Hydrogen Peroxide-Induced Akt Phosphorylation Regulates Bax Activation

Abstract

Reactive oxygen species such as hydrogen peroxide (H₂O₂) are involved in many cellular processes that positively and negatively regulate cell fate. H₂O₂, acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H₂O₂ was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H₂O₂-induced activation of PI3K/Akt influences posttranslational modification of Bax and inactivate a key component of the cell death machinery.

Introduction

Reactive oxygen species (ROS) are a natural byproduct of cellular metabolism. They are involved in various signaling pathways under normal physiological conditions [1]. The intracellular concentration of ROS is tightly regulated by cellular antioxidant defense mechanisms both enzymatically (antioxidant enzymes) and non-enzymatically (small antioxidant molecules, e.g. glutathione). An imbalance in the oxidant/antioxidant system, either due to excess ROS generation, impairment of antioxidant defense system, or both, leads to oxidative stress [2]. Chronic and sustained high toxic levels of ROS are associated with several pathological conditions including inflammatory diseases and the complications of diabetes [2–8]. In contrast, ROS are directly and indirectly involved in physiological signaling pathways [9, 10]. Low levels of ROS regulates cellular function in tumor cells [11], T cells [9], and macrophages [12].

We are interested in the underlying mechanisms by which ROS operate as second messengers. Mild increases in ROS act as second messengers in regulating survival signaling pathways [13, 14]. The pro-survival effects of low ROS levels suggest that they may be involved in neuronal preconditioning, such as hypoxic preconditioning against subsequent ischemic injury [15, 16]. This preconditioning model allows for the elucidation of subsequent survival pathways, which may serve as therapeutic targets for clinical intervention.

One of the most commonly used ROS for neuronal oxidative-stress preconditioning is H₂O₂ [17, 18]. H₂O₂ is formed by the dismutation of superoxide (O₂^•−) spontaneously or enzymatically in a reaction that is catalyzed by superoxide dismutase (SOD). In addition, many cell types produce H₂O₂ in response to growth factors such as VEGF, EGF, PDGF, and insulin that promote cell survival [19]. H₂O₂ is less reactive compared to other ROS, easily crosses membranes, and diffuses from its original site of production, all of which make H₂O₂ a candidate molecule for both inter- and intracellular signaling [20–23]. H₂O₂-mediated signaling alters the function of various proteins, including protein phosphatases, protein kinases, phospholipases, transcription factors, and ion channel proteins [24].

Exogenous H₂O₂ also activates Akt [25–27], which operates downstream of the PI3K cell survival pathway. Akt regulates downstream substrates such as Bcl-2 family proteins that mediate apoptosis. Bax is a pro-apoptotic member of the Bcl-2 family with three highly conserved BH domains (BH 1–3) and a hydrophobic C-terminal. The BH3 domain of the pro-apoptotic proteins is required for the translocation to and complex formation in, the mitochondrial membrane that leads to mitochondrial dysfunction and cell death [28–30]. Post-translational modifications (such as phosphorylation) of Bcl-2 members influences function and protein-protein interactions, and thus, play a major role in regulating cell fate. Indeed, phosphorylation abrogates Bax pro-apoptotic activity in human lung cancer [31] and in neutrophils [32].

In this study, we used SH-SY5Y human neuroblastoma cells as a model system for studying the molecular events regulating neuronal survival in response to H₂O₂. We tested the hypothesis that low levels of H₂O₂ regulate cell survival by altering the posttranslational modification of Bax through the PI3K/Akt pathway. We report that H₂O₂ protects SH-SY5Y cells from apoptosis and stimulates Akt phosphorylation in a PI3K-dependent manner. Preventing H₂O₂-induced PI3K activity with LY294002 results in Bax dephosphorylation and translocation to the mitochondria, which leads to cytochrome c release, caspase-3 activation, and apoptosis. These findings reveal a mechanism by which a H₂O₂- regulated signaling cascade promotes cell survival by inactivating a pro-apoptotic component of the cell death machinery.

