10 nM) for 1 h. *P < 0.05 vs. vehicle. Each experiment was repeated 5 times. E: representative immunoblotting for total GSK-3β and phospho-GSK-3β in mitochondrial fractions (Mito) and cytosolic fractions (Cyto). Prohibitin and β-actin were used as markers of mitochondria and cytosol, respectively. Three separate experiments showed similar results.

 
Effects of EPO on H2O2-induced translocation of BAX and Bcl-2. To prepare subcellular fractions, H9c2 cells were harvested when confluency was >90% in this series of experiments. Since H9c2 cells became more resistant to oxidant stress under this highly confluent condition (data not shown), we used 1 mM as a dose of H2O2 for testing their effects on responses of Bcl-2 family proteins. At 15 min after the addition of H2O2 to the culture medium, BAX was translocated to the mitochondria, whereas there was no discernible change in the protein level of Bcl-2 (Fig. 8A). This BAX translocation in response to H2O2 was inhibited by pretreatment with EPO (10 units/ml). Suppression of GSK-3β expression by the use of GSK-3β siRNA also inhibited BAX translocation after H2O2 treatment (Fig. 8B). Together with the finding on EPO-induced phosphorylation of mitochondrial GSK-3β (Fig. 6), these results suggest that inactivation of GSK-3β by its Ser9 phosphorylation leads to inhibition of the mitochondrial translocation of BAX in response to oxidant stress.


Figure 8
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  		Fig. 8. Effects of EPO and GSK-3β siRNA on H2O2-induced translocation of Bcl-2-associated X protein (BAX). Immunoblotting for BAX, Bcl-2, and prohibitin using mitochondrial fractions (Mito) and cytosolic fractions (Cyto) are shown. Prohibitin was used as a marker of mitochondria. BAX level in the mitochondria was increased at 15 min after exposing H9c2 cells to H2O2 (1 mM), whereas cytosolic BAX level was reduced. Pretreatment with EPO (10 units/ml; A) and transfection with GSK-3β siRNA before H2O2 challenge (B) suppressed H2O2-induced BAX translocation. Three separate experiments showed similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
EPO receptor activation has been shown to induce phosphorylation of Akt and GSK-3β in different types of cells in previous studies (17, 19, 23, 27, 28, 31, 36). This phosphorylation was abrogated by PI3K inhibitors together with EPO-induced cell protection against necrosis or apoptosis, suggesting the involvement of Akt-mediated GSK-3β phosphorylation in the cytoprotection. However, evidence indicating a crucial role of Ser9-phospho-GSK-3β in the antiapoptotic effect of EPO in cardiomyocytes has been lacking. In this study, we showed that EPO could not protect H9c2 cells with a mutant GSK-3β (S9A), which lacks Ser9 for inhibitory phosphorylation, from H2O2-induced apoptosis (Fig. 4). The contribution of inactivation of GSK-3β activity to the cytoprotection was also supported by the findings that the elimination of GSK-3β activity by mutation at K85 and reduction of GSK-3β protein level by siRNA mimicked the effect of EPO on apoptosis (Figs. 3 and 4). Slightly less protection by K85R transfection compared with that by EPO might reflect the presence of a GSK-3β-independent protective mechanism, such as bcl-xl/bcl-2-associated death promoter (BAD) phosphorylation (8), in EPO-induced protection. Nevertheless, the present study indicated for the first time that inactivation of GSK-3β by its Ser9 phosphorylation is primarily important for EPO-induced protection against oxidant stress-induced apoptosis.

Localization of GSK-3β in the mitochondria has been indicated by using immunoelectron microscopy and immunoblotting (3, 13), and proapoptotic actions of GSK-3β in the mitochondria of noncardiac cells have been suggested in earlier studies (25, 34, 35, 37). The proapoptotic function of this kinase is consistent with increased susceptibility of H9c2 cells transfected with S9A, a constitutively active and uninhibitable GSK-3β, to H2O2-induced apoptosis in the present study (Fig. 4). Recently, we found that cytosolic GSK-3β translocates to the mitochodria and binds to adenine nucleotide translocase (ANT), a major subunit of mPTP, and that the phospho-GSK-3β level closely correlates with increased tolerance of the myocardium against necrosis (28, 29). Based on these findings, we hypothesized that translocation of phospho-GSK-3β to the mitochondria is important in EPO-induced suppression of apoptosis. In contrast with the hypothesis, such translocation was not detected either by tagging GSK-3β with EGFP (Fig. 5) or by use of immunocytochemistry (Fig. 6). However, the level of Ser9-phospho-GSK-3β colocalized with mitochondria was significantly elevated after EPO treatment. Furthermore, the EPO-induced phosphorylation of GSK-3β in the mitochondria was inhibited by LY-294002. These findings suggest that Ser9 phosphorylation by EPO receptor activation occurs in GSK-3β preexisting in the mitochondria by PI3K/Akt signaling.

