Regulation Of Manganese Superoxide Dismutase Research Articles

Cloning of Mn-SOD gene and its mRNA expression difference and antioxidant enzyme activities under hypoxia stress of cobia Rachycentron canadum.

Environmental hypoxia affects the survival and development of organisms. It is also an important environmental factor that leads to oxidative damage. Hypoxia is a condition in which tissues are deprived of oxygen; reoxygenation is the phenomenon in which hypoxic tissues are exposed to oxygen. Hypoxia-reoxygenation is vital in pathogenesis, where the production of reactive oxygen species and antioxidant disparity significantly contribute to disease progression, and it is one of the most common physiological stressors in the aquaculture industry. In this study, the full length of complementary DNA (cDNA) of the manganese superoxide dismutase (Mn-SOD) gene of healthy cobia Rachycentron canadum was analysed using rapid amplification of cDNA ends. The real-time quantitative Polymerase Chain Reaction was used to measure the expression levels of Mn-SOD mRNAs in various tissues (heart, muscle, brain, liver, kidney, gill, intestine, and spleen). The 2-ΔΔCT method was used to performed the expression analysis. The experimental data were analysed using SPSS ver. 19.0 ( https://spss.software.informer.com/19.0/ ). P < 0.05 and P < 0.01 were set as significant differences. The values were articulated as mean ± standard deviation. The Mn-SOD gene cDNA sequence was 1209bp long, including a 684bp open reading frame, 42bp 5'UTR and 483bp 3'UTR, encoding 227 amino acids. Under hypoxia-reoxygen stress, the expression of Mn-SOD in brain tissue was significantly lower than in the control group after 8h of reoxygenation and higher than the control group after 24h. Hypoxia and subsequent reoxygenation triggered a disturbance in antioxidant homeostasis, displayed in the modification of GPx expression/activity in the liver: GPx was improved. These results provide valuable information on the role of Mn-SOD regulation in oxidative stress caused by hypoxia.

Sirtuin 3 Inhibits Airway Epithelial Mitochondrial Oxidative Stress in Cigarette Smoke-Induced COPD.

Mitochondrial damage in airway epithelial cells plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Sirtuin 3 (Sirt3) is a mitochondrial deacetylase regulating mitochondrial function, but its role in the pathogenesis of COPD is still unknown. The aim of the present study was to investigate the effect of Sirt3 on airway epithelial mitochondria in cigarette smoke-induced COPD. Our present study has shown serious airway inflammation, alveolar space enlargement, and mitochondrial damage of the airway epithelium in COPD rats. Compared to the control rats, Sirt3 protein expression was significantly decreased in the airway epithelium and lung tissue homogenate from COPD rats. In airway epithelial cells (BEAS-2B), cigarette smoke extract (CSE) treatment significantly decreased mRNA and protein expression of Sirt3 and manganese superoxide dismutase (MnSOD), as well as MnSOD activity in a concentration and time-dependent manner. Sirt3 siRNA further significantly intensified the decreases in MnSOD expression and activity and aggravated mitochondrial oxidative stress and cell injury when airway epithelial cells were treated with 7.5% CSE. In contrast, Sirt3 overexpression significantly prevented the decrease of MnSOD expression and activity and improved mitochondrial oxidative stress and cell injury in CSE-treated airway epithelial cells. These data suggest that Sirt3 inhibits airway epithelial mitochondrial oxidative stress possibly through the regulation of MnSOD, thereby contributing to the pathogenesis of COPD.

Manganese Superoxide Dismutase Dysfunction and the Pathogenesis of Kidney Disease.

