Manganese superoxide dismutase (MnSOD) is a mitochondrial antioxidant enzyme that is down-regulated in a majority of cancers. Due to this observation, as well as MnSOD's potent antioxidant enzymatic activity, MnSOD has been suggested as a tumor suppressor for over 30 years. However, testing this postulate has proven difficult due to the early post-natal lethality of the MnSOD constitutive knock-out mouse. We have previously used a conditional tissue-specific MnSOD knock-out mouse to study the effects of MnSOD loss on the development of various cell types, but long-term cancer development studies have not been performed. We hypothesized the complete loss of MnSOD would significantly increase the rate of tumor formation in a tissue-specific manner.
Utilizing a hematopoietic stem cell specific Cre-recombinase mouse model, we created pan-hematopoietic cell MnSOD knock-out mice. Additionally, we combined this MnSOD knock-out with two well established models of lymphoma development: B-lymphocyte specific Myc over-expression and conditional pan-hematopoietic cell p53 knock-out. Mice were allowed to age unchallenged until illness or death had occurred. Contrary to our initial hypothesis, the loss of MnSOD alone was insufficient in causing an increase in tumor formation, but did cause significant life-shortening skin pathology in a strain-dependent manner. Moreover, the loss of MnSOD in conjunction with either Myc overexpression or p53 knock-out did not accelerate tumor formation, and in fact delayed lymphomagenesis in the p53 knock-out model.
Our findings strongly suggest that MnSOD does not act as a classical tumor suppressor in hematological tissues. Additionally, the complete loss of MnSOD may actually protect from tumor development by the creation of an unfavorable redox environment for tumor progression. In summary, these results in combination with our previous work suggest that MnSOD needs to be tightly regulated for proper cellular homeostasis, and altering the activity in either direction may lead to cellular dysfunction, oncogenesis, or death.
•The free radical theory of cancer postulates that loss of MnSOD promotes cancer.•We created mouse models of malignancy with and without conditional loss of MnSOD.•We show that MnSOD loss delays the onset of p53-dependent tumor development.•Our data suggest that inhibition of MnSOD in tumor cells may slow tumor progression.
MnSOD, manganese superoxide dismutase; FoxO, Forkhead family of transcription factors; Anti-oxidant; Pro-oxidant; Cancer; Oxidative stress; Redox
Alzheimer’s disease (AD) is associated with β-amyloid accumulation, oxidative stress and mitochondrial dysfunction. However, the effects of genetic mutation of AD on oxidative status and mitochondrial manganese superoxide dismutase (MnSOD) production during neuronal development are unclear. To investigate the consequences of genetic mutation of AD on oxidative damages and production of MnSOD during neuronal development, we used primary neurons from new born wild-type (WT/WT) and APP (NLh/NLh) and PS1 (P264L) knock-in mice (APP/PS1) which incorporated humanized mutations in the genome. Increasing levels of oxidative damages, including protein carbonyl, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT), were accompanied by a reduction in mitochondrial membrane potential in both developing and mature APP/PS1 neurons compared to WT/WT neurons suggesting mitochondrial dysfunction under oxidative stress. Interestingly, developing APP/PS1 neurons were significantly more resistant to β-amyloid 1-42 treatment, whereas mature APP/PS1 neurons were more vulnerable than WT/WT neurons of the same age. Consistent with the protective function of MnSOD, developing APP/PS1 neurons have increased MnSOD protein and activity, indicating an adaptive response to oxidative stress in developing neurons. In contrast, mature APP/PS1 neurons exhibited lower MnSOD levels compared to mature WT/WT neurons indicating that mature APP/PS1 neurons lost the adaptive response. Moreover, mature APP/PS1 neurons had more co-localization of MnSOD with nitrotyrosine indicating a greater inhibition of MnSOD by nitrotyrosine. Overexpression of MnSOD or addition of MnTE-2-PyP5+ (SOD mimetic) protected against β-amyloid-induced neuronal death and improved mitochondrial respiratory function. Together, the results demonstrate that compensatory induction of MnSOD in response to an early increase in oxidative stress protects developing neurons against β-amyloid toxicity. However, continuing development of neurons under oxidative damage conditions may suppress the expression of MnSOD and enhance cell death in mature neurons.
Alzheimer’s disease; APP/PS1; MnSOD; Oxidative stress; β-amyloid; MnTE-2-PyP5+ (SOD mimetic)
Inactivation of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant, has been associated with renal disorders and often results in detrimental downstream events that are mechanistically not clear. Development of an animal model that exhibits kidney-specific deficiency of MnSOD would be extremely beneficial in exploring the downstream events that occur following MnSOD inactivation. Using Cre-Lox recombination technology, kidney-specific MnSOD deficient mice (both 100% and 50%) were generated that exhibited low expression of MnSOD in discrete renal cell types and reduced enzymatic activity within the kidney. These kidney-specific 100% KO mice possessed a normal life-span, although it was interesting that the mice were smaller. Consistent with the important role in scavenging superoxide radicals, the kidney-specific KO mice showed a significant increase in oxidative stress (tyrosine nitration) in a gene-dose dependent manner. In addition, loss of MnSOD resulted in mild renal damage (tubular dilation and cell swelling). Hence, this novel mouse model will aid in determining the specific role (local and/or systemic) governed by MnSOD within certain kidney cells. Moreover, these mice will serve as a powerful tool to explore molecular mechanisms that occur downstream of MnSOD inactivation in renal disorders or possibly in other pathologies that rely on normal renal function.
