Induction Of SESN2 Research Articles

DNMT3a-mediated upregulation of the stress inducible protein sestrin-2 contributes to malignant transformation of human bronchial epithelial cells following nickel exposure

BackgroundNickel is a confirmed human lung carcinogen. Nonetheless, the molecular mechanisms driving its carcinogenic impact on lung tissue remain poorly defined. In this study, we assessed SESN2 expression and the signaling pathways responsible for cellular transformation in human bronchial epithelial cells (HBECs) as a result of nickel exposure. MethodsWe employed the Western blotting to determine the induction of SESN2 by nickel. To clarify the signaling pathways leading to cellular transformation following nickel exposure, we applied techniques such as gene knockdown, methylation-specific PCR, and chromatin immunoprecipitation. ResultExposure to nickel results in the upregulation of SESN2 and the initiation of autophagy in human bronchial epithelial cells (HBECs). This leads to degradation of HUR protein and consequently downregulation of USP28 mRNA, PP2AC protein, β-catenin protein, and diminished VHL transcription, culminating in the accumulation of hypoxia-inducible factor-1α (HIF-1α) and the malignant transformation of these cells. Mechanistic studies revealed that the increased expression of SESN2 is attributed to the demethylation of the SESN2 promoter induced by nickel, a process facilitated by decreased DNA methyl-transferase 3 A (DNMT3a) expression, while The downregulation of VHL transcription is linked to the suppression of the PP2A-C/GSK3β/β-Catenin/C-Myc pathway. Additionally, we discovered that SESN2-mediated autophagy triggers the degradation of HUR protein, which subsequently reduces the stability of USP28 mRNA and inhibits the PP2A-C/GSK3β/β-Catenin pathway and c-Myc transcription in HBECs post nickel exposure. ConclusionOur results reveal that nickel exposure leads to the downregulation of DNMT3a, resulting in the hypomethylation of the SESN2 promoter and its protein induction. This triggers autophagy-dependent suppression of the HUR/USP28/PP2A/β-Catenin/c-Myc pathway, subsequently leading to reduced VHL transcription, accumulation of HIF-1α protein, and the malignant transformation of human bronchial epithelial cells (HBECs). Our research offers novel insights into the molecular mechanisms that underlie the lung carcinogenic effects of nickel exposure. Specifically, nickel induces aberrant DNA methylation in the SESN2 promoter region through the decrease of DNMT3a levels, which ultimately leads to HIF-1α protein accumulation and the malignant transformation of HBECs. Specifically, nickel initiates DNA-methylation of the SESN2 promoter region by decreasing DNMT3a, ultimately resulting in HIF-1α protein accumulation and malignant transformation of HBECs. This study highlights DNMT3a as a potential prognostic biomarker or therapeutic target to improve clinical outcomes in lung cancer patients.

Sestrin2 attenuates renal damage by regulating Hippo pathway in diabetic nephropathy.

Glomerular mesangial cell proliferation and extracellular matrix accumulation contribute to the progression of diabetic nephropathy (DN). As a conserved stress-inducible protein, sestrin2 (Sesn2) plays critical role in the regulation of oxidative stress, inflammation, autophagy, metabolism, and endoplasmic reticulum stress. In this study, we investigated the role of Sesn2 on renal damage in diabetic kidney using transgenic mice overexpressing Sesn2 and the effect of Sesn2 on mesangial cell proliferation and extracellular matrix accumulation in diabetic conditions and the possible molecular mechanisms involved. Sesn2 overexpression improved renal function and decreased glomerular hypertrophy, albuminuria, mesangial expansion, extracellular matrix accumulation, and TGF-β1 expression, as well as oxidative stress in diabetic mice. In vitro experiments, using human mesangial cells (HMCs), revealed that Sesn2 overexpression inhibited high glucose (HG)-induced proliferation, fibronectin and collagen IV production, and ROS generation. Meanwhile, Sesn2 overexpression restored phosphorylation levels of Lats1 and YAP and inhibited TEAD1 expression. Inhibition of Lats1 accelerated HG-induced proliferation and expression of fibronectin and collagen IV. Verteporfin, an inhibitor of YAP, suppressed HG-induced proliferation and expression of fibronectin and collagen IV. However, Sesn2 overexpression reversed Lats1 deficiency-induced Lats1 and YAP phosphorylation, nuclear expression levels of YAP and TEAD1, and proliferation and fibronectin and collagen IV expressions in HMCs exposed to HG. In addition, antioxidant NAC or tempol treatment promoted phosphorylation of Lats1 and YAP and inhibited TEAD1 expression, proliferation, and fibronectin and collagen IV accumulation in HG-treated HMCs. Taken together, Sesn2 overexpression inhibited mesangial cell proliferation and fibrosis via regulating Hippo pathway in diabetic nephropathy. Induction of Sesn2 may be a potential therapeutic target in diabetic nephropathy.