Materials and Methods

Dulbecco’s modified Eagle’s medium (DMEM), Hank’s balanced salt solution (HBSS), trypsin-EDTA and calf serum (CS) were purchased from Gibco BRL (Gaithersburg, MD, USA). LY294002 was purchased from Calbiochem, Biosciences, Inc., (Santa Cruz, CA). Antibodies for Bax (Sc-7480, and Bax N-20) were purchased from Santa Cruz Biotechnology, Inc., CA; Akt, p-Akt (serine 473), and cleaved caspase-3 (Asp175) from Cell Signaling Technologies (Beverly, MA); phospho-serine (7F12) from Alexis Biochemical (San Diego, CA); Wortmannin and PP-2A/C were purchased from Calbiochem, cytochrome c from BD Pharmingen (San Diego, CA), and anti-oxphos complex IV, subunit I (anti-cytochrome oxidase, subunit I, Cox1), Alexa Flour 488, Alexa Flour 594, and DAPI were obtained from Invitrogen (Carlsbad, CA). Horseradish peroxidase-conjugated polyclonal goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology, Inc, (Santa Cruz, CA). Enhanced chemiluminescence system using LumiGLO^™ reagents were purchased from Cell Signaling, and molecular weight standards from Amersham (Arlington Heights, IL, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell Culture

SH-SY5Y human neuroblastoma cells were cultured in DMEM with 10% CS at 37°C in a humidified atmosphere containing 10% CO₂ as described previously [33]. In all conditions, SH-SY5Y cells were seeded in 100 mm culture dishes at an initial density of 5 × 10⁶ cells/cm² and grown to approximately 80–90% confluency. Cells were serum starved for 4 h prior to treatments and then sub-cultured in serum-free DMEM for the specified times and experimental conditions. Previous work in our laboratory [34–37] and others [25] established that 20 μM LY294002 is not toxic to the cells and it effectively blocks the PI3K pathway in SH-SY5Y cells [34–37]. Cells were pre-treated with 20 μM LY294002, or Wortmannin (100 nM, and 1 μM) for 1 h then rinsed with fresh serum-free DMEM prior to treatment with a single bolus addition of 0.1 mM H₂O₂. Cells were collected at the end of 4 or 24 h treatments for subsequent immunoprecipitation, Western blotting, immunocytochemistry, or flow cytometry analyses.

Immunoprecipitation and Western Blot Analysis

SH-SY5Y cells were washed with HBSS and solubilized in ice-cold lysis buffer, containing 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 150 mM NaCl, 1 mM phenylmethyl-sulfonyl fluoride (PMSF), 10 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mM sodium orthovanadate. Lysates were collected, sonicated briefly, centrifuged for 10 min at 4°C, and protein concentration determined using the Lowry method. Proteins were first immunoprecipitated, using an anti-Bax antibody (1:1000), or p-serine antibody (1:1000), as described previously [38], and then subjected to Western blot analysis. In the phosphatase experiment, PP-2A/C was added and incubated for 30 minutes. For Western blot analysis, samples were boiled in sample buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 20 mM DTT, 2% SDS, 20% glycerol, and 0.1% bromophenol blue), and loaded on 12.5% SDS-polyacrylamide gels (SDS-PAGE), followed by transfer to nitrocellulose membranes. Membranes were blocked in 5% non-fat milk in TBS-T (20 mM Tris, 0.16 M NaCl, and 0.10% Tween-20, pH 7.4) for more than 1 h at room temperature, and incubated overnight (at 4°C) with primary antibodies (Akt, p-Akt, Bax, and p-serine, 1:1000) in 5% non-fat milk in TBS-T. Membranes were then washed with TBS-T (3 times, 10 min each), and incubated with secondary goat anti- rabbit HRP (1:1000), or goat anti- mouse HRP antibody (1:2000) at room temperature for 1 h in 5% non-fat milk in TBS-T. An enhanced chemiluminescent (ECL) detection system was used according to the manufacturer’s protocol, and immunoblots were exposed to autoradiography film (Hyperfilm-ECL, Amersham Pharmacia Biotech). In some experiments, blots were stripped by incubation in buffer containing 2% SDS, 0.1 M Tris pH 6.8, 0.1 M DTT, and probed using a different antibody.

