Cadmium induces mitochondrial ROS inactivation of XIAP pathway leading to apoptosis in neuronal cells

Abstract

Cadmium (Cd), a heavy metal pollutant, contributes to neurodegenerative disorders. Recently, we have demonstrated that Cd-induced reactive oxygen species (ROS) causes apoptosis in neuronal cells. Whether X-linked inhibitor of apoptosis protein (XIAP) is involved in Cd-induced ROS-dependent neuronal apoptosis remains unclear. Here, we show that Cd-induced ROS reduced the expression of XIAP, which resulted in up-regulation of murine double minute 2 homolog (MDM2) and down-regulation of p53, leading to apoptosis in PC12 cells and primary neurons. Inhibition of MDM2 with Nutlin-3a reversed Cd-induced reduction of p53 and substantially rescued cells from excess ROS-dependent death. Overexpression of XIAP protected against Cd induction of ROS-dependent neuronal apoptosis. Inhibition of XIAP by Embelin strengthened Cd-induced ROS and apoptosis in the cells. Furthermore, we found that Cd inactivation of XIAP pathway was attributed to Cd induction of mitochondrial ROS, as evidenced by using a mitochondrial superoxide indicator MitoSOX and a mitochondria-targeted antioxidant Mito-TEMPO. Taken together, these results indicate that Cd induces mitochondrial ROS inactivation of XIAP-MDM2-p53 pathway leading to apoptosis in neuronal cells. Our findings suggest that activators of XIAP or modulation of XIAP-MDM2-p53 pathway by antioxidants may be exploited for the prevention of Cd-induced oxidative stress and neurodegenerative diseases.

1. INTRODUCTION

Cadmium (Cd) is a well-known environmental toxic metals polluted in water, air and soil (Wang et al., 2013). People can easily expose to Cd through daily life, such as smoking and eating foods with excessive Cd (Huang et al., 2010; Perlman et al., 2012). A number of clinical reports show that Cd has toxic effects on many organs/tissues such as blood (Kocak et al., 2006), bone (Akesson et al., 2006), liver (Jomova et al., 2011), kidney (Johri et al., 2010), lung (Jiang et al., 2008), testis (Thompson et al., 2008), and brain (Lopez et al., 2003; Mendez-Armenta et al., 2007; Okuda et al., 1997), because of its long biological half-life (15 to 20 years). Cd can bind to proteins, such as albumin and certain immunoglobulins, in blood, forming metal-protein complexes, and then internalize and accumulate in neuronal cells by fluid phase endocytosis (Arvidson, 1994; Yamamoto et al., 1987). Cd can readily pass through the blood-brain barrier and accumulate in the brain, causing central nervous system (CNS) dysfunction, such as headache and vertigo, olfactory dysfunction, peripheral neuropathy, vasomotor function slowing of vasomotor functioning, equilibrium decreases, psychomotor speed, neurobehavioral defects in attention, and learning disabilities (Pihl et al., 1977; Wang, et al., 2013; Wright et al., 2006). Merging data have documented that Cd-evoked apoptotic cell death in PC12 cells, cortical neurons, anterior pituitary cells, and different brain regions is attributed to oxidative stress such as reactive oxygen species (ROS) (Chen et al., 2011; Chen et al., 2014; Jomova, et al., 2011; Lopez et al., 2006; Xu et al., 2016). Cd-elicited excessive ROS can directly damage cell proteins and nucleic acids, alter their functions, thereby disturbing related signaling pathways contributing to cell dysfunction and cell death (Genovese et al., 2008; Wang, et al., 2013). Therefore, ROS induced by Cd has been considered as a possible cause in the development of neuronal cell death and corresponding neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and Huntington’s disease (HD) (Goncalves et al., 2010; Monroe et al., 2006; Panayi et al., 2002; Wang, et al., 2013).

Inhibitor of apoptosis proteins (IAPs) is a family of caspase inhibitors to prevent cell from apoptosis (Schimmer, 2004). X-linked inhibitor of apoptosis protein (XIAP) is the most characterized member of IAPs. XIAP contains three zinc-binding baculovirus IAP repeat (BIR) domains, of which, BIR2 and BIR3 are responsible for binding and inhibiting caspase-3/7 and caspase-9, respectively (Deveraux et al., 1999; Takahashi et al., 1998). In addition, XIAP also has an additional zinc-binding motif, the really interesting new gene (RING) domain, containing ubiquitin E3 ligase activity that targets caspase-3 for degradation through proteasome (Suzuki et al., 2001). It is known that p53 acts as a tumor suppressor, orchestrating cell-cycle arrest, DNA damage and apoptosis, in response to a variety of stresses in cells, (D’Brot et al., 2017; Oren, 2003; Vaughn et al., 2007; Vousden et al., 2002; Xiong et al., 2015). Mouse double minute 2 homolog (MDM2), an E3 ubiquitin ligase, regulates the ubiquitination of p53, leading to its degradation by the proteasome (Fang et al., 2000; Ishizawa et al., 2018; Itahana et al., 2007). Recently, XIAP has been shown to be a novel E3 ubiquitin ligase of MDM2 in cancer cell lines, which down-regulates MDM2 by ubiquitination, thereby up-regulating p53 level (Huang et al., 2013). Interestingly, studies have also demonstrated that MDM2 promotes XIAP expression (Gu et al., 2016; Gu et al., 2009; Liu et al., 2015). These intriguing data suggest a complicated relationship between MDM2 and XIAP, which may impact cell fate depending on cell types and experimental conditions.