The mitochondria are a major source of reactive oxygen species (ROS). Superoxide anion (O2•–) is produced by the process of oxidative phosphorylation associated with glucose, amino acid, and fatty acid metabolism, resulting in the production of adenosine triphosphate (ATP) in the mitochondria. Excess production of reactive oxidants in the mitochondria, including O2•–, and its by-product, peroxynitrite (ONOO–), which is generated by a reaction between O2•– with nitric oxide (NO•), alters cellular function via oxidative modification of proteins, lipids, and nucleic acids. Mitochondria maintain an antioxidant enzyme system that eliminates excess ROS; manganese superoxide dismutase (Mn-SOD) is one of the major components of this system, as it catalyzes the first step involved in scavenging ROS. Reduced expression and/or the activity of Mn-SOD results in diminished mitochondrial antioxidant capacity; this can impair the overall health of the cell by altering mitochondrial function and may lead to the development and progression of kidney disease. Targeted therapeutic agents may protect mitochondrial proteins, including Mn-SOD against oxidative stress-induced dysfunction, and this may consequently lead to the protection of renal function. Here, we describe the biological function and regulation of Mn-SOD and review the significance of mitochondrial oxidative stress concerning the pathogenesis of kidney diseases, including chronic kidney disease (CKD) and acute kidney injury (AKI), with a focus on Mn-SOD dysfunction.

Role of MnSOD in AIFm2‐Mediated Apoptosis During Cardiac Ischemia/Reperfusion Injury

Role of MnSOD in AIFm2‐Mediated Apoptosis During Cardiac Ischemia/Reperfusion Injury

Estrogen Protects the Female Heart from Ischemia/Reperfusion Injury through Manganese Superoxide Dismutase Phosphorylation by Mitochondrial p38β at Threonine 79 and Serine 106

A collective body of evidence indicates that estrogen protects the heart from myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism remains incompletely understood. We have previously delineated a novel mechanism of how 17β-estradiol (E2) protects cultured neonatal rat cardiomyocytes from hypoxia/reoxygenation (H/R) by identifying a functionally active mitochondrial pool of p38β and E2-driven upregulation of manganese superoxide dismutase (MnSOD) activity via p38β, leading to the suppression of reactive oxygen species (ROS) and apoptosis. Here we investigate these cytoprotective actions of E2 in vivo. Left coronary artery ligation and reperfusion was used to produce I/R injury in ovariectomized (OVX) female mice and in estrogen receptor (ER) null female mice. E2 treatment in OVX mice reduced the left ventricular infarct size accompanied by increased activity of mitochondrial p38β and MnSOD. I/R-induced infarct size in ERα knockout (ERKO), ERβ knockout (BERKO) and ERα and β double knockout (DERKO) female mice was larger than that in wild type (WT) mice, with little difference among ERKO, BERKO, and DERKO. Loss of both ERα and ERβ led to reduced activity of mitochondrial p38β and MnSOD at baseline and after I/R. The physical interaction between mitochondrial p38β and MnSOD in the heart was detected by co-immunoprecipitation (co-IP). Threonine 79 (T79) and serine 106 (S106) of MnSOD were identified to be phosphorylated by p38β in kinase assays. Overexpression of WT MnSOD in cardiomyocytes reduced ROS generation during H/R, while point mutation of T79 and S106 of MnSOD to alanine abolished its antioxidative function. We conclude that the protective effects of E2 and ER against cardiac I/R injury involve the regulation of MnSOD via posttranslational modification of the dismutase by p38β.

CDK4-mediated MnSOD activation and mitochondrial homeostasis in radioadaptive protection

Mammalian cells are able to sense environmental oxidative and genotoxic conditions such as the environmental low-dose ionizing radiation (LDIR) present naturally on the earth’s surface. The stressed cells then can induce a so-called radioadaptive response with an enhanced cellular homeostasis and repair capacity against subsequent similar genotoxic conditions such as a high dose radiation. Manganese superoxide dismutase (MnSOD), a primary mitochondrial antioxidant in mammals, has long been known to play a crucial role in radioadaptive protection by detoxifying O2•− generated by mitochondrial oxidative phosphorylation. In contrast to the well-studied mechanisms of SOD2 gene regulation, the mechanisms underlying posttranslational regulation of MnSOD for radioprotection remain to be defined. Herein, we demonstrate that cyclin D1/cyclin-dependent kinase 4 (CDK4) serves as the messenger to deliver the stress signal to mitochondria to boost mitochondrial homeostasis in human skin keratinocytes under LDIR-adaptive radioprotection. Cyclin D1/CDK4 relocates to mitochondria at the same time as MnSOD enzymatic activation peaks without significant changes in total MnSOD protein level. The mitochondrial-localized CDK4 directly phosphorylates MnSOD at serine-106 (S106), causing enhanced MnSOD enzymatic activity and mitochondrial respiration. Expression of mitochondria-targeted dominant negative CDK4 or the MnSOD-S106 mutant reverses LDIR-induced mitochondrial enhancement and adaptive protection. The CDK4-mediated MnSOD activation and mitochondrial metabolism boost are also detected in skin tissues of mice receiving in vivo whole-body LDIR. These results demonstrate a unique CDK4-mediated mitochondrial communication that allows cells to sense environmental genotoxic stress and boost mitochondrial homeostasis by enhancing phosphorylation and activation of MnSOD.

