Hydrogen Peroxide as a Cell-Survival Signaling Molecule

Abstract

Reactive oxygen species (ROS) were seen as destructive molecules, but recently, they have been shown also to act as second messengers in varying intracellular signaling pathways. This review concentrates on hydrogen peroxide (H₂O₂), as it is a more stable ROS, and delineates its role as a survival molecule. In the first part, the production of H₂O₂ through the NADPH oxidase (Nox) family is investigated. Through careful examination of Nox proteins and their regulation, it is determined how they respond to stress and how this can be prosurvival rather than prodeath. The pathways on which H₂O₂ acts to enable its prosurvival function are then examined in greater detail. The main survival pathways are kinase driven, and oxidation of cysteines in the active sites of various phosphatases can thus regulate those survival pathways. Regulation of transcription factors such as p53, NF-κB, and AP-1 also are reviewed. Finally, prodeath proteins such as caspases could be directly inhibited through their cysteine residues. A better understanding of the prosurvival role of H₂O₂ in cells, from the why and how it is generated to the various molecules it can affect, will allow more precise targeting of therapeutics to this pathway. Antioxid. Redox Signal. 11, 2655–2671.

Introduction

R eactive oxygen species (ROS) are a group of molecules produced in cells when oxygen is metabolized. They were traditionally viewed as destructive molecules that resulted in cell death through damaging key cellular molecules such as DNA and lipids, but recently they have also been shown to contribute to cell proliferation, migration, and survival [reviewed by (126)]. This group is made up of several members, including superoxide (O₂ ^•−), nitric oxide (NO^•), hydrogen peroxide (H₂O₂), and the hydroxyl anion (OH^•). This review is focused primarily on H₂O₂. It is formed as the end product of O₂ ^•− degeneration, which can be accomplished either by a family of cellular enzymes, called superoxide dismutase (SOD), or by its spontaneous breakdown. ROS, and O₂ ^•− in particular, are produced by a variety of enzymes, including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), xanthine oxidase [for review, see (25)], lipoxygenase [for reviews, see (80, 140)], and myeloperoxidase [for reviews, see (75, 98)]. ROS also are associated with mitochondria, where they are seen mainly as a by-product of the high oxygen consumption of this organelle. This review focuses initially on Nox proteins, because they form the family of enzymes about which most is known in terms of the production of O₂ ^•−/H₂O₂.

It is not just the source of H₂O₂ that is important when considering its role as a survival molecule; it also is important to understand how it interacts with its targets molecules. The main way that H₂O₂ affects cell-signaling pathways is through oxidation of specific target molecules. If the concentration of H₂O₂ is high, then these oxidation processes may lead to irreversible damage, followed by cell death. However, this is not always the case, and H₂O₂ at low concentrations is capable of reversible inhibition of many enzymes, including phosphatases, which are the second focus of this review. We examine Nox proteins as a source of H₂O₂ and the oxidation of phosphatases to demonstrate how H₂O₂ can act as a survival molecule.

The Nox family

Seven Nox family members in mammals are known with at least 101 orthologues across 25 species (71). The seven members are Nox1-5, and dual oxidases (Duoxs) 1 and 2. Nox2 is the prototype member of this family and originally was called gp91^phox. It is highly expressed in neutrophils and macrophages, in which it produces an oxidative burst to destroy pathogens. This burst occurs when these cells engulf microbes, containing them in a phagosome, and was characterized first in the early 1960s (61). It was not until the mid-1980s that the gene responsible for the burst was cloned (128, 150). Activated Nox2 in the membrane of the phagosome produces high levels of O₂ ^•−, which is converted to H₂O₂, either through the action of SOD, or spontaneously at the low pH of this organelle (75). The O₂ ^•−/H₂O₂ can be in the micromolar concentration range in the phagosome (57), which is lethal for the engulfed microbe. Understandably, the production of this high level of H₂O₂ is strictly regulated to avoid damaging the cell itself, and this high activity level allowed the regulatory mechanisms of Nox2 to be studied comprehensively.

Nox1 was the next member to be discovered and was first cloned in 1999 (143). It is highly expressed in the colon (21), with no expression in Nox2-rich phagocytes (143). In the following 5–6 years, genetic database mining and subsequent cloning led to the discovery of the other family members. Nox3 is expressed predominantly in the octonia of the inner ear (9), and it also is found in some fetal tissues (21). Nox4 is found in many systems, with high expression in the kidney (47, 138). It can have different subcellular localizations, which can affect its function (19). Nox5, highly expressed in testes, spleen, and lymph nodes, stands apart from Nox1 to 4, as it is calcium dependent (7). Duox1 and Duox2 are found mainly in the thyroid and in airway epithelium (32), have an extracellular peroxidase-like domain, and also are calcium dependent.

Nox proteins are integral membrane proteins, with six transmembrane domains, which together are thought to form a channel to allow the successive transfer of electrons (for a review on the structure of Nox proteins, see ref. 146) (Fig. 1). They operate by transferring electrons from NADPH (converting it to NADP⁻) to FAD to heme and finally to oxygen to make O₂ ^•−. This transfer is dependent on the presence of two heme groups associated with the inner and outer cellular membrane envelope. Two His residues, in two of the transmembrane domains, are responsible for binding the hemes, and these are the most conserved regions of the Nox family. Nox1, - 2, - 3, and - 4 are similar in sequence and only differ in length by 14 amino acids, the shortest being Nox1, which is 564 amino acids in length (143), and the longest, Nox4 of 578 amino acids (138). Their amino acid homology ranges from 56% (Nox1 and Nox2), and 58% (Nox2 and Nox3), to 39% (Nox2 and Nox4) (145). Nox5 is significantly different because of its four EF domains, which confer its dependence on calcium for activation (7, 10) (Fig. 1). Duox1 and 2 both have a peroxidase-like domain on their N-terminal (36), which means that these two members of the family produce H₂O₂ and not O₂ ^•− as their final product (48, 106, 142). They also have two EF domains and so are also calcium dependent (28, 33) (Fig. 1). With the discovery of the other members of the Nox family, it became apparent that they do not always produce the lethal levels of O₂ ^•−/H₂O₂ characteristic of Nox2 in neutrophils. Because of their different locations, both within cells and within tissues, this group of proteins are now known to affect many cellular functions including growth, motility, and cell survival.

FIG. 1.

Schematic representations of the structure of Nox proteins (Fe represents binding of heme groups, and EF represents EF domains that confer calcium dependence).

The regulation of Nox proteins

Nox subunits

The activity of the best known Nox, Nox2, is dependent on its regulatory subunits, reviewed in depth by (84, 146) and summarized here. Normally, Nox2 is found at the cytoplasmic membrane with its essential partner p22^phox, and together, they form a complex called cytochrome b₅₅₈ . In the resting cell, p47^phox, p67^phox, and p40^phox form a large complex in the cytoplasm (62, 163), and it is only when the cell is stimulated that they are recruited to cytochrome b₅₅₈ at the membrane (26) (Fig. 2). During cellular stimulation, p47^phox is phosphorylated, removing its autoinhibition (60). This phosphorylation is correlated with Rac (Rac2 in myeloid cells or Rac1 in other cell types) recruitment to this complex, and together these four proteins translocate to cytochrome b₅₅₈ in the membrane, resulting in the production of O₂ ^•− and H₂O₂. The interactions between the complex members are determined by their structures (Figs. 2 and 3). p47^phox binds p22^phox (90) and p67^phox (62). In turn, p67^phox also binds Rac (78, 85). Some doubt exists over the necessity of p40^phox in vitro and in vivo (41, 79), but in vivo, p47^phox remains essential for Nox2 activation (95). In the Nox1 system, homologues exist for two of these subunits. NOXO1 is homologous to p47^phox, and both are seen as Nox-organizing proteins. NOXA1 is homologous to p67^phox, and both are the activating proteins. To date, no homologues to p40^phox or p22^phox have been discovered. In different cell systems, these regulatory proteins were found to be interchangeable between Nox1 and Nox2 (8, 149). It remains unknown what actually happens to the Nox protein when all the subunits arrive and interact with it. It is assumed that it leads to a conformational change in the protein, opening up fully the channel formed by the six transmembrane domains, and so allowing the flow of electrons and the generation of O₂ ^•−. None of the seven Nox family members have been crystallized yet, so their actual structures have yet to be solved, but models of their structures support this hypothesis (146).

FIG. 2.

The change in localization of the regulatory subunits on activation of Nox2 (I, phosphorylation, arrows indicate flow of electrons in the activated cell).

FIG. 3.

The known phosphorylation sites of the regulatory subunits (bm, binding motif; Ser, serine; Thr, threonine). Numbers are representative of specific amino acids in the proteins; numbers in bold are proven to be important for activation. Thr154i on p40^phox has been shown to be inhibitory.

The regulation of Nox3, Nox4, Nox5, Duox1, and Duox2 has been less comprehensively investigated. Nox3 requires co-expression of p22^phox alone to generate H₂O₂, and this can be enhanced by both p47^phox and p67^phox, but Rac is not required in either of these situations (156), which is somewhat at odds with data showing that Nox3 has a Rac-binding site (67). Nox3 appears to use NOXO1 as a signaling partner, as mice knockouts of NOXO1 have the same phenotype as that of Nox3 knockouts (74). Nox4 generates O₂ ^•−/H₂O₂ constitutively when it is stabilized by the coexpression of p22^phox (4). None of the other known regulatory subunits appears to play a role in Nox4 generation of O₂ ^•−/H₂O₂ (99). Nox5, in contrast to the first four family members, does not require any of the known regulatory subunits, including p22^phox (70), and its activity is dependent on calcium (7, 9). Duox1 and Duox2 also are activated by calcium and have yet to show any reliance on the known regulatory subunits. Two maturation factors are associated with Duox proteins, DUOXA1 and DUOXA2, and they are necessary for the full maturation and stabilization of the two Duox proteins (53), but not their activity per se. It remains unknown whether other homologous and nonhomologous Nox regulatory subunits could play a role in the activation of these newer family members.