Recent studies have reported that Cd down-regulates XIAP and activates caspase-3, leading to apoptosis in prostate cancer cells (Golovine et al., 2010), Mytilus edulis’ hemocytes (Granger Joly de Boissel et al., 2017) and chicken ovarian follicles granulosa cells (Jia et al., 2011), but the role of XIAP in Cd-induced neuronal cell death has not been investigated. Our group has demonstrated that Cd-induced ROS causes neuronal apoptosis by caspase-dependent and -independent mechanisms (Chen et al., 2008a). Here we further studied whether XIAP is involved in Cd-induced ROS-dependent apoptosis of neuronal cells. Our results indicate that Cd inactivates XIAP-MDM2-p53 pathway by triggering mitochondrial ROS, leading to neuronal apoptosis.

2. MATERIALS AND METHODS

2.1. Reagents

Cadmium chloride, poly-D-lysine (PDL), 4′,6-diamidino-2-phenylindole (DAPI), N-acetyl-L-cysteine (NAC), glutathione (GSH), Nutlin-3a, and protease inhibitor cocktail were purchased from Sigma (St Louis, MO, USA), whereas Embelin was provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mitochondria-targeted antioxidant Mito-TEMPO was from ALEXIS Biochemicals (San Diego, CA, USA). 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) was purchased from MP Biomedicals (Solon, OH, USA). Dulbecco’s modified Eagle medium (DMEM), 0.05% Trypsin-EDTA, NEUROBASALTM Media, and B27 Supplement were purchased from Invitrogen (Grand Island, NY, USA). Horse serum and fetal bovine serum (FBS) were supplied by Hyclone (Logan, UT, USA). MitoSox was bought from YEASEN (Shanghai, China). CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit was from Promega (Madison, WI, USA). Other chemicals were purchased from local commercial sources and were of analytical grade.

2.2. Cell Culture

Rat pheochromocytoma (PC12) cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). Because of the replicative nature and cost-effectiveness, the cell line is widely used as neuronal cell models, so it was employed in this study. For culture, PC12 cells were seeded in a PDL (0.2 μg/ml)-coated 6-well or 96-well plate and cultured in antibiotic-free DMEM with supplements (10% horse serum and 5% FBS). To verify the data observed from PC12 cells, primary neurons were also used in this study. For this, primary murine neurons were isolated from fetal mouse cerebral cortexes of 16–18 days of gestation in female ICR mice (being pregnant) as described (Chen et al., 2010), and sowed in a PDL (10 μg/ml)-coated 6-well or 96-well plate for experiments after 6 days of culture. The cells were maintained at 37°C in 5% CO₂ atmosphere. All procedures used in this study were approved by the Institutional Animal Care and Use Committee, and were in compliance with the guidelines set forth by the Guide for the Care and Use of Laboratory Animals.

2.3. Lentiviral Cloning, Production, and Infection

To make FLAG-tagged wild-type XIAP (FLAG-XIAP) construct (for XIAP overexpression) and EGFP construct (for control), PCR template for XIAP or EGFP was from PC12 cells’ cDNA generated by RT-PCR using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Bio, Kusatsu, Japan) and plasmid PX458 (Addgene, Cambridge, MA, USA), respectively. The primers used are listed in Table 1. The PCR products of FLAG-XIAP and EGFP were cloned into pSin4-EF2-IRES-Pur vector via EcoRI/BamHI double-digestion. To package lentivirus, the constructed plasmids were co-transfected together with pMD2.G and psPAX2 (Addgene, Cambridge, MA, USA) to 293TD cells using MegaTran 1.0 reagent (OriGene Technologies, Rockville, MD, USA). Each supernatant containing viral particles was collected 48 h and 60 h post-transfection and filtered through a 0.45 μm filter, and stored at −80 °C until use. At the time of use, PC12 cells, when grown to about 70% confluency, were infected with the above lentivirus-containing medium in the presence of 8 mg/ml polybrene for 12 h, and reinfected after 6 h. Finally, Uninfected cells were abolished by using 2 mg/ml puromycin for 48 h before use.

Table 1:

The sequences of primers for EGFP and FLAG-XIAP oligos

Name	Sense	Anti-sense
EGFP	5’-CCGGAATTCATGGTGAGCAAGGGCGAGGAGCT-3’	5’-CGCGGATCCGTTACTTGTACAGCTCGTCCATG-3’
FLAG-XIAP	5’-CGGAATTCCGATGGATTACAAGGATGACGACGATAAGATGACTTTTAACAGTTTTGAAGG-3’,	5’-CGGGATCCCGTTAAGACATAAAAATTTTTTGCTTGAACGTAATGA-3’

2.4. Analysis for cell viability

Since treatment with 10–20 μM Cd for 24 h is able to elicit significant apoptosis in neuronal cells (PC12, SH-SY5Y, and primary neurons) (Chen et al., 2008a; Gerspacher et al., 2009; Jiang et al., 2014), such Cd treatments were chosen for this research. PC12 cells or PC12 cells infected with lentiviral FLAG-XIAP or EGFP were seeded in a PDL-coated 96-well plate (1 × 10⁴ cells/well). After 24 h, cells were pretreated with/without an inhibitor of MDM2 binding to p53 Nutlin-3a (10 μM) (Thotala et al., 2012) or a mitochondria-targeted antioxidant Mito-TEMPO (10 μM) (Yeh et al., 2014) for 1 h, followed by Cd (10 μM) for 24 h, with 5 replicates of each treatment. Subsequently, MTS reagent (one solution reagent) (20 μl/well) was added and incubated for an additional 3 h. After that, the values of optical density (OD) at 490 nm were recorded with a Victor X3 Light Plate Reader (PerkinElmer, Waltham, MA, USA).