Immunocytochemistry

SH-SY5Y cells were seeded at a density of 5 × 10⁶ cells/cm² onto 25 mm glass coverslips arranged within the 100 mm culture dishes in order to maintain consistent culture conditions. Cells were serum starved for 4 h and exposed to experimental conditions as described previously. Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Coverslips were stained with DAPI and antibodies against Bax N-20 (1:100), Cox1 (1:50), cytochrome c (1:100), or cleaved caspase-3 (1:100) according to the experimental conditions described in the results. Samples were examined using a Nikon Diaphot 200 microscope with a 40 X objective lens. Digital images were captured with a Hamamatsu ORCA-ER CCD camera using Simple-PCI software (Compix Inc.).

Flow Cytometry

Analysis of DNA content was performed using flow cytometry as described previously [33]. After treatment, both floating and adherent (detached by trypsin-EDTA) cells were collected and rinsed in HBSS, fixed in ice cold 70% ethanol, and stored at 4°C prior to staining with 18 μg/ml propidium iodide and 40 μg/ml RNase A. DNA content of the cells was measured and separated into phases of the cell cycle based on the propidium iodide fluorescence. Apoptotic cells characteristically contain fragmented DNA, which is evident as a sub-G_o peak on the cell cycle histogram. Analyses were performed by the University of Michigan Flow Cytometry Core Facility using an Epics flow cytometry system (Coulter Cytometry, Hialeah, FL). The data were analyzed using analysis of variance (ANOVA). All results are expressed as the mean percent cell death of 3 independent experiments ± the standard error of the mean (SEM).

RESULTS

Effect of Exogenous H₂O₂ on Akt Activation and Survival Signaling

Exogenous H₂O₂ mimics the effect of endogenous receptor-induced H₂O₂, and activates multiple kinases [25–27, 39, 40]. To assess the effect of H₂O₂ on Akt activation, cells were exposed to 0.1 mM H₂O₂ for indicated times and lysates analyzed by Western blotting. Under our culture conditions, the intracellular concentration of H₂O₂ after a single bolus addition of 0.1 mM H₂O₂ is low [41]. We chose 0.1 mM H₂O₂, a condition that activates signaling but dose not lead to apoptosis. Treatment of SH-SY5Y cells with 0.1 mM H₂O₂ induced Akt phosphorylation (Ser 473) (Figure 1) with no change of Akt protein levels. Phosphorylation of Akt increased between 1 and 4 h of H₂O₂ exposure (Figure 1A). To assess the involvement of the PI3K pathway in H₂O₂–induced Akt activation, cells were pretreated with the PI3K inhibitor, LY294002. Pre-treatment of cells with LY294002 (20 μM) for 1 h prior to H₂O₂ treatment prevented H₂O₂-induced Akt phosphorylation at 4 h (Figure 1B) and 24 h (Figure 1C); data are representative of three independent experiments.

Fig. 1.

H₂O₂ stimulates phosphorylation of Akt in SH-SY5Y Cells. (A) Serum starved SH-SY5Y cells were treated with 0.1 mM H₂O₂ for the indicated times. Whole cell lysates were analyzed by Western blotting, using an anti-phospho-Akt antibody (1:1000, upper panel). Blots were then stripped and blotted for Akt protein (1:1000, lower panel). Cells were pre-treated with or without 20 μM LY294002 for 1 h, followed by H₂O₂ treatment for 4 h (B) or for 24 h (C) and immunoblotted for phospho-Akt as indicated (upper panels). Blots were then stripped and blotted for Akt protein (lower panels). Data are from one of three representative experiments.

Previous work in our laboratory has shown that the PI3K/Akt pathway is critical for cellular protection against apoptosis [42, 43]. To assess the effect of H₂O₂ –mediated PI3K/Akt-activity on survival, serum-starved cells with no treatment (control) were compared with serum-starved cells treated with H₂O₂ in the presence or absence of the PI3K pathway inhibitor LY294002. We acknowledge that H₂O₂ is not present at the end point of our experiments (24 h), however, we intended to show that a low dose of H₂O₂ protected cell death even after 24 h. Flow cytometry was used to quantitate the percentage of cells undergoing apoptosis as previously described [34, 44–47]. Treatment of cells with LY alone had no effect on the percentage of cell death when compared to untreated control cells (data not shown). H₂O₂ significantly protected SH-SY5Y cells from serum-deprivation-induced apoptosis measured by flow cytometry (Figure 2, p < 0.05). However, the PI3K inhibitor LY294002 prevented the H₂O₂ induced protection (Figure 2, p < 0.01). Our findings show that blocking basal levels of PI3K is not sufficient to block cell death induced by serum starvation (as shown in the control condition), however, H₂O₂ suppresses apoptosis via the PI3K pathway, confirmed by the fact that LY blocked the effects of H₂O₂.