Cre-Lox technology; Kidney; MnSOD; Cre recombinase; Superoxide; Nitrotyrosine
The superoxide free radical (O2•−) has been viewed as a likely major contributor to aging. If this is correct, then superoxide dismutase (SOD), which removes O2•−, should contribute to longevity assurance. In Caenorhabditis elegans, overexpression (OE) of the major cytosolic Cu/Zn-SOD, sod-1, increases life span. But is this increase caused by enhanced antioxidant defense? sod-1 OE did not reduce measures of lipid oxidation or glycation and actually increased levels of protein oxidation. The effect of sod-1 OE on life span was dependent on the DAF-16/FoxO transcription factor (TF) and, partially, on the heat shock TF HSF-1. Similarly, overexpression of sod-2 (major mitochondrial Mn-SOD) resulted in life-span extension that was daf-16 dependent. sod-1 OE increased steady-state hydrogen peroxide (H2O2) levels in vivo. However, co-overexpression of catalase did not suppress the life-span extension, arguing against H2O2 as a cause of longevity. sod-1 OE increased hsp-4 expression, suggesting increased endoplasmic reticulum (ER) stress. Moreover, longevity was partially suppressed by inactivation of ire-1 and xbp-1, mediators of the ER stress response. This suggests that high levels of SOD-1 protein may challenge protein-folding homeostasis, triggering a daf-16- and hsf-1-dependent stress response that extends life span. These findings imply that SOD overexpression increases C. elegans life span, not by removal of O2•−, but instead by activating longevity-promoting transcription factors.
► sod-1 over-expression increases levels of cellular ROS and of molecular damage. ► sod-1 over-expression effects on lifespan are dependent on DAF-16 (FoxO) and HSF-1. ► Increased lifespan is partially suppressed by inactivation of ER stress mediators. ► sod-2 (MnSOD) over-expression effects on lifespan are dependent on DAF-16.
HNE, 4-hydroxynonenal; AMPK, AMP-dependent kinase; CML, carboxymethyllysine; co-OE, co-overexpression; HSF-1, heat shock factor-1; IIS, insulin/IGF-1 signaling; NAC, N-acetylcysteine; OE, overexpression; ROS, reactive oxygen species; RNAi, RNA-mediated interference; O2•−, superoxide anion; SOD, superoxide dismutase; Aging; Caenorhabditis elegans; daf-16/FoxO; ER stress; Oxidative damage; Superoxide dismutase; Free radicals
Previously, we have shown manganese superoxide dismutase (MnSOD) activity protects quiescent human normal skin fibroblasts (NHFs) from age associated loss in proliferative capacity. The loss in proliferative capacity of aged vs. young quiescent cells is often characterized as the chronological life span, which is clearly distinct from replicative senescence. We investigate the hypothesis that MnSOD activity protects the mitochondrial morphology from age associated damage and preserves the chronological life span of quiescent fibroblasts. Aged quiescent NHFs exhibited abnormalities in mitochondrial morphology including abnormal cristae formation and increased number of vacuoles. These results correlate with the levels of cellular reactive oxygen species (ROS) and mitochondrial morphology in MnSOD homozygous and heterozygous knockout mouse embryonic fibroblasts. The abnormalities in mitochondrial morphology in aged quiescent NHFs cultured in presence of 21% oxygen concentration were more severe than NHFs cultured in 4% oxygen environment. The alteration in mitochondrial morphology was associated with a significant increase in cell population doubling: 54 h in 21% compared to 44 h in 4% oxygen environment. Overexpression of MnSOD decreased ROS levels, and preserved mitochondrial morphology in aged quiescent NHFs. These results demonstrate that MnSOD activity protects mitochondrial morphology and preserves the proliferative capacities of quiescent NHFs from age associated loss.
MnSOD; mitochondria; quiescent growth; ROS; Aging; Chronological life span
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.
Increased reactive oxygen species (ROS) such as superoxide have been implicated as causal elements of oncogenesis. A variety of cancers have displayed changes in steady state levels of key antioxidant enzymes with the mitochondrial form of superoxide dismutase (MnSOD) being commonly implicated. Increasing MnSOD expression suppresses the malignant phenotype in various cancer cell lines and suppresses tumor formation in xenograft and transgenic mouse models. We examined the impact of MnSOD expression in the development of T cell lymphoma in mice expressing pro-apoptotic Bax. Lck-Bax38/1 transgenic mice were crossed to mice overexpressing MnSOD (Lck-MnSOD) as well as MnSOD +/− mice. The effect of MnSOD on apoptosis, cell cycle, chromosomal instability (CIN), and lymphoma development was determined. The apoptotic and cell cycle phenotypes observed in thymocytes from control and Bax transgenic mice were unaffected by variations in MnSOD levels. Remarkably, increased gene dosage of MnSOD significantly decreased aneuploidy in pre-malignant thymocytes as well as the onset of tumor formation in Lck-Bax38/1 mice. The observed effects of MnSOD support a role for ROS in CIN and tumor formation in this mouse model of T cell lymphoma.
MnSOD; ROS; Apoptosis; Bcl-2; Oncogenesis; Chromosomal Instability
To test the impact of increased mitochondrial oxidative stress as a mechanism underlying aging and age-related pathologies, we generated mice with a combined deficiency in two mitochondrial-localized antioxidant enzymes, Mn superoxide dismutase (MnSOD) and glutathione peroxidase-1 (Gpx-1). We compared life span, pathology, and oxidative damage in Gpx1−/−, Sod2+/−Gpx1+/−, Sod2+/−Gpx1−/−, and wild-type control mice. Oxidative damage was elevated in Sod2+/−Gpx1−/− mice, as shown by increased DNA oxidation in liver and skeletal muscle and increased protein oxidation in brain. Surprisingly, Sod2+/−Gpx1−/− mice showed no reduction in life span, despite increased levels of oxidative damage. Consistent with the important role for oxidative stress in tumorigenesis during aging, the incidence of neoplasms was significantly increased in the older Sod2+/−Gpx1−/− mice (28–30 months). Thus, these data do not support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice and do not support the oxidative stress theory of aging.
Oxidative stress; Longevity
The manganese superoxide dismutase (MnSOD) ala16val polymorphism has been associated with various diseases including breast cancer. In the present study, we investigated levels of MnSOD protein, enzymatic activity and mRNA with respect to MnSOD genotype in several human breast carcinoma cell lines and in mouse embryonic fibroblasts (MEF), developed from the MnSOD knockout mouse, stably expressing human MnSOD-ala and MnSOD-val. In human breast cell lines, the MnSOD-ala allele was associated with increased levels of MnSOD protein and MnSOD protein per unit mRNA. In the MEF transformants, MnSOD activity correlated fairly well with MnSOD protein levels. MnSOD mRNA expression was significantly lower in MnSOD-ala versus MnSOD-val lines. MnSOD protein and activity levels were not related to MnSOD genotype in the transformed MEF, although, as observed in the human breast cell lines, the MEF human MnSOD-ala lines produced significantly more human MnSOD protein per unit mRNA than the human MnSOD-val lines. This suggests that there is more efficient production of MnSOD-ala protein compared to MnSOD-val protein. Examination of several indicators of reactive oxygen species levels, including superoxide and hydrogen peroxide, in wild type MEF and in MEF expressing similar elevated amounts of MnSOD-ala or val activity did not show differences related to the levels of MnSOD protein expression. In conclusion, in both human breast carcinoma cell lines and MEF cell lines stably transfected with human MnSOD, the MnSOD-ala allele was associated with increased production of MnSOD protein per unit mRNA indicating a possible imbalance in MnSOD protein production from the MnSOD-val mRNA.
MnSOD; breast cancer; oxidative stress; polymorphism
Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant enzyme that localizes to the mitochondria. Expression of MnSOD is essential for the survival of aerobic life. Transgenic mice expressing a luciferase reporter gene under the control of the human MnSOD promoter demonstrate that the level of MnSOD is reduced prior to the formation of cancer. Overexpression of MnSOD in transgenic mice reduces the incidences and multiplicity of papillomas in a DMBA/TPA skin carcinogenesis model. However, MnSOD deficiency does not lead to enhanced tumorigenicity of skin tissue similarly treated because MnSOD can modulate both the p53-mediated apoptosis and AP-1-mediated cell proliferation pathways. Apoptosis is associated with an increase in mitochondrial levels of p53 suggesting a link between MnSOD deficiency and mitochondrial-mediated apoptosis. Activation of p53 is preventable by application of a SOD mimetic (MnTE-2-PyP5+). Thus, p53 translocation to mitochondria and subsequent inactivation of MnSOD explain the observed mitochondrial dysfunction that leads to transcription-dependent mechanisms of p53-induced apoptosis. Administration of MnTE-2-PyP5+ following apoptosis but prior to proliferation leads to suppression of protein carbonyls and reduces the activity of AP-1 and the level of the proliferating cellular nuclear antigen, without reducing the activity of p53 or DNA fragmentation following TPA treatment. Remarkably, the incidence and multiplicity of skin tumors are drastically reduced in mice that receive MnTE-2-PyP5+ prior to cell proliferation. The results demonstrate the role of MnSOD beyond its essential role for survival and suggest a novel strategy for an antioxidant approach to cancer intervention.
MnSOD; p53; Cancer; Chemotherapy
Manganese superoxide dismutase (MnSOD) is a nuclear encoded and mitochondrial matrix localized redox enzyme that is known to regulate cellular redox environment. Cellular redox environment changes during transitions between quiescent and proliferative cycles. Human MnSOD has two poly(A) sites resulting in two transcripts: 1.5 and 4.2 kb. The present study investigates if the 3'-untranslated region (UTR) of MnSOD regulates its expression during transitions between quiescent and proliferating cycles, and in response to radiation. A preferential increase in the 1.5 kb MnSOD transcript levels was observed in quiescent cells, while the abundance of the longer transcript showed a direct correlation with the percentage of S-phase cells. Log transformed expression ratio of the longer to shorter transcript was also higher in proliferating normal and cancer cells. Deletion and reporter assays showed a significant decrease in reporter activity in constructs carrying multiple AU-rich sequence that are present in the 3'-UTR of the longer MnSOD transcript. Overexpression of the MnSOD 3'-UTR representing the longer transcript enhanced endogenous MnSOD mRNA levels, which was associated with an increase in MnSOD protein levels and a decrease in the percentage of S-phase cells. Irradiation increases the mRNA levels of the 1.5 kb MnSOD transcript, which was consistent with a significant increase in reporter activity of the construct carrying the 3'-UTR of the shorter transcript. We conclude that the 3'-UTR of MnSOD regulates MnSOD expression in response to different growth states and radiation.
