Manganese Superoxide Dismutase RNA Research Articles

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.

Tyrosine Phosphorylation Regulates Manganese Superoxide Dismutase (MnSOD) RNA-Binding Protein Activity and MnSOD Protein Expression

All cells tested contain a cytosolic protein that binds to a defined region in the 3' untranslated region of manganese superoxide dismutase (MnSOD) RNA; both the MnSOD RNA-binding protein (MnSOD-BP) and the cis element are required for efficient translation of MnSOD RNA [Chung, D. J., Wright, A. E., and Clerch, L. B. (1997) Biochemistry 37, 16298-16306]. This study was designed to test the hypothesis that MnSOD-BP activity is regulated by phosphorylation. When cell extracts from whole rat lung or a rat lung fibroblast cell line, RFL-6, were treated in vitro with a protein tyrosine phosphatase, there was a 4-fold increase in MnSOD-BP activity indicating that MnSOD-BP activity was upregulated by tyrosine dephosphorylation. RFL-6 cells treated in cell culture with herbimycin A or genistein, inhibitors of protein tyrosine kinase, had significantly more MnSOD-BP activity than cells treated with diluent. In RFL-6 cells treated with herbimycin A, the increase in MnSOD-BP activity was associated with an increase in the level of MnSOD protein without a change in MnSOD mRNA concentration. We propose that the modulation of MnSOD protein expression by the tyrosine phosphorylation state of MnSOD-BP is a potential therapeutic target for increasing MnSOD activity during periods of oxidative stress.

A Region in the 3′ UTR of MnSOD RNA Enhances Translation of a Heterologous RNA

The studies reported in this paper were designed to test the hypothesis that a cis element located in the 3′ UTR of manganese superoxide dismutase (MnSOD) RNA, designated MnSOD-response element (MnSOD-RE), is a translational enhancer in vivo. NIH/3T3 cells were transfected with a posttranscriptional reporter construct in which MnSOD-RE was placed 3′ of the coding region of chloramphenicol acetyltransferase (CAT); this construct is designated CAT-RMS. Transient transfection of CAT-RMS did not change the concentration of CAT mRNA but increased CAT activity by ∼400% compared to a control construct, CAT-V, which contains approximately the same size of non-MnSOD 3′ UTR sequence. Transfection of CAT-RMS had no effect on endogenous MnSOD protein, mRNA, or MnSOD RNA-binding protein activity. Because of its ability to increase translation of a heterologous RNA, MnSOD-RE may be useful in designing expression vectors for in vitro expression systems and in vivo gene therapy.

Insulin Antiapoptotic Signaling Involves Insulin Activation of the Nuclear Factor κB-dependent Survival Genes Encoding Tumor Necrosis Factor Receptor-associated Factor 2 and Manganese-superoxide Dismutase

We recently showed that the antiapoptotic function of insulin requires nuclear factor kappaB (NF-kappaB) activation (Bertrand, F., Atfi, A., Cadoret, A., L'Allemain, G., Robin, H., Lascols, O., Capeau, J., and Cherqui, G. (1998) J. Biol. Chem. 273, 2931-2938). Here we sought to identify the NF-kappaB-dependent survival genes that are activated by insulin to mediate this function. Insulin increased the expression of tumor necrosis factor receptor-associated factor 2 (TRAF2) mRNA and protein in Chinese hamster ovary cells overexpressing insulin receptors (IRs). This effect required (i) IR activation since it was abrogated by IR mutation at tyrosines 1162 and 1163 and (ii) NF-kappaB activation since it was abolished by overexpression of dominant-negative IkappaB-alpha(A32/36) and mimicked by overexpression of the NF-kappaB c-Rel subunit. TRAF2 contributed to insulin protection against serum withdrawal-induced apoptosis since TRAF2 overexpression mimicked insulin protection, whereas overexpression of dominant-negative TRAF2-(87-501) reduced this process. Along with its protective effect, overexpressed TRAF2 increased basal and insulin-stimulated NF-kappaB activities. All effects were inhibited by IkappaB-alpha(A32/36), suggesting that an amplification loop involving TRAF2 activation of NF-kappaB is implicated in insulin antiapoptotic signaling. We also show that insulin increased manganese-superoxide dismutase (Mn-SOD) mRNA expression through NF-kappaB activation and that Mn-SOD contributed to insulin antiapoptotic signaling since expression of antisense Mn-SOD RNA decreased this process. This study provides the first evidence that insulin activates the NF-kappaB-dependent survival genes encoding TRAF2 and Mn-SOD and thereby clarifies the role of NF-kappaB in the antiapoptotic function of insulin.