Fig. 2.

Effect of H₂O₂ on cell death. Serum deprived SH-SY5Y cells with no treatment for 24 h (control), and treated with 0.1 mM H₂O₂ for 24 h without LY pre-treatment (H₂O₂) or with pre-treatment with 20 μM LY294002 (LY+H₂O₂). DNA was stained with propidium iodide and DNA content was measured by flow cytometry. Percent cell death is shown as mean ± S.E.M. for four separate experiments. Treatment of the serum-starved cells with H₂O₂ alone was significantly different from control (*, p < 0.05). Serum-starved cells with pre-treatment with LY and then H₂O₂ addition (LY+H₂O₂) were significantly different from both control and from H₂O₂ alone (*, p < 0.01).

H₂O₂-induced Bax Phosphorylation Depends on PI3K

Previous reports suggest that Bax is inactivated through serine phosphorylation [31, 32]. Therefore, we examined how PI3K/Akt signaling alters Bax phosphorylation in cells exposed to a low level of H₂O₂. SH-SY5Y cells were treated with 0.1 mM H₂O₂ for various times, and Bax protein was detected by Western blotting. As shown in Figure 3A′, total Bax protein levels did not change during the time course of H₂O₂ treatment. Bax is immunoprecipitated, and an equal amount of sample was used for immunoblotting. Bax protein level was determined by immunoblotting for Bax (Fig. 3A) and Bax phosphorylation was determined by phosphoserine immunoblotting (Fig. 3B). Figure 3A shows that Bax protein level was not changed, while Bax phosphorylation increased (Fig. 3B). Additionally, increased Bax phosphorylation was confirmed by immunoprecipitation with an antibody to phosphoserine and subsequent immunoblotting for Bax (data not shown). Fig. 3B′ shows that inhibition of PI3K by LY blocked H₂O₂-induced Bax phosphorylation at 4 h. Inhibition of the PI3K pathway also blocked the H₂O₂ induced phosphorylation at 24h as shown in Figure 3C, and Bax protein level did not change (not shown). To further confirm Bax serine phosphorylation, we used protein phosphatase 2A (PP2A), which has recently being shown to dephosphosphorylate Bcl-2 proteins [48]. PP-2A decreased H₂O₂-induced Bax phosphorylation, as is shown in Fig. 3C (last lane). To further confirm the involvement of PI3K pathway, Wortmannin, another inhibitor of PI3K/Akt signaling pathway, was also examined. There was a dose dependent decrease in H₂O₂–induced Bax phosphorylation by Wortmannin. Wortmannin at 1 μM concentration decreased H₂O₂–induced Bax phosphorylation, while low dose Wortmannin(100 nM) had no effect (Fig. 3D). These results show that Bax phosphorylation is dependent upon PI3K activity in SH-SY5Y cells. For immunoprecipitation control, IP experiments were done without cell lysates, and no cross reactivity with the precipitating antibody was observed. IP efficiency was also confirmed by running supernatant, which collected after beads precipitation, and no bands were detected on the blots.

Fig. 3.

Bax phosphorylation in SH-SY5Y Cells. (A) Cells were treated with H₂O₂ for the indicated times. Cell lysates were immunoprecipitated with anti-Bax antibody (I.P.Bax) and then immunoblotted with Bax antibody. (A′) Whole cell lysates were analyzed by Western blotting, using Bax antibody. (B) Cells were treated with H₂O₂ for the indicated times. Cell lysates were immunoprecipitated with anti-Bax antibody (I.P.Bax) and then immunoblotted with anti-phosphoserine antibody. (B′) Cells were pre-treated with or without LY, and then treated with H₂O₂ for 4 h. Cell lysates were immunoprecipitated with Bax antibody and then immunoblotted with anti- phosphoserine antibody. (C) 24 h time course; Cell lysates from control (no treatment), H₂O₂ alone, LY + H₂O₂, and H₂O₂ + PP-2A/C were immunoprecipitated with anti- phosphoserine antibody, and then immunoblotted with Bax antibody. (D) Cells were pre-treated with or without Wortmannin (W), and then treated with H₂O₂ for24 h. Cell lysates were immunoprecipitated with anti- phosphoserine antibody and then immunoblotted with Bax antibody.