MnSOD; 3'-Untranslated region; radiation; polyadenylation; quiescence
Previous phenotyping of glucose homeostasis and insulin secretion in a mouse model of hereditary hemochromatosis (Hfe−/−) and iron overload suggested mitochondrial dysfunction. Mitochondria from Hfe−/− mouse liver exhibited decreased respiratory capacity and increased lipid peroxidation. Although the cytosol contained excess iron, Hfe−/− mitochondria contained normal iron but decreased copper, manganese, and zinc, associated with reduced activities of copper-dependent cytochrome c oxidase and manganese-dependent superoxide dismutase (MnSOD). The attenuation in MnSOD activity was due to substantial levels of unmetallated apoprotein. The oxidative damage in Hfe−/− mitochondria is due to diminished MnSOD activity, as manganese supplementation of Hfe−/− mice led to enhancement of MnSOD activity and suppressed lipid peroxidation. Manganese supplementation also resulted in improved insulin secretion and glucose tolerance associated with increased MnSOD activity and decreased lipid peroxidation in islets. These data suggest a novel mechanism of iron-induced cellular dysfunction, namely altered mitochondrial uptake of other metal ions.
Superoxide is widely regarded as the primary reactive oxygen species (ROS) which initiates downstream oxidative stress. Increased oxidative stress contributes, in part, to many disease conditions such as cancer, atherosclerosis, ischemia/reperfusion, diabetes, aging, and neurodegeneration. Manganese superoxide dismutase (MnSOD) catalyzes the dismutation of superoxide into hydrogen peroxide which can then be further detoxified by other antioxidant enzymes. MnSOD is critical in maintaining the normal function of mitochondria, thus its inactivation is thought to lead to compromised mitochondria. Previously, our laboratory observed increased mitochondrial biogenesis in a novel kidney-specific MnSOD knockout mouse. The current study used transient siRNA mediated MnSOD knockdown of normal rat kidney (NRK) cells as the in vitro model, and confirmed functional mitochondrial biogenesis evidenced by increased PGC1α expression, mitochondrial DNA copy numbers and integrity, electron transport chain protein CORE II, mitochondrial mass, oxygen consumption rate, and overall ATP production. Further mechanistic studies using mitoquinone (MitoQ), a mitochondria-targeted antioxidant and L-NAME, a nitric oxide synthase (NOS) inhibitor demonstrated that peroxynitrite (at low micromolar levels) induced mitochondrial biogenesis. These findings provide the first evidence that low levels of peroxynitrite can initiate a protective signaling cascade involving mitochondrial biogenesis which may help to restore mitochondrial function following transient MnSOD inactivation.
•MnSOD knockdown in NRK cells results in a transient loss of MnSOD activity, increased nitrotyrosine and mitochondrial superoxide.•MnSOD knockdown in NRK cells results in a transient induction of mitochondrial biogenesis.•Nitric oxide synthase inhibition and Mitoquinone blocks mitochondrial biogenesis after MnSOD knockdown.•Low doses of peroxynitrite induce biogenesis in NRK cells.
MnSOD; Peroxynitrite; siRNA; mtDNA; Respiration; Mitochondrial biogenesis
In recent years, the intracellular reactive oxygen species (ROS) levels gained increasing attention as a critical regulator of cellular proliferation. We investigated the hypothesis that manganese superoxide dismutase (MnSOD) activity regulates proliferative and quiescent growth by modulating cellular ROS levels. Decreasing MnSOD activity favored proliferation in mouse embryonic fibroblasts (MEFs), while increasing MnSOD activity facilitated proliferating cells’ transitions into quiescence. MnSOD (+/−) and (−/−) MEFs demonstrated increased superoxide steady-state levels; these fibroblasts failed to exit from the proliferative cycle, and showed increasing cyclin D1 and cyclin B1 protein levels. MnSOD (+/−) MEFs exhibited an increase in the percentage of G2 cells compared to MnSOD (+/+) MEFs. Overexpression of MnSOD in MnSOD (+/−) MEFs suppressed superoxide levels and G2 accumulation, decreased cyclin B1 protein levels, and facilitated cells’ transit into quiescence. While ROS are known to regulate differentiation and cell death pathways both of which are irreversible processes, our results show MnSOD activity and therefore, mitochondria-derived ROS levels regulate cellular proliferation and quiescence, which are reversible processes essential to prevent aberrant proliferation and subsequent exhaustion of normal cell proliferative capacity. These results support the hypothesis that MnSOD activity regulates a mitochondrial “ROS-Switch” favoring a superoxide-signaling regulating proliferation and a hydrogen peroxide-signaling supporting quiescence.
