Perfluorooctane sulfonate induces apoptosis via activation of FoxO3a and upregulation of proapoptotic Bcl-2 proteins in PC12 cells

Abstract

Perfluorooctane sulfonate (PFOS), a kind of organic pollutant widely found in the environment and biota, could alter normal brain development and produce cognitive dysfunction. For the past years, the neurotoxic effects of PFOS have been shown. Recent studies have proven that PFOS can induce neuronal apoptosis and cause neurotoxicity, but the regulatory proteins referred to the process have not been clarified. In this study, PC12 cells were used to investigate the changes of the expression of apoptosis-related proteins, forkhead box O3 (FoxO3a) and pro-apoptotic Bcl-2 proteins. We detected that the levels of cleaved caspase-3 and cleaved PARP were up-regulated obviously in PFOS-treated PC12 cells by using Western blotting, and that the apoptotic rate of PC12 cells was increased significantly by using flow cytometry, verifying that PFOS could induce neuronal apoptosis. Western blot analysis and immunofluorescence revealed obvious up-regulation of the expression of FoxO3a and proapoptotic Bcl-2 proteins. In addition, knockdown of FoxO3a gene inhibited Bim expression and apoptosis. According to the data, we believe that FoxO3a may play a crucial role in PFOS-induced neurotoxicity.

INTRODUCTION

Perfluorooctane sulfonate (PFOS) is one of the most important members of the perfluorinated compounds family, which has been used widely in industrial and agricultural production and daily life because of its unique chemical properties (Renner, 2001; Lehmler, 2005). PFOS can adhere to various protein molecules in the liver, kidney and blood, and accumulate in the organism for a long time (Conder et al., 2008), which causes diversiform toxic effects in animals and humans, such as hepatotoxicity, immunotoxicity, reproductive toxicity, developmental toxicity and even carcinogenic effects (Ankley et al., 2005; Butenhoff et al., 2012; Dong et al., 2012; Qazi et al., 2013). On account of its biological accumulation and potential toxicity, PFOS was included among persistent organic pollutants (POPs) in 2009. Several recent studies have proven that PFOS in animal blood, liver and kidney can cross the blood-brain barrier and accumulate in the brain (Broadbent et al., 2004; Chang et al., 2009; Sato et al., 2009). PFOS exposure influences the expression of proteins associated with brain development and causes irreversible neuronal damage and neurological deficits (Johansson et al., 2008, 2009). In addition, studies have shown that PFOS induced the apoptosis of SH-SY5Y cells and astrocytes, indicating that PFOS has significant neurotoxicity (Chen et al., 2014; Dong et al., 2015). Nevertheless, the specific mechanism of neurotoxicity of PFOS remains unknown.

The Forkhead box O (FoxO) family is considered to be a key transcription factor involved in the regulation of intracellular events, including cellular metabolism, proliferation, differentiation, apoptosis, stress resistance and aging (Weigel et al., 1989; Accili and Arden, 2004). Forkhead box O3 (FoxO3a) transcription factor has a unique and critical role in these aspects (Nho and Hergert, 2014). Under stress conditions, FoxO3a gives play to neuroprotective effects by inducing stress resistance, thereby preventing the occurrence of motor neuron disease (Mojsilovic-Petrovic et al., 2009). For instance, in Parkinson’s disease, FoxO3a is essential for the neuroprotective effect of erythropoietin (EPO) through the AKT signaling pathway (Ghosh Choudhury et al., 2003). However, studies also have found that FoxO3a can cause neuronal death by promoting the transcription of apoptotic proteins under many pathological conditions, leading to neurotoxicity (Li et al., 2009; Xu et al., 2014). Studies have shown that FoxO3a can regulate apoptotic protein Bim in specific circumstances (Gilley et al., 2003; Herold et al., 2013). The transcription factor FoxO3a is combined with the promoter of Bim to regulate Bim expression. Bim is one of the BH3-only Bcl-2 family members, and it can antagonize all the antiapoptotic Bcl-2 proteins (Chen et al., 2005). The upregulation of Bim expression induced mitochondria to release cytochrome C and activated caspase proteins, leading to apoptosis (Gilley et al., 2003). Furthermore, Bim was also indicated to be able to directly trigger Bax / Bak to release cytochrome C, inducing apoptosis (Kim et al., 2006). In addition, studies have found that PFOS-induced astrocyte proinflammatory responses may lead to adverse effects of PFOS on the central nervous system (Chen et al., 2018). Studies have also shown that PFOS promotes the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in HBMEC (Wang et al., 2011). However, whether FoxO3a plays a similar role in the process of PFOS-induced neuronal apoptosis and neurotoxicity is still not clear.