Inhibition of PI3K Causes Bax Activation and Translocation to the Mitochondria

Bax activation and localization are involved in apoptosis [49, 50]. Upon stimulation, Bax undergoes conformational rearrangement, resulting in activation and translocation to the mitochondria [51]. Therefore, we examined Bax intracellular localization following H₂O₂ treatment (Figure 4). SH-SY5Y cells were pretreated + LY294002 for 1 h and then treated with 0.1 mM H₂O₂ for 24 h, followed by immunostaining with N-20 to detect active Bax, Cox1 to identify mitochondria, and DAPI to label nuclei (Figure 4). Bax was diffusely expressed in the cytoplasm of the healthy SH-SY5Y cells and showed punctate staining in apoptotic cells. Bax remained cytoplasmic following treatment with 0.1 mM H₂O₂ for 4 h (data not shown) or 24 h (Figure 4D–4F) and DAPI staining confirmed intact nuclei (Figure 4D). Inhibition of PI3K by pretreatment of cells with 20 μM LY294002 for 1 h resulted in Bax translocation from the cytoplasm to the mitochondria (Figure 4G–4I).

Fig. 4.

Bax translocation to the mitochondria following PI3K inhibition. (A–C) Control SH-SY5Y cells; (D–F) cells treated with 0.1 mM H₂O₂ for 24 h; (G–I) cells pre-treated with PI3K inhibitor (20 μM of LY294002, LY) and then treated with 0.1 mM H₂O₂ for 24 h (LY + H₂O₂). Cells were double immunostained with Bax (N-20, red), Cox1 (green) and nuclei were visualized by DAPI (blue). Asterisks indicate cells in inserts. Normal healthy control cells are shown in A and C. In H₂O₂ only treatments (D–F), cells are healthy with intact nuclei and diffuse Bax staining in the cytoplasm. PI3K inhibition resulted in punctate Bax staining (G) and Bax translocation to mitochondria (H) as is evident by yellow signal in the merged image (I). Bars = 10 μm.

PI3K Inhibition Induces Cytochrome c Release into the Cytoplasm and Caspase-3 Activation

Mitochondrial Bax triggers cytochrome c release from mitochondria, resulting in apoptosome formation, caspase activation, and apoptosis [52, 53]. Therefore, we next investigated cytochrome c release (Figure 5) in SH-SY5Y cells following H₂O₂ treatment with or without pre-treatment of LY294002. In healthy cells, cytochrome c staining was punctate (Figure 5A) and cells contained intact nuclei (Figure 5B). Treatment with 0.1 mM H₂O₂ alone for 24 h did not change cytochrome c localization; healthy cells are shown with punctuate cytochrome c (Figure 5C) and intact nuclei (Figure 5D). However, pre-treatment of cells with LY294002 for 1 h resulted in cytochrome c release into the cytoplasm (Figure 5E) and condensed nuclei typical of apoptosis (Figure 5F).

Fig. 5.

Cytochrome c release into the cytoplasm following PI3K inhibition. (A, B) Control cells; (C, D) cells treated with 0.1 mM H₂O₂ for 24 h; (E, F) cells pre-treated with 20 μM of LY294002 (LY), followed by 0.1 mM H₂O₂ treatment for 24 h (LY + H₂O₂). Cells were immunostained with cytochrome c (green) and nuclei were visualized by DAPI staining (blue). Asterisks indicate cells in inserts. Healthy cells (A, B and C, D) show punctate cytochrome c staining. Apoptotic cells with diffused cytochrome c (E), and condensed nuclei (F) are shown by red arrowheads. Bars = 10μm.

