Autophagy induction by SIRT6 is involved in oxidative stress-induced neuronal damage
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SIRT6 is a NAD+-dependent histone deacetylase and has been implicated in the regulation of genomic stability, DNA repair, metabolic homeostasis and several diseases. The effect of SIRT6 in cerebral ischemia and oxygen/glucose deprivation (OGD) has been reported, however the role of SIRT6 in oxidative stress damage remains unclear. Here we used SH-SY5Y neuronal cells and found that overexpression of SIRT6 led to decreased cell viability and increased necrotic cell death and reactive oxygen species (ROS) production under oxidative stress. Mechanistic study revealed that SIRT6 induced autophagy via attenuation of AKT signaling and treatment with autophagy inhibitor 3-MA or knockdown of autophagy-related protein Atg5 rescued H2O2-induced neuronal injury. Conversely, SIRT6 inhibition suppressed autophagy and reduced oxidative stress-induced neuronal damage. These results suggest that SIRT6 might be a potential therapeutic target for neuroprotection.
KeywordsSIRT6 oxidative stress neuronal damage autophagy AKT
Sirtuins are a family of NAD+-dependent histone deacetylases involved in diverse cellular functions including inflammation, energy metabolism, stress resistance and cancer (Haigis and Sinclair, 2010). There are seven mammalian sirtuins with varied subcellular localizations and enzymatic activities (Michishita et al., 2005). SIRT6 is predominantly localized in the nucleus, functioning as an ADP-ribosyltransferase and NAD+-dependent deacetylase (Liszt et al., 2005; Michishita et al., 2008; Feldman et al., 2013). SIRT6 plays an important role in genomic stability, DNA repair, metabolic homeostasis and diseases such as obesity, cardiac hypertrophy and cancer (Liu et al., 2013; Kugel and Mostoslavsky, 2014). A recent study has reported that SIRT6 is associated with high mobility group box-1 (HMGB1) release after cerebral ischemia, but knockdown of SIRT6 has no effect on neuronal cell death induced by oxygen and glucose deprivation (OGD) (Lee et al., 2013b). Since oxidative stress is a key mediator of tissue damage after ischemia-reperfusion, the role of SIRT6 in oxidative stress-induced neuronal injury remains to be investigated.
Autophagy is a catabolic process which involves degradation of unnecessary cellular components or dysfunctional organelles in response to metabolic demand and diverse stress (Kroemer et al., 2010). Whether the autophagic process is beneficial or detrimental remains controversial. The term ‘autophagic cell death’ has been introduced to describe cell death that can be suppressed by inhibition of the autophagy (Galluzzi et al., 2012). Autophagy is regulated by diverse signaling pathways and stressful conditions including nutrient deprivation, hypoxia and oxidative stress (He and Klionsky, 2009). Reactive oxygen species (ROS) serve as triggers of autophagy since antioxidants treatment could to some extent rescue the autophagic programme (Filomeni et al., 2014). In this study, we aimed to uncover the role of SIRT6 in oxidative stress-induced cell death and the possible mechanisms like autophagy.
Oxidative stress causes an acute reduction in SIRT6 expression
SIRT6 enhances oxidative stress-induced neuronal cell death
SIRT6 induces autophagy under oxidative stress
SIRT6 attenuates AKT signaling under oxidative stress
SIRT6 inhibition reduces neuronal cell death and attenuates autophagy under oxidative stress
Previous studies have reported that SIRT6 levels are downregulated under stressful conditons such as cellular senescence or oxygen and glucose deprivation (Lee et al., 2013b; Liu et al., 2014; Takasaka et al., 2014). Similar to the above results, we found that SIRT6 levels showed an acute decrease following H2O2 treatment, but restored to almost basal levels during longer time. This might be explained by the reason that we detected SIRT6 levels upon a transient stimuli, since cells were replaced with fresh medium and cultured for different time after exposure to H2O2. The acute decrease in both protein and mRNA levels indicates that SIRT6 expression might be regulated by proteasomal degradation or micro-RNAs. Ubiquitin ligase CHIP could prevent degradation of SIRT6 through noncanonically ubiquitinating SIRT6 at K170 (Ronnebaum et al., 2013). CHIP protein is reduced under oxidative stress (Lee et al., 2013a), thus it is possible that inhibition of SIRT6 degradation by CHIP will be removed after H2O2 treatment. Another ubiquitin-specific peptidase USP10 could also deubiquitinate SIRT6 protecting it from proteasomal degradation (Lin et al., 2013). Activation of AKT1 phosphorylates SIRT6 leading to interaction and ubiquitination of SIRT6 by MDM2 (Thirumurthi et al., 2014), thus oxidative stress-induced AKT activation will cause ubiquitination-mediated degradation of SIRT6. Furthermore, micro-RNAs targeting SIRT6 have also been reported, including miR-33b, miR-34a and miR-766 (Davalos et al., 2011; Lefort et al., 2013; Sharma et al., 2013). Ischemia/Reperfusion (IR) induces miR-34a expression in the rat hepatocytes (Huang et al., 2014), which makes the possibility that upregulation of miR-34a under oxidative stress might lead to decrease in SIRT6 levels.
