Upregulation Of Manganese Superoxide Dismutase Research Articles

Redox up-regulated expression of rat liver manganese superoxide dismutase and Bcl-2 by thyroid hormone is associated with inhibitor of κB-α phosphorylation and nuclear factor-κB activation

Recently, we demonstrated that 3,3',5-triiodothyronine (T3) induces oxidative stress in rat liver, with enhancement in the DNA binding of nuclear factor-kappaB (NF-kappaB) and the NF-kappaB-dependent expression of tumor necrosis factor-alpha (TNF-alpha). In this study, we show that T3 administration (daily doses of 0.1 mg/kg i.p. for three consecutive days) elicited a calorigenic response and higher liver O2 consumption rates, with increased serum levels of TNF-alpha (ELISA), liver inhibitor of kappaB (IkappaB-alpha) phosphorylation (Western blot analysis), and hepatic NF-kappaB DNA binding (EMSA) at 56-72 h after treatment. Within this time interval, liver manganese superoxide dismutase (MnSOD) activity and the protein expression of MnSOD and Bcl-2 are enhanced. These changes are abrogated by the administration of alpha-tocopherol (100 mg/kg i.p.) prior to T3. It is concluded that T3 treatment leads to the redox upregulation of MnSOD and Bcl-2 in rat liver, in association with TNF-alpha release and activation of the IkappaB-alpha kinase/NF-kappaB cascade, which may constitute a protective mechanism against free radical toxicity involving cell death signaling.

Induction of Manganese-Superoxide Dismutase by YS 51, a Synthetic 1-(β-Naphtylmethyl)6,7-Dihydroxy- 1,2,3,4-Tetrahydroisoquinoline Alkaloid

The effect of YS 51, a synthetic 1-(β-naphtylmethyl)6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline alkaloid, on the expression of manganese-superoxide dismutase (Mn-SOD), an antioxidant enzyme, was examined in sheep pulmonary artery endothelial cells (SPAEC) and a human cervical carcinoma cell line (Hela). YS 51 alone or in combination with cytokines enhanced the expression of Mn-SOD mRNA in SPAEC and Hela cells. YS 51 also showed synergistic effects on the induction of Mn-SOD mRNA with phorbol-12-myristate-13-acetate (TPA) and/or tumor necrosis factor-α (TNF-α). In Hela cells, the induction of Mn-SOD mRNA by YS 51 was in a time- and dose-dependent manner and the expression of Mn-SOD mRNA was increased to a maximum of 4-fold in 9 h. Enhancement of Mn-SOD mRNA by YS 51 was completely abolished by actinomycin D but not cycloheximide, suggesting that the induction of Mn-SOD mRNA byYS 51 is independent of new protein synthesis. Pretreatment of curcumin, an inhibitor of c-jun N-terminal kinase (JNK), dose-dependently suppressed the induction of Mn-SOD mRNA by YS 51, but not by 2′-amino-3′-methoxyflavone (PD98059) and 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazol (SB203580), inhibitors of mitogen-activated protein kinase. Also, YS 51 induced the phosphorylation activity of JNK in a time-dependent manner without affecting the phosphorylation activity of the extracellular signal-regulated kinase 1 (ERK1) and p38 MAP kinase. These results implicated that the JNK pathway appears to play a crucial role in mediating the YS 51-induced Mn-SOD gene expression, and that up-regulation of Mn-SOD would contribute to the anti-inflammatory actions mediated by YS 51.

P53-induced up-regulation of MnSOD and GPx but not catalase increases oxidative stress and apoptosis.

p53-mediated apoptosis may involve the induction of redox-controlling genes, resulting in the production of reactive oxygen species. Microarray expression analysis of doxorubicin exposed, related human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53-dependent up-regulation of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences were identified in human MnSOD and GPx promoter regions. A 3-fold increase in the MnSOD promoter activity was observed after the induction of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing p53 under the control of a tetracycline-regulated promoter. An increased protein expression of endogenous MnSOD and GPx also positively correlated with the level of p53 induction in TR9-7 cells. However, catalase (CAT) protein expression remained unaltered after p53 induction. We also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We found increased MnSOD enzymatic activity, MnSOD mRNA expression, and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele when compared with homozygous mutant p53 isogenic cells. The CAT protein level was unchanged in these cells. We observed both the release of cytochrome C and Ca(2+) from the mitochondria into the cytoplasm and an increased frequency of apoptotic cells after p53 induction in the TR9-7 cells that coincided with an increased expression of MnSOD and GPx, and the level of reactive oxygen species. The increase in apoptosis was reduced by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of MnSOD and GPx, but not CAT, produces an imbalance in antioxidant enzymes and oxidative stress.