The 3' untranslated region of manganese superoxide dismutase RNA contains a translational enhancer element.

A redox-sensitive protein that binds to the 3' untranslated region (UTR) of manganese superoxide dismutase (MnSOD) RNA has been described previously [Fazzone, H., Wangner, A., and Clerch, L. B. (1993) J. Clin. Invest. 92, 1278-1281; Chung, D. J., and Clerch, L. B. (1997) Am. J. Physiol. 16, L714-L719]. In the present study, cross-competition gel retardation and RNase H assays were used to identify a 41-base region located 111 bases downstream of the stop codon as the 3' UTR cis element involved in protein binding. The base sequence of this region is approximately 75% conserved among the 3' UTRs of rat, mouse, cow, and human MnSOD mRNAs at approximately the same distance downstream of the stop codon. The role of this protein-binding region in RNA translation was assessed in an in vitro rabbit reticulocyte lysate system. Translation of MnSOD RNA from which the 3' UTR element was deleted decreased 60% compared with translation of MnSOD RNA containing the 3' UTR cis element. In the presence of a specific competitor oligoribonucleotide that inhibits MnSOD RNA protein-binding activity, translation of MnSOD RNA containing the 3' UTR was decreased by 65%. Thus, both the cis element and RNA protein-binding activity were required for more efficient translation of the MnSOD. An analysis of ribosomal profiles suggests the MnSOD RNA-binding protein participates in the formation of the translation initiation complex. When MnSOD RNA-binding activity was inhibited, initiation complex formation was decreased by 50%. From the data obtained in this study, we propose that the 3' UTR cis element of MnSOD through its interaction with MnSOD RNA-binding protein may function as a translational enhancer.

Early divergent lung antioxidant enzyme expression in response to lipopolysaccharide.

Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, interacts with eukaryotic cells, causing changes in gene expression and a rapid increase in the formation of superoxide and H2O2. We now report that, within 6 h of treating rats with LPS, there was a divergent response in the lung of manganese superoxide dismutase (Mn-SOD) and catalase expression without a change in expression of copper-zinc superoxide dismutase or glutathione peroxidase. The activity and mRNA concentration of Mn-SOD increased during the time catalase mRNA concentration and activity decreased. These divergent changes and activation of nuclear factor-kappa B (NF-kappa B) were preceded by a fall, 1 h after LPS treatment, in the RNA binding activity of two redox-sensitive proteins: Mn-SOD RNA binding protein and catalase RNA binding protein. The rapid onset of these changes, the bacteriostatic properties of H2O2, and the signaling function of NF-kappa B suggest that the divergent expression of Mn-SOD and catalase is a coordinated component of the acute phase reaction to bacterial invasion.