Upstream regulation of Nox proteins

Although the preceding paragraphs detailed the immediate regulation of the Nox family members, a wider view must be taken to understand how H₂O₂ can operate as a survival signal through Nox proteins. For H₂O₂ to act as a survival signal, it must be generated when a cell is stressed. Decoursey and Ligeti (29) summarized the known upstream regulation of Nox2, but they focused on this particular family member, and as this is a developing area of Nox biochemistry, more work has been published since then on it and the other family members. They considered the phosphorylation status of p47^phox and the activity of Rac to be the key determining factors, and although p22^phox (124, 125), p40^phox (15, 42), and p67^phox (34, 38) also can be phosphorylated (Fig. 3), their phosphorylation statuses are not thought to play a pivotal role in Nox activity.

When p47^phox is phosphorylated, its autoinhibition is removed, resulting in its being able to bind to the cytoplasmic tail of p22^phox and allowing activation of the entire Nox complex (54, 115). Phosphorylation is generally under the control of kinases, and protein kinase C (PKC) has been shown to be critical for Nox2 activity in neutrophils (27, 123) and also can activate Nox1 (20, 22) and Nox3 (156) (Fig. 4). Two other kinases have been shown to phosphorylate p47^phox: p38 mitogen-activated protein kinase (89) and p21-activated kinase-1 (127). But what activates these kinases in the case of Nox proteins? Phospholipase A₂ (PLA₂) activation causes the release of arachidonic acid, which is known to activate PKC directly (72), and the inhibition of PLA₂ inhibits Nox activity (2, 152). Recently, Kambayashi et al. (65) showed that diacylglycerol, produced through the action of phospholipase C, can activate Nox through its well-known activation of PKC. Oxidative stress can cause a direct activation of some types of PKC (11), but this has not yet been linked to Nox activity. Many different ways can be used to stimulate PKC and the other kinases capable of activating Nox, so it is highly unlikely that one single pathway exists between stress stimuli and Nox activation, which would lead to H₂O₂ production. This also shows that many ways exist to activate Nox proteins, and so illustrates that possibly many different types of stresses exist to which Nox could respond.

FIG. 4.

The possible pathways of activation of Nox1 and Nox2. (A) General activation pathways. (B) Specific examples of some of the factors involved (PLC, phospholipase C; DAG, diacylglycerol; PLA₂, phospholipase A₂).

The second key determinant of Nox activity, according to Decoursey and Ligeti (29), is the status of the Rac associated with the complex (Fig. 4), but so far this is proved for only Nox1 and Nox2. Rac is a small RhoGTPase and can be bound to guanosine diphosphate (GDP), making it inactive, or to guanosine triphosphate (GTP), making it active. Two groups of molecules control which of these is bound to Rac, guanine nucleotide exchange factors (GEFs), which promote the exchange from GDP to GTP, increasing the activity of Rac, whereas guanine-activating proteins (GAPs) do the opposite and switch it off. Seventy different GEFs and 80 GAPs are expressed in various cells (45), and so these might be quite cell-type and stimulus-type specific in their control of Nox activity. For example, Vav1 is upstream of Nox2 signaling in a murine microglial cell line (127), whereas Tiam1 plays a role in Nox-mediated signaling in keratinocytes (129), and βPix1 can bind to Nox1 and is involved in its activation (113, 114). Recently, Umoto et al. (157) showed that the clustering of Fc-γ-receptors on neutrophils resulted in the activation of Vav, and therefore also in Nox2 activation, illustrating nicely the role of GEFs in Nox activation.

Rac is known to play a role controlling actin through the formation of membrane ruffles (108); therefore, it might be that the activation of Rac binds it to p67^phox and also results in actin changes, thereby moving the entire complex to the membrane and inducing Nox activation. Rac activity is not necessarily the main or only way in which the complex could translocate, as both p47^phox and p40^phox bind moesin, which is an actin-binding protein, and so could also be responsible for the translocation (164). It has been shown that the phosphorylation of p47^phox and the activation of Rac are required in tandem for the maximal production of H₂O₂ through Nox2 (1) and Nox1 (20), and one publication even linked Vav to PKC(theta) activation (161), implying that it could have a dual role in Nox activation. Because of the large number of GEFs and GAPs, and the variability in their structures, it is difficult to pick out any common traits that might show which of them respond to specific stress stimuli and to determine how they would do so. This large number could also allow specific targeting of certain Nox proteins under certain conditions by therapeutics.

As mentioned earlier, little evidence exists of p47^phox or Rac playing a role in regulating Nox4 or Nox5 or Duox1/2 (40). This is reinforced by the recent data of Kao et al. (67), which showed that a conserved Rac binding site is found on Nox1, Nox2, and Nox3, but not on the others. For Nox5, Duox1 and Duox2 calcium triggers their generation of O₂ ^•− (7, 28, 33). In the case of Nox5, when calcium binds to the protein, the N-terminal undergoes a large conformational change, presumably then allowing the transfer of electrons (10). Calcium concentrations in the low micromolar range were enough to trigger activation of Nox5 (10) and Duox1 and Duox2 (5, 105). Therefore, any stimulus that increases intracellular calcium concentrations is likely to result in increased O₂ ^•−/H₂O₂ production. Nox5 may also be phosphorylated, during which it becomes more sensitive to calcium (63), and this phosphorylation may be under the control of PKC (135) or of c-Abl (37). When calmodulin binds to the opposite end of the molecule from the calcium-binding domains, sensitivity to calcium increases (151). A recent publication showed an interaction between Duox1 and NOXA1 and that this interaction is inhibitory of activity (111). Nox4 stands out as being constitutively active when expressed with p22^phox (47, 99) and capable of stimulation to produce extra O₂ ^•−/H₂O₂ (51, 81, 112), but as yet very little is known about its regulation and how it could be activated by a stress stimulus, even though it has been linked to several different signaling pathways in cells involving different stresses. So although many structural similarities are found between the Nox family members, the differences in their regulation make it highly unlikely that some overriding controlling factor exists for all Nox proteins and hence O₂ ^•−/H₂O₂. Conversely, it probably means that they can be stimulated by a variety of stressors and respond in subtle ways to varying therapeutic treatments.

When discussing H₂O₂ as a survival molecule, it is important to know how quickly the Nox signaling complex can be deactivated, as this determines the amount of H₂O₂ produced. The deactivation depends mainly on the dephosphorylation of p47^phox, typically accomplished by phosphatases and the binding of Rac to GDP, through the activity of GAPs. Although much work has shown that phosphatases are important (for review, see ref. 29), little progress has occurred in identifying which specific phosphatase(s) is involved, as most of the studies to date have used general phosphatase inhibitors. It is likely that protein phosphatase 1 and protein phosphatase 2B or both are involved (14). The second step is the nature of the nucleotide bound to Rac, which is controlled by GAPs, but little is known about how these interact with Nox2 (59, 148), and nothing with Nox1. Other regulatory mechanisms of Nox proteins are being discovered. A recent publication demonstrated that the binding of NOXA1 to 14-3-3 proteins results in the inhibition of Nox1 activity (73), and it is likely with time that more of these types of interactions will be revealed. It is assumed that once the calcium levels in cells decrease, the activity of Nox5, Duox1 and Duox2 also decreases. Again very little is known about the deactivation of Nox3 and Nox4. Overall the deactivation of the Nox signaling complex remains an underinvestigated area in the Nox field, but it could prove to be very important when trying to control the concentration of H₂O₂, and its possible effects, in cells.

In comparing studies on the prodeath against the prosurvival effects of Nox proteins, it becomes apparent that the final outcome on the cell is probably due to several factors (e.g., the amount of O₂ ^•−/H₂O₂ produced, the location of the O₂ ^•−/H₂O₂ production, and the duration of O₂ ^•−/H₂O₂ production). It is widely believed that a shorter/smaller production of H₂O₂ is prosurvival, whereas longer or larger amounts (or both) would be prodeath, and this was proved in a p53 system (130), but not yet for Nox proteins. One way in which H₂O₂ probably acts as a survival factor is through preconditioning, which relies on a system receiving a smaller stimulus before it experiences a major stress. A well-known example of this is ischemic preconditioning, in which the heart is stopped for a few times of short duration before it is stopped for a number of hours as a person goes onto a bypass machine during heart surgery (104). Nox activity has been shown to act as a preconditioning factor in a model of ischemic preconditioning (133). For a preconditioning stimulus to work, it must be of a similar nature to the main one, so it is likely that this prosurvival effect of H₂O₂ generated by Nox proteins is true only when the cell is under oxidative stress. We recently showed that this is the case in retinal cells, as a prosurvival burst of H₂O₂ occurs only when these cells are treated with an oxidative stress, and not with other types of stressors (96).

Other sources of ROS

As mentioned in the opening paragraph of this review, Nox proteins are not the only enzymes capable of forming ROS. The mitochondria are the organelles with the largest oxygen-consumption rate within cells and hence the largest producers of ROS, but because of their many redox systems, they contribute little to the overall redox status of cells (141). Only if mitochondria malfunction do they tend to play a role in the oxidation status of proteins elsewhere in the cell. Nonmitochondrial sources of ROS other than Nox proteins are known, but their biochemistry is less well studied, and so their relevance to survival signaling remains unknown. Overall, they tend to be expressed by specific cell types only. For example, xanthine oxidase is expressed mainly in the liver and epithelia and can produce O₂ ^•− and uric acid from xanthine and oxygen (25). Lipoxygenases, cyclooxygenases, and nitric oxide synthases tend to produce ROS indirectly, through the formation of reactive intermediates, when they form their main respective products of leukotrienes (80), prostaglandins (144), and nitric oxide (117). These intermediates can then interact with oxygen to form ROS. Nitric oxide synthase, in certain circumstances, may be uncoupled, and then it generates O₂ ^•− directly (160). The smaller space allocation in this review to these many other enzymes capable of generating ROS reflects simply the paucity of knowledge about them, particularly in relation to cell-survival signaling, but does not reflect their possible importance to this key cellular pathway.

H₂O₂-driven cell-survival pathways

Up to this point, the main sources of H₂O₂ in cells have been outlined, with particular focus on Nox proteins. To understand how H₂O₂ can act as a survival molecule, it is important to understand how it can interact with its target molecules, and this is the focus of the remaining sections. The scientific evidence to date is in two separate halves, reflected in the dichotomy of this review, with one half showing that Nox proteins can produce ROS, and the other demonstrating that ROS can promote cell-survival signaling. A limited number of articles bridge this gap and show precisely how Nox proteins can promote cell survival, and these are highlighted in the next section. However, all possible ways that H₂O₂ can interact with prosurvival downstream targets are mentioned, as it is likely that Nox-derived H₂O₂ can play a role in these events.