Induced expression of manganese superoxide dismutase by non-toxic concentrations of oxidized low-density lipoprotein (oxLDL) protects against oxLDL-mediated cytotoxicity.

Oxidized low-density lipoprotein (oxLDL) affects macrophages and plays a critical role in the development of atherosclerosis. In the present paper, we demonstrate that high concentrations of oxLDL provoked apoptosis of human Mono-Mac-6 cells, which was blocked by diphenylene-iodonium (DPI), an inhibitor of flavin-containing enzymes, such as NADPH oxidase, suggesting the involvement of reactive oxygen species (ROS). Importantly, pre-treatment of cells with low concentrations of oxLDL prevented apoptosis in response to high concentrations of oxLDL by up-regulating manganese superoxide dismutase (MnSOD). DPI prevented expression of MnSOD by oxLDL, whereas inhibitors of cytochrome P450 (methoxalen) or xanthine oxidase (allopurinol) did not, thus pointing to a role of NADPH-oxidase-derived ROS in oxLDL-induced MnSOD expression. Transfection of cells with MnSOD antisense, but not scrambled antisense, oligonucleotides significantly attenuated oxLDL-mediated MnSOD expression and hindered cytoprotective effects of non-toxic oxLDL concentrations. Our findings suggest that up-regulation of MnSOD by low concentrations of oxLDL is critical for protection towards oxLDL-mediated cytotoxicity.

Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), Fort Lauderdale, Florida, May 5-10, 2002.

Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), Fort Lauderdale, Florida, May 5-10, 2002.

The c-Rel transcription factor can both induce and inhibit apoptosis in the same cells via the upregulation of MnSOD.

Rel/NF-kappaB transcription factors are involved in several physiological processes, including the regulation of apoptosis. These factors were shown to exhibit pro- or anti-apoptotic activities in different cellular models, but at present, the mechanisms underlying these opposite effects are poorly understood. In this study, we show that the constitutive expression of a transcriptionally active member of the Rel/NF-kappaB family, c-Rel, first induces a resistance against TNFalpha-induced apoptosis and later increases the level of spontaneous apoptosis of HeLa cells. Both the anti- and pro-apoptotic effects increase with the level of c-Rel overexpression. The up-regulation by c-Rel of the manganese superoxide dismutase (MnSOD) could explain both the rapid anti-apoptotic effect and the delayed pro-apoptotic one. Indeed, the enzymatic activity of MnSOD is to transform the toxic O(2)(*)(-) in H(2)O(2). Hence, on one hand, its induction helps cells to resist against the apoptogenic burst of O(2)(*)(-) produced upon TNFalpha stimulation, but on the other hand, it leads to a progressive H(2)O(2) accumulation that ultimately results in apoptosis. These results indicate that the anti- and pro-apoptotic effects of Rel/NF-kappaB factors are not necessarily alternative but can occur successively in the same cell, via the up-regulation of the same target gene.

Activation of signal transducer and activator of transcription 3 protects cardiomyocytes from hypoxia/reoxygenation-induced oxidative stress through the upregulation of manganese superoxide dismutase.

Mice with cardiac-specific overexpression of signal transducer and activator of transcription 3 (STAT3) are resistant to doxorubicin-induced damage. The STAT3 signal may be involved in the detoxification of reactive oxygen species (ROS). The effects of leukemia inhibitory factor (LIF) or adenovirus-mediated transfection of constitutively activated STAT3 (caSTAT3) on the intracellular ROS formation induced by hypoxia/reoxygenation (H/R) were examined using rat neonatal cardiomyocytes. Either LIF treatment or caSTAT3 significantly suppressed the increase of H/R-induced ROS evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. To assess whether ROS are really involved in H/R-induced cardiomyocyte injury, the amount of creatine phosphokinase in cultured medium was examined. Both LIF treatment and caSTAT3 significantly decreased H/R-induced creatine phosphokinase release. These results indicate that the gp130/STAT3 signal protects H/R-induced cardiomyocyte injury by scavenging ROS generation. To investigate the mechanism of scavenging ROS, the effects of LIF on the induction of antioxidant enzymes were examined. LIF treatment significantly increased the expression of manganese superoxide dismutase (MnSOD) mRNA, whereas the expression of the catalase and glutathione peroxidase genes were unaffected. This induction of MnSOD mRNA expression was completely blocked by adenovirus-mediated transfection of dominant-negative STAT3. Moreover, caSTAT3 augmented MnSOD mRNA and its enzyme activity. In addition, the antisense oligodeoxyribonucleotide to MnSOD significantly inhibited both LIF and caSTAT3-mediated protective effects. The activation of STAT3 induces a protective effect on H/R-induced cardiomyocyte damage, mainly by inducting MnSOD. The STAT3-mediated signal is proposed as a therapeutical target of ROS-induced cardiomyocyte injury.