Oxidative stress induces autophagy in different type of cells such as cardiomyocytes, auditory cells, endothelial progenitor cells and embryonic stem cells (Dutta et al., 2013; Ou et al., 2014; Hayashi et al., 2015; Tang et al., 2015). Here we confirmed that oxidative stress could induce autophagy in neuronal cells, as H2O2 treatment upregulated LC3-II levels in a time course-dependent manner. The role of autophagy in the oxidative stress damage remains controversial. Curcumin protects vascular endothelial cell from H2O2-induced cytotoxicity through induction of autophagy (Han et al., 2012), whereas adiponectin inhibits H2O2-mediated autophagosome formation and prevents loss of cell viability in cardiomyocytes (Essick et al., 2013). In this study, we showed that SIRT6-mediated autophagy contributes to oxidative stress-induced neuronal injury, since inhibition of autophagy by specific inhibitor 3-MA or knockdown of Atg5 could rescue the detrimental effect of SIRT6 on cell survival. Recently it has been reported that rAAV-mediated SIRT6 overexpression downregulated cell viability in primary neurons under oxidative stress (Cardinale et al., 2014), which agrees with our present results.
The mammalian target of rapamycin (mTOR) is a crucial negative regulator of autophagy and AKT is an upstream signaling molecule for mTOR activation (Arico et al., 2001; Manning et al., 2002). Previous studies have shown the corelation between SIRT6 and AKT signaling. SIRT6 extends lifespan in male mice or prevents cardiac hypertrophy via the attenuation of IGF-AKT pathways (Kanfi et al., 2012; Sundaresan et al., 2012). Recently it has been reported that autophagy induction by SIRT6 through attenuation of insulin-like growth factor signaling is involved in human bronchial epithelial cell senescence (Takasaka et al., 2014). In this study, SIRT6 enhances oxidative stress-induced autophagy associated with decreased phosphorylation of AKT, which is in line with the above observations. Moreover, activation of AKT1 phosphorylates and promotes the degradation of SIRT6 (Thirumurthi et al., 2014), thus there may exist a positive-feedback loop between SIRT6 and AKT. Ras-Raf1-MEK cascade is another signaling pathway regulating autophagy (Pattingre et al., 2003; Furuta et al., 2004). We showed that oxidative stress causes a dramatic increase in phosphorylation of ERK1/2, but SIRT6 overexpression does not affect ERK1/2 signaling pathway.
In conclusion, we demonstrated that autophagy induction by SIRT6 is involved in oxidative stress-induced neuronal cell death, which could be attributed to attenuation of AKT signaling. Therefore, regulation of autophagy by controlling SIRT6 levels might be a promising therapeutic target for ischemia and other brain diseases.
MATERIALS AND METHODS
Hydrogen peroxide (H2O2) (323381) and 3-methyladenine (3-MA) (M9281) were purchased from Sigma Aldrich (St. Louis, MO). MYR-AKT (#9005) and EGFP-LC3 (#21073) plasmids were from Addgene. Atg5 and Sirt6 siRNAs were from GenePharma (Shanghai, China).
SH-SY5Y human neuroblastoma cells were from Institute of Neurology, Ruijin Hospital (Shanghai, China). Differentiated PC12 cells were from Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Cells were maintained in Dulbecco’s Modified Eagle Medium (HyClone) containing 10% fetal bovine serum (HyClone), 100 units/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified incubator under 95% air and 5% CO2.
Construction of stably transfected cells
Human SIRT6 cDNA construct was cloned into pCDH-CMV-MCS-EF1-copGFP backbone. HEK293T cells were transfected with the pCDH constructs accompanied by pMD.2G/psPAX2 plasmids and mature lentivirus was obtained by ultracentrifugation. SH-SY5Y cells were transfected with the vector or SIRT6 lentivirus and stably transfected cells were selected by flow cytometry.
Lactate dehydrogenase (LDH) assay
For intracellular LDH assay, cells were incubated with lysis buffer for 20 min. Cell lysates were mixed with reaction buffer containing 7.5 mmol/L sodium pyruvate and 1.5 mmol/L NADH at a ratio of 1:3, and the absorbance at 340 nm was measured over 30 s using a microplate reader (Synergy2, BioTek). For LDH release assay, cell culture medium was mixed with reaction buffer at a ratio of 1:1 and the absorbance at 340 nm was measured as above.