Sestrin2 protects against traumatic brain injury by reinforcing the activation of Nrf2 signaling.

Sestrin2 (SESN2) is stress-inducible protein that confers cytoprotective effects against various noxious stimuli. Accumulating evidence has documented that SESN2 has potent anti-apoptosis and anti-oxidative stress functions. However, whether it provides neuroprotection in traumatic brain injury (TBI) models remains unexplored. The purpose of this study was to explore the regulatory effect of SESN2 on TBI using in vivo and in vitro models. We found that TBI resulted in a marked induction of SESN2 in the cerebral cortex tissues of mice. SESN2 overexpression in the brain by in vivo gene transfer significantly decreased neurological deficit, brain edema, and neuronal apoptosis of mice with TBI. Moreover, the overexpression of SESN2 significantly decreased the oxidative stress induced by TBI in mice. In vitro studies of TBI demonstrated that SESN2 overexpression decreased apoptosis and oxidative stress in scratch-injured cortical neurons. Notably, SESN2 overexpression increased the nuclear levels of nuclear factor-erythroid 2-related factor 2 (Nrf2) and enhanced the activation of Nrf2 antioxidant signaling in in vivo and in vitro models of TBI. In addition, the inhibition of Nrf2 significantly abolished SESN2-mediated neuroprotective effects in vivo and in vitro. In conclusion, these results of our work demonstrate that SESN2 protects against TBI by enhancing the activation of Nrf2 antioxidant signaling.

Protective effect of sestrin2 against iron overload and ferroptosis-induced liver injury

Ferroptosis is the non-apoptotic form of cell death caused by small molecules or conditions that inhibit glutathione biosynthesis or resulting in iron-dependent accumulation of lipid peroxidation by lipid reactive oxygen species (ROS). Sestrin2 (Sesn2), a conserved antioxidant protein, is responsive to various stresses including genotoxic, metabolic, and oxidative stresses and acts to restore homeostatic balance. Sesn2 expression was reported to be regulated via stress-responsive transcription factors including p53, Nrf2, and HIF-1α. However, the role of Sesn2 in regulating ferroptosis is not known. In the current study, we investigated whether ferroptosis inducing compounds including erastin, sorafenib, and buthionine sulfoximine affect Sesn2 expression and the role of Sesn2 in cytoprotection against ferroptosis-mediated cell death. Our data demonstrate that ferroptosis inducers significantly increased Sesn2 in hepatocytes in a dose- and time-dependent manner. Treatment with erastin upregulated Sesn2 mRNA levels and luciferase reporter gene activity, and erastin-mediated Sesn2 induction was transcriptionally regulated by NF-E2-related factor 2 (Nrf2). Furthermore, deletion of the antioxidant response element (ARE) in the Sesn2 promoter or Nrf2 knockout or knockdown abolished erastin-induced Sesn2 expression. In cells expressing Sesn2, erastin-induced cell death, ROS formation, and glutathione depletion were almost completely inhibited compared to that in control cells. Treatment with phenylhydrazine in mice, well-reported iron overload liver injury model, increased ALT and AST levels and altered histological features, which were almost completely inhibited by adenoviral Sesn2 infection. Collectively, our results suggest that ferroptosis-mediated Sesn2 induction is dependent on Nrf2 and plays a protective role against iron overload and ferroptosis-induced liver injury.