Differential Regulation of Manganese Superoxide Dismutase Activity by Alcohol and TNF in Human Hepatoma Cells

Increasing evidence implicates free radical processes in the pathogenesis of ethanol-induced liver injury. One of the antioxidant defense systems in mammalian cells is the mitochondrial enzyme manganese superoxide dismutase (MnSOD). MnSOD activity is increased by agents that cause oxidative stress. One such agent is the cytokine tumor necrosis factor-α (TNF). Increased serum/tissue TNF levels have been observed in alcoholic liver disease, and TNF has been postulated to play a role in ethanol-induced liver injury. Substantial evidence suggests that ethanol itself can cause oxidative stress. In order to investigate the mechanism of the cellular adaptive response to ethanol-induced oxidative stress, the effects of short-term ethanol exposure on MnSOD RNA, protein, and activity were determined in a human hepatoma cell line (HepG2). We found that exposure to ethanol (25 mMconcentration) for 72 h increased the protein level and enzyme activity of MnSOD. However, examination of the mRNA levels of the enzyme showed no corresponding increase. Long-term administration of ethanol (10 weeks) did not significantly increase MnSOD protein and MnSOD activity. MnSOD activity was significantly increased by TNF. Thus it appears that both TNF and ethanol are capable of increasing MnSOD activity presumably via enhanced oxidative stress. However, unlike TNF, acute ethanol administration increases the activity of MnSOD without increasing MnSOD mRNA. The increase in MnSOD after a short-term dose of ethanol is diminished with repeated ethanol administrations. These findings are compatible with the view that chronic exposure to ethanol suppresses the cellular adaptive response to oxidative stress. If this adaptive response of MnSOD is lessened, it may have implica tions in the increased toxicity due to prolonged ethanol exposure.

Effects of recombinant superoxide dismutase on manganese superoxide dismutase gene expression in gerbil hippocampus after ischemia.

We reported that recombinant human superoxide dismutase ameliorates delayed neuronal death in the postischemic gerbil hippocampus. Since postischemic induction of copper-zinc superoxide dismutase messenger RNA was abolished by this treatment, oxygen radicals generated on reperfusion may induce the expression of this gene. In the present study we examined whether oxygen radicals also induce the expression of manganese superoxide dismutase messenger RNA in the postischemic brain. We induced transient cerebral ischemia by occluding the bilateral common carotid arteries of gerbils. Recombinant human superoxide dismutase (8 x 10(5) U/kg) or apo-superoxide dismutase was administered intravenously 1 minute before a 5-minute occlusion of the carotid arteries. We analyzed both copper-zinc and manganese superoxide dismutase RNA by in situ hybridization histochemistry and by Northern and dot blot analyses using radioisotope-labeled oligonucleotide probes. Hybridization with the manganese superoxide dismutase messenger RNA occurred at the limit of detection in normal CA1 neurons. We observed striking increases in the labeling of CA1 up to 24 hours after 5 minutes of ischemia. The hybridization occurred anew in glial cells of the CA1 layer during 3 to 7 days. Pretreatment with recombinant human superoxide dismutase had no effect on the postischemic induction of manganese superoxide dismutase messenger RNA, whereas the same treatment significantly attenuated (P < .01) the increase in copper-zinc superoxide dismutase messenger RNA. Our results demonstrated temporal postischemic induction of manganese superoxide dismutase messenger RNA. The inducer may not be superoxide radicals but may be other chemical mediators such as cytokines.

Rat lung contains a developmentally regulated manganese superoxide dismutase mRNA-binding protein.

It has become increasingly clear that RNA-binding proteins play an important role in the regulation of gene expression. The presence in rat lung of a specific, redox-sensitive catalase RNA-binding protein was recently reported (Clerch, L. B., and D. Massaro, 1992. J. Biol. Chem. 267:2853). In order to determine if specific manganese superoxide dismutase (MnSOD) RNA-binding proteins exist, we tested whether protein in rat lung extract would bind to 32P-labeled MnSOD RNA. Using a gel mobility shift assay we show rat lung protein forms specific complexes with a 216 b fragment of the 3' untranslated region of MnSOD RNA and the binding requires the presence of free sulfhydryl groups. Competition studies indicate MnSOD RNA-binding protein is different from catalase RNA-binding protein. Furthermore, unlike catalase RNA-binding protein, rat lung MnSOD RNA-binding protein activity is developmentally regulated; there is less MnSOD RNA-protein binding activity in adult rat lung extract compared to prenatal or neonatal rat lung extracts. We conclude the lung contains developmentally regulated MnSOD mRNA-binding protein that is redox sensitive.

Regulation of Manganese Superoxide Dismutase and Other Antioxidant Genes in Normal and Leukemic Hematopoietic Cells and Their Relationship to Cytotoxicity by Tumor Necrosis Factor

Myeloid cells are a major source of superoxide and other oxygen metabolites. As a protective mechanism, cells express antioxidant enzymes including manganese superoxide dismutase (Mn-SOD), copper-zinc SOD (Cu/Zn-SOD), and glutathione peroxidase (GSX-PX). Even though hematopoietic cells are a major source of oxidants, little is known of their expression of antioxidants. We found that seven myeloid leukemic cell lines blocked at different stages of differentiation constitutively expressed Mn-SOD, Cu/Zn-SOD, and GSX-PX RNAs. Level of Mn-SOD activities paralleled levels of Mn-SOD RNA. Terminal differentiation of native HL-60 cells to either granulocytes or macrophages did not alter levels of Mn-SOD RNA but markedly decreased cell division. Myeloid leukemic lines sensitive to cytotoxic effects of tumor necrosis factor (TNF) as well as normal peripheral blood lymphocytes and monocytes, dramatically increased their levels of Mn-SOD RNA in the presence of TNF. In contrast, Cu/Zn-SOD and GSX-PX RNA levels did not increase in these same cells. TNF-resistant leukemic lines had higher constitutive levels of Mn-SOD RNA and activity; and these levels did not change in the presence of TNF. Antisense but not random oligonucleotides to Mn-SOD markedly increased the sensitivity to the inhibitory effects of TNF for both the native HL-60 (TNF-sensitive) and K562 (TNF-resistant) cell lines. Further studies showed that the antisense oligonucleotides entered the cells and resulted in decreased levels of Mn-SOD RNA. The data suggest that Mn-SOD may provide protection against cytotoxicity of TNF in hematopoietic cells.

Regulation of manganese superoxide dismutase and other antioxidant genes in normal and leukemic hematopoietic cells and their relationship to cytotoxicity by tumor necrosis factor

Myeloid cells are a major source of superoxide and other oxygen metabolites. As a protective mechanism, cells express antioxidant enzymes including manganese superoxide dismutase (Mn-SOD), copper-zinc SOD (Cu/Zn-SOD), and glutathione peroxidase (GSX-PX). Even though hematopoietic cells are a major source of oxidants, little is known of their expression of antioxidants. We found that seven myeloid leukemic cell lines blocked at different stages of differentiation constitutively expressed Mn-SOD, Cu/Zn-SOD, and GSX-PX RNAs. Level of Mn-SOD activities paralleled levels of Mn-SOD RNA. Terminal differentiation of native HL-60 cells to either granulocytes or macrophages did not alter levels of Mn-SOD RNA but markedly decreased cell division. Myeloid leukemic lines sensitive to cytotoxic effects of tumor necrosis factor (TNF) as well as normal peripheral blood lymphocytes and monocytes, dramatically increased their levels of Mn- SOD RNA in the presence of TNF. In contrast, Cu/Zn-SOD and GSX-PX RNA levels did not increase in these same cells. TNF-resistant leukemic lines had higher constitutive levels of Mn-SOD RNA and activity; and these levels did not change in the presence of TNF. Antisense but not random oligonucleotides to Mn-SOD markedly increased the sensitivity to the inhibitory effects of TNF for both the native HL-60 (TNF-sensitive) and K562 (TNF-resistant) cell lines. Further studies showed that the antisense oligonucleotides entered the cells and resulted in decreased levels of Mn-SOD RNA. The data suggest that Mn-SOD may provide protection against cytotoxicity of TNF in hematopoietic cells.