H₂O₂ affects cell-signaling pathways mainly through oxidation of specific molecules. If the concentration of H₂O₂ is high, then these oxidation processes may lead to irreversible damage, followed by cell death. However, this is not always the case, and H₂O₂ is capable of reversible inhibition of many proteins (i.e., phosphatases) along the main activation cascade of survival pathways (Fig. 5). First we provide a quick overview of the different survival pathways and how they can be redox regulated by phosphatases.

FIG. 5.

Redox-sensitive signaling pathways for regulation of cell survival. Many of the signaling protein are redox sensitive; they control survival at the level of transduction, transcription, or execution. Oxidative stress not only serves as a type of stimulus to trigger the stress-response signaling pathway, but it also modulates cell fate through direct oxidative modification of those signaling molecules.

Key survival pathways

Mitogen-activated protein kinases (MAPK) constitute a large kinase network that regulates physiologic processes such as cell growth, differentiation, and apoptosis. Three MAPK pathways have been characterized: the extracellular signal-regulated kinase (ERK) pathway, the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase, and the p38 pathway. MAPK operates in a cascade fashion with MAPKKK phosphorylating and activating MAPKK, which then activates MAPK. Generally ERK1/2 signaling promotes cell survival under mild oxidative stress, whereas JNK and p38 can induce apoptosis by involving direct phosphorylation of pro-/antiapoptotic Bcl-2 family members, transactivation of transcription factor AP-1, and stabilization of p53.

Signal transduction via phosphoinositide 3-kinase (PI3K) plays an important role in regulating proliferation, survival, and motility. A moderate level of ROS activates PI3K signaling and promotes cell survival. PI3K/protein kinase B (Akt) transduces the signal for cell survival mainly through phosphorylation of target molecules by Akt. This results in inactivation of proapoptotic proteins and activation of transcription factors that target the expression of antiapoptotic proteins.

Reliance on extracellular matrix (ECM) contact for cell survival is common in untransformed adherent cells; apoptosis induced by loss of ECM attachment is called anoikis and is a redox-regulated event (for a review, see ref. 24). Redox regulation of epidermal growth factor receptor (EGFR) signaling plays a crucial role in the protection from anoikis, as is shown in different cell types (49, 86, 119). During cell spreading, integrin activation through ECM-cell contact induces a significant increase in the intracellular level of ROS. This increase in ROS level is dependent on the activity of small GTPase Rac-1 (49), which could suggest that Nox1 or Nox2 is involved. The tyrosine kinase Src is the main target of the ROS and is consequently the main player in the crosstalk between ECM contact and generation of prosurvival signal. When Src is oxidized and fully activated, it allows the specific ligand-independent phosphorylation and activation of EGFR, downstream of which ERK and Akt signaling pathways are activated. They will eventually induce the phosphorylation of the proapoptotic protein Bim, which is then degraded, leading to the inhibition of apoptosis (116).

The survival pathways transduce their survival signal through phosphorylation of key proteins, as a consequence of which phosphatases are potent negative regulators, and, in particular, multiple steps along the PI3K/Akt survival pathway are negatively regulated by phosphatases.

Negative regulation by phosphatases

The phosphatase superfamily can be categorized into three smaller subfamilies based on substrate specificity: (a) the classic protein phosphatases, which are tyrosine specific (protein tyrosine phosphatases, PTPs); (b) the dual-specificity phosphatases (DSPs) which can dephosphorylate phosphotyrosine-, phosphoserine-, and phosphothreonine-containing substrates; and (c) serine/threonine (Ser/Thr)-specific phosphatases, which are further divided into two major classes (Fig. 6). Type I phosphatases include protein phosphatase (PP) 1; type II phosphatases include the spontaneously active phosphatases (e.g., PP2A), calcium-dependent phosphatases (e.g., PP2B), and magnesium-dependent (e.g., PP2C) classes of phosphatases.

FIG. 6.

Classification of the protein phosphatase superfamily. PP-superfamily can be categorized into three smaller subfamilies based on substrate specificity: (a) the classic protein phosphatase, which is a tyrosine-specific phosphatase (PTP); (b) the dual-specificity phosphatases (DSPs), which can dephosphorylate phosphotyrosine-, phosphoserine-, and phosphothreonine-containing susbstrate; and (c) serine/threonine (Ser/Thr)-specific phosphatases, which are further divided into two major classes. Type I phosphatase includes PP1; type II phosphatase includes spontaneous active phosphatase (PP2A), calcium-dependent (PP2B), or magnesium-dependent (PP2C) classes of phosphatases.

The regulation of cell function through redox-sensitive cysteine residues has been most convincingly demonstrated in PTPs. They are encoded by 38 genes in humans and belong to a larger family of cysteine-dependent phosphatases that totals approximately 100 human members and numerous pseudogenes (for review, see ref. 3). All PTPs contain an essential cysteine residue in the signature motif C(X)₅R that exists as a thiolate anion at neutral pH (30). This thiolate anion contributes to the formation of a thiol-phosphate intermediate in the catalytic mechanism of PTPs. Oxidation of the active-site cysteine of PTPs to a sulfenic derivative leads to enzymatic inactivation; however, this modification can be reversed by incubation with thiol compounds (Fig. 7). In some cases, the sulfhydryl group is open to further irreversible oxidation if no cysteine derivatives or thiols are close enough to facilitate the formation of a disulfide bridge. The addition of another oxygen molecule or two additional oxygen molecules results in the formation of sulfinic and sulfonic acid, respectively. These oxidative modifications are irreversible, and the phosphatase will be unable to become active again, even in a reducing environment (Fig. 7). It is highly likely that all phosphatases are sensitive to oxidative inhibition to some degree, as they all require a reduced cysteine for catalysis. Reversible oxidation was first demonstrated for PTP-1B during epidermal growth factor (87) and insulin (97) signaling. The same regulation was then demonstrated for low-molecular-weight PTP (LMW-PTP) during platelet-derived growth factor stimulation (23). Both PTP-1B and LMW-PTP rescued their phosphatase activity because of a re-reduction 30 min after receptor activation (12, 18). Very few articles in the literature are able to make a direct link between oxidation of PTP and Nox; nevertheless, it has been reported that the Src-homology 2 domain (SHP-2) PTP is reversibly oxidized in response to PDGF, and that requires association with the PDGFR (100). Because PDGF is involved in Nox1 regulation (69), it is likely that Nox is at least indirectly responsible for SHP-2 redox inactivation. More recently Sharma et al. (136) showed that IL-4 receptors can activate Nox1 and Nox5, thereby increasing ROS production, and consequently inactivate PTP-1B, which physically associates with and deactivates IL-4 receptors (136), thus revealing a role for ROS in cytokine crosstalk.

FIG. 7.

The regulation of cell function through redox-sensitive cysteine residues. There are different ways to inactivate the cysteine residue of a protein. Oxidation of the active-site cysteine to a sulfenic derivative leads to enzymatic inactivation. This modification can be reversed by incubation with thiol compounds. The addition of another oxygen molecule or two additional oxygen molecules results in the formation of sulfinic and sulfonic acid, respectively. These oxidative modifications are irreversible and will not become active again even in a reducing environment.

The dual specific phosphatases (DSP) also can be oxidized. In contrast with the classic PTP member like PTP-1B that forms a sulfenyl amide linkage between the active-site cysteine and an adjacent main-chain nitrogen (131), oxidation of phosphatase and tensin homologue (PTEN) in vitro with H₂O₂ leads to the formation of a disulfide bond between the active-site cysteine (cys-124) and another cysteine residue (cys-71) that is close by in its three-dimensional structure. This disulfide bond prevents further irreversible oxidation of the cysteine residues and PTEN can retain its phosphatase ability. PTEN has been demonstrated in many studies to be the main phosphatase negatively regulating the PI3K/Akt pathway. PTEN can dephosphorylate the lipid PIP₃ to PIP₂, preventing the recruitment of PH-containing proteins to the plasma membrane. This results in a decrease in the survival signal transduced by Akt. The importance of PTEN in regulating this pathway is highlighted by its classification as a tumor suppressor. Moreover, Savintsky et al. (134) provided evidence for the degradation of cdc25 phosphatase (another DSP), which is the result of H₂O₂ -induced disulfide bond formation between the active-site cysteine and another invariant cysteine residue.

The Ser/Thr phosphatases also are sensitive to redox modifications. It was demonstrated in vitro that H₂O₂ can reversibly block some of the main Ser/Thr phosphatases, such as PP2A, and PP1α (110, 122). PP1 and PP2A contain redox-sensitive cysteine residues (39, 55). Structure-based analysis has identified a potential disulfide oxidoreductase active site, Cys-X-X-Cys, in members of the PP1 subfamily (39). It is still not clear whether the oxidation of this pair of cysteine residues can result in PP1 inactivation in vivo. The Ser/Thr phosphatases (the main members being PP1, PP2A, PP2B, and PP2C) dephosphorylate serine and threonine, which are the main phosphorylation events in the transduction of the PI3K/Akt survival pathway. Various studies demonstrated links between these phosphatases and the PI3K/Akt pathway. For example, the calcium-activated PP2B has been shown to be a direct phosphatase of Akt, Gsk-3β, and Bad (76, 88, 165) (Fig. 8). PP1α has been shown to dephosphorylate Akt and Bad, while PP2A has been demonstrated to be a key Akt phosphatase. PP2A can dephosphorylate Akt on both threonine 308 and serine 473, blocking the PI3K/Akt pathway (Fig. 8). Immunoprecipitation studies have shown that PP2A can colocalize with Akt. Recently, a novel phosphatase belonging to the PP2C family of phosphatases was shown to dephosphorylate Akt on serine 473 only. This novel phosphatase, PHLPP, contains a PH domain that localizes it in the vicinity of activated Akt. Expression of PHLPP in cells derived from a small cell lung cancer that have constitutively active Akt (phosphorylated on serine 473 and threonine 409) resulted in a 50% decrease in phosphor-serine 473 Akt levels. This resulted in a correlating decrease in the phosphorylation levels of an Akt substrate and an increase in the basal levels of apoptosis in this cell line (44). These experiments highlight the importance of the phosphorylated serine residue in Akt signaling and demonstrate a clear link between the increased activity of PHLPP with an induction of apoptosis via the PI3K/Akt pathway. Inactivation of PP2A was shown in cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1 (55), which can both induce H₂O₂ production. TNF-α has been shown to regulate Nox1, Nox4 (168), and recently NOXO1 (83) and IL-1 were shown to enhance Nox2 expression, which can provide a direct link between Nox-generated ROS and inactivation of PP2A.

FIG. 8.

Redox regulation of PI3K/Akt. PI3K/Akt transduces the signal for cell survival mainly through phosphorylation of target molecules by Akt. Under oxidative stress, this pathway is activated by oxidative inactivation of cysteine in the active site of phosphatases, allowing constitutive activation of tyrosine kinase receptor and PI3K. This results in Akt phosphorylation, which in turn allows phosphorylation of several targets involved in survival mechanisms. Moreover, the PI3K/Akt pathway can promote cellular production of ROS through activation of Rac and NADPH oxidase.

Activation of transcription factors

Abundant evidence exists for the regulation of gene expression by ROS. Nox-dependent ROS have been shown to induce, for example, the expression of TNF-α (118), TGF-β1, and angiotensin II (56). Most studies investigating the mechanism of ROS-upregulated mRNA have shown that the upregulation can occur through redox-sensitive second-messenger systems [MAPK activation (50)] or through transcription factors including NF-κB, hypoxia-inducible factor (HIF)-1α, p53, and AP-1, which contain redox-sensitive cysteine residues in their DNA-binding domains (147). In most cases, thiol oxidation of these proteins inhibits their DNA-binding activities.

p53

p53 is a transcription factor as well as a tumor-suppressor gene. Recent work demonstrated how the redox potential of a tumor cell can be elevated during carcinogenesis, as a consequence of the loss of wild-type p53. However, under normal physiologic conditions, p53 was recently shown to be able to reduce the redox potential within a cell by regulating the expression of antioxidative genes such as glutathione peroxidase, SOD2, and the mammalian sestrins sestrin1 and sestrin2 (sestrins are involved in the regeneration of oxidized peroxiredoxin). This differential control of redox levels within a cell is attributed to the levels of p53 itself. Thus, low levels of p53 suppress ROS within the cell whereas higher levels of p53 promote ROS accumulation. Studies in p53^−/− mice have demonstrated that the antioxidant function of p53 may directly contribute to the prevention of tumor development (109, 130). In cancer cells deficient in wild-type p53, lack of the antioxidative properties of p53 has the ability to increase the redox stress within the cell, which in turn can increase the rate of mutagenicity and also modulate redox-sensitive pathways. This may have important implications in the activation of redox-sensitive survival pathways. For instance, Li et al. (91) showed that, in endothelial cells that express Nox2 and Nox4, Nox2-derived ROS participate in cell-cycle regulation and apoptosis through modulation of p21cip1 and p53 expression (91).

NF-κB

The heterodimeric protein NF-κB is a ubiquitous redox-regulated transcription factor that remains sequestered in the cytoplasm as an inactive complex with its inhibitory counterpart IκB. Exposure to oxidative stimuli leads to phosphorylation and subsequent proteasomal degradation of IκBα, thereby releasing free NF-κB dimers for translocation to the nucleus. Experimental evidence suggests that ROS have paradoxic effects on NF-κB regulation. ROS can either activate or inhibit NF-κB activity, depending on the ROS levels, types of stimuli, and cell types. A moderate level of ROS generally leads to NF-κB activation (Fig. 9). Conversely, a high level of ROS could inactivate NF-κB, leading to cell death. In the nucleus, direct oxidation of the redox-sensitive cysteine 62 of the p50 subunit inhibits its availability to bind DNA (153). This oxidation is reversible, and DNA binding can be restored. Besides direct structural modification, DNA-binding activity of NF-κB can be modulated by chromatin remodelling (121). Thus, the enzyme histone deacetylase (HDAC), which catalyzes the removal of an acetyl group from histone, can be inactivated by oxidative stress, allowing histone acetylation, chromatin uncoiling, and increased accessibility for NF-κB (120). In the cytosol, NF-κB is sequestered as a complex formed with its inhibitor IκB; its activation is then regulated by phosphorylation of NF-κB itself or phosphorylation of its inhibitor. Under certain conditions, H₂O₂ is able to activate NF-κB activity directly through phosphorylation of IκB-kinase (IκK) (64) or indirectly through activation of Akt or MEKK1 (or both), which then phosphorylates and activates IκK. The transactivation of NF-κB induced by Akt or MEKK1 has been shown to be really important in NF-κB antiapoptotic effects (107, 158). Active IκK phosphorylates IκB and liberates active NF-κB from the complex to translocate to the nucleus. Phosphorylated IκK is then degraded by the proteasome. Because the proteasome system is also redox sensitive, ROS can regulate NF-κB activity by affecting IκK stability. One example of the direct effect of Nox on NF-κB was shown using siRNA against Nox2, which decreased insulin-induced O₂ ^•− production and inhibited the turn-on of NF-κB as well as p38MAPK and ERK 1/2 (132).

FIG. 9.

Redox regulation of NF-κB. In the nucleus, direct oxidation of the cysteine in the DNA-binding domain can restrain NF-κB activity. In contrast, enzyme histone deacetylase (HDAC) can be inactivated by oxidative stress, allowing chromatin remodeling and increased accessibility for NF-κB. In cytosol, NF-κB and IκB form a complex that is inactive; NF-κB activation can be regulated through phosphorylation of NF-κB itself or phosphorylation of its inhibitor IκB. Increased ROS can activate IκB-kinase (IκK) either directly or indirectly through activation of Akt or MEKK1 or both. Phosphorylated IκK is thus activated and can phosphorylate IκB, which liberates active NF-κB from the complex and translocates to the nucleus. Phosphorylated IκB is then degraded by the proteasome. The proteasome system is also redox sensitive; ROS can then regulate NF-κB activity by affecting the stability of IκB.

AP-1

AP-1 is another redox-sensitive transcription factor that plays a critical role in both cell growth and apoptosis. Under specific conditions, AP-1 activation can lead to cell death, whereas under other circumstances, AP-1 can promote cell proliferation. The AP-1 family consists of several proteins, including Jun, Fos, Maf, and ATF subfamilies (6). Activation of AP-1 is regulated at both the transcript and protein levels. The MAPKs play a major role in controlling activation of AP-1 proteins through phosphorylation. All three classes of MAPKs are involved in regulation of AP-1. c-Fos is a substrate of ERK, and ATF2 is regulated by JNK and p38 kinases (68). Oxidative stress can activate c-Jun and ATF2 through phosphorylation by JNK and p38, respectively. Redox regulation of the JNK and p38 pathway is well described (for review, see refs. 154, 155). Like that of NF-κB, transcriptional activation of AP-1 can be regulated by chromatin remodelling (102). Oxidative stress is known to promote AP-1 activity through histone acetylation by inhibition of HDAC (120). Furthermore, the intracellular level of AP-1 can be regulated by redox-mediated mechanisms at the levels of transcription and protein turnover. Recent reports show that expression of c-Jun can be transcriptionally repressed by HDAC or degraded by the proteasome through MEKK1-induced ubiquitination. This downregulation of c-Jun plays an important role in apoptosis induction by oxidative stress (166, 167). As described for NF-κB, several binding sites for AP-1 have been shown on Nox family members [e.g., the 5' region of human Nox1 (82) gene and p67^phox]. Binding sites for AP-1 transcription factors were found in the first intron of p67^phox, and they are essential for p67^phox expression (46, 92).

HIF

Hypoxia-inducible factor (HIF) was first described as a transcription factor that regulates cellular metabolism under hypoxic stress. However, HIF also can be activated by nonhypoxic stimuli. Active HIF requires heterodimeric formation of two subunits, HIF-α and HIF-β, which then translocate to the nucleus. Under normoxia, a variety of stimuli stabilize HIF, in part through increased ROS/RNS production. However, attenuation of ROS by antioxidant molecule or by genetic downregulation of Nox4 was found to decrease HIF expression. (52, 77, 137). Under hypoxia, both ROS and RNS were found to inhibit HIF-1 DNA-binding activity and HIF-1 accumulation; however, the mechanisms are still unclear (17).

Caspases

The redox regulation of transcription factors and signal-transduction pathways may affect cell survival through a series of molecular processes resulting in the activation or inhibition of cell death–execution molecules. Under certain conditions, ROS may affect directly the activity of those cell-death effectors. Several apoptotic effectors are redox sensitive, and their functions can be directly modulated by intracellular ROS; these include caspases, the Bcl-2 family of proteins, and cytochrome c.

A hallmark of apoptosis is the activation of caspases, which requires sequential proteolysis of the initiator caspases and effector caspases. Apoptotic stimuli can trigger caspase activation through either the extrinsic (death receptor–mediated activation) or the intrinsic (mitochondria-mediated activation) pathways. Activation of the caspase cascade ultimately leads to the cleavage of different substrata, such as PARP, DNA fragmentation, and cell death. ROS can directly affect caspase function. The reduced state of the cysteine in the active site is necessary for the catalytic activity of caspases; thus, depending on the degree of intracellular oxidative stress, caspases can be activated or inhibited. By using different concentrations of exogenous H₂O₂, Hampton et al. (58) demonstrated that a low dose of H₂O₂ can activate caspases and induce apoptosis, but a high dose of H₂O₂ inhibits caspases, and cells undergo necrosis. A recent study by Peshavariya et al. (116) showed that in endothelial cells, Nox2 and Nox4 have two different roles. Although inhibition of Nox4 has no effect on caspase activation, Nox2 inhibition with siRNA caused an increase in caspase 3/7 activity. Therefore, in endothelial cells, Nox4-derived H₂O₂ promotes proliferation, whereas Nox2 maintains the cytoskeleton and prevents apoptosis to support cell survival.

Relevance to Disease

Most of the studies quoted to date have come either from cell-culture experiments, in which various elements of the survival pathways were artificially expressed, or from a totally in vitro situation, in which cell lysates only were used in the experiments. The question therefore arises whether H₂O₂ produced from Nox can act as a survival molecule in disease models. Nox proteins are already known to play a large role in a prodeath manner in certain diseases (for review, see ref. 13), particularly chronic granulomatous disease, in which a mutation in Nox2 or p22^phox leads to a loss of immune function in those with the disease. But in the last decade or so, more evidence has accumulated for their contribution to prosurvival cell signaling in certain situations. Vaquero et al. (159) showed that H₂O₂ generated by Nox4, in particular, is prosurvival in pancreatic cancer cells, which has since been confirmed in several studies (35, 101, 169). This also was found to be true of other cancer models, for example, Nox1 is increased in prostate cancer (93) and in certain colon cancers (43), and Nox5 is involved in hairy cell leukemia (66) and prostate cancer (16). This has also been found in hepatocytes expressing Nox1, 2, and 4 (103); astrocytes, in which Nox4 is important (94, 139); and in various cell types of the cardiovascular system (31). Even in monocytes and macrophages, ROS generated by Nox proteins are found to have a prosurvival effect (162).

By understanding precisely how the production of H₂O₂ is generated by Nox, we can begin to use this knowledge to control the pathway, and so help in the development of therapeutics. These proteins are of course regulated at a transcriptional level [reviewed in (13)], but this is outside the scope of this review. Overall, a subtle change in the level of Nox activity is likely to be the key, and not some overall inhibition/activation. Therapeutics, based on the information in this review, might be able (a) to stop Nox activity relatively quickly, so that only a short burst would occur; or (b) to generate a small burst of H₂O₂ some time before a known large oxidative stress were to happen. We are slowly getting glimpses as to how this might be possible. It is feasible that manipulating the two main controlling factors of Nox activation, which are the phosphorylation of p47^phox and activation of Rac, or their regulators, could control Nox activity with more finesse than by targeting Nox alone. This will be possible only for Nox1 and 2. In the case of Nox5, Duox1, and Duox2, this control would be through controlling calcium levels and also phosphorylation of the proteins involved. For Nox4, the most work is to be done to determine its regulators, and therefore how to control it in disease situations. The downstream targets of H₂O₂ could also be targeted, once they have been identified in individual diseases.

Concluding Remarks

The dual nature of ROS has recently emerged, showing that they are not necessarily destructive but can also be closely regulated molecules acting as second messengers in many different signaling pathways. H₂O₂ is one of the main ROS capable of acting as a second messenger because of it is relative stability. The Nox family of proteins plays a major role in the production of H₂O₂, which mediates the regulation of survival pathways. Nox proteins are multimeric proteins, with different subunits, such as p47^phox and p67^phox, which allows a very tight and quick regulation of H₂O₂ production. Although much is known about the archetypical Nox, Nox2, and its closest homologue, Nox1, much remains to be learned about the regulation of the newer members of the Nox family; for example, very little is known about any regulatory proteins associated with Nox4.

When considering the role of H₂O₂ as a survival molecule, it is important not only to determine its source, but also to understand how H₂O₂ interacts with its target molecules. The main way that H₂O₂ affects cell-signaling pathways is through oxidation of specific molecules. If the concentration of H₂O₂ is high, then these oxidation processes may lead to irreversible damage, followed by cell death. However, this is not always the case, and H₂O₂ is capable of reversible inhibition of many enzymes, including phosphatases. As a consequence of survival pathways transducing their survival signal through phosphorylation of key proteins, phosphatases are potent negative regulators. The PI3K/Akt survival pathway is a good example of such, as multiple steps are negatively regulated by phosphatases.

It is important to realize that ROS-mediated regulation of cell survival is a very complex cascade of events. Activation of Nox proteins will modulate ROS production, which will activate various intracellular signaling pathways, inducing survival factors such as insulin, PDGF, TNF-α, and Bcr-Abl. In turn, those survival factors have been shown to be Nox regulators producing complex feedback loops. A comprehensive understanding of the regulation of the redox regulation of survival pathways, as well as of the regulation of ROS production by Nox, is essential when considering ROS as survival molecules and eventually developing redox-based therapeutic strategies in the many ROS-related diseases.

Footnotes

Acknowledgments

The first two authors contributed equally to this work. This work was supported by Science Foundation Ireland, Children Leukemia Research Project, and the Irish Cancer Society.

Abbreviations Used

References

Abo

, Pick

, Hall

, Totty

, Teahan

, Segal

. Activation of the NADPH Gtp-binding protein P21rac1. Nature, 353:668–670. 1991.

Aebischer

, Pasche

, Jorg

. nanomolar arachidonic-acid influences the respiratory burst in eosinophils and neutrophils induced by Gtp-binding protein: a comparative study of the respiratory burst in bovine eosinophils and neutrophils. Eur J Biochem, 218:669–677. 1993.

Alonso

, Sasin

, Bottini

, Friedberg

, Osterman

, Godzik

, Hunter

, Dixon

, Mustelin

. Protein tyrosine phosphatases in the human genome. Cell, 117:699–711. 2004.

Ambasta

, Kumar

, Griendling

, Schmidt

, Busse

, Brandes

. Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem, 279:45935–45941. 2004.

Ameziane-El-Hassani

, Morand

, Boucher

, Frapart

, Apostolou

, Agnandji

, Gnidehou

, Ohayon

, Noel-Hudson

, Francon

, Lalaoui

, Virion

, Dupuy

. Dual oxidase-2 has an intrinsic Ca²⁺-dependent H₂O₂-generating activity. J Biol Chem, 280:30046–30054. 2005.

Angel

, Karin

. The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta, 1072:129–157. 1991.

Banfi

, Molnar

, Maturana

, Steger

, Hegedus

, Demaurex

, Krause

. A Ca²⁺-activated NADPH oxidase in testis, spleen, and lymph nodes. J Biol Chem, 276:37594–37601. 2001.

Banfi

, Clark

, Steger

, Krause

. Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. J Biol Chem, 278:3510–3513. 2003.

Banfi

, Malgrange

, Knisz

, Steger

, Dubois-Dauphin

, Krause

. NOX3, a superoxide-generating NADPH oxidase of the inner ear. J Biol Chem, 279:46065–46072. 2004.

10.

Banfi

, Tirone

, Durussel

, Knisz

, Moskwa

, Molnar

, Krause

, Cox

. Mechanism of Ca²⁺ activation of the NADPH oxidase 5 (NOX5) J Biol Chem, 279:18583–18591. 2004.

11.

Barnett

, Madgwick

, Takemoto

. Protein kinase C as a stress sensor. Cell Signal, 19:1820–1829. 2007.

12.

Barrett

, DeGnore

, Konig

, Fales

, Keng

, Zhang

, Yim

, Chock

. Regulation of PTP1B via glutathionylation of the active site cysteine 215. Biochemistry, 38:6699–6705. 1999.

13.

Bedard

, Krause

. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev, 87:245–313. 2007.

14.

Bengisgarber

, Gruener

. Involvement of protein-kinase-c and of protein phosphatase-1 and/or phosphatase-2a in p47 phox phosphorylation in formylmet-Leu-phe stimulated neutrophils: studies with selective inhibitors ro-31-8220 and alyculin-A. Cell Signal, 7:721–732. 1995.

15.

Bouin

, Grandvaux

, Vignais

, Fuchs

. p40(phox) is phosphorylated on threonine 154 and serine 315 during activation of the phagocyte NADPH oxidase: implication of a protein kinase C type kinase in the phosphorylation process. J Biol Chem, 273:30097–30103. 1998.

16.

Brar

, Corbin

, Kennedy

, Hemendinger

, Thornton

, Bommarius

, Arnold

, Whorton

, Sturrock

, Huecksteadt

, Quinn

, Krenitsky

, Ardie

, Lambeth

, Hoidal

. NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells. Am J Physiol Cell Physiol, 285:C353–C369. 2003.

17.

Brune

, Zhou

. The role of nitric oxide (NO) in stability regulation of hypoxia inducible factor-1alpha (HIF-1alpha) Curr Med Chem, 10:845–855. 2003.

18.

Caselli

, Marzocchini

, Camici

, Manao

, Moneti

, Pieraccini

, Ramponi

. The inactivation mechanism of low molecular weight phosphotyrosine-protein phosphatase by H₂O₂ . J Biol Chem, 273:32554–32560. 1998.

19.

Chen

, Kirber

, Xiao

, Yang

, Keaney

. Regulation of ROS signal transduction by NADPH oxidase 4 localization. J Cell Biol, 181:1129–1139. 2008.

20.

Cheng

, Diebold

, Hughes

, Lambeth

. Nox1-dependent reactive oxygen generation is regulated by Rac1. J Biol Chem, 281:17718–17726. 2006.

21.

Cheng

, Cao

, Xu

, Van Meir

, Lambeth

. Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene, 269:131–140. 2001.

22.

Cheng

, Lambeth

. NOXO1, regulation of lipid binding, localization, and activation of Nox1 by the phox homology (PX) domain. J Biol Chem, 279:4737–4742. 2004.

23.

Chiarugi

, Fiaschi

, Taddei

, Talini

, Giannoni

, Raugei

, Ramponi

. Two vicinal cysteines confer a peculiar redox regulation to low molecular weight protein tyrosine phosphatase in response to platelet-derived growth factor receptor stimulation. J Biol Chem, 276:33478–33487. 2001.

24.

Chiarugi

, Giannoni

. Anoikis: a necessary death program for anchorage-dependent cells. Biochem Pharmacol, 76:1352–1364. 2008.

25.

Chung

, Baek

, Song

, Kim

, Huh

, Shim

, Kim

, Lee

. Xanthine dehydrogenase xanthine oxidase and oxidative stress. Age, 20:127–140. 1997.

26.

Clark

, Volpp

, Leidal

, Nauseef

. Cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma-membrane during cell activation. J Clin Invest, 85:714–721. 1990.

27.

Curnutte

, Erickson

, Ding

, Badwey

. Reciprocal interactions between protein-kinase-c and components of the NADPH oxidase complex may regulate superoxide production by neutrophils stimulated with a phorbol ester. J Biol Chem, 269:10813–10819. 1994.

28.

De Deken

, Wang

, Many

, Costagliola

, Libert

, Vassart

, Dumont

, Miot

. Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family. J Biol Chem, 275:23227–23233. 2000.

29.

DeCoursey

, Ligeti

. Regulation and termination of NADPH oxidase activity. Cell Mol Life Sci, 62:2173–2193. 2005.

30.

Denu

, Dixon

. Protein tyrosine phosphatases: mechanisms of catalysis and regulation. Curr Opin Chem Biol, 2:633–641. 1998.

31.

Doerries

, Grote

, Hilfiker-Kleiner

, Luchtefeld

, Schaefer

, Holland

, Sorrentino

, Manes

, Schieffer

, Drexler

, Landmesser

. Critical role of the NAD(P)H oxidase subunit p47(phox) for left ventricular remodeling/dysfunction and survival after myocardial infarction. Circ Res, 100:894–903. 2007.

32.

Donko

, Peterfi

, Sum

, Leto

, Geiszt

. Dual oxidases. Phil Trans R Soc Lond B Biol Sci, 360:2301–2308. 2005.

33.

Dupuy

, Ohayon

, Valent

, Noel-Hudson

, Deme

, Virion

. Purification of a novel flavoprotein involved in the thyroid NADPH oxidase: cloning of the porcine and human cDNAs. J Biol Chem, 274:37265–37269. 1999.

34.

Dusi

, Rossi

. Activation of NADPH oxidase of human neutrophils involves the phosphorylation and the translocation of cytosolic p67phox. Biochem J, 296:367–371. 1993.

35.

Edderkaoui

, Hong

, Vaquero

, Lee

, Fischer

, Friess

, Buchler

, Lerch

, Pandol

, Gukovskaya

. Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase. Am J Physiol Gastrointest Liver Physiol, 289:G1137–G1147. 2005.

36.

Edens

, Sharling

, Cheng

, Shapira

, Kinkade

, Lee

, Edens

, Tang

, Sullards

, Flaherty

, Benian

, Lambeth

. Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox. J Cell Biol, 154:879–891. 2001.

37.

El Jamali

, Valente

, Lechleiter

, Gamez

, Pearson

, Nauseef

, Clark

. Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl. Free Radic Biol Med, 44:868–881. 2008.

38.

El Benna

, Dang

PMC

, Gaudry

, Fay

, Morel

, Hakim

, Gougerot Pocidalo

. Phosphorylation of the respiratory burst oxidase subunit p67(phox) during human neutrophil activation: regulation by protein kinase C-dependent and independent pathways. J Biol Chem, 272:17204–17208. 1997.

39.

Fetrow

, Siew

, Skolnick

. Structure-based functional motif identifies a potential disulfide oxidoreductase active site in the serine/threonine protein phosphatase-1 subfamily. FASEB J, 13:1866–1874. 1999.

40.

Fortemaison

, Miot

, Dumont

, Dremier

. Regulation of H₂O₂ generation in thyroid cells does not involve Rac1 activation. Eur J Endocrinol, 152:127–133. 2005.

41.

Freeman

, Lambeth

. NADPH oxidase activity is independent of p47(phox) in vitro (vol 271, pg 22578, 1996) J Biol Chem, 272:7566–7566. 1997.

42.

Fuchs

, Bouin

, Rabilloud

, Vignais

. The 40-kDa component of the phagocyte NADPH oxidase (p40phox) is phosphorylated during activation in differentiated HL60 cells. Eur J Biochem, 249:531–539. 1997.

43.

Fukuyama

, Rokutan

, Sano

, Miyake

, Shimada

, Tashiro

. Overexpression of a novel superoxide-producing enzyme, NADPH oxidase 1, in adenoma and well differentiated adenocarcinoma of the human colon. Cancer Lett, 221:97–104. 2005.

44.

Gao

, Furnari

, Newton

. PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol Cell, 18:13–24. 2005.

45.

Garcia-Mata

, Burridge

. Catching a GEF by its tail. Trends Cell Biol, 17:36–43. 2007.

46.

Gauss

, Bunger

, Quinn

. AP-1 is essential for p67(phox) promoter activity. J Leukoc Biol, 71:163–172. 2002.

47.

Geiszt

, Kopp

, Varnai

, Leto

. Identification of renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci U S A, 97:8010–8014. 2000.

48.

Geiszt

, Witta

, Baffi

, Lekstrom

, Leto

. Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense. FASEB J, 17:1502–1504. 2003.

49.

Giannoni

, Buricchi

, Grimaldi

, Parri

, Cialdai

, Taddei

, Raugei

, Ramponi

, Chiarugi

. Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival. Cell Death Differ, 15:867–878. 2008.

50.

, Gipp

, Mulcahy

, Jones

. H₂O₂-dependent activation of GCLC-ARE4 reporter occurs by mitogen-activated protein kinase pathways without oxidation of cellular glutathione or thioredoxin-1. J Biol Chem, 279:5837–5845. 2004.

51.

Gorin

, Ricono

, Kim

, Bhandari

, Choudhury

, Abboud

. Nox4 mediates angiotensin II-induced mesangial cell hypertrophy via activation of Akt/protein kinase B. J Am Soc Nephrol, 14:91A–91A. 2003.

52.

Gorlach

, Diebold

, Schini-Kerth

, Berchner-Pfannschmidt

, Roth

, Brandes

, Kietzmann

, Busse

. Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: role of the p22(phox)-containing NADPH oxidase. Circ Res, 89:47–54. 2001.

53.

Grasberger

, Refetoff

. Identification of the maturation factor for dual oxidase: evolution of a eukaryotic operon equivalent. J Biol Chem, 281:18269–18272. 2006.

54.

Groemping

, Lapouge

, Smerdon

, Rittinger

. Molecular basis of phosphorylation-induced activation of the NADPH oxidase. Cell, 113:343–355. 2003.

55.

Guy

, Philp

, Tan

. Activation of protein kinases and the inactivation of protein phosphatase 2A in tumour necrosis factor and interleukin-1 signal-transduction pathways. Eur J Biochem, 229:503–511. 1995.

56.

, Lee

. Reactive oxygen species amplify glucose signaling in renal cells cultured under high glucose and in diabetic kidney. Nephrology (Carlton), 10,suppl:S7–S10. 2005.

57.

Hampton

, Kettle

, Winterbourn

. Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils. Infect Immun, 64:3512–3517. 1996.

58.

Hampton

, Orrenius

. Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis. FEBS Lett, 414:552–556. 1997.

59.

Heyworth

, Knaus

, Settleman

, Curnutte

, Bokoch

. Regulation of NADPH oxidase activity by rac GTPase-activating protein(S) Mol Biol Cell, 4:1217–1223. 1993.

60.

Inanami

, Johnson

, McAdara

, El Benna

, Faust

LRP

, Newburger

, Babior

. Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47(phox) on serine 303 or 304. J Biol Chem, 273:9539–9543. 1998.

61.

Iyer

, Islam

, Quastel

. Biochemical aspects of phagocytosis. Nature, 192:535–542. 1961.

62.

Iyer

, Pearson

, Nauseef

, Clark

. Evidence for a readily dissociable complex of p47phox and p67phox in cytosol of unstimulated human neutrophils. J Biol Chem, 269:22405–22411. 1994.

63.

Jagnandan

, Church

, Banfi

, Stuehr

, Marrero

, Fulton

DJR

. Novel mechanism of activation of NADPH oxidase 5: calcium sensitization via phosphorylation. J Biol Chem, 282:6494–6507. 2007.

64.

Kamata

, Manabe

, Oka

, Kamata

, Hirata

. Hydrogen peroxide activates IkappaB kinases through phosphorylation of serine residues in the activation loops. FEBS Lett, 519:231–237. 2002.

65.

Kambayashi

, Takekoshi

, Tanino

, Watanabe

, Nakano

, Hitomi

, Takigawa

, Ogino

, Yamamoto

. Various molecular species of diacylglycerol hydroperoxide activate human neutrophils via PKC activation. J Clin Biochem Nutr, 41:68–75. 2007.

66.

Kamiguti

, Serrander

, Lin

, Harris

, Cawley

, Allsup

, Slupsky

, Krause

, Zuzel

. Expression and activity of NOX5 in the circulating malignant B cells of hairy cell leukemia. J Immunol, 175:8424–8430. 2005.

67.

Kao

, Gianni

, Bohl

, Taylor

, Bokoch

. Identification of a conserved Rac-binding site on NADPH oxidases supports a direct GTPase regulatory mechanism. J Biol Chem, 283:12736–12746. 2008.

68.

Karin

. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem, 270:16483–16486. 1995.

69.

Katsuyama

, Fan

, Yabe-Nishimura

. NADPH oxidase is involved in prostaglandin F2alpha-induced hypertrophy of vascular smooth muscle cells: induction of NOX1 by PGF2alpha. J Biol Chem, 277:13438–13442. 2002.

70.

Kawahara

, Ritsick

, Cheng

, Lambeth

. Point mutations in the proline-rich region of p22(phox) are dominant inhibitors of Nox1- and Nox2-dependent reactive oxygen generation. J Biol Chem, 280:31859–31869. 2005.

71.

Kawahara

, Quinn

, Lambeth

. Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes. BMC Evol Biol, 7:104. 2007.

72.

Khan

, Blob

, Hannun

. Arachidonic-acid and free fatty-acids as 2nd-messengers and the role of protein-kinase-C. Cell Signal, 7:171–184. 1995.

73.

Kim

, Diebold

, Babior

, Knaus

, Bokoch

. Regulation of Nox1 activity via protein kinase A-mediated phosphorylation of NoxA1 and 14-3-3 binding. J Biol Chem, 282:34787–34800. 2007.

74.

Kiss

, Knisz

, Zhang

, Baltrusaitis

, Sigmund

, Thalmann

, Smith

RJH

, Verpy

, Banfi

. Inactivation of NADPH oxidase organizer 1 results in severe imbalance. Curr Biol, 16:208–213. 2006.

75.

Klebanoff

. Myeloperoxidase: friend and foe. J Leukoc Biol, 77:598–625. 2005.

76.

Klumpp

, Maurer

, Zhu

, Aichele

, Pinna

, Krieglstein

. Protein kinase CK2 phosphorylates BAD at threonine-117. Neurochem Int, 45:747–752. 2004.

77.

Knowles

, Raval

, Harris

, Ratcliffe

. Effect of ascorbate on the activity of hypoxia-inducible factor in cancer cells. Cancer Res, 63:1764–1768. 2003.

78.

Koga

, Terasawa

, Nunoi

, Takeshige

, Inagaki

, Sumimoto

. Tetratricopeptide repeat (TPR) motifs of p67(phox) participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase. J Biol Chem, 274:25051–25060. 1999.

79.

Koshkin

, Lotan

, Pick

. The cytosolic component p47(phox) is not a sine qua non participant in the activation of NADPH oxidase but is required for optimal superoxide production. J Biol Chem, 271:30326–30329. 1996.

80.

Kuhn

, Thiele

. The diversity of the lipoxygenase family: many sequence data but little information on biological significance. FEBS Lett, 449:7–11. 1999.

81.

Kuroda

, Nakagawa

, Yamasaki

, Nakamura

, Takeya

, Kuribayashi

, Imajoh-Ohmi

, Igarashi

, Shibata

, Sueishi

, Sumimoto

. The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells. Genes Cells, 10:1139–1151. 2005.

82.

Kuwano

, Kawahara

, Yamamoto

, Teshima-Kondo

, Tominaga

, Masuda

, Kishi

, Morita

, Rokutan

. Interferon-gamma activates transcription of NADPH oxidase 1 gene and upregulates production of superoxide anion by human large intestinal epithelial cells. Am J Physiol Cell Physiol, 290:C433–C443. 2006.

83.

Kuwano

, Tominaga

, Kawahara

, Sasaki

, Takeo

, Nishida

, Masuda

, Kawai

, Teshima-Kondo

, Rokutan

. Tumor necrosis factor alpha activates transcription of the NADPH oxidase organizer 1 (NOXO1) gene and upregulates superoxide production in colon epithelial cells. Free Radic Biol Med, 45:1642–1652. 2008.

84.

Lambeth

, Kawahara

, Diebold

. Regulation of Nox and Duox enzymatic activity and expression. Free Radic Biol Med, 43:319–331. 2007.

85.

Lapouge

, Smith

SJM

, Walker

, Gamblin

, Smerdon

, Rittinger

. Structure of the TPR domain of p67(phox) in complex with Rac center dot GTP. Mol Cell, 6:899–907. 2000.

86.

Le Gall

, Chambard

, Breittmayer

, Grall

, Pouyssegur

, Van Obberghen-Schilling

. The p42/p44 MAP kinase pathway prevents apoptosis induced by anchorage and serum removal. Mol Biol Cell, 11:1103–1112. 2000.

87.

Lee

, Kwon

, Kim

, Rhee

. Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor. J Biol Chem, 273:15366–15372. 1998.

88.

Lee

, Seo

, Kim

, Kang

, Kim

, Juhnn

. Membrane depolarization induces the undulating phosphorylation/dephosphorylation of glycogen synthase kinase 3beta, and this dephosphorylation involves protein phosphatases 2A and 2B in SH-SY5Y human neuroblastoma cells. J Biol Chem, 280:22044–2252. 2005.

89.

Lemarie

, Bourdonnay

, Morzadec

, Fardel

, Lernhet

. Inorganic arsenic activates reduced NADPH oxidase in human primary macrophages through a Rho kinase/p38 kinase pathway. J Immunol, 180:6010–6017. 2008.

90.

Leto

, Adams

, Demendez

. Assembly of the phagocyte NADPH oxidase: binding of Src homology-3 domains to proline-rich targets. Proc Natl Acad Sci U S A, 91:10650–10654. 1994.

91.

, Fan

, George

, Brooks

. Nox2 regulates endothelial cell cycle arrest and apoptosis via p21cip1 and p53. Free Radic Biol Med, 43:976–986. 2007.

92.

, Valente

, Wang

, Gamez

, Clark

. Transcriptional regulation of the p67phox gene: role of AP-1 in concert with myeloid-specific transcription factors. J Biol Chem, 276:39368–39378. 2001.

93.

Lim

, Sun

, Lambeth

, Marshall

, Amin

, Chung

, Petros

, Arnold

. Increased NOX1 and hydrogen peroxide in prostate cancer. Prostate, 62:200–207. 2005.

94.

Liu

, Kang

, Zheng

. NADPH oxidase produces reactive oxygen species and maintains survival of rat astrocytes. Cell Biochem Funct, 23:93–100. 2005.

95.

Lomax

, Leto

, Nunoi

, Gallin

, Malech

. Recombinant 47-kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease. Science, 245:409–412. 1989.

96.

Mackey

, Sanvicens

, Groeger

, Doonan

, Wallace

, Cotter

. Redox survival signaling in retina-derived 661W cells. Cell Death Differ, 15:1291–1303. 2008.

97.

Mahadev

, Zilbering

, Zhu

, Goldstein

. Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade. J Biol Chem, 276:21938–21942. 2001.

98.

Malle

, Furtmuller

, Sattler

, Obinger

. Myeloperoxidase: a target for new drug development? Br J Pharmacol, 152:838–854. 2007.

99.

Martyn

, Frederick

, von Loehneysen

, Dinauer

, Knaus

. Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases. Cell Signal, 18:69–82. 2006.

100.

Meng

, Fukada

, Tonks

. Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol Cell, 9:387–399. 2002.

101.

Mochizuki

, Furuta

, Mitsushita

, Shang

Ito

, Yokoo

, Yamaura

, Ishizone

, Nakayama

, Konagai

, Hirose

, Kiyosawa

, Kamata

. Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells. Oncogene, 25:3699–3707. 2006.

102.

Monks

, Xie

, Tikoo

, Lau

. ROS-induced histone modifications and their role in cell survival and cell death. Drug Metab Rev, 38:755–767. 2006.

103.

Murillo

, Carmona-Cuenca

, Del Castillo

, Ortiz

, Roncero

, Sanchez

, Fernandez

, Fabregat

. Activation of NADPH oxidase by transforming growth factor-beta in hepatocytes mediates up-regulation of epidermal growth factor receptor ligands through a nuclear factor-kappa B-dependent mechanism. Biochem J, 405:251–259. 2007.

104.

Murry

, Jennings

, Reimer

. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation, 74:1124–1136. 1986.

105.

Nakamura

, Ogihara

, Ohtaki

. Activation by Atp of calcium-dependent NADPH-oxidase generating hydrogen-peroxide in thyroid plasma-membranes. J Biochem (Tokyo), 102:1121–1132. 1987.

106.

Nakamura

, Makino

, Tanaka

, Ishimura

, Ohtaki

. Mechanism of H₂O₂ production in porcine thyroid-cells: evidence for intermediary formation of superoxide anion by NADPH-dependent H₂O₂-generating machinery. Biochemistry, 30:4880–4886. 1991.

107.

Nawata

, Yujiri

, Nakamura

, Ariyoshi

, Takahashi

, Sato

, Oka

, Tanizawa

. MEK kinase 1 mediates the antiapoptotic effect of the Bcr-Abl oncogene through NF-kappaB activation. Oncogene, 22:7774–7780. 2003.

108.

Nobes

, Hall

. Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell, 81:53–62. 1995.

109.

O'Connor

, Wallace

, O'Brien

, Cotter

. A novel antioxidant function for the tumor-suppressor gene p53 in the retinal ganglion cell. Invest Ophthalmol Vis Sci, 49:4237–4244. 2008.

110.

O'Loghlen

, Perez-Morgado

, Salinas

, Martin

. Reversible inhibition of the protein phosphatase 1 by hydrogen peroxide: potential regulation of eIF2 alpha phosphorylation in differentiated PC12 cells. Arch Biochem Biophys, 417:194–202. 2003.

111.

Pacquelet

, Lehmann

, Luxen

, Regazzoni

, Frausto

, Noack

, Knaus

. Inhibitory action of NoxA1 on dual oxidase activity in airway cells. J Biol Chem, 283:24649–24658. 2008.

112.

Park

, Jung

, Park

, Kim

, Lee

, Bae

. Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B. J Immunol, 173:3589–3593. 2004.

113.

Park

, Lee

, Park

, Lee

, Ryu

, Lee

, Rhee

, Bae

. Sequential activation of phosphatidylinositol 3-kinase, beta Pix, Rac1, and Nox1 in growth factor-induced production of H₂O₂ . Mol Cell Biol, 24:4384–4394. 2004.

114.

Park

, Park

, Bae

. Molecular interaction of NADPH oxidase 1 with beta Pix and Nox organizer 1. Biochem Biophys Res Commun, 339:985–990. 2006.

115.

Park

, Babior

. Activation of the leukocyte NADPH oxidase subunit p47(phox) by protein kinase C: a phosphorylation-dependent change in the conformation of the C-terminal end of p47(phox) Biochemistry, 36:11292–11292. 1997.

116.

Peshavariya

, Dusting

, Jiang

, Halmos

, Sobey

, Drummond

, Selemidis

. NADPH oxidase isoform selective regulation of endothelial cell proliferation and survival. Naunyn Schmiedebergs Arch Pharmacol, 380:193–204. 2009.

117.

Puddu

, Puddu

, Cravero

, Rosati

, Muscari

. The molecular sources of reactive oxygen species in hypertension. Blood Press, 17:70–77. 2008.

118.

Qin

, Liu

, Qian

, Hong

, Block

. Microglial NADPH oxidase mediates leucine enkephalin dopaminergic neuroprotection. Ann N Y Acad Sci, 1053:107–120. 2005.

119.

Quadros

, Peruzzi

, Kari

, Rodeck

. Complex regulation of signal transducers and activators of transcription 3 activation in normal and malignant keratinocytes. Cancer Res, 64:3934–3939. 2004.

120.

Rahman

, Gilmour

, Jimenez

, MacNee

. Oxidative stress and TNF-alpha induce histone acetylation and NF-kappaB/AP-1 activation in alveolar epithelial cells: potential mechanism in gene transcription in lung inflammation. Mol Cell Biochem, 234–235:239–248. 2002.

121.

Rahman

, Marwick

, Kirkham

. Redox modulation of chromatin remodeling: impact on histone acetylation and deacetylation, NF-kappaB and pro-inflammatory gene expression. Biochem Pharmacol, 68:1255–1267. 2004.

122.

Rao

, Clayton

. Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation. Biochem Biophys Res Commun, 293:610–616. 2002.

123.

Reeves

, Dekker

, Forbes

, Wientjes

, Grogan

, Pappin

DJC

, Segal

. Direct interaction between p47(phox) and protein kinase C: evidence for targeting of protein kinase C by p47(phox) in neutrophils. Biochem J, 344:859–866. 1999.

124.

Regier

, Waite

, Wallin

, McPhail

. A phosphatidic acid-activated protein kinase and conventional protein kinase C isoforms phosphorylate p22(phox) NADPH oxidase component. J Biol Chem, 274:36601–36608. 1999.

125.

Regier

, Greene

, Sergeant

, Jesaitis

, McPhail

. Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms: correlation of NADPH oxidase activity and p22phox phosphorylation. J Biol Chem, 275:28406–28412. 2000.

126.