Brain-Derived Neurotrophic Factor Elevates Activating Transcription Factor 4 (ATF4) in Neurons and Promotes ATF4-Dependent Induction of Sesn2.

Activating transcription factor 4 (ATF4) plays important physiologic roles in the brain including regulation of learning and memory as well as neuronal survival and death. Yet, outside of translational regulation by the eIF2α-dependent stress response pathway, there is little information about how its levels are controlled in neurons. Here, we show that brain-derived neurotrophic factor (BDNF) promotes a rapid and sustained increase in neuronal ATF4 transcripts and protein levels. This increase is dependent on tropomyosin receptor kinase (TrkB) signaling, but independent of levels of phosphorylated eIF2α. The elevation in ATF4 protein occurs both in nuclei and processes. Transcriptome analysis revealed that ATF4 mediates BDNF-promoted induction of Sesn2 which encodes Sestrin2, a protector against oxidative and genotoxic stresses and a mTor complex 1 inhibitor. In contrast, BDNF-elevated ATF4 did not affect expression of a number of other known ATF4 targets including several with pro-apoptotic activity. The capacity of BDNF to elevate neuronal ATF4 may thus represent a means to maintain this transcription factor at levels that provide neuroprotection and optimal brain function without risk of triggering neurodegeneration.

SESN2/sestrin 2 induction-mediated autophagy and inhibitory effect of isorhapontigenin (ISO) on human bladder cancers

ABSTRACTIsorhapontigenin (ISO) is a new derivative of stilbene isolated from the Chinese herb Gnetum cleistostachyum. Our recent studies have revealed that ISO treatment at doses ranging from 20 to 80 μM triggers apoptosis in multiple human cancer cell lines. In the present study, we evaluated the potential effect of ISO on autophagy induction. We found that ISO treatment at sublethal doses induced autophagy effectively in human bladder cancer cells, which contributed to the inhibition of anchorage-independent growth of cancer cells. In addition, our studies revealed that ISO-mediated autophagy induction occurred in a SESN2 (sestrin 2)-dependent and BECN1 (Beclin 1, autophagy related)-independent manner. Furthermore, we identified that ISO treatment induced SESN2 expression via a MAPK8/JNK1 (mitogen-activated protein kinase 8)/JUN-dependent mechanism, in which ISO triggered MAPK8-dependent JUN activation and facilitated the binding of JUN to a consensus AP-1 binding site in the SESN2 promoter region, thereby led to a significant transcriptional induction of SESN2. Importantly, we found that SESN2 expression was dramatically downregulated or even lost in human bladder cancer tissues as compared to their paired adjacent normal tissues. Collectively, our results demonstrate that ISO treatment induces autophagy and inhibits bladder cancer growth through MAPK8-JUN-dependent transcriptional induction of SESN2, which provides a novel mechanistic insight into understanding the inhibitory effect of ISO on bladder cancers and suggests that ISO might act as a promising preventive and/or therapeutic drug against human bladder cancer.

Mitochondrial dysfunction induces SESN2 gene expression through Activating Transcription Factor 4

