Growing evidence suggests that NADPH oxidase (Nox)-derived reactive oxygen species (ROS) play important roles in regulating cytokine signaling. We have explored how TNF-a induction of Nox-dependent ROS influences NF-kB activation. Cellular stimulation by TNF-a induced NADPH-dependent superoxide production in the endosomal compartment, and this ROS was required for IKK-mediated activation of NF-kB. Inhibiting endocytosis reduced the ability of TNF-a to induce both NADPH-dependent endosomal superoxide and NF-kB, supporting the notion that redox-dependent signaling of the receptor occurs in the endosome. Molecular analyses demonstrated that endosomal H 2 O 2 was critical for the recruitment of TRAF2 to the TNFR1=TRADD complex after endocytosis. Studies using both Nox2 siRNA and Nox2-knockout primary fibroblasts indicated that Nox2 was critical for TNF-a-mediated induction of endosomal superoxide. Redox-active endosomes that form after TNF-a or IL-1b induction recruit several common proteins (Rac1, Nox2, p67 phox , SOD1), while also retaining specificity for ligand-activated receptor effectors. Our studies suggest that TNF-a and IL-1b signaling pathways both can use Nox2 to facilitate redox activation of their respective receptors at the endosomal level by promoting the redox-dependent recruitment of TRAFs. These studies help to explain how cellular compartmentalization of redox signals can be used to direct receptor activation from the plasma membrane.

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...

Redox reactions have been established as major biological players in many cellular signaling pathways. Here we review mechanisms of redox signaling with an emphasis on redox-active signaling endosomes. Signals are transduced by relatively few reactive oxygen species (ROS), through very specific redox modifications of numerous proteins and enzymes. Although ROS signals are typically associated with cellular injury, these signaling pathways are also critical for maintaining cellular health at homeostasis. An important component of ROS signaling pertains to localization and tightly regulated signal transduction events within discrete microenvironments of the cell. One major aspect of this specificity is ROS compartmentalization within membrane-enclosed organelles such as redoxosomes (redox-active endosomes) and the nuclear envelope. Among the cellular proteins that produce superoxide are the NADPH oxidases (NOXes), transmembrane proteins that are implicated in many types of redox signaling. NOXes produce superoxide on only one side of a lipid bilayer; as such, their orientation dictates the compartmentalization of ROS and the local control of signaling events limited by ROS diffusion and/or movement through channels associated with the signaling membrane. NOX-dependent ROS signaling pathways can also be self-regulating, with molecular redox sensors that limit the local production of ROS required for effective signaling. ROS regulation of the Rac-GTPase, a required co-activator of many NOXes, is an example of this type of sensor. A deeper understanding of redox signaling pathways and the mechanisms that control their specificity will provide unique therapeutic opportunities for aging, cancer, ischemia-reperfusion injury, and neurodegenerative diseases.

Recent studies have implicated enhanced Nox2-mediated reactive oxygen species (ROS) by microglia in the pathogenesis of motor neuron death observed in familial amyotrophic lateral sclerosis (ALS). In this context, ALS mutant forms of SOD1 enhance Rac1 activation, leading to increased Nox2-dependent microglial ROS production and neuron cell death in mice. It remains unclear if other genetic mutations that cause ALS also function through similar Nox-dependent pathways to enhance ROS-mediate motor neuron death. In the present study, we sought to understand whether alsin, which is mutated in an inherited juvenile form of ALS, functionally converges on Rac1-dependent pathways acted upon by SOD1G93A to regulate Noxdependent ROS production. Our studies demonstrate that glial cell expression of SOD1 G93A or wild type alsin induces ROS production, Rac1 activation, secretion of TNF␣, and activation of NFB, leading to decreased motor neuron survival in co-culture. Interestingly, coexpression of alsin, or shRNA against Nox2, with SOD1 G93A in glial cells attenuated these proinflammatory indicators and protected motor neurons in co-culture, although shRNAs against Nox1 and Nox4 had little effect. SOD1G93A expression dramatically enhanced TNF␣-mediated endosomal ROS in glial cells in a Rac1-dependent manner and alsin overexpression inhibited SOD1 G93A -induced endosomal ROS and Rac1 activation. SOD1 G93A expression enhanced recruitment of alsin to the endomembrane compartment in glial cells, suggesting that these two proteins act to modulate Nox2-dependent endosomal ROS and proinflammatory signals that modulate NFB. These studies suggest that glial proinflammatory signals regulated by endosomal ROS are influenced by two gene products known to cause ALS.Amyotrophic lateral sclerosis (ALS) is a lethal degenerative neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord (1, 2). The majority of ALS patients have onset of disease between 40 and 50 years of age and about 50% of patients die within 3 years. The majority of ALS cases are categorized as sporadic with no family history of disease. In this context, the causative genes and environmental factors that initiate the disease process remain poorly defined. Only ϳ10% of ALS cases have a clearly inherited genetic component and hence are classified as familial ALS (1, 2).The best-characterized forms of familial ALS include those caused by mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1) 2 (3). Approximately 20% of familial ALS cases are caused by a variety of dominant SOD1 mutations (1, 3). There remains great uncertainty as to the primary mechanism(s) by which mutant SOD1 leads to pathology observed in ALS (1, 4). Proposed mechanisms include toxicity associated with misfolding of mutant SOD1, such as ER stress and inhibition of the proteasome, enhanced proinflammatory ROS production, altered axonal transport, excitotoxicity caused by glutamate mishandling, and mitochondrial damage (1, 4). Relevant to the studi...

