Mitochondrial superoxide targets energy metabolism to modulate epigenetic regulation of NRF2-mediated transcription.

Mitochondria are central to the metabolic circuitry that generates superoxide radicals/anions (O2•-) as a by-product of oxygen metabolism. By regulating superoxide levels, manganese superoxide dismutase plays important roles in numerous biochemical and molecular events essential for the survival of aerobic life. In this study, we used MitoParaquat (mPQ) to generate mitochondria-specific O2•- and stable isotope-resolved metabolomics tracing in primary human epidermal keratinocytes to investigate how O2•- generated in mitochondria regulates gene expression. The results reveal that isocitrate is blocked from conversion to α-ketoglutarate and that acetyl-coenzyme A (CoA) accumulates, which is consistent with a reduction in oxygen consumption rate and inactivation of isocitrate dehydrogenase (IDH) activity. Since acetyl-CoA is linked to histone acetylation and gene regulation, we determined the effect of mPQ on histone acetylation. The results demonstrate an increase in histone H3 acetylation at lysines 9 and 14. Suppression of IDH increased histone acetylation, providing a direct link between metabolism and epigenetic alterations. The activity of histone acetyltransferase p300 increased after mPQ treatment, which is consistent with histone acetylation. Importantly, mPQ selectively increased the nuclear levels and activity of the oxidative stress-sensitive nuclear factor erythroid 2-related factor 2. Together, the results establish a new paradigm that recognizes O2•- as an initiator of metabolic reprogramming that activates epigenetic regulation of gene transcription in response to mitochondrial dysfunction.

UVB-induced inactivation of manganese-containing superoxide dismutase promotes mitophagy via ROS-mediated mTORC2 pathway activation.

Mitochondria are major sites of energy metabolism that influence numerous cellular events, including immunity and cancer development. Previously, we reported that the mitochondrion-specific antioxidant enzyme, manganese-containing superoxide dismutase (MnSOD), has dual roles in early- and late-carcinogenesis stages. However, how defective MnSOD impacts the chain of events that lead to cell transformation in pathologically normal epidermal cells that have been exposed to carcinogens is unknown. Here, we show that UVB radiation causes nitration and inactivation of MnSOD leading to mitochondrial injury and mitophagy. In keratinocytes, exposure to UVB radiation decreased mitochondrial oxidative phosphorylation, increased glycolysis and the expression of autophagy-related genes, and enhanced AKT Ser/Thr kinase (AKT) phosphorylation and cell growth. Interestingly, UVB initiated a prosurvival mitophagy response by mitochondria-mediated reactive oxygen species (ROS) signaling via the mammalian target of the mTOR complex 2 (mTORC2) pathway. Knockdown of rictor but not raptor abrogated UVB-induced mitophagy responses. Furthermore, fractionation and proximity-ligation assays reveal that ROS-mediated mTOC2 activation in mitochondria is necessary for UVB-induced mitophagy. Importantly, pretreatment with the MnSOD mimic MnTnBuOE-2-PyP5+ (MnP) attenuates mTORC2 activation and suppresses UVB-induced mitophagy. UVB radiation exposure also increased cell growth as assessed by soft-agar colony survival and cell growth assays, and pretreatment with MnP or the known autophagy inhibitor 3-methyladenine abrogated UVB-induced cell growth. These results indicate that MnSOD is a major redox regulator that maintains mitochondrial health and show that UVB-mediated MnSOD inactivation promotes mitophagy and thereby prevents accumulation of damaged mitochondria.

DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling.

Autophagy is a highly regulated evolutionarily conserved metabolic process induced by stress and energy deprivation. Here, we show that DNA polymerase gamma (Polγ) deficiency activates a selective prosurvival autophagic response via mitochondria-mediated reactive oxygen species (ROS) signaling and the mammalian target of rapamycin complex 2 (mTORC2) activities. In keratinocytes, Polγ deficiency causes metabolic adaptation that triggers cytosolic sensing of energy demand for survival. Knockdown of Polγ causes mitochondrial stress, decreases mitochondrial energy production, increases glycolysis, increases the expression of autophagy-associated genes, and enhances AKT phosphorylation and cell proliferation. Deficiency of Polγ preferentially activates mTORC2 formation to increase autophagy and cell proliferation, and knocking down Rictor abrogates these responses. Overexpression of Rictor, but not Raptor, reactivates autophagy in Polγ-deficient cells. Importantly, inhibition of ROS by a mitochondria-selective ROS scavenger abolishes autophagy and cell proliferation. These results identify Rictor as a critical link between mitochondrial stress, ROS, and autophagy. They represent a major shift in our understanding of the prosurvival role of the mTOR complexes and highlight mitochondria-mediated ROS as a prosurvival autophagy regulator during cancer development.