ABSTRACTWe found that inhibitors of mitochondrial respiratory chain complexes III (myxothiazol) and I (piericidin A) in some epithelial carcinoma cell lines induce transcription of the p53-responsive SESN2 gene that plays an important role in stress response and homeostatic regulation. However, the effect did not depend on p53 because i) there was no induction of p53 after the treatment with piericidin A; ii) after the treatment with myxothiazol the peak of SESN2 gene upregulation occurred as early as 5h, before the onset of p53 activation (13h); iii) a supplementation with uridine that abolishes the p53 activation in response to myxothiazol did not abrogate the induction of SESN2 transcripts; iv) in the p53 negative HCT116 p53 -/- cells SESN2 transcription could be also induced by myxothiazol. In response to the respiratory chain inhibitors we observed an induction of ATF4, the key transcription factor of the integrated stress response (ISR). We found that the induction of SESN2 transcripts could be prevented by the ISR inhibitory small molecule ISRIB. Also, by inhibiting or overexpressing ATF4 with specific shRNA or ATF4-expressing constructs, respectively, we have confirmed the role of ATF4 in the SESN2 gene upregulation induced by mitochondrial dysfunction. At a distance of 228 bp upstream from the SESN2 transcription start site we found a candidate sequence for the ATF4 binding site and confirmed its requirement for the induction of SESN2 in luciferase reporter experiments. We suggest that the upregulation of SESN2 by mitochondrial dysfunction provides a homeostatic feedback that attenuates biosynthetic processes during temporal losses of energy supply from mitochondria thereby assisting better adaptation and viability of cells in hostile environments.

Compound C Increases Sestrin2 Expression <i>via</i> Mitochondria-Dependent ROS Production

Compound C is a widely used chemical inhibitor that down-regulates AMP-activated protein kinase (AMPK) activity. However, it has been suggested that compound C exerts AMPK-independent effects in various cells. Here, we investigated whether compound C induces Sestrin2 (SESN2), an antioxidant enzyme induced by diverse stress. In addition, the mechanism responsible for SESN2 induction by compound C was determined. Our results showed that compound C increased SESN2 protein expression in HepG2 cells in a concentration- and time-dependent manner. The induction of SESN2 mRNA was also observed in cells treated with compound C. Increase of SESN2 luciferase activity confirmed transcriptional regulation by compound C and this substance also increased nuclear factor erythroid 2 (NF-E2)-related factor-2 (Nrf2) phosphorylation, which implies that Nrf2 was involved in SESN2 induction. Next, we sought to demonstrate whether production of reactive oxygen species (ROS) accompanied SESN2 expression. Compound C increased ROS production, but this effect was prevented by pretreatment with antioxidants or the mitochondrial complex I inhibitor. Moreover, cyclosporin A, an inhibitor of pore formation in the mitochondrial membrane, attenuated compound C-induced SESN2 induction. However, overexpression of a constitutively active form of AMPK was not able to abolish SESN2 induction by compound C, which implies that its action is independent of AMPK inhibition. In conclusion, this is the first study demonstrating that compound C alters mitochondrial function and induces ROS production, which ultimately leads to phosphorylation of Nrf2 and induction of SESN2.

ATF6‐dependent Sestrin2 induction protects from ER stress‐mediated liver injury

Endoplasmic reticulum (ER) stress is characterized by the accumulation of misfolded proteins in the ER and it leads to a variety of liver damage including hepatic steatosis, alcohol‐induced liver disease, viral hepatitis. Sestrin 2 (Sesn2) is stress‐inducible protein and responses to hypoxia and oxidative stress. The present study investigated the role of Sesn2 on ER stress and its underlying mechanisms in hepatocytes. The treatment of thapsigargin (Tg) and tunicamycin (Tm), the representative ER stress inducer, led to increase of Sesn2 protein expression, mRNA and luciferase gene activity in HepG2 cells. Among signaling molecules in response to ER stress, ectopic expression of ATF6 increased Sesn2 promoter driven luciferase activity and Sesn2 protein expression. In addition, dominant negative mutant of ATF6 blocked the Tg or Tm‐mediated Sesn2 induction. ER stress‐mediated cytotoxicity was reduced in Sesn2 over‐expressed HepG2 cells. Furthermore, we investigated whether Sesn2 protects Tm‐induced liver injury in mice infected with a recombinant adenovirus Sesn2 (Ad‐Sesn2). Injection of Ad‐Sesn2 less severe hepatic injury as supported by decrease in ALT, AST, LDH, and hepatocyte degeneration. Thus, our result indicates that ATF6 is essential for ER stress‐mediated Sesn2 induction, and the Sesn2 induction protects hepatocytes from ER stress‐induced liver injury.