Free Fatty Acids, Lipopolysaccharide and IL-1α Induce Adipocyte Manganese Superoxide Dismutase Which Is Increased in Visceral Adipose Tissues of Obese Rodents

Excess fat storage in adipocytes is associated with increased generation of reactive oxygen species (ROS) and impaired activity of antioxidant mechanisms. Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme involved in detoxification of ROS, and objective of the current study is to analyze expression and regulation of MnSOD in obesity. MnSOD is increased in visceral but not subcutaneous fat depots of rodents kept on high fat diets (HFD) and ob/ob mice. MnSOD is elevated in visceral adipocytes of fat fed mice and exposure of differentiating 3T3-L1 cells to lipopolysaccharide, IL-1α, saturated, monounsaturated and polyunsaturated free fatty acids (FFA) upregulates its level. FFA do not alter cytochrome oxidase 4 arguing against overall induction of mitochondrial enzymes. Upregulation of MnSOD in fat loaded cells is not mediated by IL-6, TNF or sterol regulatory element binding protein 2 which are induced in these cells. MnSOD is similarly abundant in perirenal fat of Zucker diabetic rats and non-diabetic animals with similar body weight and glucose has no effect on MnSOD in 3T3-L1 cells. To evaluate whether MnSOD affects adipocyte fat storage, MnSOD was knocked-down in adipocytes for the last three days of differentiation and in mature adipocytes. Knock-down of MnSOD does neither alter lipid storage nor viability of these cells. Heme oxygenase-1 which is induced upon oxidative stress is not altered while antioxidative capacity of the cells is modestly reduced. Current data show that inflammation and excess triglyceride storage raise adipocyte MnSOD which is induced in epididymal adipocytes in obesity.

Mitochondrial p38β and Manganese Superoxide Dismutase Interaction Mediated by Estrogen in Cardiomyocytes

AimsWhile etiology behind the observed acceleration of ischemic heart disease in postmenopausal women is poorly understood, collective scientific data suggest cardioprotective effects of the endogenous female sex hormone, estrogen. We have previously shown that 17β-estradiol (E2) protects cardiomyocytes exposed to hypoxia-reoxygenation (H/R) by inhibiting p38α - p53 signaling in apoptosis and activating pro-survival p38β mitogen activated protein kinase (p38β MAPK), leading to suppression of reactive oxygen species (ROS) post H/R. However, little is known about the mechanism behind the antioxidant actions of E2-dependent p38β. The aim of this study is to determine whether the cytoprotection by estrogen involves regulation of manganese superoxide dismutase (MnSOD), a major mitochondrial ROS scavenging enzyme, via cardiac p38β.Methods and ResultsWe identified mitochondrial p38β by immunocytochemistry and by immunoblotting in mitochondria isolated from neonatal cardiomyocytes of Sprague-Dawley rats. E2 facilitated the mitochondrial localization of the active form of the kinase, phosphorylated p38β (p-p38β). E2 also reduced the H/R-induced mitochondrial membrane potential decline, augmented the MnSOD activity and suppressed anion superoxide generation, while the dismutase protein expression remained unaltered. Co-immunoprecipitation studies showed physical association between MnSOD and p38β. p38β phosphorylated MnSOD in an E2-dependent manner in in-vitro kinase assays.ConclusionThis work demonstrates for the first time a mitochondrial pool of active p38β and E2-mediated phosphorylation of MnSOD by the kinase. The results shed light on the mechanism behind the cytoprotective actions of E2 in cardiomyocytes under oxidative stress.