2.5. DAPI and TUNEL staining

PC12 cells and/or primary neurons, or PC12 cells infected with lentiviral FLAG-XIAP or EGFP were seeded at a density of 5 × 10⁵ cells/well in a 6-well plate containing a PDL-coated glass coverslip per well. After 24 h, cells were pretreated with/without Nutlin-3a (10 μM), an XIAP inhibitor Embelin (20 μM) (Dai et al., 2011) and/or Mito-TEMPO (10 μM) for 1 h, followed by Cd (10 μM) for 24 h, with 5 replicates of each treatment. Subsequently, the cells with fragmented and condensed nuclei were stained by adding DAPI (4 μg/ml in deionized water) as described (Chen et al., 2008b). For the cells pretreated with/without Embelin (20 μM) or Nutlin-3a (10 μM) for 1 h and then exposed to Cd (10 and/or 20 μM) for 24 h, after DAPI staining, the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining was used according to the protocol given by the manufacturer’s In Situ Cell Death Detection Kit® (Roche, Mannheim, Germany).Finally, the stained cell photos were taken using a fluorescence microscope (Leica DMi8, Wetzlar, Germany) connected to a digital camera. For quantitative analysis of the fluorescence intensity using TUNEL staining, the integral optical density (IOD) was determined by Image-Pro Plus 6.0 software (Media Cybernetics Inc., Newburyport, MA, USA).

2.6. Immunofluorescence and imaging

PC12 cells and primary neurons were seeded at a density of 5 × 10⁵ cells/well in a 6-well plate containing a PDL-coated glass coverslip per well. The next day, cells were exposed to Cd (10 μM) for 0–24 h, or pre-incubated with/without an antioxidant and ROS scavenger NAC (5 mM) (Chen et al., 2008a; Chen et al., 2011) or a thiol-reducing antioxidant GSH (5 mM) (Fan et al., 2013) for 1 h and then exposed to Cd (10 and/or 20 μM) for 24 h. Then, the cells on the cover-slips were fixed with 4% paraformaldehyde and blocked with 3% normal goat serum with 0.3% Triton X-100 for 1 h, incubated with rabbit anti-TOM20 antibody (Abcam Technology, Cambridge, UK, 1:50, diluted in PBS containing 1% BSA) or anti-XIAP antibody (Sciben Biotech, Nanjing, China, 1:50, diluted in PBS containing 1% BSA) overnight at 4°C. After incubation, cover-slips were washed three times (5 min per time) with PBS and then incubated with FITC-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, CA, USA, 1:500, diluted in PBS containing 1% BSA) for 1 h at room temperature. After three rinses, slides were fixed in glycerol/PBS (1:1, v/v) containing 2.5% 1,4-diazabis-(2,2,2)octane. Cell images were obtained under a fluorescence microscope (Leica DMi8, Wetzlar, Germany) connected to a digital camera. IOD for fluorescence intensity was quantitatively analyzed by Image-Pro Plus 6.0 software as described above.

2.7. Intracellular and mitochondrial ROS imaging

According to the information provided by the suppliers, CM-H₂DCFDA and MitoSOX are able to trace intracellular ROS and mitochondrial superoxide levels, respectively. CM-H₂DCFDA is a stable non-fluorescent molecule that can be passively diffused into cells where it is oxidized by ROS to emit green fluorescence. MitoSOX is a superoxide indicator dye, which can specifically recognize mitochondrial superoxide and produce red fluorescence in live cells. In brief, PC12 cells and primary neurons, or PC12 cells infected with lentiviral FLAG-XIAP or EGFP were seeded at a density of 5 × 10⁵ cells/well in a 6-well plate containing a PDL-coated glass coverslip per well. The next day, cells were exposed to Cd (10 μM) for 0–24 h, or pretreated with/without NAC (5 mM), GSH (5 mM), Embelin (20 μM), Nutlin-3a (10 μM) or Mito-TEMPO (10 μM) for 1 h, followed by exposure to Cd (10 and/or 20 μM) for 24 h, with 5 replicates of each treatment. Subsequently, the cells were incubated with CM-H₂DCFDA (10 μM) for 1 h or with MitoSOX (5 μM) for 10 min at 37°C, immersed three times with PBS, and then photographed under a fluorescence microscope, followed by quantifying the IOD of fluorescence intensity as described above.

2.8. Western blot analysis

The indicated cells, after treatments with Cd (10 and/or 20 μM) for 4 h (Xu et al., 2016; Zhang et al., 2017), were washed with cold PBS, and then on ice, lysed in the radioimmunoprecipitation assay buffer [50 mM Tris, pH 7.2; 150 mM NaCl; 1% sodium deoxycholate; 0.1% sodium dodecyl sulfate (SDS); 1% Triton X-100; 10 mM NaF; 1 mM Na₃VO₄; protease inhibitor cocktail (1:1000)]. Lysates were sonicated for 10 s and centrifuged at 16000 × g for 2 min at 4°C. The supernatants were collected and then Western blotting was performed as described previously (Chen et al., 2008b). In brief, lysates containing equivalent amounts of protein were separated on 7–12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were incubated with PBS containing 0.05% Tween 20 and 5% nonfat dry milk to block nonspecific binding, and then with primary antibodies against poly (ADP-ribose) polymerase (PARP), p53 (Cell Signaling Technology, Danvers, MA, USA), MDM2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FLAG (Sigma, St Louis, MO, USA), cleaved-caspase-3, XIAP, β-tubulin (Sciben Biotech, Nanjing, China) overnight at 4°C, respectively, followed by incubating with appropriate secondary antibodies including horseradish peroxidase-coupled goat anti-rabbit IgG or goat anti-mouse IgG (Pierce, Rockford, IL, USA) overnight at 4°C. Immunoreactive bands were visualized by using enhanced chemiluminescence solution (Sciben Biotech, Nanjing, China).