Sestrin2–AMPK activation protects mitochondrial function against glucose deprivation-induced cytotoxicity

Sestrin2 (SESN2) regulates redox-homeostasis and apoptosis in response to various stresses. Although the antioxidant effects of SESN2 have been well established, the roles of SESN2 in mitochondrial function and metabolic stress have not yet been elucidated. In this study, we investigated the role of SESN2 in mitochondrial dysfunction under glucose deprivation and related signaling mechanisms. Glucose deprivation significantly upregulated SESN2 expression in hepatocyte-derived cells. Antioxidant treatments repressed SESN2 induction under glucose deprivation, this result suggested that reactive oxygen species (ROS) production was involved in SESN2 induction. Moreover, NF-E2-related factor-2 (Nrf2) phosphorylation was accompanied in induction of SESN2 by glucose deprivation. To elucidate the functional role of SESN2, we examined cells that stably overexpressed SESN2. Overexpression of SESN2 inhibited glucose deprivation-induced ROS production and cell death. In addition, under glucose deprivation, the changes in mitochondrial membrane potential, ADP/ATP ratio, and mitochondrial DNA content were significantly restored in SESN2-overexpressing cells. Moreover, siRNA knockdown of SESN2 failed to prevent mitochondrial permeability transition by glucose depletion. Mechanistic investigation showed that glucose deprivation significantly increased AMP-activated protein kinase (AMPK) activation. The recovery of mitochondrial function under glucose deprivation in SESN2-overexpressing cells was not seen in SESN2-overexpressing cells transfected with a dominant-negative AMPK; this result suggested that AMPK activation was responsible for SESN2-mediated mitochondrial protection against glucose deprivation. Treatment with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, an AMPK activator) also provided cytoprotective effects against glucose deprivation. Our findings provide evidence for the functional importance of SESN2–AMPK activation in the protection of mitochondria and cells against glucose deprivation-induced metabolic stress.

Regulation of Toll-like receptor-mediated Sestrin2 induction by AP-1, Nrf2, and the ubiquitin-proteasome system in macrophages.

The Sestrin2 (Sesn2) is an evolutionary conserved enzyme that scavenges reactive oxygen species and regulates autophagy through the AMPK-mTOR pathway. The present study was aimed at determining whether Toll-like receptor (TLR) signaling regulates Sesn2 expression and identifying the underlying molecular mechanism. Lipopolysaccharide (LPS), a representative TLR4 ligand, significantly increased the levels of Sesn2 protein in macrophages. LPS also increased Sesn2 mRNA levels and luciferase reporter activity; however, the mRNA levels of Sesn1 were not affected by LPS. Moreover, treatment of macrophages with other TLR ligands (eg, polyI:C or peptidoglycan) also induced Sesn2 expression. We found that LPS-mediated Sesn2 induction was transcriptionally regulated by AP-1 and Nrf2, and that overexpression of c-Jun or Nrf2 increased Sesn2 protein levels and Sesn2 promoter-driven luciferase reporter activity. Moreover, deletion of the antioxidant response element (ARE) in the Sesn2 promoter or Nrf2 knockout abolished LPS-mediated induction of Sesn2. LPS induced Sesn2 gene expression through p38 and PI3K activation. Surprisingly, treatment with the proteasome inhibitor MG132, but not the lysosomal inhibitor chloroquine, caused Sesn2 to accumulate in the cells. In the presence of MG132, we observed that Sesn2 was ubiquitinated. However, LPS treatment attenuated Sesn2 ubiquitination induced by MG132, which resulted in Sesn2 accumulation. Mice treated with D-galactosamine (Gal)/LPS exhibited enhanced Sesn2 expression in the liver. Moreover, infection with a recombinant adenovirus encoding Sens2 markedly reduced the number of Gal/LPS-induced TUNEL-positive cells. Our results suggest that TLR-mediated Sesn2 induction is dependent on AP-1, Nrf2, and the inhibition of ubiquitin-mediated degradation of Sesn2 and might protect cells against endotoxin toxicity.

Resveratrol attenuates methylglyoxal-induced mitochondrial dysfunction and apoptosis by Sestrin2 induction

Methylglyoxal is found in high levels in the blood and other tissues of diabetic patients and exerts deleterious effects on cells and tissues. Previously, we reported that resveratrol, a polyphenol in grapes, induced the expression of Sestrin2 (SESN2), a novel antioxidant protein, and inhibited hepatic lipogenesis. This study investigated whether resveratrol protects cells from the methylglyoxal-induced toxicity via SESN2 induction. Methylglyoxal significantly induced cell death in HepG2 cells. However, cells pretreated with resveratrol were rescued from methylglyoxal-induced apoptosis. Resveratrol attenuated glutathione (GSH) depletion and ROS production promoted by methylglyoxal. Moreover, mitochondrial damage was observed by methylglyoxal treatment, but resveratrol restored mitochondrial function, as evidenced by the observed lack of mitochondrial permeability transition and increased ADP/ATP ratio. Resveratrol treatment inhibited SESN2 depletion elicited by methylglyoxal. SESN2 overexpression repressed methylglyoxal-induced mitochondrial dysfunction and apoptosis. Likewise, rotenone-induced cytotoxicity was not observed in SESN2 overexpressed cells. Furthermore, siRNA knockdown of SESN2 reduced the ability of resveratrol to prevent methylglyoxal-induced mitochondrial permeability transition. In addition, when mice were exposed to methylglyoxal after infection of Ad-SESN2, the plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and GSH depletion by methylglyoxal in liver was reduced in Ad-SESN2 infected mice. Our results demonstrated that resveratrol is capable of protecting cells from methylglyoxal-induced mitochondrial dysfunction and oxidative stress via SESN2 induction.

Upregulation of sestrin-2 expression protects against endothelial toxicity of angiotensin II.

Sestrin-2 (SESN2) is involved in the cellular response to different stress conditions. However, the function of SESN2 in the cardiovascular system remains unknown. In the present study, we tested whether SESN2 has a beneficial effect on vascular endothelial damage induced by angiotensin II (AngII). Firstly, we found that AngII induces expression of SESN2 in human umbilical vein endothelial cells (HUVECs) in a time-dependent and dose-dependent manner. We also found that knockdown of SESN2 using small RNA interference promotes cellular toxicity of AngII, as well as a reduction in cell viability, exacerbation of oxidative stress, and stimulation of apoptosis. In addition, our results show that the c-Jun NH (2)-terminal kinase (JNK)/c-Jun pathway is activated by AngII. Inhibiting the activity of the JNK pathway abolishes the increase in SESN2 induced by AngII. Importantly, overexpression of c-Jun promotes luciferase activity of the SESN2 promoter. These findings suggest that the inductive effect of SESN2 is mediated by the JNK/c-Jun pathway. Our results indicate that the induction of SESN2 acts as a compensatory response to AngII for survival, implying that stimulating expression of SESN2 might be an effective pharmacological target for the treatment of AngII-associated cardiovascular diseases.

