Control of Hepatic Nuclear Superoxide Production by Glucose 6-Phosphate Dehydrogenase and NADPH Oxidase-4

Abstract

Redox-regulated signal transduction is coordinated by spatially controlled production of reactive oxygen species within subcellular compartments. The nucleus has long been known to produce superoxide (O 2 . ); however, the mechanisms that control this function remain largely unknown. We have characterized molecular features of a nuclear superoxide-producing system in the mouse liver. Using electron paramagnetic resonance, we investigated whether several NADPH oxidases (NOX1, 2, and 4) and known activators of NOX (Rac1, Rac2, p22 phox , and p47 phox ) contribute to nuclear O 2 . production in isolated hepatic nuclei.Our findings demonstrate that NOX4 most significantly contributes to hepatic nuclear O 2 . production that utilizes NADPH as an electron donor. Although NOX4 protein immunolocalized to both nuclear membranes and intranuclear inclusions, fluorescent detection of NADPH-dependent nuclear O 2 . predominantly localized to the perinuclear space. Interestingly, NADP ؉ and G6P also induced nuclear O 2 . production, suggesting that intranuclear glucose-6-phosphate dehydrogenase (G6PD) can control NOX4 activity through nuclear NADPH production. Using G6PD mutant mice and G6PD shRNA, we confirmed that reductions in nuclear G6PD enzyme decrease the ability of hepatic nuclei to generate O 2 . in response to NADP ؉ and G6P.NOX4 and G6PD protein were also observed in overlapping microdomains within the nucleus. These findings provide new insights on the metabolic pathways for substrate regulation of nuclear O 2 . production by NOX4.Reactive oxygen species (ROS) 2 such as superoxide (O 2 . ) and H 2 O 2 play important roles in cellular oxidative stress as well as in the regulation of cellular signal transduction in the healthy state. Understanding the regulatory pathways that control cellular ROS at specific sites in the cell is vital to determining their function in cell signaling (1). Important for the regulation of redox signaling is the controlled production of ROS at specific intracellular sites such as mitochondria and endosomes. ROS production and transport at these locations are tightly regulated and linked to effector redox signals (2-6). Another, much less studied, site of ROS production is the nucleus. Despite the fact that NADPH-dependent O 2 . production by the nucleus was discovered over three decades ago (7-10), the regulation and function of nuclear O 2 . remain largely uncharacterized.Over the years, several enzymes including cytochrome P450 and many others have been suggested as candidate sources of nuclear O 2 . . More recently, endothelial nuclei disrupted by sonication (11), and endothelial nuclear protein extracts (12) have both been shown to produce ROS that is, at least in part, NOX4-dependent. NOX4 has been localized in nuclei of vascular smooth muscle cells, but its subnuclear localization (such as within specific nuclear membranes) remains unclear (13). Nuclear NOX4 has also been implicated in DNA damage resulting from both hemangioendothelioma formation (14) and hepatitis C infection (15...

Areca nut exposure increases secretion of tumor‐promoting cytokines in gingival fibroblasts that trigger DNA damage in oral keratinocytes

“…In our study, NOX4 expression increased in cytokine‐treated IHOK, whereas NOX1 expression was reduced. It has been reported that NOX1 is regulated by recruitment of cytosolic subunits to redoxosomes, whereas NOX4‐dependent ROS generation is confined to the nuclear envelope 46, 47. Considering that intracellular signaling by cytokine binding proceeds by endocytosis, NOX1 may be primarily affected by cytokine binding, resulting in a decrease of NOX1 activity.…”

NADPH Oxidase-generated Hydrogen Peroxide Induces DNA Damage in Mutant FLT3-expressing Leukemia Cells

“…histone deacetylase (33). Also, NOX4, localized in the perinuclear space was also the source of nuclear superoxide generation in hepatocytes (38). Interestingly, exposure of mice to ionizing radiation increased the expression of NOX4 in hematopoietic stem cells (10,39).…”

Nox4 NADPH Oxidase Mediates Peroxynitrite-dependent Uncoupling of Endothelial Nitric-oxide Synthase and Fibronectin Expression in Response to Angiotensin II

“…For this reason, the Nox oxidases that have been linked to eNOS uncoupling in vascular pathology are the superoxide-generating homologues Nox1 and Nox2 (57)(58)(59)(60). These considerations are mitigated by the numerous studies in cardiac, vascular, or renal cells and tissue demonstrating that Nox4 produces detectable amount of superoxide (20,21,40,(61)(62)(63). Importantly, several of these reports measured superoxide generation using a DHE-and HPLC-based assay as in the present work or via electron paramagnetic/spin resonance, "gold standards" for superoxide measurement (61)(62)(63).…”

“…These considerations are mitigated by the numerous studies in cardiac, vascular, or renal cells and tissue demonstrating that Nox4 produces detectable amount of superoxide (20,21,40,(61)(62)(63). Importantly, several of these reports measured superoxide generation using a DHE-and HPLC-based assay as in the present work or via electron paramagnetic/spin resonance, "gold standards" for superoxide measurement (61)(62)(63). Therefore, even if Nox4 produces less superoxide than hydrogen peroxide, it is plausible that the readily diffusible and membrane-permeable NO is still able to react with these moderate amounts of superoxide to form the peroxynitrite required for the initiation of NOS uncoupling.…”