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.     

SESN2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting NLRP3 activation in macrophages

Proper regulation of mitophagy for mitochondrial homeostasis is important in various inflammatory diseases. However, the precise mechanisms by which mitophagy is activated to regulate inflammatory responses remain largely unknown. The NLRP3 (NLR family, pyrin domain containing 3) inflammasome serves as a platform that triggers the activation of CASP1 (caspase 1) and secretion of proinflammatory cytokines. Here, we demonstrate that SESN2 (sestrin 2), known as stress-inducible protein, suppresses prolonged NLRP3 inflammasome activation by clearance of damaged mitochondria through inducing mitophagy in macrophages. SESN2 plays a dual role in inducing mitophagy in response to inflammasome activation. First, SESN2 induces “mitochondrial priming” by marking mitochondria for recognition by the autophagic machinery. For mitochondrial preparing, SESN2 facilitates the perinuclear-clustering of mitochondria by mediating aggregation of SQSTM1 (sequestosome 1) and its binding to lysine 63 (Lys63)-linked ubiquitins on the mitochondrial surface. Second, SESN2 activates the specific autophagic machinery for degradation of primed mitochondria via an increase of ULK1 (unc-51 like kinase 1) protein levels. Moreover, increased SESN2 expression by extended LPS (lipopolysaccharide) stimulation is mediated by NOS2 (nitric oxide synthase 2, inducible)-mediated NO (nitric oxide) in macrophages. Thus, Sesn2-deficient mice displayed defective mitophagy, which resulted in hyperactivation of inflammasomes and increased mortality in 2 different sepsis models. Our findings define a unique regulatory mechanism of mitophagy activation for immunological homeostasis that protects the host from sepsis.     

Association of platelet-derived growth factor receptor β accumulation with increased oxidative stress and cellular injury in sestrin 2 silenced human glioblastoma cells

Sestrin 2 (SESN2) is a stress-inducible protein required for maintaining redox homeostasis. However, its mode of action remains to be clarified. In this study, we found that SESN2 is induced in human glioblastoma U87 cells following ionizing radiation (IR). SESN2 silencing not only results in increased oxidative stress but also sensitizes U87 cells to IR. Intriguingly, we found SESN2 silencing significantly increases the expression of platelet-derived growth factor receptor β (PDGFRβ). Using a double knockdown technique, we showed that inhibition of PDGFRβ accumulation in SESN2-silencing cells protects the cells from the deleterious effects induced by SESN2 silencing. Taken together, we revealed that PDGFRβ accumulation is associated with increased oxidative stress and cellular damage in SESN2 silenced human glioblastoma U87 cells.     

Sestrin 2 attenuates sepsis‐associated encephalopathy through the promotion of autophagy in hippocampal neurons

Sepsis‐associated encephalopathy (SAE) has typically been associated with a poor prognosis. Although sestrin 2 (SESN2) plays a crucial role in metabolic regulation and the stress response, its expression and functional roles in SAE are still unclear. In the present study, SAE was established in mice through caecal ligation and puncture (CLP). The adeno‐associated virus 2 (AAV2)‐mediated SESN2 expression (ie overexpression and knockdown) system was injected into the hippocampi of mice with SAE, and subsequently followed by electron microscopic analysis, the Morris water maze task and pathological examination. Our results demonstrated an increase of SESN2 in the hippocampal neurons of mice with SAE, 2‐16 hours following CLP. AAV2‐mediated ectopic expression of SESN2 attenuated brain damage and loss of learning and memory functions in mice with SAE, and these effects were associated with lower pro‐inflammatory cytokines in the hippocampus. Mechanistically, SESN2 promoted unc‐51‐like kinase 1 (ULK1)‐dependent autophagy in hippocampal neurons through the activation of the AMPK/mTOR signalling pathway. Finally, AMPK inhibition by SBI‐0206965 blocked SESN2‐mediated attenuation of SAE in mice. In conclusion, our findings demonstrated that SESN2 might be a novel pharmacological intervention strategy for SAE treatment through promotion of ULK1‐dependent autophagy in hippocampal neurons.     