To investigate whether cytochrome c release results in caspase-3 activation, control and H₂O₂ treated (± LY294002) SH-SY5Y cells were stained with an antibody to detect cleaved caspase-3 fragments. Caspase-3 was not activated by H₂O₂ (0.1 mM) treatment alone and DAPI staining confirmed that the nuclei are intact and healthy (Figure 6D–6F). However, pre-treatment with LY294002 (20 μM, 1 h) resulted in caspase-3 cleavage (Figure 6G) and condensed nuclei (Figure 6H–I).

Fig. 6.

Inhibition of PI3K pathway lead to caspase-3 activation. (A–C) Control; (D–F) SH-SY5Y cells treated with 0.1 mM H₂O₂ for 24 h; (G–I) 20 μM of LY294002 (LY) followed by 0.1 mM H₂O₂ for 24 h (LY + H₂O₂). Cells were immunostained with cleaved caspase-3 (red), and nuclei were visualized by DAPI staining (blue). Caspase-3 activation is shown by the presence of red staining in G. Red arrowheads indicate cleaved caspase-3 staining in cells with condensed nuclei (I). Merged fluorescence signals are overlaid onto phase images (C, F, I) Bars = 10μm.

DISCUSSION

The current study demonstrates that H₂O₂–mediated Akt activation, downstream of PI3K, results in Bax phosphorylation and inactivation. Phosphorylation of Bax regulates cell survival by preventing Bax translocation to the mitochondria. Inhibition of the PI3K pathway by LY294002 results in Bax dephosphorylation and activation, followed by Bax translocation to the mitochondria, leading to cytochrome c release, caspase-3 activation and cell death.

Exogenous H₂O₂ increases Akt phosphorylation via the PI3K pathway in neuroblastoma cells. Involvement of PI3K in Akt phosphorylation was confirmed when the PI3K inhibitor LY294002 blocked Akt phosphorylation. Increasing evidence suggests that ROS, H₂O₂ in particular, mediate protein phosphorylation [13, 19, 25, 54–56], and H₂O₂–mediated Akt phosphorylation is reported in vascular smooth muscle cells [26], and in different cell types such as HeLa cells, epithelial cancer cells, and fibroblasts [25]. Our findings support the concept that protein phosphorylation is redox sensitive and is altered by ROS generation [1, 57–59]. Since the PI3K/Akt pathway is known to be important for cell survival in multiple systems [25, 60–62], we further examined cell survival in our system.

We show that mild oxidation induced by low levels of H₂O₂ promotes cell survival, and correlates with Akt activation. However, inhibition of the PI3K pathway and Akt down-regulation enhances cell death in SH-SY5Y cells. These findings are in agreement with other reports that Akt activation inhibits the apoptosis induced by serum withdrawal in lymphoid cells, fibroblasts, and neurons [63–67]. To characterize the mechanism of cell survival in our system, we examined downstream events of Akt activation. It is established that Bcl-2 family proteins, downstream of PI3K/Akt, are the key regulators of cell fate. In this regard we further investigated the effect of H₂O₂–mediated Akt upregulation on the proapoptotic protein Bax. Specifically, we examined whether Bax phosphorylation and activation is regulated by Akt.

The novel finding reported in this study is that H₂O₂ regulates Bax phosphorylation downstream of the PI3K/Akt pathway and contributes to cell survival. This association between H₂O₂-induced Akt activation and Bax phosphorylation, and suppression of apoptosis has not previously been reported. We show that H₂O₂-induced Bax phosphorylation is blocked by PI3K inhibitor LY294002, confirming that PI3K/Akt signaling regulates Bax phosphorylation. Recent studies indicate that Bcl-2 family proteins, downstream of PI3K/Akt, play a vital role in regulating cellular survival/death at the mitochondrial level [44, 50, 68–72]. Nechushtan and colleagues reported that structural re-arrangement in the C-terminal region of Bax is responsible for Bax activation [73]. It has also been shown that the phosphorylation state of Bax is a critical regulator of survival in human lung cancer cells [31] and neutrophils [32]. Our findings highlight a critical role for the PI3K/Akt pathway in regulating Bax activity and promoting cell survival following mild oxidation by H₂O₂. Taken together, these findings support our previous work regarding PI3K/Akt regulation of cell survival in neuronal cells [36, 62] and demonstrate that Bax plays a significant role in regulating neuronal death [50].

