FUsed in sarcoma is a novel regulator of manganese superoxide dismutase gene transcription.

AIMS: FUsed in sarcoma (FUS) is a multifunctional DNA/RNA-binding protein that possesses diverse roles, such as RNA splicing, RNA transport, DNA repair, translation, and transcription. The network of enzymes and processes regulated by FUS is far from being fully described. In this study, we have focused on the mechanisms of FUS-regulated manganese superoxide dismutase (MnSOD) gene transcription. RESULTS: Here we demonstrate that FUS is a component of the transcription complex that regulates the expression of MnSOD. Overexpression of FUS increased MnSOD expression in a dose-dependent manner and knockdown of FUS by siRNA led to the inhibition of MnSOD gene transcription. Reporter analyses, chromatin immunoprecipitation assay, electrophoretic mobility shift assay, affinity chromatography, and surface plasmon resonance analyses revealed the far upstream region of MnSOD promoter as an important target of FUS-mediated MnSOD transcription and confirmed that FUS binds to the MnSOD promoter and interacts with specificity protein 1 (Sp1). Importantly, overexpression of familial amyotropic lateral sclerosis (fALS)-linked R521G mutant FUS resulted in a significantly reduced level of MnSOD expression and activity, which is consistent with the decline in MnSOD activity observed in fibroblasts from fALS patients with the R521G mutation. R521G-mutant FUS abrogates MnSOD promoter-binding activity and interaction with Sp1. INNOVATION AND CONCLUSION: This study identifies FUS as playing a critical role in MnSOD gene transcription and reveals a previously unrecognized relationship between MnSOD and mutant FUS in fALS.

Curbing cancer's sweet tooth: is there a role for MnSOD in regulation of the Warburg effect?

Reactive oxygen species (ROS), while vital for normal cellular function, can have harmful effects on cells, leading to the development of diseases such as cancer. The Warburg effect, the shift from oxidative phosphorylation to glycolysis, even in the presence of adequate oxygen, is an important metabolic change that confers many growth and survival advantages to cancer cells. Reactive oxygen species are important regulators of the Warburg effect. The mitochondria-localized antioxidant enzyme manganese superoxide dismutase (MnSOD) is vital to survival in our oxygen-rich atmosphere because it scavenges mitochondrial ROS. MnSOD is important in cancer development and progression. However, the significance of MnSOD in the regulation of the Warburg effect is just now being revealed, and it may significantly impact the treatment of cancer in the future.

Manganese superoxide dismutase regulation and cancer.

Mitochondria are the power plants of the eukaryotic cell and the integrators of many metabolic activities and signaling pathways important for the life and death of a cell. Normal aerobic cells use oxidative phosphorylation to generate ATP, which supplies energy for metabolism. To drive ATP production, electrons are passed along the electron transport chain, with some leaking as superoxide during the process. It is estimated that, during normal respiration, intramitochondrial superoxide concentrations can reach 10⁻¹² M. This extremely high level of endogenous superoxide production dictates that mitochondria are equipped with antioxidant systems that prevent consequential oxidative injury to mitochondria and maintain normal mitochondrial functions. The major antioxidant enzyme that scavenges superoxide anion radical in mitochondria is manganese superoxide dismutase (MnSOD). Extensive studies on MnSOD have demonstrated that MnSOD plays a critical role in the development and progression of cancer. Many human cancer cells harbor low levels of MnSOD proteins and enzymatic activity, whereas some cancer cells possess high levels of MnSOD expression and activity. This apparent variation in MnSOD level among cancer cells suggests that differential regulation of MnSOD exists in cancer cells and that this regulation may be linked to the type and stage of cancer development. This review summarizes current knowledge of the relationship between MnSOD levels and cancer with a focus on the mechanisms regulating MnSOD expression.

Manganese superoxide dismutase: beyond life and death.

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-PyP(5+)). 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-PyP(5+) 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-PyP(5+) 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.

Manganese superoxide dismutase vs. p53: regulation of mitochondrial ROS.

Coordination of mitochondrial and nuclear activities is vital for cellular homeostasis, and many signaling molecules and transcription factors are regulated by mitochondria-derived reactive oxygen species (ROS) to carry out this interorganellar communication. The tumor suppressor p53 regulates myriad cellular functions through transcription-dependent and -independent mechanisms at both the nucleus and mitochondria. p53 affect mitochondrial ROS production, in part, by regulating the expression of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD). Recent evidence suggests mitochondrial regulation of p53 activity through mechanisms that affect ROS production, and a breakdown of communication amongst mitochondria, p53, and the nucleus can have broad implications in disease development.

Nucleophosmin blocks mitochondrial localization of p53 and apoptosis.