In the process of PFOS-induced neuronal apoptosis, whether FoxO3a regulates pro-apoptotic Bcl-2 proteins is unknown. To determine the effect of FoxO3a and proapoptotic Bcl-2 proteins in PFOS-induced neuronal apoptosis, we constructed an in vitro model.

MATERIALS AND METHODS

Chemicals

The following chemicals were bought from their providers: PFOS (potassium salt, purity 98%) (Sigma Aldrich, St. Louis, MO, USA); DMSO (Sigma Aldrich).

Cell culture and treatment

The undifferentiated Rat pheochromocytoma (PC12) cells were obtained from the China Academy of Sciences (Shanghai, China), and grown in 1 g/L glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan City, UT, USA) with 10% fetal bovine serum (FBS; Gibco, California, USA), 5% horse serum, and glutamine (Sigma Aldrich). Cells were maintained in a humidified incubator of 95% O2 and 5% CO2 at 37°C. After 1 day of incubation, the medium was replaced with nerve growth factor (NGF; Sigma Aldrich)-containing medium. The cells were cultured in a cell bottle for several days, during which cell culture medium was changed every other day, and the cells were subcultured until the cells were fully differentiated. The differentiated PC12 cells were treated with 0.05%DMSO or different concentrations of PFOS (10, 50, 100, 150 and 200 μM) for 48 hr, or with 100 μM PFOS for 0, 12, 24, 36, 48 hr (Liao et al., 2009; Liao et al., 2008). PFOS was dissolved in DMSO. The differentiated PC12 cells were transiently transfected with non-specific siRNA or Foxo3a siRNA before PFOS exposure.

Cell counting kit-8 (CCK-8) assay

PC12 cells were seeded into 96-well plates at a density of 1.0 × 10³ cells/well, and cultured overnight in a cell incubator. Cells were stimulated respectively with DMSO or different doses of PFOS (10, 50, 100, 150 and 200 μM), and then each group was divided into different points in time (0, 12, 24, 36, 48 hr). Next, we added 100 μL of CCK-8 (Dojindo, Kumamoto, Japan) reagent dilution (CCK8 / DMEM: 1/9) in each well of the plates and incubated for two hours at 37°C in an incubator. The OD value was measured by the spectrophotometer (450 nm). After that, the viability of PC12 cells was assessed according to the specification of the CCK-8 assay. An assay was implemented using triplicate independent cell cultures.

Western blot analysis

Cells were fleetly washed with ice-cold PBS, then lysed with the sodium lauryl sulfate loading buffer. Subsequently, the protein samples were collected. The concentration of each protein sample was mensurated by Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). The protein samples were loaded directly onto a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrophoresis and transfer membrane. Immediately, the protein membrane was placed in a pre-prepared closed solution (bovine serum albumin (BSA) or 5% skim milk in PBS containing 0.5% Tween 20 (PBS-T)), and shook slowly on the table concentrator at room temperature for 2 hr. Next, the membrane was incubated with primary antibody overnight at 4°C: rabbit monoclonal anti-caspase-3 (1:1000; Cell Signaling, Boston, USA), rabbit monoclonal anti-PARP (1:1000; Cell Signaling), GAPDH (1:1,000; Sigma Aldrich), rabbit anti-foxO3a (1:1000; Cell Signaling), Bak (1:1000; Santa Cruz, CA, USA), Bax (1:1000; Santa Cruz), Bim (1:1000; Santa Cruz). After washing with PBS-T buffer three times, the membrane was incubated with secondary antibody (Pierce Company, Massachusetts, USA) at room temperature for 2 hr. Finally, the membrane was developed using an enhanced chemiluminescence system (Santa Cruz).