2.9. Statistical analysis

All data were expressed as mean values ± standard error (mean ± SE). Student’s t-test for non-paired replicates was used to identify statistically significant differences between treatment means. Group variability and interaction were compared using either one-way or two-way ANOVA followed by Bonferroni’s post-tests to compare replicate means. The criterion for the statistical significance was P < 0.05.

3. RESULTS

3.1. Cd induces ROS-dependent inactivation of XIAP-MDM2-p53 pathway with a concomitant activation of caspase pathway in neuronal cells

XIAP, a caspase inhibitor, can down-regulate MDM2 by ubiquitination, thereby increasing p53 level, which is defined as the XIAP-MDM2-p53 pathway (Huang et al., 2013; Schimmer, 2004). To investigate whether this pathway is involved in Cd-induced neuronal apoptosis, PC12 cells and primary neurons were exposed to 0–20 μM Cd for 4 h or 10 μM Cd for 0–24 h. Western blot analysis showed that Cd treatment reduced the protein levels of XIAP and p53, and increased the protein levels of MDM2 and cleaved caspase-3/PARP dose- and time-dependently in the cells (Fig.1A–D). Especially there existed dramatic changes of the proteins in the cells treated with 10 μM Cd for 4 h, consistent with our previous reports (Xu et al., 2016; Zhang et al., 2017).

Fig. 1.

Cd induces inactivation of XIAP-MDM2-p53 pathway and activation of caspase-3 concentration- and time-dependently in neuronal cells. PC12 cells and primary neurons were exposed to 0–20 μM Cd for 4 h, or 10 μM Cd for 0–24 h. (A and C) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B and D) The blots for XIAP, MDM2, p53, cleaved-caspase-3 and cleaved-PARP were semi-quantified. For (B) and (D), all data were expressed as means ± SE (n = 3). *P < 0.05, **P < 0.01, different from control group.

Our group has documented dose- and time-dependent increases of Cd-induced intracellular ROS levels, and unveiled that Cd induction of ROS causes neuronal apoptosis by caspase-dependent and -independent mechanisms (Chen et al., 2008a; Chen et al., 2011). In line with the above finding, here, we also observed that 24 h-treatment with Cd (10 and 20 μM) significantly increased production of ROS (Fig. 2A and B) and 4 h-Cd exposure triggered cleavages of caspase-3 and PARP in PC12 cells and primary neurons (Fig. 2C and D), whereas pretreatment with N-acetyl-L-cysteine (NAC), an antioxidant and ROS scavenger (Chen et al., 2008a; Chen et al., 2011), obviously inhibited the events (Fig. 2A–D). Moreover, we noted that Cd inhibited XIAP protein expression, increased the protein level of MDM2 and correspondingly reduced the protein level of p53, which was blocked by NAC in the cells (Fig. 2C and D). Further, our immunofluorescence staining revealed that Cd treatment resulted in a drastic decrease in XIAP (in green) in PC12 and primary neurons, which was significantly rescued by NAC pretreatment (Fig. 2E and F). To gain more insights into the event that Cd induces ROS-dependent inactivation of XIAP pathway, we extended our studies using glutathione (GSH), another antioxidant (Diaz-Vivancos et al., 2015; Fan et al., 2013; Rushworth et al., 2014). Pretreatment of PC12 cells and primary neurons with GSH dramatically inhibited Cd-induced ROS and cleavages of caspase-3 and PARP (Fig. 2G–I), and obviously ameliorated the protein levels of XIAP, MDM2 and p53 in response to Cd (Fig. 2H and I). GSH potently rescued the reduction of XIAP in Cd-exposed PC12 cells and primary neurons as well (Fig. 2J). These results indicate that Cd induces ROS-dependent inactivation of XIAP-MDM2-p53 pathway with a concomitant activation of caspase pathway in neuronal cells.

Fig. 2.

Cd induces ROS-dependent inactivation of XIAP pathway with a concomitant activation of caspase pathway in neuronal cells. PC12 cells and primary neurons were pretreated with/without NAC (5 mM) or GSH (5 mM) for 1 h and then exposed to Cd (10 and/or 20 μM) for 4 h (for Western blotting) or 24 h (for immunofluorescence staining and cell ROS imaging). (A) Cell ROS was imaged using an oxidant-sensitive probe CM-H2DCFDA. Scale bar: 20 μm. (B and G) IOD for cell ROS fluorescence intensity was quantitatively analyzed. (C and H) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (D and I) The blots for XIAP, MDM2, p53, cleaved-caspase-3 and cleaved-PARP were semi-quantified. (E) Expression of XIAP was stained by immunofluorescence (in green). Scale bar: 20 μm. (F and J) IOD for fluorescence intensity of XIAP expression was determined. For (B), (D), (F), (G), (I) and (J), all data were expressed as means ± SE (n = 3–5). ^aP < 0.05, different from control group; ^bP < 0.05, different from 10 μM Cd group; ^cP < 0.05, different from 20 μM Cd group.

To define whether the MDM2-p53 pathway plays a role in Cd-induced neuronal apoptosis, PC12 cells and primary neurons were exposed to Cd (10 μM) for 4 h or 24 h following pretreatment with/without Nutlin-3a (10 μM), an inhibitor that blocks the binding of MDM2 to p53 (Thotala et al., 2012), for 1 h. As expected, pretreatment with Nutlin-3a did not affect the protein level of XIAP, but reversed Cd-reduced p53 level in the cells (Fig. 3A and B). Interestingly, Nutlin-3a substantially rescued cells from cell death, as evidenced by the findings that Nutlin-3a rendered high resistance to Cd-evoked cleavage of caspase-3 (Fig. 3A and B), cell viability reduction (Fig. 3C) and apoptosis (Fig. 3D–F). In line with this, a lower Cd-induced ROS level was also found in the cells treated with Nutlin-3a (Fig. 3G). Collectively, the results suggest that the XIAP-MDM2-p53 pathway may be involved in Cd-induced ROS-dependent neuronal apoptosis.