In this report, we demonstrate the involvement of PI3K signaling in Bax regulation and suppression of apoptosis. Following treatment with low levels of H₂O₂, Bax remains in the cytoplasm, however, inhibition of the PI3K pathway by LY294002 results in Bax activation and translocation to the mitochondria. Dephosphorylation of Bax alters its structure and allows its insertion into the mitochondria. Our study supports previous findings of our laboratory [50] and others [51] that activated Bax translocates to the mitochondria. Bax is a key regulator of mitochondrial integrity and alters mitochondrial membrane stability [74]. Some studies suggested that mitochondrial translocation of Bax creates pores in the outer membrane of mitochondria that allow cytochrome c release into the cytosol [52]. Therefore, we examined whether Bax translocation to the mitochondria results in cytochrome c release in SH-SY5Y cells.

In our study, cytochrome c is retained in the mitochondria after exposure of the cells to a low level of H₂O₂. However, PI3K inhibition resulted in diffused cytochrome c staining, an indication of cytochrome c release from the mitochondria into the cytoplasm. Cytochrome c is a peripheral protein of the mitochondrial inner membrane, which is released into the cytosol after mitochondrial damage. Our findings support the concept that activated Bax is involved in a multi-step apoptotic pathway [49, 75] that results in the loss of mitochondrial membrane integrity and the release of apoptotic molecules such as cytochrome c [52, 76]. We further characterized the apoptotic pathway by examining caspase-3 activation.

In SH-SY5Y cells Bax translocation to the mitochondria and cytochrome c release was followed by caspase-3 activation. Our study confirms other findings that Bax activation and translocation induces apoptosis [49, 50]. Mitochondrial Bax triggers cytochrome c release from mitochondria, resulting in apoptosome formation, caspase activation, and apoptosis [52, 53, 77]. This is the final commitment to cell death in many neuronal and non-neuronal systems [78]. Activated caspase-3, is detected both in vitro, and in animal models of Alzheimer’s, Huntington’s and Parkinson’s disease [79–82]. Together, our findings support a model where translocation of activated Bax to the mitochondria is followed by cytochrome c release, caspase-3 activation, and apoptosis (Figure 7).

Fig. 7.

Activation of Akt by H₂O₂ regulates the activity of the pro-apoptotic Protein Bax. H₂O₂, that produces a mild oxidative environment, activates the PI3K/Akt survival pathway (blue arrows). When Akt is activated by H₂O₂, Bax is located in the cytoplasm in its phosphorylated (inactive) form and the cell survives (blue arrows). Inhibition of the PI3K pathway results in dephosphorylation of Bax and its translocation to the mitochondria (red arrows). Insertion of Bax into the mitochondria leads to cytochrome c release (possibly through Bax pore formation), caspase-3 activation, and apoptosis (red arrows). Abbreviations: MOM, mitochondrial outer membrane; MIM: mitochondrial inner membrane; MIS, mitochondrial intermembrane space.

In summary, we demonstrated that the cell survival/death pathway is redox sensitive and confirmed a stepwise mechanism by which H₂O₂-induced stimulation of Akt promotes survival through posttranslational modification of the proapoptotic protein Bax. Our findings show that a low level of H₂O₂ activates Akt via the PI3K pathway and induces Bax phosphorylation, and as a result Bax remains in the cytoplasm in its inactive conformation. However, inhibition of the PI3K pathway results in dephosphorylation of its downstream proteins Akt and Bax, resulting in Bax translocation to the mitochondria, cytochrome c release, and caspase-3 activation (as illustrated in Figure 7). Further studies will address whether post-translational modification of Bax alters its protein-protein interactions with other Bcl-2 family members, or mitochondrial proteins. These interactions will impact cell survival through regulation of mitochondrial function. The strong association between cellular redox state and the regulation of apoptosis is an exciting new area of investigation. Insight into these mechanisms could yield novel therapeutic strategies to regulate cell survival for many disorders that involve the dysregulation of apoptosis, including cancer and neurodegenerative diseases.

Abbreviations

DMEM

Dulbecco’s Modified Eagle’s Medium

DTT

dithiothreitol

EDTA

ethylene diamine tetra acetic acid

H₂O₂

hydrogen peroxide

PI-3K

phosphatidylinositol 3-kinase

ROS

reactive oxygen species

SDS- PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

S.E.M

standard error of the mean