Regulation of MnSOD Enzymatic Activity by Sirt3 Connects the Mitochondrial Acetylome Signaling Networks to Aging and Carcinogenesis

It is a well-established scientific observation that mammalian cells contain fidelity or watchdog proteins that maintain the correct function of cellular organelles. Over the past several years, the Sirtuin deacetylase family protein Sirt3 has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species (ROS) scavenging proteins. Loss of function or genetic mutation of these fidelity proteins has been shown to create a cellular environment that is permissive for the development of cellular damage associated with processes such as aging and carcinogenesis. Mitochondria are the primary organelles that direct oxidative metabolism for the production of ATP; however, this is also a significant source of ROS. Thus, it is reasonable to propose that mitochondria should contain proteins that would signal downstream target molecules and/or ROS scavenger enzymes to maintain mitochondrial and cellular homeostatic poise. It is also reasonable to hypothesize that the mitochondria contain fidelity proteins similar to those found in the nucleus and cytoplasm. We discuss a new role of Sirt3 in the direction of the primary superoxide scavenger protein, manganese superoxide dismutase (MnSOD), and how the acetylation or deacetylation of several specific lysines appears to direct MnSOD enzymatic dismutase activity. Aberrant downstream regulation of MnSOD by Sirt3 may be a potential source of cellular damage that accumulates with aging to create a tumor-permissive phenotype. Future studies can explore the role of MnSOD in age-related illness using this new mechanism of enzymatic regulation.

MnSOD in Oxidative Stress Response-Potential RegulationviaMitochondrial Protein Influx

The mitochondrial antioxidant manganese superoxide dismutase (MnSOD) is encoded by genomic DNA and its dismutase function is fully activated in the mitochondria to detoxify free radical O2(•-) generated by mitochondrial respiration. Accumulating evidence shows an extensive communication between the mitochondria and cytoplasm under oxidative stress. Not only is the MnSOD gene upregulated by oxidative stress, but MnSOD activity can be enhanced via the mitochondrial protein influx (MPI). A cluster of MPI containing cytoplasmic/nuclear proteins, such as cyclins, cyclin-dependent kinases, and p53 interact with and alter MnSOD activity. These proteins modulate MnSOD superoxide scavenging activity via post-translational modifications in the mitochondria. In addition to well-established pathways in gene expression, recent findings suggest that MnSOD enzymatic activity can also be enhanced by phosphorylation of specific motifs in mitochondria. This review attempts to discuss the pre- and post-translational regulation of MnSOD, and how these modifications alter MnSOD activity, which induces a cell adaptive response to oxidative stress. MnSOD is biologically significant to aerobic cells. Its role in protecting the cells against the deleterious effects of reactive oxygen species is evident. However, the exact network of MnSOD-associated cellular adaptive reaction to oxidative stress and its post-translational modifications, especially its enzymatic enhancement via phosphorylation, is not yet fully understood. The broad discussion of the multiple aspects of MnSOD regulation, including gene expression, protein modifications, and enzymatic activity, will shed light onto the unknown mechanisms that govern the prosurvival networks involved in cellular and mitochondrial adaptive response to genotoxic environment.

Type 5 Adenylyl Cyclase Increases Oxidative Stress by Transcriptional Regulation of Manganese Superoxide Dismutase via the SIRT1/FoxO3a Pathway