A Role for Manganese Superoxide Dismutase in Radioprotection of Hematopoietic Stem Cells by Interleukin-1

Pretreatment with interleukin-1 (IL-1) has been shown to protect mice from the myelotoxicity associated with irradiation via a mechanism potentially mediated through the induction of the antioxidant enzyme manganese superoxide dismutase (MnSOD). In this study, we have compared the ability of IL-1 to induce MnSOD mRNA in murine bone marrow cells and human cell lines with its ability to protect these cells against the damaging effects of ionizing radiation. Bone marrow cells obtained from mice 6 hours after a single injection of IL-1 demonstrate a dose-dependent increase in the expression of MnSOD RNA. In this same study, IL-1 was also shown to be radioprotective when given to mice 20 hours before lethal irradiation. Similarly, in vitro treatment with IL-1 of bone marrow cells isolated from 5-fluorouracil-treated mice results in elevated levels of MnSOD RNA. Pretreatment with IL-1 also protected bone marrow long-term culture-initiating cells capable of reconstituting irradiated stromal cultures from an irradiation insult. Furthermore, IL-1-treated human bone marrow cells display both elevated MnSOD RNA and protein levels when compared with media controls. The human A375 melanoma, A549 adenocarcinoma, and factor-dependent TF-1 leukemic cell lines demonstrate low basal MnSOD RNA levels that increase following treatment with IL-1. For the A375 cells, this correlates with increased MnSOD protein expression and radioprotection by IL-1 using a colony assay. In contrast, the chronic myelogenous leukemic cell line, K562, displays a high basal MnSOD RNA level, and this RNA expression is not further increased by IL-1 treatment. In addition, these cells are comparatively radioresistant and are not further protected by IL-1 treatment. Finally, the Mo-7 cell line displays a low basal level of MnSOD RNA that correlates with a high sensitivity to irradiation and IL-1 pretreatment has no effect on MnSOD RNA levels. Our results indicate that increased radioprotection by IL-1 correlates with the induction of the antioxidant enzyme MnSOD and this induction may be an important factor in IL-1 radioprotection.

A role for manganese superoxide dismutase in radioprotection of hematopoietic stem cells by interleukin-1

Pretreatment with interleukin-1 (IL-1) has been shown to protect mice from the myelotoxicity associated with irradiation via a mechanism potentially mediated through the induction of the antioxidant enzyme manganese superoxide dismutase (MnSOD). In this study, we have compared the ability of IL-1 to induce MnSOD mRNA in murine bone marrow cells and human cell lines with its ability to protect these cells against the damaging effects of ionizing radiation. Bone marrow cells obtained from mice 6 hours after a single injection of IL-1 demonstrate a dose- dependent increase in the expression of MnSOD RNA. In this same study, IL-1 was also shown to be radioprotective when given to mice 20 hours before lethal irradiation. Similarly, in vitro treatment with IL-1 of bone marrow cells isolated from 5-fluorouracil-treated mice results in elevated levels of MnSOD RNA. Pretreatment with IL-1 also protected bone marrow long-term culture-initiating cells capable of reconstituting irradiated stromal cultures from an irradiation insult. Furthermore, IL-1-treated human bone marrow cells display both elevated MnSOD RNA and protein levels when compared with media controls. The human A375 melanoma, A549 adenocarcinoma, and factor-dependent TF-1 leukemic cell lines demonstrate low basal MnSOD RNA levels that increase following treatment with IL-1. For the A375 cells, this correlates with increased MnSOD protein expression and radioprotection by IL-1 using a colony assay. In contrast, the chronic myelogenous leukemic cell line, K562, displays a high basal MnSOD RNA level, and this RNA expression is not further increased by IL-1 treatment. In addition, these cells are comparatively radioresistant and are not further protected by IL-1 treatment. Finally, the Mo-7 cell line displays a low basal level of MnSOD RNA that correlates with a high sensitivity to irradiation and IL-1 pretreatment has no effect on MnSOD RNA levels. Our results indicate that increased radioprotection by IL-1 correlates with the induction of the antioxidant enzyme MnSOD and this induction may be an important factor in IL-1 radioprotection.