MnSOD; Cyclin D1; Cyclin B1; ROS; Cell Cycle; Antioxidant Enzymes; superoxide; hydrogen peroxide
It has been suggested that oxidative stress and mitochondrial DNA damage play important roles in breast cancer carcinogenesis. Manganese superoxide dismutase (MnSOD) is a major enzyme that is responsible for the detoxification of reactive oxygen species in the mitochondria. A T → C substitution in the MnSOD gene results in a Val → Ala change at the -9 position of the mitochondrial targeting sequence (Val-9Ala), which alters the protein secondary structure and thus affects transport of MnSOD into the mitochondria.
We evaluated this genetic polymorphism in association with breast cancer risk using data from the Shanghai Breast Cancer Study, a population-based case–control study conducted in urban Shanghai from 1996 to 1998. The MnSOD Val-9Ala polymorphism was examined in 1125 breast cancer cases and 1197 age-frequency-matched control individual.
Breast cancer risk was slightly elevated in women with Ala/Ala genotype (odds ratio [OR] 1.3, 95% confidence interval [CI] 0.7–2.3), particularly among premenopausal women (OR 1.8, 95% CI 0.9–3.7), as compared with those with Val/Val genotype. The increased risk with the Ala/Ala genotype was stronger among premenopausal women with a higher body mass index (OR 2.5, 95% CI 0.9–7.0) and more years of menstruation (OR 2.6, 95% CI 0.8–8.0). The risk among premenopausal women was further increased twofold to threefold among those with a low intake of fruits, vegetables, vitamin supplements, selenium, or antioxidant vitamins, including carotenes and vitamins A, C, and E. However, the frequency of the Ala allele was low (14%) in the study population, and most of the ORs provided above were not statistically significant.
The present study provides some evidence that genetic polymorphism in the MnSOD gene may be associated with increased risk of breast cancer among Chinese women with high levels of oxidative stress or low intake of antioxidants. Studies with a larger sample size are needed to confirm the findings.
antioxidant; breast cancer; case-control study; MnSOD; polymorphism
The biocontrol potential of entomopathogenic fungi against arthropod pests depends on not only their virulence to target pests but tolerance to outdoor high temperature and solar UV irradiation. Two Beauveria bassiana superoxide dismutases (SODs), BbSod2 and BbSod3, were characterized as cytosolic and mitochondrial manganese-cored isoenzymes (MnSODs) dominating the total SOD activity of the fungal entomopathogen under normal growth conditions. To probe their effects on the biocontrol potential of B. bassiana, ΔBbSod2, ΔBbSod3, and three hairpin RNA-interfered (RNAi) mutants with the transcripts of both BbSod2 and BbSod3 being suppressed by 91–97% were constructed and assayed for various phenotypic parameters in conjunction with ΔBbSod2/BbSod2, ΔBbSod3/BbSod3 and wild-type (control strains). In normal cultures, the knockout and RNAi mutants showed significant phenotypic alterations, including delayed sporulation, reduced conidial yields, and impaired conidial quality, but little change in colony morphology. Their mycelia or conidia became much more sensitive to menadione or H2O2-induced oxidative stress but had little change in sensitivity to the hyperosmolarity of NaCl and the high temperature of 45°C. Accompanied with the decreased antioxidative capability, conidial tolerances to UV-A and UV-B irradiations were reduced by 16.8% and 45.4% for ΔBbSod2, 18.7% and 44.7% for ΔBbSod3, and ∼33.7% and ∼63.8% for the RNAi mutants, respectively. Their median lethal times (LT50s) against Myzus persicae apterae, which were topically inoculated under a standardized spray, were delayed by 18.8%, 14.5% and 37.1%, respectively. Remarkably, the effects of cytosolic BbSod2 and mitochondrial BbSod3 on the phenotypic parameters important for the fungal bioncontrol potential were additive, well in accordance with the decreased SOD activities and the increased superoxide levels in the knockout and RNAi mutants. Our findings highlight for the first time that the two MnSODs co-contribute to the biocontrol potential of B. bassiana by mediating cellular antioxidative response.
Manganese superoxide dismutase (MnSOD) in the mitochondria plays an important role in cellular defense against oxidative damage. Homozygous MnSOD knockout (Sod2−/−) mice are neonatal lethal, indicating the essential role of MnSOD in early development. To investigate the potential cellular abnormalities underlying the aborted development of Sod2−/− mice, we examined the growth of isolated mouse embryonic fibroblasts (MEF) from Sod2−/− mice. We found that the proliferation of Sod2−/− MEFs was significantly decreased when compared with wild type MEFs despite the absence of morphological differences. The Sod2−/− MEFs produced less cellular ATP, had lower O2 consumption, generated more superoxide, and expressed less Prdx3 protein. Furthermore, the loss of MnSOD dramatically altered several markers involved in cell proliferation and growth, including decreased growth stimulatory function of mTOR signaling and enhanced growth inhibitory function of GSK-3β signaling. Interestingly, the G protein coupled receptor-mediated intracellular Ca2+ ([Ca2+]i) signal transduction was also severely suppressed in Sod2−/− MEFs. Finally, the ratio of LC3-II/LC3-I, an index of autophagic activity, was increased in Sod2−/− MEFs, consistent with a reduction of mTOR signal transduction. These data demonstrate that MnSOD deficiency results in alterations in several key signaling pathways, which may contribute to the lethal phenotype of Sod2−/− mice.
MnSOD; oxidative stress; ROS; signal transduction
Manganese superoxide dismutase is a nuclear encoded primary antioxidant enzyme localized exclusively in the mitochondrial matrix. Genotoxic agents, such as UV radiation, generates oxidative stress and cause mitochondrial DNA (mtDNA) damage. The mitochondrial DNA polymerase (Polγ), a major constituent of nucleoids, is responsible for the replication and repair of the mitochondrial genome. Recent studies suggest that mitochondria contain fidelity proteins and MnSOD constitutes an integral part of the nucleoid complex. However, it is not known whether or how MnSOD participates in the mitochondrial repair processes. Using skin tissue from C57/BL6 mice exposed to UVB radiation, we demonstrate that MnSOD plays a critical role in preventing mtDNA damage by protecting the function of Polγ. Q-PCR analysis shows an increase in mtDNA damage after UVB exposure. Immunofluorescence and immunoblotting studies demonstrate p53 translocation to mitochondria and interaction with Polγ after UVB exposure. The mtDNA immunoprecipitation assay with Polγ and p53 antibodies in p53+/+ and p53−/− mice demonstrates an interaction between MnSOD, p53, and Polγ. The results suggest that these proteins form a complex for the repair of UVB-associated mtDNA damage. The data also demonstrate that UVB exposure injures the mtDNA D-loop in a p53-dependent manner. Using MnSOD-deficient mice we demonstrate that UVB-induced mtDNA damage is MnSOD-dependent. Exposure to UVB results in nitration and inactivation of Polγ, which is prevented by addition of the MnSOD mimetic MnIIITE-2-PyP5+. These results demonstrate for the first time that MnSOD is a fidelity protein that maintains the activity of Polγ by preventing UVB-induced nitration and inactivation of Polγ. The data also demonstrate that MnSOD plays a role along with p53 to prevent mtDNA damage.
Fidelity gene; Polymerase gamma; p53; MnSOD; Oxidative/nitrative stresses; MnIIITE-2-PyP5+
The loss of dopaminergic neurons induced by the parkinsonian toxins paraquat, rotenone and 1-methyl-4-phenylpyridinium (MPP+) is associated with oxidative stress. However, controversial reports exist regarding the source/compartmentalization of reactive oxygen species (ROS) generation and its exact role in cell death. We aimed to determine in detail the role of superoxide anion (O2•−), oxidative stress and their subcellular compartmentalization in dopaminergic cell death induced by parkinsonian toxins. Oxidative stress and ROS formation was determined in the cytosol, intermembrane (IMS) and mitochondrial matrix compartments, using dihydroethidine derivatives, the redox sensor roGFP, as well as electron paramagnetic resonance spectroscopy. Paraquat induced an increase in ROS and oxidative stress in both the cytosol and mitochondrial matrix prior to cell death. MPP+ and rotenone primarily induced an increase in ROS and oxidative stress in the mitochondrial matrix. No oxidative stress was detected at the level of the IMS. In contrast to previous studies, overexpression of manganese superoxide dismutase (MnSOD) or copper/zinc SOD (CuZnSOD) had no effect on ROS steady state levels, lipid peroxidation, loss of mitochondrial membrane potential (ΔΨm) and dopaminergic cell death induced by MPP+ or rotenone. In contrast, paraquat-induced oxidative stress and cell death were selectively reduced by MnSOD overexpression, but not by CuZnSOD or manganese-porphyrins. However, MnSOD also failed to prevent ΔΨm loss. Finally, paraquat, but not MPP+ or rotenone, induced the transcriptional activation the redox-sensitive antioxidant response elements (ARE) and nuclear factor kappa-B (NF-κB). These results demonstrate a selective role of mitochondrial O2•− in dopaminergic cell death induced by paraquat, and show that toxicity induced by the complex I inhibitors rotenone and MPP+ does not depend directly on mitochondrial O2•− formation.
Paraquat; MPP+; rotenone; roGFP; SOD; MnSOD; CuZnSOD; porphyrins; pesticides; environmental; Parkinson’s disease
It is well documented that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) can activate manganese superoxide dismutase (MnSOD) expression. However, it is unclear how repeated exposure to TPA following a single application of tumor initiator 7,12-dimethylbenz-(a)-anthracene causes tumor development. We generated transgenic mice expressing human MnSOD promoter-and enhancer-driven luciferase reporter gene and used a noninvasive imaging system to investigate the effects of TPA on MnSOD expression in vivo. Our data indicate that TPA initially activates MnSOD expression, but this positive effect declines after repeated applications. Changes in MnSOD expression in vivo were verified by measuring MnSOD mRNA and protein levels. Using chromatin immunoprecipitation coupled to Western analysis of the transcription factors known to be essential for the constitutive and TPA-induced transcription of MnSOD, we found that TPA treatment leads to both activation and inactivation of MnSOD gene transcription. During the activation phase, the levels of p50, p65, specificity protein 1 (Sp1) and nucleophosmin (NPM) increase after TPA treatments. Sustained treatments with TPA lead to further increase of p50 but not p65, Sp1 or NPM, suggesting that excess p50 may have inhibitory effects leading to the suppression of MnSOD. Alteration of p50 levels by expressing p50 cDNA or p50 small interfering RNA in mouse epithelial (JB6) cells confirms that p50 is inhibitory to MnSOD transcription. These findings identify p50 as having a negative effect on MnSOD induction upon repeated applications of TPA and provide an insight into a cause for the reduction of MnSOD expression during early stages of skin carcinogenesis.
The oxidative stress theory of aging postulates that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS) generated during normal metabolism. Superoxide dismutases (SODs) counteract this process by detoxifying superoxide. It has previously been shown that elimination of either cytoplasmic or mitochondrial SOD in yeast, flies, and mice results in decreased lifespan. In this experiment, we examine the effect of eliminating each of the five individual sod genes present in Caenorhabditis elegans. In contrast to what is observed in other model organisms, none of the sod deletion mutants shows decreased lifespan compared to wild-type worms, despite a clear increase in sensitivity to paraquat- and juglone-induced oxidative stress. In fact, even mutants lacking combinations of two or three sod genes survive at least as long as wild-type worms. Examination of gene expression in these mutants reveals mild compensatory up-regulation of other sod genes. Interestingly, we find that sod-2 mutants are long-lived despite a significant increase in oxidatively damaged proteins. Testing the effect of sod-2 deletion on known pathways of lifespan extension reveals a clear interaction with genes that affect mitochondrial function: sod-2 deletion markedly increases lifespan in clk-1 worms while clearly decreasing the lifespan of isp-1 worms. Combined with the mitochondrial localization of SOD-2 and the fact that sod-2 mutant worms exhibit phenotypes that are characteristic of long-lived mitochondrial mutants—including slow development, low brood size, and slow defecation—this suggests that deletion of sod-2 extends lifespan through a similar mechanism. This conclusion is supported by our demonstration of decreased oxygen consumption in sod-2 mutant worms. Overall, we show that increased oxidative stress caused by deletion of sod genes does not result in decreased lifespan in C. elegans and that deletion of sod-2 extends worm lifespan by altering mitochondrial function.
In this paper, we examine the oxidative stress theory of aging using C. elegans as a model system. This theory proposes that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS). To test this theory, we examined the effect of deleting each of the five individual superoxide dismutase (SOD) genes on lifespan and sensitivity to oxidative stress. Since SOD acts to detoxify ROS, the oxidative stress theory predicts that deletion of sod genes should increase oxidative stress and decrease lifespan. However, in contrast to yeast, flies, and mice, where loss of either cytoplasmic or mitochondrial SOD results in decreased lifespan, we find that none of the sod deletion mutants in C. elegans exhibits a shortened lifespan despite increased sensitivity to oxidative stress. Surprisingly, we find that sod-2 mutant worms have extended lifespan and even worms with the primary cytoplasmic, mitochondrial, and extracellular sod genes deleted can live longer than wild-type worms. By examining genetic interactions with other genes known to extend lifespan and by comparing the phenotype of worms lacking sod-2 to that of known long-lived mitochondrial mutants such as clk-1 or isp-1, we provide evidence that the loss of sod-2 extends lifespan through alteration of mitochondrial function.
Epileptic seizures are a common feature associated with inherited mitochondrial diseases. This study investigated the role of mitochondrial oxidative stress in epilepsy resulting from mitochondrial dysfunction using cross-bred mutant mice lacking mitochondrial manganese superoxide dismutase (MnSOD or SOD2) and a lipophilic metalloporphyrin catalytic antioxidant. Video-EEG monitoring revealed that in the second to third week of postnatal life (P14–P21) B6D2F2 Sod2−/− mice exhibited frequent spontaneous motor seizures providing evidence that oxidative stress-induced mitochondrial dysfunction may contribute to epileptic seizures. To confirm the role of mitochondrial oxidative stress in epilepsy a newly developed lipophilic metalloporphyrin, AEOL 11207, with high potency for catalytic removal of endogenously generated reactive oxygen species was utilized. AEOL 11207-treated Sod2−/− mice showed a significant decrease in both the frequency and duration of spontaneous seizures but no effect on seizure severity. A significant increase in the average lifespan of AEOL 11207-treated Sod2−/− mice compared to vehicle-treated Sod2−/− mice was also observed. Indices of mitochondrial oxidative stress and damage (aconitase inactivation, 3-nitrotyrosine formation, and depletion of reduced coenzyme A) and ATP levels affecting neuronal excitability were significantly attenuated in the brains of AEOL 11207-treated Sod2−/− mice compared to vehicle-treated Sod2−/− mice. The occurrence of epileptic seizures in Sod2−/− mice and the ability of catalytic antioxidant therapy to attenuate seizure activity, mitochondrial dysfunction, and ATP levels suggest that ongoing mitochondrial oxidative stress can contribute to epilepsy associated with mitochondrial dysfunction and disease.
Seizures; EEG; antioxidant; mitochondrial encephalopathy; glutathione; 3-nitrotyrosine; aconitase; complexes; ATP
Oxidative stress in the RPE is widely accepted as a contributing factor to AMD. We have previously shown that ribozyme-mediated reduction in the antioxidant enzyme manganese superoxide dismutase (MnSOD) leads to some of the features of geographic atrophy in mice. To develop a mouse model independent of viral injection, we used a conditional knockout of the Sod2 gene in the RPE to elevate mitochondrial oxidative stress in that cell layer.
Experimental mice in which exon 3 of Sod2 was flanked by loxP sites were also transgenic for PVMD2-rtTA and tetO-PhCMV
cre, so that cre recombinase was expressed only in the RPE. Pups of this genotype (Sod2flox/floxVMD2cre) were induced to express cre recombinase by feeding doxycycline-laced chow to nursing dams. Controls included mice of this genotype not treated with doxycycline and doxycycline-treated Sod2flox/flox mice lacking the cre transgene. Expression of cre in the RPE was verified by immunohistochemistry, and deletion of Sod2 exon 3 in the RPE was confirmed by PCR. Mice were followed up over a period of 9 months by spectral-domain optical coherence tomography (SD-OCT), digital fundus imaging, and full-field ERG. Following euthanasia, retinas were examined by light and electron microscopy or by immunohistochemistry. Contour length of rod outer segments and thickness of the RPE layer were measured by unbiased stereology.
Following doxycycline induction of cre, Sod2flox/flox cre mice demonstrated increased signs of oxidative stress in the RPE and accumulation of autofluorescent material by age 2 months. They showed a gradual decline in the ERG response and thinning of the outer nuclear layer (by SD-OCT), which were statistically significant by 6 months. In addition, OCT and electron microscopy revealed increased porosity of the choroid. At the same interval, hypopigmented foci appeared in fundus micrographs, and vascular abnormalities were detected by fluorescein angiography. By 9 months, the RPE layer in Sod2flox/flox cre mice was thicker than in nontransgenic littermates, and the rod outer segments were significantly longer over most of the retina, although localized atrophy of photoreceptors was also obvious in some eyes.
Conditional tissue-specific reduction in MnSOD induced oxidative stress in mouse RPE, leading to RPE dysfunction, damage to the choroid, and death of photoreceptor cells. The RPE oxidative stress did not cause drusen-like deposits, but the model recapitulated certain key aspects of the pathology of dry AMD and may be useful in testing therapies.
The RPE-specific deletion of mitochondrial superoxide dismutase in mice leads to oxidative stress in the RPE, accumulation of autofluorescent material, a decline in the ERG response, and localized death of photoreceptors. This model may be useful in understanding the mechanism of geographic atrophy.
retinal degeneration; mouse model; reactive oxygen species; manganese superoxide dismutase; retinal pigment epithelium
Proliferating cells consume more glucose to cope with the bioenergetics and biosynthetic demands of rapidly dividing cells as well as to counter a shift in cellular redox environment. This study investigates the hypothesis that manganese superoxide dismutase (MnSOD) regulates cellular redox flux and glucose consumption during the cell cycle. A direct correlation was observed between glucose consumption and percentage of S-phase cells in MnSOD wild type fibroblasts, which was absent in MnSOD homozygous knockout fibroblasts. Results from electron paramagnetic resonance spectroscopy and flow cytometry assays showed a significant increase in cellular superoxide levels in S-phase cells, which was associated with an increase in glucose and oxygen consumption, and a decrease in MnSOD activity. Mass spectrometry results showed a complex pattern of MnSOD-methylation at both lysine (68, 89, 122, and 202) and arginine (197 and 216) residues. MnSOD protein carrying a K89A mutation had significantly lower activity compared to wild type MnSOD. Computational-based simulations indicate that lysine and arginine methylation of MnSOD during quiescence would allow greater accessibility to the enzyme active site as well as increase the positive electrostatic potential around and within the active site. Methylation-dependent changes in the MnSOD conformation and subsequent changes in the electrostatic potential around the active site during quiescence vs. proliferation could increase the accessibility of superoxide, a negatively charged substrate. These results support the hypothesis that MnSOD regulates a “metabolic switch” during progression from quiescent through the proliferative cycle. We propose MnSOD as a new molecular player contributing to the “Warburg effect.”
MnSOD; methylation; glucose; cell cycle; Warburg effect
Angiotensin II (Ang II) produces oxidative stress and endothelial dysfunction in blood vessels. The vasculature from females may be protected against deleterious effects of Ang II. We tested the hypothesis that manganese superoxide dismutase (MnSOD) protects against Ang II-induced endothelial dysfunction. Experiments were performed in C57Bl/6, wild-type (MnSOD+/+) and MnSOD deficient (MnSOD+/−) mice treated systemically with vehicle or Ang II. Basilar arteries were isolated from C57Bl/6 mice treated for one week with a non-pressor dose of Ang II (0.28 mg/kg × day). Ang II treatment produced superoxide-mediated impairment of responses to the endothelium-dependent vasodilator acetylcholine (Ach)(P<0.05). In male, but not female MnSOD+/+ mice, Ang II modestly inhibited responses to ACh (P<0.05). In contrast, Ang II selectively impaired these responses by up to 70% in male MnSOD+/− mice (P<0.05) and this effect was reversed by tempol (P<0.05). Ang II had no effect on ACh responses in MnSOD+/− female mice. Vascular superoxide levels following treatment with an inhibitor of CuZn- and extracellular-SOD, were higher in Ang II-treated vs vehicle-treated MnSOD+/− mice. Thus, a non-pressor dose of Ang II produces endothelial dysfunction in male mice only, suggesting that the female vasculature is protected from Ang II. In male, but not female mice, MnSOD deficiency enhanced endothelial dysfunction, suggesting that MnSOD normally protects the vasculature during disease states in which Ang II contributes to vascular dysfunction.
genetically-altered mice; cerebral arteries; mitochondria; oxidative stress