For reasons that remain unclear, whether type 5 adenylyl cyclase (AC5), 1 of 2 major AC isoforms in heart, is protective or deleterious in response to cardiac stress is controversial. To reconcile this controversy we examined the cardiomyopathy induced by chronic isoproterenol in AC5 transgenic (Tg) mice and the signaling mechanisms involved. Chronic isoproterenol increased oxidative stress and induced more severe cardiomyopathy in AC5 Tg, as left ventricular ejection fraction fell 1.9-fold more than wild type, along with greater left ventricular dilation and increased fibrosis, apoptosis, and hypertrophy. Oxidative stress induced by chronic isoproterenol, detected by 8-OhDG was 15% greater, P=0.007, in AC5 Tg hearts, whereas protein expression of manganese superoxide dismutase (MnSOD) was reduced by 38%, indicating that the susceptibility of AC5 Tg to cardiomyopathy may be attributable to decreased MnSOD expression. Consistent with this, susceptibility of the AC5 Tg to cardiomyopathy was suppressed by overexpression of MnSOD, whereas protection afforded by the AC5 knockout (KO) was lost in AC5 KO×MnSOD heterozyous KO mice. Elevation of MnSOD was eliminated by both sirtuin and MEK inhibitors, suggesting both the SIRT1/FoxO3a and MEK/ERK pathway are involved in MnSOD regulation by AC5. Overexpression of AC5 exacerbates the cardiomyopathy induced by chronic catecholamine stress by altering regulation of SIRT1/FoxO3a, MEK/ERK, and MnSOD, resulting in oxidative stress intolerance, thereby shedding light on new approaches for treatment of heart failure.

Bidirectional Regulation of Manganese Superoxide Dismutase (MnSOD) on the Radiosensitivity of Esophageal Cancer Cells

The mitochondrial antioxidant protein manganese superoxide dismutase (MnSOD) may represent a new type of tumor suppressor protein. Overexpression of the cDNA of this gene by plasmid or recombinant lentiviral transfection in various types of cancer leads to growth suppression both in vitro and in vivo. We previously determined that changes in MnSOD expression had bidirectional effects on adriamycin (ADR) when combined with nitric oxide (NO). Radiation induces free radicals in a manner similar to ADR, so we speculated that MnSOD combined with NO would also have a bidirectional effect on cellular radiosensitivity. To examine this hypothesis, TE-1 human esophageal squamous carcinoma cells were stably transfected using lipofectamine with a pLenti6-DEST plasmid containing human MnSOD cDNA at moderate to high overexpression levels or with no MnSOD insert. Blastidicin-resistant colonies were isolated, grown, and maintained in culture. We found that moderate overexpression of MnSOD decreased growth rates, plating efficiency, and increased apoptosis. However, high overexpression increased growth rates, plating efficiency, and decreased apoptosis. When combined with NO, moderate overexpression of MnSOD increased the radiosensitivity of esophageal cancer cells, whereas high MnSOD overexpression had the opposite effect. This finding suggests a potential new method to kill certain radioresistant tumors and to provide radioresistance to normal cells.

Abstract A175: Role of MnSOD and hydrogen peroxide in rapamycin-induced cytostasis.