Oxidative stress-induced JNK/AP-1 signaling is a major pathway involved in selective apoptosis of myelodysplastic syndrome cells by Withaferin-A.

Myelodysplastic syndromes (MDS) are a diverse group of malignant clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, dysplastic cell morphology in one or more hematopoietic lineages, and a risk of progression to acute myeloid leukemia (AML). Approximately 50% of MDS patients respond to current FDA-approved drug therapies but a majority of responders relapse within 2-3 years. There is therefore a compelling need to identify potential new therapies for MDS treatment. We utilized the MDS-L cell line to investigate the anticancer potential and mechanisms of action of a plant-derived compound, Withaferin A (WFA), in MDS. WFA was potently cytotoxic to MDS-L cells but had no significant effect on the viability of normal human primary bone marrow cells. WFA also significantly reduced engraftment of MDS-L cells in a xenotransplantation model. Through transcriptome analysis, we identified reactive oxygen species (ROS)-activated JNK/AP-1 signaling as a major pathway mediating apoptosis of MDS-L cells by WFA. We conclude that the molecular mechanism mediating selective cytotoxicity of WFA on MDS-L cells is strongly associated with induction of ROS. Therefore, pharmacologic manipulation of redox biology could be exploited as a selective therapeutic target in MDS.

Manganese superoxide dismutase is a p53-regulated gene that switches cancers between early and advanced stages.

Manganese superoxide dismutase (MnSOD) plays a critical role in the survival of aerobic life, and its aberrant expression has been implicated in carcinogenesis and tumor resistance to therapy. However, despite extensive studies in MnSOD regulation and its role in cancer, when and how the alteration of MnSOD expression occurs during the process of tumor development in vivo are unknown. Here, we generated transgenic mice expressing a luciferase reporter gene under the control of human MnSOD promoter-enhancer elements and investigated the changes of MnSOD transcription using the 7,12-dimethylbenz(α)anthracene (DMBA)/12-O-tetradecanoylphorbol-l3-acetate (TPA) multistage skin carcinogenesis model. The results show that MnSOD expression was suppressed at a very early stage but increased at late stages of skin carcinogenesis. The suppression and subsequent restoration of MnSOD expression were mediated by two transcription-factors, Sp1 and p53. Exposure to DMBA and TPA activated p53 and decreased MnSOD expression via p53-mediated suppression of Sp1 binding to the MnSOD promoter in normal-appearing skin and benign papillomas. In squamous cell carcinomas, Sp1 binding increased because of the loss of functional p53. We used chromatin immunoprecipitation, electrophoretic mobility shift assay, and both knockdown and overexpression of Sp1 and p53 to verify their roles in the expression of MnSOD at each stage of cancer development. The results identify MnSOD as a p53-regulated gene that switches between early and advanced stages of cancer. These findings also provide strong support for the development of means to reactivate p53 for the prevention of tumor progression.

Manganese superoxide dismutase versus p53: the mitochondrial center.

Mitochondria are important sites of myriad metabolic activities. The actions of mitochondria must be carefully synchronized with other processes in the cell to maintain cellular homeostasis. Interorganellar communication between mitochondria and the nucleus is key for coordination of these cellular functions. Numerous signaling proteins and transcription factors are affected by reactive oxygen species and aid interorganellar communication. p53 is an important tumor suppressing protein that regulates many cellular activities, such as cell cycle regulation, DNA repair, and programmed cell death. p53 carries out these functions through both transcription-dependent and transcription-independent routes at mitochondria and the nucleus. Manganese superoxide dismutase (MnSOD), a p53-regulated gene that is a vital antioxidant enzyme localized in the matrix of mitochondria, scavenges reactive oxygen species. Recent studies suggest that mitochondria can regulate p53 activity and that assaults on the cell that affect mitochondrial ROS production and mitochondrial function can influence p53 activity. Cross-talk between mitochondria and p53 is important in normal cellular functions, and a breakdown in communication among mitochondria, p53, and the nucleus may have serious consequences in disease development.

Nuclear factor kappaB- and specificity protein 1-dependent p53-mediated bi-directional regulation of the human manganese superoxide dismutase gene.