Flow cytometry

PE Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lake, NJ, USA) was used to confirm PFOS-induced apoptosis in PC12 cells by flow cytometry. Treated cells were collected, washed twice with ice-cold PBS and then resuspended in 100 µL binding buffer. Afterwards, 5 µL PE Annexin V and 5 µL 7-Amino-Actinomycin (7-ADD) were added to the 100 µL binding buffer, and then incubated for 15 min at room temperature in the dark. Subsequently, 400 µL binding buffer was added to the sample. Eventually, the cells were analyzed using flow cytometry analyzer (BD Biosciences) and Cell Quest software (BD Biosciences).

Immunofluorescent staining

Small discs of planted with cells were reaped after treatment in use or not of PFOS or FoxO3a siRNA (small interfering RNA (siRNA) against FOXO3a was purchased from Santa Cruz Biotechnology, Inc. The sequences were as follows: 5′-GCUGUCUCCAUGGACAAUATT-3′ and 5′-CCACCCGAAUUGGCAGAUUTT-3′.) for 48 hr, fixed on the slide and implemented to immunofluorescence. Concisely, the harvested small discs were washed using ice-cold PBS and fixed with 4% paraformaldehyde for 15-20 min. Then the small discs were washed with ice-cold PBS and permeabilized with 0.1% Triton X-100 for 10 min, subsequently preincubated with 5% BSA for 60 min. Small discs were stained with rabbit anti-FoxO3a (1:100; Cell Signaling), Bim (1:100; Santa Cruz), followed by incubation with secondary antibody (1:250; Santa Cruz). Finally, the cells were observed and analyzed with an inverted confocal laser fluorescence microscope (Leica, Microsystems, GmbH, Weztlar, Germany).

Small interfering RNA (siRNA) and transfection

Cells were transfected with FoxO3a siRNA and non-specific siRNA using Lipofectamine 2000 (Invitrogen, Shanghai, China) according to the manufacturer’s specification. Then the cells were stimulated with 100 μM PFOS after 48 hr of transfection.

Statistical analysis

Each experiment was repeated at least three times, and then data were presented as mean ± SD for multiple independent experiments. The data were analyzed using SPSS 20.0 software. The relative expression levels of two groups were analyzed using the unpaired T test. The relative expression levels of more than two groups were analyzed using one-way analysis of variance. P value of less than 0.05 were considered statistically significant.

RESULTS

Effects of PFOS on PC12 cells viability and morphological changes

To investigate the effect of PFOS on cell viability and morphology, PC12 cells were treated with different concentrations of PFOS (0, 10, 50, 100, 150, and 200 μM) for different times (0, 12, 24, 36, 48 hr). As shown in Fig. 1A, CCK-8 assay showed PFOS treatment led to gradually declined activity in PC12 cells in a dose- and time-dependent manner. Subsequently, PC12 cells were exposed to different concentrations of PFOS (0, 10, 50, 100, 150, and 200 μM) for 48 hr. It was observed that, with the increasing concentration of PFOS, the volume of PC12 cells gradually increased, synaptic retraction and reduction, and even became vacuolated (Fig. 1B). Meanwhile, the viability of PC12 cells treated with 100 μM PFOS for 48 hr was less than 60%. Therefore, we believed that the IC50 concentration of PFOS is 100 μM and the optimal time point is 48 hr.

Fig. 1

Effects of PFOS on PC12 cell viability and morphological changes. A) PC12 cells were stimulated with different concentrations of PFOS (0, 10, 50, 100, 150, and 200 μM) for different times (0, 12, 24, 36, 48 hr), and the activity of PC12 cells was detected by the CCK-8 assay. B) PC12 cells were treated with different concentrations of PFOS (0, 10, 50, 100, 150, and 200 μM) for 48 hr and then their morphology was observed by light microscope (× 200).