Abstract Existing treatments for mantle cell lymphoma (MCL) are non-curative, demonstrating a need for a refined treatment approach. Recent clinical trials have shown promising results with the use of mammalian target of rapamycin (mTOR) inhibitors. While cyclin D1 can be regulated through the mTOR pathway, treatment with mTOR inhibitors often inhibits MCL growth without altering cyclin D1 levels of expression. Thus, the anti-tumor activity of mTOR inhibitors is likely mediated through other key targets. In other malignancies, recent data suggests that increased oxidative stress plays a role in the growth suppressive mechanisms of mTOR inhibitors. Initial studies in our lab indicated that a 48 hour rapamycin treatment of MCL cell lines increased overall levels of reactive oxygen species (ROS). Specifically, we found evidence of increased hydrogen peroxide (H2O2) levels in the cells. One potential source of H2O2 is manganese superoxide dismutase (MnSOD), which is a known tumor suppressor in several cell types. MnSOD catalyzes the dismutation of superoxide to H2O2 in the mitochondria. Thus, it removes one form of ROS while generating another. We hypothesized that the anti-tumor effect of mTOR inhibitors in MCL is mediated by an accumulation of ROS, specifically H2O2, due to an increase in MnSOD protein expression. We investigated the effect of a 48 hour rapamycin treatment on MnSOD in two MCL cell lines and genetic manipulation of MnSOD in Jurkat cells, a T lymphocyte cell line. Rapamycin treatment increased MnSOD in the MCL cell lines as well as Jurkat cells. Overall, sensitivity of cells to rapamycin correlated with high MnSOD levels. Analysis of the results showed that rapamycin treatment increased MnSOD expression in parallel with H2O2. Over-expression of MnSOD in Jurkat cells elevated the level of H2O2 and resulted in a cytotoxic, rather than cytostatic, effect of rapamycin. Treatment with the general ROS scavenger, N-acetyl cysteine, reduced overall ROS, cellular levels of H2O2, and the growth inhibitory effect of rapamycin. These findings indicate that the rapamycin-induced cytostatic effect is characterized by increased levels of MnSOD and H2O2, which is necessary for the full growth inhibitory effect of rapamycin. Rapamycin and its derivatives are generally well accepted as inhibitors of the mTORC1 complex upon initial treatment, but recent studies in other cell lines have shown that long term treatment can have an inhibitory effect on the mTORC2 complex and its downstream targets, such as Akt. Our results found that treatment with rapamycin resulted in a loss of S473 phosphorylation of Akt in the MCL cell lines. Increased levels of MnSOD following treatment of Jurkat cells were found to be due to inhibition of the mTORC2 complex. These results suggest that in MCL cells treated with rapamycin for 48 hours the mTORC2 complex is inhibited. Multiple transcription factors that have been linked to MnSOD regulation are affected by the Akt pathway. By understanding the key signaling molecules and affected pathways in the anti-tumor effects of rapamycin, we may be able to identify additional predictive markers to improve the therapeutic value of mTOR inhibitors, or study drug combinations that will enhance the effect of ROS-induced cytotoxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A175.

Abstract 1606: Altered regulation of manganese superoxide dismutase is an important pre-carcinogenesis event mediated by p53

Abstract Cancer cells have aberrant manganese superoxide dismutase (MnSOD) activity and MnSOD deficient mice exhibit abnormal mitochondrial pathology. Recent data from human and animal studies indicate that the activity of MnSOD and the tumor suppressor gene, p53, are important for the maintenance of cellular energy metabolism. In addition, these critical proteins regulate oxidative stress to control the switch from oxidative phosphorylation to the aerobic glycolysis that is observed in cancer cells (Warburg's effect). It has been well-documented that MnSOD level is reduced in transformed cells and benign cancer tissues but increased in some aggressive cancers. However, there is little information concerning the regulation of MnSOD during carcinogenesis in vivo. Furthermore, when and how the alteration of MnSOD transcription occurs during the process of tumor development is unclear. To address these questions, we employed three complementary approaches in vitro and in vivo. First, we generated transgenic mice expressing luciferase reporter gene under the control of human MnSOD promoter/enhancer elements for investigating changes in MnSOD expression in animals by a non-invasive in vivo imaging. Second we used the 7-12-dimethylbenz(a)anthracene (DMBA)/TPA treated multistage skin carcinogenesis model to generate tumors and identify transcription factors associated with the expression of MnSOD in pathologically normal-appearing skin tissue, papillomas and squamous cell carcinoma. Third, we used Chromatin-immunoprecipation, EMSA, RNAi mediated knock-down and over-expression of candidates transcription factors in cultured cells to verify their roles in regulation of MnSOD at each stage of cancer development. The results demonstrate that reduction of MnSOD occurred very early in the carcinogenesis process and preceded the formation of any detectable tumor. The reduction of MnSOD transcription continued until papillomas were detected. However, as the benign tumor progressed into carcinoma, MnSOD expression was drastically increased. Contributing factors leading to the suppression and de-repression of MnSOD expression included Sp1, members of the NF-κB family, and p53. Among these important transcription factors, p53 appeared to play a major role in the switch from suppressed expression of MnSOD in the early carcinogenesis process to overexpression of MnSOD at late stages of cancer development. These results have identified MnSOD as a p53 regulated gene during cancer development and progression and highlighted the importance of mitochondrial oxidative stress in carcinogenesis at the early stages and chemo- and radio-resistance at the late stages of cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1606. doi:10.1158/1538-7445.AM2011-1606

Nuclear Factor κB- and Specificity Protein 1-dependent p53-mediated Bi-directional Regulation of the Human Manganese Superoxide Dismutase Gene