Upregulation of Sestrin-2 Expression via P53 Protects Against 1-Methyl-4-Phenylpyridinium (MPP+) Neurotoxicity

Sestrin-2 (SESN2) is a conserved antioxidant protein that is activated upon oxidative stress and protects cells against reactive oxygen species (ROS). However, the role of SESN2 in neurodegenerative diseases, especially in Parkinson's disease (PD), has not yet been elucidated. In this study, we found that expression of SESN2 is elevated in the midbrain of patients with PD. Moreover, in vitro experiments display that the drug 1-methyl-4-phenylpyridinium (MPP+) induces the expression of SESN2 in SH-SY5Y cells in a time- and dose-dependent manner. Our results show that p53 is activated by MPP+. Importantly, inhibition of p53 using small RNA interferences abolishes the increased SESN2 levels induced by MPP+, suggesting that the inductive effect of MPP+ on SESN2 is mediated by p53. Furthermore, knockdown of SESN2 using small RNA interferences promotes MPP+-related neurotoxicity by attenuating oxidative stress, mitochondrial dysfunction, and apoptosis. All these data imply that the induction of SESN2 produces a protective effect in PD.

Nrf2-ARE pathway regulates induction of Sestrin-2 expression

The Sestrin2 (Sesn2) gene encodes a conserved antioxidant protein that is induced on oxidative stress and protects cells against reactive oxygen species. NF-E2-related factor-2 (Nrf2) is an essential transcription factor that regulates expression of a variety of antioxidant genes via binding to the antioxidant-response element (ARE), but the role of Nrf2 in Sesn2 gene expression has not been elucidated yet. The present study investigated whether the Nrf2-ARE pathway regulates Sesn2 gene expression and the identification of the molecular mechanism. The Nrf2 activators, tert-butylhydroquinone (t-BHQ) and sulforaphane (SFN), up-regulated Sesn2 expression in a dose- and time-dependent manner in hepatocytes. Also, t-BHQ increased Sesn2 mRNA and luciferase gene activity, whereas the levels of Sesn1 and Sesn3 mRNA were not affected by t-BHQ treatment. The specific role of Nrf2 in Sesn2 induction was verified by using Nrf2 overexpression plasmid and Nrf2 knockout or knockdown cells. In silico analysis of the 5′ upstream region of Sesn2 gene identified a putative ARE sequence. Deletion of the putative ARE demonstrated that the ARE from −550 to −539bp in the human Sesn2 promoter was critical for the Nrf2-mediated response. Moreover, SFN injection increased Sesn2 mRNA and protein levels in the livers of mice. Knockdown experiments with Sesn2 siRNA showed that Sesn2 is required for the Nrf2-mediated cytoprotective activity against hydrogen peroxide. Our results suggest that the Nrf2-ARE pathway is critical for Sesn2 gene expression and might protect against oxidative stress.

Free radical biology and medicine

The Sestrin2 (Sesn2) gene encodes a conserved antioxidant protein that is induced on oxidative stress and protects cells against reactive oxygen species. NF-E2-related factor-2 (Nrf2) is an essential transcription factor that regulates expression of a variety of antioxidant genes via binding to the antioxidant-response element (ARE), but the role of Nrf2 in Sesn2 gene expression has not been elucidated yet. The present study investigated whether the Nrf2-ARE pathway regulates Sesn2 gene expression and the identification of the molecular mechanism. The Nrf2 activators, tert-butylhydroquinone (t-BHQ) and sulforaphane (SFN), up-regulated Sesn2 expression in a dose- and time-dependent manner in hepatocytes. Also, t-BHQ increased Sesn2 mRNA and luciferase gene activity, whereas the levels of Sesn1 and Sesn3 mRNA were not affected by t-BHQ treatment. The specific role of Nrf2 in Sesn2 induction was verified by using Nrf2 overexpression plasmid and Nrf2 knockout or knockdown cells. In silico analysis of the 5 0 upstream region of Sesn2 gene identified a putative ARE sequence. Deletion of the putative ARE demonstrated that the ARE from � 550 to � 539 bp in the human Sesn2 promoter was critical for the Nrf2-mediated response. Moreover, SFN injection increased Sesn2 mRNA and protein levels in the livers of mice. Knockdown experiments with Sesn2 siRNA showed that Sesn2 is required for the Nrf2-mediated cytoprotective activity against hydrogen peroxide. Our results suggest that the Nrf2-ARE pathway is critical for Sesn2 gene expression and might protect against oxidative stress.