Nitric oxide has been proposed to be transported by hemoglobin as a third respiratory gas and to elicit vasodilation by an oxygenlinked (allosteric) mechanism. For hemoglobin to transport nitric oxide bioactivity it must capture nitric oxide as iron nitrosyl hemoglobin rather than destroy it by dioxygenation. Once bound to the heme iron, nitric oxide has been reported to migrate reversibly from the heme group of hemoglobin to the ␤-93 cysteinyl residue, in response to an oxygen saturation-dependent conformational change, to form an S-nitrosothiol. However, such a transfer requires redox chemistry with oxidation of the nitric oxide or ␤-93 cysteinyl residue. In this article, we examine the ability of nitric oxide to undergo this intramolecular transfer by cycling human hemoglobin between oxygenated and deoxygenated states. Under various conditions, we found no evidence for intramolecular transfer of nitric oxide from either cysteine to heme or heme to cysteine. In addition, we observed that contaminating nitrite can lead to formation of iron nitrosyl hemoglobin in deoxygenated hemoglobin preparations and a radical in oxygenated hemoglobin preparations. Using 15 N-labeled nitrite, we clearly demonstrate that nitrite chemistry could explain previously reported results that suggested apparent nitric oxide cycling from heme to thiol. Consistent with our results from these experiments conducted in vitro, we found no arterial͞venous gradient of iron nitrosyl hemoglobin detectable by electron paramagnetic resonance spectroscopy. Our results do not support a role for allosterically controlled intramolecular transfer of nitric oxide in hemoglobin as a function of oxygen saturation. How is the activity of the endothelium-derived relaxation factor, nitric oxide (NO), preserved if an abundant scavenger, hemoglobin (Hb), reacts so quickly with it? This question has puzzled many investigators ever since the identification of NO as the endothelium-derived relaxation factor was made (1-4). NO reacts with Hb via the following reactions:where Fe(II) and Fe(III) refer to the ferrous and ferric forms of the iron in the heme group of Hb, respectively. The rate constants for these reactions are 2.6 ϫ 10 7 M Ϫ1 ⅐s Ϫ1 at 20°C (5) and 6-9 ϫ 10 7 M Ϫ1 ⅐s Ϫ1 at 20°C (6, 7) for Eqs. 1 and 2, respectively. The second reaction destroys NO activity through the formation of nitrate. The activity of NO is potentially conserved by the reaction described by Eq. 1, but to function it must come off the heme and get out of the RBC (where the Hb concentration is Ϸ0.02 M in heme) before undergoing the fast reactions described by Eqs. 1 and 2. Thus, scavenging of NO in blood can be predicted to be a significant NO sink and poses the problem of how NO can function as the endothelium-derived relaxation factor (8).One possible resolution of this paradox would be that NO produced in endothelial cells acts only locally and is thus immune to scavenging by Hb, but a variety of evidence indicating increased vasoconstriction when free Hb or Hb substitutes are in...