Tumor suppressor p53 is known to activate certain sets of genes while suppressing others. However, whether p53 can both activate and suppress the same gene is unclear. To address this question, concentration-dependent p53 effect on the manganese superoxide dismutase (MnSOD) gene was investigated. By transfecting p53 in PC-3 cells, we demonstrate that low concentrations of p53 increase while high concentrations suppress MnSOD expression. The physiological relevance of this effect was determined in vitro and in vivo using combined UVB-mediated activation and small interference RNA-mediated suppression of p53. Results were consistent with the bi-directional effect of p53 on MnSOD expression. MnSOD-promoter/enhancer analysis demonstrates that p53 is suppressive to the promoter activity regardless of the presence or absence of putative p53 binding sites. However, a low level of p53 increases MnSOD gene transcription in the presence of the intronic-enhancer element, and this effect is dependent on nuclear-factor kappaB (NF-kappaB) binding sites. Expression of p53 enhances nuclear levels of p65 with corresponding increase in the DNA-binding activity of NF-kappaB as detected by electrophoretic mobility shift and chromatin immunoprecipitation assays. Transfection of p65 small interference RNA reduces the positive effect of p53 on MnSOD gene transcription. These data suggest that p65 can overcome the negative effect of p53 on MnSOD expression. However, when the level of p53 was further increased, the suppressive effect of p53 outweighed the positive effect of p65 and led to the suppression of MnSOD gene transcription. These results demonstrated that p53 can both suppress and induce MnSOD expression depending on the balance of promoter and enhancer binding transcription factors.

Radioprotection by short-term oxidative preconditioning: Role of manganese superoxide dismutase

Manganese superoxide dismutase (MnSOD) is vital to the protection of mitochondria and cells against oxidative stress. Earlier, we demonstrated that catalytically active homo-tetramer of MnSOD can be stabilized by oxidative cross-linking. Here we report that this effect may be translated into increased radioresistance of mouse embryonic cells (MECs) by pre-exposure to oxidative stress. Pre-treatment of MECs with antimycin A, rotenone or H 2O 2 increased their survival after irradiation. Using MnSOD siRNA, we show that MECs with decreased MnSOD levels displayed a lowered ability to preconditioning. Thus oxidative preconditioning may be used for targeted regulation of MnSOD. Structured summary MINT- 7288408: MnSOD (uniprotkb: P04179) and MnSOD (uniprotkb: P04179) physically interact (MI: 0915) by zymography (MI: 0512)

Carbon monoxide provides antioxidant protection in hepatic sinusoids during a remote inflammatory stress by reducing carbonylated MnSOD

Although the anti‐inflammatory benefits of exogenous CO are well recognized, its antioxidant potential remains unknown. We examined the role of CO in regulating hepatic oxidant generation using bilateral hindlimb ischemia/reperfusion (I/R) as a model of systemic inflammation. Immediately following the onset of I/R, mice were treated with exogenous CO (methylene chloride or gaseous CO). Bilateral hindlimb I/R caused significant liver damage and lipid peroxidation. In vivo digital microscopy with the oxidant‐sensitive probe dihydrorhodamine confirmed the generation of hepatic oxidants. Treatment with CO significantly reduced all aspects of liver damage and oxidant formation. Due to its chemistry, CO is not likely a direct oxidant scavenger. Thus we investigated whether the antioxidant benefit of exogenous CO occurred via regulation of manganese superoxide dismutase (MnSOD) activity. As with direct I/R, MnSOD protein expression was unchanged yet its activity was diminished. Concomitant to this functional MnSOD demise was an increase in its carbonylation ‐ an inactivating oxidative protein modification. Treatment with CO reduced the carbonylation of MnSOD and restored its activity. These results suggest that exogenous CO indirectly reduces the hepatic oxidant stress that accompanies a remote inflammatory insult by diminishing MnSOD carbonylation, and thereby restoring MnSOD activity.

