Expression Of Manganous Superoxide Dismutase Research Articles

Chronic Exercise Training Improved Aortic Endothelial and Mitochondrial Function via an AMPKα2-Dependent Manner.

Chronic exercise training is known to protect the vasculature; however, the underlying mechanisms remain obscure. The present study hypothesized that exercise may improve aortic endothelial and mitochondrial function through an adenosine monophosphate-activated protein kinase α2 (AMPKα2)-dependent manner. Ten-week-old AMPKα2 knockout (AMPKα2−/−) mice and age-matched wild-type (WT) mice were subjected to daily treadmill running for 6 weeks, and the thoracic aorta from these mice were used for further examination. Our results showed that exercise significantly promoted vasodilatation and increased expression and phosphorylation of endothelial nitric oxide synthase (eNOS), concomitant with increased AMPKα2 expression in WT mice. These effects were not observed in AMPKα2−/− mice. Furthermore, exercise training increased thoracic aortic mitochondrial content as indicated by increased Complex I and mitochondrial DNA (mtDNA) in WT mice but not in AMPKα2−/− mice. This may be caused by decreased mitochondrial autophagy since the expression of BH3 domain-containing BCL2 family members BNIP3-like (BNIP3L) and LC3B were decreased in WT mice with exercise. And these changes were absent with AMPKα2 deletion in mice. Importantly, exercise increased the expression of manganous superoxide dismutase (MnSOD) and catalase, suggesting that mitochondrial antioxidative capacity was increased. Notably, the improved antioxidative capacity was lost in AMPKα2−/− mice with exercise. In conclusion, this study illustrated that AMPKα2 plays a critical role in exercise-related vascular protection via increasing endothelial and mitochondrial function in the artery.

C/EBPβ regulates TNF induced MnSOD expression and protection against apoptosis

The CCAAT enhancer binding protein-beta (C/EBPbeta) is a critical regulator of many cellular processes. Exposure of C/EBPbeta-deficient fibroblasts to tumor necrosis factor-alpha (TNF) resulted in their death due to apoptosis. While, the expression of Bad, Bcl-2, Bcl-x, CAS, and hILP/XIAP, as well as the nuclear translocation of NF-kappaB was normal in C/EBPbeta-deficient cells, induction of manganous superoxide dismutase (MnSOD) gene did not occur. Ectopic expression of C/EBPbeta in C/EBPbeta-deficient fibroblasts prevented TNF-induced apoptosis. C/EBPbeta complemented cells were able to induce MnSOD in response to TNF, ruling out the possibilities that C/EBPbeta could render protection by regulating early apoptotic gene expression and/or NF-kappaB p65 expression. Moreover, C/EBPbeta-deficient cells stably transfected with an MnSOD expression vector bypassed the requirement of C/EBPbeta in protection against TNF-induced cell death, suggesting that C/EBPbeta protects TNF-induced apoptotic cell death through its role in activating MnSOD expression. Mechanistically, C/EBPbeta was required for induced NF-kappaB p65 binding to MnSOD's intronic TNF response element and indispensable for histone acetylation of the element in response to TNF. These results suggest a role for C/EBPbeta in MnSOD regulation through remodeling of local chromatin structure.

Role of the Intronic Enhancer in Tumor Necrosis Factor-mediated Induction of Manganous Superoxide Dismutase

Manganous superoxide dismutase (Mn-SOD), a tumor necrosis factor (TNF)-inducible gene product, plays an important role in removing superoxide anions produced inside mitochondria. Two regulatory regions, the proximal promoter region (PPR), which is upstream from the transcription initiation site, and the TNF-responsive element (TNFRE), which is inside intron 2, are responsible for Mn-SOD expression. To understand how each of these regions contributes to the transcription of Mn-SOD, quantitative reverse transcription-PCR, chromatin immunoprecipitations, and in vivo nuclease sensitivity assays were performed on the murine Mn-SOD gene. These assays demonstrate that Sp1 and nuclear factor (NF)-kappaB p65 are required for Mn-SOD induction by TNF. Sp1 bound the PPR constitutively, whereas NF-kappaB p65 and C/EBP-beta bound the TNFRE only after TNF treatment. Binding of C/EBP-beta to the TNFRE was dependent on the presence of NF-kappaB p65. The chromatin structure within the TNFRE became more accessible to nuclease digestion after TNF treatment. This accessibility required Sp1 and NF-kappaB p65. Treatment of cells with an inhibitor of histone deacetylation, or transient transfection with coactivator-expressing plasmids, enhanced the expression of Mn-SOD. NF-kappaB p65 binding was required for acetylation of histones H3 and H4 at the PPR and the TNFRE. Together, these data suggest communication between the PPR and the TNFRE which involves chromatin remodeling and histone acetylation during the induction process of Mn-SOD in response to TNF.