PFOS-induced PC12 cell apoptosis

To verify that PFOS could induce apoptosis in PC12 cells. PC12 cells were exposed to different concentrations of PFOS (0, 10, 50, 100, 150, and 200 μM) for 48 hr. PC12 cells exposed to PFOS demonstrated that a significant increase in cleaved casepase3, accompanied by a decrease in caspase3 in a dose-dependent manner by Western blotting (Fig. 2A). Consistent with the previous results, the level of cleaved PARP which is an apoptotic marker was visibly up-regulated, accompanied by down-regulation in PARP (Fig. 2B). Furthermore, we also detected that apoptotic indexes were significantly increased in a dose-dependent manner by flow cytometry analysis with PE Annexin V/7-Amino-Actinomycin (7-ADD) double staining (Fig. 2C). These results suggested that PFOS induces PC12 cell apoptosis.

Fig. 2

PFOS-induced PC12 cell apoptosis. PC12 cells were exposed to different concentrations of PFOS (0, 10, 50, 100, 150, and 200 μM) for 48 hr. A) Levels of caspase3 and cleaved caspase3 were detected using Western blot analysis. Histograms of the ratio of caspase3 and cleaved caspase3 to GAPDH at different concentrations of PFOS. B) Levels of PARP and cleaved PARP were detected using Western blot analysis. Histograms of the ratio of PARP and cleaved PARP to GAPDH at different concentrations of PFOS. C) The apoptotic percentage of PC12 cells was analyzed by flow cytometry analysis. Histograms of the percentage of apoptotic cells at different concentrations of PFOS. The data are presented as means ± SD. n = 3, *P < 0.05, **P < 0.01, significantly different from control.

PFOS-induced FoxO3a and proapoptotic Bcl-2 proteins expression

To elucidate the function of PFOS in neuronal apoptosis, PC12 cells were processed with different concentrations of PFOS (0, 10, 50, 100, 150, and 200 μM) for 48 hr. The expression of FoxO3a and pro-apoptotic Bcl2 proteins (Bak, Bax, Bim) were up-regulated in a dose-dependent manner in PC12 cells. Also, their protein levels were significantly higher in PC12 cells with PFOS treatment of not less than 100 μM than the control group (in PC12 cells the concentrations of PFOS not less than 100 μM than that in the control group) (Fig. 3). Furthermore, we implemented immunofluorescence assay to reveal the translocation of FoxO3a and the alter of Bim expression after PFOS exposure. Figure 4 shows that FoxO3a was transferred from the cytoplasm to the nucleus (a, d) and Bim staining was more pronounced (g, j) after 48 hr with 100 μM PFOS treatment. These results indicate that PFOS induces the expression of FoxO3a and pro-apoptotic Bcl2 proteins in PC12 cells.

Fig. 3

Effects of PFOS on FoxO3a and proapoptotic Bcl-2 protein expression. PC12 cells were treated with different concentrations of PFOS (0, 10, 50, 100, 150, and 200 μM) for 48 hr. A) Level of FoxO3a was detected using Western blot analysis. Histogram of the ratio of FoxO3a to GAPDH at different concentrations of PFOS. B) Levels of Bim, Bax and Bak were determined using Western blot analysis. Histograms of the ratio of Bim, Bax and Bak to GAPDH at different concentrations of PFOS. The data are presented as means ± SD. n = 3, *P < 0.05, **P < 0.01, significantly different from control.

Fig. 4

Effects of PFOS on FoxO3a and Bim. To observe the levels of FoxO3a and Bim by using a fluorescence microscopy. Immunofluorescence staining of FoxO3a (red; a and d) and Bim (green; g and j) in PC12 cells treated with 0 or 100 μM PFOS for 48 hr. Besides, the nucleus was stained with Hoechst (blue; b, e, h, and k). Scale bars: 50 μm.