Activation of p53 is an important mechanism in apoptosis. However, whether the presence of p53 in mitochondria plays an important role in p53-mediated apoptosis is unclear. Here, we demonstrate that overexpression of NPM (nucleophosmin) significantly suppresses 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated apoptosis, in part, by blocking the mitochondrial localization of p53. Within 1 h following TPA treatment of skin epithelial (JB6) cells, p53 accumulated in mitochondria. Expression of NPM enhances p53 levels in the nucleus but reduces p53 levels in mitochondria, as detected by immunocytochemistry and Western blot analysis. The suppressive effect of NPM on p53 mitochondrial localization is also observed in TPA-treated primary epithelial cells and in JB6 cells treated with doxorubicin. NPM enhances the expression of p53 target gene p21 and bax. However, the increase in Bax level in the absence of p53 in mitochondria did not lead to an increase in TPA-induced apoptosis, suggesting that the presence of p53 in mitochondria is important. Suppression of NPM by NPM small interfering RNA leads to an increase of p53 levels in mitochondria and apoptosis. Furthermore, suppression of NPM in tumor cells with a high constitutive level of NPM results in p53 translocation to mitochondria and enhances TPA-mediated apoptosis. The results demonstrate the effect of NPM on p53 localization in mitochondria and apoptosis. Together, the data indicate that the presence of p53 in mitochondria plays an important role in stress-induced apoptosis and suggest that NPM may protect cells from apoptosis by reducing the mitochondrial level of p53.

Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells.

The physicochemical properties of nanomaterials differ from those of the bulk material of the same composition. However, little is known about the underlying effects of these particles in carcinogenesis. The purpose of this study was to determine the mechanisms involved in the carcinogenic properties of nanoparticles using aluminum oxide (Al(2)O(3)/alumina) nanoparticles as the prototype. Well-established mouse epithelial JB6 cells, sensitive to neoplastic transformation, were used as the experimental model. We demonstrate that alumina was internalized and maintained its physicochemical composition inside the cells. Alumina increased cell proliferation (53%), proliferating cell nuclear antigen (PCNA) levels, cell viability and growth in soft agar. The level of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, was elevated, suggesting a redox signaling event. In addition, the levels of reactive oxygen species and the activities of the redox sensitive transcription factor activator protein-1 (AP-1) and a longevity-related protein, sirtuin 1 (SIRT1), were increased. SIRT1 knockdown reduces DNA synthesis, cell viability, PCNA levels, AP-1 transcriptional activity and protein levels of its targets, JunD, c-Jun and BcL-xl, more than controls do. Immunoprecipitation studies revealed that SIRT1 interacts with the AP-1 components c-Jun and JunD but not with c-Fos. The results identify SIRT1 as an AP-1 modulator and suggest a novel mechanism by which alumina nanoparticles may function as a potential carcinogen.

Chronic exposure to 12-O-tetradecanoylphorbol-13-acetate represses sod2 induction in vivo: the negative role of p50.

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 non-invasive 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.

Specificity protein 1-dependent p53-mediated suppression of human manganese superoxide dismutase gene expression.

Manganese superoxide dismutase (MnSOD) is a primary antioxidant enzyme necessary for the survival of aerobic life. Previously, we demonstrated that specificity protein 1 (Sp1) is essential for the basal transcription of the MnSOD gene. We also identified nucleophosmin (NPM), an RNA-binding protein, as an important co-activator of NF-kappaB in the induction of MnSOD by cytokine and tumor promoter. Here, using chromatin immunoprecipitation (ChIP) analysis, we demonstrate that Sp1 and NPM interact in vivo to enhance NF-kappaB-mediated MnSOD induction. Interaction between NPM and Sp1 or NF-kappaB at the promoter and enhancer of the MnSOD gene in vivo were verified by the presence of the PCR products from the promoter and enhancer elements in the ChIP assay. Unexpectedly, we also found p53, another transcription factor, to be a component of the complex detected by ChIP assay. The presence of p53 in this transcription complex was verified by immunoprecipitation of p53 proteins with antibody to Sp1 in nuclear extracts. Using a vector expressing full-length p53 cDNA, we demonstrated that p53 overexpression suppresses MnSOD mRNA and protein levels. Consistent with the negative role of p53 in the expression of the MnSOD gene, expression of small interfering RNA for p53 leads to an increase of MnSOD mRNA and protein levels. Using ChIP assays and immunoprecipitation, we further demonstrated that p53 interacts with Sp1 to suppress both the constitutive and 12-O-tetradecanoylphorbol-13-acetate-stimulated expression of the MnSOD gene. Inhibition of the MnSOD gene by p53 was abolished when Sp1 sites on the MnSOD promoter were mutated or when the Sp1 protein was reduced by siRNA approaches. Because expression of MnSOD protects against cell death, our findings reveal a previously unrecognized mechanism of p53-mediated cell death and demonstrate an intricate relationship between the positive and negative control of MnSOD expression.