Fig. 3.

Inhibition of MDM2 protects against Cd induction of ROS-dependent neuronal apoptosis. PC12 cells and primary neurons were pretreated with Nutlin-3a (10 μM) for 1 h and then exposed to Cd (10 μM) for 4 h (for Western blotting) or 24 h (for DAPI staining, cell viability analysis and cell ROS imaging). (A) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) The blots for XIAP, MDM2, p53 and cleaved-caspase-3 were semi-quantified. (C) Cell viability was determined by the MTS assay. (D) Apoptotic cells were evaluated by nuclear fragmentation and condensation (arrows) using DAPI staining (upper panel) and concurrently by in situ detection of fragmented DNA (in green) using TUNEL staining (lower panel). Scale bar: 20 μm. (E and F) The percentages of cells with fragmented nuclei and the number of TUNEL-positive cells were quantified. (G) Cell ROS was quantified using an oxidant-sensitive probe CM-H2DCFDA. For (B), (C), (E), (F) and (G), all data were expressed as means ± SE (n = 3–5). ^aP < 0.05, different from control group; ^bP < 0.05, different from 10 μM Cd group.

3.2. Overexpression of XIAP protects against Cd induction of ROS-dependent neuronal apoptosis

To determine the role of the XIAP-MDM2-p53 pathway in Cd-induced neuronal apoptosis, XIAP was overexpressed in PC12 cells. Infection with lentiviral FLAG-tagged wild-type XIAP (FLAG-XIAP), but not lentiviral EGFP (control), resulted in a robust expression of FLAG-tagged XIAP (Fig. 4A). Interestingly, overexpression of XIAP significantly suppressed MDM2 expression and elevated p53 expression in PC12 cells treated with Cd and/or Nutlin-3a (Fig. 4A and B). Noticeably, pretreatment with Nutlin-3a failed to further up-regulate p53 expression in FLAG-XIAP-overexpressing cells (Fig. 4A and B), implying that the inhibitory effect of overexpressed XIAP alone on MDM2 might reach a maximal level. Consistent with this, overexpression of XIAP potently blocked Cd-induced cleaved-caspase-3 (Fig. 4A and B), cell viability reduction (Fig. 4C) and apoptosis (Fig. 4D and E), as well as ROS production (Fig. 4F) in the cells regardless of pretreatment with/without Nutlin-3a. These findings demonstrate that Cd-downregulated XIAP contributes to Cd-induced ROS-dependent apoptosis in neuronal cells.

Fig. 4.

Overexpression of XIAP attenuates Cd induction of ROS-dependent neuronal apoptosis. PC12 cells, infected with lentiviral FLAG-tagged wild-type XIAP (FLAG-XIAP) or EGFP (as control), were pretreated with Nutlin-3a (10 μM) for 1 h and then exposed to Cd (10 μM) for 4 h (for Western blotting) or 24 h (for DAPI staining, cell viability analysis and cell ROS imaging). (A) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) The blots for XIAP, MDM2, p53 and cleaved-caspase-3 were semi-quantified. (C) Cell viability was determined by the MTS assay. (D) Apoptotic cells were evaluated by nuclear fragmentation and condensation (arrows) using DAPI staining. Scale bar: 20 μm. (E) The percentages of cells with fragmented nuclei were quantified. (F) Cell ROS was quantified using an oxidant-sensitive probe CM-H2DCFDA. For (B), (C), (E) and (F), all data were expressed as means ± SE (n = 3–5). ^aP < 0.05, different from control group; ^bP < 0.05, different from 10 μM Cd group; ^cP < 0.05, FLAG-XIAP group versus EGFP control group.

3.3. Pharmacological inhibition of XIAP strengthens Cd-induced ROS and apoptosis in neuronal cells

To further corroborate the role of XIAP in Cd-induced apoptosis in neuronal cells, PC12 cells and primary neurons were pretreated with/without Embelin (20 μM), an inhibitor that blocks XIAP binding to caspase (Dai et al., 2011; Nikolovska-Coleska et al., 2004), for 1 h and then exposed to Cd (10 or 20 μM) for 4 h or 24 h. As shown in Fig. 5A and B, pretreatment with Embelin alone decreased the protein level of XIAP, and a stronger inhibitory effect on XIAP expression was observed in co-treatment with Embelin/Cd. Consistently, MDM2 level increased with the decrease of XIAP, and correspondingly, p53 level decreased with the increase of MDM2, in line with the findings in Fig. 2C and D. Importantly, Embelin potentiated Cd-induced cleavages of caspase-3 and PARP (Fig. 5A and B). Furthermore, the quantified data for DAPI/TUNEL staining revealed that Embelin significantly increased the percentage of the cells with nuclear fragmentation and condensation, as well as the number of TUNEL-positive cells with fragmented DNA in the cells in response to Cd exposure, compared with the control group (Fig. 5C and D). Of note, pretreatment with Embelin also markedly resulted in the basic and Cd-induced ROS production in PC12 cells and primary neurons (Fig. 5E). These results support that XIAP plays a critical role for neuronal survival, and inhibition of XIAP enhances Cd-induced ROS and apoptosis in neuronal cells.

Fig. 5.

Pharmacological inhibition of XIAP strengthens Cd-induced ROS contributing to apoptosis in neuronal cells. PC12 cells and primary neurons were pretreated with/without Embelin (20 μM) for 1 h and then exposed to Cd (10 and 20 μM) for 4 h (for Western blotting) or 24 h (for DAPI and TUNEL staining, and cell ROS imaging). (A) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) The blots for XIAP, MDM2, p53, cleaved-caspase-3 and cleaved-PARP were semi-quantified. (C and D) The percentages of cells with fragmented nuclei and the number of TUNEL-positive cells were quantified. (E) Cell ROS was quantified using an oxidant-sensitive probe CM-H2DCFDA. For (B), (C), (D) and (E), all data were expressed as means ± SE (n = 3–5). ^aP < 0.05, different from control group; ^bP < 0.05, different from 10 μM Cd group; ^cP < 0.05, different from 20 μM Cd group.

3.4. Cd induces mitochondrial ROS-mediated inactivation of XIAP leading to neuronal apoptosis.

Our previous studies have reported that Cd induces mitochondrial ROS leading to neuronal apoptosis (Xu, et al., 2016; Zhang et al., 2017), so we next asked whether excess ROS formed in the mitochondria interferes with the mitochondrial dynamics and whether inactivation of XIAP is attributed to mitochondrial ROS induction in Cd-exposed neuronal cells. Firstly, TOM20, a mitochondrial membrane protein for presenting mitochondrial integrity (Hossain et al., 2009), was evaluated in PC12 cells and primary neurons exposed to Cd (10 μM) for 0–24 h, using immunofluorescence staining. Images and quantification showed that Cd treatment reduced the expression of TOM20 time-dependently (Fig. 6A and B), implying that the integrity and function of mitochondria are impaired. MitoSOX, a mitochondrial superoxide indicator, was used to define mitochondrial ROS in PC12 cells and primary neurons exposed to Cd for 0–24 h. In line with manifestation of TOM20, we observed that MitoSOX red fluorescence increased with Cd exposure time in the cells (Fig. 6C and D). Next, PC12 cells and primary neurons were pretreated with/without Mito-TEMPO (10 μM), a mitochondria-targeted antioxidant (Yeh et al., 2014), or in combination with Embelin (20 μM) for 1 h. We found that pretreatment with Mito-TEMPO alone substantially ameliorated Cd-affected XIAP, MDM2, p53, cleaved-caspase-3 and cleaved-PARP in the cells (Fig. 7A and B), and conferred profound resistance to the inhibitory effect of Embelin on XIAP (Fig. 7A and B). Consistently, Mito-TEMPO also showed potent attenuation of Cd-elicited apoptosis and ROS production and reversed Embelin’s elevation of Cd-evoked these events, as evidenced by DAPI staining, as well as intracellular and mitochondrial ROS imaging (Fig. 7C–E). In agreement with the above findings, overexpression of XIAP using lentiviral FLAG-XIAP obviously prevented Cd-induced cleavage of caspase-3, cell viability reduction, apoptosis and intracellular and mitochondrial ROS elevations (Fig. 8A–G). However, due to the predominant effect of XIAP overexpression, addition of Mito-TEMPO did not exhibit more significant protection (Fig. 8A–G). Taken together, our data support the idea that Cd induces massive mitochondrial ROS, which can impair mitochondrial dynamics and mediate inactivation of XIAP leading to neuronal apoptosis.

Fig. 6.

Cd impairs mitochondrial dynamics and induces mitochondrial ROS. PC12 cells and primary neurons were exposed to 10 μM Cd for 0–24 h. (A and B) Expression of TOM20 was stained by immunofluorescence (in green) and quantified. Scale bar: 20 μm. (C and D) Mitochondrial ROS (in red) was imaged and quantified using a mitochondrial superoxide indicator MitoSOX. Scale bar: 20 μm. For (B) and (D), all data were expressed as means ± SE (n = 5). **P < 0.01, different from control group.

Fig. 7.

Cd induces mitochondrial ROS-mediated inactivation of XIAP leading to neuronal apoptosis. PC12 cells and primary neurons were pretreated with/without Embelin (20 μM) and/or Mito-TEMPO (10 μM) for 1 h and then exposed to Cd (10 μM) for 4 h (for Western blotting) or 24 h (for DAPI staining and cell ROS imaging). (A) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) The blots for XIAP, MDM2, p53, cleaved-caspase-3 and cleaved-PARP were semi-quantified. (C) Apoptotic cells were evaluated by nuclear fragmentation and condensation using DAPI staining. (D) Cell ROS was quantified using an oxidant-sensitive probe CM-H2DCFDA. (E) Mitochondrial ROS level was quantified using a mitochondrial superoxide indicator MitoSOX. For (B), (C), (D) and (E) all data were expressed as means ± SE (n = 3–5). ^aP < 0.05, different from control group; ^bP < 0.05, different from 10 μM Cd group; ^cP < 0.05, different from Cd/Embelin group or Cd/Mito-TEMPO group.

Fig. 8.

Overexpression of XIAP attenuates Cd induction of mitochondrial ROS related to neuronal apoptosis. PC12 cells, infected with lentiviral FLAG-XIAP or EGFP (as control), were pretreated with Mito-TEMPO (10 μM) for 1 h and then exposed to Cd (10 μM) for 4 h (for Western blotting) or 24 h (for DAPI staining, cell viability analysis and ROS imaging). (A) Total cell lysates were subjected to Western blotting using indicated antibodies. The blots were probed for β-tubulin as a loading control. Similar results were observed in at least three independent experiments. (B) The blots for XIAP, MDM2, p53 and cleaved-caspase-3 were semi-quantified. (C) Cell viability was evaluated using MTT assay. (D) Apoptotic cells were evaluated by nuclear fragmentation and condensation (arrows) using DAPI staining. Scale bar: 20 μm. (E) The percentages of cells with fragmented nuclei were quantified. (F) Cell ROS was quantified using an oxidant-sensitive probe CM-H2DCFDA. (G) Mitochondrial ROS level was quantified using a mitochondrial superoxide indicator MitoSOX. For (B), (C), (E), (F) and (G) all data were expressed as means ± SE (n = 3–5). ^aP < 0.05, different from control group; ^bP < 0.05, different from 10 μM Cd group; ^cP < 0.05, FLAG-XIAP group versus EGFP control group.

4. DISCUSSION

Cd can easily penetrate the blood–brain barrier and cause CNS neurotoxicity, including AD, PD and ALS, through either acute or chronic exposure (Goncalves, et al., 2010; Monroe, et al., 2006; Panayi, et al., 2002; Pihl, et al., 1977; Wang, et al., 2013; Wright, et al., 2006). A large number of studies have confirmed that Cd-induced ROS is one of the causes of Cd-induced neurotoxicity by (Hossain et al., 2009; Lopez, et al., 2006; Wei et al., 2015). Studies have demonstrated that Cd treatment can alter the expression of genes related to cellular protection and damage control (e.g. those encoding metallothioneins, anti-oxidant proteins and heat shock proteins) and many other genes involved in signaling and metabolism in cells (Koizumi and Yamada, 2003). In addition, Cd-induced excess ROS can directly disrupt cell proteins, disturb their functions, and activate or inhibit related signaling pathways, thereby leading to neuronal cell dysfunction and cell death (Genovese et al., 2008; Wang et al., 2013). Previously, we have reported that Cd regulates intracellular proteins’ activity and their signaling pathways, such as inactivation of PTEN, AMPK, PP5 and PP2A proteins, and activation of Akt/mTOR, JNK and Erk1/2 pathways, by evoking intracellular and mitochondrial ROS, ultimately leading to apoptosis in neuronal cells (Chen et al., 2008a; Chen et al., 2011; Chen et al., 2014; Xu, et al., 2016; Zhang, et al., 2017). However, whether or how XIAP, an X-linked inhibitor of apoptosis protein, is involved in Cd-induced neuronal apoptosis remains unknown. Here, for the first time, we discovered that: i) Cd down-regulated the expression of XIAP by evoking intracellular ROS in neuronal cells; ii) Overexpression of XIAP protected cells from Cd-induced ROS and apoptosis; iii) Pharmacological inhibition of XIAP enhanced Cd neurotoxicity in neuronal cells; iv) Cd inactivated the XIAP pathway contributing to apoptosis by inducing mitochondrial ROS in neuronal cells.

Many data have shown that XIAP down-regulates MDM2 by ubiquitination, thereby increasing p53 level (Huang et al., 2013; Schimmer, 2004). p53 exerts a key action in orchestrating cell-cycle arrest, DNA damage and apoptosis in cells in response to a variety of stresses (D’Brot et al., 2017; Oren, 2003; Vaughn et al., 2007; Vousden et al., 2002; Xiong et al., 2015). Initially, XIAP is defined as an inhibitor of apoptosis because its BIR domain binds and inhibits caspase (Deveraux, et al., 1999; Eckelman et al., 2006; Takahashi, et al., 1998). In neurological studies, XIAP is generally considered to be a negative regulator of the caspase cascade (Huesmann et al., 2006; Unsain et al., 2013). Our group has demonstrated that Cd neurotoxicity is attributed to Cd induction of ROS-mediated neuronal apoptosis (Chen, et al., 2008a; Chen, et al., 2008b; Chen, et al., 2011). We therefore postulated that the XIAP-MDM2-p53 pathway might be involved in Cd-elicited ROS and apoptosis in neuronal cells. In this study, we firstly identified that Cd dose- and time-dependently reduced the expression of XIAP, up-regulated MDM2 and down-regulated p53 in PC12 cells and primary neurons (Fig. 1), implying inactivation of the XIAP-MDM2-p53 pathway. Subsequently, we demonstrated that Cd indeed induced ROS-dependent inactivation of XIAP-MDM2-p53 pathway, leading to neuronal apoptosis. This is supported by the findings that NAC, an antioxidant that acts as a precursor to GSH synthesis to achieve antioxidant function (Rushworth, et al., 2014), or GSH, a thiol-reducing antioxidant (Fan et al., 2013), substantially blocked Cd-induced excess ROS and subsequently inactivated XIAP-MDM2-p53 pathway, as well as cleavages of caspase-3 and PARP in PC12 cells and primary neurons (Fig. 2). Importantly, Inhibition of MDM2 with Nutlin-3a reversed Cd-induced reduction of p53 and powerfully rescued cells from massive ROS-dependent death (Fig. 3). Overexpression of XIAP greatly protected cells from Cd-induced ROS-dependent apoptosis (Fig. 4). Also, inhibition of XIAP by Embelin strengthened Cd-induced ROS and apoptosis in neuronal cells (Fig. 5). Collectively, these data potently support that Cd-mediated neurotoxicity is at least partially attributed to Cd-elicited ROS, as an upstream signal, inactivating the XIAP-MDM2-p53 pathway, and suggest that down-regulated XIAP or up-regulated MDM2 may have a feedforward effect on Cd-induced ROS generation.

It has been described that protein kinase RNA-like endoplasmic reticulum kinase (PERK) down-regulates XIAP synthesis through eukaryotic initiation factor 2α (eIF2α) and promotes XIAP degradation through transcription factor 4 (ATF4) (Hiramatsu et al., 2014). Also, Cd treatment can induce endoplasmic reticulum (ER) stress and activate the PERK-eIF2α-ATF4 pathway (Liu et al., 2019; Yokouchi et al., 2007). Whether Cd-induced ROS results in decreased protein levels of XIAP by activating the PERK-eIF2α-ATF4 pathway remains to be determined.