There are four lines of evidence suggesting that Ser9 phosphorylation of GSK-3β in the mitochondria contributes to protection of cardiomyocytes against apoptosis by elevation of the threshold for mPTP opening. First, we recently found that Ser9-phospho-GSK-3β forms complex with ANT, which was associated with reduction of ANT binding to cyclophilin D, a factor sensitizing mPTP for opening (29). Second, in a study by Juhaszova et al. (16), EPO elevated the threshold for mPTP opening in response to laser irradiation in isolated cardiomyocytes. Third, earlier studies (9, 30) have shown that opening of the mPTP triggers apoptosis via translocation of BAX, and BAX translocation to the mitochondria by H2O2 in the present study was inhibited by EPO (Fig. 8). Finally, pharmacological inhibitors of mPTP (cyclosporine A and bongkrekic acid) have been shown to suppress H2O2-induced apoptosis (12, 32, 33). Collectively, these findings support the notion that interaction of Ser9-phosho-GSK-3β in the mitochondria with mPTP, leading to elevation of the threshold for mPTP opening, underlies the protection of cardiomyocytes afforded by EPO against oxidant-induced apoptosis.

Other possible mechanisms for phospho-GSK-3β to suppress apoptosis are reduced GSK-3β-mediated phosphorylation of myeloid cell leukemia sequence-1 (MCL-1), reduced p53-GSK-3β interaction, and reduced expression of transcriptional factor Gadd153. In noncardiac cells, at least, GSK-3β directly phosphorylates MCL-1, which leads to degradation of this antiapoptotic Bcl-2 family protein (25, 37). GSK-3β is also known to promote proapoptotic functions of p53 in both the nucleus and mitochondria (34, 35). Furthermore, a recent study by Menon et al. (26) showed that the expression of a proapoptotic factor, Gadd153, is increased in association with nuclear translocation of GSK-3β in cardiomyocytes undergoing β-adrenergic receptor-stimulated apoptosis. Thus inactivation of GSK-3β could preserve antiapoptotic MCL-1 and suppress proapoptotic functions of p53 and expression of Gadd153. However, in our preliminary experiments, IGF-I, which induces Akt and GSK-3β phosphorylation to levels higher than those induced by EPO, did not preserve the MCL-1 level in the mitochondrial fraction. We also could not detect significant changes in p53 level and its intracellular distribution after H2O2 treatment in H9c2 cells (data not shown). Thus it is unlikely that the effects of GSK-3β phosphorylation on MCL-1 and p53 were responsible for EPO-induced protection against apoptosis in the present preparation. The effect of GSK-3β inactivation on the expression of Gadd153 and its function in oxidant-induced apoptosis remains to be examined.

In apoptosis experiments of the present study, H9c2 cell were incubated with EPO for 1 h before H2O2 challenge, since the time course data indicate that levels of phospho-Akt and phospho-GSK-3β reach plateaus at 1 h after EPO treatment (Fig. 1). Furthermore, phosphorylation of GSK-3β in the mitochondria was significantly enhanced at 1 h after EPO treatment (Fig. 6). Namely, H9c2 cells were challenged with H2O2, when protective mechanisms by EPO had already been turned on. BAX translocation at 15 min after the onset of H2O2 treatment and subsequent apoptosis were inhibited in EPO-treated cells. Taken together, these results suggest that activation of phospho-GSK-3β-mediated protective mechanisms before oxidant stress significantly suppresses the development of apoptosis in H9c2 cells during subsequent 6 h of oxidant stress.

A limitation of the present study is that the mechanism by which GSK-3β in the mitochondria is phosphorylated after EPO receptor activation was not elucidated. Inhibition of EPO-induced GSK-3β phosphorylation by LY-294002 (Fig. 6, B and D) suggests a primary role of PI3K/Akt in the GSK-3β phosphorylation, but the possibility of the involvement of other Ser/Thr protein kinases upstream of GSK-3β cannot be excluded. Translocation of Akt to the mitochondria (3, 19) and activation of PKC-{varepsilon} or mitogen-activated protein kinase in the mitochondria (1) are possible explanations for the GSK-3β phosphorylation in the mitochondria, and these possibilities need to be tested in future studies.

In the present study, we examined the role of GSK-3β in the protection of H9c2 cells against acute apoptosis. However, inactivation of GSK-3β is probably not the only mechanism for suppression of cardiomyocyte apoptosis associated with heart failure by EPO administration. In fact, a recent study by Chen et al. (7) showed that EPO treatment restored Bcl-2 expression and preserved ventricular function in doxorubicin-induced heart failure. Furthermore, Akt activated by EPO could prevent apoptosis by modulation of BAD (8), procaspase-9 (5), and/or cAMP-responsive element-binding protein (11). The relative importance of GSK-3β inactivation compared with other Akt-mediated mechanisms in chronic treatment with EPO may warrant further investigation.

In summary, Ser9 phosphorylation of GSK-3β was necessary for the protection afforded by EPO against H2O2-induced apoptosis in H9c2 cells, and this phosphorylation of GSK-3β was induced PI3K dependently in the mitochondria without the recruitment of GSK-3β into the mitochondria. These observations support the notion that inactivation of mitochondrial GSK-3β by Ser9 phosphorylation is primarily important for antiapoptosis resistance of cardiomyocytes afforded by EPO receptor activation.


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 ABSTRACT
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This study was supported by a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science (No. 18590781) and grants from Sapporo Medical University Academic Foundation and from Chugai Pharmaceutical.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Miura, Second Dept. of Internal Medicine, Sapporo Medical Univ. School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan (e-mail: miura{at}sapmed.ac.jp)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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