A novel Arabidopsis thaliana protein protects tumor cells from tumor necrosis factor-induced apoptosis

Recently we have cloned and characterized a novel, oxidative stress-induced Arabidopsis thaliana gene ( oxy5), and showed that expression of oxy5 protects bacterial cells from death caused by oxidative stress. As oxidative stress is one pathway of TNF cytotoxicity, we investigated whether the encoded protein could also protect human tumor cells from TNF killing. We stably transfected the oxy5 gene into TNF-sensitive HeLa D98 cells (D98/O.5), and found that all examined transfectants were highly TNF-resistant in the absence of the protein synthesis inhibitor cycloheximide. The acquired TNF resistance of these clones was accompanied by a sharp decrease in the intracellular formation of reactive oxygen species, suggesting the activation of antioxidant enzymes like superoxide dismutases (SODs). Indeed, D98/O.5 clones showed an increased manganous superoxide dismutase (MnSOD) mRNA and protein expression in the absence or presence of TNF stimulation, whereas the expression of the Cu/ZnSOD was not affected. Furthermore, the elevated MnSOD expression in the D98/O.5 clones correlated well with an increased antioxidative activity, which was specifically due to MnSOD as measured by the suppression of xanthine oxidase. Our results demonstrate a novel role for a plant-derived protein in resistance to TNF cytotoxicity, and that the Arabidopsis thaliana protein Oxy5 can exert its protective function across evolutionary boundaries through activation of antioxidant enzymes like MnSOD.

Protein kinase C inhibitors augment tumor-necrosis-factor-induced apoptosis in normal human diploid cells.

In this report we show augmentation of tumor necrosis factor (TNF)-induced apoptosis by protein kinase C (PKC) inhibitors in human embryonic lung fibroblast (HEL) cells. Staurosporine (STS) reportedly potentiates TNF-mediated cytotoxicity in cancer cell lines via inhibition of PKC. We investigated whether STS or another potent PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), augmented TNF-induced apoptosis in normal human diploid cells and examined the effects of the PKC inhibitors on the expression of endogenous TNF (enTNF) and manganous superoxide dismutase (MnSOD) as possible cell resistance factors opposing TNF-induced apoptosis. Both PKC inhibitors augmented TNF-mediated DNA fragmentation in HEL cells, which are normally TNF resistant and constitutively express high amounts of enTNF and MnSOD activity. Neither the number nor the affinity of TNF receptors were altered by STS and H7. The expression of enTNF and MnSOD was suppressed by the presence of STS or H7 along with TNF. The results indicate that PKC inhibitors augment TNF-induced apoptosis not only in tumor cells but also in normal cells by inhibitory effects on cellular resistance factors such as enTNF and MnSOD.

Interleukin 1α-induced expression of manganous superoxide dismutase reduces myocardial reperfusion injury in the rat

