Managing odds in stem cells: insights into the role of mitochondrial antioxidant enzyme MnSOD

Reactive oxygen species (ROS) have been poised at a straddled state of being beneficiary as well detrimental depending on its threshold levels. Maintaining the homeostasis of ROS is imperative for normal cellular physiology, wherein physiological concentrations of ROS are involved in cell signaling and elevated ROS contribute to the development of various diseases. Superoxide dismutases (SODs), enzymes involved in dismutation of superoxide anion to hydrogen peroxide, arrive as a first line of defense when there is perturbation in the homeostasis of ROS. As mitochondria are the main site of superoxide production, among SODs, mitochondrial manganese SOD (MnSOD) is the primary antioxidant enzyme that protects cells from ROS. Most importantly, knockout of MnSOD leads to postnatal lethality and tissue-specific conditional knockout in brain resulted in death of mice, conclusively portraying the essential role of MnSOD in development. Although MnSOD has been extensively discussed with the purview of tumor biology and aging, understanding the crucial role of MnSOD in stem cell physiology is still at its infant stage. Ever increasing progress in stem cell research has recently unveiled the essential role of MnSOD in self-renewal and differentiation of stem cells. In this review, we will conglomerate the current aspects by which MnSOD can contribute to embryonic stem cells’ and adult stem cells’ functions and interpret the necessity of understanding MnSOD for further stem cell mediated applications.     

Manganese superoxide dismutase activity regulates transitions between quiescent and proliferative growth

In recent years, the intracellular reactive oxygen species (ROS) levels have 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 (MEF), 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 G(2) cells compared to MnSOD +/+ MEFs. Overexpression of MnSOD in MnSOD +/- MEFs suppressed superoxide levels and G(2) 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.     

Water restriction increases renal inner medullary manganese superoxide dismutase (MnSOD)

Oxidative stress damages cells. NaCl and urea are high in renal medullary interstitial fluid, which is necessary to concentrate urine, but which causes oxidative stress by elevating reactive oxygen species (ROS). Here, we measured the antioxidant enzyme superoxide dismutases (SODs, MnSOD, and Cu/ZnSOD) and catalase in mouse kidney that might mitigate the oxidative stress. MnSOD protein increases progressively from the cortex to the inner medulla, following the gradient of increasing NaCl and urea. MnSOD activity increases proportionately, but MnSOD mRNA does not. Water restriction, which elevates renal medullary NaCl and urea, increases MnSOD protein, accompanied by a proportionate increase in MnSOD enzymatic activity in the inner medulla, but not in the cortex or the outer medulla. In contrast, Cu/ZnSOD and TNF-α (an important regulator of MnSOD) do not vary between the regions of the kidney, and expression of catalase protein actually decreases from the cortex to the inner medulla. Water restriction increases activity of mitochondrial enzymes that catalyze production of ROS in the inner medulla, but reduces NADPH oxidase activity there. We also examined the effect of high NaCl and urea on MnSOD in Madin-Darby canine kidney (MDCK) cells. High NaCl and high urea both increase MnSOD in MDCK cells. This increase in MnSOD protein apparently depends on the elevation of ROS since it is eliminated by the antioxidant N-acetylcysteine, and it occurs without raising osmolality when ROS are elevated by antimycin A or xanthine oxidase plus xanthine. We conclude that ROS, induced by high NaCl and urea, increase MnSOD activity in the renal inner medulla, which moderates oxidative stress.     

Induction of apoptosis by adenovirus-mediated manganese superoxide dismutase overexpression in SV-40-transformed human fibroblasts

Although mitochondrial reactive oxygen species (ROS) have been implicated both as an initiator and as an effector of apoptosis, the exact role of mitochondrial ROS has been difficult to establish due to the lack of an appropriate experimental system where ROS could be specifically generated from mitochondria and subsequent effects on cells analyzed. In this study, a manganese superoxide dismutase (MnSOD) activity-mediated apoptosis model was established and characterized. It was shown that despite early increases in the steady-state levels of ROS upon MnSOD overexpression, cellular oxidative damage was decreased significantly at later time points. Alterations in levels of peroxiredoxin (Prxn1) protein preceded the onset of apoptosis after MnSOD overexpression. A time course study demonstrated that increases in MnSOD activity prior to the onset of apoptosis correlated with alterations in the levels of nitration of tyrosine residue(s) of MnSOD protein. A direct correlation between MnSOD activity and the degree of apoptosis was demonstrated using a mutant MnSOD with decreased activity. The current study supports a causative role of mitochondrial ROS leading to the onset of apoptosis. The MnSOD activity-mediated apoptosis model described here could be further utilized to study mitochondrial apoptotic pathways.     