Down-regulation of Bim expression and PFOS-induced cell apoptosis by knockdown of FoxO3a expression

The above experiments showed that the expression of FoxO3a and Bim were significantly altered in PFOS-induced apoptosis. Studies have shown that FoxO3a plays a crucial role in regulating cell proliferation, apoptosis and metabolism. To confirm the effect of FoxO3a on PFOS-induced apoptosis and Bim expression, PC12 cells were transfected with FoxO3a siRNA or non-specific siRNA, and then treated with 100 μM PFOS for 48 hr, we detected that the levels of FoxO3a and Bim were decreased in FoxO3a siRNA transfected PC12 cells, regardless of treatment with PFOS, using Western blot analysis (Fig. 5A). In addition, we found that PFOS-induced nuclear transport of FoxO3a was blocked by FoxO3a siRNA using immunofluorescence assay (Fig. 5C). Subsequently, we examined the effect of FoxO3a siRNA on apoptosis. PC12 cells were treated with the same procedures. Flow cytometry analysis showed that the apoptotic index of the FoxO3a siRNA group was significantly lower than the other groups (Fig. 5B). These results revealed that FoxO3a plays a critical influence on Bim expression and PFOS-induced PC12 cell apoptosis.

Fig. 5

Down-regulation of Bim expression and PFOS-induced cell apoptosis by knockdown of FoxO3a expression. PC12 cells were treated with different reagents (non-specific siRNA, FoxO3a siRNA, and treatment or untreatment with 100 μM PFOS). A) Levels of FoxO3a and Bim were measured by using Western blot analysis. Histograms of the ratio of FoxO3a and Bim to GAPDH at different groups. B) The apoptotic percentage of PC12 cells was analyzed by flow cytometry analysis. C) Immunofluorescence staining of FoxO3a (red; a, e and i), Bim (green; b, f and j) and Hoechst (blue; b, e, h and k) in treated PC12 cell. Scale bars: 50 μm. The data are presented as means ± SD. n = 3, *P < 0.05, **P < 0.01, significantly different from control.

DISCUSSION

PFOS was proved to be a new persistent environmental pollutant after polychlorinated biphenyl (PCB), organophosphorus pesticides (OPPs) and dioxin. PFOS induces neuronal apoptosis, and is neurotoxic [13]. However, the specific mechanism of PFOS-mediated neuronal apoptosis remains unclear. Our study demonstrated that PFOS up-regulated the expression levels of FoxO3a and pro-apoptotic Bcl-2 proteins, and led to neuronal apoptosis. In addition, we discovered that knockdown of FoxO3a expression restrained Bim transcription and neuronal apoptosis. In brief, these results demonstrated that PFOS-induced neuronal apoptosis is significantly associated with FoxO3a.

PFOS accumulates in the organism for a long time, because of its unique chemical properties (Conder et al., 2008). It has been found that PFOS in animal blood, liver and kidney can cross the blood-brain barrier and accumulate in the brain (Broadbent et al., 2004; Chang et al., 2009; Sato et al., 2009). PFOS in the brain affects the expression of proteins associated with brain development, and causes irreversible neuronal damage and neurological deficits (Johansson et al., 2008, 2009). For instance, PFOS exposure leads to spatial memory deficits in adult male mice (Fuentes et al., 2007); the mice were exposed to PFOS in the embryonic stage, occurring cognitive impairment after birth (Fuentes et al., 2007). Further research found that the long-term PFOS exposure in mice could down-regulate the expression of genes associated with long-term potentiation (LTP) (Lee et al., 2012), induce apoptosis of hippocampal cells (Long et al., 2013), and lead to neurological impairment of the hippocampus, which caused the defects of spatial learning and memory, cognition and other functions in the hippocampus (Lee et al., 2012; Long et al., 2013). Notably, studies have shown that neuronal apoptosis may also be involved in PFOS-induced hippocampal disorders (Lee et al., 2012). The current study confirmed that high concentration of PFOS induced apoptosis in PC12 cells; it may be an important mechanism that PFOS caused hippocampal disorders and neurotoxicity.