Distinct Functions of CCAAT Enhancer-binding Protein Isoforms in the Regulation of Manganese Superoxide Dismutase during Interleukin-1β Stimulation

The mitochondrial antioxidant enzyme manganese superoxide dismutase (Mn-SOD) is crucial in maintaining cellular and organismal homeostasis. Mn-SOD expression is tightly regulated in a manner that synchronizes its cytoprotective functions during inflammatory challenges. Induction of Mn-SOD gene expression by the proinflammatory cytokine IL-1beta is mediated through a complex intronic enhancer element. To identify and characterize the transcription factors required for Mn-SOD enhancer function, a yeast one-hybrid assay was utilized, and two CCAAT enhancer-binding protein (C/EBP) members, C/EBP beta and C/EBP delta, were identified. These two transcription factors responded to IL-1beta treatment with distinct expression profiles, different temporal yet inducible interactions with the endogenous Mn-SOD enhancer, and also opposite effects on Mn-SOD transcription. C/EBP beta is expressed as three isoforms, LAP* (liver-activating protein), LAP, and LIP (liver-inhibitory protein). Our functional analysis demonstrated that only the full-length C/EBP beta/LAP* served as a true activator for Mn-SOD, whereas LAP, LIP, and C/EBP delta functioned as potential repressors. Finally, our systematic mutagenesis of the unique N-terminal 21 amino acids further solidified the importance of LAP* in the induction of Mn-SOD and emphasized the crucial role of this isoform. Our data demonstrating the physiological relevance of the N-terminal peptide also provide a rationale for revisiting the role of LAP* in the regulation of other genes and in pathways such as lipogenesis and development.

Down-regulation of Manganese-Superoxide Dismutase through Phosphorylation of FOXO3a by Akt in Explanted Vascular Smooth Muscle Cells from Old Rats

Manganese-superoxide dismutase (MnSOD) is one of the major cellular antioxidant defense systems. To study the effect of age on the regulation of MnSOD in the vasculature, we compared MnSOD expression and its transcriptional regulation in explanted vascular smooth muscle cells (VSMC) isolated from old (24 months old) versus young (6 months old) rats and grown in a normal (5 mM) or high (12.5 and 25 mM) glucose or tumor necrosis factor alpha (5 ng/ml) environment to induce oxidative stress. Both MnSOD protein and activity were reduced in VSMC from old compared with young animals. FOXO3a, a member of the family of Forkhead transcription factors, interacted with the promoter of the rat MnSOD gene at a specific binding site. Inhibition of FOXO3a transcription with small interfering RNA led to a reduction in MnSOD gene expression. VSMC from old rats had increased phosphorylated FOXO3a at Ser(253), which paralleled the reduction of MnSOD protein. Treatment of VSMC with 5 nm insulin-like growth factor-1 induced phosphorylation of Akt and FOXO3a over time, repressing FOXO3a DNA binding and consequently MnSOD gene expression. Furthermore, Akt activity was selectively increased in VSMC from the old, supporting the hypothesis that increased age-related Akt activity might be responsible for the phosphorylation and inactivation of FOXO3a, which in turn down-regulates MnSOD transcription.

FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK.

Forkhead transcription factors of the FOXO class are negatively regulated by PKB/c-Akt in response to insulin/IGF signalling, and are involved in regulating cell cycle progression and cell death. Here we show that, in contrast to insulin signalling, low levels of oxidative stress generated by treatment with H2O2 induce the activation of FOXO4. Upon treatment of cells with H2O2, the small GTPase Ral is activated and this results in a JNK-dependent phosphorylation of FOXO4 on threonine 447 and threonine 451. This Ral-mediated, JNK-dependent phosphorylation is involved in the nuclear translocation and transcriptional activation of FOXO4 after H2O2 treatment. In addition, we show that this signalling pathway is also employed by tumor necrosis factor alpha to activate FOXO4 transcriptional activity. FOXO members have been implicated in cellular protection against oxidative stress via the transcriptional regulation of manganese superoxide dismutase and catalase gene expression. The results reported here, therefore, outline a homeostasis mechanism for sustaining cellular reactive oxygen species that is controlled by signalling pathways that can convey both negative (PI-3K/PKB) and positive (Ras/Ral) inputs.