Tumor suppressor p53 is known to activate certain sets of genes while suppressing others. However, whether p53 can both activate and suppress the same gene is unclear. To address this question, concentration-dependent p53 effect on the manganese superoxide dismutase (MnSOD) gene was investigated. By transfecting p53 in PC-3 cells, we demonstrate that low concentrations of p53 increase while high concentrations suppress MnSOD expression. The physiological relevance of this effect was determined in vitro and in vivo using combined UVB-mediated activation and small interference RNA-mediated suppression of p53. Results were consistent with the bi-directional effect of p53 on MnSOD expression. MnSOD-promoter/enhancer analysis demonstrates that p53 is suppressive to the promoter activity regardless of the presence or absence of putative p53 binding sites. However, a low level of p53 increases MnSOD gene transcription in the presence of the intronic-enhancer element, and this effect is dependent on nuclear-factor kappaB (NF-kappaB) binding sites. Expression of p53 enhances nuclear levels of p65 with corresponding increase in the DNA-binding activity of NF-kappaB as detected by electrophoretic mobility shift and chromatin immunoprecipitation assays. Transfection of p65 small interference RNA reduces the positive effect of p53 on MnSOD gene transcription. These data suggest that p65 can overcome the negative effect of p53 on MnSOD expression. However, when the level of p53 was further increased, the suppressive effect of p53 outweighed the positive effect of p65 and led to the suppression of MnSOD gene transcription. These results demonstrated that p53 can both suppress and induce MnSOD expression depending on the balance of promoter and enhancer binding transcription factors.

Mutations in the SOD2 promoter reveal a molecular basis for an activating protein 2-dependent dysregulation of manganese superoxide dismutase expression in cancer cells.

A primary antioxidant enzyme in mitochondria, manganese superoxide dismutase (MnSOD), plays a critical role in the survival of aerobic life. It is well documented that, compared with normal cell counterparts, MnSOD level is decreased in neoplastic transformed cells but is increased in aggressive cancers. However, the underlying mechanism for the observed dysregulation of MnSOD in cancer is unknown. We have identified previously a unique set of mutations located in the promoter region of the SOD2 gene in several types of cancer cells. We found that a C-to-T transition at -102 and an insertion of A at -93 down-regulate MnSOD transcription by interrupting the formation of a single-stranded loop that is essential for a high level of promoter activity. Here, we show that the additional downstream mutation, C-to-G transversion at -38, creates a binding site for the transcription factors specificity protein 1 (Sp1) and activating protein 2 (AP-2). The promoter function is regulated by the relative levels of Sp1 and AP-2. In cytokine-induced expression of the SOD2 gene, Sp1 cooperates with a transcriptional complex containing nuclear factor-kappaB and nucleophosmin. The presence of AP-2 attenuates this induction. Our results suggest that the high level of MnSOD observed in aggressive cancer cells may be due, in part, to the absence of AP-2 transcriptional repression.

The role of a single-stranded nucleotide loop in transcriptional regulation of the human sod2 gene.

Manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, is necessary for survival of aerobic life. Previously, we demonstrated that a Sp1-based promoter is essential for constitutive transcription and a NF-kappaB-based intronic enhancer is responsible for cytokine-mediated induction. Here we show that nucleophosmin (NPM), a RNA-binding protein, binds to an 11G single-stranded loop in the promoter region and serves to integrate the Sp1 and NF-kappaB responses. Disruption of the loop structure causes a reduction of both constitutive and inductive transcription due to loss of the binding motif for NPM. Interaction of NF-kappaB.NPM.Sp1 facilitated by binding of NPM to the loop structure in the promoter region appears to comprise the basic complex for the transcriptional stimulation. These results suggest a novel molecular mechanism for communication between the enhancer and the GC-rich promoter.

Identification of nucleophosmin as an NF-kappaB co-activator for the induction of the human SOD2 gene.

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme essential for the survival of life. We have reported that NF-kappaB is essential but not sufficient for the synergistic induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines. To further identify transcription factors and co-activators that participate in the induction of MnSOD, we used NF-kappaB affinity chromatography to isolate potential NF-kappaB interacting proteins. Proteins eluted from the NF-kappaB affinity column were subjected to proteomic analysis and verified by Western analysis. Nucleophosmin (NPM), a nucleolar phosphoprotein, is the most abundant single protein identified. Co-immunoprecipitation studies suggest a physical interaction between NPM and NF-kappaB proteins. To verify the role of NPM on MnSOD gene transcription, cells were transfected with constructs expressing NPM in sense or antisense orientation as well as interference RNA. The results indicate that an increase NPM expression leads to increased MnSOD gene transcription in a dose-dependent manner. Consistent with this, expression of small interfering RNA for NPM leads to inhibition of MnSOD gene transcription but does not have any effect on the expression of interleukin-8, suggesting that the effect of NPM is selective. These results identify NPM as a partner of the NF-kappaB transcription complex in the induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines.