Adam J. Case

Elevated mitochondrial superoxide disrupts normal T cell development, impairing adaptive immune responses to an influenza challenge

Reactive oxygen species (ROS) are critical in a broad spectrum of cellular processes including signaling, tumor progression, and innate immunity. The essential nature of ROS signaling in the immune systems of Drosophila and zebrafish has been demonstrated; however, the role of ROS, if any, in mammalian adaptive immune system development and function remains unknown. This work provides the first clear demonstration that thymus-specific elevation of mitochondrial superoxide (O(2)(•-)) disrupts normal T cell development and impairs the function of the mammalian adaptive immune system. To assess the effect of elevated mitochondrial superoxide in the developing thymus, we used a T-cell-specific knockout of manganese superoxide dismutase (i.e., SOD2) and have thus established a murine model to examine the role of mitochondrial superoxide in T cell development. Conditional loss of SOD2 led to increased superoxide, apoptosis, and developmental defects in the T cell population, resulting in immunodeficiency and susceptibility to the influenza A virus H1N1. This phenotype was rescued with mitochondrially targeted superoxide-scavenging drugs. These findings demonstrate that loss of regulated levels of mitochondrial superoxide lead to aberrant T cell development and function, and further suggest that manipulations of mitochondrial superoxide levels may significantly alter clinical outcomes resulting from viral infection.

Mitochondrial-localized NADPH oxidase 4 is a source of superoxide in angiotensin II-stimulated neurons

Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O₂∙-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O₂∙--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O₂∙- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O₂∙- in neuron mitochondria that contributes to ANG II intraneuronal signaling.

NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells

Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formation in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect.

Aberrant promoter CpG methylation is a mechanism for impaired PHD3 expression in a diverse set of malignant cells.

Background The prolyl-hydroxylase domain family of enzymes (PHD1-3) plays an important role in the cellular response to hypoxia by negatively regulating HIF-α proteins. Disruption of this process can lead to up-regulation of factors that promote tumorigenesis. We observed decreased basal expression of PHD3 in prostate cancer tissue and tumor cell lines representing diverse tissues of origin. Furthermore, some cancer lines displayed a failure of PHD3 mRNA induction when introduced to a hypoxic environment. This study explores the mechanism by which malignancies neither basally express PHD3 nor induce PHD3 under hypoxic conditions. Methodology/Principal Findings Using bisulfite sequencing and methylated DNA enrichment procedures, we identified human PHD3 promoter hypermethylation in prostate, breast, melanoma and renal carcinoma cell lines. In contrast, non-transformed human prostate and breast epithelial cell lines contained PHD3 CpG islands that were unmethylated and responded normally to hypoxia by upregulating PHD3 mRNA. Only treatment of cells lines containing PHD3 promoter hypermethylation with the demethylating drug 5-aza-2′-deoxycytidine significantly increased the expression of PHD3. Conclusions/Significance We conclude that expression of PHD3 is silenced by aberrant CpG methylation of the PHD3 promoter in a subset of human carcinoma cell lines of diverse origin and that this aberrant cytosine methylation status is the mechanism by which these cancer cell lines fail to upregulate PHD3 mRNA. We further show that a loss of PHD3 expression does not correlate with an increase in HIF-1α protein levels or an increase in the transcriptional activity of HIF, suggesting that loss of PHD3 may convey a selective advantage in some cancers by affecting pathway(s) other than HIF.

Loss of SOD3 (EcSOD) Expression Promotes an Aggressive Phenotype in Human Pancreatic Ductal Adenocarcinoma

Purpose: Pancreatic ductal adenocarcinoma (PDA) cells are known to produce excessive amounts of reactive oxygen species (ROS), particularly superoxide, which may contribute to the aggressive and refractory nature of this disease. Extracellular superoxide dismutase (EcSOD) is an antioxidant enzyme that catalyzes the dismutation of superoxide in the extracellular environment. This study tests the hypothesis that EcSOD modulates PDA growth and invasion by modifying the redox balance in PDA. Experimental Design: We evaluated the prognostic significance of EcSOD in a human tissue microarray (TMA) of patients with PDA. EcSOD overexpression was performed in PDA cell lines and animal models of disease. The impact of EcSOD on PDA cell lines was evaluated with Matrigel invasion in combination with a superoxide-specific SOD mimic and a nitric oxide synthase (NOS) inhibitor to determine the mechanism of action of EcSOD in PDA. Results: Loss of EcSOD expression is a common event in PDA, which correlated with worse disease biology. Overexpression of EcSOD in PDA cell lines resulted in decreased invasiveness that appeared to be related to reactions of superoxide with nitric oxide. Pancreatic cancer xenografts overexpressing EcSOD also demonstrated slower growth and peritoneal metastasis. Overexpression of EcSOD or treatment with a superoxide-specific SOD mimic caused significant decreases in PDA cell invasive capacity. Conclusions: These results support the hypothesis that loss of EcSOD leads to increased reactions of superoxide with nitric oxide, which contributes to the invasive phenotype. These results allow for the speculation that superoxide dismutase mimetics might inhibit PDA progression in human clinical disease. Clin Cancer Res; 21(7); 1741–51. ©2015 AACR.

Human chondrosarcoma cells acquire an epithelial-like gene expression pattern via an epigenetic switch: Evidence for mesenchymal-epithelial transition during sarcomagenesis

Chondrocytes are mesenchymally derived cells that reportedly acquire some epithelial characteristics; however, whether this is a progression through a mesenchymal to epithelial transition (MET) during chondrosarcoma development is still a matter of investigation. We observed that chondrosarcoma cells acquired the expression of four epithelial markers, E-cadherin,desmocollin 3, maspin, and 14-3-3σ, all of which are governed epigenetically through cytosine methylation. Indeed, loss of cytosine methylation was tightly associated with acquired expression of both maspin and 14-3-3σ in chondrosarcomas. In contrast, chondrocyte cells were negative for maspin and 14-3-3σ and displayed nearly complete DNA methylation. Robust activation of these genes was also observed in chondrocyte cells following 5-aza-dC treatment. We also examined the transcription factor snail which has been reported to be an important mediator of epithelial to mesenchymal transitions (EMTs). In chondrosarcoma cells snail is downregulated suggesting a role for loss of snail expression in lineage maintenance. Taken together, these results document an epigenetic switch associated with an MET-like phenomenon that accompanies chondrosarcoma progression.

Manganese superoxide dismutase depletion in murine hematopoietic stem cells perturbs iron homeostasis, globin switching, and epigenetic control in erythrocyte precursorcells

Heme synthesis partially occurs in the mitochondrial matrix; thus there is a high probability that enzymes and intermediates important in the production of heme will be exposed to metabolic by-products including reactive oxygen species. In addition, the need for ferrous iron for heme production, Fe/S coordination, and other processes occurring in the mitochondrial matrix suggests that aberrant fluxes of reactive oxygen species in this compartment might perturb normal iron homeostasis. Manganese superoxide dismutase (Sod2) is an antioxidant enzyme that governs steady-state levels of the superoxide in the mitochondrial matrix. Using hematopoietic stem cell-specific conditional Sod2 knockout mice we observed increased superoxide concentrations in red cell progeny, which caused significant pathologies including impaired erythrocytes and decreased ferrochelatase activity. Animals lacking Sod2 expression in erythroid precursors also displayed extramedullary hematopoiesis and systemic iron redistribution. Additionally, the increase in superoxide flux in erythroid precursors caused abnormal gene regulation of hematopoietic transcription factors, globins, and iron-response genes. Moreover, the erythroid precursors also displayed evidence of global changes in histone posttranslational modifications, a likely cause of at least some of the aberrant gene expression noted. From a therapeutic translational perspective, mitochondrially targeted superoxide-scavenging antioxidants partially rescued the observed phenotype. Taken together, our findings illuminate the superoxide sensitivity of normal iron homeostasis in erythrocyte precursors and suggest a probable link between mitochondrial redox metabolism and epigenetic control of nuclear gene regulation during mammalian erythropoiesis.

Manganese superoxide dismutase is dispensable for post-natal development and lactation in the murine mammary gland

Abstract Mammary gland development is a multistage process requiring tightly regulated spatial and temporal signalling pathways. Many of these pathways have been shown to be sensitive to oxidative stress. Understanding that the loss of manganese superoxide dismutase (Sod2) leads to increased cellular oxidative stress, and that the loss or silencing of this enzyme has been implicated in numerous pathologies including those of the mammary gland, we sought to examine the role of Sod2 in mammary gland development and function in situ in the mouse mammary gland. Using Cre-recombination driven by the mouse mammary tumor virus (MMTV) promoter, we created a mammary-specific post-natal conditional Sod2 knock-out mouse model. Surprisingly, while substantial decreases in Sod2 were noted throughout both virgin and lactating adult mammary glands, no significant changes in developmental structures either pre- or post-pregnancy were observed histologically. Moreover, mothers lacking mammary gland expression of Sod2 were able to sustain equal numbers of litters, equal pups per litter, and equal pup weights as were control animals. Overall, our results demonstrate that loss of Sod2 expression is not universally toxic to all cell types and that excess mitochondrial superoxide can apparently be tolerated during the development and function of post-natal mammary glands.

Over-expressed copper/zinc superoxide dismutase localizes to mitochondria in neurons inhibiting the angiotensin II-mediated increase in mitochondrial superoxide

Angiotensin II (AngII) is the main effector peptide of the renin–angiotensin system (RAS), and contributes to the pathogenesis of cardiovascular disease by exerting its effects on an array of different cell types, including central neurons. AngII intra-neuronal signaling is mediated, at least in part, by reactive oxygen species, particularly superoxide (O2•−). Recently, it has been discovered that mitochondria are a major subcellular source of AngII-induced O2•−. We have previously reported that over-expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix-localized O2•− scavenging enzyme, inhibits AngII intra-neuronal signaling. Interestingly, over-expression of copper/zinc superoxide dismutase (CuZnSOD), which is believed to be primarily localized to the cytoplasm, similarly inhibits AngII intra-neuronal signaling and provides protection against AngII-mediated neurogenic hypertension. Herein, we tested the hypothesis that CuZnSOD over-expression in central neurons localizes to mitochondria and inhibits AngII intra-neuronal signaling by scavenging mitochondrial O2•−. Using a neuronal cell culture model (CATH.a neurons), we demonstrate that both endogenous and adenovirus-mediated over-expressed CuZnSOD (AdCuZnSOD) are present in mitochondria. Furthermore, we show that over-expression of CuZnSOD attenuates the AngII-mediated increase in mitochondrial O2•− levels and the AngII-induced inhibition of neuronal potassium current. Taken together, these data clearly show that over-expressed CuZnSOD in neurons localizes in mitochondria, scavenges AngII-induced mitochondrial O2•−, and inhibits AngII intra-neuronal signaling.

Maintenance of mitochondrial genomic integrity in the absence of manganese superoxide dismutase in mouse liver hepatocytes.

Manganese superoxide dismutase, encoded by the Sod2 gene, is a ubiquitously expressed mitochondrial antioxidant enzyme that is essential for mammalian life. Mice born with constitutive genetic knockout of Sod2 do not survive the neonatal stage, which renders the longitudinal study of the biochemical and metabolic effects of Sod2 loss difficult. However, multiple studies have demonstrated that tissue-specific knockout of Sod2 in murine liver yields no observable gross pathology or injury to the mouse. We hypothesized that Sod2 loss may have sub-pathologic effects on liver biology, including the acquisition of reactive oxygen species-mediated mitochondrial DNA mutations. To evaluate this, we established and verified a hepatocyte-specific knockout of Sod2 in C57/B6 mice using Cre-LoxP recombination technology. We utilized deep sequencing to identify possible mutations in Sod2−/− mitochondrial DNA as compared to wt, and both RT-PCR and traditional biochemical assays to evaluate baseline differences in redox-sensitive pathways in Sod2−/− hepatocytes. Surprisingly, no mutations in Sod2−/− mitochondrial DNA were detected despite measurable increases in dihydroethidium staining in situ and concomitant decreases in complex II activity indicative of elevated superoxide in the Sod2−/− hepatocytes. In contrast, numerous compensatory alterations in gene expression were identified that suggest hepatocytes have a remarkable capacity to adapt and overcome the loss of Sod2 through transcriptional means. Taken together, these results suggest that murine hepatocytes have a large reserve capacity to cope with the presence of additional mitochondrial reactive oxygen species.