We investigated the effects of pretreatment with interleukin (IL)-1 alpha on the expression of manganous (Mn) superoxide dismutase (SOD) mRNA and reperfusion-induced arrhythmias and the size of myocardial infarct in rats. Male Wistar rats received 10 mg intraperitoneal injections of human recombinant IL-1 alpha. Their hearts were thereafter isolated at 6, 12, 24, 36 h. A Northern analysis showed that Mn-SOD mRNA was mainly expressed in the heart and slightly in kidney, but not in any other organs. The expression of Mn-SOD mRNA peaked at 6 h after the injection of IL-1 alpha. The Mn-SOD protein content was most increased 12 h after injection. In the isolated heart model, the rats were pretreated with IL-1 alpha 24 h earlier and their hearts were perfused by the Langendorff method. After 20 min of ischemia which was induced by a ligation of a coronary artery, reperfusion-induced arrhythmias were observed. There were no significant differences in the incidence of ventricular arrhythmias between the IL-1 alpha pretreated and the untreated hearts. IL-1 alpha pretreatment significantly reduced the mean duration of the ventricular arrhythmias and also delayed the onset of arrhythmias. The effect of IL-1 alpha pretreatment was also investigated in a 30-min model of ischemia followed by a 3-min reperfusion in anesthetized rats. The infarct size expressed as a percentage of the area at risk was significantly reduced in the IL-1 alpha pretreated hearts compared with the untreated hearts. The left ventricular systolic pressure increased significantly in rat hearts pretreated with IL-1 alpha. Our results therefore showed that the pretreatment with IL-1 alpha induced the overexpression of Mn-SOD mRNA in the rat hearts and also suggested that pretreatment with IL-1 alpha 24 h before ischemia reduced the risk of ischemia-reperfusion injury.

ADP-Ribosylation Inhibitors Inhibit Cellular RNA Synthesis but Do Not Affect Expression of Manganous Superoxide Dismutase or Heat Shock Protein 70 in Tumor Necrosis Factor αSensitive and -Resistant Tumor Cells

We have shown that the cytotoxic response of TNF-sensitive L929 cells and TNF-resistant EMT-6 cells to TNF-alpha can be modulated by ADP-ribosylation inhibitors independently of ADP-ribosylation rates. To explore the possibility that these inhibitors modulate TNF cytotoxicity by interfering with cellular protective mechanisms, we evaluated their effects on general RNA synthesis and on mRNA expression of two proposed protective genes, manganous superoxide dismutase (MnSOD) and heat shock protein 70 (hsp70). We found that ADP-ribosylation inhibitors could inhibit general RNA synthesis in a dose-dependent fashion to a similar extent in both EMT-6 and L929 cells, although these inhibitors increased or decreased the sensitivity of the cells to TNF, respectively. In EMT-6 cells, combination of actinomycin D with these inhibitors further inhibited the RNA synthesis rate, and it actually decreased the TNF sensitivity of the EMT-6 cells. Furthermore, the expression of MnSOD or hsp70 was not regulated by these inhibitors. Thus, TNF resistance must depend on other mechanisms in addition to the expression of these protective genes.

Protective roles of cytokines against radiation: Induction of mitochondrial MnSOD

Oxidative stress such as radiation can trigger the production of cytokines including tumor necrosis factor (TNF) and lymphotoxin (LT). The increased cytokine levels may in turn induce the synthesis of protein(s) that protect against subsequent killing by oxidative stress. Indeed, pretreatment of animals with TNF or LT can protect them against lethal doses of radiation and the alopecia that results from anticancer drugs. TNF or LT can specifically and selectively induce the expression of manganous superoxide dismutase (MnSOD). MnSOD, identified as one of the protective proteins, is a mitochondrial enzyme that scavenges superoxide radicals (O 2 −). TNF-R1 but not TNF-R2 is responsible for TNF and LT's induction of MnSOD. Paradoxically, the TNF-R1 is also the receptor that mediates the production of oxygen free radicals and apoptosis. Overexpression of MnSOD but not CuZn-SOD or EC-SOD enhances cellular resistance to radiation. Conversely, overexpression of antisense MnSOD RNA diminishes resistance. Transfection of cells with MnSOD lacking the mitochondrial matrix signal does not provide protection against radiation. However, insertion of the mitochondrial signal sequence into CuZn-SOD or EC-SOD results in significant protection. TNF or LT does not induce MnSOD in tumor cells; nor do they protect these cells against radiation. Actually, TNF or LT pretreatment can sensitize tumor cells to killing by radiation.