Potential role of mitochondrial ROS in Sestrin2 degradation

Sestrin2 (SESN2) is a stress-inducible antioxidant protein that controls redox-homeostasis via its oxidoreductase activity. Although the induction mechanism of SESN2 has been reported, the mechanism for regulation of SESN2 protein stability has not yet been elucidated. In the present study, we investigated the role of mitochondrial reactive oxygen species (ROS) in the degradation of SESN2 and the related mechanism. First, we found that rotenone, an inhibitor of mitochondrial complex I, significantly lowered the SESN2 protein level in hepatocytes. In addition, antimycin A (an inhibitor of mitochondrial complex III) and Mn-TBAP (a superoxide dismutase mimetic) were also shown to reduce SESN2. Rotenone decreased mitochondrial ROS; this result implied that mitochondrial ROS production was involved in SESN2 regulation. However, the level of SESN2 mRNA was unchanged by rotenone treatment. Next, SESN2 protein stability was observed after treatment with cycloheximide and rotenone. Decreased SESN2 protein stability was detected in the presence of cycloheximide, and rotenone accelerated this SESN2 decay. MG132 (a proteasome inhibitor) treatment causes significant accumulation of SESN2 protein, but rotenone still reduced SESN2 levels in the presence of MG132. However, chloroquine, a lysosomal inhibitor, restored rotenone-reduced SESN2 levels. These results suggest that mitochondrial ROS prevent SESN2 protein degradation, and this is dependent on lysosomal activity.     

Reduced expression of FASN through SREBP‐1 down‐regulation is responsible for hypoxic cell death in HepG2 cells

Cells under hypoxic stress either activate an adaptive response or undergo cell death. Although some mechanisms have been reported, the exact mechanism behind hypoxic cell death remains unclear. Recently, increased expression of fatty acid synthase (FASN) has been observed in various human cancers. In highly proliferating cells, tumor‐associated FASN is considered necessary for both membrane lipids production and post‐translational protein modification, but the exact mechanisms are not fully understood. Further, FASN overexpression is associated with aggressive and malignant cancer diseases and FASN inhibition induces apoptosis in cancer cells. For this reason, FASN is emerging as a key target for the potential diagnosis and treatment of various cancers. Here, we observed decreased FASN expression under hypoxic cell death conditions in HepG2 cells. Thus, we examined the effect of decreased FASN expression on hypoxia‐induced cell death in HepG2 cells and also investigated the mechanism responsible for reduction of FASN expression under hypoxic cell death conditions. As a result, reduction of FASN expression resulted in hypoxic cell death via malonyl‐CoA accumulation. In addition, SREBP‐1 restored FASN reduction and hypoxia‐induced apoptosis. Taken together, we suggest that hypoxic cell death is promoted by the reduced expression of FASN through SREBP‐1 down‐regulation. J. Cell. Biochem. 113: 3730–3739, 2012. © 2012 Wiley Periodicals, Inc.     

Aerobic exercise ameliorates insulin resistance in C57BL/6 J mice via activating Sestrin3

Skeletal muscle insulin resistance (IR) is closely linked to hyperglycemia and metabolic disorders. Regular exercise enhances insulin sensitivity in skeletal muscle, but its underlying mechanisms remain unknown. Sestrin3 (SESN3) is a stress-inducible protein that protects against obesity-induced hepatic steatosis and insulin resistance. Regular exercise training is known to increase SESN3 expression in skeletal muscle. The purpose of this study was to explore whether SESN3 mediates the metabolic effects of exercise in the mouse model of high-fat diet (HFD)-induced IR. SESN3^?/? mice exhibited severer body weight gain, ectopic lipid accumulation, and dysregulation of glucose metabolism after long-term HFD feeding compared with the wild-type (WT) mice. Moreover, we found that SESN3 deficiency weakened the effects of exercise on reducing serum insulin levels and improving glucose tolerance in mice. Exercise training increased pAKT-S473 and GLUT4 expression, accompanied by enhanced pmTOR-S2481 (an indicator of mTORC2 activity) in WT quadriceps that were less pronounced in SESN3^?/? mice. SESN3 overexpression in C2C12 myotubes further confirmed that SESN3 played an important role in skeletal muscle glucose metabolism. SESN3 overexpression increased the binding of Rictor to mTOR and pmTOR-S2481 in C2C12 myotubes. Moreover, SESN3 overexpression resulted in an elevation of glucose uptake and a concomitant increase of pAKT-S473 in C2C12 myotubes, whereas these effects were diminished by downregulation of mTORC2 activity. Taken together, SESN3 is a crucial protein in amplifying the beneficial effects of exercise on insulin sensitivity in skeletal muscle and systemic glucose levels. SESN3/mTORC2/AKT pathway mediated the effects of exercise on skeletal muscle insulin sensitivity.     