Rhee

. H₂O₂, a necessary evil for cell signaling. Science, 312:1882–1883. 2006.

127.

Roepstorff

, Rasmussen

, Sawada

, Cudre-Maroux

, Salmon

, Bokoch

, van Deurs

, Vilhardt

. Stimulus-dependent regulation of the phagocyte NADPH oxidase by a VAV1, Rac1, and PAK1 signaling axis. J Biol Chem, 283:7983–7993. 2008.

128.

Royerpokora

, Kunkel

, Monaco

, Goff

, Newburger

, Baehner

, Cole

, Curnutte

, Orkin

. Cloning the gene for an inherited human disorder—chronic granulomatous disease—on the basis of its chromosomal location. Nature, 322:32–38. 1986.

129.

Rygiel

, Mertens

, Strumane

, van der Kammen

, Collard

. The Rac activator Tiam1 prevents keratinocyte apoptosis by controlling ROS-mediated ERK phosphorylation. J Cell Sci, 121:1183–1192. 2008.

130.

Sablina

, Budanov

, Ilyinskaya

, Agapova

, Kravchenko

, Chumakov

. The antioxidant function of the p53 tumor suppressor. Nat Med, 11:1306–1313. 2005.

131.

Salmeen

, Barford

. Functions and mechanisms of redox regulation of cysteine-based phosphatases. Antioxid Redox Signal, 7:560–577. 2005.

132.

San Jose

, Bidegain

, Robador

, Diez

, Fortuno

, Zalba

. Insulin-induced NADPH oxidase activation promotes proliferation and matrix metalloproteinase activation in monocytes/macrophages. Free Radic Biol Med, 46:1058–1067. 2009.

133.

Sanchez

, Escobar

, Pedrozo

, Macho

, Domenech

, Hartel

, Hidalgo

, Donoso

. Exercise and tachycardia increase NADPH oxidase and ryanodine receptor-2 activity: possible robe in cardioprotection. Cardiovasc Res, 77:380–386. 2008.

134.

Savitsky

, Finkel

. Redox regulation of Cdc25C. J Biol Chem, 277:20535–20540. 2002.

135.

Serrander

, Jaquet

, Bedard

, Plastre

, Hartley

, Arnaudeau

, Demaurex

, Schlegel

, Krause

. NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation. Biochimie, 89:1159–1167. 2007.

136.

Sharma

, Chakraborty

, Wang

, Min

, Tremblay

, Kawahara

, Lambeth

, Haque

. Redox regulation of interleukin-4 signaling. Immunity, 29:551–564. 2008.

137.

Shatrov

, Sumbayev

, Zhou

, Brune

. Oxidized low-density lipoprotein (oxLDL) triggers hypoxia-inducible factor-1alpha (HIF-1alpha) accumulation via redox-dependent mechanisms. Blood, 101:4847–4849. 2003.

138.

Shiose

, Kuroda

, Tsuruya

, Hirai

, Hirakata

, Naito

, Hattori

, Sakaki

, Sumimoto

. A novel superoxide-producing NAD(P)H oxidase in kidney. J Biol Chem, 276:1417–1423. 2001.

139.

Shono

, Yokoyama

, Uesaka

, Kuroda

, Takeya

, Yamasaki

, Amano

, Mizoguchi

, Suzuki

, Niiro

, Miyamoto

, Akashi

, Iwaki

, Sumimoto

, Sasaki

. Enhanced expression of NADPH oxidase Nox4 in human gliomas and its roles in cell proliferation and survival. Int J Cancer, 123:787–792. 2008.

140.

Silverman

, Drazen

. The biology of 5-lipoxygenase: function, structure, and regulatory mechanisms. Proc Assoc Am Physicians, 111:525–536. 1999.

141.

St-Pierre

, Buckingham

, Roebuck

, Brand

. Topology of superoxide production from different sites in the mitochondrial electron transport chain. J Biol Chem, 277:44784–44790. 2002.

142.

Sugawara

, Sugawara

, Wen

, Giulivi

. Generation of oxygen free radicals in thyroid cells and inhibition of thyroid peroxidase. Exp Biol Med, 227:141–146. 2002.

143.

Suh

, Arnold

, Lassegue

, Shi

, Xu

, Sorescu

, Chung

, Griendling

, Lambeth

. Cell transformation by the superoxide-generating oxidase Mox1. Nature, 401:79–82. 1999.

144.

Suleyman, H, Demircan

, Karagoz

. Anti-inflammatory and side effects of cyclooxygenase inhibitors. Pharmacol Rep, 59:247–258. 2007.

145.

Sumimoto

, Miyano

, Takeya

. Molecular composition and regulation of the Nox family NAD(P)H oxidases. Biochem Biophys Res Commun, 338:677–686. 2005.

146.

Sumimoto

. Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species. FEBS J, 275:3249–3277. 2008.

147.

Sun

, Oberley

. Redox regulation of transcriptional activators. Free Radic Biol Med, 21:335–348. 1996.

148.

Szaszi

, Korda

, Wolfl

, Paclet

, Morel

, Ligeti

. Possible role of Rac-GTPase-activating protein in the termination of superoxide production in phagocytic cells. Free Radic Biol Med, 27:764–772. 1999.

149.

Takeya

, Ueno

, Kami

, Taura

, Kohjima

, Izaki

, Nunoi

, Sumimoto

. Novel human homologues of p47(phox) and p67(phox) participate in activation of superoxide-producing NADPH oxidases. J Biol Chem, 278:25234–25246. 2003.

150.

Teahan

, Rowe

, Parker

, Totty

, Segal

. The X-linked chronic granulomatous disease gene codes for the beta-chain of cytochrome-b-245. Nature, 327:720–721. 1987.

151.

Tirone

, Cox

. NADPH oxidase 5 (NOX5) interacts with and is regulated by calmodulin. FEBS Lett, 581:1202–1208. 2007.

152.

Tithof

, Peters-Golden

, Ganey

. Distinct phospholipases A(2) regulate the release of arachidonic acid for eicosanoid production and superoxide anion generation in neutrophils. J Immunol, 160:953–960. 1998.

153.

Toledano

, Leonard

. Modulation of transcription factor NF-kappa B binding activity by oxidation-reduction in vitro. Proc Natl Acad Sci U S A, 88:4328–4332. 1991.

154.

Torres

. Mitogen-activated protein kinase pathways in redox signaling. Front Biosci, 8:d369–d391. 2003.

155.

Trachootham

, Lu

, Ogasawara

, Nilsa

, Huang

. Redox regulation of cell survival. Antioxid Redox Signal, 10:1343–1374. 2008.

156.

Ueno

, Takeya

, Miyano

, Kikuchi

, Sumimoto

. The NADPH oxidase Nox3 constitutively produces superoxide in a p22(Phox)-dependent manner. J Biol Chem, 280:23328–23339. 2005.

157.

Utomo

, Cullere

, Glogauer

, Swat

, Mayadas

. Vav proteins in neutrophils are required for Fc gamma R-mediated signaling to Rac GTPases and nicotinamide adenine dinucleotide phosphate oxidase component p40(phox) J Immunol, 177:6388–6397. 2006.

158.

Vandermoere

, El Yazidi-Belkoura

, Adriaenssens

, Lemoine

, Hondermarck

. The antiapoptotic effect of fibroblast growth factor-2 is mediated through nuclear factor-kappaB activation induced via interaction between Akt and IkappaB kinase-beta in breast cancer cells. Oncogene, 24:5482–5491. 2005.

159.

Vaquero

, Edderkaoui

, Pandol

, Gukovsky

, Gukovskaya

. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells. J Biol Chem, 279:34643–34654. 2004.

160.

Vasquez-Vivar

, Kalyanaraman

, Martasek

, Hogg

, Masters

BSS

, Karoui

, Tordo

, Pritchard

. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A, 95:9220–9225. 1998.

161.

Villalba

, Bi

, Hu

, Altman

, Bushway

, Reits

, Neefjes

, Beire

, Abraham

, Altman

. Translocation of PKC0 in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C. J Cell Biol, 157:253–263. 2002.

162.

Wang

, Zeigler

, Lam

, Hunter

, Eubank

, Khramtsov

, Tridandapani

, Sen

, Marsh

. The role of the NADPH oxidase complex, p38 MAPK, and Akt in regulating human monocyte/macrophage survival. Am J Respir Cell Mol Biol, 36:68–77. 2007.

163.

Wientjes

, Hsuan

, Totty

, Segal

. P40(phox), a 3rd cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. Biochem J, 296:557–561. 1993.

164.

Wientjes

, Reeves

, Soskic

, Furthmayr

, Segal

. The NADPH oxidase components p47(phox) and p40(phox) bind to moesin through their PX domain. Biochem Biophys Res Commun, 289:382–388. 2001.

165.

, Zu

, Warren

, Wallace

, Ip

. Delineating the mechanism by which selenium deactivates Akt in prostate cancer cells. Mol Cancer Ther, 5:246–252. 2006.

166.

Xia

, Wang

, Liu

, Yung

, Hunter

, Lu

. c-Jun downregulation by HDAC3-dependent transcriptional repression promotes osmotic stress-induced cell apoptosis. Mol Cell, 25:219–232. 2007.

167.

Xia

, Wang

, Xu

, Johnson

, Hunter

, Lu

. MEKK1 mediates the ubiquitination and degradation of c-Jun in response to osmotic stress. Mol Cell Biol, 27:510–517. 2007.

168.

Yoshida

, Tsunawaki

. Expression of NADPH oxidases and enhanced H(2)O(2)-generating activity in human coronary artery endothelial cells upon induction with tumor necrosis factor-alpha. Int Immunopharmacol, 8:1377–1385. 2008.

169.

, Kim

. Diphenyleneiodonium suppresses apoptosis in cerulein-stimulated pancreatic acinar cells. Int J Biochem Cell Biol, 39:2063–2075. 2007.

Hydrogen Peroxide as a Cell-Survival Signaling Molecule

Abstract

Introduction

The Nox family

The regulation of Nox proteins

Nox subunits

Upstream regulation of Nox proteins

Other sources of ROS

H2O2-driven cell-survival pathways

Key survival pathways

Negative regulation by phosphatases

Activation of transcription factors

p53

NF-κB

AP-1

HIF

Caspases

Relevance to Disease

Concluding Remarks

Footnotes

Acknowledgments

Abbreviations Used

References

H₂O₂-driven cell-survival pathways