Overexpression of manganous superoxide dismutase (MnSOD) in pulmonary endothelial cells confers resistance to hyperoxia

Treatment of cells or organisms with agents that increase the expression of MnSOD confers resistance to certain types of oxidative damage. However, since these treatments also affect other cellular systems with antioxidant capabilities, the role of MnSOD remains uncertain. To better determine whether increased MnSOD expression confers increased resistance to oxidant stress, a eukaryotic expression vector harboring a mouse MnSOD cDNA was constructed. Bovine lung microvessel endothelial cells were co-transfected with the MnSOD expression vector and pSV2-neo, which contains the neor gene and provides a dominant selectable marker. Control clones were generated by transfecting the cells with psV2-neo alone. Stably transfected cell lines were selected and cell lines overexpressing MnSOD were confirmed by Northern blotting, immunoblot analysis, and activity gels. The activities of copper/zinc superoxide dismutase, catalase, and glutathione peroxidase were examined, and no increase in activity of any of these enzymes was detected. Cells were exposed to hyperoxic challenge by treatment with 95% O2 and 5% CO2 for 24 h. Viability was assessed by a clonogenic assay. The cell lines that overexpressed MnSOD showed a twofold increase in survival compared to control cells. These results demonstrate a significant resistance to hyperoxia induced oxidative stress in endothelial cells overexpressing MnSOD.

Tumor necrosis factor-α pretreatment is protective in a rat model of myocardial ischemia-reperfusion injury

We have demonstrated that tumor necrosis factor-α (TNF-α) pretreatment protected the rat heart from ischemia-reperfusion injury. This effect was monitored by assaying for lactate dehydrogenase (LDH), an enzyme whose release correlates with loss of cell membrane integrity. Intact hearts removed from rats pretreated with TNF released significantly lower amounts of LDH compared to control hearts after 20 min. of total global ischemia followed by reperfusion. Hearts from TNF-α-pretreated animals contained higher levels of manganous superoxide dismutase (MnSOD) mRNA than hearts from untreated rats. Because oxygen free radicals have been implicated as a major cause of reperfusion damage and the function of MnSOD is to detoxify superoxide anions in the mitochondria, a possible protective mechanism for TNF-α may be to induce expression of MnSOD in the heart and thus confer resistance to oxygen free radicals generated during reperfusion.

Tumor necrosis factor alpha selectively sensitizes human immunodeficiency virus-infected cells to heat and radiation.

We report here that infection of the human T-cell line HUT-78 with human immunodeficiency virus (HIV) increases its sensitivity to heat and radiation toxicity. A possible explanation for this result may be the reduced expression of manganous superoxide dismutase (MnSOD) in HIV-infected cells compared to uninfected cells. Tumor necrosis factor alpha (TNF-alpha) further sensitizes HIV-infected cells but not uninfected cells to heat and radiation. This is consistent with the ability of TNF-alpha to induce the expression of MnSOD in uninfected but not in HIV-infected cells. HIV-infected HUT-78 cell lines engineered to overexpress MnSOD are more resistant to heat and radiation than HIV-infected cells that do not overexpress MnSOD. However, treatment with TNF-alpha still sensitizes these cells to heat and radiation.

The E1b oncogene of adenovirus confers cellular resistance to cytotoxicity of tumor necrosis factor and monoclonal anti-Fas antibody

The cell lines KB8, 16, and 18 are KB cells which constitutively express adenovirus type 2 (Ad2) E1a, E1a plus E1b, and E1b genes, respectively. We show here that KB18 cells are completely resistant to cytolysis by tumor necrosis factor (TNF) or anti-Fas, although KB8 and KB or KB16 are highly and moderately sensitive, respectively. The levels of receptors for TNF and anti-Fas of KB18 were almost the same as compared with those of KB or other KB-cell lines. Expression of manganous superoxide dismutase (MnSOD) mRNA in KB18 was about 20-fold higher than that in KB or KB8 cells. KB, HT29, and A673 cells infected with dl337 (an Ad5 mutant defective in E1b function) are highly sensitive to TNF or anti-Fas, although wild-type Ad2-infected cells are resistant. Our results indicate that the E1b oncogene can confer cellular resistance to cytolysis by either TNF or anti-Fas in both KB cells and adenovirus-infected human cell lines through influencing intracellular events including regulation of MnSOD genes. Furthermore, we describe how anti-Fas mimics only the cytolytic activity of TNF, whereas TNF also has many other biological activities.