Attenuation and augmentation of ischaemia-related neuronal death by tumour necrosis factor-alpha in vitro.

Upregulation of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF) occurs rapidly in the brain following ischaemia, although it is unclear whether this represents a neurotoxic or neuroprotective response. We have investigated whether TNF has different actions in the pre- and postischaemic periods in a tissue culture model of cerebral ischaemia. Organotypic hippocampal slice cultures were prepared from 8-10-day-old rats and maintained in vitro for 14 days. Neuronal damage was induced by either 1 h oxygen-glucose deprivation or 3 h exposure to NMDA or the superoxide generator duroquinone, and assessed after 24 h by propidium iodide fluorescence. TNF pretreatment was neuroprotective against both oxygen-glucose deprivation and duroquinone. This effect was associated with an activation of the transcription factor NFkappaB and upregulation of manganese superoxide dismutase, and was prevented by a free radical scavenger. When addition of TNF was delayed until the postinsult period, an exacerbation of neurotoxicity occurred, which was also prevented by a free radical scavenger. The actions of TNF are determined by whether TNF is present before or after an ischaemia-related insult. Both actions are mediated through the production of free radicals, and the response to TNF is determined by whether a cell is metabolically competent to respond by synthesis of antioxidant defences.

Role of heme oxygenase-1 in the regulation of manganese superoxide dismutase gene expression in oxidatively-challenged astroglia.

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme that reduces superoxide anion to hydrogen peroxide in cell mitochondria. MnSOD is overexpressed in normal aging brain and in various central nervous system disorders; however, the mechanisms mediating the upregulation of MnSOD under these conditions remain poorly understood. We previously reported that cysteamine (CSH) and other pro-oxidants rapidly induce the heme oxygenase-1 (HO-1) gene in cultured rat astroglia followed by late upregulation of MnSOD in these cells. In the present study, we demonstrate that antecedent upregulation of HO-1 is necessary and sufficient for subsequent induction of the MnSOD gene in neonatal rat astroglia challenged with CSH or dopamine, and in astroglial cultures transiently transfected with full-length human HO-1 cDNA. Treatment with potent antioxidants attenuates MnSOD expression in HO-1-transfected astroglia, strongly suggesting that intracellular oxidative stress signals MnSOD gene induction in these cells. Activation of this HO-1-MnSOD axis may play an important role in the pathogenesis of Alzheimer disease, Parkinson disease and other free radical-related neurodegenerative disorders. In these conditions, compensatory upregulation of MnSOD may protect mitochondria from oxidative damage accruing from heme-derived free iron and carbon monoxide liberated by the activity of HO-1.

COCAINE-INDUCED INCREASE OF Mn-SOD IN ADULT RAT LIVER CELLS

Cultured adult rat liver cells (non-induced) were exposed to two different concentrations (2 μg/ml and 4 μg/ml) of cocaine-HCl for 1–3 hours. Cocaine-treated rat liver cells showed a significant increase in manganese superoxide dismutase (Mn-SOD) activity with time. Coinciding the rise in Mn-SOD activity was the Mn-SOD mRNA transcript, which also demonstrated a time-dependent elevation in its level (p < 0.005). At the end of 1 and 3 hr. of cocaine exposure, the increase in Mn-SOD activity was 23% (p < 0.05) and 39% (p < 0.005), respectively. In contrast, activities of catalase (CAT) and glutathione peroxidase (GSH-px) were significantly diminished. Induction of Mn-SOD mRNA by cocaine (4 μg/ml) was inhibited by actinomycin D (4 μM), revealing that the up regulation of Mn-SOD involved transcriptional activation. The increase in Mn-SOD was accompanied by a decrease in liver cell integrity, indicated by increased lactate dehydrogenase (LDH) release by rat liver cells. These results suggest that antioxidant enzymes may play a critical role in cocaine-induced liver injury.

Increased manganese superoxide dismutase activity, protein, and mRNA levels and concurrent induction of tumor necrosis factor alpha in radiation-initiated Syrian hamster cells.

The levels of the antioxidant enzyme manganese superoxide dismutase (Mn-SOD) are frequently decreased in tumor cells and increased in normal cells upon treatment with ionizing radiation. We studied Mn-SOD at different stages during the neoplastic conversion of radiation-initiated Syrian hamster embryo HDR-3 cells. Mn-SOD activity and the concentration of Mn-SOD protein and mRNA increased gradually during the malignant transformation of HDR-3 cells after radiation exposure; fully neoplastic cells showed greater Mn-SOD levels than preneoplastic and normal 84-3 cells. Inhibitors of superoxide (SO) anion production (thenoyltrifluoroacetone and rotenone) decreased the concentration of Mn-SOD mRNA, raising the possibility that the generation of SO radicals participated in the upregulation of Mn-SOD in cells transformed by exposure to radiation. We observed an increase in the concentration of tumor necrosis factor alpha (TNF alpha) in HDR-3 cells relative to mock-irradiated cells. Together with the observation that TNF alpha stimulates the production of SO by mitochondria and increases the level of Mn-SOD mRNA in other experimental systems, our results suggest that as normal 84-3 cells undergo malignant transformation induced by ionizing radiation they produce TNF alpha, to which the cells respond by increasing the concentration of Mn-SOD mRNA and protein and the activity of the enzyme.

Redox perturbations in cysteamine-stressed astroglia: Implications for inclusion formation and gliosis in the aging brain

The aminothiol compound, cysteamine (CSH), induces astrocyte hypertrophy (gliosis) and the appearance of autofluorescent, peroxidase-positive cytoplasmic granules in these cells akin to changes that occur spontaneously in astroglia of the aging periventricular brain. Paradoxically, CSH damages astroglial mitochondria (granule precursors) while protecting these cells from subsequent H 2O 2 and mechanoenzymatic stress. In this study, in vitro CSH administration significantly increased manganese superoxide dismutase (MnSOD) activity in cultured astroglia. Immunoblot and Northern analyses indicated that MnSOD protein and mRNA levels were increased in cultured astrocytes after 3–6 days of CSH treatment. Systemic administration of CSH also significantly augmented MnSOD activity in the intact diencephalon. CSH caused a pronounced (6-fold), but transient, increase in the level of reduced glutathione (GSH) in cultured astrocytes. In contrast, catalase and glutathione reductase (GR) activities were suppressed, whereas copper-zinc superoxide dismutase (CuZnSOD) activity remained unchanged both in cultured astroglia and in the intact diencephalon following CSH treatment. Glutathione peroxidase (GP) activity was increased after 3 and 48 h of CSH treatment and then declined below control levels in cultured astrocytes. CSH inhibited the formation of thiobarbituric acid-reactive products (TBAR) in whole astrocyte monolayers, although it promoted TBAR formation in suspensions of isolated astroglial mitochondria. CSH-related oxidative stress may accelerate aging-related changes in astroglial mitochondria while conferring cytoprotection to these cells by stimulating the upregulation of various heat shock proteins and MnSOD. These cytoprotective responses may facilitate astrocyte survival and the development of reactive gliosis in the face of concomitant neuronal degeneration. CSH-treated astrocytes may serve as a model for the (dys)regulation of neuroglial MnSOD and other antioxidant enzymes in the aging and degenerating nervous system.

Induction of MnSOD gene expression in a hepatic model of TNF-alpha toxicity does not result in increased protein.

The model of toxic liver injury was used to examine the role of manganese superoxide dismutase (MnSOD) expression in cellular resistance to tumor necrosis factor (TNF)-alpha toxicity. The effects of the hepatotoxin D-galactosamine (GalN) and lipopolysaccharide (LPS) on hepatic and splenic TNF-alpha and MnSOD expression were studied. Treatment with GalN and LPS alone or in combination led to equivalent increases in hepatic and splenic TNF-alpha gene expression. Hepatic MnSOD mRNA levels were not affected by GalN or GalN with LPS but were increased 13-fold by LPS alone. Splenic MnSOD mRNA levels were increased twofold by GalN and 12-fold by either LPS alone or GalN plus LPS. The determination of MnSOD protein content, however, revealed no changes in hepatic or splenic steady-state levels of the protein with any of the treatments, despite the marked increases in MnSOD gene expression. Hepatic MnSOD enzyme activity was also unchanged by LPS or GalN plus LPS administration. Biosynthesis of MnSOD protein in rat hepatocytes isolated from an in vivo LPS-treated rat was unchanged compared with control. MnSOD mRNA levels were increased when GalN treatment was combined with uridine rescue, but again no change in protein was seen. The lack of any increase in MnSOD protein after GalN or LPS administration indicates that MnSOD upregulation is not involved in cellular resistance against TNF-alpha cytotoxicity in the liver in vivo.