Sestrin2 modulates AMPK subunit expression and its response to ionizing radiation in breast cancer cells

Background

The sestrin family of stress-responsive genes (SESN1-3) are suggested to be involved in regulation of metabolism and aging through modulation of the AMPK-mTOR pathway. AMP-activated protein kinase (AMPK) is an effector of the tumour suppressor LKB1, which regulates energy homeostasis, cell polarity, and the cell cycle. SESN1/2 can interact directly with AMPK in response to stress to maintain genomic integrity and suppress tumorigenesis. Ionizing radiation (IR), a widely used cancer therapy, is known to increase sestrin expression, and acutely activate AMPK. However, the regulation of AMPK expression by sestrins in response to IR has not been studied in depth.

Methods and Findings

Through immunoprecipitation we observed that SESN2 directly interacted with the AMPKα1β1γ1 trimer and its upstream regulator LKB1 in MCF7 breast cancer cells. SESN2 overexpression was achieved using a Flag-tagged SESN2 expression vector or a stably-integrated tetracycline-inducible system, which also increased AMPKα1 and AMPKβ1 subunit phosphorylation, and co-localized with phosphorylated AMPKα-Thr127 in the cytoplasm. Furthermore, enhanced SESN2 expression increased protein levels of LKB1 and AMPKα1β1γ1, as well as mRNA levels of LKB1, AMPKα1, and AMPKβ1. Treatment of MCF7 cells with IR elevated AMPK expression and activity, but this effect was attenuated in the presence of SESN2 siRNA. In addition, elevated SESN2 inhibited IR-induced mTOR signalling and sensitized MCF7 cells to IR through an AMPK-dependent mechanism.

Conclusions

Our results suggest that in breast cancer cells SESN2 is associated with AMPK, it is involved in regulation of basal and IR-induced expression and activation of this enzyme, and it mediates sensitization of cancer cells to IR.     

ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF-1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2alpha or by a dominant-negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.     

Exercise improves glucose uptake in murine myotubes through the AMPKα2-mediated induction of Sestrins

Exercise training increases insulin sensitivity. Over the past decades, considerable progress has been made in understanding the molecular basis for this important effect of physical exercise. However, the underlying mechanism is still not fully described. Recent studies have revealed that the stress responsive protein family Sestrins (SESNs) may play an important role in improving insulin sensitivity of skeletal muscle under exercise training. In this study, we aim to better understand the relationship between SESNs and AMPK in response to exercise training and the possible mechanism by which SESNs mediate glucose metabolism. We used wild type, AMPKα2^+/? and AMPKα2^?/? C57BL/6 mice to reveal the pathway by which 6?weeks of exercise training induced SESNs. We explored the mechanism through which SESNs regulated glucose metabolism in vitro by overexpressing or inhibiting SESNs, and inhibiting AMPK or autophagy in myotubes. We found that a 6-week exercise training regime improved oxidative metabolism, activated the insulin signaling pathway and increased the level of SESN2 and SESN3 in an AMPKα2-dependent manner. Overexpression of SESN3 or SESN2 and SESN3 together increased glucose uptake, activated the insulin signaling pathway, and promoted GLUT4 translocation in myotubes. Although inhibition of SESNs had no effect on glucose uptake, SESNs could reverse reduced glucose uptake following autophagy inhibition, and may be downstream effectors of AMPK responses in myotubes. Taken together our data show that SESNs are induced by AMPKα2 after exercise training, and SESNs, specifically SESN3, play a key role in exercise training-mediated glucose metabolism in skeletal muscle.     