FoxO3a (Forkhead-like protein-1, FKH R-L1) gene is a transcription factor, found in recent years, and was extensively studied as one of the members of the O subfamily of Fox transcription factor. Studies have found that FoxO3a is widely expressed in the tissues and organs of the body, and plays an important role in neurobiology. Under stress conditions, FoxO3a gives play to neuroprotective effects by inducing stress resistance (Mojsilovic-Petrovic et al., 2009). For example, FoxO3a is essential for the neuroprotective effect of erythropoietin (EPO) through the AKT signaling pathway in Parkinson’s disease (Ghosh Choudhury et al., 2003). Nevertheless, studies also have found that FoxO3a induces neuronal death by promoting the transcription of apoptotic proteins, in many pathologic conditions, leading to neurotoxicity (Li et al., 2009; Xu et al., 2014). Studies have shown that FoxO3a induces apoptosis by regulating many downstream target genes: activating the caspase system through multiple pathways such as FasL and Bcl-2 (Le-Niculescu et al., 1999); reducing the expression of cyclin B / D2 by activating p27kip1, and inducing cell arrest in G1, G2 phase (Medema et al., 2000); moreover, the expression of the apoptotic protein Bim is increased rapidly (Obexer et al., 2007) and so on. Therefore, we investigated the effects of PFOS on FoxO3a expression, and FoxO3a on PFOS-induced neuronal apoptosis. In this study, FoxO3a expression was obviously up-regulated in a dose-dependent manner in PC12 cells (Fig. 3A). In addition, after knockout of the FoxO3a gene, we found that the percentage of apoptotic cells was significantly decreased (Fig. 5B). Therefore, we believe that PFOS-induced neuronal apoptosis is significantly related to the expression of FoxO3a.

Bim is a BH3-only protein; this BH3 domain is the region where Bim binds to the anti-apoptotic members of the Bcl-2 subfamily, and plays an important role in Bim-induced apoptosis (Sionov et al., 2015). The BH3-only pro-apoptotic proteins impact on the upstream of the multi-domain pro-apoptotic proteins, which have three BH domains (BH1, BH2 and BH3), such as Bak, Bax and Bok. Dual knockout of Bax and Bak usually leads to accomplishing resistance to apoptosis mediated by the intrinsic apoptotic pathway (Rathmell et al., 2002). Furthermore, Bim directly activates the pro-apoptotic molecules, and prompts it to insert the outer membrane of the mitochondria, the cytochrome C was released by altering the permeability of the outer membrane of the mitochondria, then cytochrome C and apoptotic protease activating factor-1 (Apaf1) formed apoptotic body, which acts on caspase, and generates the cascade reaction of caspase, leading to apoptosis (Huang and Strasser, 2000; Westphal et al., 2011). Studies have indicated that Bim expression is regulated by FoxO transcription factor in neurons (Gilley et al., 2003). Therefore, we investigated whether PFOS affects Bim expression, and the effect of FoxO3a on Bim expression. In our study, the expression of pro-apoptotic Bcl2 proteins was significantly increased after PFOS exposure (Fig. 3B). Moreover, we also found that Bim expression was reduced significantly after knockout of FoxO3a gene (Fig. 5A). These show that PFOS exposure up-regulated the expression of pro-apoptotic Bcl2 proteins, which was regulated by FoxO3a gene.

In short, our results suggested that the levels of FoxO3a and pro-apoptotic Bcl2 proteins were significantly increased after PFOS exposure in PC12 cells, and FoxO3a was involved in PFOS-induced PC12 cell apoptosis. Furthermore, our results also suggested that knockout of the FoxO3a gene protected neurons from PFOS-induced apoptosis. Taken together, our findings revealed the mechanism of PFOS-induced neuronal apoptosis, which may be helpful in preventing PFOS-induced neurological disorders.

ACKNOWLEDGMENTS

The present study was supported by the Six Talent Peaks Foundation [WSN-061], the Scientific Research Program of Jiangsu Province Health Department [H201423], the Science and Technology Program of Nantong City [MS22015071], the Maternal and Child Health Research Project of Jiangsu Provincial Health and Family Planning Commission [F201751].

Conflict of interest

The authors declare that there is no conflict of interest.

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