CCK-8 cell viability assay
Cell viability was measured by using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). In brief, cells were seeded in 96-well plates at the density of 1 × 104/well. After treatment, CCK-8 solution was added to the medium at a dilution of 1:10 and cells were incubated at 37°C for 4 h. The absorbance at 450 nm was measured using a microplate reader (Synergy2, BioTek).
Annexin V/7-AAD assay
The degrees of both apoptosis and necrosis were measured using ApoScreen Annexin V kit (Southern-Biotech, Birmingham, AL, USA). Briefly, cells were resuspended in pre-chilled binding buffer at concentrations of 5 × 106 cells/mL. Annexin V solution was added into cell suspension at a dilution of 1:10. After incubation on ice for 15 min, 7-AAD solution was added into cell suspension at a dilution of 1:40. Further analysis was performed on a flow cytometer (BD FACSAriaII).
Dihydroethidium (DHE) staining
Reactive oxygen species (ROS) levels were detected by DHE staining. In brief, after treatment cells were washed once with PBS, followed by incubation with 5 µmol/L DHE (Beyotime Institute of Biotechnology, Jiangsu Province, China) at 37°C for 20 min. Cells were then washed once with PBS again and the absorbance at 545 nm was obtained using a laser-scanning microscope (Leica TCS SP5 II, Germany).
Assessment of autophagosome formation
Cells were seeded on coverslips in 24-well plates and transfected with EGFP-LC3 plasmid (0.5 µg for each well of a 24-well plate). Twenty-four hours after transfection, cells were exposed to H2O2 for 1 h and cultured for another 24 h. Cells were subsequently washed with PBS and fixed in 4% paraformaldehyde for 10 min. Finally slides were mounted with cover slips and epifluorescence images were taken using a confocal microscope (Leica, Solms, Germany). To quantify autophagosome formation, the number of EGFP-LC3 puncta per cell was counted in multiple fields.
Total RNA was extracted from cells by RNAiso Plus (TaKaRa, Dalian, China) and reverse-transcribed to cDNA by PrimeScript RT reagent kit (TaKaRa). Quantitative real-time PCR was used to determine Sirt6 mRNA levels by SYBR Premix Ex Taq (TaKaRa) and the primers below: human SIRT6 (forward 5′-cccggatcaacggctctatc-3′ and reverse 5′-gccttcacccttttggggg-3′); human GAPDH (forward 5′-atggggaaggtgaaggtcg-3′ and reverse 5′-ggggtcattgatggcaacaata-3′).
Cells were lysed using RIPA buffer supplemented with protease inhibitors. 40 μg of total protein was used for Western blot analysis based on standard procedures. Protein signals were detected by ECL system. The primary antibodies were listed as follows: SIRT6 (1:1000) (Abcam, New Territories, HK); LC3 (1:2000), p62 (1:2000) (Sigma, MO, USA); AKT1 (1:1000), phos-AKT1 (1:1000) (Epitomics); ERK1/2 (1:1000), phos-ERK1/2 (1:2000), ATG5 (1:1000) (Cell Signaling Technology, Danvers, USA); beta-Tubulin (1:2000) (Abmart); Actin (1:500) (Santa Cruz Biotechnology, CA, USA).
Plasmid or siRNA transfection
Cells were seeded at 40,000 cells per 24-well plate or 200,000 cells per 6-well plate. Cells were grown for 24 h and transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols. 4–6 h later, cells were replaced with fresh medium and cultured for 48 h to conduct further experiments. siRNA sequences were as follows: hAtg5 siRNA#1 (5′-GACCAUGCAAUGGUGGCUU-3′); hAtg5 siRNA#2 (5′-GCAGUGGCUGAGUGAACAU-3′); hAtg5 siRNA#3 (5′-GAUGCAAUUGAAGCUCAUU-3′); hSirt6 siRNA#1 (5′-GAAUGUGCCAAGUGUAAGATT-3′); hSirt6 siRNA #2 (5′-GGCUCUGCACCGUGGCUAATT-3′).
Data were presented as mean ± SEM and analysed by ANOVA followed by Tukey’s post hoc test. P values less than 0.05 were considered statistically significant.
We are grateful to Mrs. Jin Xu for her assistance in flow cytometry.
3-MA, 3-methyladenine; HBEC, human bronchial epithelial cell; HMGB1, high mobility group box-1; IR, ischemia/reperfusion; LDH, lactate dehydrogenase; Mtor, mammalian target of rapamycin; OGD, oxygen/glucose deprivation; ROS, reactive oxygen species.
COMPLIANCE WITH ETHICS GUIDELINES
Jiaxiang Shao, Xiao Yang, Tengyuan Liu, Tingting Zhang, Qian Reuben Xie and Weiliang Xia declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by the any of the authors.
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