Roles of mitochondria-generated reactive oxygen species on X-ray-induced apoptosis in a human hepatocellular carcinoma cell line,HLE

Abstract

HLE, a human hepatocellular carcinoma cell line was transiently transfected with normal human MnSOD and MnSOD without a mitochondrial targeting signal (MTS). Mitochondrial reactive oxygen species (ROS), lipid peroxidation and apoptosis were examined as a function of time following 18.8 Gy X-ray irradiation. Our results showed that the level of mitochondrial ROS increased and reached a maximum level 2 hours after X-ray irradiation. Authentic MnSOD, but not MnSOD lacking MTS, protected against mitochondrial ROS, lipid peroxidation and apoptosis. In addition, the levels of mitochondrial ROS were consistently found to always correlate with the levels of authentic MnSOD in mitochondria. These results suggest that only when MnSOD is located in mitochondria is it efficient in protecting against cellular injuries by X-ray irradiation and that mitochondria are the critical sites of X-ray-induced cellular oxidative injuries.     

Activation of matrix metalloproteinase-2 by overexpression of manganese superoxide dismutase in human breast cancer MCF-7 cells involves reactive oxygen species

Matrix metalloproteinases (MMPs) participate in cell migration and remodeling processes by affecting the extracellular matrix. MMP-2 is thought to be involved in cancer cell invasiveness. It has been proposed that the activity of MMP-2 can be modulated by intracellular reactive oxygen species (ROS)/reactive nitrogen species. We hypothesized that manganese superoxide dismutase (MnSOD) could mediate MMP-2 activity by changing the intracellular ROS level and that nitric oxide ((.)NO) may be involved in this process. Human breast cancer MCF-7 cells were stably transfected with plasmids containing MnSOD cDNA. A 2-30-fold increase of MnSOD protein and activity was observed in four clones. Our data demonstrated that overexpression of MnSOD stimulated the activation of MMP-2 with a corresponding elevation of ROS. A decrease in ROS by ebselen, a glutathione peroxidase mimetic, or by transduction of adenovirus containing human catalase or glutathione peroxidase cDNA abolished the effect of MnSOD on MMP-2 activation. Treatment of MCF-7 cells with antimycin A or rotenone increased intracellular ROS production and MMP-2 activation simultaneously. Our data also showed a suppression of endothelial nitric-oxide synthase expression that was accompanied by decreased (.)NO production in MnSOD-overexpressing cells. However, the changes in endothelial nitric-oxide synthase and (.)NO did not correlate with the MnSOD activity. Corresponding changes of MMP-2 activity after the addition of a NOS inhibitor (N(G)-amino-l-arginine) or a (.)NO donor ((Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate) to the cells suggested the possibility that (.)NO may be involved in the MnSOD-mediated MMP-2 activation pathway. These results indicate that MnSOD induces MMP-2 activity by regulation of intracellular ROS and imply that signaling pathways involving (.)NO may also be involved in the MnSOD mediation of MMP-2 activity.     

Roles of mitochondria-generated reactive oxygen species on X-ray-induced apoptosis in a human hepatocellular carcinoma cell line, HLE

HLE, a human hepatocellular carcinoma cell line was transiently transfected with normal human MnSOD and MnSOD without a mitochondrial targeting signal (MTS). Mitochondrial reactive oxygen species (ROS), lipid peroxidation and apoptosis were examined as a function of time following 18.8 Gy X-ray irradiation. Our results showed that the level of mitochondrial ROS increased and reached a maximum level 2 hours after X-ray irradiation. Authentic MnSOD, but not MnSOD lacking MTS, protected against mitochondrial ROS, lipid peroxidation and apoptosis. In addition, the levels of mitochondrial ROS were consistently found to always correlate with the levels of authentic MnSOD in mitochondria. These results suggest that only when MnSOD is located in mitochondria is it efficient in protecting against cellular injuries by X-ray irradiation and that mitochondria are the critical sites of X-ray-induced cellular oxidative injuries.     

内源性活性氧水平及锰型超氧化物歧化酶的表达与胃癌细胞分化、增殖相关

检测了不同分化的胃癌细胞株内MnSOD基因的表达及胞内活性氧限（ROS)的水平。同时通过基因转染观察上调或下调MnSOD基因表达对SGC790l胃癌细胞胞内ROS水平及增殖能力的影响。用电穿孔法将人反义和正义MnSOD cDNA真核表达载体pHβA—SOD(-)／pHβA—SOD（ )转入790l细胞,用含G418的RPMIl640培养基筛选稳定表达克隆。然后用RT-PCR鉴定MnDSOD基因的表达。同时用RT-PCR方法检测正常胃粘膜组织及MKN-28、SGC790l、BGC823、HGC-27四株高、中、低、未分化胃癌细胞株内的MnSoD的mRNA表达。利用DCFH-DA荧光染色方法检测不同分化胃癌细胞株及790l转染细胞株内的ROS水平。四唑蓝比色法(MTT)绘制MKN-28、SGC790l、BGC823、HGC-27四株不同分化胃癌细胞及正义、反义、空载MnSOD转染790l细胞的生长曲线。发现不同分化胃癌细胞内的MnSOD普遍呈低表达且与分化程度平行,不同分化胃癌细胞株胞内ROS水平随着MnSOD表达的下调逐步上升,细胞增殖加快。较之MnSOD空载790l,正义、反义MnSOD转染的790l细胞中该基因的表达出现明显上调及下调,胞内ROS水平较对照细胞也相应有显著降低和升高。正义株增殖受抑,反义株增殖加快。表明胃癌细胞内MnSOD的表达与肿瘤的分化程度呈负相关。可通过改变胞内RoS水平改变MnSOD基因的表达,从而调节胃癌细胞的生长。     

The Hsp60 folding machinery is crucial for manganese superoxide dismutase folding and function

Abstract

Even though the deleterious effects of increased reactive oxygen species (ROS) levels have been implicated in a variety of neurodegenerative disorders, the triggering events that lead to the increased ROS and successive damages are still ill-defined. Mitochondria are the key organelles controlling the ROS balance, being their main source and also counteracting them by the action of the ROS scavenging system. Mitochondria, moreover, control the presence of ROS-damaged proteins by action of the protein quality control (PQC) system. One of its components is the mitochondrial chaperone Hsp60 assisting the folding of a subset of mitochondrial matrix proteins. Mutations in Hsp60 cause a late onset form of the neurodegenerative disease hereditary spastic paraplegia (SPG13). In this study, we aimed to address the molecular consequences of Hsp60 shortage. We here demonstrate that a heterozygous knockout Hsp60 model that recapitulates features of the human disease and exhibits increased oxidative stress in neuronal tissues. Moreover, we indicate that the increase of ROS is, at least in part, due to impaired folding of the manganese superoxide dismutase (MnSOD), a key antioxidant enzyme. We observed that the Hsp60 and MnSOD proteins interact. Based on these results, we propose that MnSOD is a substrate of the Hsp60 folding machinery and that under conditions of diminished availability of Hsp60, MnSOD is impaired in reaching the native state. This suggests a possible link between Hsp60-dependent PQC and the ROS scavenging systems that may have the function to increase ROS production under conditions of folding stress.     

Overexpression of Manganese Superoxide Dismutase Promotes the Survival of Prostate Cancer Cells Exposed to Hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1?h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Overexpression of manganese superoxide dismutase promotes the survival of prostate cancer cells exposed to hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1 h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Redox-regulation of Erk1/2-directed phosphatase by reactive oxygen species: role in signaling TPA-induced growth arrest in ML-1 cells

Extracellular signal-regulated kinase (Erk)1/2 activity signals myeloid cell differentiation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Previously, we reported that Erk1/2 activation (phosphorylation) induced by TPA required reactive oxygen species (ROS) as a second messenger. Here, we hypothesized that ROS generated in response to TPA inhibit Erk1/2-directed phosphatase activity, which leads to an increase phosphorylation of Erk1/2 to signal p21(WAF1/Cip1)-mediated growth arrest in ML-1 cells. Incubation of ML-1 cells with TPA resulted in a marked accumulation of phosphorylated Erk1/2, and is subsequent to H2O2 generation. Interestingly, post-TPA-treatment with N-acetylcysteine (NAC) stimulated a marked and a rapid dephosphorylation of Erk1/2, suggesting a regeneration of Erk1/2-directed phospahatase activity by NAC. ROS generation in ML-1 cells induced by TPA was suggested to occur in the mitochondrial electron transport chain (METC) based on the following observations: (i) undifferentiated ML-1 cells not only lack p67-phox and but also express a low level of p47-phox key components required for NADPH oxidase enzymatic activity, (ii) pretreatment with DPI, an inhibitor of NADH- and NADPH-dependent enzymes, or rhein, an inhibitor of complex I, blocked the ROS generation, and (iii) examination of the microarray analysis data and Western blot analysis data revealed an induction of MnSOD expression at both mRNA and protein levels in response to TPA. MnSOD is a key member of the mitochondrial defense system against mitochondrial-derived superoxide. Together, this study suggested that TPA stimulated ROS generation as a second messenger to activate Erk1/2 via a redox-mediated inhibition of Erk1/2-directed phosphatase in ML-1 cells.     

Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK(-) bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles.     

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

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