Autophagy is a double-edged sword, promoting cell survival or cell death, depending on environmental cues (Mizushima et al., 2008). Recently, it has been reported that E3 ubiquitin ligase activity of the XIAP RING domain is involved in the regulation of intracellular autophagy in cancer cells (Huang et al., 2018; Huang, et al., 2013), so XIAP can regulate both autophagy and apoptosis (Merlo et al., 2013). Our recent data have demonstrated that Cd accumulates autophagosomes-dependent apoptosis through impairing autophagic flux in neuronal cells (Zhang et al., 2019). Whether XIAP exerts as a promoter of autophagic flux to rescue cells from death remains to be investigated in the future.

The relationship between MDM2 and XIAP is controversial. Some studies have suggested that MDM2 is positively correlated with XIAP (Gu, et al., 2016), whereas others have shown that XIAP acts as a new E3 ubiquitin ligase for MDM2, promoting the degradation of MDM2 through proteasome (Huang, et al., 2013). Obviously, our results are in line with the latter’s viewpoint. p53 is a critical intracellular signaling molecule, which is involved in the regulation of many physiological activities such as cell cycle arrest, apoptosis, and senescence (Oren, 2003; Vaughn, et al., 2007). p53 is the downstream target of MDM2 and can be targeted by MDM2 for degradation (Haupt et al., 1997). In response to radiation or oxidative stress, p53 can be activated by the ATM/ATR pathway, upregulate p21^Cip1, which leads cell cycle arrest, allowing cell to repair damaged DNA for survival (Oren, 2003; Vousden, et al., 2002). It has also been described that cytoplasmic p21^Cip1 interacted with apoptosis signal-regulating kinase 1 (ASK1) to inhibit activation of ASK1-JNK signaling and blocked apoptosis induced by various stimuli (Asada et al., 1999; Huang et al., 2003). Our previous work has revealed that Cd induces neuronal apoptosis in part by activating ASK1-JNK pathway (Chen, et al., 2008a). In this study, we found that Nutlin-3a, an inhibitor that disrupts the MDM2-p53 interaction, up-regulated p53 and prevented Cd-induced cell death in neuronal cells. Further research is needed to elucidate whether XIAP promotes cell survival by upregulating p21^Cip1, and subsequently inactivating ASK1-JNK cascade in non-mitotic neuronal cells.

The functional integrity of mitochondria is pivotal for neuronal cell survival and homeostasis (Karbowski et al., 2012). Under pathological conditions, excess ROS can be formed in neuronal mitochondria, resulting in decreased mitochondrial biogenesis (Lu et al., 2012; Seo et al., 2010). As a feedback loop, mitochondria dysfunction further promotes ROS induction, and thus leads to deep deterioration of the cells (Woo et al., 2011). It has been confirmed in our previous studies that Cd neurotoxicity is implicated in mitochondrial ROS (Xu, et al., 2016; Zhang, et al., 2017). In this study, using immunofluorescence staining for TOM20, a MitoSOX red dye for monitoring mitochondrial superoxide and a mitochondria-targeted antioxidant Mito-TEMPO, we demonstrated that Cd evoked excessive mitochondrial ROS, which indeed impaired mitochondrial dynamics and mediated inactivation of XIAP-MDM2-p53 pathway, finally causing neuronal apoptosis. Because ROS family comprises oxygen radicals, including O₂⁻•, hydroxyl (•OH), peroxyl (RO₂•), alkoxyl (RO•), and certain nonradicals that are oxidizing agents and/or are readily converted into radicals, such as hypochlorous acid (HOCl), ozone (O₃), singlet oxygen (¹O₂), and H₂O₂ (Zhou et al., 2015), it is still necessary to further define which one is involved in Cd-inactivated XIAP-MDM2-p53 pathway contributing to neuronal apoptosis.

In conclusion, we have shown that Cd down-regulated the protein level of XIAP, contributing to apoptosis by induction of mitochondrial ROS in neuronal cells. Mechanistically, Cd-induced mitochondrial ROS triggered the downregulation of XIAP, which resulted in increase in MDM2 and consequent decrease in p53, leading to neuronal apoptosis (Fig. 9). The results highlight that Cd-induced mitochondrial ROS promotes neuronal apoptosis in part by inactivating XIAP-MDM2-p53 pathway. Our findings suggest that activator of XIAP or modulation of XIAP-MDM2-p53 pathway by antioxidants is a promising approach against Cd-induced neurotoxicity.

Fig. 9.

Diagram illustrating how Cd induces neuronal apoptosis by inactivating XIAP-MDM2-p53 pathway. Cd-induced mitochondrial ROS triggered the downregulation of XIAP, which resulted in increase in MDM2 and consequent decrease in p53, leading to neuronal apoptosis.

Highlights.

• Cd induces mitochondrial ROS, causing inactivation of XIAP-MDM2-p53 pathway in neuronal cells.

• ROS-inactivated XIAP-MDM2-p53 pathway plays a critical role in Cd-induced apoptosis.

• Modulation of XIAP-MDM2-p53 pathway is a promising approach against Cd-induced neurotoxicity.

Abbreviations

AD

Alzheimer disease

Cd

cadmium

CM-H₂DCFDA

5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate

CNS

central nervous system

DAPI

4′, 6-diamidino-2-phenylindole

DMEM

Dulbecco’s Modified Eagle’s Medium

FBS

fetal bovine serum

GSH

glutathione

HD

Huntington’s disease

IOD

Integral Optical Density

MDM2

Mouse double minute 2 homolog

NAC

N-acetyl-L-cysteine

PBS

phosphate buffered saline

PD

Parkinson disease

PDL

poly-D-lysine

ROS

reactive oxygen species

Ser/Thr

serine/threonine

TOM20

translocase of the outer mitochondrial membrane 20

TUNEL

the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling

XIAP

X-linked inhibitor of apoptosis protein