Role of gp91phox-containing NADPH oxidase in left ventricular remodeling induced by intermittent hypoxic stress

Intermittent hypoxia due to sleep apnea syndrome is associated with cardiovascular diseases. However, the precise mechanisms by which intermittent hypoxic stress accelerates cardiovascular diseases are largely unclear. The aim of this study was to investigate the role of gp91(phox)-containing NADPH oxidase in the development of left ventricular (LV) remodeling induced by intermittent hypoxic stress in mice. Male gp91(phox)-deficient (gp91(-/-)) mice (n = 26) and wild-type (n = 39) mice at 7-12 wk of age were exposed to intermittent hypoxia (30 s of 4.5-5.5% O(2) followed by 30 s of 21% O(2) for 8 h/day during daytime) or normoxia for 10 days. Mean blood pressure and LV systolic and diastolic function were not changed by intermittent hypoxia in wild-type or gp91(-/-) mice, although right ventricular systolic pressure tended to be increased. In wild-type mice, intermittent hypoxic stress significantly increased the diameter of cardiomyocytes and interstitial fibrosis in LV myocardium. Furthermore, intermittent hypoxic stress increased superoxide production, 4-hydroxy-2-nonenal protein, TNF-alpha and transforming growth factor-beta mRNA, and NF-kappaB binding activity in wild-type, but not gp91(-/-), mice. These results suggest that gp91(phox)-containing NADPH oxidase plays a crucial role in the pathophysiology of intermittent hypoxia-induced LV remodeling through an increase of oxidative stress.     

Cigarette smoke exposure induces ROS-mediated autophagy by regulating sestrin,AMPK, and mTOR level in mice

Many pathological conditions linked to cigarette smoking are caused by the production of reactive oxygen species (ROS). The present study was conducted to analyze the effect of ROS on the lungs of Swiss mice exposed to cigarette smoking, focusing on autophagy-mediated mechanisms, and investigate the involvement of SESN2, AMPK, and mTOR signaling. Mice were exposed to cigarette smoke (CS) for 7, 15, 30, 45, and 60 days; the control group was not exposed to CS. Only mice exposed to CS for 45 days were selected for subsequent N-acetylcysteine (NAC) supplementation and smoke cessation analyses. Exposure to CS increased the production of ROS and induced molecular changes in the autophagy pathway, including an increase in phosphorylated AMPK and ULK1, reduction in phosphorylated mTOR, and increases in SESN2, ATG12, and LC3B levels. NAC supplementation reduced ROS levels and reversed all molecular changes observed upon CS treatment, suggesting the involvement of oxidative stress in inducing autophagy upon CS exposure. When exposure to CS was stopped, there were decreases in the levels of oxidative stress, AMPK and ULK1 phosphorylation, and autophagy-initiating molecules and increase in mTOR phosphorylation. In conclusion, these results suggest the involvement of ROS, SESN2, AMPK, and mTOR in the CS-induced autophagic process in the lung.     

Protective Role of miR-34c in Hypoxia by Activating Autophagy through BCL2 Repression

Hypoxia leads to significant cellular stress that has diverse pathological consequences such as cardiovascular diseases and cancers. MicroRNAs (miRNAs) are one of regulators of the adaptive pathway in hypoxia. We identified a hypoxia-induced miRNA, miR-34c, that was significantly upregulated in hypoxic human umbilical cord vein endothelial cells (HUVECs) and in murine blood vessels on day 3 of hindlimb ischemia (HLI). miR-34c directly inhibited BCL2 expression, acting as a toggle switch between apoptosis and autophagy in vitro and in vivo. BCL2 repression by miR-34c activated autophagy, which was evaluated by the expression of LC3-II. Overexpression of miR-34c inhibited apoptosis in HUVEC as well as in a murine model of HLI, and increased cell viability in HUVEC. Importantly, the number of viable cells in the blood vessels following HLI was increased by miR-34c overexpression. Collectively, our findings show that miR-34c plays a protective role in hypoxia, suggesting a novel therapeutic target for hypoxic and ischemic diseases in the blood vessels.